Applications of copolymer for rapid identification of bacteria in blood culture broths using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

Ashizawa, Kazuho; Murata, Syota; Terada, Takashi; Ito, Daisuke; Bunya, Masaru; Watanabe, Koji; Teruuchi, Yoko; Tsuchida, Sachio; Satoh, Mamoru; Nishimura, Motoi; Matsushita, Kazuyuki; Sugama, Yuji; Nomura, Fumio

2017-08-01

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be used to identify pathogens in blood culture samples. However, sample pretreatment is needed for direct identification of microbes in blood culture bottles. Conventional protocols are complex and time-consuming. Therefore, in this study, we developed a method for collecting bacteria using polyallylamine-polystyrene copolymer for application in wastewater treatment technology. Using representative bacterial species Escherichia coli and Staphylococcus capitis, we found that polyallylamine-polystyrene can form visible aggregates with bacteria, which can be identified using MALDI-TOF MS. The processing time of our protocol was as short as 15min. Hemoglobin interference in MALDI spectra analysis was significantly decreased in our method compared with the conventional method. In a preliminary experiment, we evaluated the use of our protocol to identify clinical isolates from blood culture bottles. MALDI-TOF MS-based identification of 17 strains from five bacterial species (E. coli, Klebsiella pneumoniae, Enterococcus faecalis, S. aureus, and S. capitis) collected by our protocol was satisfactory. Prospective large-scale studies are needed to further evaluate the clinical application of this novel and simple method of collecting bacteria in blood culture bottles. Copyright © 2017 Elsevier B.V. All rights reserved.

A real-time, quantitative PCR protocol for assessing the relative parasitemia of Leucocytozoon in waterfowl

USGS Publications Warehouse

Smith, Matthew M.; Schmutz, Joel A.; Apelgren, Chloe; Ramey, Andy M.

2015-01-01

Microscopic examination of blood smears can be effective at diagnosing and quantifying hematozoa infections. However, this method requires highly trained observers, is time consuming, and may be inaccurate for detection of infections at low levels of parasitemia. To develop a molecular methodology for identifying and quantifying Leucocytozoon parasite infection in wild waterfowl (Anseriformes), we designed a real-time, quantitative PCR protocol to amplify Leucocytozoon mitochondrial DNA using TaqMan fluorogenic probes and validated our methodology using blood samples collected from waterfowl in interior Alaska during late summer and autumn (n = 105). By comparing our qPCR results to those derived from a widely used nested PCR protocol, we determined that our assay showed high levels of sensitivity (91%) and specificity (100%) in detecting Leucocytozoon DNA from host blood samples. Additionally, results of a linear regression revealed significant correlation between the raw measure of parasitemia produced by our qPCR assay (Ct values) and numbers of parasites observed on blood smears (R2 = 0.694, P = 0.003), indicating that our assay can reliably determine the relative parasitemia levels among samples. This methodology provides a powerful new tool for studies assessing effects of haemosporidian infection in wild avian species.

Erythrocyte deformability and aggregation responses to intermittent and continuous artificial gravity exposure

NASA Astrophysics Data System (ADS)

Marijke, Grau; Vera, Abeln; Tobias, Vogt; Wilhelm, Bloch; Stefan, Schneider

2017-02-01

Artificial gravity protocols are used to improve g-tolerance of aviators and discussed as countermeasure during prolonged space flight. Little is known about the impact of artificial gravity on the red blood cells (RBC). The purpose of the study was to test how artificial gravity affects RBC deformability and aggregation, which are important determinants of microcirculation. Nine male subjects were exposed to two hypergravity protocols using a short arm human centrifuge: a continuous (CONT) protocol with constant +2 Gz for 30 min and an intermittent (INTER) protocol with repeated intervals of +2 Gz and rest. Blood was sampled pre and post interventions to measure basal blood parameters, RBC nitrite, RBC deformability, aggregation, and to determine the shear rate balancing aggregation and disaggregation (γ at dIsc min). To test for orthostasis effects, five male subjects were asked to stay for 46 min, corresponding to the length of the centrifuge protocols, with blood sampling pre and post intervention. Artificial gravity programs did not affect basal blood parameters or RBC nitrite levels; a marker for RBC deformability influencing nitric oxide. The INTER program did not affect any of the tested parameters. The CONT program did not remarkably affect RBC deformability or γ at dIsc min but significantly aggravated aggregation. Orthostasis effects were thus excluded. The results indicate that continuous artificial gravity, especially with higher g-forces applied, may negatively affect the RBC system and that for a prolonged space flight intermittent but not continuous artificial gravity might represent an appropriate countermeasure.

An affordable method to obtain cultured endothelial cells from peripheral blood

PubMed Central

Bueno-Betí, Carlos; Novella, Susana; Lázaro-Franco, Macarena; Pérez-Cremades, Daniel; Heras, Magda; Sanchís, Juan; Hermenegildo, Carlos

2013-01-01

The culture of endothelial progenitor cells (EPC) provides an excellent tool to research on EPC biology and vascular regeneration and vasculogenesis. The use of different protocols to obtain EPC cultures makes it difficult to obtain comparable results in different groups. This work offers a systematic comparison of the main variables of most commonly used protocols for EPC isolation, culture and functional evaluation. Peripheral blood samples from healthy individuals were recovered and mononuclear cells were cultured. Different recovery and culture conditions were tested: blood volume, blood anticoagulant, coating matrix and percentage of foetal bovine serum (FBS) in culture media. The success of culture procedure, first colonies of endothelial cells appearance time, correlation with number of circulating EPC (cEPC) and functional comparison with human umbilical vein endothelial cells (HUVEC) were studied. The use of heparin, a minimum blood volume of 30 ml, fibronectin as a coating matrix and endothelial growing media-2 supplemented with 20% FBS increased the success of obtaining EPC cultures up to 80% of the processed samples while reducing EPC colony appearance mean time to a minimum of 13 days. Blood samples exhibiting higher cEPC numbers resulted in reduced EPC colony appearance mean time. Cells isolated by using this combination were endothelial cell-like EPCs morphological and phenotypically. Functionally, cultured EPC showed decreased growing and vasculogenic capacity when compared to HUVEC. Thus, above-mentioned conditions allow the isolation and culture of EPC with smaller blood volumes and shorter times than currently used protocols. PMID:24118735

Performance of Identifiler Direct and PowerPlex 16 HS on the Applied Biosystems 3730 DNA Analyzer for processing biological samples archived on FTA cards.

PubMed

Laurin, Nancy; DeMoors, Anick; Frégeau, Chantal

2012-09-01

Direct amplification of STR loci from biological samples collected on FTA cards without prior DNA purification was evaluated using Identifiler Direct and PowerPlex 16 HS in conjunction with the use of a high throughput Applied Biosystems 3730 DNA Analyzer. In order to reduce the overall sample processing cost, reduced PCR volumes combined with various FTA disk sizes were tested. Optimized STR profiles were obtained using a 0.53 mm disk size in 10 μL PCR volume for both STR systems. These protocols proved effective in generating high quality profiles on the 3730 DNA Analyzer from both blood and buccal FTA samples. Reproducibility, concordance, robustness, sample stability and profile quality were assessed using a collection of blood and buccal samples on FTA cards from volunteer donors as well as from convicted offenders. The new developed protocols offer enhanced throughput capability and cost effectiveness without compromising the robustness and quality of the STR profiles obtained. These results support the use of these protocols for processing convicted offender samples submitted to the National DNA Data Bank of Canada. Similar protocols could be applied to the processing of casework reference samples or in paternity or family relationship testing. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Blood gas testing and related measurements: National recommendations on behalf of the Croatian Society of Medical Biochemistry and Laboratory Medicine.

PubMed

Dukić, Lora; Kopčinović, Lara Milevoj; Dorotić, Adrijana; Baršić, Ivana

2016-10-15

Blood gas analysis (BGA) is exposed to risks of errors caused by improper sampling, transport and storage conditions. The Clinical and Laboratory Standards Institute (CLSI) generated documents with recommendations for avoidance of potential errors caused by sample mishandling. Two main documents related to BGA issued by the CLSI are GP43-A4 (former H11-A4) Procedures for the collection of arterial blood specimens; approved standard - fourth edition, and C46-A2 Blood gas and pH analysis and related measurements; approved guideline - second edition. Practices related to processing of blood gas samples are not standardized in the Republic of Croatia. Each institution has its own protocol for ordering, collection and analysis of blood gases. Although many laboratories use state of the art analyzers, still many preanalytical procedures remain unchanged. The objective of the Croatian Society of Medical Biochemistry and Laboratory Medicine (CSMBLM) is to standardize the procedures for BGA based on CLSI recommendations. The Working Group for Blood Gas Testing as part of the Committee for the Scientific Professional Development of the CSMBLM prepared a set of recommended protocols for sampling, transport, storage and processing of blood gas samples based on relevant CLSI documents, relevant literature search and on the results of Croatian survey study on practices and policies in acid-base testing. Recommendations are intended for laboratory professionals and all healthcare workers involved in blood gas processing.

Blood gas testing and related measurements: National recommendations on behalf of the Croatian Society of Medical Biochemistry and Laboratory Medicine

PubMed Central

Dukić, Lora; Kopčinović, Lara Milevoj; Dorotić, Adrijana; Baršić, Ivana

2016-01-01

Blood gas analysis (BGA) is exposed to risks of errors caused by improper sampling, transport and storage conditions. The Clinical and Laboratory Standards Institute (CLSI) generated documents with recommendations for avoidance of potential errors caused by sample mishandling. Two main documents related to BGA issued by the CLSI are GP43-A4 (former H11-A4) Procedures for the collection of arterial blood specimens; approved standard – fourth edition, and C46-A2 Blood gas and pH analysis and related measurements; approved guideline – second edition. Practices related to processing of blood gas samples are not standardized in the Republic of Croatia. Each institution has its own protocol for ordering, collection and analysis of blood gases. Although many laboratories use state of the art analyzers, still many preanalytical procedures remain unchanged. The objective of the Croatian Society of Medical Biochemistry and Laboratory Medicine (CSMBLM) is to standardize the procedures for BGA based on CLSI recommendations. The Working Group for Blood Gas Testing as part of the Committee for the Scientific Professional Development of the CSMBLM prepared a set of recommended protocols for sampling, transport, storage and processing of blood gas samples based on relevant CLSI documents, relevant literature search and on the results of Croatian survey study on practices and policies in acid-base testing. Recommendations are intended for laboratory professionals and all healthcare workers involved in blood gas processing. PMID:27812301

The UK Biobank sample handling and storage protocol for the collection, processing and archiving of human blood and urine.

PubMed

Elliott, Paul; Peakman, Tim C

2008-04-01

UK Biobank is a large prospective study in the UK to investigate the role of genetic factors, environmental exposures and lifestyle in the causes of major diseases of late and middle age. Extensive data and biological samples are being collected from 500,000 participants aged between 40 and 69 years. The biological samples that are collected and how they are processed and stored will have a major impact on the future scientific usefulness of the UK Biobank resource. The aim of the UK Biobank sample handling and storage protocol is to specify methods for the collection and storage of participant samples that give maximum scientific return within the available budget. Processing or storage methods that, as far as can be predicted, will preclude current or future assays have been avoided. The protocol was developed through a review of the literature on sample handling and processing, wide consultation within the academic community and peer review. Protocol development addressed which samples should be collected, how and when they should be processed and how the processed samples should be stored to ensure their long-term integrity. The recommended protocol was extensively tested in a series of validation studies. UK Biobank collects about 45 ml blood and 9 ml of urine with minimal local processing from each participant using the vacutainer system. A variety of preservatives, anti-coagulants and clot accelerators is used appropriate to the expected end use of the samples. Collection of other material (hair, nails, saliva and faeces) was also considered but rejected for the full cohort. Blood and urine samples from participants are transported overnight by commercial courier to a central laboratory where they are processed and aliquots of urine, plasma, serum, white cells and red cells stored in ultra-low temperature archives. Aliquots of whole blood are also stored for potential future production of immortalized cell lines. A standard panel of haematology assays is completed on whole blood from all participants, since such assays need to be conducted on fresh samples (whereas other assays can be done on stored samples). By the end of the recruitment phase, 15 million sample aliquots will be stored in two geographically separate archives: 9.5 million in a -80 degrees C automated archive and 5.5 million in a manual liquid nitrogen archive at -180 degrees C. Because of the size of the study and the numbers of samples obtained from participants, the protocol stipulates a highly automated approach for the processing and storage of samples. Implementation of the processes, technology, systems and facilities has followed best practices used in manufacturing industry to reduce project risk and to build in quality and robustness. The data produced from sample collection, processing and storage are highly complex and are managed by a commercially available LIMS system fully integrated with the entire process. The sample handling and storage protocol adopted by UK Biobank provides quality assured and validated methods that are feasible within the available funding and reflect the size and aims of the project. Experience from recruiting and processing the first 40,000 participants to the study demonstrates that the adopted methods and technologies are fit-for-purpose and robust.

Direct and long-term detection of gene doping in conventional blood samples.

PubMed

Beiter, T; Zimmermann, M; Fragasso, A; Hudemann, J; Niess, A M; Bitzer, M; Lauer, U M; Simon, P

2011-03-01

The misuse of somatic gene therapy for the purpose of enhancing athletic performance is perceived as a coming threat to the world of sports and categorized as 'gene doping'. This article describes a direct detection approach for gene doping that gives a clear yes-or-no answer based on the presence or absence of transgenic DNA in peripheral blood samples. By exploiting a priming strategy to specifically amplify intronless DNA sequences, we developed PCR protocols allowing the detection of very small amounts of transgenic DNA in genomic DNA samples to screen for six prime candidate genes. Our detection strategy was verified in a mouse model, giving positive signals from minute amounts (20 μl) of blood samples for up to 56 days following intramuscular adeno-associated virus-mediated gene transfer, one of the most likely candidate vector systems to be misused for gene doping. To make our detection strategy amenable for routine testing, we implemented a robust sample preparation and processing protocol that allows cost-efficient analysis of small human blood volumes (200 μl) with high specificity and reproducibility. The practicability and reliability of our detection strategy was validated by a screening approach including 327 blood samples taken from professional and recreational athletes under field conditions.

Assessment of four protocols for rapid bacterial identification from positive blood culture pellets by matrix-assisted laser desorption ionization-time of flight mass spectrometry (Vitek® MS).

PubMed

Thomin, Jean; Aubin, Guillaume Ghislain; Foubert, Fabrice; Corvec, Stéphane

2015-08-01

In this study, we developed and compared four protocols to prepare a bacterial pellet from 944 positive blood cultures for direct MALDI-TOF mass spectrometry Vitek® MS analysis. Protocol 4, tested on 200 monomicrobial samples, allowed 83% of bacterial identification. This easy, fast, cheap and accurate method is promising in daily practice, especially to limit broad range antibiotic treatment. Copyright © 2015 Elsevier B.V. All rights reserved.

Bioindicators in the MIDUS National Study: Protocol, Measures, Sample, and Comparative Context

PubMed Central

Love, Gayle Dienberg; Seeman, Teresa E.; Weinstein, Maxine; Ryff, Carol D.

2010-01-01

Objectives MIDUS is a national study of health and aging among individuals aged 25 to 74 at baseline(1995/96). Longitudinal survey assessments (2004/05), were followed by biological assessments on a subsample aged 35–85. To facilitate public use, we describe the protocol, measures, and sample. Methods Respondents traveled to clinics for a two-day data collection protocol that included fasting blood specimens, 12-hour urine specimen, medical history, physical exam, bone densitometry, a laboratory challenge (heart rate variability, blood pressure, respiration, salivary cortisol). Results Response rates for the biological protocol (N = 1,255) were 39.3%, or 43.1% (adjusting for those who could not be located or contacted). Reasons for non-participation were travel, family obligations, and being too busy. Respondents were comparable to the recruitment pool on most demographic characteristics and health assessments. Discussion Strengths of the protocol vis-à-vis other similar studies include opportunities to link biological factors with diverse content from other MIDUS projects. PMID:20876364

What a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into population-based research.

PubMed

McDade, Thomas W; Williams, Sharon; Snodgrass, J Josh

2007-11-01

Logistical constraints associated with the collection and analysis of biological samples in community-based settings have been a significant impediment to integrative, multilevel bio-demographic and biobehavioral research. However recent methodological developments have overcome many of these constraints and have also expanded the options for incorporating biomarkers into population-based health research in international as well as domestic contexts. In particular using dried blood spot (DBS) samples-drops of whole blood collected on filter paper from a simple finger prick-provides a minimally invasive method for collecting blood samples in nonclinical settings. After a brief discussion of biomarkers more generally, we review procedures for collecting, handling, and analyzing DBS samples. Advantages of using DBS samples-compared with venipuncture include the relative ease and low cost of sample collection, transport, and storage. Disadvantages include requirements for assay development and validation as well as the relatively small volumes of sample. We present the results of a comprehensive literature review of published protocols for analysis of DBS samples, and we provide more detailed analysis of protocols for 45 analytes likely to be of particular relevance to population-level health research. Our objective is to provide investigators with the information they need to make informed decisions regarding the appropriateness of blood spot methods for their research interests.

High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA.

PubMed

Lundblom, Klara; Macharia, Alex; Lebbad, Marianne; Mohammed, Adan; Färnert, Anna

2011-08-08

Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome. High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/μl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene. High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of P. falciparum parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection. High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots.

Collecting and Storing Blood Samples From Patients With Cancer

ClinicalTrials.gov

2011-12-08

Brain and Central Nervous System Tumors; Chronic Myeloproliferative Disorders; Leukemia; Lymphoma; Lymphoproliferative Disorder; Multiple Myeloma and Plasma Cell Neoplasm; Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Neoplasms; Nonmalignant Neoplasm; Unspecified Adult Solid Tumor, Protocol Specific; Unspecified Childhood Solid Tumor, Protocol Specific

A real-time, quantitative PCR protocol for assessing the relative parasitemia of Leucocytozoon in waterfowl.

PubMed

Smith, Matthew M; Schmutz, Joel; Apelgren, Chloe; Ramey, Andrew M

2015-04-01

Microscopic examination of blood smears can be effective at diagnosing and quantifying hematozoa infections. However, this method requires highly trained observers, is time consuming, and may be inaccurate for detection of infections at low levels of parasitemia. To develop a molecular methodology for identifying and quantifying Leucocytozoon parasite infection in wild waterfowl (Anseriformes), we designed a real-time, quantitative PCR protocol to amplify Leucocytozoon mitochondrial DNA using TaqMan fluorogenic probes and validated our methodology using blood samples collected from waterfowl in interior Alaska during late summer and autumn (n=105). By comparing our qPCR results to those derived from a widely used nested PCR protocol, we determined that our assay showed high levels of sensitivity (91%) and specificity (100%) in detecting Leucocytozoon DNA from host blood samples. Additionally, results of a linear regression revealed significant correlation between the raw measure of parasitemia produced by our qPCR assay (Ct values) and numbers of parasites observed on blood smears (R(2)=0.694, P=0.003), indicating that our assay can reliably determine the relative parasitemia levels among samples. This methodology provides a powerful new tool for studies assessing effects of haemosporidian infection in wild avian species. Published by Elsevier B.V.

Evaluation of limited blood sampling population input approaches for kinetic quantification of [18F]fluorothymidine PET data.

PubMed

Contractor, Kaiyumars B; Kenny, Laura M; Coombes, Charles R; Turkheimer, Federico E; Aboagye, Eric O; Rosso, Lula

2012-03-24

Quantification of kinetic parameters of positron emission tomography (PET) imaging agents normally requires collecting arterial blood samples which is inconvenient for patients and difficult to implement in routine clinical practice. The aim of this study was to investigate whether a population-based input function (POP-IF) reliant on only a few individual discrete samples allows accurate estimates of tumour proliferation using [18F]fluorothymidine (FLT). Thirty-six historical FLT-PET data with concurrent arterial sampling were available for this study. A population average of baseline scans blood data was constructed using leave-one-out cross-validation for each scan and used in conjunction with individual blood samples. Three limited sampling protocols were investigated including, respectively, only seven (POP-IF7), five (POP-IF5) and three (POP-IF3) discrete samples of the historical dataset. Additionally, using the three-point protocol, we derived a POP-IF3M, the only input function which was not corrected for the fraction of radiolabelled metabolites present in blood. The kinetic parameter for net FLT retention at steady state, Ki, was derived using the modified Patlak plot and compared with the original full arterial set for validation. Small percentage differences in the area under the curve between all the POP-IFs and full arterial sampling IF was found over 60 min (4.2%-5.7%), while there were, as expected, larger differences in the peak position and peak height.A high correlation between Ki values calculated using the original arterial input function and all the population-derived IFs was observed (R2 = 0.85-0.98). The population-based input showed good intra-subject reproducibility of Ki values (R2 = 0.81-0.94) and good correlation (R2 = 0.60-0.85) with Ki-67. Input functions generated using these simplified protocols over scan duration of 60 min estimate net PET-FLT retention with reasonable accuracy.

Direct identification of bacteria from charcoal-containing blood culture bottles using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

PubMed

Wüppenhorst, N; Consoir, C; Lörch, D; Schneider, C

2012-10-01

Several protocols for direct matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) from positive blood cultures are currently used to speed up the diagnostic process of bacteraemia. Identification rates are high and results are accurate for the BACTEC™ system and for charcoal-free bottles. Only a few studies have evaluated protocols for charcoal-containing BacT/ALERT bottles reaching substantially lower identification rates. We established a new protocol for sample preparation from aerobic and anaerobic positive charcoal-containing BacT/ALERT blood culture bottles and measured the protein profiles (n = 167). Then, we integrated this protocol in the routine workflow of our laboratory (n = 212). During the establishment of our protocol, 74.3 % of bacteria were correctly identified to the species level, in 23.4 %, no result and in 2.4 %, a false identification were obtained. Reliable criteria for correct species identification were a score value ≥1.400 and a best match on rank 1-3 of the same species. Identification rates during routine workflow were 77.8 % for correct identification, 20.8 % for not identified samples and 1.4 % for discordant identification. In conclusion, our results indicate that MALDI-TOF MS is possible, even from charcoal-containing blood cultures. Reliable criteria for correct species identification are a score value ≥1.400 and a best match on rank 1-3 of a single species.

High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA

PubMed Central

2011-01-01

Background Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome. Methods High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/μl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene. Results High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of P. falciparum parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection. Conclusions High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots. PMID:21824391

Rapid Microbial Sample Preparation from Blood Using a Novel Concentration Device

PubMed Central

Boardman, Anna K.; Campbell, Jennifer; Wirz, Holger; Sharon, Andre; Sauer-Budge, Alexis F.

2015-01-01

Appropriate care for bacteremic patients is dictated by the amount of time needed for an accurate diagnosis. However, the concentration of microbes in the blood is extremely low in these patients (1–100 CFU/mL), traditionally requiring growth (blood culture) or amplification (e.g., PCR) for detection. Current culture-based methods can take a minimum of two days, while faster methods like PCR require a sample free of inhibitors (i.e., blood components). Though commercial kits exist for the removal of blood from these samples, they typically capture only DNA, thereby necessitating the use of blood culture for antimicrobial testing. Here, we report a novel, scaled-up sample preparation protocol carried out in a new microbial concentration device. The process can efficiently lyse 10 mL of bacteremic blood while maintaining the microorganisms’ viability, giving a 30‑μL final output volume. A suite of six microorganisms (Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, Pseudomonas aeruginosa, and Candida albicans) at a range of clinically relevant concentrations was tested. All of the microorganisms had recoveries greater than 55% at the highest tested concentration of 100 CFU/mL, with three of them having over 70% recovery. At the lowest tested concentration of 3 CFU/mL, two microorganisms had recoveries of ca. 40–50% while the other four gave recoveries greater than 70%. Using a Taqman assay for methicillin-sensitive S. aureus (MSSA)to prove the feasibility of downstream analysis, we show that our microbial pellets are clean enough for PCR amplification. PCR testing of 56 spiked-positive and negative samples gave a specificity of 0.97 and a sensitivity of 0.96, showing that our sample preparation protocol holds great promise for the rapid diagnosis of bacteremia directly from a primary sample. PMID:25675242

Collecting, archiving and processing DNA from wildlife samples using FTA® databasing paper

PubMed Central

Smith, LM; Burgoyne, LA

2004-01-01

Background Methods involving the analysis of nucleic acids have become widespread in the fields of traditional biology and ecology, however the storage and transport of samples collected in the field to the laboratory in such a manner to allow purification of intact nucleic acids can prove problematical. Results FTA® databasing paper is widely used in human forensic analysis for the storage of biological samples and for purification of nucleic acids. The possible uses of FTA® databasing paper in the purification of DNA from samples of wildlife origin were examined, with particular reference to problems expected due to the nature of samples of wildlife origin. The processing of blood and tissue samples, the possibility of excess DNA in blood samples due to nucleated erythrocytes, and the analysis of degraded samples were all examined, as was the question of long term storage of blood samples on FTA® paper. Examples of the end use of the purified DNA are given for all protocols and the rationale behind the processing procedures is also explained to allow the end user to adjust the protocols as required. Conclusions FTA® paper is eminently suitable for collection of, and purification of nucleic acids from, biological samples from a wide range of wildlife species. This technology makes the collection and storage of such samples much simpler. PMID:15072582

Identification of phlebotomine sand fly blood meals by real-time PCR.

PubMed

Sales, Kamila Gaudêncio da Silva; Costa, Pietra Lemos; de Morais, Rayana Carla Silva; Otranto, Domenico; Brandão-Filho, Sinval Pinto; Cavalcanti, Milena de Paiva; Dantas-Torres, Filipe

2015-04-16

Phlebotomine sand flies are blood-feeding insects of great medical and veterinary significance acting as vectors of Leishmania parasites. Studying the blood-feeding pattern of these insects may help in the understanding of their interactions with potential reservoir hosts of Leishmania parasites. In this study, we developed real time PCR assays for the identification of sand fly blood meal. Six pairs of primers were designed based on cytochrome b gene sequences available in GenBank of the following potential hosts: dog, cat, horse, chicken, black rat, and human. Firstly, SYBR Green-based real time PCR assays were conducted using a standard curve with eight different concentrations (i.e., 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg per 2 μl) of DNA samples extracted from EDTA blood samples from each target animal. Then, DNA samples extracted from field-collected engorged female sand flies belonging to three species (i.e., Lutzomyia longipalpis, L. migonei and L. lenti) were tested by the protocols standardized herein. Additionally, female sand flies were experimentally fed on a black rat (Rattus rattus) and used for evaluating the time course of the detection of the protocol targeting this species. The protocols performed well with detection limits of 10 pg to 100 fg. Field-collected female sand flies were fed on blood from humans (73%), chickens (23%), dogs (22%), horses (15%), black rats (11%) and cats (2%). Interestingly, 76.1% of the L. longipalpis females were positive for human blood. In total, 48% of the tested females were fed on single sources, 31% on two and 12% on three. The analysis of the time course showed that the real time PCR protocol targeting the black rat DNA was able to detect small amounts of the host DNA up to 5 days after the blood meal. The real time PCR assays standardized herein successfully detected small amounts of host DNA in female sand flies fed on different vertebrate species and, specifically for the black rats, up to 5 days after the blood meal. These assays represent promising tools for the identification of blood meal in field-collected female sand flies.

Assessing basophil activation by using flow cytometry and mass cytometry in blood stored 24 hours before analysis.

PubMed

Mukai, Kaori; Gaudenzio, Nicolas; Gupta, Sheena; Vivanco, Nora; Bendall, Sean C; Maecker, Holden T; Chinthrajah, Rebecca S; Tsai, Mindy; Nadeau, Kari C; Galli, Stephen J

2017-03-01

Basophil activation tests (BATs) have promise for research and for clinical monitoring of patients with allergies. However, BAT protocols vary in blood anticoagulant used and temperature and time of storage before testing, complicating comparisons of results from various studies. We attempted to establish a BAT protocol that would permit analysis of blood within 24 hours of obtaining the sample. Blood from 46 healthy donors and 120 patients with peanut allergy was collected into EDTA or heparin tubes, and samples were stored at 4°C or room temperature for 4 or 24 hours before performing BATs. Stimulation with anti-IgE or IL-3 resulted in strong upregulation of basophil CD203c in samples collected in EDTA or heparin, stored at 4°C, and analyzed 24 hours after sample collection. However, a CD63 hi population of basophils was not observed in any conditions in EDTA-treated samples unless exogenous calcium/magnesium was added at the time of anti-IgE stimulation. By contrast, blood samples collected in heparin tubes were adequate for quantification of upregulation of basophil CD203c and identification of a population of CD63 hi basophils, irrespective of whether the specimens were analyzed by means of conventional flow cytometry or cytometry by time-of-flight mass spectrometry, and such tests could be performed after blood was stored for 24 hours at 4°C. BATs to measure upregulation of basophil CD203c and induction of a CD63 hi basophil population can be conducted with blood obtained in heparin tubes and stored at 4°C for 24 hours. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Collection, Storage, and Preparation of Human Blood Cells

PubMed Central

Dagur, Pradeep K.; McCoy, J. Philip

2015-01-01

Human peripheral blood is often studied by flow cytometry in both the research and clinical laboratories. The methods for collection, storage, and preparation of peripheral blood will vary depending on the cell lineage to be examined as well as the type of assay to be performed. This unit presents protocols for collection of blood, separation of leukocytes from whole blood by lysis of erythrocytes, isolating mononuclear cells by density gradient separation, and assorted non-flow sorting methods, such as magnetic bead separations, for enriching specific cell populations, including monocytes, T lymphocytes, B lymphocytes, neutrophils,, , and platelets prior to flow cytometric analysis. A protocol is also offered for cryopreservation of cells since clinical research often involves retrospective flow cytometric analysis of samples stored over a period of months or years. PMID:26132177

Distribution of Parvovirus B19 DNA in Blood Compartments and Persistence of Virus in Blood Donors

PubMed Central

Lee, Tzong-Hae; Kleinman, Steven H.; Wen, Li; Montalvo, Lani; Todd, Deborah S.; Wright, David J.; Tobler, Leslie H.; Busch, Michael P.

2013-01-01

Introduction Because the receptor for Parvovirus B19 (B19V) is on erythrocytes, we investigated B19V distribution in blood by in-vitro spiking experiments and evaluated viral compartmentalization and persistence in natural infection. Methods Two whole blood protocols (ultracentrifugation and a rapid RBC lysis/removal protocol) were evaluated using quantitative real-time PCR. Whole blood (WB) was spiked with known concentrations of B19V and recovery in various blood fractions was determined. The rapid RBC lysis/removal protocol was then used to compare B19V concentrations in 104 paired whole blood and plasma samples collected longitudinally from 43 B19V infected donors with frozen specimens in the REDS Allogeneic Donor and Recipient Repository (RADAR). Results In B19V spiking experiments, ~one-third of viral DNA was recovered in plasma and two-thirds was loosely bound to erythrocytes. In the IgM positive stage of infection in blood donors when plasma B19V DNA concentrations were > 100 IU/mL, median DNA concentrations were ~30-fold higher in WB than in plasma. In contrast, when IgM was absent and when the B19V DNA concentration was lower, the median whole blood to plasma ratio was ~1. Analysis of longitudinal samples demonstrated persistent detection of B19V in WB but declining ratios of WB/plasma B19V with declining plasma VL levels and loss of IgM-reactivity. Conclusions The WB/plasma B19V DNA ratio varies by stage of infection. Further study is required to determine if this is related to the presence of circulating DNA-positive erythrocytes derived from B19V infected erythroblasts, B19V-specific IgM mediated binding of virus to cells, or other factors. PMID:21303368

DNA elution from buccal cells stored on Whatman FTA Classic Cards using a modified methanol fixation method.

PubMed

Johanson, Helene C; Hyland, Valentine; Wicking, Carol; Sturm, Richard A

2009-04-01

We describe here a method for DNA elution from buccal cells and whole blood both collected onto Whatman FTA technology, using methanol fixation followed by an elution PCR program. Extracted DNA is comparable in quality to published Whatman FTA protocols, as judged by PCR-based genotyping. Elution of DNA from the dried sample is a known rate-limiting step in the published Whatman FTA protocol; this method enables the use of each 3-mm punch of sample for several PCR reactions instead of the standard, one PCR reaction per sample punch. This optimized protocol therefore extends the usefulness and cost effectiveness of each buccal swab sample collected, when used for nucleic acid PCR and genotyping.

Heparin removal by ecteola-cellulose pre-treatment enables the use of plasma samples for accurate measurement of anti-Yellow fever virus neutralizing antibodies.

PubMed

Campi-Azevedo, Ana Carolina; Peruhype-Magalhães, Vanessa; Coelho-Dos-Reis, Jordana Grazziela; Costa-Pereira, Christiane; Yamamura, Anna Yoshida; Lima, Sheila Maria Barbosa de; Simões, Marisol; Campos, Fernanda Magalhães Freire; de Castro Zacche Tonini, Aline; Lemos, Elenice Moreira; Brum, Ricardo Cristiano; de Noronha, Tatiana Guimarães; Freire, Marcos Silva; Maia, Maria de Lourdes Sousa; Camacho, Luiz Antônio Bastos; Rios, Maria; Chancey, Caren; Romano, Alessandro; Domingues, Carla Magda; Teixeira-Carvalho, Andréa; Martins-Filho, Olindo Assis

2017-09-01

Technological innovations in vaccinology have recently contributed to bring about novel insights for the vaccine-induced immune response. While the current protocols that use peripheral blood samples may provide abundant data, a range of distinct components of whole blood samples are required and the different anticoagulant systems employed may impair some properties of the biological sample and interfere with functional assays. Although the interference of heparin in functional assays for viral neutralizing antibodies such as the functional plaque-reduction neutralization test (PRNT), considered the gold-standard method to assess and monitor the protective immunity induced by the Yellow fever virus (YFV) vaccine, has been well characterized, the development of pre-analytical treatments is still required for the establishment of optimized protocols. The present study intended to optimize and evaluate the performance of pre-analytical treatment of heparin-collected blood samples with ecteola-cellulose (ECT) to provide accurate measurement of anti-YFV neutralizing antibodies, by PRNT. The study was designed in three steps, including: I. Problem statement; II. Pre-analytical steps; III. Analytical steps. Data confirmed the interference of heparin on PRNT reactivity in a dose-responsive fashion. Distinct sets of conditions for ECT pre-treatment were tested to optimize the heparin removal. The optimized protocol was pre-validated to determine the effectiveness of heparin plasma:ECT treatment to restore the PRNT titers as compared to serum samples. The validation and comparative performance was carried out by using a large range of serum vs heparin plasma:ECT 1:2 paired samples obtained from unvaccinated and 17DD-YFV primary vaccinated subjects. Altogether, the findings support the use of heparin plasma:ECT samples for accurate measurement of anti-YFV neutralizing antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.

HIV-1 viral load measurement in venous blood and fingerprick blood using Abbott RealTime HIV-1 DBS assay.

PubMed

Tang, Ning; Pahalawatta, Vihanga; Frank, Andrea; Bagley, Zowie; Viana, Raquel; Lampinen, John; Leckie, Gregor; Huang, Shihai; Abravaya, Klara; Wallis, Carole L

2017-07-01

HIV RNA suppression is a key indicator for monitoring success of antiretroviral therapy. From a logistical perspective, viral load (VL) testing using Dried Blood Spots (DBS) is a promising alternative to plasma based VL testing in resource-limited settings. To evaluate the analytical and clinical performance of the Abbott RealTime HIV-1 assay using a fully automated one-spot DBS sample protocol. Limit of detection (LOD), linearity, lower limit of quantitation (LLQ), upper limit of quantitation (ULQ), and precision were determined using serial dilutions of HIV-1 Virology Quality Assurance stock (VQA Rush University), or HIV-1-containing armored RNA, made in venous blood. To evaluate correlation, bias, and agreement, 497 HIV-1 positive adult clinical samples were collected from Ivory Coast, Uganda and South Africa. For each HIV-1 participant, DBS-fingerprick, DBS-venous and plasma sample results were compared. Correlation and bias values were obtained. The sensitivity and specificity were analyzed at a threshold of 1000 HIV-1 copies/mL generated using the standard plasma protocol. The Abbott HIV-1 DBS protocol had an LOD of 839 copies/mL, a linear range from 500 to 1×10 7 copies/mL, an LLQ of 839 copies/mL, a ULQ of 1×10 7 copies/mL, and an inter-assay SD of ≤0.30 log copies/mL for all tested levels within this range. With clinical samples, the correlation coefficient (r value) was 0.896 between DBS-fingerprick and plasma and 0.901 between DBS-venous and plasma, and the bias was -0.07 log copies/mL between DBS-fingerprick and plasma and -0.02 log copies/mL between DBS-venous and plasma. The sensitivity of DBS-fingerprick and DBS-venous was 93%, while the specificity of both DBS methods was 95%. The results demonstrated that the Abbott RealTime HIV-1 assay with DBS sample protocol is highly sensitive, specific and precise across a wide dynamic range and correlates well with plasma values. The Abbott RealTime HIV-1 assay with DBS sample protocol provides an alternative sample collection and transfer option in resource-limited settings and expands the utility of a viral load test to monitor HIV-1 ART treatment for infected patients. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Characterizing biological variability in livestock blood cholinesterase activity for biomonitoring organophosphate nerve agent exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Halbrook, R.S.; Shugart, L.R.; Watson, A.P.

1992-09-01

A biomonitoring protocol, using blood cholinesterase (ChE) activity in livestock as a monitor of potential organophosphate nerve agent exposure during the planned destruction of US unitary chemical warfare agent stockpiles, is described. The experimental design included analysis of blood ChE activity in individual healthy sheep, horses, and dairy and beef cattle during a 10- to 12-month period. Castrated and sexually intact males, pregnant and lactating females, and adult and immature animals were examined through at least one reproductive cycle. The same animals were used throughout the period of observation and were not exposed to ChE-inhibiting organophosphate or carbamate compounds. Amore » framework for an effective biomonitoring protocol within a monitoring area includes establishing individual baseline blood ChE activity for a sentinel group of 6 animals on the bases of blood samples collected over a 6-month period, monthly collection of blood samples for ChE-activity determination during monitoring, and selection of adult animals as sentinels. Exposure to ChE-inhibiting compounds would be suspected when all blood ChE activity of all animals within the sentinel group are decreased greater than 20% from their own baseline value. Sentinel species selection is primarily a logistical and operational concern; however, sheep appear to be the species of choice because within-individual baseline ChE activity and among age and gender group ChE activity in sheep had the least variability, compared with data from other species. This protocol provides an effective and efficient means for detecting abnormal depressions in blood ChE activity in livestock and can serve as a valuable indicator of the extent of actual plume movement and/or deposition in the event of organophosphate nerve agent release.« less

Rapid DNA extraction protocol for detection of alpha-1 antitrypsin deficiency from dried blood spots by real-time PCR.

PubMed

Struniawski, R; Szpechcinski, A; Poplawska, B; Skronski, M; Chorostowska-Wynimko, J

2013-01-01

The dried blood spot (DBS) specimens have been successfully employed for the large-scale diagnostics of α1-antitrypsin (AAT) deficiency as an easy to collect and transport alternative to plasma/serum. In the present study we propose a fast, efficient, and cost effective protocol of DNA extraction from dried blood spot (DBS) samples that provides sufficient quantity and quality of DNA and effectively eliminates any natural PCR inhibitors, allowing for successful AAT genotyping by real-time PCR and direct sequencing. DNA extracted from 84 DBS samples from chronic obstructive pulmonary disease patients was genotyped for AAT deficiency variants by real-time PCR. The results of DBS AAT genotyping were validated by serum IEF phenotyping and AAT concentration measurement. The proposed protocol allowed successful DNA extraction from all analyzed DBS samples. Both quantity and quality of DNA were sufficient for further real-time PCR and, if necessary, for genetic sequence analysis. A 100% concordance between AAT DBS genotypes and serum phenotypes in positive detection of two major deficiency S- and Z- alleles was achieved. Both assays, DBS AAT genotyping by real-time PCR and serum AAT phenotyping by IEF, positively identified PI*S and PI*Z allele in 8 out of the 84 (9.5%) and 16 out of 84 (19.0%) patients, respectively. In conclusion, the proposed protocol noticeably reduces the costs and the hand-on-time of DBS samples preparation providing genomic DNA of sufficient quantity and quality for further real-time PCR or genetic sequence analysis. Consequently, it is ideally suited for large-scale AAT deficiency screening programs and should be method of choice.

Cryopreservation of human whole blood allows immunophenotyping by flow cytometry up to 30days after cell isolation.

PubMed

Paredes, R Madelaine; Tadaki, Douglas K; Sooter, Amanda; Gamboni, Fabia; Sheppard, Forest

2018-01-01

Immunophenotyping of whole blood (WB) by flow cytometry (FC) is used clinically to assess a patient's immune status and also in biomedical research. Current protocols recommend storage of immunolabeled samples at 4°C with FC analysis to be completed within seven days. This data acquisition window can be extended to up to one year post-labeling, but this requires cryopreservation of the samples at ultra-low temperatures (≤-80°C or in liquid nitrogen). In this study we optimized a standardized cryopreservation protocol to enable preservation of immunolabeled, human WB samples at -20°C for FC and tested its effectiveness after 0, 5, 15 or 30days. Analysis of stored samples shows that this protocol effectively preserves immunolabeled WB samples and that the duration of storage has no effect on morphology, viability or frequency of WB cell subpopulations, and that the intensity of fluorescent signal from labeled extracellular markers is fully preserved for at least 15days, and up to 30days for some markers. We demonstrate that using this protocol, we are able to differentiate resting versus activated WB cells as demonstrated by detection of significantly increased expression of CD11b by myeloid cells in WB samples pretreated with LPS (100μg/mL for 12h). Finally, we show that this method allows for labeling and detection of the intracellular cytokine (IL-8) up to 30days following cryopreservation from myeloid cells, in previously labeled and cryopreserved WB samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Protocol for the Meningococcal Control Programme - Canadian Forces Base Cornwallis and Canadian Forces Base St. Jean 1980/81,

DTIC Science & Technology

1980-11-01

hours, then overnight in a refrigerator or cold room at 40C. Fol- lowing clot retraction , the serum may be decanted and spun, or the whole sample spun...Drogram. The SIMP programme may involve one or all of the following: collection of post nasal swabs, blood sera samples, air samples and surface samples...outlined in para 16. 16. Culturing of Swabs Swabs shall be placed on Columbia Blood Agar (4 or 5% sheep RBC’s) supplemented with IsoVitalex (Baltimore

21 CFR 660.46 - Samples; protocols; official release.

Code of Federal Regulations, 2014 CFR

2014-04-01

... with 125I or unlyophilized HBsAg-coated red blood cells means a sample from each lot of diagnostic test...) One sample until written notification of official release is no longer required under paragraph (c)(2... notification of official release is no longer required under paragraph (c)(2) of this section. The sample...

21 CFR 660.46 - Samples; protocols; official release.

Code of Federal Regulations, 2013 CFR

2013-04-01

... with 125I or unlyophilized HBsAg-coated red blood cells means a sample from each lot of diagnostic test...) One sample until written notification of official release is no longer required under paragraph (c)(2... notification of official release is no longer required under paragraph (c)(2) of this section. The sample...

21 CFR 660.46 - Samples; protocols; official release.

Code of Federal Regulations, 2012 CFR

2012-04-01

... with 125I or unlyophilized HBsAg-coated red blood cells means a sample from each lot of diagnostic test...) One sample until written notification of official release is no longer required under paragraph (c)(2... notification of official release is no longer required under paragraph (c)(2) of this section. The sample...

Direct identification of bacteria in positive blood culture bottles by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry.

PubMed

La Scola, Bernard; Raoult, Didier

2009-11-25

With long delays observed between sampling and availability of results, the usefulness of blood cultures in the context of emergency infectious diseases has recently been questioned. Among methods that allow quicker bacterial identification from growing colonies, matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry was demonstrated to accurately identify bacteria routinely isolated in a clinical biology laboratory. In order to speed up the identification process, in the present work we attempted bacterial identification directly from blood culture bottles detected positive by the automate. We prospectively analysed routine MALDI-TOF identification of bacteria detected in blood culture by two different protocols involving successive centrifugations and then lysis by trifluoroacetic acid or formic acid. Of the 562 blood culture broths detected as positive by the automate and containing one bacterial species, 370 (66%) were correctly identified. Changing the protocol from trifluoroacetic acid to formic acid improved identification of Staphylococci, and overall correct identification increased from 59% to 76%. Lack of identification was observed mostly with viridans streptococci, and only one false positive was observed. In the 22 positive blood culture broths that contained two or more different species, only one of the species was identified in 18 samples, no species were identified in two samples and false species identifications were obtained in two cases. The positive predictive value of bacterial identification using this procedure was 99.2%. MALDI-TOF MS is an efficient method for direct routine identification of bacterial isolates in blood culture, with the exception of polymicrobial samples and viridans streptococci. It may replace routine identification performed on colonies, provided improvement for the specificity of blood culture broths growing viridans streptococci is obtained in the near future.

Automated processing of first-pass radioisotope ventriculography data to determine essential central circulation parameters

NASA Astrophysics Data System (ADS)

Krotov, Aleksei; Pankin, Victor

2017-09-01

The assessment of central circulation (including heart function) parameters is vital in the preventive diagnostics of inherent and acquired heart failures and during polychemotherapy. The protocols currently applied in Russia do not fully utilize the first-pass assessment (FPRNA) and that results in poor data formalization, while the FPRNA is the one of the fastest, affordable and compact methods among other radioisotope diagnostics protocols. A non-imaging algorithm basing on existing protocols has been designed to use the readings of an additional detector above vena subclavia to determine the total blood volume (TBV), not requiring blood sampling in contrast to current protocols. An automated processing of precordial detector readings is presented, in order to determine the heart strike volume (SV). Two techniques to estimate the ejection fraction (EF) of the heart are discussed.

LTR real-time PCR for HIV-1 DNA quantitation in blood cells for early diagnosis in infants born to seropositive mothers treated in HAART area (ANRS CO 01).

PubMed

Avettand-Fènoël, Véronique; Chaix, Marie-Laure; Blanche, Stéphane; Burgard, Marianne; Floch, Corinne; Toure, Kadidia; Allemon, Marie-Christine; Warszawski, Josiane; Rouzioux, Christine

2009-02-01

HIV-1 diagnosis in babies born to seropositive mothers is one of the challenges of HIV epidemics in children. A simple, rapid protocol was developed for quantifying HIV-1 DNA in whole blood samples and was used in the ANRS French pediatric cohort in conditions of prevention of mother-to-child transmission. A quantitative HIV-1 DNA protocol (LTR real-time PCR) requiring small blood volumes was developed. First, analytical reproducibility was evaluated on 172 samples. Results obtained on blood cell pellets and Ficoll-Hypaque separated mononuclear cells were compared in 48 adult HIV-1 samples. Second, the protocol was applied to HIV-1 diagnosis in infants in parallel with plasma HIV-RNA quantitation. This prospective study was performed in children born between May 2005 and April 2007 included in the ANRS cohort. The assay showed good reproducibility. The 95% detection cut-off value was 6 copies/PCR, that is, 40 copies/10(6) leukocytes. HIV-DNA levels in whole blood were highly correlated with those obtained after Ficoll-Hypaque separation (r = 0.900, P < 0.0001). A total of 3,002 specimens from 1,135 infants were tested. The specificity of HIV-DNA and HIV-RNA assays was 100%. HIV-1 infection was diagnosed in nine infants before age 60 days. HIV-DNA levels were low, underlining the need for sensitive assays when highly active antiretroviral therapy (HAART) has been given. The performances of this HIV-DNA assay showed that it is adapted to early diagnosis in children. The results were equivalent to those of HIV-RNA assay. HIV-DNA may be used even in masked primary infection in newborns whose mothers have received HAART. (c) 2008 Wiley-Liss, Inc.

Can we Replace Arterial Blood Gas Analysis by Pulse Oximetry in Neonates with Respiratory Distress Syndrome, who are Treated According to INSURE Protocol?

PubMed Central

Niknafs, Pedram; Norouzi, Elahe; Bahman Bijari, Bahareh; Baneshi, Mohammad Reza

2015-01-01

Neonates with respiratory distress syndrome (RDS), who are treated according to INSURE protocol; require arterial blood gas (ABG) analysis to decide on appropriate management. We conducted this study to investigate the validity of pulse oximetry instead of frequent ABG analysis in the evaluation of these patients. From a total of 193 blood samples obtained from 30 neonates <1500 grams with RDS, 7.2% were found to have one or more of the followings: acidosis, hypercapnia, or hypoxemia. We found that pulse oximetry in the detection of hyperoxemia had a good validity to appropriately manage patients without blood gas analysis. However, the validity of pulse oximetry was not good enough to detect acidosis, hypercapnia, and hypoxemia. PMID:25999627

Automated extraction of DNA from blood and PCR setup using a Tecan Freedom EVO liquid handler for forensic genetic STR typing of reference samples.

PubMed

Stangegaard, Michael; Frøslev, Tobias G; Frank-Hansen, Rune; Hansen, Anders J; Morling, Niels

2011-04-01

We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO liquid handler mounted with the Te-MagS magnetic separation device (Tecan, Männedorf, Switzerland). The protocols were validated for accredited forensic genetic work according to ISO 17025 using the Qiagen MagAttract DNA Mini M48 kit (Qiagen GmbH, Hilden, Germany) from fresh whole blood and blood from deceased individuals. The workflow was simplified by returning the DNA extracts to the original tubes minimizing the risk of misplacing samples. The tubes that originally contained the samples were washed with MilliQ water before the return of the DNA extracts. The PCR was setup in 96-well microtiter plates. The methods were validated for the kits: AmpFℓSTR Identifiler, SGM Plus and Yfiler (Applied Biosystems, Foster City, CA), GenePrint FFFL and PowerPlex Y (Promega, Madison, WI). The automated protocols allowed for extraction and addition of PCR master mix of 96 samples within 3.5h. In conclusion, we demonstrated that (1) DNA extraction with magnetic beads and (2) PCR setup for accredited, forensic genetic short tandem repeat typing can be implemented on a simple automated liquid handler leading to the reduction of manual work, and increased quality and throughput. Copyright © 2011 Society for Laboratory Automation and Screening. Published by Elsevier Inc. All rights reserved.

Comparison of human umbilical cord blood processing with or without hydroxyethyl starch.

PubMed

Souri, Milad; Nikougoftar Zarif, Mahin; Rasouli, Mahboobeh; Golzadeh, Khadijeh; Nakhlestani Hagh, Mozhdeh; Ezzati, Nasim; Atarodi, Kamran

2017-11-01

Umbilical cord blood (UCB) processing with hydroxyethyl starch (HES) is the most common protocol in the cord blood banks. The quality of UCB volume reduction was guaranteed by minimum manipulation of cord blood samples in the closed system. This study aimed to analyze and compare cell recovery and viability of UCB processed using the Sepax automated system in the presence and absence of HES. Thirty UCB bags with a total nucleated cell (TNC) count of more than 2.5 × 10 9 were divided in two bags with equal volume. HES solution was added to one bag and another was intact. Both bags were processed with the Sepax. To determine cell recovery, viability, and potential of colony-forming cells (CFCs), preprocessing, postprocessing, and thawing samples were analyzed. The mean TNC recovery after processing and after thaw was significantly better with the HES method (p < 0.01 for the postprocessing step and p < 0.05 for the postthaw step). There were no significant differences to mononucleated cells (MNCs) and CD34+ cell recovery between the two methods after processing and after thaw. TNC and MNC viability was significantly higher without HES after processing and after thaw (p < 0.01). The results of the CFC assay were similar for both methods after processing and after thaw. These results showed that processing of UCB using the Sepax system with the without-HES protocol due to the lower manipulation of samples could be used as an eligible protocol to reduce the volume of UCB. © 2017 AABB.

Blood cell mRNAs and microRNAs: optimized protocols for extraction and preservation.

PubMed

Eikmans, Michael; Rekers, Niels V; Anholts, Jacqueline D H; Heidt, Sebastiaan; Claas, Frans H J

2013-03-14

Assessing messenger RNA (mRNA) and microRNA levels in peripheral blood cells may complement conventional parameters in clinical practice. Working with small, precious samples requires optimal RNA yields and minimal RNA degradation. Several procedures for RNA extraction and complementary DNA (cDNA) synthesis were compared for their efficiency. The effect on RNA quality of freeze-thawing peripheral blood cells and storage in preserving reagents was investigated. In terms of RNA yield and convenience, quality quantitative polymerase chain reaction signals per nanogram of total RNA and using NucleoSpin and mirVana columns is preferable. The SuperScript III protocol results in the highest cDNA yields. During conventional procedures of storing peripheral blood cells at -180°C and thawing them thereafter, RNA integrity is maintained. TRIzol preserves RNA in cells stored at -20°C. Detection of mRNA levels significantly decreases in degraded RNA samples, whereas microRNA molecules remain relatively stable. When standardized to reference targets, mRNA transcripts and microRNAs can be reliably quantified in moderately degraded (quality index 4-7) and severely degraded (quality index <4) RNA samples, respectively. We describe a strategy for obtaining high-quality and quantity RNA from fresh and stored cells from blood. The results serve as a guideline for sensitive mRNA and microRNA expression assessment in clinical material.

Collaborating to Compete: Blood Profiling Atlas in Cancer (BloodPAC) Consortium.

PubMed

Grossman, R L; Abel, B; Angiuoli, S; Barrett, J C; Bassett, D; Bramlett, K; Blumenthal, G M; Carlsson, A; Cortese, R; DiGiovanna, J; Davis-Dusenbery, B; Dittamore, R; Eberhard, D A; Febbo, P; Fitzsimons, M; Flamig, Z; Godsey, J; Goswami, J; Gruen, A; Ortuño, F; Han, J; Hayes, D; Hicks, J; Holloway, D; Hovelson, D; Johnson, J; Juhl, H; Kalamegham, R; Kamal, R; Kang, Q; Kelloff, G J; Klozenbuecher, M; Kolatkar, A; Kuhn, P; Langone, K; Leary, R; Loverso, P; Manmathan, H; Martin, A-M; Martini, J; Miller, D; Mitchell, M; Morgan, T; Mulpuri, R; Nguyen, T; Otto, G; Pathak, A; Peters, E; Philip, R; Posadas, E; Reese, D; Reese, M G; Robinson, D; Dei Rossi, A; Sakul, H; Schageman, J; Singh, S; Scher, H I; Schmitt, K; Silvestro, A; Simmons, J; Simmons, T; Sislow, J; Talasaz, A; Tang, P; Tewari, M; Tomlins, S; Toukhy, H; Tseng, H R; Tuck, M; Tzou, A; Vinson, J; Wang, Y; Wells, W; Welsh, A; Wilbanks, J; Wolf, J; Young, L; Lee, Jsh; Leiman, L C

2017-05-01

The cancer community understands the value of blood profiling measurements in assessing and monitoring cancer. We describe an effort among academic, government, biotechnology, diagnostic, and pharmaceutical companies called the Blood Profiling Atlas in Cancer (BloodPAC) Project. BloodPAC will aggregate, make freely available, and harmonize for further analyses, raw datasets, relevant associated clinical data (e.g., clinical diagnosis, treatment history, and outcomes), and sample preparation and handling protocols to accelerate the development of blood profiling assays. © 2017 Authors Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

Cleaning the IceMole: collection of englacial samples from Blood Falls, Antarctica

NASA Astrophysics Data System (ADS)

Mikucki, J.; Digel, I.; Chua, M.; Davis, J.; Ghosh, D.; Lyons, W. B.; Welch, K. A.; Purcell, A.; Francke, G.; Feldmann, M.; Espe, C.; Heinen, D.; Dachwald, B.; Kowalski, J.; Tulaczyk, S. M.

2016-12-01

The Minimally Invasive Direct Glacial Access project (MIDGE) used a maneuverable thermoelectric melting probe called the IceMole to collect the first englacial samples of brine from Blood Falls, Antarctica. In order to maintain the scientific integrity of samples collected and minimize impact to this specially protected ecosystem, microbial and chemical contamination of the IceMole needed to be minimized. Guidelines have been established for research in Antarctic subglacial systems by the scientific and regulatory community and have been detailed by the "Code of Conduct for the Exploration and Research of Subglacial Aquatic Environments" put forth by the Scientific Committee on Antarctic Research (SCAR) Action Group, and was submitted to the Antarctic Treaty System. This Code of Conduct (CoC) recognizes the ecological importance and pristine nature of subglacial habitats and recommends a path forward towards clean exploration. Similarly, the US and European space agencies (NASA and ESA) have detailed instrument preparation protocols for the exploration of icy worlds in our solar system for planetary protection. Given the synergistic aims of these two groups we have adopted protocols from both subglacial and space exploration approaches. Here we present our approach to cleaning the IceMole in the field and report on ability to reduce the bioload inherent on the melter. Specifically our protocol reduced the exterior bio-load by an order of magnitude, to levels common in most clean rooms, and 1-3 orders of magnitude below that of Taylor Glacier ice surrounding Blood Falls. Our results indicate that the collection of englacial samples for microbiological analysis is feasible with melting probes.

Evaluation of a rapid agglutination method for detection of equine red cell surface antigens (Ca and Aa) as part of pretransfusion testing.

PubMed

Owens, Sean D; Snipes, Joy; Magdesian, K Gary; Christopher, Mary M

2008-03-01

Blood typing before transfusion minimizes the risk of transfusion reactions and prevents immunization of the recipient against incompatible RBC antigens. The major RBC antigens that warrant identification before packed RBC or whole blood transfusions in horses are Ca and Aa. Standard blood-typing protocols are time-consuming (2.5-3.0 hours) and impractical in emergency settings. The purpose of this study was to determine whether equine RBCs could be typed for Ca and Aa antigens using sera from horses with RBC antibodies in a modified rapid (15 minute) blood-typing protocol. Serum was obtained from a horse with anti-Ca antibodies and from another horse with anti-Aa antibodies. The presence of agglutinating antibodies was confirmed with antibody screening. Venous blood samples, collected in citrate-phosphate-dextrose, were obtained from 21 horses of various breeds. Samples were blood typed in the Veterinary Medical Teaching Hospital Hematology Laboratory using standard methodology. Washed RBCs from each of the 21 horses were incubated individually with anti-Ca and anti-Aa sera at dilutions of 1:4, 1:8, and 1:16 for 15 and 30 minutes at room temperature and 37 degrees C. Of the 21 horses, 13 were identified as Aa+/Ca+, four were Aa+/Ca-, two were Aa-/Ca+, and two were Aa-/Ca-. All 17 Aa-positive horses had a positive agglutination reaction at all dilutions of anti-Aa serum, incubation times, and temperatures, while all Aa-negative horses were negative. Each Ca-positive horse had a positive agglutination reaction at all incubation time points and temperatures up to the 1:16 dilution of the anti-Ca serum. All Ca-negative horses were negative at all times, temperatures, and dilutions of anti-Ca serum. Use of the modified protocol on 26 hospitalized horses resulted in accurate typing, based on complete antibody screens. These results support the hypothesis that equine RBCs can be blood typed using a rapid (15 minute) protocol, at room temperature, for the presence of Ca and Aa antigens using equine-derived antisera. This technique may be beneficial for pretransfusion testing of equine patients in an emergency setting.

Noninvasive fetal RhCE genotyping from maternal blood.

PubMed

Geifman-Holtzman, O; Grotegut, C A; Gaughan, J P; Holtzman, E J; Floro, C; Hernandez, E

2009-01-01

The successful prevention of RhD disease has brought attention to other red blood cells' antigens causing alloimmunisation including RhC/c and RhE/e. Prenatal diagnosis of fetal Rh genotype from maternal blood is in clinical use in Europe but not in the USA. To estimate the collective reported diagnostic accuracy of fetal RhCE genotyping from peripheral maternal blood and compare the results of genotyping when fetal cells and free fetal DNA (FfDNA) are used. English-written literature describing fetal RhCE determination from maternal blood using fetal cells or FfDNA was performed using medical subject headings and text words. The sources included Pubmed (1966-2007), Ovid (1966-2007), CINAHL, The Cochrane Library, ACP Journal Club and OCLC. Key words were prenatal diagnosis, fetal RhCE, fetal DNA in maternal blood and alloimmunisation. A study was considered eligible if it described fetal RhCE type determination using maternal peripheral blood reported in the English literature. Abstracts were excluded. From each study, we determined the number of samples tested, fetal RhCE genotype, the source of the fetal DNA, gestational age, presence of alloimmunisation and confirmation of fetal RhCE type. Exclusions and inclusions were noted. We calculated composite estimates of accuracy using a weighted random effects model. We assessed the papers against an international quality, STARD checklist which is standards for reporting studies of diagnostic accuracy. We identified 20 protocols in six English-written publications reporting fetal RhC/c (seven protocols) and/or E/e (13 protocols) genotyping using DNA obtained from maternal blood for a total of 369 samples. For RhC/c, 176 samples were tested and for RhE/e, 193 samples were tested. Accuracy was determined for each study and for all studies. The combined accuracy of fetal genotype was 96.3% for RhC/c and 98.2% for RhE/e. Only a few samples of the sorted cells were found to be a source for accurate diagnosis, but plasma was consistently the best source of fetal RhCE genotyping in 147/147 (100%) for RhC/c and 168/168 (100%) for RhE/e. The combined accuracy of noninvasive fetal RhC/c or RhE/e determination using maternal peripheral blood is 96.3% and 98.2%, respectively. FfDNA in maternal plasma is a better source for genotyping reported to be 100% correct for both RHCE genotypes. Further studies and reports of accuracy from laboratories performing the tests are required before prenatal determination of fetal RhC/c or RhE/e genotypes from maternal blood can safely replace the current methods used in the management of the RhC/c or RhE alloimmunised pregnancies.

Development of an improved rapid BACpro® protocol and a method for direct identification from blood-culture-positive bottles using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

Yonezawa, Takatoshi; Watari, Tomohisa; Ashizawa, Kazuho; Hanada, Daisuke; Yanagiya, Takako; Watanabe, Naoki; Terada, Takashi; Tomoda, Yutaka; Fujii, Satoshi

2018-05-01

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been incorporated into pathogenic bacterial identification methods and has improved their rapidity. Various methods have been reported to directly identify bacteria with MALDI-TOF MS by pretreating culture medium in blood culture bottles. Rapid BACpro® (Nittobo Medical Co., Ltd.) is a pretreatment kit for effective collection of bacteria with cationic copolymers. However, the Rapid BACpro® pretreatment kit is adapted only for MALDI Biotyper (Bruker Daltonics K.K.), and there has been a desire to expand its use to VITEK MS (VMS; bioMerieux SA). We improved the protocol and made it possible to analyze with VMS. The culture medium bacteria collection method was changed to a method with centrifugation after hemolysis using saponin; the cationic copolymer concentration was changed to 30% of the original concentration; the sequence with which reagents were added was changed; and a change was made to an ethanol/formic acid extraction method. The improved protocol enhanced the identification performance. When VMS was used, the identification rate was 100% with control samples. With clinical samples, the identification agreement rate with the cell smear method was 96.3%. The improved protocol is effective in blood culture rapid identification, being both simpler and having an improved identification performance compared with the original. Copyright © 2018 Elsevier B.V. All rights reserved.

Performance Characteristics of Plasma Amyloid β 40 and 42 Assays

PubMed Central

Okereke, Olivia I.; Xia, Weiming; Irizarry, Michael C.; Sun, Xiaoyan; Qiu, Wei Q.; Fagan, Anne M.; Mehta, Pankaj D.; Hyman, Bradley T.; Selkoe, Dennis J.; Grodstein, Francine

2009-01-01

Background Identifying biomarkers of Alzheimer disease (AD) risk will be critical to effective AD prevention. Levels of circulating amyloid β (Aβ) 40 and 42 may be candidate biomarkers. However, properties of plasma Aβ assays must be established. Methods Using five different protocols, blinded samples were used to assess: intra-assay reproducibility; impact of EDTA vs. heparin anticoagulant tubes; and effect of time-to-blood processing. In addition, percent recovery of known Aβ concentrations in spiked samples was assessed. Results Median intra-assay coefficients of variation (CVs) for the assay protocols ranged from 6–24% for Aβ-40, and 8–14% for Aβ-42. There were no systematic differences in reproducibility by collection method. Plasma concentrations of Aβ (particularly Aβ-42) appeared stable in whole blood kept in ice packs and processed as long as 24 hours after collection. Recovery of expected concentrations was modest, ranging from -24% to 44% recovery of Aβ-40, and 17% to 61% of Aβ-42. Conclusions Across five protocols, plasma Aβ-40 and Aβ-42 levels were measured with generally low error, and measurements appeared similar in blood collected in EDTA vs. heparin. While these preliminary findings suggest that measuring plasma Aβ-40 and Aβ-42 may be feasible in varied research settings, additional work in this area is necessary. PMID:19221417

[Identification of occult disseminated tumor cells by recombinant herpes simplex virus expressing GFP (HSV(GFP))].

PubMed

Han, Xiang-ping; Shi, Gui-lan; Wang, Cheng-feng; Li, Jie; Zhang, Jian-wei; Zhang, Yu; Zhang, Shu-ren; Liu, Bin-lei

2012-12-01

To develop a novel rapid protocol for the detection of occult disseminated tumor cells by a recombinant herpes simplex virus expressing GFP (HSV(GFP)). Tumor cells of seven cell lines were exposed to HSV(GFP) and then examined for GFP expression by fluorescence microscopy. Various numbers of tumor cells (10, 100, 1000, 10 000) were mixed into 2 ml human whole blood, separated with lymphocytes separation medium, exposed to HSV(GFP), incubated at 37°C for 6 - 24 h and then counted for the number of green cells under the fluorescence microscope. Some clinical samples including peripheral blood, pleural effusion, ascites, spinal fluid from tumor-bearing patients were screened using this protocol in parallel with routine cytological examination. HSV(GFP) was able to infect all 7 tumor cell lines indicating that the HSV(GFP) can be used to detect different types of tumor cells. The detection sensitivity was 10 cancer cells in 2 ml whole blood. In the clinical samples, there were 4/15 positive by routine cytological examination but 11/15 positive by HSV(GFP), indicating a higher sensitivity of this new protocol. Recombinant herpes simplex virus-mediated green fluorescence is a simple and sensitive technique for the identification of occult disseminated cancer cells including circulating tumor cells (CTCs).

Performance of Stochastic Targeted Blood Glucose Control Protocol by virtual trials in the Malaysian intensive care unit.

PubMed

Jamaludin, Ummu K; M Suhaimi, Fatanah; Abdul Razak, Normy Norfiza; Md Ralib, Azrina; Mat Nor, Mohd Basri; Pretty, Christopher G; Humaidi, Luqman

2018-08-01

Blood glucose variability is common in healthcare and it is not related or influenced by diabetes mellitus. To minimise the risk of high blood glucose in critically ill patients, Stochastic Targeted Blood Glucose Control Protocol is used in intensive care unit at hospitals worldwide. Thus, this study focuses on the performance of stochastic modelling protocol in comparison to the current blood glucose management protocols in the Malaysian intensive care unit. Also, this study is to assess the effectiveness of Stochastic Targeted Blood Glucose Control Protocol when it is applied to a cohort of diabetic patients. Retrospective data from 210 patients were obtained from a general hospital in Malaysia from May 2014 until June 2015, where 123 patients were having comorbid diabetes mellitus. The comparison of blood glucose control protocol performance between both protocol simulations was conducted through blood glucose fitted with physiological modelling on top of virtual trial simulations, mean calculation of simulation error and several graphical comparisons using stochastic modelling. Stochastic Targeted Blood Glucose Control Protocol reduces hyperglycaemia by 16% in diabetic and 9% in nondiabetic cohorts. The protocol helps to control blood glucose level in the targeted range of 4.0-10.0 mmol/L for 71.8% in diabetic and 82.7% in nondiabetic cohorts, besides minimising the treatment hour up to 71 h for 123 diabetic patients and 39 h for 87 nondiabetic patients. It is concluded that Stochastic Targeted Blood Glucose Control Protocol is good in reducing hyperglycaemia as compared to the current blood glucose management protocol in the Malaysian intensive care unit. Hence, the current Malaysian intensive care unit protocols need to be modified to enhance their performance, especially in the integration of insulin and nutrition intervention in decreasing the hyperglycaemia incidences. Improvement in Stochastic Targeted Blood Glucose Control Protocol in terms of u en model is also a must to adapt with the diabetic cohort. Copyright © 2018 Elsevier B.V. All rights reserved.

Evaluation of Streck BCT and PAXgene Stabilised Blood Collection Tubes for Cell-Free Circulating DNA Studies in Plasma.

PubMed

Warton, Kristina; Yuwono, Nicole L; Cowley, Mark J; McCabe, Mark J; So, Alwin; Ford, Caroline E

2017-10-01

Blood samples for studies of circulating DNA in disease are often collected in clinical settings where prompt processing of samples is not possible. In order to avoid problems associated with leukocyte lysis after prolonged blood storage, stabilised blood tubes have been developed containing preservatives that prevent cell lysis. We evaluated Streck BCT tubes and PAXgene ccfDNA tubes, as well as standard EDTA blood collection tubes, in terms of DNA yield and fragment size. Blood was collected in EDTA, Streck BCT or PAXgene ccfDNA tubes and stored for 1 h at 4 °C, or 4 days at room temperature. DNA was extracted using the QIAamp Circulating Nucleic Acids kit, and visualised on an agarose gel or quantitated by qPCR. Ratios of a 247-base and a 115-base amplicon of the Alu repetitive element were used to infer size distribution. While plasma DNA in EDTA tube blood samples increased by ~10- to 20-fold after 4 days of storage at room temperature, both Streck BCT tubes and PAXgene ccfDNA tubes maintained stable plasma DNA concentrations. A slight decrease in DNA yield following 1 h of blood storage at 4 °C was observed in Streck BCT and PAXgene ccfDNA tubes relative to EDTA tubes. This decrease was reversed by increasing the proteinase digest step of the DNA extraction protocol to 60 min, as recommended by Streck tube product literature. Visualisation of the extracted DNA on an agarose gel showed that after 4 days of room temperature storage, samples collected in EDTA tubes contained abundant high-molecular weight DNA, which was partially fragmented in a ladder pattern. A slight increase in high-molecular weight DNA in samples stored for 4 days at room temperature in Streck BCT tubes was also observed, but this was not reflected in a change in large and small Alu fragment ratios as measured by qPCR. Tubes containing preservative to prevent cell lysis can extend the scope for blood collection in clinical settings; however, slight differences between samples collected in different tube types underscore the requirement for standardised protocols, as well as attention to sample handling.

Establishment of Protocols for Global Metabolomics by LC-MS for Biomarker Discovery.

PubMed

Saigusa, Daisuke; Okamura, Yasunobu; Motoike, Ikuko N; Katoh, Yasutake; Kurosawa, Yasuhiro; Saijyo, Reina; Koshiba, Seizo; Yasuda, Jun; Motohashi, Hozumi; Sugawara, Junichi; Tanabe, Osamu; Kinoshita, Kengo; Yamamoto, Masayuki

2016-01-01

Metabolomics is a promising avenue for biomarker discovery. Although the quality of metabolomic analyses, especially global metabolomics (G-Met) using mass spectrometry (MS), largely depends on the instrumentation, potential bottlenecks still exist at several basic levels in the metabolomics workflow. Therefore, we established a precise protocol initially for the G-Met analyses of human blood plasma to overcome some these difficulties. In our protocol, samples are deproteinized in a 96-well plate using an automated liquid-handling system, and conducted either using a UHPLC-QTOF/MS system equipped with a reverse phase column or a LC-FTMS system equipped with a normal phase column. A normalization protocol of G-Met data was also developed to compensate for intra- and inter-batch differences, and the variations were significantly reduced along with our normalization, especially for the UHPLC-QTOF/MS data with a C18 reverse-phase column for positive ions. Secondly, we examined the changes in metabolomic profiles caused by the storage of EDTA-blood specimens to identify quality markers for the evaluation of the specimens' pre-analytical conditions. Forty quality markers, including lysophospholipids, dipeptides, fatty acids, succinic acid, amino acids, glucose, and uric acid were identified by G-Met for the evaluation of plasma sample quality and established the equation of calculating the quality score. We applied our quality markers to a small-scale study to evaluate the quality of clinical samples. The G-Met protocols and quality markers established here should prove useful for the discovery and development of biomarkers for a wider range of diseases.

Metabolic profiling of body fluids and multivariate data analysis.

PubMed

Trezzi, Jean-Pierre; Jäger, Christian; Galozzi, Sara; Barkovits, Katalin; Marcus, Katrin; Mollenhauer, Brit; Hiller, Karsten

2017-01-01

Metabolome analyses of body fluids are challenging due pre-analytical variations, such as pre-processing delay and temperature, and constant dynamical changes of biochemical processes within the samples. Therefore, proper sample handling starting from the time of collection up to the analysis is crucial to obtain high quality samples and reproducible results. A metabolomics analysis is divided into 4 main steps: 1) Sample collection, 2) Metabolite extraction, 3) Data acquisition and 4) Data analysis. Here, we describe a protocol for gas chromatography coupled to mass spectrometry (GC-MS) based metabolic analysis for biological matrices, especially body fluids. This protocol can be applied on blood serum/plasma, saliva and cerebrospinal fluid (CSF) samples of humans and other vertebrates. It covers sample collection, sample pre-processing, metabolite extraction, GC-MS measurement and guidelines for the subsequent data analysis. Advantages of this protocol include: •Robust and reproducible metabolomics results, taking into account pre-analytical variations that may occur during the sampling process•Small sample volume required•Rapid and cost-effective processing of biological samples•Logistic regression based determination of biomarker signatures for in-depth data analysis.

[Study on the reproducibility of ACTH concentrations in plasma of horses with and without equine Cushing syndrome].

PubMed

Gehlen, Heidrun; Bradaric, Zrinkja

2013-01-01

The evaluation of plasma ACTH and the dexamethasone suppression test are considered the methods of choice to evaluate the course of therapy of pituitary pars intermedia dysfunction (PPID). Sampling protocols as well as vacutainers for analysis differ between the laboratories. To evaluate the reproducability of plasma ACTH measurement between four different laboratories (A, B, C, D) in Germany as well as within the laboratories themselves, ten horses with previously diagnosed PPID and four healthy horses were sampled and analyzed. Each laboratory received two differently labeled samples of each horse which had been drawn at the same time (blinded samples). Sampling was performed in the morning at the same time. The sampling vacutainers (with and without addition of coagulation and proteinase inhibitors) and postage of the samples was performed according to laboratory standards. In one laboratory the influence of the time of centrifugation (immediately after taking blood versus after one hour) was determined. The samples were processed and analyzed according to laboratory protocols. Determination of ACTH levels was performed using chemiluminescence immunoassay. In total 132 blood samples were analyzed. The results of doubled blood samples of the same horse showed a standard deviation ranging from +/- 6 to +/- 27 pg/ml within the laboratories (Ø 19,29 pg/ml). The standard deviation of the repeatability of the variation coefficient was 13,48%. Blood samples of the same horse resulted in ACTH levels of 121 pg/ml in the first probe and in < 5 pg/ml in the second probe. Standard deviation of measured ACTH values between the laboratories was +/- 26,4 pg/ml (Ø 27,44 pg/ml). The standard deviation of the reproducibility of the variation coefficient was 18,36%. In a 20 year old gelding the lowest ACTH value was 60.9 pg/ml whereas the highest measured value was 108 pg/ml. Immediate centrifugation of blood samples resulted in significantly higher ACTH values at an average of 11.6 pg/ml. The additional use of proteinase inhibitors (aprotinine) showed no influence on ACTH levels in this study.

Evaluating anesthetic protocols for functional blood flow imaging in the rat eye

NASA Astrophysics Data System (ADS)

Moult, Eric M.; Choi, WooJhon; Boas, David A.; Baumann, Bernhard; Clermont, Allen C.; Feener, Edward P.; Fujimoto, James G.

2017-01-01

The purpose of this study is to evaluate the suitability of five different anesthetic protocols (isoflurane, isoflurane-xylazine, pentobarbital, ketamine-xylazine, and ketamine-xylazine-vecuronium) for functional blood flow imaging in the rat eye. Total retinal blood flow was measured at a series of time points using an ultrahigh-speed Doppler OCT system. Additionally, each anesthetic protocol was qualitatively evaluated according to the following criteria: (1) time-stability of blood flow, (2) overall rate of blood flow, (3) ocular immobilization, and (4) simplicity. We observed that different anesthetic protocols produced markedly different blood flows. Different anesthetic protocols also varied with respect to the four evaluated criteria. These findings suggest that the choice of anesthetic protocol should be carefully considered when designing and interpreting functional blood flow studies in the rat eye.

Protocol for the use of a rapid real-time PCR method for the detection of HIV-1 proviral DNA using double-stranded primer.

PubMed

Pau, Chou-Pong; Wells, Susan K; Granade, Timothy C

2012-01-01

This chapter describes a real-time PCR method for the detection of HIV-1 proviral DNA in whole blood samples using a novel double-stranded primer system. The assay utilizes a simple commercially available DNA extraction method and a rapid and easy-to-perform real-time PCR protocol to consistently detect a minimum of four copies of HIV-1 group M proviral DNA in as little as 90 min after sample (whole blood) collection. Co-amplification of the human RNase P gene serves as an internal control to monitor the efficiency of both the DNA extraction and amplification. Once the assay is validated properly, it may be suitable as an alternative confirmation test for HIV-1 infections in a variety of HIV testing venues including the mother-to-child transmission testing sites, clinics, and diagnostic testing centers.

Human blood RNA stabilization in samples collected and transported for a large biobank

PubMed Central

2012-01-01

Background The Norwegian Mother and Child Cohort Study (MoBa) is a nation-wide population-based pregnancy cohort initiated in 1999, comprising more than 108.000 pregnancies recruited between 1999 and 2008. In this study we evaluated the feasibility of integrating RNA analyses into existing MoBa protocols. We compared two different blood RNA collection tube systems – the PAXgene™ Blood RNA system and the Tempus™ Blood RNA system - and assessed the effects of suboptimal blood volumes in collection tubes and of transportation of blood samples by standard mail. Endpoints to characterize the samples were RNA quality and yield, and the RNA transcript stability of selected genes. Findings High-quality RNA could be extracted from blood samples stabilized with both PAXgene and Tempus tubes. The RNA yields obtained from the blood samples collected in Tempus tubes were consistently higher than from PAXgene tubes. Higher RNA yields were obtained from cord blood (3 – 4 times) compared to adult blood with both types of tubes. Transportation of samples by standard mail had moderate effects on RNA quality and RNA transcript stability; the overall RNA quality of the transported samples was high. Some unexplained changes in gene expression were noted, which seemed to correlate with suboptimal blood volumes collected in the tubes. Temperature variations during transportation may also be of some importance. Conclusions Our results strongly suggest that special collection tubes are necessary for RNA stabilization and they should be used for establishing new biobanks. We also show that the 50,000 samples collected in the MoBa biobank provide RNA of high quality and in sufficient amounts to allow gene expression analyses for studying the association of disease with altered patterns of gene expression. PMID:22988904

PCR-based detection of Toxoplasma gondii DNA in blood and ocular samples for diagnosis of ocular toxoplasmosis.

PubMed

Bourdin, C; Busse, A; Kouamou, E; Touafek, F; Bodaghi, B; Le Hoang, P; Mazier, D; Paris, L; Fekkar, A

2014-11-01

PCR detection of Toxoplasma gondii in blood has been suggested as a possibly efficient method for the diagnosis of ocular toxoplasmosis (OT) and furthermore for genotyping the strain involved in the disease. To assess this hypothesis, we performed PCR with 121 peripheral blood samples from 104 patients showing clinical and/or biological evidence of ocular toxoplasmosis and from 284 (258 patients) controls. We tested 2 different extraction protocols, using either 200 μl (small volume) or 2 ml (large volume) of whole blood. Sensitivity was poor, i.e., 4.1% and 25% for the small- and large-volume extractions, respectively. In comparison, PCR with ocular samples yielded 35.9% sensitivity, while immunoblotting and calculation of the Goldmann-Witmer coefficient yielded 47.6% and 72.3% sensitivities, respectively. Performing these three methods together provided 89.4% sensitivity. Whatever the origin of the sample (ocular or blood), PCR provided higher sensitivity for immunocompromised patients than for their immunocompetent counterparts. Consequently, PCR detection of Toxoplasma gondii in blood samples cannot currently be considered a sufficient tool for the diagnosis of OT, and ocular sampling remains necessary for the biological diagnosis of OT. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Challenging traditional activity limits after coronary artery bypass graft surgery: a simulated lawn-mowing activity.

PubMed

Adams, Jenny; Pullum, Gwen; Stafford, Pamala; Hanners, Nava; Hartman, Julie; Strauss, Danielle; Hubbard, Matt; Lawrence, Anne; Anderson, Valerie; McCullough, Tiffany

2008-01-01

Physician advice and restrictions to patients following a cardiac event can, in some instances, lead patients to be fearful regarding their activities even to the point of inactivity. The purpose of this study was to test whether lawn mowing, one of the activities most strongly discouraged after coronary artery bypass surgery, could be safely performed in a supervised setting. Subjects participated in a 6-session simulated lawn-mowing protocol, calibrated to match the push and pull forces of using an outdoor nonpropelled lawn mower. Plain chest radiographs were taken before and after the protocol period. During each session, subjects' sternums were carefully palpated and electrocardiograms, heart rates, and blood pressures were monitored. None of the 13 subjects experienced adverse arrhythmia events or detrimental heart rate, blood pressure, or sternal palpation findings that led to study discontinuation. The radiographs taken after protocol completion showed stable sternal wires with no evidence of sternal dehiscence. Simulated lawn mowing did not negatively affect the sternal incision, electrocardiogram findings, blood pressure, or heart rate in this small sample.

Comparison of three blood transfusion guidelines applied to 31 feline donors to minimise the risk of transfusion-transmissible infections.

PubMed

Marenzoni, Maria Luisa; Lauzi, Stefania; Miglio, Arianna; Coletti, Mauro; Arbia, Andrea; Paltrinieri, Saverio; Antognoni, Maria Teresa

2017-08-01

Objectives The increased demand for animal blood transfusions creates the need for an adequate number of donors. At the same time, a high level of blood safety must be guaranteed and different guidelines (GLs) deal with this topic. The aim of this study was to evaluate the appropriateness of different GLs in preventing transfusion-transmissible infections (TTI) in Italian feline blood donors. Methods Blood samples were collected from 31 cats enrolled as blood donors by the owners' voluntary choice over a period of approximately 1 year. Possible risk factors for TTI were recorded. Based on Italian, European and American GLs, specific TTI, including haemoplasmas, feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV), Anaplasma phagocytophilum, Ehrlichia species, Bartonella species, Babesia species, Theileria species, Cytauxzoon species, Leishmania donovani sensu lato and feline coronavirus (FCoV) were screened. Rapid antigen and serological tests and biomolecular investigations (PCR) were used. Several PCR protocols for haemoplasma and FeLV DNA were compared. Results The presence of at least one recognised risk factor for TTI was reported in all cats. Results for FIV and FeLV infections were negative using rapid tests, whereas five (16.1%) cats were positive for FCoV antibodies. Four (12.9%) cats were PCR positive for haemoplasma DNA and one (3.2%) for FeLV provirus, the latter being positive only using the most sensitive PCR protocol applied. Other TTI were not detected using PCR. Conclusions and relevance Blood safety increases by combining the recommendations of different GLs. To reduce the risk of TTI, sensitive tests are needed and the choice of the best protocol is a critical step in improving blood safety. The cost and time of the screening procedures may be reduced if appropriate tests are selected. To this end, the GLs should include appropriate recruitment protocols and questionnaire-based risk profiles to identify suitable donors.

Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics.

PubMed

van Ginkel, Joost H; van den Broek, Daan A; van Kuik, Joyce; Linders, Dorothé; de Weger, Roel; Willems, Stefan M; Huibers, Manon M H

2017-10-01

In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell-free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for detecting rare mutational targets. In order to perform ddPCR on blood samples, a standardized procedure for processing and analyzing blood samples is necessary to facilitate implementation into clinical practice. Therefore, we assessed the technical sample workup procedure for ddPCR on blood plasma samples. Blood samples from healthy individuals, as well as lung cancer patients were analyzed. We compared different methods and protocols for sample collection, storage, centrifugation, isolation, and quantification. Cell-free DNA (cfDNA) concentrations of several wild-type targets and BRAF and EGFR-mutant ctDNA concentrations quantified by ddPCR were primary outcome measurements. Highest cfDNA concentrations were measured in blood collected in serum tubes. No significant differences in cfDNA concentrations were detected between various time points of up to 24 h until centrifugation. Highest cfDNA concentrations were detected after DNA isolation with the Quick cfDNA Serum & Plasma Kit, while plasma isolation using the QIAamp Circulating Nucleic Acid Kit yielded the most consistent results. DdPCR results on cfDNA are highly dependent on multiple factors during preanalytical sample workup, which need to be addressed during the development of this diagnostic tool for cancer diagnostics in the future. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Implementation of a Multidisciplinary Bleeding and Transfusion Protocol Significantly Decreases Perioperative Blood Product Utilization and Improves Some Bleeding Outcomes.

PubMed

Timpa, Joseph G; O'Meara, L Carlisle; Goldberg, Kellen G; Phillips, Jay P; Crawford, Jack H; Jackson, Kimberly W; Alten, Jeffrey A

2016-03-01

Perioperative transfusion of blood products is associated with increased morbidity and mortality after pediatric cardiac surgery. We report the results of a quality improvement project aimed at decreasing perioperative blood product administration and bleeding after pediatric cardiopulmonary bypass (CPB) surgery. A multidisciplinary team evaluated baseline data from 99 consecutive CPB patients, focusing on the variability in transfusion management and bleeding outcomes, to create a standardized bleeding and transfusion management protocol. A total of 62 subsequent patients were evaluated after implementation of the protocol: 17 with single pass hemoconcentrated (SPHC) blood transfusion and 45 with modified ultrafiltration (MUF). Implementation of the protocol with SPHC blood led to significant decrease in transfusion of every blood product in the cardiovascular operating room and first 6 hours in cardiovascular intensive care unit ([CVICU] p < .05). Addition of MUF to the protocol led to further decrease in transfusion of all blood products compared to preprotocol. Patients <2 months old had 49% decrease in total blood product administration: 155 mL/kg preprotocol, 117 mL/kg protocol plus SPHC, and 79 mL/kg protocol plus MUF (p < .01). There were significant decreases in postoperative bleeding in the first hour after CVICU admission: 6 mL/kg preprotocol, 3.8 mL/kg protocol plus SPHC, and 2 mL/kg protocol plusMUF (p = .02). There was also significantly decreased incidence of severe postoperative bleeding (>10 mL/kg) in the first CVICU hour for protocol plus MUF patients (p < .01). Implementation of a multidisciplinary bleeding and transfusion protocol significantly decreases perioperative blood product transfusion and improves some bleeding outcomes.

TECHNICAL ADVANCES: Effects of genotyping protocols on success and errors in identifying individual river otters (Lontra canadensis) from their faeces.

PubMed

Hansen, Heidi; Ben-David, Merav; McDonald, David B

2008-03-01

In noninvasive genetic sampling, when genotyping error rates are high and recapture rates are low, misidentification of individuals can lead to overestimation of population size. Thus, estimating genotyping errors is imperative. Nonetheless, conducting multiple polymerase chain reactions (PCRs) at multiple loci is time-consuming and costly. To address the controversy regarding the minimum number of PCRs required for obtaining a consensus genotype, we compared consumer-style the performance of two genotyping protocols (multiple-tubes and 'comparative method') in respect to genotyping success and error rates. Our results from 48 faecal samples of river otters (Lontra canadensis) collected in Wyoming in 2003, and from blood samples of five captive river otters amplified with four different primers, suggest that use of the comparative genotyping protocol can minimize the number of PCRs per locus. For all but five samples at one locus, the same consensus genotypes were reached with fewer PCRs and with reduced error rates with this protocol compared to the multiple-tubes method. This finding is reassuring because genotyping errors can occur at relatively high rates even in tissues such as blood and hair. In addition, we found that loci that amplify readily and yield consensus genotypes, may still exhibit high error rates (7-32%) and that amplification with different primers resulted in different types and rates of error. Thus, assigning a genotype based on a single PCR for several loci could result in misidentification of individuals. We recommend that programs designed to statistically assign consensus genotypes should be modified to allow the different treatment of heterozygotes and homozygotes intrinsic to the comparative method. © 2007 The Authors.

Energy dispersive X-ray fluorescence spectrometry for the direct multi-element analysis of dried blood spots

NASA Astrophysics Data System (ADS)

Marguí, E.; Queralt, I.; García-Ruiz, E.; García-González, E.; Rello, L.; Resano, M.

2018-01-01

Home-based collection protocols for clinical specimens are actively pursued as a means of improving life quality of patients. In this sense, dried blood spots (DBS) are proposed as a non-invasive and even self-administered alternative to sampling whole venous blood. This contribution explores the potential of energy dispersive X-ray fluorescence spectrometry for the simultaneous and direct determination of some major (S, Cl, K, Na), minor (P, Fe) and trace (Ca, Cu, Zn) elements in blood, after its deposition onto clinical filter papers, thus giving rise to DBS. For quantification purposes the best strategy was to use matrix-matched blood samples of known analyte concentrations. The accuracy and precision of the method were evaluated by analysis of a blood reference material (Seronorm™ trace elements whole blood L3). Quantitative results were obtained for the determination of P, S, Cl, K and Fe, and limits of detection for these elements were adequate, taking into account their typical concentrations in real blood samples. Determination of Na, Ca, Cu and Zn was hampered by the occurrence of high sample support (Na, Ca) and instrumental blanks (Cu, Zn). Therefore, the quantitative determination of these elements at the levels expected in blood samples was not feasible. The methodology developed was applied to the analysis of several blood samples and the results obtained were compared with those reported by standard techniques. Overall, the performance of the method developed is promising and it could be used to determine the aforementioned elements in blood samples in a simple, fast and economic way. Furthermore, its non-destructive nature enables further analyses by means of complementary techniques to be carried out.

The impact of lymphocyte isolation on induced DNA damage in human blood samples measured by the comet assay.

PubMed

Bausinger, Julia; Speit, Günter

2016-09-01

The comet assay is frequently used in human biomonitoring for the detection of exposure to genotoxic agents. Peripheral blood samples are most frequently used and tested either as whole blood or after isolation of lymphocytes (i.e. peripheral blood mononuclear cells, PBMC). To investigate a potential impact of lymphocyte isolation on induced DNA damage in human blood samples, we exposed blood ex vivo to mutagens with different modes of genotoxic action. The comet assay was performed either directly with whole blood at the end of the exposure period or with lymphocytes isolated directly after exposure. In addition to the recommended standard protocol for lymphocyte isolation, a shortened protocol was established to optimise the isolation procedure. The results indicate that the effects of induced DNA strand breaks and alkali-labile sites induced by ionising radiation and alkylants, respectively, are significantly reduced in isolated lymphocytes. In contrast, oxidative DNA base damage (induced by potassium bromate) and stable bulky adducts (induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide; BPDE) seem to be less affected. Our findings suggest that in vivo-induced DNA damage might also be reduced in isolated lymphocytes in comparison with the whole blood depending of the types of DNA damage induced. Because only small genotoxic effects can generally be expected in human biomonitoring studies with the comet assay after occupational and environmental exposure to genotoxic agents, any loss might be relevant and should be avoided. The possibility of such effects and their potential impact on variability of comet assay results in human biomonitoring should be considered when performing or evaluating such kind of studies. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Toward Rare Blood Cell Preservation for RNA Sequencing.

PubMed

Vickovic, Sanja; Ahmadian, Afshin; Lewensohn, Rolf; Lundeberg, Joakim

2015-07-01

Cancer is driven by various events leading to cell differentiation and disease progression. Molecular tools are powerful approaches for describing how and why these events occur. With the growing field of next-generation DNA sequencing, there is an increasing need for high-quality nucleic acids derived from human cells and tissues-a prerequisite for successful cell profiling. Although advances in RNA preservation have been made, some of the largest biobanks still do not employ RNA blood preservation as standard because of limitations in low blood-input volume and RNA stability over the whole gene body. Therefore, we have developed a robust protocol for blood preservation and long-term storage while maintaining RNA integrity. Furthermore, we explored the possibility of using the protocol for preserving rare cell samples, such as circulating tumor cells. The results of our study confirmed that gene expression was not impacted by the preservation procedure (r(2) > 0.88) or by long-term storage (r(2) = 0.95), with RNA integrity number values averaging over 8. Similarly, cell surface antigens were still available for antibody selection (r(2) = 0.95). Lastly, data mining for fusion events showed that it was possible to detect rare tumor cells among a background of other cells present in blood irrespective of fixation. Thus, the developed protocol would be suitable for rare blood cell preservation followed by RNA sequencing analysis. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Rapid DNA extraction from dried blood spots on filter paper: potential applications in biobanking.

PubMed

Choi, Eun-Hye; Lee, Sang Kwang; Ihm, Chunhwa; Sohn, Young-Hak

2014-12-01

Dried blood spot (DBS) technology is a microsampling alternative to traditional plasma or serum sampling for pharmaco- or toxicokinetic evaluation. DBS technology has been applied to diagnostic screening in drug discovery, nonclinical, and clinical settings. We have developed an improved elution protocol involving boiling of blood spots dried on Whatman filter paper. The purpose of this study was to compare the quality, purity, and quantity of DNA isolated from frozen blood samples and DBSs. We optimized a method for extraction and estimation of DNA from blood spots dried on filter paper (3-mm FTA card). A single DBS containing 40 μL blood was used. DNA was efficiently extracted in phosphate-buffered saline (PBS) or Tris-EDTA (TE) buffer by incubation at 37°C overnight. DNA was stable in DBSs that were stored at room temperature or frozen. The housekeeping genes GAPDH and beta-actin were used as positive standards for polymerase chain reaction (PCR) validation of general diagnostic screening. Our simple and convenient DBS storage and extraction methods are suitable for diagnostic screening by using very small volumes of blood collected on filter paper, and can be used in biobanks for blood sample storage.

Preparation and Application of an Innovative Thrombocyte/Leukocyte-Enriched Plasma to Promote Tissue Repair in Chelonians

PubMed Central

Di Ianni, Francesco; Merli, Elisa; Burtini, Francesca; Conti, Virna; Pelizzone, Igor; Di Lecce, Rosanna; Parmigiani, Enrico; Squassino, Gian Paolo; Del Bue, Maurizio; Lucarelli, Enrico; Ramoni, Roberto; Grolli, Stefano

2015-01-01

Platelet concentrates are widely used in mammalian regenerative medicine to improve tissue healing. Chelonians (Testudines) would benefit from the application of thrombocyte preparations to regenerate damaged tissues, since traumatic injuries are leading causes of morbidity and mortality for both wild-living and domesticated animals. The aim of this study was to establish a protocol that optimized the recovery of the thrombocytes from blood samples and to show the efficacy of thrombocyte-enriched plasma in chelonians. Peripheral blood samples were obtained from Testudo spp. (n = 12) and Trachemys scripta elegans (n = 10). Blood cells were fractionated by sodium diatrizoate-sodium polysucrose density gradient using a two-step centrifugation protocol. Thrombocytes and leukocytes were isolated and resuspended to obtain thrombocyte-leucocyte rich plasma (TLRP). The mean recovery of leukocytes and thrombocytes was 48.9% (±4.0 SEM, n = 22) of the whole blood cell content. No statistically significant difference was observed between blood samples collected from different turtle species. The ability of TLRP to form a gel was evaluated by adding variable concentrations of calcium gluconate at room temperature and at 37°C. A reliable and consistent clotting of the TLRP was obtained in glass tubes and dishes by adding 5-20% v/v of a 100 mg/ml solution of calcium gluconate. Furthermore, in order to test the clinical efficacy of TLRP, a preliminary evaluation was performed on four turtles (Testudo spp.) with traumatic injuries. In all the four animals, a successful clinical outcome was observed. The results demonstrated that a thrombocyte-enriched plasma, comparable to mammalian platelet rich plasma, can be prepared from chelonian blood samples. Furthermore, although the low number of cases presented does not allow definitive conclusions from a clinical point of view, their outcome suggests that TLRP application could be further investigated to improve the healing process of both soft and hard tissue injuries in chelonians. PMID:25901960

Host-Associated Metagenomics: A Guide to Generating Infectious RNA Viromes

PubMed Central

Robert, Catherine; Pascalis, Hervé; Michelle, Caroline; Jardot, Priscilla; Charrel, Rémi; Raoult, Didier; Desnues, Christelle

2015-01-01

Background Metagenomic analyses have been widely used in the last decade to describe viral communities in various environments or to identify the etiology of human, animal, and plant pathologies. Here, we present a simple and standardized protocol that allows for the purification and sequencing of RNA viromes from complex biological samples with an important reduction of host DNA and RNA contaminants, while preserving the infectivity of viral particles. Principal Findings We evaluated different viral purification steps, random reverse transcriptions and sequence-independent amplifications of a pool of representative RNA viruses. Viruses remained infectious after the purification process. We then validated the protocol by sequencing the RNA virome of human body lice engorged in vitro with artificially contaminated human blood. The full genomes of the most abundant viruses absorbed by the lice during the blood meal were successfully sequenced. Interestingly, random amplifications differed in the genome coverage of segmented RNA viruses. Moreover, the majority of reads were taxonomically identified, and only 7–15% of all reads were classified as “unknown”, depending on the random amplification method. Conclusion The protocol reported here could easily be applied to generate RNA viral metagenomes from complex biological samples of different origins. Our protocol allows further virological characterizations of the described viral communities because it preserves the infectivity of viral particles and allows for the isolation of viruses. PMID:26431175

Screening for intermediate and severe forms of thalassaemia in discarded red blood cells: optimization and feasibility.

PubMed

George, Elizabeth; Lai, Mei I; Teh, Lai Kuan; Ramasamy, Rajesh; Goh, Ern Huei; Asokan, Kamalan; Tan, J A M A; Vasudevan, Maithili; Low, Sharon

2011-12-01

Detection and quantification of Hb subtypes of human blood is integral to presumptive identification of thalassaemias. It has been used in neonatal screening of thalassaemia and Hb variants. The use of discarded red blood cells following processing of the cord blood for stem cells provides readily available diagnostic material for thalassaemia screening. In this study, we determined the range of Hb subtypes in 195 consecutive cord blood samples collected for cord blood banking. The 'cord blood samples' analysed were those of the remaining red blood cells after the cord blood was processed for stem cell storage. Quantification of Hb subtypes by high performance liquid chromatography (HPLC) was done on BioRad Variant II Hb testing system. Only 73 (36.5%) of the samples could be analyzed neat without dilution. With a 1:300 dilution with wash solution the acceptable area as recommended by the manufacturer for reading of a C-gram within the 1 to 3 million ranges were achieved in all. Eighteen (9%) 12 showed classical Hb Barts (y4) prerun peaks were confirmed by Sebia Hydrasys automated Hb gel electrophoresis and quantified by Sebia Capillarys 2 capillary electrophoresis. Only 1 (0.5%) was presumptively identified with HbH disease. Due to the limited number of samples no beta-thalassaemia major, Hb E beta-thalassaemia and Hb Barts hydrops fetalis were found. The HPLC assay was possible at a cost US$ 5 per sample and a turnover time of 10 samples per hour without technical difficulties. This study reports an effective and valuable protocol for thalassaemia screening in red blood cells which would otherwise be discarded during cord blood processing. Cord blood with severe and intermediate forms of thalassaemia can be preselected and not stored.

Toward Reliable Lipoprotein Particle Predictions from NMR Spectra of Human Blood: An Interlaboratory Ring Test.

PubMed

Monsonis Centelles, Sandra; Hoefsloot, Huub C J; Khakimov, Bekzod; Ebrahimi, Parvaneh; Lind, Mads V; Kristensen, Mette; de Roo, Niels; Jacobs, Doris M; van Duynhoven, John; Cannet, Claire; Fang, Fang; Humpfer, Eberhard; Schäfer, Hartmut; Spraul, Manfred; Engelsen, Søren B; Smilde, Age K

2017-08-01

Lipoprotein profiling of human blood by 1 H nuclear magnetic resonance (NMR) spectroscopy is a rapid and promising approach to monitor health and disease states in medicine and nutrition. However, lack of standardization of measurement protocols has prevented the use of NMR-based lipoprotein profiling in metastudies. In this study, a standardized NMR measurement protocol was applied in a ring test performed across three different laboratories in Europe on plasma and serum samples from 28 individuals. Data was evaluated in terms of (i) spectral differences, (ii) differences in LPD predictions obtained using an existing prediction model, and (iii) agreement of predictions with cholesterol concentrations in high- and low-density lipoproteins (HDL and LDL) particles measured by standardized clinical assays. ANOVA-simultaneous component analysis (ASCA) of the ring test spectral ensemble that contains methylene and methyl peaks (1.4-0.6 ppm) showed that 97.99% of the variance in the data is related to subject, 1.62% to sample type (serum or plasma), and 0.39% to laboratory. This interlaboratory variation is in fact smaller than the maximum acceptable intralaboratory variation on quality control samples. It is also shown that the reproducibility between laboratories is good enough for the LPD predictions to be exchangeable when the standardized NMR measurement protocol is followed. With the successful implementation of this protocol, which results in reproducible prediction of lipoprotein distributions across laboratories, a step is taken toward bringing NMR more into scope of prognostic and diagnostic biomarkers, reducing the need for less efficient methods such as ultracentrifugation or high-performance liquid chromatography (HPLC).

Spectral features of the body fluids of patients with benign and malignant prostate tumours

NASA Astrophysics Data System (ADS)

Atif, M.; Devanesan, S.; Farhat, K.; Rabah, D.; AlSalhi, M. S.; Masilamani, V.

2013-05-01

In this study, we present the results of fluorescence spectra of blood and urine to detect and discriminate between samples drawn from benign and malignant prostate patients and we find a very good demarcation in terms of spectral features. This preliminary study was carried out as a proof of concept, with limited samples of blood and urine from known cases of patients of BPH and CaP. In the near future it is expected that a detailed clinical validation will be done to establish it as a reliable cancer diagnosis protocol.

An evaluation of direct PCR amplification

PubMed Central

Hall, Daniel E.; Roy, Reena

2014-01-01

Aim To generate complete DNA profiles from blood and saliva samples deposited on FTA® and non-FTA® paper substrates following a direct amplification protocol. Methods Saliva samples from living donors and blood samples from deceased individuals were deposited on ten different FTA® and non-FTA® substrates. These ten paper substrates containing body fluids were kept at room temperature for varying lengths of time ranging from one day to approximately one year. For all assays in this research, 1.2 mm punches were collected from each substrate containing one type of body fluid and amplified with reagents provided in the nine commercial polymerase chain reaction (PCR) amplification kits. The substrates were not subjected to purification reagent or extraction buffer prior to amplification. Results Success rates were calculated for all nine amplification kits and all ten substrates based on their ability to yield complete DNA profiles following a direct amplification protocol. Six out of the nine amplification kits, and four out of the ten paper substrates had the highest success rates overall. Conclusion The data show that it is possible to generate complete DNA profiles following a direct amplification protocol using both standard (non-direct) and direct PCR amplification kits. The generation of complete DNA profiles appears to depend more on the success of the amplification kit rather than the than the FTA®- or non-FTA®-based substrates. PMID:25559837

METHOD FOR MICRORNA ISOLATION FROM CLINICAL SERUM SAMPLES

PubMed Central

Li, Yu; Kowdley, Kris V.

2012-01-01

MicroRNAs are a group of intracellular non-coding RNA molecules that have been implicated in a variety of human diseases. Due to their high stability in blood, microRNAs released into circulation could be potentially utilized as non-invasive biomarkers for diagnosis or prognosis. Current microRNA isolation protocols are specifically designed for solid tissues and are impractical for biomarker development utilizing small-volume serum samples on a large scale. Thus, a protocol for microRNA isolation from serum is needed to accommodate these conditions in biomarker development. To establish such a protocol, we developed a simplified approach to normalize sample input by using single synthetic spike-in microRNA. We evaluated three commonly used commercial microRNA isolation kits for the best performance by comparing RNA quality and yield. The manufacturer’s protocol was further modified to improve the microRNA yield from 200 μL of human serum. MicroRNAs isolated from a large set of clinical serum samples were tested on the miRCURY LNA real-time PCR panel and confirmed to be suitable for high-throughput microRNA profiling. In conclusion, we have established a proven method for microRNA isolation from clinical serum samples suitable for microRNA biomarker development. PMID:22982505

Adjustment of Cell-Type Composition Minimizes Systematic Bias in Blood DNA Methylation Profiles Derived by DNA Collection Protocols

PubMed Central

Shiwa, Yuh; Hachiya, Tsuyoshi; Furukawa, Ryohei; Ohmomo, Hideki; Ono, Kanako; Kudo, Hisaaki; Hata, Jun; Hozawa, Atsushi; Iwasaki, Motoki; Matsuda, Koichi; Minegishi, Naoko; Satoh, Mamoru; Tanno, Kozo; Yamaji, Taiki; Wakai, Kenji; Hitomi, Jiro; Kiyohara, Yutaka; Kubo, Michiaki; Tanaka, Hideo; Tsugane, Shoichiro; Yamamoto, Masayuki; Sobue, Kenji; Shimizu, Atsushi

2016-01-01

Differences in DNA collection protocols may be a potential confounder in epigenome-wide association studies (EWAS) using a large number of blood specimens from multiple biobanks and/or cohorts. Here we show that pre-analytical procedures involved in DNA collection can induce systematic bias in the DNA methylation profiles of blood cells that can be adjusted by cell-type composition variables. In Experiment 1, whole blood from 16 volunteers was collected to examine the effect of a 24 h storage period at 4°C on DNA methylation profiles as measured using the Infinium HumanMethylation450 BeadChip array. Our statistical analysis showed that the P-value distribution of more than 450,000 CpG sites was similar to the theoretical distribution (in quantile-quantile plot, λ = 1.03) when comparing two control replicates, which was remarkably deviated from the theoretical distribution (λ = 1.50) when comparing control and storage conditions. We then considered cell-type composition as a possible cause of the observed bias in DNA methylation profiles and found that the bias associated with the cold storage condition was largely decreased (λadjusted = 1.14) by taking into account a cell-type composition variable. As such, we compared four respective sample collection protocols used in large-scale Japanese biobanks or cohorts as well as two control replicates. Systematic biases in DNA methylation profiles were observed between control and three of four protocols without adjustment of cell-type composition (λ = 1.12–1.45) and no remarkable biases were seen after adjusting for cell-type composition in all four protocols (λadjusted = 1.00–1.17). These results revealed important implications for comparing DNA methylation profiles between blood specimens from different sources and may lead to discovery of disease-associated DNA methylation markers and the development of DNA methylation profile-based predictive risk models. PMID:26799745

Adjustment of Cell-Type Composition Minimizes Systematic Bias in Blood DNA Methylation Profiles Derived by DNA Collection Protocols.

PubMed

Shiwa, Yuh; Hachiya, Tsuyoshi; Furukawa, Ryohei; Ohmomo, Hideki; Ono, Kanako; Kudo, Hisaaki; Hata, Jun; Hozawa, Atsushi; Iwasaki, Motoki; Matsuda, Koichi; Minegishi, Naoko; Satoh, Mamoru; Tanno, Kozo; Yamaji, Taiki; Wakai, Kenji; Hitomi, Jiro; Kiyohara, Yutaka; Kubo, Michiaki; Tanaka, Hideo; Tsugane, Shoichiro; Yamamoto, Masayuki; Sobue, Kenji; Shimizu, Atsushi

2016-01-01

Differences in DNA collection protocols may be a potential confounder in epigenome-wide association studies (EWAS) using a large number of blood specimens from multiple biobanks and/or cohorts. Here we show that pre-analytical procedures involved in DNA collection can induce systematic bias in the DNA methylation profiles of blood cells that can be adjusted by cell-type composition variables. In Experiment 1, whole blood from 16 volunteers was collected to examine the effect of a 24 h storage period at 4°C on DNA methylation profiles as measured using the Infinium HumanMethylation450 BeadChip array. Our statistical analysis showed that the P-value distribution of more than 450,000 CpG sites was similar to the theoretical distribution (in quantile-quantile plot, λ = 1.03) when comparing two control replicates, which was remarkably deviated from the theoretical distribution (λ = 1.50) when comparing control and storage conditions. We then considered cell-type composition as a possible cause of the observed bias in DNA methylation profiles and found that the bias associated with the cold storage condition was largely decreased (λ adjusted = 1.14) by taking into account a cell-type composition variable. As such, we compared four respective sample collection protocols used in large-scale Japanese biobanks or cohorts as well as two control replicates. Systematic biases in DNA methylation profiles were observed between control and three of four protocols without adjustment of cell-type composition (λ = 1.12-1.45) and no remarkable biases were seen after adjusting for cell-type composition in all four protocols (λ adjusted = 1.00-1.17). These results revealed important implications for comparing DNA methylation profiles between blood specimens from different sources and may lead to discovery of disease-associated DNA methylation markers and the development of DNA methylation profile-based predictive risk models.

A semi-nested real-time PCR method to detect low chimerism percentage in small quantity of hematopoietic stem cell transplant DNA samples.

PubMed

Aloisio, Michelangelo; Bortot, Barbara; Gandin, Ilaria; Severini, Giovanni Maria; Athanasakis, Emmanouil

2017-02-01

Chimerism status evaluation of post-allogeneic hematopoietic stem cell transplantation samples is essential to predict post-transplant relapse. The most commonly used technique capable of detecting small increments of chimerism is quantitative real-time PCR. Although this method is already used in several laboratories, previously described protocols often lack sensitivity and the amount of the DNA required for each chimerism analysis is too high. In the present study, we compared a novel semi-nested allele-specific real-time PCR (sNAS-qPCR) protocol with our in-house standard allele-specific real-time PCR (gAS-qPCR) protocol. We selected two genetic markers and analyzed technical parameters (slope, y-intercept, R2, and standard deviation) useful to determine the performances of the two protocols. The sNAS-qPCR protocol showed better sensitivity and precision. Moreover, the sNAS-qPCR protocol requires, as input, only 10 ng of DNA, which is at least 10-fold less than the gAS-qPCR protocols described in the literature. Finally, the proposed sNAS-qPCR protocol could prove very useful for performing chimerism analysis with a small amount of DNA, as in the case of blood cell subsets.

An Improved In-house MALDI-TOF MS Protocol for Direct Cost-Effective Identification of Pathogens from Blood Cultures.

PubMed

Zhou, Menglan; Yang, Qiwen; Kudinha, Timothy; Sun, Liying; Zhang, Rui; Liu, Chang; Yu, Shuying; Xiao, Meng; Kong, Fanrong; Zhao, Yupei; Xu, Ying-Chun

2017-01-01

Background: Bloodstream infection is a major cause of morbidity and mortality in hospitalized patients worldwide. Delays in the identification of microorganisms often leads to a poor prognosis. The application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) directly to blood culture (BC) broth can potentially identify bloodstream infections earlier, and facilitate timely management. Methods: We developed an "in-house" (IH) protocol for direct MALDI-TOF MS based identification of organisms in positive BCs. The IH protocol was initially evaluated and improved with spiked BC samples, and its performance was compared with the commercial Sepsityper™ kit using both traditional and modified cut-off values. We then studied in parallel the performance of the IH protocol and the colony MS identifications in positive clinical BC samples using only modified cut-off values. All discrepancies were investigated by "gold standard" of gene sequencing. Results: In 54 spiked BC samples, the IH method showed comparable results with Sepsityper™ after applying modified cut-off values. Specifically, accurate species and genus level identification was achieved in 88.7 and 3.9% of all the clinical monomicrobial BCs (284/301, 94.4%), respectively. The IH protocol exhibited superior performance for Gram negative bacteria than for Gram positive bacteria (92.8 vs. 82.4%). For anaerobes and yeasts, accurate species identification was achieved in 80.0 and 90.0% of the cases, respectively. For polymicrobial cultures (17/301, 5.6%), MALDI-TOF MS correctly identified a single species present in all the polymicrobial BCs under the Standard mode, while using the MIXED method, two species were correctly identified in 52.9% of the samples. Comparisons based on BC bottle type, showed that the BACTEC™ Lytic/10 Anaerobic/F culture vials performed the best. Conclusion: Our study provides a novel and effective sample preparation method for MALDI-TOF MS direct identification of pathogens from positive BC vials, with a lower cost ($1.5 vs. $ 7) albeit a slightly more laborious extracting process (an extra 15 min) compared with Sepsityper™ kit.

[THE COMPARISON OF RESULTS OF DETECTION OF MINIMAL RESIDUAL DISEASE IN PERIPHERAL BLOOD AND MARROW IN CHILDREN OF THE FIRST YEAR OF LIFE WITH ACUTE LYMPHOBLASTIC LEUCOSIS].

PubMed

Tsaur, G A; Riger, T O; Popov, A M; Nasedkina, T V; Kustanovich, A M; Solodovnikov, A G; Streneva, O V; Shorikov, E V; Tsvirenko, S V; Saveliev, L I; Fechina, L G

2015-04-01

The occurrence of minimal residual disease is an important prognostic factor under acute lymphoblastic leucosis in children and adults. In overwhelming majority of research studies bone marrow is used to detect minimal residual disease. The comparative characteristic of detection of minimal residual disease in peripheral blood and bone marrow was carried out. The prognostic role of occurrence of minimal residual disease in peripheral blood and bone marrow under therapy according protocol MLL-Baby was evaluated. The analysis embraced 142 pair samples from 53 patients with acute lymphoblastic leucosis and various displacements of gene MLL younger than 365 days. The minimal residual disease was detected by force of identification of chimeric transcripts using polymerase chain reaction in real-time mode in 7 sequential points of observation established by protocol of therapy. The comparability of results of qualitative detection of minimal residual disease in bone marrow and peripheral blood amounted to 84.5%. At that, in all 22 (15.5%) discordant samples minimal residual disease was detected only in bone marrow. Despite of high level of comparability of results of detection of minimal residual disease in peripheral blood and bone marrow the occurrence of minimal residual disease in peripheral blood at various stages of therapy demonstrated no independent prognostic significance. The established differences had no relationship with sensitivity of method determined by value of absolute expression of gene ABL. Most likely, these differences reflected real distribution of tumor cells. The results of study demonstrated that application of peripheral blood instead of bone marrow for monitoring of minimal residual disease under acute lymphoblastic leucosis in children of first year of life is inappropriate. At the same time, retention of minimal residual disease in TH4 in bone marrow was an independent and prognostic unfavorable factor under therapy of acute lymphoblastic leucosis of children of first year of life according protocol MLL-Baby (OO=7.326, confidence interval 2.378-22.565).

Influences of two high intensity interval exercise protocols on the main determinants of blood fluidity in overweight men.

PubMed

Ahmadizad, Sajad; Bassami, Minoo; Hadian, Mohsen; Eslami, Maryam

2016-01-01

Acute effects of continuous exercise on the markers of blood fluidity have been addressed in different populations and the changes are intensity related. However, the effect of different high intensity interval exercise (HIIE) on these variables is unclear. This study is designed to determine the effects of two different HIIE with different work/rest ratios but the same energy expenditure on the main determinants of blood fluidity. Ten overweight men (age, 26.3±1.7 yrs) completed two HIIE protocols on two separate occasions with one week intervening. The two HIIE encompassed performing: 1) 6 intervals of 2 min activity at 85% of VO2max interspersed by 2 min active recovery at 30% of VO2max (ratio 1 to 1, HIIE1/1), and 2) 6 intervals of 30 s activity at 110% of VO2max interspersed by 4 min active recovery at 40% of VO2max (ratio 1 to 8, HIIE1/8). Each exercise trial was followed by 30 min rest. Venous blood samples were obtained before exercise, immediately after exercise and after recovery and analyzed for blood and plasma viscosity, fibrinogen and red blood cell indices. The HIIE1/1 protocol led to higher reduction (P < 0.01) in plasma volume changes compared to HIIE1/8 (9.9% vs 5.7%). Moreover, increases in blood viscosity, plasma viscosity, hematocrit, RBC count and mean arterial blood pressure observed following HIIE1/1 were significantly (P < 0.05) higher than HIIE1/8 ; whereas, the changes in fibrinogen concentration neither were significant in response to both trials nor were significantly different between two protocols (P > 0.05). However, the changes in all variables during exercise were transient and returned to the baseline levels after 30 min recovery. It is concluded that the HIIE protocol with lower intensity and shorter rest intervals (higher work to rest ratio) clearly results in more physiological strain than HIIE with higher intensity but longer rest intervals (lower work to rest ratio) in overweight individuals, and that the work to rest ratio could be as important as exercise intensity when considering the hemorheological variables during HIIE.

A nonlethal sampling method to obtain, generate and assemble whole blood transcriptomes from small, wild mammals.

PubMed

Huang, Zixia; Gallot, Aurore; Lao, Nga T; Puechmaille, Sébastien J; Foley, Nicole M; Jebb, David; Bekaert, Michaël; Teeling, Emma C

2016-01-01

The acquisition of tissue samples from wild populations is a constant challenge in conservation biology, especially for endangered species and protected species where nonlethal sampling is the only option. Whole blood has been suggested as a nonlethal sample type that contains a high percentage of bodywide and genomewide transcripts and therefore can be used to assess the transcriptional status of an individual, and to infer a high percentage of the genome. However, only limited quantities of blood can be nonlethally sampled from small species and it is not known if enough genetic material is contained in only a few drops of blood, which represents the upper limit of sample collection for some small species. In this study, we developed a nonlethal sampling method, the laboratory protocols and a bioinformatic pipeline to sequence and assemble the whole blood transcriptome, using Illumina RNA-Seq, from wild greater mouse-eared bats (Myotis myotis). For optimal results, both ribosomal and globin RNAs must be removed before library construction. Treatment of DNase is recommended but not required enabling the use of smaller amounts of starting RNA. A large proportion of protein-coding genes (61%) in the genome were expressed in the blood transcriptome, comparable to brain (65%), kidney (63%) and liver (58%) transcriptomes, and up to 99% of the mitogenome (excluding D-loop) was recovered in the RNA-Seq data. In conclusion, this nonlethal blood sampling method provides an opportunity for a genomewide transcriptomic study of small, endangered or critically protected species, without sacrificing any individuals. © 2015 John Wiley & Sons Ltd.

Automation of DNA and miRNA co-extraction for miRNA-based identification of human body fluids and tissues.

PubMed

Kulstein, Galina; Marienfeld, Ralf; Miltner, Erich; Wiegand, Peter

2016-10-01

In the last years, microRNA (miRNA) analysis came into focus in the field of forensic genetics. Yet, no standardized and recommendable protocols for co-isolation of miRNA and DNA from forensic relevant samples have been developed so far. Hence, this study evaluated the performance of an automated Maxwell® 16 System-based strategy (Promega) for co-extraction of DNA and miRNA from forensically relevant (blood and saliva) samples compared to (semi-)manual extraction methods. Three procedures were compared on the basis of recovered quantity of DNA and miRNA (as determined by real-time PCR and Bioanalyzer), miRNA profiling (shown by Cq values and extraction efficiency), STR profiles, duration, contamination risk and handling. All in all, the results highlight that the automated co-extraction procedure yielded the highest miRNA and DNA amounts from saliva and blood samples compared to both (semi-)manual protocols. Also, for aged and genuine samples of forensically relevant traces the miRNA and DNA yields were sufficient for subsequent downstream analysis. Furthermore, the strategy allows miRNA extraction only in cases where it is relevant to obtain additional information about the sample type. Besides, this system enables flexible sample throughput and labor-saving sample processing with reduced risk of cross-contamination. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of an adult insulin infusion protocol at an academic medical center.

PubMed

Petrov, Katerina I; Burns, Tammy L; Drincic, Andjela

2012-05-01

Acknowledging evidence of possible detrimental effects of tightly controlled blood glucose levels, the American Association of Clinical Endocrinologists and the American Diabetes Association published a consensus statement recommending less strict control for most diabetic patients. As a result of these recommendations, our academic center at Creighton University Medical Center revised its adult insulin infusion protocol to target blood glucose levels ranging from 120 to 180 mg/dL for regular (standard) glycemic control and 80 to 120 mg/dL for tight control; previous targets had ranged from 80 to 180 mg/dL and 70 to 110 mg/dL, respectively. The primary objective was to evaluate the time that blood glucose values were within the target range for patients receiving the new protocol, compared with patients receiving the previous protocol. Our study was designed to evaluate the effectiveness and safety of the revised protocol. Using a retrospective chart review, we collected data for 4 months from patients on the old insulin protocol (May to August 2009) and for 4 months from patients on the new protocol (September to December 2009). Secondary endpoints included the number of hypoglycemic episodes (blood glucose below 70 mg/dL) and severe hypoglycemic episodes (blood glucose 40 mg/dL or lower) experienced by patients receiving the new insulin protocol compared with those receiving the former protocol. Patient characteristics were similar at baseline. Blood glucose values stayed within the target range for a significantly shorter time with the new protocol than with the former protocol (44.6% vs. 56.8%, respectively; P < 0.001), probably because of the narrower target range in the revised protocol. No statistically significant differences in hypoglycemia were observed after the protocol was changed. Hypoglycemia occurred in 31% of the former-protocol patients compared with 18% of the revised-protocol patients. Severe hypoglycemia was experienced by 2.1% of patients on the old protocol and by 3.1% of patients on the new protocol. Rates of severe hypoglycemia were low (2.6%) with the original protocol. Patients' blood glucose levels were within the target range for a shorter time with the new protocol. Fewer episodes of hypoglycemia were recorded with the new protocol, but rates of severe hypoglycemia were similar with both protocols.

Working towards accreditation by the International Standards Organization 15189 Standard: how to validate an in-house developed method an example of lead determination in whole blood by electrothermal atomic absorption spectrometry.

PubMed

Garcia Hejl, Carine; Ramirez, Jose Manuel; Vest, Philippe; Chianea, Denis; Renard, Christophe

2014-09-01

Laboratories working towards accreditation by the International Standards Organization (ISO) 15189 standard are required to demonstrate the validity of their analytical methods. The different guidelines set by various accreditation organizations make it difficult to provide objective evidence that an in-house method is fit for the intended purpose. Besides, the required performance characteristics tests and acceptance criteria are not always detailed. The laboratory must choose the most suitable validation protocol and set the acceptance criteria. Therefore, we propose a validation protocol to evaluate the performance of an in-house method. As an example, we validated the process for the detection and quantification of lead in whole blood by electrothermal absorption spectrometry. The fundamental parameters tested were, selectivity, calibration model, precision, accuracy (and uncertainty of measurement), contamination, stability of the sample, reference interval, and analytical interference. We have developed a protocol that has been applied successfully to quantify lead in whole blood by electrothermal atomic absorption spectrometry (ETAAS). In particular, our method is selective, linear, accurate, and precise, making it suitable for use in routine diagnostics.

Validation of the fully automated A&D TM-2656 blood pressure monitor according to the British Hypertension Society Protocol.

PubMed

Zeng, Wei-Fang; Liu, Ming; Kang, Yuan-Yuan; Li, Yan; Wang, Ji-Guang

2013-08-01

The present study aimed to evaluate the accuracy of the fully automated oscillometric upper-arm blood pressure monitor TM-2656 according to the British Hypertension Society (BHS) Protocol 1993. We recruited individuals until there were 85 eligible participants and their blood pressure could meet the blood pressure distribution requirements specified by the BHS Protocol. For each individual, we sequentially measured the systolic and diastolic blood pressures using a mercury sphygmomanometer (two observers) and the TM-2656 device (one supervisor). Data analysis was carried out according to the BHS Protocol. The device achieved grade A. The percentage of blood pressure differences within 5, 10, and 15 mmHg was 62, 85, and 96%, respectively, for systolic blood pressure, and 71, 93, and 99%, respectively, for diastolic blood pressure. The average (±SD) of the device-observer differences was -2.1±7.8 mmHg (P<0.0001) and -1.1±5.8 mmHg (P<0.0001) for systolic and diastolic blood pressures, respectively. The A&D upper-arm blood pressure monitor TM-2656 has passed the requirements of the BHS Protocol, and can thus be recommended for blood pressure measurement.

Muscle contraction duration and fibre recruitment influence blood flow and oxygen consumption independent of contractile work during steady-state exercise in humans.

PubMed

Richards, Jennifer C; Crecelius, Anne R; Kirby, Brett S; Larson, Dennis G; Dinenno, Frank A

2012-06-01

We tested the hypothesis that, among conditions of matched contractile work, shorter contraction durations and greater muscle fibre recruitment result in augmented skeletal muscle blood flow and oxygen consumption ( ) during steady-state exercise in humans. To do so, we measured forearm blood flow (FBF; Doppler ultrasound) during 4 min of rhythmic hand-grip exercise in 24 healthy young adults and calculated forearm oxygen consumption ( ) via blood samples obtained from a catheter placed in retrograde fashion into a deep vein draining the forearm muscle. In protocol 1 (n = 11), subjects performed rhythmic isometric hand-grip exercise at mild and moderate intensities during conditions in which time-tension index (isometric analogue of work) was held constant but contraction duration was manipulated. In this protocol, shorter contraction durations led to greater FBF (184 ± 25 versus 164 ± 25 ml min(-1)) and (23 ± 3 versus 17 ± 2 ml min(-1); both P < 0.05) among mild workloads, whereas this was not the case for moderate-intensity exercise. In protocol 2 (n = 13), subjects performed rhythmic dynamic hand-grip exercise at mild and moderate intensities in conditions of matched total work, but muscle fibre recruitment was manipulated. In this protocol, greater muscle fibre recruitment led to significantly greater FBF (152 ± 15 versus 127 ± 13 ml min(-1)) and (20 ± 2 versus 17 ± 2 ml min(-1); both P < 0.05) at mild workloads, and there was a trend for similar responses at the moderate intensity but this was not statistically significant. In both protocols, the ratio of the change in FBF to change in was similar across all exercise intensities and manipulations, and the strongest correlation among all variables was between and blood flow. Our collective data indicate that, among matched workloads, shorter contraction duration and greater muscle fibre recruitment augment FBF and during mild-intensity forearm exercise, and that muscle blood flow is more closely related to metabolic cost ( ) rather than contractile work per se during steady-state exercise in humans.

Sensitivity of different Trypanosoma vivax specific primers for the diagnosis of livestock trypanosomosis using different DNA extraction methods.

PubMed

Gonzales, J L; Loza, A; Chacon, E

2006-03-15

There are several T. vivax specific primers developed for PCR diagnosis. Most of these primers were validated under different DNA extraction methods and study designs leading to heterogeneity of results. The objective of the present study was to validate PCR as a diagnostic test for T. vivax trypanosomosis by means of determining the test sensitivity of different published specific primers with different sample preparations. Four different DNA extraction methods were used to test the sensitivity of PCR with four different primer sets. DNA was extracted directly from whole blood samples, blood dried on filter papers or blood dried on FTA cards. The results showed that the sensitivity of PCR with each primer set was highly dependant of the sample preparation and DNA extraction method. The highest sensitivities for all the primers tested were determined using DNA extracted from whole blood samples, while the lowest sensitivities were obtained when DNA was extracted from filter paper preparations. To conclude, the obtained results are discussed and a protocol for diagnosis and surveillance for T. vivax trypanosomosis is recommended.

Rapid method for direct identification of bacteria in urine and blood culture samples by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: intact cell vs. extraction method.

PubMed

Ferreira, L; Sánchez-Juanes, F; Muñoz-Bellido, J L; González-Buitrago, J M

2011-07-01

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a fast and reliable technology for the identification of microorganisms with proteomics approaches. Here, we compare an intact cell method and a protein extraction method before application on the MALDI plate for the direct identification of microorganisms in both urine and blood culture samples from clinical microbiology laboratories. The results show that the intact cell method provides excellent results for urine and is a good initial method for blood cultures. The extraction method complements the intact cell method, improving microorganism identification from blood culture. Thus, we consider that MALDI-TOF MS performed directly on urine and blood culture samples, with the protocols that we propose, is a suitable technique for microorganism identification, as compared with the routine methods used in the clinical microbiology laboratory. © 2010 The Authors. Clinical Microbiology and Infection © 2010 European Society of Clinical Microbiology and Infectious Diseases.

Evaluation of Methods to Improve the Extraction and Recovery of DNA from Cotton Swabs for Forensic Analysis

PubMed Central

Adamowicz, Michael S.; Stasulli, Dominique M.; Sobestanovich, Emily M.; Bille, Todd W.

2014-01-01

Samples for forensic DNA analysis are often collected from a wide variety of objects using cotton or nylon tipped swabs. Testing has shown that significant quantities of DNA are retained on the swab, however, and subsequently lost. When processing evidentiary samples, the recovery of the maximum amount of available DNA is critical, potentially dictating whether a usable profile can be derived from a piece of evidence or not. The QIAamp DNA Investigator extraction kit was used with its recommended protocol for swabs (one hour incubation at 56°C) as a baseline. Results indicate that over 50% of the recoverable DNA may be retained on the cotton swab tip, or otherwise lost, for both blood and buccal cell samples when using this protocol. The protocol’s incubation time and temperature were altered, as was incubating while shaking or stationary to test for increases in recovery efficiency. An additional step was then tested that included periodic re-suspension of the swab tip in the extraction buffer during incubation. Aliquots of liquid blood or a buccal cell suspension were deposited and dried on cotton swabs and compared with swab-less controls. The concentration of DNA in each extract was quantified and STR analysis was performed to assess the quality of the extracted DNA. Stationary incubations and those performed at 65°C did not result in significant gains in DNA yield. Samples incubated for 24 hours yielded less DNA. Increased yields were observed with three and 18 hour incubation periods. Increases in DNA yields were also observed using a swab re-suspension method for both cell types. The swab re-suspension method yielded an average two-fold increase in recovered DNA yield with buccal cells and an average three-fold increase with blood cells. These findings demonstrate that more of the DNA collected on swabs can be recovered with specific protocol alterations. PMID:25549111

Evidence of Mycobacterium tuberculosis complex bacteraemia in intradermal skin test positive cattle detected using phage-RPA.

PubMed

Swift, Benjamin M C; Convery, Thomas W; Rees, Catherine E D

2016-10-02

Bovine tuberculosis is a zoonotic infectious disease caused by Mycobacterium bovis that affects cattle and can cause tuberculosis in a range of wildlife animals. A bacteriophage-based method combined with PCR (phage-PCR) has been recently used to detect and identify viable pathogenic mycobacteria in the peripheral blood mononuclear cells (PBMCs) of animals suffering from paratuberculosis. To adapt this method for the detection of M. bovis in blood, a new isothermal DNA amplification protocol using Recombinase Polymerase Amplification (RPA) was developed and was found to be able to detect M. bovis BCG within 48 h, with a limit of detection of approximately 10 cells per ml of blood for artificially inoculated blood samples. When blood samples (2 ml) from a Single Comparative Cervical Intradermal Tuberculin (SCCIT)- negative beef herd were tested, Mycobacterium tuberculosis complex (MTC) cells were not detected from any (45) of the blood samples. However when blood samples from SCCIT-positive animals were tested, viable MTC bacteria were detected in 66 % (27/41) of samples. Of these 41 animals sampled, 32 % (13) had visible lesions. In the visible lesion (VL) group, 85 % (11/13) had detectable levels of MTC whereas only 57 % (16/28) of animals which had no visible lesions (NVL) were found to have detectable mycobacteraemia. These results indicated that this simple, rapid method can be applied for the study of M. bovis infections. The frequency with which viable mycobacteria were detected in the peripheral blood of SCCIT-positive animals changes the paradigm of this disease.

Gene expression profiling of whole blood: Comparison of target preparation methods for accurate and reproducible microarray analysis

PubMed Central

Vartanian, Kristina; Slottke, Rachel; Johnstone, Timothy; Casale, Amanda; Planck, Stephen R; Choi, Dongseok; Smith, Justine R; Rosenbaum, James T; Harrington, Christina A

2009-01-01

Background Peripheral blood is an accessible and informative source of transcriptomal information for many human disease and pharmacogenomic studies. While there can be significant advantages to analyzing RNA isolated from whole blood, particularly in clinical studies, the preparation of samples for microarray analysis is complicated by the need to minimize artifacts associated with highly abundant globin RNA transcripts. The impact of globin RNA transcripts on expression profiling data can potentially be reduced by using RNA preparation and labeling methods that remove or block globin RNA during the microarray assay. We compared four different methods for preparing microarray hybridization targets from human whole blood collected in PAXGene tubes. Three of the methods utilized the Affymetrix one-cycle cDNA synthesis/in vitro transcription protocol but varied treatment of input RNA as follows: i. no treatment; ii. treatment with GLOBINclear; or iii. treatment with globin PNA oligos. In the fourth method cDNA targets were prepared with the Ovation amplification and labeling system. Results We find that microarray targets generated with labeling methods that reduce globin mRNA levels or minimize the impact of globin transcripts during hybridization detect more transcripts in the microarray assay compared with the standard Affymetrix method. Comparison of microarray results with quantitative PCR analysis of a panel of genes from the NF-kappa B pathway shows good correlation of transcript measurements produced with all four target preparation methods, although method-specific differences in overall correlation were observed. The impact of freezing blood collected in PAXGene tubes on data reproducibility was also examined. Expression profiles show little or no difference when RNA is extracted from either fresh or frozen blood samples. Conclusion RNA preparation and labeling methods designed to reduce the impact of globin mRNA transcripts can significantly improve the sensitivity of the DNA microarray expression profiling assay for whole blood samples. While blockage of globin transcripts during first strand cDNA synthesis with globin PNAs resulted in the best overall performance in this study, we conclude that selection of a protocol for expression profiling studies in blood should depend on several factors, including implementation requirements of the method and study design. RNA isolated from either freshly collected or frozen blood samples stored in PAXGene tubes can be used without altering gene expression profiles. PMID:19123946

Human DNA extraction from whole saliva that was fresh or stored for 3, 6 or 12 months using five different protocols

PubMed Central

GARBIERI, Thais Francini; BROZOSKI, Daniel Thomas; DIONÍSIO, Thiago José; SANTOS, Carlos Ferreira; NEVES, Lucimara Teixeira das

2017-01-01

Abstract Saliva when compared to blood collection has the following advantages: it requires no specialized personnel for collection, allows for remote collection by the patient, is painless, well accepted by participants, has decreased risks of disease transmission, does not clot, can be frozen before DNA extraction and possibly has a longer storage time. Objective and Material and Methods This study aimed to compare the quantity and quality of human DNA extracted from saliva that was fresh or frozen for three, six and twelve months using five different DNA extraction protocols: protocol 1 – Oragene™ commercial kit, protocol 2 – QIAamp DNA mini kit, protocol 3 – DNA extraction using ammonium acetate, protocol 4 – Instagene™ Matrix and protocol 5 – Instagene™ Matrix diluted 1:1 using proteinase K and 1% SDS. Briefly, DNA was analyzed using spectrophotometry, electrophoresis and PCR. Results Results indicated that time spent in storage typically decreased the DNA quantity with the exception of protocol 1. The purity of DNA was generally not affected by storage times for the commercial based protocols, while the purity of the DNA samples extracted by the noncommercial protocols typically decreased when the saliva was stored longer. Only protocol 1 consistently extracted unfragmented DNA samples. In general, DNA samples extracted through protocols 1, 2, 3 and 4, regardless of storage time, were amplified by human specific primers whereas protocol 5 produced almost no samples that were able to be amplified by human specific primers. Depending on the protocol used, it was possible to extract DNA in high quantities and of good quality using whole saliva, and furthermore, for the purposes of DNA extraction, saliva can be reliably stored for relatively long time periods. Conclusions In summary, a complicated picture emerges when taking into account the extracted DNA’s quantity, purity and quality; depending on a given researchers needs, one protocol’s particular strengths and costs might be the deciding factor for its employment. PMID:28403355

An in vitro study of the effects of low-level laser radiation on human blood

NASA Astrophysics Data System (ADS)

Siposan, Dan G.; Lukacs, Adalbert

2003-12-01

In the last time the study of the effects of LLLR on the blood is considered to be a subject of great importance in elucidating the mechanisms of action between LLLR and biologic tissues. Different methods of therapy by blood irradiation have been developed and used in clinical purposes with benefic effects. This study investigates some in vitro effects of LLLR on some selected rheologic indices of human blood. After establishing whether or not damaging effects could appear due to laser irradiation of the blood, we tried to find a new method for rejuvenating the blood preserved in MacoPharma-type bags. Blood samples were obtained from adult regular donors (volunteers). HeNe laser and laser diodes were used as radiation source, in a wide range of wavelengths, power densities, doses and other parameters of irradiation protocol. In the first series of experiments we established that LLLR does not alter the fresh blood from healthy donors, for doses between 0 and 10 J/cm3 and power densities between 30 and 180 mW/cm3. In the second series of experiments we established that LLLR does have, in some specific conditions, a revitalizing effect on the erythrocytes in preserved blood. We concluded that laser irradiation of the preserved blood, following a selected protocol of irradiation, could be used as a new method to improve the performances of preservation: prolonging the period of storage and blood rejuvenation before transfusion.

Capillary and venous samples of total creatine kinase are similar after eccentric exercise.

PubMed

Knoblauch, Mark A; O'Connor, Daniel P; Clarke, Mark S F

2010-12-01

Circulating creatine kinase (CK) levels are often monitored as an indirect biomarker of muscle damage after resistive exercise. The purpose of the present investigation was to evaluate whether capillary whole-blood sampling, a simpler and less invasive method for obtaining a venous blood sample, would allow for a reliable measurement of total CK compared to venipuncture. Fifteen untrained subjects performed 50 maximal eccentric elbow extensions to induce muscle damage of the biceps brachii. Capillary (fingerstick) and venous whole-blood samples were collected contemporaneously at baseline and again at 24, 48, 72, and 96 hours post-exercise. Using a commercial CK analysis kit with a protocol modification to account for a reduced sample size, total CK activity of the capillary and venous samples was analyzed concurrently via spectrophotometry. Results indicated a 0.997 correlation between sampling sites for total CK, with disagreement between the venous and capillary samples estimated at <12% across the range of CK values. These findings indicate capillary sampling for total CK activity provides a valid alternative to venipuncture and should be considered by researchers, clinicians, and strength and conditioning specialists as an alternate sampling technique when indirectly evaluating muscle damage after exercise.

A review of blood sample handling and pre-processing for metabolomics studies.

PubMed

Hernandes, Vinicius Veri; Barbas, Coral; Dudzik, Danuta

2017-09-01

Metabolomics has been found to be applicable to a wide range of clinical studies, bringing a new era for improving clinical diagnostics, early disease detection, therapy prediction and treatment efficiency monitoring. A major challenge in metabolomics, particularly untargeted studies, is the extremely diverse and complex nature of biological specimens. Despite great advances in the field there still exist fundamental needs for considering pre-analytical variability that can introduce bias to the subsequent analytical process and decrease the reliability of the results and moreover confound final research outcomes. Many researchers are mainly focused on the instrumental aspects of the biomarker discovery process, and sample related variables sometimes seem to be overlooked. To bridge the gap, critical information and standardized protocols regarding experimental design and sample handling and pre-processing are highly desired. Characterization of a range variation among sample collection methods is necessary to prevent results misinterpretation and to ensure that observed differences are not due to an experimental bias caused by inconsistencies in sample processing. Herein, a systematic discussion of pre-analytical variables affecting metabolomics studies based on blood derived samples is performed. Furthermore, we provide a set of recommendations concerning experimental design, collection, pre-processing procedures and storage conditions as a practical review that can guide and serve for the standardization of protocols and reduction of undesirable variation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA damage in blood cells exposed to low-level lasers.

PubMed

Sergio, Luiz Philippe da Silva; Silva, Ana Paula Almeida da; Amorim, Philipi Freitas; Campos, Vera Maria Araújo; Magalhães, Luis Alexandre Gonçalves; de Paoli, Flavia; de Souza da Fonseca, Adenilson

2015-04-01

In regenerative medicine, there are increasing applications of low-level lasers in therapeutic protocols for treatment of diseases in soft and in bone tissues. However, there are doubts about effects on DNA, and an adequate dosimetry could improve the safety of clinical applications of these lasers. This work aimed to evaluate DNA damage in peripheral blood cells of Wistar rats induced by low-level red and infrared lasers at different fluences, powers, and emission modes according to therapeutic protocols. Peripheral blood samples were exposed to lasers and DNA damage was accessed by comet assay. In other experiments, DNA damage was accessed in blood cells by modified comet assay using formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III enzymes. Data show that exposure to low-level red and infrared lasers induce DNA damage depending on fluence, power and emission mode, which are targeted by Fpg and endonuclease III. Oxidative DNA damage should be considered for therapeutic efficacy and patient safety in clinical applications based on low-level red and infrared lasers. © 2015 Wiley Periodicals, Inc.

Whole blood flow cytometry measurements of in vivo platelet activation in critically-Ill patients are influenced by variability in blood sampling techniques.

PubMed

Rondina, Matthew T; Grissom, Colin K; Men, Shaohua; Harris, Estelle S; Schwertz, Hansjorg; Zimmerman, Guy A; Weyrich, Andrew S

2012-06-01

Flow cytometry is often used to measure in vivo platelet activation in critically-ill patients. Variability in blood sampling techniques, which may confound these measurements, remains poorly characterized. Platelet activation was measured by flow cytometry performed on arterial and venous blood from 116 critically-ill patients. We determined how variability in vascular sampling site, processing times, and platelet counts influenced levels of platelet-monocyte aggregates (PMA), PAC-1 binding (for glycoprotein (GP) IIbIIIa), and P-selectin (P-SEL) expression. Levels of PMA, but not PAC-1 binding or P-SEL expression, were significantly affected by variability in vascular sampling site. Average PMA levels were approximately 60% higher in whole blood drawn from an arterial vessel compared to venous blood (16.2±1.8% vs. 10.7±1.2%, p<0.05). Levels of PMA in both arterial and venous blood increased significantly during ex vivo processing delays (1.7% increase for every 10 minute delay, p<0.05). In contrast, PAC-1 binding and P-SEL expression were unaffected by processing delays. Levels of PMA, but not PAC-1 binding or P-SEL expression, were correlated with platelet count quartiles (9.4±1.6% for the lowest quartile versus 15.4±1.6% for the highest quartile, p<0.05). In critically-ill patients, variability in vascular sampling site, processing times, and platelet counts influence levels of PMA, but not PAC-1 binding or P-SEL expression. These data demonstrate the need for rigorous adherence to blood sampling protocols, particularly when levels of PMA, which are most sensitive to variations in blood collection, are measured for detection of in vivo platelet activation. Copyright © 2011 Elsevier Ltd. All rights reserved.

The UK Biobank sample handling and storage validation studies.

PubMed

Peakman, Tim C; Elliott, Paul

2008-04-01

and aims UK Biobank is a large prospective study in the United Kingdom to investigate the role of genetic factors, environmental exposures and lifestyle in the causes of major diseases of late and middle age. It involves the collection of blood and urine from 500 000 individuals aged between 40 and 69 years. How the samples are collected, processed and stored will have a major impact on the future scientific usefulness of the UK Biobank resource. A series of validation studies was recommended to test the robustness of the draft sample handling and storage protocol. Samples of blood and urine were collected from 40 healthy volunteers and either processed immediately according to the protocol or maintained at specified temperatures (4 degrees C for all tubes with the exception of vacutainers containing acid citrate dextrose that were maintained at 18 degrees C) for 12, 24 or 36 h prior to processing. A further sample was maintained for 24 h at 4 degrees C, processed and the aliquots frozen at -80 degrees C for 20 days and then thawed under controlled conditions. The stability of the samples was compared for the different times in a wide variety of assays. The samples maintained at 4 degrees C were stable for at least 24 h after collection for a wide range of assays. Small but significant changes were observed in metabonomic studies in samples maintained at 4 degrees C for 36 h. There was no degradation of the samples for a range of biochemical assays after short-term freezing and thawing under controlled conditions. Whole blood maintained at 18 degrees C for 24 h in vacutainers containing acid citrate dextrose is suitable for viral immortalization techniques. The validation studies reported in this supplement provide justification for the sample handling and storage procedures adopted in the UK Biobank project.

[Effect of Morinda lucida Benth. (Rubiaceae) and Newbouldia leavis P. Beauv. (Bignoniaceae) on sickling of red blood cells].

PubMed

Joppa, K M; Vovor, A; Eklu-Gadegbeku, K; Agbonon, A; Aklikokou, K; Gbeassor, M

2008-06-01

The purpose of this study was to evaluate the In vitro anti-sickling activity of two plants widely used for treatment of sickle cell disease in Togo, i.e., Morinda lucida et Newbouldia leavis. A concentration-dependent decrease in the rate of sickling was observed after incubation of red blood cells with plant extracts and 2% sodium metabisulfite as compared to incubation with 0.9% NaCl. On samples with a SS blood genotype the inhibition rate of Morinda lucida was 17.30% at a concentration of 1 mg/ml and 92.31% at a concentration of 30 mg/ml. On samples with an AS blood genotype, the inhibition rate of Morinda lucida 48.10% at a concentration of 1 mg/ml and 99.34% at a concentration of 30 mg/ml. Using Newbouldia leavis the inhibition rates at concentrations of 1 mg/ml and 30 mg/ml were 15.66% and 90.42% respectively on samples with a SS blood genotype and 64.03% and 99.02% respectively on samples with an AS blood genotype. The study protocol appeared to be adequate for both SS and AS blood genotypes since the Pearson correlation coefficient between rates measured on the two types of samples was 0.92 for Newuboulida and 0.89 for Morinda. These findings show that these two plants have clear-cut in vitro anti-sickling activity and support their use in traditional medicine.

The Symptomatic Persian Gulf Veterans Protocol: An Analysis of Risk Factors with an Immunologic and Neuropsychiatric Assessment.

DTIC Science & Technology

1999-01-01

Evaluation of peripheral blood mononuclear cells, urine and throat cultures samples for the presence of mycoplasma or ureaplasma organisms by...c) by PCR with a universal mycoplasma primer. Mycoplasma or ureaplasma were detected frequently in urine and throat swabs, however no significant

Immunosuppressant therapeutic drug monitoring by LC-MS/MS: workflow optimization through automated processing of whole blood samples.

PubMed

Marinova, Mariela; Artusi, Carlo; Brugnolo, Laura; Antonelli, Giorgia; Zaninotto, Martina; Plebani, Mario

2013-11-01

Although, due to its high specificity and sensitivity, LC-MS/MS is an efficient technique for the routine determination of immunosuppressants in whole blood, it involves time-consuming manual sample preparation. The aim of the present study was therefore to develop an automated sample-preparation protocol for the quantification of sirolimus, everolimus and tacrolimus by LC-MS/MS using a liquid handling platform. Six-level commercially available blood calibrators were used for assay development, while four quality control materials and three blood samples from patients under immunosuppressant treatment were employed for the evaluation of imprecision. Barcode reading, sample re-suspension, transfer of whole blood samples into 96-well plates, addition of internal standard solution, mixing, and protein precipitation were performed with a liquid handling platform. After plate filtration, the deproteinised supernatants were submitted for SPE on-line. The only manual steps in the entire process were de-capping of the tubes, and transfer of the well plates to the HPLC autosampler. Calibration curves were linear throughout the selected ranges. The imprecision and accuracy data for all analytes were highly satisfactory. The agreement between the results obtained with manual and those obtained with automated sample preparation was optimal (n=390, r=0.96). In daily routine (100 patient samples) the typical overall total turnaround time was less than 6h. Our findings indicate that the proposed analytical system is suitable for routine analysis, since it is straightforward and precise. Furthermore, it incurs less manual workload and less risk of error in the quantification of whole blood immunosuppressant concentrations than conventional methods. © 2013.

Molecular – genetic variance of RH blood group system within human population of Bosnia and Herzegovina

PubMed Central

Lasić, Lejla; Lojo-Kadrić, Naida; Silajdžić, Elma; Pojskić, Lejla; Hadžiselimović, Rifat; Pojskić, Naris

2013-01-01

There are two major theories for inheritance of Rh blood group system: Fisher – Race theory and Wiener theory. Aim of this study was identifying frequency of RHDCE alleles in Bosnian – Herzegovinian population and introduction of this method in screening for Rh phenotype in B&H since this type of analysis was not used for blood typing in B&H before. Rh blood group was typed by Polymerase Chain Reaction, using the protocols and primers previously established by other authors, then carrying out electrophoresis in 2-3% agarose gel. Percentage of Rh positive individuals in our sample is 84.48%, while the percentage of Rh negative individuals is 15.52%. Inter-rater agreement statistic showed perfect agreement (K=1) between the results of Rh blood system detection based on serological and molecular-genetics methods. In conclusion, molecular – genetic methods are suitable for prenatal genotyping and specific cases while standard serological method is suitable for high-throughput of samples. PMID:23448604

Identification of forensic samples by using an infrared-based automatic DNA sequencer.

PubMed

Ricci, Ugo; Sani, Ilaria; Klintschar, Michael; Cerri, Nicoletta; De Ferrari, Francesco; Giovannucci Uzielli, Maria Luisa

2003-06-01

We have recently introduced a new protocol for analyzing all core loci of the Federal Bureau of Investigation's (FBI) Combined DNA Index System (CODIS) with an infrared (IR) automatic DNA sequencer (LI-COR 4200). The amplicons were labeled with forward oligonucleotide primers, covalently linked to a new infrared fluorescent molecule (IRDye 800). The alleles were displayed as familiar autoradiogram-like images with real-time detection. This protocol was employed for paternity testing, population studies, and identification of degraded forensic samples. We extensively analyzed some simulated forensic samples and mixed stains (blood, semen, saliva, bones, and fixed archival embedded tissues), comparing the results with donor samples. Sensitivity studies were also performed for the four multiplex systems. Our results show the efficiency, reliability, and accuracy of the IR system for the analysis of forensic samples. We also compared the efficiency of the multiplex protocol with ultraviolet (UV) technology. Paternity tests, undegraded DNA samples, and real forensic samples were analyzed with this approach based on IR technology and with UV-based automatic sequencers in combination with commercially-available kits. The comparability of the results with the widespread UV methods suggests that it is possible to exchange data between laboratories using the same core group of markers but different primer sets and detection methods.

Blood transport method for chromosome analysis of residents living near Semipalatinsk nuclear test site.

PubMed

Rodzi, Mohd; Ihda, Shozo; Yokozeki, Masako; Takeichi, Nobuo; Tanaka, Kimio; Hoshi, Masaharu

2009-12-01

A study was conducted to compare the storage conditions and transportation period for blood samples collected from residents living in areas near the Semipalatinsk nuclear test site (SNTS). Experiments were performed to simulate storage and shipping environments. Phytohaemagglutinin (PHA)-stimulated blood was stored in 15-ml tubes (condition A: current transport method) in the absence or in 50-ml flasks (condition B: previous transport method) in the presence of RPMI-1640 and 20% fetal bovine serum (FBS). Samples were kept refrigerated at 4 degrees C and cell viability was assessed after 3, 8, 12 and 14 days of storage. RPMI-1640, 20% FBS and further PHA were added to blood samples under condition A in 50-ml flasks for culture. Whole-blood samples under condition B were directly incubated without further sub-culturing process, neither media nor PHA were added, to adopt a similar protocol to that employed in the previous transport method. Samples in condition A and condition B were incubated for 48 hr at 37 degrees C and their mitotic index was determined. The results showed that viable lymphocytes were consistent in both storage conditions but the mitotic index was higher in condition A than in condition B. Although further confirmation studies have to be carried out, previous chromosomal studies and the present experiment have shown that PHA-stimulated blood could be stored without culture medium for up to 8 days under condition A. The present results will be useful for cytogenetic analysis of blood samples that have been transported long distances wherever a radiation accident has occurred.

Immune system changes during simulated planetary exploration on Devon Island, high arctic

PubMed Central

Crucian, Brian; Lee, Pascal; Stowe, Raymond; Jones, Jeff; Effenhauser, Rainer; Widen, Raymond; Sams, Clarence

2007-01-01

Background Dysregulation of the immune system has been shown to occur during spaceflight, although the detailed nature of the phenomenon and the clinical risks for exploration class missions have yet to be established. Also, the growing clinical significance of immune system evaluation combined with epidemic infectious disease rates in third world countries provides a strong rationale for the development of field-compatible clinical immunology techniques and equipment. In July 2002 NASA performed a comprehensive immune assessment on field team members participating in the Haughton-Mars Project (HMP) on Devon Island in the high Canadian Arctic. The purpose of the study was to evaluate the effect of mission-associated stressors on the human immune system. To perform the study, the development of techniques for processing immune samples in remote field locations was required. Ten HMP-2002 participants volunteered for the study. A field protocol was developed at NASA-JSC for performing sample collection, blood staining/processing for immunophenotype analysis, whole-blood mitogenic culture for functional assessments and cell-sample preservation on-location at Devon Island. Specific assays included peripheral leukocyte distribution; constitutively activated T cells, intracellular cytokine profiles, plasma cortisol and EBV viral antibody levels. Study timepoints were 30 days prior to mission start, mid-mission and 60 days after mission completion. Results The protocol developed for immune sample processing in remote field locations functioned properly. Samples were processed on Devon Island, and stabilized for subsequent analysis at the Johnson Space Center in Houston. The data indicated that some phenotype, immune function and stress hormone changes occurred in the HMP field participants that were largely distinct from pre-mission baseline and post-mission recovery data. These immune changes appear similar to those observed in astronauts following spaceflight. Conclusion The immune system changes described during the HMP field deployment validate the use of the HMP as a ground-based spaceflight/planetary exploration analog for some aspects of human physiology. The sample processing protocol developed for this study may have applications for immune studies in remote terrestrial field locations. Elements of this protocol could possibly be adapted for future in-flight immunology studies conducted during space missions. PMID:17521440

Pre-analytical effects of blood sampling and handling in quantitative immunoassays for rheumatoid arthritis.

PubMed

Zhao, Xiaoyan; Qureshi, Ferhan; Eastman, P Scott; Manning, William C; Alexander, Claire; Robinson, William H; Hesterberg, Lyndal K

2012-04-30

Variability in pre-analytical blood sampling and handling can significantly impact results obtained in quantitative immunoassays. Understanding the impact of these variables is critical for accurate quantification and validation of biomarker measurements. Particularly, in the design and execution of large clinical trials, even small differences in sample processing and handling can have dramatic effects in analytical reliability, results interpretation, trial management and outcome. The effects of two common blood sampling methods (serum vs. plasma) and two widely-used serum handling methods (on the clot with ambient temperature shipping, "traditional", vs. centrifuged with cold chain shipping, "protocol") on protein and autoantibody concentrations were examined. Matched serum and plasma samples were collected from 32 rheumatoid arthritis (RA) patients representing a wide range of disease activity status. Additionally, a set of matched serum samples with two sample handling methods was collected. One tube was processed per manufacturer's instructions and shipped overnight on cold packs (protocol). The matched tube, without prior centrifugation, was simultaneously shipped overnight at ambient temperatures (traditional). Upon delivery, the traditional tube was centrifuged. All samples were subsequently aliquoted and frozen prior to analysis of protein and autoantibody biomarkers. Median correlation between paired serum and plasma across all autoantibody assays was 0.99 (0.98-1.00) with a median % difference of -3.3 (-7.5 to 6.0). In contrast, observed protein biomarker concentrations were significantly affected by sample types, with median correlation of 0.99 (0.33-1.00) and a median % difference of -10 (-55 to 23). When the two serum collection/handling methods were compared, the median correlation between paired samples for autoantibodies was 0.99 (0.91-1.00) with a median difference of 4%. In contrast, significant increases were observed in protein biomarker concentrations among certain biomarkers in samples processed with the 'traditional' method. Autoantibody quantification appears robust to both sample type (plasma vs. serum) and pre-analytical sample collection/handling methods (protocol vs. traditional). In contrast, for non-antibody protein biomarker concentrations, sample type had a significant impact; plasma samples generally exhibit decreased protein biomarker concentrations relative to serum. Similarly, sample handling significantly impacted the variability of protein biomarker concentrations. When biomarker concentrations are combined algorithmically into a single test score such as a multi-biomarker disease activity test for rheumatoid arthritis (MBDA), changes in protein biomarker concentrations may result in a bias of the score. These results illustrate the importance of characterizing pre-analytical methodology, sample type, sample processing and handling procedures for clinical testing in order to ensure test accuracy. Copyright © 2012 Elsevier B.V. All rights reserved.

Sonoclot evaluation of whole blood coagulation in healthy adult dogs.

PubMed

Babski, Danielle M; Brainard, Benjamin M; Krimer, Paula M; Ralph, Alan G; Pittman, Jennifer R; Koenig, Amie

2012-12-01

To establish a standard protocol for analysis of canine whole blood and generate reference intervals for healthy dogs using the Sonoclot analyzer, and to compare Sonoclot values to standard and viscoelastic coagulation tests. Prospective study. Veterinary University research facility and teaching hospital. Twelve healthy random source dogs and 52 healthy dogs from the general veterinary school population. Blood sampling for viscoelastic coagulation testing. Blood was collected from 12 healthy adult dogs by jugular venipuncture. After a rest period at room temperature of 30, 60, or 120 minutes, 340 μL of citrated blood was added to 20 μL of 0.2 M CaCl(2) in 1 of 2 cuvette types warmed to 37° C. Cuvettes contained a magnetic stir-bar with glass beads (gbACT+) or only a magnetic stir-bar (nonACT). Reference interval samples were collected from 52 healthy adult dogs and analyzed in duplicate. The ACT, CR, and PF were not affected by duration of rest period for either cuvette type. ACT variability was decreased when using gbACT+ cuvettes (P < 0.05). In normal dogs reference intervals (mean ± 2 SD) using gbACT+ cuvettes were: ACT 56.0-154.0 seconds, CR 14.85-46.0, and PF 2.1-4.05. ACT correlated to TEG R-time, K-time, and angle, while CR correlated with all TEG parameters. Fibrinogen correlated with ACT, CR, and PF. Sonoclot did not correlate with other common coagulation tests. Sonoclot provides viscoelastic evaluation of canine whole blood coagulation and correlated to several TEG parameters and fibrinogen. A standard protocol and reference intervals were established. © Veterinary Emergency and Critical Care Society 2012.

Protocol and standard operating procedures for common use in a worldwide multicenter study on reference values.

PubMed

Ozarda, Yesim; Ichihara, Kiyoshi; Barth, Julian H; Klee, George

2013-05-01

The reference intervals (RIs) given in laboratory reports have an important role in aiding clinicians in interpreting test results in reference to values of healthy populations. In this report, we present a proposed protocol and standard operating procedures (SOPs) for common use in conducting multicenter RI studies on a national or international scale. The protocols and consensus on their contents were refined through discussions in recent C-RIDL meetings. The protocol describes in detail (1) the scheme and organization of the study, (2) the target population, inclusion/exclusion criteria, ethnicity, and sample size, (3) health status questionnaire, (4) target analytes, (5) blood collection, (6) sample processing and storage, (7) assays, (8) cross-check testing, (9) ethics, (10) data analyses, and (11) reporting of results. In addition, the protocol proposes the common measurement of a panel of sera when no standard materials exist for harmonization of test results. It also describes the requirements of the central laboratory, including the method of cross-check testing between the central laboratory of each country and local laboratories. This protocol and the SOPs remain largely exploratory and may require a reevaluation from the practical point of view after their implementation in the ongoing worldwide study. The paper is mainly intended to be a basis for discussion in the scientific community.

Superiority of SDS lysis over saponin lysis for direct bacterial identification from positive blood culture bottle by MALDI-TOF MS.

PubMed

Caspar, Yvan; Garnaud, Cécile; Raykova, Mariya; Bailly, Sébastien; Bidart, Marie; Maubon, Danièle

2017-05-01

Fast species diagnosis has an important health care impact, as rapid and specific antibacterial therapy is of clear benefit for patient's outcome. Here, a new protocol for species identification directly from positive blood cultures is proposed. Four in-house protocols for bacterial identification by MS directly from clinical positive blood cultures evaluating two lytic agents, SDS and saponin, and two protein extraction schemes, fast (FP) and long (LP) are compared. One hundred and sixty-eight identification tests are carried out on 42 strains. Overall, there are correct identifications to the species level in 90% samples for the SDS-LP, 60% for the SDS-FP, 48% for the saponin LP, and 43% for the saponin FP. Adapted scores allowed 92, 86, 72, and 53% identification for SDS-LP, SDS-FP, saponin LP, and saponin FP, respectively. Saponin lysis is associated with a significantly lower score compared to SDS (0.87 [0.83-0.92], p-value < 0.001). This study supports the use of SDS lysis instead of saponin lysis and the application of this rapid and cost-effective protocol in daily routine for microbiological agents implicated in septicemia. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of two aerobic exercise training protocols on parameters of oxidative stress in the blood and liver of obese rats.

PubMed

Delwing-de Lima, Daniela; Ulbricht, Ariene Sampaio Souza Farias; Werlang-Coelho, Carla; Delwing-Dal Magro, Débora; Joaquim, Victor Hugo Antonio; Salamaia, Eloise Mariani; de Quevedo, Silvana Rodrigues; Desordi, Larissa

2017-12-08

We evaluated the effects of moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) protocols on the alterations in oxidative stress parameters caused by a high-fat diet (HFD), in the blood and liver of rats. The HFD enhanced thiobarbituric acid reactive substances (TBA-RS) and protein carbonyl content, while reducing total sulfhydryl content and catalase (CAT) and glutathione peroxidase (GSH-Px) activities in the blood. Both training protocols prevented an increase in TBA-RS and protein carbonyl content, and prevented a reduction in CAT. HIIT protocol enhanced SOD activity. In the liver, HFD didn't alter TBA-RS, total sulfhydryl content or SOD, but increased protein carbonyl content and CAT and decreased GSH-Px. The exercise protocols prevented the increase in protein carbonyl content and the MICT protocol prevented an alteration in CAT. In conclusion, HFD elicits oxidative stress in the blood and liver and both protocols prevented most of the alterations in the oxidative stress parameters.

Simultaneous validation of the Grandway MD2301 digital automatic blood pressure monitor by the British Hypertension Society and the Association for the Advancement of Medical Instrumentation/the International Organization for Standardization protocols.

PubMed

Huang, Jinhua; Wang, Yun; Liu, Zhaoying; Wang, Yuling

2017-02-01

The aim of this study was to determine the accuracy of the Grandway MD2301 digital automatic blood pressure monitor by the British Hypertension Society (BHS) and the Association for the Advancement of Medical Instrumentation (AAMI)/the International Organization for Standardization (ISO) protocols. A total of 85 participants were included for evaluation based on the requirements of the BHS and the AAMI/ISO protocols. The validation procedure and data analysis followed the protocols precisely. The device achieved A/A grading for the BHS protocol and maintained A/A grading throughout the low, medium and high blood pressure ranges. The device also fulfilled the requirement of the AAMI/ISO protocol with device-observer differences of -0.9±5.6 and 0.8±5.2 mmHg for systolic and diastolic blood pressure, respectively, for criterion 1, and -0.9±4.7 and 0.8±4.2 mmHg, respectively, for criterion 2. The Grandway MD2301 digital automatic blood pressure monitor achieved A/A grade of the BHS protocol and passed the requirements of the AAMI/ISO protocol in adults.

Oligonucleotide Microarray Analysis of Dietary-Induced Hyperlipidemia Gene Expression Profiles in Miniature Pigs

PubMed Central

Takahashi, Junko; Waki, Shiori; Matsumoto, Rena; Odake, Junji; Miyaji, Takayuki; Tottori, Junichi; Iwanaga, Takehiro; Iwahashi, Hitoshi

2012-01-01

Background Hyperlipidemia animal models have been established, but complete gene expression profiles of the transition from normal lipid levels have not been obtained. Miniature pigs are useful model animals for gene expression studies on dietary-induced hyperlipidemia because they have a similar anatomy and digestive physiology to humans, and blood samples can be obtained from them repeatedly. Methodology Two typical dietary treatments were used for dietary-induced hyperlipidemia models, by using specific pathogen-free (SPF) Clawn miniature pigs. One was a high-fat and high-cholesterol diet (HFCD) and the other was a high-fat, high-cholesterol, and high-sucrose diet (HFCSD). Microarray analyses were conducted from whole blood samples during the dietary period and from white blood cells at the end of the dietary period to evaluate the transition of expression profiles of the two dietary models. Principal Findings Variations in whole blood gene expression intensity within the HFCD or the HFCSD group were in the same range as the controls provide with normal diet at all periods. This indicates uniformity of dietary-induced hyperlipidemia for our dietary protocols. Gene ontology- (GO) based functional analyses revealed that characteristics of the common changes between HFCD and HFCSD were involved in inflammatory responses and reproduction. The correlation coefficient between whole blood and white blood cell expression profiles at 27 weeks with the HFCSD diet was significantly lower than that of the control and HFCD diet groups. This may be due to the effects of RNA originating from the tissues and/or organs. Conclusions No statistically significant differences in fasting plasma lipids and glucose levels between the HFCD and HFCSD groups were observed. However, blood RNA analyses revealed different characteristics corresponding to the dietary protocols. In this study, whole blood RNA analyses proved to be a useful tool to evaluate transitions in dietary-induced hyperlipidemia gene expression profiles in miniature pigs. PMID:22662175

Direct and quantitative analysis of underivatized acylcarnitines in serum and whole blood using paper spray mass spectrometry

PubMed Central

Yang, Qian; Manicke, Nicholas E.; Wang, He; Petucci, Christopher; Cooks, R. Graham

2013-01-01

A simple protocol for rapid quantitation of acylcarnitines in serum and whole blood has been developed using paper spray mass spectrometry. Dried serum and whole blood containing a mixture of ten acylcarnitines at various concentrations were analyzed as spots from paper directly without any sample pretreatment, separation, or derivatization. The composition of the spray solvent was found to be a critical factor: for serum samples, spray solvent of methanol/water/formic acid (80:20:0.1) gave the best signal intensity while for blood samples which contain more matrix components, acetonitrile/water (90:10) was a much more suitable spray solvent. For the paper type and size used, 0.5 μL of sample provided an optimal signal for both serum and whole blood samples. For quantitative profiling, the limits of quantitation obtained from both serum and blood were much lower than the clinically validated cutoff values for diagnosis of fatty acid oxidation disorders in newborn screening. Linearity (R2>0.95) and reproducibility (RSD ~10 %) were achieved in the concentration ranges from 100 nM to 5 μM for the C2 acylcarnitine, and for other acylcarnitines, these values were from 10 to 500 nM. Acylcarnitine profiles offer an effective demonstration of the fact that paper spray mass spectrometry is an appropriate, simple, rapid method with high sensitivity and high reproducibility applicable to newborn screening tests. PMID:22760507

Transcutaneous bilirubinometry reduces the need for blood sampling in neonates with visible jaundice.

PubMed

Mishra, S; Chawla, D; Agarwal, R; Deorari, A K; Paul, V K; Bhutani, V K

2009-12-01

We determined usefulness of transcutaneous bilirubinometry to decrease the need for blood sampling to assay serum total bilirubin (STB) in the management of jaundiced healthy Indian neonates. Newborns, > or =35 weeks' gestation, with clinical evidence of jaundice were enrolled in an institutional approved randomized clinical trial. The severity of hyperbilirubinaemia was determined by two non-invasive methods: i) protocol-based visual assessment of bilirubin (VaB) and ii) transcutaneous bilirubin (TcB) determination (BiliCheck). By a random allocation, either method was used to decide the need for blood sampling, which was defined to be present if assessed STB by allocated method exceeded 80% of hour-specific threshold values for phototherapy (2004 AAP Guidelines). A total of 617 neonates were randomized to either TcB (n = 314) or VaB (n = 303) groups with comparable gestation, birth weight and postnatal age. Need for blood sampling to assay STB was 34% lower (95% CI: 10% to 51%) in the TcB group compared with VaB group (17.5% vs 26.4% assessments; risk difference: -8.9%, 95% CI: -2.4% to -15.4%; p = 0.008). Routine use of transcutaneous bilirubinometry compared with systematic visual assessment of bilirubin significantly reduced the need for blood sampling to assay STB in jaundiced term and late-preterm neonates. (ClinicalTrials.gov number, NCT00653874).

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

PubMed Central

van Duin, Richard W B; Verzijl, Annemarie; Reiss, Irwin K; Duncker, Dirk J; Merkus, Daphne

2016-01-01

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise. Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling. After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage. The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study. PMID:26889804

Three-dimensional architecture and cell composition of a Choukroun's platelet-rich fibrin clot and membrane.

PubMed

Dohan Ehrenfest, David M; Del Corso, Marco; Diss, Antoine; Mouhyi, Jaafar; Charrier, Jean-Baptiste

2010-04-01

Platelet-rich fibrin (PRF; Choukroun's technique) is a second-generation platelet concentrate for surgical use. This easy protocol allows the production of leukocyte and platelet-rich fibrin clots and membranes starting from 10-ml blood samples. The purposes of this study were to determine the cell composition and three-dimensional organization of this autologous biomaterial and to evaluate the influence of different collection tubes (dry glass or glass-coated plastic tubes) and compression procedures (forcible or soft) on the final PRF-membrane architecture. After centrifugation, blood analyses were performed on the residual waste plasmatic layers after collecting PRF clots. The PRF clots and membranes were processed for examination by light microscopy and scanning electron microscopy. Approximately 97% of the platelets and >50% of the leukocytes were concentrated in the PRF clot and showed a specific three-dimensional distribution, depending on the centrifugation forces. Platelets and fibrin formed large clusters of coagulation in the first millimeters of the membrane beyond the red blood cell base. The fibrin network was very mature and dense. Moreover, there was no significant difference in the PRF architecture between groups using the different tested collection tubes and compression techniques, even if these two parameters could have influenced the growth factor content and biologic matrix properties. The PRF protocol concentrated most platelets and leukocytes from a blood harvest into a single autologous fibrin biomaterial. This protocol offers reproducible results as long as the main production principles are respected.

Acute hormonal responses in elite junior weightlifters.

PubMed

Kraemer, W J; Fry, A C; Warren, B J; Stone, M H; Fleck, S J; Kearney, J T; Conroy, B P; Maresh, C M; Weseman, C A; Triplett, N T

1992-02-01

To date, no published studies have demonstrated resistance exercise-induced increases in serum testosterone in adolescent males. Furthermore, few data are available on the effects of training experience and lifting performance on acute hormonal responses to weightlifting in young males. Twenty-eight junior elite male Olympic-style weightlifters (17.3 +/- 1.4 yrs) volunteered for the study. An acute weightlifting exercise protocol using moderate to high intensity loads and low volume, characteristic of many weightlifting training sessions, was examined. The exercise protocol was directed toward the training associated with the snatch lift weightlifting exercise. Blood samples were obtained from a superficial arm vein at 7 a.m. (for baseline measurements), and again at pre-exercise, 5 min post-, and 15 min post-exercise time points for determination of serum testosterone, cortisol, growth hormone, plasma beta-endorphin, and whole blood lactate. The exercise protocol elicited significant (p less than or equal to 0.05) increases in each of the hormones and whole blood lactate compared to pre-exercise measures. While not being significantly older, subsequent analysis revealed that subjects with greater than 2 years training experience exhibited significant exercise-induced increases in serum testosterone from pre-exercise to 5 min post-exercise (16.2 +/- 6.2 to 21.4 +/- 7.9 nmol.l-1), while those with less than or equal to 2 years training showed no significant serum testosterone differences. None of the other hormones or whole blood lactate appear to be influenced by training experience.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-invasive surveillance for Plasmodium in reservoir macaque species.

PubMed

Siregar, Josephine E; Faust, Christina L; Murdiyarso, Lydia S; Rosmanah, Lis; Saepuloh, Uus; Dobson, Andrew P; Iskandriati, Diah

2015-10-12

Primates are important reservoirs for human diseases, but their infection status and disease dynamics are difficult to track in the wild. Within the last decade, a macaque malaria, Plasmodium knowlesi, has caused disease in hundreds of humans in Southeast Asia. In order to track cases and understand zoonotic risk, it is imperative to be able to quantify infection status in reservoir macaque species. In this study, protocols for the collection of non-invasive samples and isolation of malaria parasites from naturally infected macaques are optimized. Paired faecal and blood samples from 60 Macaca fascicularis and four Macaca nemestrina were collected. All animals came from Sumatra or Java and were housed in semi-captive breeding colonies around West Java. DNA was extracted from samples using a modified protocol. Nested polymerase chain reactions (PCR) were run to detect Plasmodium using primers targeting mitochondrial DNA. Sensitivity of screening faecal samples for Plasmodium was compared to other studies using Kruskal Wallis tests and logistic regression models. The best primer set was 96.7 % (95 % confidence intervals (CI): 83.3-99.4 %) sensitive for detecting Plasmodium in faecal samples of naturally infected macaques (n = 30). This is the first study to produce definitive estimates of Plasmodium sensitivity and specificity in faecal samples from naturally infected hosts. The sensitivity was significantly higher than some other studies involving wild primates. Faecal samples can be used for detection of malaria infection in field surveys of macaques, even when there are no parasites visible in thin blood smears. Repeating samples from individuals will improve inferences of the epidemiology of malaria in wild primates.

Virtual Global Transplant Laboratory Standard Operating Procedures for Blood Collection, PBMC Isolation, and Storage.

PubMed

Higdon, Lauren E; Lee, Karim; Tang, Qizhi; Maltzman, Jonathan S

2016-09-01

Research on human immune responses frequently involves the use of peripheral blood mononuclear cells (PBMC) immediately, or at significantly delayed timepoints, after collection. This requires PBMC isolation from whole blood and cryopreservation for some applications. It is important to standardize protocols for blood collection, PBMC isolation, cryopreservation, and thawing that maximize survival and functionality of PBMC at the time of analysis. This resource includes detailed protocols describing blood collection tubes, isolation of PBMC using a density gradient, cryopreservation of PBMC, and thawing of cells as well as preparation for functional assays. For each protocol, we include important considerations, such as timing, storage temperatures, and freezing rate. In addition, we provide alternatives so that researchers can make informed decisions in determining the optimal protocol for their application.

Exogenous bacterial contamination of donor blood.

PubMed

Rock, G; Westwood, J C

1977-05-07

The Canadian Red Cross blood transfusion service has followed a set protocol for phlebotomy and collection of a unit of blood. Recent requirements for automated testing have necessitated that a second tube of blood be obtained from the blood line following collection of the unit. Evaluation of the techniques used, however, has indicated the possibility of bacterial contamination from the skin of donors, from insertion of the needle through an unsterile rubber stopper, and through backflow from a nonsterile vacuum tube. To test these possibilities swabs were taken from skin and stoppers of vacuum tubes. Further, vacuum tubes were deliberately contaminated with Escherichia coli. The normal sampling procedure, which involves stripping the donor line to refill and mix the blood, was then followed. This resulted in contamination of the segments and even the blood bag. These findings led to modification of the standard bleeding technique, whereby stripping was eliminated and sterile vacuum tubes were to be used at all times.

University of South Carolina CB Support, Basic Research in Materials and Techniques for Optical Computing Standoff Sensors

DTIC Science & Technology

2004-10-01

chloroform-soaked swab prior to making electrical contact with directly related to the oxidation and reduction potential of the an alligator clip. In...other cases, no cleaning protocol was used emitting layers.’.’ Wrighton et al.𔃺 examined the cyclic and a direct connection via an alligator clip was...applied to optical spectra of complex mix- samples requires techniques of simple multivariate patterntame (gasoline, blood , environmental samples

Effect of cold air inhalation and isometric exercise on coronary blood flow and myocardial function in humans

PubMed Central

Muller, Matthew D.; Gao, Zhaohui; Drew, Rachel C.; Herr, Michael D.; Leuenberger, Urs A.

2011-01-01

The effects of cold air inhalation and isometric exercise on coronary blood flow are currently unknown, despite the fact that both cold air and acute exertion trigger angina in clinical populations. In this study, we used transthoracic Doppler echocardiography to measure coronary blood flow velocity (CBV; left anterior descending coronary artery) and myocardial function during cold air inhalation and handgrip exercise. Ten young healthy subjects underwent the following protocols: 5 min of inhaling cold air (cold air protocol), 5 min of inhaling thermoneutral air (sham protocol), 2 min of isometric handgrip at 30% of maximal voluntary contraction (grip protocol), and 5 min of isometric handgrip at 30% maximal voluntary contraction while breathing cold air (cold + grip protocol). Heart rate, blood pressure, inspired air temperature, CBV, myocardial function (tissue Doppler imaging), O2 saturation, and pulmonary function were measured. The rate-pressure product (RPP) was used as an index of myocardial O2 demand, whereas CBV was used as an index of myocardial O2 supply. Compared with the sham protocol, the cold air protocol caused a significantly higher RPP, but there was a significant reduction in CBV. The cold + grip protocol caused a significantly greater increase in RPP compared with the grip protocol (P = 0.045), but the increase in CBV was significantly less (P = 0.039). However, myocardial function was not impaired during the cold + grip protocol relative to the grip protocol alone. Collectively, these data indicate that there is a supply-demand mismatch in the coronary vascular bed when cold ambient air is breathed during acute exertion but myocardial function is preserved, suggesting an adequate redistribution of blood flow. PMID:21940852

Study of microtip-based extraction and purification of DNA from human samples for portable devices

NASA Astrophysics Data System (ADS)

Fotouhi, Gareth

DNA sample preparation is essential for genetic analysis. However, rapid and easy-to-use methods are a major challenge to obtaining genetic information. Furthermore, DNA sample preparation technology must follow the growing need for point-of-care (POC) diagnostics. The current use of centrifuges, large robots, and laboratory-intensive protocols has to be minimized to meet the global challenge of limited access healthcare by bringing the lab to patients through POC devices. To address these challenges, a novel extraction method of genomic DNA from human samples is presented by using heat-cured polyethyleneimine-coated microtips generating a high electric field. The microtip extraction method is based on recent work using an electric field and capillary action integrated into an automated device. The main challenges to the method are: (1) to obtain a stable microtip surface for the controlled capture and release of DNA and (2) to improve the recovery of DNA from samples with a high concentration of inhibitors, such as human samples. The present study addresses these challenges by investigating the heat curing of polyethyleneimine (PEI) coated on the surface of the microtip. Heat-cured PEI-coated microtips are shown to control the capture and release of DNA. Protocols are developed for the extraction and purification of DNA from human samples. Heat-cured PEI-coated microtip methods of DNA sample preparation are used to extract genomic DNA from human samples. It is discovered through experiment that heat curing of a PEI layer on a gold-coated surface below 150°C could inhibit the signal of polymerase chain reaction (PCR). Below 150°C, the PEI layer is not completely cured and dissolved off the gold-coated surface. Dissolved PEI binds with DNA to inhibit PCR. Heat curing of a PEI layer above 150°C on a gold-coated surface prevents inhibition to PCR and gel electrophoresis. In comparison to gold-coated microtips, the 225°C-cured PEI-coated microtips improve the recovery of DNA to 45% efficiency. Furthermore, the 225°C-cured PEI-coated microtips recover more DNA than gold-coated microtips when the surface is washed. Heat-cured (225°C) PEI-coated microtips are used for the recovery of human genomic DNA from whole blood. A washing protocol is developed to remove inhibiting particles bound to the PEI-coated microtip surface after DNA extraction. From 1.25 muL of whole blood, an average of 1.83 ng of human genomic DNA is captured, purified, and released using a 225°C-cured PEI-coated microtip in less than 30 minutes. The extracted DNA is profiled by short tandem repeat analysis (STR). For forensic and medical applications, genomic DNA is extracted from dried samples using heat-cured PEI-coated microtips that are integrated into an automated device. DNA extraction from dried samples is critical for forensics. The use of dried samples in the medical field is increasing because dried samples are convenient for storage, biosafety, and contamination. The main challenge is the time required to properly extract DNA in a purified form. Typically, a 1 hour incubation period is required to complete this process. Overnight incubation is sometimes necessary. To address this challenge, a pre-extraction washing step is investigated to remove inhibiting particles from dried blood spots (DBS) before DNA is released from dried form into solution for microtip extraction. The developed protocol is expanded to extract DNA from a variety of dried samples including nasal swabs, buccal swabs, and other forensic samples. In comparison to a commercial kit, the microtip-based extraction reduced the processing time from 1.5 hours to 30 minutes or less with an equivalent concentration of extracted DNA from dried blood spots. The developed assay will benefit genetic studies on newborn screening, forensic investigation, and POC diagnostics.

DNA Fingerprinting Using PCR: A Practical Forensic Science Activity

ERIC Educational Resources Information Center

Choi, Hyun-Jung; Ahn, Jung Hoon; Ko, Minsu

2008-01-01

This paper describes a forensic science simulation programme applicable for use in colleges. Students were asked to find a putative suspect by DNA fingerprinting using a simple protocol developed in this study. DNA samples were obtained from a hair root and a drop of blood, common sources of DNA in forensic science. The DNA fingerprinting protocol…

Vascular Access Port Implantation and Serial Blood Sampling in a Gottingen Minipig (Sus scrofa domestica) Model of Acute Radiation Injury

PubMed Central

Moroni, Maria; Coolbaugh, Thea V; Mitchell, Jennifer M; Lombardini, Eric; Moccia, Krinon D; Shelton, Larry J; Nagy, Vitaly; Whitnall, Mark H

2011-01-01

Threats of nuclear and other radiologic exposures have been increasing, but no countermeasure for acute radiation syndrome has been approved by regulatory authorities. Because of their similarity to humans in regard to physiology and anatomy, we are characterizing Gottingen minipigs as a model to aid the development of radiation countermeasures. Irradiated minipigs exhibit immunosuppression, severe thrombocytopenia, vascular leakage, and acute inflammation. These complications render serial acquisition of blood samples problematic. Vascular access ports (VAP) facilitate serial sampling, but their use often is complicated by infections and fibrin deposition. We demonstrate here the successful use of VAP for multiple blood samplings in irradiated minipigs. Device design and limited postoperative prophylactic antimicrobial therapy before irradiation were key to obtaining serial sampling, reducing swelling, and eliminating infection and skin necrosis at the implantation site. Modifications of previous protocols included the use of polydioxanone sutures instead of silk; eliminating chronic port access; single-use, sterile, antireflux prefilled syringes for flushing; strict aseptic weekly maintenance of the device, and acclimating animals to reduce stress. VAP remained functional in 19 of 20 irradiated animals for as long as 3 mo. The remaining VAP failed due to a small leak in the catheter, leading to clot formation. VAP-related sepsis occurred in 2 minipigs. Blood sampling did not cause detectable stress in nonanesthetized sham-irradiated animals, according to leukograms and clinical signs. PMID:21333166

Evaluation of viremia, proviral load and cytokine profile in naturally feline immunodeficiency virus infected cats treated with two different protocols of recombinant feline interferon omega.

PubMed

Leal, Rodolfo O; Gil, Solange; Duarte, Ana; McGahie, David; Sepúlveda, Nuno; Niza, Maria M R E; Tavares, Luís

2015-04-01

This study assesses viremia, provirus and blood cytokine profile in naturally FIV-infected cats treated with two distinct protocols of interferon omega (rFeIFN-ω). Samples from FIV-cats previously submitted to two single-arm studies were used: 7/18 received the licensed/subcutaneous protocol (SC) while 11/18 were treated orally (PO). Viremia, provirus and blood mRNA expression of interleukin (IL)-1, IL-4, IL-6, IL-10, IL-12p40, Interferon-γ and Tumor Necrosis Factor-α were monitored by Real-Time qPCR. Concurrent plasma levels of IL-6, IL-12p40 and IL-4 were assessed by ELISA. IL-6 plasma levels decreased in the SC group (p = 0.031). IL-6 mRNA expression (p = 0.037) decreased in the PO group, albeit not sufficiently to change concurrent plasma levels. Neither viremia nor other measured cytokines changed with therapy. Proviral load increased in the SC group (p = 0.031), which can be justified by a clinically irrelevant increase of lymphocyte count. Independently of the protocol, rFeIFN-ω seems to act on innate immunity by reducing pro-inflammatory stimulus. Copyright © 2015 Elsevier Ltd. All rights reserved.

The NHLBI Retrovirus Epidemiology Donor Studies (REDS and REDS-II): Twenty years of research to advance blood product safety and availability

PubMed Central

Kleinman, Steven; King, Melissa R; Busch, Michael P; Murphy, Edward L; Glynn, Simone A.

2012-01-01

The Retrovirus Epidemiology Donor Study (REDS), conducted from 1989–2001, and the Retrovirus Epidemiology Donor Study-II (REDS-II), conducted from 2004–2012, were National Heart Lung and Blood Institute (NHLBI) funded multicenter programs focused on improving blood safety and availability in the United States. REDS-II also included international study sites in Brazil and China. The three major research domains of REDS/REDS-II have been infectious disease risk evaluation, blood donation availability, and blood donor characterization. Both programs have made significant contributions to transfusion medicine research methodology by the use of mathematical modeling, large-scale donor surveys, innovative methods of repository sample storage, and establishing an infrastructure that responded to potential emerging blood safety threats such as XMRV. Blood safety studies have included protocols evaluating epidemiologic and/or laboratory aspects of HIV, HTLV I/II, HCV, HBV, WNV, CMV, HHV-8, B19V, malaria, CJD, influenza, and T. cruzi infections. Other analyses have characterized: blood donor demographics, motivations to donate, factors influencing donor return, behavioral risk factors, donors’ perception of the blood donation screening process, and aspects of donor deferral. In REDS-II, two large-scale blood donor protocols examined iron deficiency in donors and the prevalence of leukocyte antibodies. This review describes the major study results from over 150 peer-reviewed articles published by these two REDS programs. In 2011, a new seven year program, the Recipient Epidemiology and Donor Evaluation Study-III (REDS-III), was launched. REDS-III expands beyond donor-based research to include studies of blood transfusion recipients in the hospital setting, and adds a third country, South Africa, to the international program. PMID:22633182

A Real-World Setting Study: Which Glucose Meter Could Be the Best for POCT Use? An Easy and Applicable Protocol During the Hospital Routine.

PubMed

Mancini, Alessio; Esposto, Giampaolo; Manfrini, Silvana; Rilli, Silvia; Tinti, Gessica; Carta, Giuseppe; Petrolati, Laura; Vidali, Matteo; Barocci, Simone

2018-05-01

The aim of this retrospective study is to evaluate the reliability and robustness of six glucose meters for point-of-care testing in our wards using a brand-new protocol. During a 30-days study period a total of 50 diabetes patients were subjected to venous blood sampling and glucose meter blood analysis. The results of six glucose meters were compared with our laboratory reference assay. GlucoMen Plus (Menarini) with the 82% of acceptable results was the most robust glucose meter. Even if the Passing-Bablok analysis demonstrates the presence of constant systematic errors and the Bland-Altman test highlighted a possible overestimation, the surveillance error grid analysis showed that this glucose meter can be used safely. We proved that portable glucose meters are not always reliable in routinely clinical settings.

Validation of the Pangao PG-800B26 upper arm blood pressure monitor in the general population according to the European Society of Hypertension and the British Hypertension Society protocols.

PubMed

Chen, Liang; Li, Jiyu; Wen, Jing; Guo, Changfeng; Zhang, Jingying; Yu, Zhen

2018-02-01

This study aimed to evaluate the accuracy of the automated oscillometric upper arm blood pressure monitor Pangao PG-800B26 for home blood pressure monitoring according to the European Society of Hypertension International Protocol (ESH-IP) revision 2010 and the British Hypertension Society (BHS) protocols. Systolic and diastolic blood pressures were measured sequentially in 33 and 85 adults, respectively, on the basis of the ESH-IP and BHS protocols using a mercury sphygmomanometer (two observers) and the device (one supervisor). The procedures and analysis methods of the protocols were followed precisely. The device fulfilled the criteria of the ESH-IP, with device-observer differences of 1.01±5.16 and -0.58±4.17 mmHg for systolic and diastolic blood pressure, respectively. Furthermore, the A/A grade of the BHS protocol was also achieved for overall grading and for the three pressure levels, with average differences of 0.85±6.35 and -0.15±5.65 mmHg for systolic and diastolic blood pressure, respectively, which also fulfilled the requirements of the Association for the Advancement of Medical Instrumentation. The Pangao PG-800B26 fulfilled the criteria of the ESH-IP 2010 and achieved the A/A grade of the BHS protocol, and hence can be recommended for home use in adults.

Validation of the Pangao PG-800A36 automatic wrist blood pressure monitor according to the European Society of Hypertension and the British Hypertension Society protocols.

PubMed

Zhao, Hairong; Qiao, Weichang; Zhang, Rui; Cui, Peng; Hou, Fanglin; Zhang, Wenli

2018-02-01

The aim of this study was to validate the PG-800A36 automatic wrist blood pressure monitor according to the European Society of Hypertension International Protocol (ESH-IP) revision 2010 and the British Hypertension Society (BHS) protocols. A total of 33 participants were initially included on the basis of the ESH-IP, followed by examination of 85 participants according to the BHS protocol. The procedures and analysis methods of the protocols were followed precisely with left arm/wrist sequential measurements by two trained observers using a mercury sphygmomanometer and one supervisor using the device. The device passed the ESH-IP with an average difference of 1.45±6.46 mmHg for systolic blood pressure and 1.25±5.10 mmHg for diastolic blood pressure. Furthermore, the A/A grade of the BHS protocol was achieved with an average difference of 1.84±6.94 mmHg for systolic blood pressure and 1.15±6.49 mmHg for diastolic blood pressure, and thus, the device also fulfilled the requirements of the Association for the Advancement of Medical Instrumentation. The Pangao PG-800A36 passed the requirements of the ESH-IP revision 2010 and achieved the A/A grade of the BHS protocol, which can be recommended for self-measurement in the general population.

FAST: Size-Selective, Clog-Free Isolation of Rare Cancer Cells from Whole Blood at a Liquid-Liquid Interface.

PubMed

Kim, Tae-Hyeong; Lim, Minji; Park, Juhee; Oh, Jung Min; Kim, Hyeongeun; Jeong, Hyunjin; Lee, Sun Ju; Park, Hee Chul; Jung, Sungmok; Kim, Byung Chul; Lee, Kyusang; Kim, Mi-Hyun; Park, Do Youn; Kim, Gwang Ha; Cho, Yoon-Kyoung

2017-01-17

Circulating tumor cells (CTCs) have great potential to provide minimally invasive ways for the early detection of cancer metastasis and for the response monitoring of various cancer treatments. Despite the clinical importance and progress of CTC-based cancer diagnostics, most of the current methods of enriching CTCs are difficult to implement in general hospital settings due to complex and time-consuming protocols. Among existing technologies, size-based isolation methods provide antibody-independent, relatively simple, and high throughput protocols. However, the clogging issues and lower than desired recovery rates and purity are the key challenges. In this work, inspired by antifouling membranes with liquid-filled pores in nature, clog-free, highly sensitive (95.9 ± 3.1% recovery rate), selective (>2.5 log depletion of white blood cells), rapid (>3 mL/min), and label-free isolation of viable CTCs from whole blood without prior sample treatment is achieved using a stand-alone lab-on-a-disc system equipped with fluid-assisted separation technology (FAST). Numerical simulation and experiments show that this method provides uniform, clog-free, ultrafast cell enrichment with pressure drops much less than in conventional size-based filtration, at 1 kPa. We demonstrate the clinical utility of the point-of-care detection of CTCs with samples taken from 142 patients suffering from breast, stomach, or lung cancer.

Automated processing of whole blood samples for the determination of immunosuppressants by liquid chromatography tandem-mass spectrometry.

PubMed

Vogeser, Michael; Spöhrer, Ute

2006-01-01

Liquid chromatography tandem-mass spectrometry (LC-MS/MS) is an efficient technology for routine determination of immunosuppressants in whole blood; however, time-consuming manual sample preparation remains a significant limitation of this technique. Using a commercially available robotic pipetting system (Tecan Freedom EVO), we developed an automated sample-preparation protocol for quantification of tacrolimus in whole blood by LC-MS/MS. Barcode reading, sample resuspension, transfer of whole blood aliquots into a deep-well plate, addition of internal standard solution, mixing, and protein precipitation by addition of an organic solvent is performed by the robotic system. After centrifugation of the plate, the deproteinized supernatants are submitted to on-line solid phase extraction, using column switching prior to LC-MS/MS analysis. The only manual actions within the entire process are decapping of the tubes, and transfer of the deep-well plate from the robotic system to a centrifuge and finally to the HPLC autosampler. Whole blood pools were used to assess the reproducibility of the entire analytical system for measuring tacrolimus concentrations. A total coefficient of variation of 1.7% was found for the entire automated analytical process (n=40; mean tacrolimus concentration, 5.3 microg/L). Close agreement between tacrolimus results obtained after manual and automated sample preparation was observed. The analytical system described here, comprising automated protein precipitation, on-line solid phase extraction and LC-MS/MS analysis, is convenient and precise, and minimizes hands-on time and the risk of mistakes in the quantification of whole blood immunosuppressant concentrations compared to conventional methods.

Validation protocols for blood pressure-measuring devices: status quo and development needs.

PubMed

Beime, Beate; Deutsch, Cornelia; Gomez, Timothy; Zwingers, Thomas; Mengden, Thomas; Bramlage, Peter

2016-02-01

Hypertension is a major risk factor for cardiovascular morbidity and mortality. Therefore, blood pressure self-measuring devices have emerged as valuable tools in patient care and the accuracy of these instruments is of fundamental importance. For this reason, several validation procedures for assessing the efficacy of blood pressure monitoring devices have been developed, including protocols by the Association for the Advancement of Medical Instrumentation, the British Hypertension Society, the German Hypertension League (Prüfsiegelprotokoll), and the International Protocol of the Working Group on Blood Pressure Monitoring of the European Society of Hypertension. In the past, most of the protocols have been reviewed and modified because of experiences gained during the validation studies carried out. However, each shows distinct differences, that is number and characteristics of patients required, blood pressure ranges, and the length of the validation procedure, which may result in unique advantages and/or limitations associated with their use. The continued standardization and evolution of these guidelines is essential to ensure the efficacy of blood pressure-measuring devices marketed for clinical and home use. Here, we aimed to compare four currently used validation protocols and to initiate a discussion on potential future improvements.

Validate or falsify: Lessons learned from a microscopy method claimed to be useful for detecting Borrelia and Babesia organisms in human blood.

PubMed

Aase, Audun; Hajdusek, Ondrej; Øines, Øivind; Quarsten, Hanne; Wilhelmsson, Peter; Herstad, Tove K; Kjelland, Vivian; Sima, Radek; Jalovecka, Marie; Lindgren, Per-Eric; Aaberge, Ingeborg S

2016-01-01

A modified microscopy protocol (the LM-method) was used to demonstrate what was interpreted as Borrelia spirochetes and later also Babesia sp., in peripheral blood from patients. The method gained much publicity, but was not validated prior to publication, which became the purpose of this study using appropriate scientific methodology, including a control group. Blood from 21 patients previously interpreted as positive for Borrelia and/or Babesia infection by the LM-method and 41 healthy controls without known history of tick bite were collected, blinded and analysed for these pathogens by microscopy in two laboratories by the LM-method and conventional method, respectively, by PCR methods in five laboratories and by serology in one laboratory. Microscopy by the LM-method identified structures claimed to be Borrelia- and/or Babesia in 66% of the blood samples of the patient group and in 85% in the healthy control group. Microscopy by the conventional method for Babesia only did not identify Babesia in any samples. PCR analysis detected Borrelia DNA in one sample of the patient group and in eight samples of the control group; whereas Babesia DNA was not detected in any of the blood samples using molecular methods. The structures interpreted as Borrelia and Babesia by the LM-method could not be verified by PCR. The method was, thus, falsified. This study underlines the importance of doing proper test validation before new or modified assays are introduced.

Application of real-time PCR and melting curve analysis in rapid Diego blood group genotyping.

PubMed

Novaretti, M C Z; Ruiz, A S; Dorlhiac-Llacer, P E; Chamone, D A F

2010-01-01

The paucity of appropriate reagents for serologic typing of the Diego blood group antigens has prompted the development of a real-time PCR and melting curve analysis for Diego blood group genotyping. In this study, we phenotyped 4326 donor blood samples for Di(a) using semiautomated equipment. All 157 Di(a+) samples were then genotyped by PCR using sequence-specific primers (PCR-SSP) for DI*02 because of anti-Di(b) scarcity. Of the 4326 samples, we simultaneously tested 160 samples for Di(a) and Di(b) serology, and DI*01 and DI*02 by PCR-SSP and by real-time PCR. We used the same primers for Diego genotyping by real-time PCR and PCR-SSP. Melting curve profiles obtained using the dissociation software of the real-time PCR apparatus enabled the discrimination of Diego alleles. Of the total samples tested, 4169 blood donors, 96.4 percent (95% confidence interval [CI], 95.8-96.9%), were homozygous for DI*02 and 157, 3.6 percent (95% CI, 3.1%-4.2%), were heterozygous DI*01/02. No blood donor was found to be homozygous for DI*01 in this study. The calculated DI*01 and DI*02 allele frequencies were 0.0181 (95% CI, 0.0173-0.0189) and 0.9819 (95% CI, 0.9791-0.9847), respectively, showing a good fit for the Hardy-Weinberg equilibrium. There was full concordance among Diego phenotype results by PCR-SSP and real-time PCR. DI*01 and DI*02 allele determination with SYBR Green I and thermal cycler technology are useful methods for Diego determination. The real-time PCR with SYBR Green I melting temperature protocol can be used as a rapid screening tool for DI*01 and DI*02 blood group genotyping.

Restrictive Versus Massive Fluid Resuscitation Strategy (REFILL study), influence on blood loss and hemostatic parameters in obstetric hemorrhage: study protocol for a randomized controlled trial.

PubMed

de Lange, Natascha; Schol, Pim; Lancé, Marcus; Woiski, Mallory; Langenveld, Josje; Rijnders, Robbert; Smits, Luc; Wassen, Martine; Henskens, Yvonne; Scheepers, Hubertina

2018-03-06

Postpartum hemorrhage (PPH) is associated with maternal morbidity and mortality and has an increasing incidence in high-resource countries, despite dissemination of guidelines, introduction of skills training, and correction for risk factors. Current guidelines advise the administration, as fluid resuscitation, of almost twice the amount of blood lost. This advice is not evidence-based and could potentially harm patients. All women attending the outpatient clinic who are eligible will be informed of the study; oral and written informed consent will be obtained. Where there is more than 500 ml blood loss and ongoing bleeding, patients will be randomized to care as usual, fluid resuscitation with 1.5-2 times the amount of blood loss or fluid resuscitation with 0.75-1.0 times the blood loss. Blood loss will be assessed by weighing all draping. A blood sample, for determining hemoglobin concentration, hematocrit, thrombocyte concentration, and conventional coagulation parameters will be taken at the start of the study, after 60 min, and 12-18 h after delivery. In a subgroup of women, additional thromboelastometric parameters will be obtained. Our hypothesis is that massive fluid administration might lead to a progression of bleeding due to secondary coagulation disorders. In non-pregnant individuals with massive blood loss, restrictive fluid management has been shown to prevent a progression to dilution coagulopathy. These data, however, cannot be extrapolated to women in labor. Our objective is to compare both resuscitation protocols in women with early, mild PPH (blood loss 500-750 ml) and ongoing bleeding, taking as primary outcome measure the progression to severe PPH (blood loss > 1000 ml). Netherlands Trial Register, NTR 3789 . Registered on 11 January 2013.

Evaluation of empirical treatment for blood culture-negative endocarditis.

PubMed

Menu, Estelle; Gouriet, Frédérique; Casalta, Jean-Paul; Tissot-Dupont, Hervé; Vecten, Maude; Saby, Ludivine; Hubert, Sandrine; Salaun, Erwan; Theron, Alexis; Grisoli, Dominique; Lavoute, Cécile; Collart, Frédéric; Habib, Gilbert; Raoult, Didier

2017-01-01

Much progress has been made in understanding the main causes of blood culture-negative endocarditis (BCNE). Few studies concerning BCNE treatment (due to previous antibiotics used or fastidious pathogens) are available. We performed this study to evaluate the effectiveness of our therapeutic protocol in BCNE, based on compliance with the protocol, outcome and 1 year mortality. We collected prospectively and analysed retrospectively cases of BCNE between 2002 and 2014, using a simplified and standardized protocol developed by our multidisciplinary team. We apply two kinds of protocols to treat BCNE, which include only four intravenous antimicrobial agents: amoxicillin, vancomycin, gentamicin and amphotericin B. We had 177 patients with definite BCNE. There were 154 (87.0%) patients treated with both appropriate antimicrobial agents and appropriate duration of treatment. We analysed the causes of inappropriate treatment in 13 (7.3%) cases and inappropriate duration in 10 (5.6%) cases. The treatment changes were justified in all cases except one of discharge against medical advice. The fatality rate was 5.1% (nine cases) and all deaths occurred in the group of patients who were treated with appropriate treatment; however, four deaths were not attributable to empirical treatment failure. Concerning the other deaths, the lack of surgical management, in association with empirical treatment, could explain our protocol's failure, such as poorly tolerated surgery. Our protocol is efficient and our mortality rate was low, compared with the literature review. This may result from a strategy that uses a sampling procedure and a standardized protocol at the same time. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Prevalence of Malaria, Dengue, and Chikungunya Significantly Associated with Mosquito Breeding Sites

PubMed Central

Islam, Mohammad Nazrul; ZulKifle, Mohammad; Sherwani, Arish Mohammad Khan; Ghosh, Susanta Kumar; Tiwari, Satyanarayan

2011-01-01

Objectives: To observe the prevalence of malaria, dengue, and chikungunya and their association with mosquito breeding sites. Methods: The study was observational and analytical. A total of 162 houses and 670 subjects were observed during the study period. One hundred forty-two febrile patients were eligible for the study. After obtaining informed consent from all febrile patients, 140 blood samples were collected to diagnose malaria, dengue, and chikungunya. Larval samples were collected by the standard protocol that follows. Correlation of data was performed by Pearson correlation test. Results: Forty-seven blood samples were found positive: 33 for chikungunya, 3 for dengue, and 11 for malaria. Fifty-one out of 224 larval samples were found positive. Out of the 51 positive samples, 37 were positive for Aedes, 12 were positive for Anopheles, and two were positive for Culex larvae. Interpretation and Conclusion: Mosquito-borne fevers, especially malaria, dengue, and chikungunya, have shown a significant relationship with mosquito breeding sites. PMID:23610486

Global Metabolic Stress of Isoeffort Continuous and High Intensity Interval Aerobic Exercise: A Comparative 1H NMR Metabonomic Study.

PubMed

Zafeiridis, Andreas; Chatziioannou, Anastasia Chrysovalantou; Sarivasiliou, Haralambos; Kyparos, Antonios; Nikolaidis, Michalis G; Vrabas, Ioannis S; Pechlivanis, Alexandros; Zoumpoulakis, Panagiotis; Baskakis, Constantinos; Dipla, Konstantina; Theodoridis, Georgios A

2016-12-02

The overall metabolic/energetic stress that occurs during an acute bout of exercise is proposed to be the main driving force for long-term training adaptations. Continuous and high-intensity interval exercise protocols (HIIE) are currently prescribed to acquire the muscular and metabolic benefits of aerobic training. We applied 1 H NMR-based metabonomics to compare the overall metabolic perturbation and activation of individual bioenergetic pathways of three popular aerobic exercises matched for effort/strain. Nine men performed continuous, long-interval (3 min), and short-interval (30 s) bouts of exercise under isoeffort conditions. Blood was collected before and after exercise. The multivariate PCA and OPLS-DA models showed a distinct separation of pre- and postexercise samples in three protocols. The two models did not discriminate the postexercise overall metabolic profiles of the three exercise types. Analysis focused on muscle bioenergetic pathways revealed an extensive upregulation of carbohydrate-lipid metabolism and the TCA cycle in all three protocols; there were only a few differences among protocols in the postexercise abundance of molecules when long-interval bouts were performed. In conclusion, continuous and HIIE exercise protocols, when performed with similar effort/strain, induce comparable global metabolic response/stress despite their marked differences in work-bout intensities. This study highlights the importance of NMR metabonomics in comprehensive monitoring of metabolic consequences of exercise training in the blood of athletes and exercising individuals.

Biobanking human endometrial tissue and blood specimens: standard operating procedure and importance to reproductive biology research and diagnostic development.

PubMed

Sheldon, Elizabeth; Vo, Kim Chi; McIntire, Ramsey A; Aghajanova, Lusine; Zelenko, Zara; Irwin, Juan C; Giudice, Linda C

2011-05-01

To develop a standard operating procedure (SOP) for collection, transport, storage of human endometrial tissue and blood samples, subject and specimen annotation, and establishing sample priorities. The SOP synthesizes sound scientific procedures, the literature on ischemia research, sample collection and gene expression profiling, good laboratory practices, and the authors' experience of workflow and sample quality. The National Institutes of Health, University of California, San Francisco, Human Endometrial Tissue and DNA Bank. Women undergoing endometrial biopsy or hysterectomy for nonmalignant indications. Collecting, processing, storing, distributing endometrial tissue and blood samples under approved institutional review board protocols and written informed consent from participating subjects. Standard operating procedure. The SOP addresses rigorous and consistent subject annotation, specimen processing and characterization, strict regulatory compliance, and a reference for researchers to track collection and storage times that may influence their research. The comprehensive and systematic approach to the procurement of human blood and endometrial tissue in this SOP ensures the high quality, reliability, and scientific usefulness of biospecimens made available to investigators by the National Institutes of Health, University of California, San Francisco, Human Endometrial Tissue and DNA Bank. The detail and perspective in this SOP also provides a blueprint for implementation of similar collection programs at other institutions. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

MALDI-TOF mass spectrometry for rapid diagnosis of postoperative endophthalmitis.

PubMed

Mailhac, Adriane; Durand, Harmonie; Boisset, Sandrine; Maubon, Danièle; Berger, Francois; Maurin, Max; Chiquet, Christophe; Bidart, Marie

2017-01-30

This study describes an innovative strategy for rapid detection and identification of bacteria causing endophthalmitis, combining the use of an automated blood culture system with MALDI-TOF mass spectrometry methodology. Using this protocol, we could identify 96% of 45 bacterial strains isolated from vitreous samples collected in acute post-operative endophthalmitis patients. Copyright © 2016 Elsevier B.V. All rights reserved.

Regulation of human retinal blood flow by endothelin-1.

PubMed

Polak, Kaija; Luksch, Alexandra; Frank, Barbara; Jandrasits, Kerstin; Polska, Elzbieta; Schmetterer, Leopold

2003-05-01

There is evidence from in vitro and animal studies that endothelin is a major regulator of retinal blood flow. We set out to characterize the role of the endothelin-system in the blood flow control of the human retina. Two studies in healthy subjects were performed. The study design was randomized, placebo-controlled, double-masked, balanced, two-way crossover in protocol A and three way-way crossover in protocol B. In protocol A 18 healthy male subjects received intravenous endothelin-1 (ET-1) in a dose of 2.5 ng kg (-1)min(-1) for 30 min or placebo on two different study days and retinal vessel diameters were measured. In protocol B 12 healthy male subjects received ET-1 in stepwise increasing doses of 0, 1.25, 2.5 and 5 ng kg (-1)min(-1) (each infusion step over 20 min) in co-infusion with the specific ET(A)-receptor antagonist BQ123 (60 microg min (-1)) or placebo or BQ123 alone investigating retinal vessel diameters, retinal blood velocity and retinal blood flow. Measurements of retinal vessel size were done with the Zeiss retinal vessel analyzer. Measurements of blood velocities were done with bi-directional laser Doppler velocimetry. From these measurements retinal blood flow was calculated. In protocol A exogenous ET-1 tended to decrease retinal arterial diameter, but this effect was not significant versus placebo. No effect on retinal venous diameter was seen. In protocol B retinal venous blood velocity and retinal blood flow was significantly reduced after administration of exogenous ET-1. These effects were significantly blunted when BQ-123 was co-administered. By contrast, BQ-123 alone had no effect on retinal hemodynamic parameters. Concluding, BQ123 antagonizes the effects of exogenously administered ET-1 on retinal blood flow in healthy subjects. In addition, the results of the present study are compatible with the hypothesis that ET-1 exerts its vasoconstrictor effects in the retina mainly on the microvessels.

Comparison of Physiological and Perceptual Responses Between Continuous and Intermittent Cycling

PubMed Central

Brasil, Roxana M.; Barreto, Ana C.; Nogueira, Leandro; Santos, Edil; Novaes, Jefferson S.; Reis, Victor M.

2011-01-01

The present study tested the hypothesis that the exercise protocol (continuous vs. intermittent) would affect the physiological response and the perception of effort during aquatic cycling. Each protocol was divided on four stages. Heart rate, arterial blood pressure, blood lactate concentration, central and peripheral rate of perceived exertion were collected in both protocols in aquatic cycling in 10 women (values are mean ± SD): age=32.8 ± 4.8 years; height=1.62 ± 0.05 cm; body mass=61.60 ± 5.19 kg; estimated body fat=27.13 ± 4.92%. Protocols were compared through two way ANOVA with Scheffé’s post-hoc test and the test of Mann- Whitney for rate of perceived exertion with α=0.05. No systematic and consistent differences in heart rate, arterial blood pressure, double product and blood lactate concentration were found between protocols. On the other hand, central rate of perceived exertion was significantly higher at stage four during continuous protocol compared with intermittent protocol (p=0.01), while the peripheral rate of perceived exertion presented higher values at stages three (p=0.02) and four (p=0.00) in the continuous protocol when compared to the results found in intermittent protocol. These findings suggest that although the aquatic cycling induces similar physiologic demands in both protocols, the rate of perceived exertion may vary according to the continuous vs. intermittent nature of the exercise. PMID:23487483

What are the keys to successful adrenal venous sampling (AVS) in patients with primary aldosteronism?

PubMed

Young, William F; Stanson, Anthony W

2009-01-01

Adrenal venous sampling (AVS) is the criterion standard to distinguish between unilateral and bilateral adrenal disease in patients with primary aldosteronism. The keys to successful AVS include appropriate patient selection, careful patient preparation, focused technical expertise, defined protocol, and accurate data interpretation. The use of AVS should be based on patient preferences, patient age, clinical comorbidities, and the clinical probability of finding an aldosterone-producing adenoma. AVS is optimally performed in the fasting state in the morning. AVS is an intricate procedure because the right adrenal vein is small and may be difficult to locate - the success rate depends on the proficiency of the angiographer. The key factors that determine the successful catheterization of both adrenal veins are experience, dedication and repetition. With experience, and focusing the expertise to 1 or 2 radiologists at a referral centre, the AVS success rate can be as high as 96%. A centre-specific, written protocol is mandatory. The protocol should be developed by an interested group of endocrinologists, radiologists and laboratory personnel. Safeguards should be in place to prevent mislabelling of the blood tubes in the radiology suite and to prevent sample mix-up in the laboratory.

A new morphologically distinct avian malaria parasite that fails detection by established polymerase chain reaction-based protocols for amplification of the cytochrome B gene.

PubMed

Zehtindjiev, Pavel; Križanauskienė, Asta; Bensch, Staffan; Palinauskas, Vaidas; Asghar, Muhammad; Dimitrov, Dimitar; Scebba, Sergio; Valkiūnas, Gediminas

2012-06-01

Plasmodium polymorphum n. sp. (Haemosporida, Plasmodiidae) was found in the skylark, Alauda arvensis (Passeriformes: Alaudidae), during autumnal migration in southern Italy. This organism is illustrated and described based on the morphology of its blood stages. The most distinctive feature of this malaria parasite is the clear preference of its blood stages (trophozoites, meronts, and gametocytes) for immature red blood cells, including erythroblasts. Based on preference of erythrocytic meronts for immature red blood cells, P. polymorphum is most similar to species of the subgenus Huffia . This parasite can be readily distinguished from all other bird malaria parasites, including Plasmodium ( Huffia ) spp., due to preferential development and maturation of its gametocytes in immature red blood cells, a unique character for avian Plasmodium spp. In addition, the margins of nuclei in blood stages of P. polymorphum are markedly smooth and distinct; this is also a distinct diagnostic feature of this parasite. Plasmodium polymorphum has been recorded only in the skylark; it is probably a rare parasite, whose host range and geographical distribution remain unclear. Microscopic examination detected a light infection of Plasmodium relictum (lineage GRW11, parasitemia of <0.01%) in the same sample with P. polymorphum ; the latter parasite clearly predominated (3.5% parasitemia). However, experienced researchers were unable to detect sequences of mitochondrial cytochrome b gene (cyt b ) of P. polymorphum from the microscopically positive sample by using published and newly designed primers for DNA amplification of avian Plasmodium spp. The light parasitemia of P. relictum was easily detectable using several polymerase chain reaction (PCR)-based assays, but P. polymorphum was undetectable in all applied assays. Quantitative PCR also showed the presence of light parasitemia (0.06%) of the lineage GRW11 in this sample. This supports the conclusion that the morphologically distinct parasite observed along with P. relictum and predominant in the sample is genetically dissimilar from the lineage GRW11 based on cyt b sequence. In samples with co-infections, general PCR protocols tend to favor the amplification of the parasite with the higher parasitemia or the amplification with the best matching sequence to the primers. Because the parasitemia of P. polymorphum was >50-fold higher than that of P. relictum and several different primers were tested, we suggest that the failure to amplify P. polymorphum is a more complex problem than why co-infections are commonly overlooked in PCR-based studies. We suggest possible explanations of these results and call for additional research on evolution of mitochondrial genome of hemosporidian parasites.

Dark blood late enhancement imaging.

PubMed

Kellman, Peter; Xue, Hui; Olivieri, Laura J; Cross, Russell R; Grant, Elena K; Fontana, Marianna; Ugander, Martin; Moon, James C; Hansen, Michael S

2016-11-07

Bright blood late gadolinium enhancement (LGE) imaging typically achieves excellent contrast between infarcted and normal myocardium. However, the contrast between the myocardial infarction (MI) and the blood pool is frequently suboptimal. A large fraction of infarctions caused by coronary artery disease are sub-endocardial and thus adjacent to the blood pool. It is not infrequent that sub-endocardial MIs are difficult to detect or clearly delineate. In this present work, an inversion recovery (IR) T2 preparation was combined with single shot steady state free precession imaging and respiratory motion corrected averaging to achieve dark blood LGE images with good signal to noise ratio while maintaining the desired spatial and temporal resolution. In this manner, imaging was conducted free-breathing, which has benefits for image quality, patient comfort, and clinical workflow in both adults and children. Furthermore, by using a phase sensitive inversion recovery reconstruction the blood signal may be made darker than the myocardium (i.e., negative signal values) thereby providing contrast between the blood and both the MI and remote myocardium. In the proposed approach, a single T1-map scout was used to measure the myocardial and blood T1 using a MOdified Look-Locker Inversion recovery (MOLLI) protocol and all protocol parameters were automatically calculated from these values within the sequence thereby simplifying the user interface. The contrast to noise ratio (CNR) between MI and remote myocardium was measured in n = 30 subjects with subendocardial MI using both bright blood and dark blood protocols. The CNR for the dark blood protocol had a 13 % loss compared to the bright blood protocol. The CNR between the MI and blood pool was positive for all dark blood cases, and was negative in 63 % of the bright blood cases. The conspicuity of subendocardial fibrosis and MI was greatly improved by dark blood (DB) PSIR as well as the delineation of the subendocardial border. Free-breathing, dark blood PSIR LGE imaging was demonstrated to improve the visualization of subendocardial MI and fibrosis in cases with low contrast with adjacent blood pool. The proposed method also improves visualization of thin walled fibrous structures such as atrial walls and valves, as well as papillary muscles.

Understanding biological mechanisms underlying adverse birth outcomes in developing countries: protocol for a prospective cohort (AMANHI bio-banking) study.

PubMed

Baqui, Abdullah H; Khanam, Rasheda; Rahman, Mohammad Sayedur; Ahmed, Aziz; Rahman, Hasna Hena; Moin, Mamun Ibne; Ahmed, Salahuddin; Jehan, Fyezah; Nisar, Imran; Hussain, Atiya; Ilyas, Muhammad; Hotwani, Aneeta; Sajid, Muhammad; Qureshi, Shahida; Zaidi, Anita; Sazawal, Sunil; Ali, Said M; Deb, Saikat; Juma, Mohammed Hamad; Dhingra, Usha; Dutta, Arup; Ame, Shaali Makame; Hayward, Caroline; Rudan, Igor; Zangenberg, Mike; Russell, Donna; Yoshida, Sachiyo; Polašek, Ozren; Manu, Alexander; Bahl, Rajiv

2017-12-01

The AMANHI study aims to seek for biomarkers as predictors of important pregnancy-related outcomes, and establish a biobank in developing countries for future research as new methods and technologies become available. AMANHI is using harmonised protocols to enrol 3000 women in early pregnancies (8-19 weeks of gestation) for population-based follow-up in pregnancy up to 42 days postpartum in Bangladesh, Pakistan and Tanzania, with collection taking place between August 2014 and June 2016. Urine pregnancy tests will be used to confirm reported or suspected pregnancies for screening ultrasound by trained sonographers to accurately date the pregnancy. Trained study field workers will collect very detailed phenotypic and epidemiological data from the pregnant woman and her family at scheduled home visits during pregnancy (enrolment, 24-28 weeks, 32-36 weeks & 38+ weeks) and postpartum (days 0-6 or 42-60). Trained phlebotomists will collect maternal and umbilical blood samples, centrifuge and obtain aliquots of serum, plasma and the buffy coat for storage. They will also measure HbA1C and collect a dried spot sample of whole blood. Maternal urine samples will also be collected and stored, alongside placenta, umbilical cord tissue and membrane samples, which will both be frozen and prepared for histology examination. Maternal and newborn stool (for microbiota) as well as paternal and newborn saliva samples (for DNA extraction) will also be collected. All samples will be stored at -80°C in the biobank in each of the three sites. These samples will be linked to numerous epidemiological and phenotypic data with unique study identification numbers. AMANHI biobank proves that biobanking is feasible to implement in LMICs, but recognises that biobank creation is only the first step in addressing current global challenges.

Translating evidence-based protocol of wound drain management for total joint arthroplasty into practice: A quasi-experimental study.

PubMed

Tsang, Lap Fung; Cheng, Hang Cheong; Ho, Hon Shuen; Hsu, Yung Chak; Chow, Chiu Man; Law, Heung Wah; Fong, Lup Chau; Leung, Lok Ming; Kong, Ivy Ching Yan; Chan, Chi Wai; Sham, Alice So Yuen

2016-05-01

Although various drains have long been used in total joint replacement, evidence suggests inconsistent practice exists in the use of drainage systems including intermittently applying suction or free of drainage suction, and variations in the optimal timing for wound drain removal. A comprehensive systematic review of available evidence up to 2013 was conducted in a previous study and a protocol was adapted for clinical application according to the summary of the retrieved information (Tsang, 2015). To determine if the protocol could reduce blood loss and blood transfusion after operation and to develop a record form so as to enhance communication of drainage record amongst surgeons and nurses. A quasi-experimental time-series design was undertaken. In the conventional group, surgeons ordered free drainage if the drain output was more than 300 ml. The time of removal of the drain was based on their professional judgement. In the protocol group the method of drainage was dependant of the drainage output as was the timing of the removal of the drain. A standardized record form was developed to guide operating room and orthopaedic ward nurses to manage the drainage system. The drain was removed significantly earlier in the protocol group. Blood loss rate at the first hour of post-operation was extremely low in the protocol group due to clamping effect. Blood loss in volume during the first three hours in the protocol group was significantly lower than that in the conventional group. Only in 11.1% and 4% of cases was it necessary to clamp at the three and four hour post-operative hours. No clamping was required at the two and eight hour postoperative period. There was no significant difference in blood loss during the removal of the drain and during blood transfusion, which was required for patients upon removal of the drain in the two groups. This is the first clinical study to develop an evidence-based protocol to manage wound drain effectively in Hong Kong. Total blood loss and blood transfusions were not significantly different between the conventional and protocol groups. A standard documentation document is beneficial to enhance communication between doctors and nurses as well as to monitor and observe drainage effectively. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effect of risk-stratified, protocol-based perioperative chemoprophylaxis on nosocomial infection rates in a series of 31 927 consecutive neurosurgical procedures (1994-2006).

PubMed

Sharma, Manish S; Vohra, Ashma; Thomas, Ponnamma; Kapil, Arti; Suri, Ashish; Chandra, P Sarat; Kale, Shashank S; Mahapatra, Ashok K; Sharma, Bhawani S

2009-06-01

Although the use of prophylactic antibiotics has been shown to significantly decrease the incidence of meningitis after neurosurgery, its effect on extra-neurosurgical-site infections has not been documented. The authors explore the effect of risk-stratified, protocol-based perioperative antibiotic prophylaxis on nosocomial infections in an audit of 31 927 consecutive routine and emergency neurosurgical procedures. Infection rates were objectively quantified by bacteriological positivity on culture of cerebrospinal fluid (CSF), blood, urine, wound swab, and tracheal aspirate samples derived from patients with clinicoradiological features of sepsis. Infections were recorded as pulmonary, wound, blood, CSF, and urinary. The total numbers of hospital-acquired infections and the number of patients infected were also recorded. A protocol of perioperative antibiotic prophylaxis of variable duration stratified by patient risk factors was introduced in 2000, which was chosen as the historical turning point. The chi test was used to compare infection rates. A P value of <0.05 was considered significant. A total of 31 927 procedures were performed during the study period 1994-2006; 5171 culture-proven hospital-acquired infections (16.2%) developed in 3686 patients (11.6%). The most common infections were pulmonary (4.4%), followed by bloodstream (3.5%), urinary (3.0%), CSF (2.9%), and wound (2.5%). The incidence of positive tracheal, CSF, blood, wound, and urine cultures decreased significantly after 2000. Chemoprophylaxis, however, altered the prevalent bacterial flora and may have led to the emergence of methicillin-resistant Staphylococcus aureus. A risk-stratified protocol of perioperative antibiotic prophylaxis may help to significantly decrease not only neurosurgical, but also extra-neurosurgical-site body fluid bacteriological culture positivity.

DNA recovery from microhymenoptera using six non-destructive methodologies with considerations for subsequent preparation of museum slides.

PubMed

Guzmán-Larralde, Adriana J; Suaste-Dzul, Alba P; Gallou, Adrien; Peña-Carrillo, Kenzy I

2017-01-01

Because of the tiny size of microhymenoptera, successful morphological identification typically requires specific mounting protocols that require time, skills, and experience. Molecular taxonomic identification is an alternative, but many DNA extraction protocols call for maceration of the whole specimen, which is not compatible with preserving museum vouchers. Thus, non-destructive DNA isolation methods are attractive alternatives for obtaining DNA without damaging sample individuals. However, their performance needs to be assessed in microhymenopterans. We evaluated six non-destructive methods: (A) DNeasy® Blood & Tissue Kit; (B) DNeasy® Blood & Tissue Kit, modified; (C) Protocol with CaCl 2 buffer; (D) Protocol with CaCl 2 buffer, modified; (E) HotSHOT; and (F) Direct PCR. The performance of each DNA extraction method was tested across several microhymenopteran species by attempting to amplify the mitochondrial gene COI from insect specimens of varying ages: 1 day, 4 months, 3 years, 12 years, and 23 years. Methods B and D allowed COI amplification in all insects, while methods A, C, and E were successful in DNA amplification from insects up to 12 years old. Method F, the fastest, was useful in insects up to 4 months old. Finally, we adapted permanent slide preparation in Canada balsam for every technique. The results reported allow for combining morphological and molecular methodologies for taxonomic studies.

Accuracy of LightCycler(R) SeptiFast for the detection and identification of pathogens in the blood of patients with suspected sepsis: a systematic review protocol.

PubMed

Dark, Paul; Wilson, Claire; Blackwood, Bronagh; McAuley, Danny F; Perkins, Gavin D; McMullan, Ronan; Gates, Simon; Warhurst, Geoffrey

2012-01-01

Background There is growing interest in the potential utility of molecular diagnostics in improving the detection of life-threatening infection (sepsis). LightCycler® SeptiFast is a multipathogen probe-based real-time PCR system targeting DNA sequences of bacteria and fungi present in blood samples within a few hours. We report here the protocol of the first systematic review of published clinical diagnostic accuracy studies of this technology when compared with blood culture in the setting of suspected sepsis. Methods/design Data sources: the Cochrane Database of Systematic Reviews, the Database of Abstracts of Reviews of Effects (DARE), the Health Technology Assessment Database (HTA), the NHS Economic Evaluation Database (NHSEED), The Cochrane Library, MEDLINE, EMBASE, ISI Web of Science, BIOSIS Previews, MEDION and the Aggressive Research Intelligence Facility Database (ARIF). diagnostic accuracy studies that compare the real-time PCR technology with standard culture results performed on a patient's blood sample during the management of sepsis. three reviewers, working independently, will determine the level of evidence, methodological quality and a standard data set relating to demographics and diagnostic accuracy metrics for each study. Statistical analysis/data synthesis: heterogeneity of studies will be investigated using a coupled forest plot of sensitivity and specificity and a scatter plot in Receiver Operator Characteristic (ROC) space. Bivariate model method will be used to estimate summary sensitivity and specificity. The authors will investigate reporting biases using funnel plots based on effective sample size and regression tests of asymmetry. Subgroup analyses are planned for adults, children and infection setting (hospital vs community) if sufficient data are uncovered. Dissemination Recommendations will be made to the Department of Health (as part of an open-access HTA report) as to whether the real-time PCR technology has sufficient clinical diagnostic accuracy potential to move forward to efficacy testing during the provision of routine clinical care. Registration PROSPERO-NIHR Prospective Register of Systematic Reviews (CRD42011001289).

Early blood glucose profile and neurodevelopmental outcome at two years in neonatal hypoxic-ischaemic encephalopathy

PubMed Central

2011-01-01

Background To examine the blood glucose profile and the relationship between blood glucose levels and neurodevelopmental outcome in term infants with hypoxic-ischaemic encephalopathy. Methods Blood glucose values within 72 hours of birth were collected from 52 term infants with hypoxic-ischaemic encephalopathy. Hypoglycaemia [< 46.8 mg/dL (2.6 mmol/L)] and hyperglycaemia [> 150 mg/dL (8.3 mmol/L)] were correlated to neurodevelopmental outcome at 24 months of age. Results Four fifths of the 468 blood samples were in the normoglycaemic range (392/468:83.8%). Of the remaining 76 samples, 51.3% were in the hypoglycaemic range and (48.7%) were hyperglycaemic. A quarter of the hypoglycaemic samples (28.2%:11/39) and a third of the hyperglycaemic samples (32.4%:12/37) were recorded within the first 30 minutes of life. Mean (SD) blood glucose values did not differ between infants with normal and abnormal outcomes [4.89(2.28) mmol/L and 5.02(2.35) mmol/L, p value = 0.15] respectively. In term infants with hypoxic-ischaemic encephalopathy, early hypoglycaemia (between 0-6 hours of life) was associated with adverse outcome at 24 months of age [OR = 5.8, CI = 1.04-32)]. On multivariate analysis to adjust for grade of HIE this association was not statistically significant. Late hypoglycaemia (6-72 hours of life) was not associated with abnormal outcome [OR = 0.22, CI (0.04-1.14)]. The occurrence of hyperglycaemia was not associated with adverse outcome. Conclusion During the first 72 hours of life, blood glucose profile in infants with hypoxic-ischaemic encephalopathy varies widely despite a management protocol. Early hypoglycaemia (0-6 hours of life) was associated with severe HIE, and thereby; adverse outcome. PMID:21294901

Combining image-derived and venous input functions enables quantification of serotonin-1A receptors with [carbonyl-11C]WAY-100635 independent of arterial sampling.

PubMed

Hahn, Andreas; Nics, Lukas; Baldinger, Pia; Ungersböck, Johanna; Dolliner, Peter; Frey, Richard; Birkfellner, Wolfgang; Mitterhauser, Markus; Wadsak, Wolfgang; Karanikas, Georgios; Kasper, Siegfried; Lanzenberger, Rupert

2012-08-01

image- derived input functions (IDIFs) represent a promising technique for a simpler and less invasive quantification of PET studies as compared to arterial cannulation. However, a number of limitations complicate the routine use of IDIFs in clinical research protocols and the full substitution of manual arterial samples by venous ones has hardly been evaluated. This study aims for a direct validation of IDIFs and venous data for the quantification of serotonin-1A receptor binding (5-HT(1A)) with [carbonyl-(11)C]WAY-100635 before and after hormone treatment. Fifteen PET measurements with arterial and venous blood sampling were obtained from 10 healthy women, 8 scans before and 7 after eight weeks of hormone replacement therapy. Image-derived input functions were derived automatically from cerebral blood vessels, corrected for partial volume effects and combined with venous manual samples from 10 min onward (IDIF+VIF). Corrections for plasma/whole-blood ratio and metabolites were done separately with arterial and venous samples. 5-HT(1A) receptor quantification was achieved with arterial input functions (AIF) and IDIF+VIF using a two-tissue compartment model. Comparison between arterial and venous manual blood samples yielded excellent reproducibility. Variability (VAR) was less than 10% for whole-blood activity (p>0.4) and below 2% for plasma to whole-blood ratios (p>0.4). Variability was slightly higher for parent fractions (VARmax=24% at 5 min, p<0.05 and VAR<13% after 20 min, p>0.1) but still within previously reported values. IDIFs after partial volume correction had peak values comparable to AIFs (mean difference Δ=-7.6 ± 16.9 kBq/ml, p>0.1), whereas AIFs exhibited a delay (Δ=4 ± 6.4s, p<0.05) and higher peak width (Δ=15.9 ± 5.2s, p<0.001). Linear regression analysis showed strong agreement for 5-HT(1A) binding as obtained with AIF and IDIF+VIF at baseline (R(2)=0.95), after treatment (R(2)=0.93) and when pooling all scans (R(2)=0.93), with slopes and intercepts in the range of 0.97 to 1.07 and -0.05 to 0.16, respectively. In addition to the region of interest analysis, the approach yielded virtually identical results for voxel-wise quantification as compared to the AIF. Despite the fast metabolism of the radioligand, manual arterial blood samples can be substituted by venous ones for parent fractions and plasma to whole-blood ratios. Moreover, the combination of image-derived and venous input functions provides a reliable quantification of 5-HT(1A) receptors. This holds true for 5-HT(1A) binding estimates before and after treatment for both regions of interest-based and voxel-wise modeling. Taken together, the approach provides less invasive receptor quantification by full independence of arterial cannulation. This offers great potential for the routine use in clinical research protocols and encourages further investigation for other radioligands with different kinetic characteristics. Copyright © 2012 Elsevier Inc. All rights reserved.

77 FR 41408 - Agency Information Collection Activities; Submission for Office of Management and Budget Review...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-13

... protocols for specific licensed biological products: Sec. Sec. 660.6 (21 CFR 660.6) (Antibody to Hepatitis B Surface Antigen); 660.36 (21 CFR 660.36) (Reagent Red Blood Cells); and 660.46 (21 CFR 660.46) (Hepatitis... samples from each lot of Antibody to Hepatitis B Surface Antigen product, and Sec. 660.6(b) provides the...

The National Heart, Lung, and Blood Institute retrovirus epidemiology donor studies (Retrovirus Epidemiology Donor Study and Retrovirus Epidemiology Donor Study-II): twenty years of research to advance blood product safety and availability.

PubMed

Kleinman, Steven; King, Melissa R; Busch, Michael P; Murphy, Edward L; Glynn, Simone A

2012-10-01

The Retrovirus Epidemiology Donor Study (REDS), conducted from 1989 to 2001, and the REDS-II, conducted from 2004 to 2012, were National Heart, Lung, and Blood Institute-funded, multicenter programs focused on improving blood safety and availability in the United States. The REDS-II also included international study sites in Brazil and China. The 3 major research domains of REDS/REDS-II have been infectious disease risk evaluation, blood donation availability, and blood donor characterization. Both programs have made significant contributions to transfusion medicine research methodology by the use of mathematical modeling, large-scale donor surveys, innovative methods of repository sample storage, and establishing an infrastructure that responded to potential emerging blood safety threats such as xenotropic murine leukemia virus-related virus. Blood safety studies have included protocols evaluating epidemiologic and/or laboratory aspects of human immunodeficiency virus, human T-lymphotropic virus 1/2, hepatitis C virus, hepatitis B virus, West Nile virus, cytomegalovirus, human herpesvirus 8, parvovirus B19, malaria, Creutzfeldt-Jakob disease, influenza, and Trypanosoma cruzi infections. Other analyses have characterized blood donor demographics, motivations to donate, factors influencing donor return, behavioral risk factors, donors' perception of the blood donation screening process, and aspects of donor deferral. In REDS-II, 2 large-scale blood donor protocols examined iron deficiency in donors and the prevalence of leukocyte antibodies. This review describes the major study results from over 150 peer-reviewed articles published by these 2 REDS programs. In 2011, a new 7-year program, the Recipient Epidemiology and Donor Evaluation Study-III, was launched. The Recipient Epidemiology and Donor Evaluation Study-III expands beyond donor-based research to include studies of blood transfusion recipients in the hospital setting and adds a third country, South Africa, to the international program. Copyright © 2012 Elsevier Inc. All rights reserved.

An in silico method to identify computer-based protocols worthy of clinical study: An insulin infusion protocol use case

PubMed Central

Wong, Anthony F; Pielmeier, Ulrike; Haug, Peter J; Andreassen, Steen

2016-01-01

Objective Develop an efficient non-clinical method for identifying promising computer-based protocols for clinical study. An in silico comparison can provide information that informs the decision to proceed to a clinical trial. The authors compared two existing computer-based insulin infusion protocols: eProtocol-insulin from Utah, USA, and Glucosafe from Denmark. Materials and Methods The authors used eProtocol-insulin to manage intensive care unit (ICU) hyperglycemia with intravenous (IV) insulin from 2004 to 2010. Recommendations accepted by the bedside clinicians directly link the subsequent blood glucose values to eProtocol-insulin recommendations and provide a unique clinical database. The authors retrospectively compared in silico 18 984 eProtocol-insulin continuous IV insulin infusion rate recommendations from 408 ICU patients with those of Glucosafe, the candidate computer-based protocol. The subsequent blood glucose measurement value (low, on target, high) was used to identify if the insulin recommendation was too high, on target, or too low. Results Glucosafe consistently provided more favorable continuous IV insulin infusion rate recommendations than eProtocol-insulin for on target (64% of comparisons), low (80% of comparisons), or high (70% of comparisons) blood glucose. Aggregated eProtocol-insulin and Glucosafe continuous IV insulin infusion rates were clinically similar though statistically significantly different (Wilcoxon signed rank test P = .01). In contrast, when stratified by low, on target, or high subsequent blood glucose measurement, insulin infusion rates from eProtocol-insulin and Glucosafe were statistically significantly different (Wilcoxon signed rank test, P < .001), and clinically different. Discussion This in silico comparison appears to be an efficient nonclinical method for identifying promising computer-based protocols. Conclusion Preclinical in silico comparison analytical framework allows rapid and inexpensive identification of computer-based protocol care strategies that justify expensive and burdensome clinical trials. PMID:26228765

First external quality assurance program of the Italian HLA-B*57:01 Network assessing the performance of clinical virology laboratories in HLA-B*57:01 testing.

PubMed

Meini, Genny; Dello Russo, Cinzia; Allice, Tiziano; Barresi, Renata; D'Arrigo, Roberta; Falasca, Francesca; Lipsi, Maria Rosaria; Paolucci, Stefania; Zanussi, Stefania; Antonetti, Raffaele; Baldanti, Fausto; Basaglia, Giancarlo; Bruzzone, Bianca; Polilli, Ennio; Ghisetti, Valeria; Pucillo, Leopoldo Paolo; Turriziani, Ombretta; Pirazzoli, Antonella; Navarra, Pierluigi; Zazzi, Maurizio

2016-05-01

Since the HLA-B*57:01 allele is strongly associated with abacavir hypersensitivity reaction, testing for the presence of HLA-B*57:01 is mandatory before administration of abacavir. While HLA-B*57:01 testing is usually provided by pharmacogenetics, genetics or blood transfusion services, clinical virology laboratories can be an optimal opportunity for HLA-B*57:01 testing since they receive blood samples for routine HIV monitoring and have the expertise for convenient and less expensive PCR-based point mutation assays. The Italian HLA-B*57:01 Network gathers accredited clinical virology laboratories offering HLA-B*57:01 testing in Italy with the aim to share protocols, test new methods, develop and maintain external quality assurance (EQA) programs. A panel of 9HLA-B*57:01-positive and 16HLA-B*57:01-negative frozen blood samples were blindly distributed to 10 units including 9 clinical virology laboratories and one reference pharmacology laboratory. Each laboratory was free to use its own routine method for DNA extraction and HLA-B*57:01 testing. DNA was extracted by automated workstations in 6 units and by manual spin columns in 4. Eight units used the Duplicα Real Time HLA-B*57:01 kit by Euroclone and two units used two different PCR homemade protocols. All the 10 units correctly identified all the 25 samples. The first HLA-B*57:01 EQA program run in Italy showed that clinical virology units are equipped and proficient for providing HLA-B*57:01 testing by inexpensive assays easy to integrate into their routine. Copyright © 2016 Elsevier B.V. All rights reserved.

Assessing immune competence in pigs by immunization with tetanus toxoid.

PubMed

Gimsa, U; Tuchscherer, A; Gimsa, J; Tuchscherer, M

2018-01-01

Immune competence can be tested by challenging organisms with a set of infectious agents. However, disease control requirements impose restrictions on the infliction of infections upon domestic pigs. Alternatively, vaccinations induce detectable immune responses that reflect immune competence. Here, we tested this approach with tetanus toxoid (TT) in young domestic pigs. To optimize the vaccination protocol, we immunized the pigs with a commercial TT vaccine at the age of 21 or 35 days. Booster immunizations were performed either 14 or 21 days later. TT-specific antibodies in plasma as well as lymphoproliferative responses were determined both 7 and 14 days after booster immunization using ELISA and lymphocyte transformation tests, respectively. In addition, general IgG and IgM plasma concentrations and mitogen-induced proliferation were measured. The highest TT-specific antibody responses were detected when blood samples were collected 1 week after a booster immunization conducted 21 days after primary immunization. The pigs' age at primary immunization did not have a significant influence on TT-specific antibody responses. Similarly, the TT-specific proliferative responses were highest when blood samples were collected 1 week after booster immunization, while age and time of primary and booster immunization were irrelevant in our setup. While general IgG and IgM plasma levels were highly age dependent, there were no significant age effects for TT-specific immune responses. In addition, mitogen-induced proliferation was independent of immunization as well as blood sampling protocols. In summary, our model of TT vaccination provides an interesting approach for the assessment of immune competence in young pigs. The detected vaccination effects were not biased by age, even though our data were acquired from immune systems that were under development during our tests.

Results of a protocol of transfusion threshold and surgical technique on transfusion requirements in burn patients.

PubMed

O'Mara, Michael S; Hayetian, Fernando; Slater, Harvey; Goldfarb, I William; Tolchin, Eric; Caushaj, Philip F

2005-08-01

Blood loss and high rates of transfusion in burn centers remains an area of ongoing concern. Blood use brings the risk of infection, adverse reaction, and immunosuppression. A protocol to reduce blood loss and blood use was implemented. Analysis included 3-year periods before and after institution of the protocol. All patients were transfused for a hemoglobin below 8.0 gm/dL. Operations per admission did not change during the two time periods (0.78 in each). Overall units transfused per operation decreased from 1.56+/-0.06 to 1.25+/-0.14 units after instituting the protocol (p<0.05). Also, units transfused per admission decreased from 1.21+/-0.15 to 0.96+/-0.06 units of blood (p<0.05). This was noticed particularly in burns of less than 20% surface area, declining from 386 to 46 units after protocol institution, from 0.37 to 0.04 units per admission, and from 0.79 to 0.08 units per operation in this group of smallest burns. There was no change noted in the larger burns. This study suggests that a defined protocol of hemostasis, technique, and transfusion trigger should be implemented in the process of burn excision and grafting. This will help especially those patients with the smallest burns, essentially eliminating transfusion need in that group.

Challenges in standardization of blood pressure measurement at the population level.

PubMed

Tolonen, Hanna; Koponen, Päivikki; Naska, Androniki; Männistö, Satu; Broda, Grazyna; Palosaari, Tarja; Kuulasmaa, Kari

2015-04-10

Accurate blood pressure measurements are needed in clinical practice, intervention studies and health examination surveys. Blood pressure measurements are sensitive: their accuracy can be affected by measurement environment, behaviour of the subject, measurement procedures, devices used for the measurement and the observer. To minimize errors in blood pressure measurement, a standardized measurement protocol is needed. The European Health Examination Survey (EHES) Pilot project was conducted in 2009-2012. A pilot health examination survey was conducted in 12 countries using a standardized protocol. The measurement protocols used in each survey, training provided for the measurers, measurement data, and observations during site visits were collected and evaluated to assess the level of standardization. The EHES measurement protocol for blood pressure was followed accurately in all 12 pilot surveys. Most of the surveys succeeded in organizing a quiet and comfortable measurement environment, and staff instructed survey participants appropriately before examination visits. In all surveys, blood pressure was measured three times, from the right arm in a sitting posture. The biggest variation was in the device used for the blood pressure measurement. It is possible to reach a high level of standardization for blood pressure measurements across countries and over time. A detailed, standardized measurement protocol, and adequate training and monitoring during the fieldwork and centrally organized quality assessment of the data are needed. The recent EU regulation banning the sale of mercury sphygmomanometer in European Union Member States has set new challenges for the standardization of measurement devices since the validity of oscillometric measurements is device-specific and performance of aneroid devices depends very much on calibration.

Validation of the OMRON M7 (HEM-780-E) blood pressure measuring device in a population requiring large cuff use according to the International Protocol of the European Society of Hypertension.

PubMed

El Feghali, Ramzi N; Topouchian, Jirar A; Pannier, Bruno M; El Assaad, Hiba A; Asmar, Roland G

2007-06-01

A high percentage of hypertensive patients present an arm circumference of over 32 cm; the use of a large cuff is therefore recommended. Validation studies are usually performed in the general population using a standard-size cuff. The aim of this study was to assess the accuracy of the Omron M7 device in a population with an arm circumference ranging from 32 to 42 cm. A validation study was performed according to the International Protocol of the European Society of Hypertension. This protocol is divided into two phases: the first phase is performed on 15 selected participants (45 pairs of blood-pressure measurements); if the device passes this phase, 18 supplementary participants are included (54 pairs of blood-pressure measurements), making a total number of 33 participants (99 pairs of blood-pressure measurements), on whom the analysis is performed. For each participant, four blood-pressure measurements were performed simultaneously by two trained observers, using mercury sphygmomanometers fitted with a Y tube; the measurements alternated with three by the test device. The difference between the blood-pressure value given by the device and that obtained by the two observers (mean of the two observations) was calculated for each measure. The 99 pairs of blood-pressure differences were classified into three categories (

Use of a plastic insulin dosage guide to correct blood glucose levels out of the target range and for carbohydrate counting in subjects with type 1 diabetes.

PubMed

Kaufman, F R; Halvorson, M; Carpenter, S

1999-08-01

To improve glycemic control, a hand-held plastic Insulin Dosage Guide was developed to correct blood glucose levels outside of the target range. Protocol 1: Some 40 children (mean age 10.6+/-4.6 years) were randomly assigned for 3 months to use a written-on-paper algorithm or the Insulin Dosage Guide to correct abnormal blood glucose levels. Mean HbA1c and blood glucose levels and time to teach insulin dosage correction were compared. Protocol 2: The Insulin Dosage Guide was used by 83 subjects (mean age 11.4+/-4.3 years) for 1 year, and mean HbA1c levels, blood glucose levels, and number of consecutive high blood glucose values taken before and after the year were compared. Protocol 3: Some 20 patients (mean age 10.1+/-3.7 years) using rapid-acting insulin and 64 patients (mean age 15.9+/-3.6 years) using an insulin pump and rapid-acting insulin used the Insulin Dosage Guide and had mean blood glucose levels, HbA1c, and percentage of blood glucose levels outside of the target range determined. Protocol 1: There was a significant reduction in mean HbA1c (P = 0.04) and blood glucose levels (P = 0.05) and in the time needed to teach how to correct blood glucose values using the Insulin Dosage Guide compared with the paper algorithm. Protocol 2: There was a decrease in mean HbA1c levels (P = 0.0001) and a decrease in the mean number of consecutive blood glucose levels (P = 0.001) over the 1-year time period. Protocol 3: With rapid-acting insulin, there was a significant increase in the percentage of blood glucose levels within the target range (1 month, P = 0.04; at 3 months, P = 0.03). With the insulin pump, there was a high rate (90%) of blood glucose levels in the target range during pump initiation when the Insulin Dosage Guide was used. This inexpensive hand-held plastic card, which is portable and easy to use, may help patients improve glycemia and successfully manage diabetes.

Monitoring of brain oxygen saturation (INVOS) in a protocol to direct blood transfusions during cardiac surgery: a prospective randomized clinical trial.

PubMed

Vretzakis, George; Georgopoulou, Stavroula; Stamoulis, Konstantinos; Tassoudis, Vassilios; Mikroulis, Dimitrios; Giannoukas, Athanasios; Tsilimingas, Nikolaos; Karanikolas, Menelaos

2013-06-07

Blood transfusions are common in cardiac surgery, but have been associated with increased morbidity and long-term mortality. Efforts to reduce blood product use during cardiac surgery include fluid restriction to minimize hemodilution, and protocols to guide transfusion decisions. INVOS is a modality that monitors brain tissue oxygen saturation, and could be useful in guiding decisions to transfuse. However, the role of INVOS (brain tissue oxygen saturation) as part of an algorithm to direct blood transfusions during cardiac surgery has not been evaluated. This study was conducted to investigate the value of INVOS as part of a protocol for blood transfusions during cardiac surgery. Prospective, randomized, blinded clinical trial, on 150 (75 per group) elective cardiac surgery patients. The study was approved by the Institution Ethics committee and all patients gave written informed consent. Data were initially analyzed based on "intention to treat", but subsequently were also analyzed "per protocol". When protocol was strictly followed ("per protocol analysis"), compared to the control group, significantly fewer patients monitored with INVOS received any blood transfusions (46 of 70 patients in INVOS group vs. 55 of 67 patients in the control group, p = 0.029). Similarly, patients monitored with INVOS received significantly fewer units of red blood cell transfusions intraoperatively (0.20 ± 0.50 vs. 0.52 ± 0.88, p = 0.008) and overall during hospital stay (1.31 ± 1.20 vs. 1.82 ± 1.46, p = 0.024). When data from all patients (including patient with protocol violation) were analyzed together ("intention to treat analysis"), the observed reduction of blood transfusions in the INVOS group was still significant (51 of 75 patients transfused in the INVOS group vs. 63 of 75 patients transfused in the control group, p = 0.021), but the overall number of units transfused per patient did not differ significantly between the groups (1.55 ± 1.97 vs. 1.84 ± 1.41, p = 0.288). Our data suggest that INVOS could be a useful tool as part of an algorithm to guide decisions for blood transfusion in cardiac surgery. Additional data from rigorous, well designed studies are needed to further evaluate the role of INVOS in guiding blood transfusions in cardiac surgery, and circumvent the limitations of this study.

Dense concentric circle scanning protocol for measuring pulsatile retinal blood flow in rats with Doppler optical coherence tomography

NASA Astrophysics Data System (ADS)

Tan, Bingyao; Hosseinaee, Zohreh; Bizheva, Kostadinka

2017-11-01

The variability in the spatial orientation of retinal blood vessels near the optic nerve head (ONH) results in imprecision of the measured Doppler angle and therefore the pulsatile blood flow (BF), when those parameters are evaluated using Doppler OCT imaging protocols based on dual-concentric circular scans. Here, we utilized a dense concentric circle scanning protocol and evaluated its precision for measuring pulsatile retinal BF in rats for different numbers of the circular scans. An spectral domain optical coherence tomography (SD-OCT) system operating in the 1060-nm spectral range with image acquisition rate of 47,000 A-scans/s was used to acquire concentric circular scans centered at the rat's ONH, with diameters ranging from 0.8 to 1.0 mm. A custom, automatic blood vessel segmentation algorithm was used to track the spatial orientation of the retinal blood vessels in three dimensions, evaluate the spatially dependent Doppler angle and calculate more accurately the axial BF for each major retinal blood vessel. Metrics such as retinal BF, pulsatility index, and resistance index were evaluated for each and all of the major retinal blood vessels. The performance of the proposed dense concentric circle scanning protocols was compared with that of the dual-circle scanning protocol. Results showed a 3.8±2.2 deg difference in the Doppler angle calculation between the two approaches, which resulted in ˜7% difference in the calculated retinal BF.

Fast carotid artery MR angiography with compressed sensing based three-dimensional time-of-flight sequence.

PubMed

Li, Bo; Li, Hao; Dong, Li; Huang, Guofu

2017-11-01

In this study, we sought to investigate the feasibility of fast carotid artery MR angiography (MRA) by combining three-dimensional time-of-flight (3D TOF) with compressed sensing method (CS-3D TOF). A pseudo-sequential phase encoding order was developed for CS-3D TOF to generate hyper-intense vessel and suppress background tissues in under-sampled 3D k-space. Seven healthy volunteers and one patient with carotid artery stenosis were recruited for this study. Five sequential CS-3D TOF scans were implemented at 1, 2, 3, 4 and 5-fold acceleration factors for carotid artery MRA. Blood signal-to-tissue ratio (BTR) values for fully-sampled and under-sampled acquisitions were calculated and compared in seven subjects. Blood area (BA) was measured and compared between fully sampled acquisition and each under-sampled one. There were no significant differences between the fully-sampled dataset and each under-sampled in BTR comparisons (P>0.05 for all comparisons). The carotid vessel BAs measured from the images of CS-3D TOF sequences with 2, 3, 4 and 5-fold acceleration scans were all highly correlated with that of the fully-sampled acquisition. The contrast between blood vessels and background tissues of the images at 2 to 5-fold acceleration is comparable to that of fully sampled images. The images at 2× to 5× exhibit the comparable lumen definition to the corresponding images at 1×. By combining the pseudo-sequential phase encoding order, CS reconstruction, and 3D TOF sequence, this technique provides excellent visualizations for carotid vessel and calcifications in a short scan time. It has the potential to be integrated into current multiple blood contrast imaging protocol. Copyright © 2017. Published by Elsevier Inc.

Concentrations of nonesterified fatty acids and glucose in blood of periparturient dairy cows are indicative of pregnancy success at first insemination.

PubMed

Garverick, H A; Harris, M N; Vogel-Bluel, R; Sampson, J D; Bader, J; Lamberson, W R; Spain, J N; Lucy, M C; Youngquist, R S

2013-01-01

Greater blood concentrations of nonesterified fatty acids (NEFA) and lesser blood concentrations of glucose are indicative of the normal process of nutrient partitioning that occurs in early postpartum dairy cows. The objective was to determine the relationship between blood NEFA and glucose concentrations and subsequent conception at first insemination in postpartum dairy cows. Holstein (n=148) and Guernsey (n=8) dairy cows were blood sampled at approximately d 10, 7, and 3 prepartum, on the day of calving and 3, 7, 14, and 21 d postpartum for measurement of NEFA and glucose concentrations. Serum and plasma were harvested and used for measurement of NEFA and glucose concentrations, respectively. Cows were given a presynchronization treatment (2 injections of PGF(2α) 14 d apart) with the second PGF(2α) injection occurring 14 d before the initiation of the timed AI (TAI) protocol. Blood for determination of progesterone concentrations was collected at each presynchronization injection and at the initiation of the TAI protocol that was used for first insemination (74±7 d postpartum). Cows were considered noncycling if serum progesterone concentrations at the 2 presynchronization PGF(2α) injections (d 37 and 51±7 postpartum) and at the initiation of the TAI protocol (d 65±7 postpartum) were ≤1 ng/mL, and there was no indication of ovulation or presence of a corpus luteum by ultrasound examination at the initiation of the TAI protocol. Pregnancy was determined at 33 d and again at 61 d after first insemination by using ultrasound. Across all days, serum NEFA and plasma glucose concentrations were not different between cows that ovulated before the initiation of the TAI program (cycling) compared with those that did not ovulate (noncycling). Serum NEFA concentrations, however, were less and plasma glucose concentrations were greater during the early postpartum period for cows that subsequently became pregnant at first insemination compared with those that failed to become pregnant. Logistic regressions were used to predict the probability of pregnancy based on NEFA and glucose concentrations from individual days. The prediction with the greatest likelihood ratio was for d 3 postpartum NEFA and glucose concentrations. Nutritional status during the early postpartum period (within 1 wk after calving), as indicated by blood NEFA and glucose concentrations, may affect subsequent fertility by a mechanism that is independent from interval to first ovulation. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Validation of two automatic devices for self-measurement of blood pressure according to the International Protocol of the European Society of Hypertension: the Omron M6 (HEM-7001-E) and the Omron R7 (HEM 637-IT).

PubMed

Topouchian, Jirar A; El Assaad, Mohamed A; Orobinskaia, Ludmila V; El Feghali, Ramzi N; Asmar, Roland G

2006-06-01

Two electronic devices for self-measurement of blood pressure - a brachial monitor, the Omron M6, and a wrist monitor, the Omron R7 - were evaluated in two separate studies according to the International Protocol of the European Society of Hypertension. The International Validation Protocol is divided into two phases: the first phase is performed on 15 selected participants (45 pairs of blood pressure measurements); if the device passes this phase, 18 supplementary participants are included (54 pairs of blood pressure measurements) making a total number of 33 participants (99 pairs of blood pressure measurements) on whom the final validation is performed. The same methodology recommended by the European Society of Hypertension protocol was applied for both studies. In each study and for each participant, four blood pressure measurements were taken simultaneously by two trained observers using mercury sphygmomanometers alternately with three measurements taken by the tested device. The difference between the blood pressure value given by the device and that obtained by the two observers (mean of the two observers) was calculated for each measure. The 99 pairs of blood pressure differences were classified into three categories (

LPS-induced microvascular leukocytosis can be assessed by blue-field entoptic phenomenon.

PubMed

Kolodjaschna, Julia; Berisha, Fatmire; Lung, Solveig; Schaller, Georg; Polska, Elzbieta; Jilma, Bernd; Wolzt, Michael; Schmetterer, Leopold

2004-08-01

Administration of low doses of Escherichia coli endotoxin [a lipopolysaccharide (LPS)] to humans enables the study of inflammatory mechanisms. The purpose of the present study was to investigate whether the blue-field entoptic technique may be used to quantify the increase in circulating leukocytes in the ocular microvasculature after LPS infusion. In addition, combined laser Doppler velocimetry and retinal vessel size measurement were used to study red blood cell movement. Twelve healthy male volunteers received 20 IU/kg iv LPS as a bolus infusion. Outcome parameters were measured at baseline and 4 h after LPS administration. In the first protocol (n = 6 subjects), ocular hemodynamic effects were assessed with the blue-field entoptic technique, the retinal vessel analyzer, and laser Doppler velocimetry. In the second protocol (n = 6 subjects), white blood cell (WBC) counts from peripheral blood samples and blue-field entoptic technique measurements were performed. LPS caused peripheral blood leukocytosis and increased WBC density in ocular microvessels (by 49%; P = 0.036) but did not change WBC velocity. In addition, retinal venous diameter was increased (by 9%; P = 0.008), but red blood cell velocity remained unchanged. The LPS-induced changes in retinal WBC density and leukocyte counts were significantly correlated (r = 0.87). The present study indicates that the blue-field entoptic technique can be used to assess microvascular leukocyte recruitment in vivo. In addition, our data indicate retinal venous dilation in response to endotoxin.

Collection and processing of whole blood for transformation of peripheral blood mononuclear cells and extraction of DNA: the Type 1 Diabetes Genetics Consortium

PubMed Central

Rosinger, Silke; Nutland, Sarah; Mickelson, Eric; Varney, Michael D; Boehm, Bernard O; Olsem, Gary J; Hansen, John A; Nicholson, Ian; Hilner, Joan E; Perdue, Letitia H; Pierce, June J; Akolkar, Beena; Nierras, Concepcion; Steffes, Michael W

2010-01-01

Background and Purpose To yield large amounts of DNA for many genotype analyses and to provide a renewable source of DNA, the Type 1 Diabetes Genetics Consortium (T1DGC) harvested DNA and peripheral blood mononuclear cells (PBMCs) from individuals with type 1 diabetes and their family members in several regions of the world. Methods DNA repositories were established in Asia-Pacific, Europe, North America, and the United Kingdom. To address region-specific needs, different methods and sample processing techniques were used among the laboratories to extract and to quantify DNA and to establish Epstein-Barr virus transformed cell lines. Results More than 98% of the samples of PBMCs were successfully transformed. Approximately 20–25 µg of DNA were extracted per mL of whole blood. Extraction of DNA from the cell pack ranged from 92 to 165 µg per cell pack. In addition, the extracted DNA from whole blood or transformed cells was successfully utilized in each regional human leukocyte antigen genotyping laboratory and by several additional laboratories performing consortium-wide genotyping projects. Limitations Although the isolation of PBMCs was consistent among sites, the measurement of DNA was difficult to harmonize. Conclusions DNA repositories can be established in different regions of the world and produce similar amounts of high-quality DNA for a variety of high-throughput genotyping techniques. Furthermore, even with the distances and time necessary for transportation, highly efficient transformation of PBMCs is possible. For future studies/trials involving several laboratories in different locations, the T1DGC experience includes examples of protocols that may be applicable. In summary, T1DGC has developed protocols that would be of interest to any scientific organization attempting to overcome the logistical problems associated with studies/trials spanning multiple research facilities, located in different regions of the world. PMID:20595244

Development of new method and protocol for cryopreservation related to embryo and oocytes freezing in terms of fertilization rate: A comparative study including review of literature.

PubMed

Barik, Mayadhar; Bajpai, Minu; Patnaik, Santosh; Mishra, Pravash; Behera, Priyamadhaba; Dwivedi, Sada Nanda

2016-01-01

Cryopreservation is basically related to meritorious thin samples or small clumps of cells that are cooled quickly without loss. Our main objective is to establish and formulate an innovative method and protocol development for cryopreservation as a gold standard for clinical uses in laboratory practice and treatment. The knowledge regarding usefulness of cryopreservation in clinical practice is essential to carry forward the clinical practice and research. We are trying to compare different methods of cryopreservation (in two dozen of cells) at the same time we compare the embryo and oocyte freezing interms of fertilization rate according to the International standard protocol. The combination of cryoprotectants and regimes of rapid cooling and rinsing during warming often allows successful cryopreservation of biological materials, particularly cell suspensions or thin tissue samples. Examples include semen, blood, tissue samples like tumors, histological cross-sections, human eggs and human embryos. Although presently many studies have reported that the children born from frozen embryos or "frosties," show consistently positive results with no increase in birth defects or development abnormalities is quite good enough and similar to our study (50-85%). We ensure that cryopreservation technology provided useful cell survivability, tissue and organ preservation in a proper way. Although it varies according to different laboratory conditions, it is certainly beneficial for patient's treatment and research. Further studies are needed for standardization and development of new protocol.

Overview of Five-Years of Experience Performing Non-Invasive Fetal Sex Assessment in Maternal Blood

PubMed Central

Perlado-Marina, Sara; Bustamante-Aragones, Ana; Horcajada, Laura; Trujillo-Tiebas, Maria Jose; Lorda-Sanchez, Isabel; Ruiz Ramos, Marta; Plaza, Javier; Rodriguez de Alba, Marta

2013-01-01

Since the discovery of the presence of fetal DNA in maternal blood, non-invasive fetal sex determination has been the test most widely translated into clinical practice. To date there is no agreement between the different laboratories performing such tests in relation to which is the best protocol. As a consequence there are almost as many protocols as laboratories offering the service, using different methodologies and thus obtaining different diagnostic accuracies. By the end of 2007, after a validation study performed in 316 maternal samples collected between the 5th and 12th week of gestation, the fetal sex determination was incorporated into clinical practice in our Service. The test is performed in the first trimester of pregnancy, and it is offered as part of the genetic counseling process for couples at risk of X-linked disorders. As a general rule and in order to avoid misdiagnosis, two samples at different gestational ages are tested per patient. The analysis is performed by the study of the SRY gene by RT-PCR. Two hundred and twenty six pregnancies have been tested so far in these 5 years. Neither false positives nor false negatives diagnoses have been registered, thus giving a diagnostic accuracy of 100%. PMID:26835681

Validation of the Microlife BP A200 Comfort and W2 Slim automated blood pressure monitors in a general adult population according to the European Society of Hypertension and the ANSI/AAMI/ISO 81060-2: 2013 protocols.

PubMed

Bing, Sen; Chen, Kang; Hou, Hong; Zhang, Weijuan; Li, Linyi; Wei, Jiao; Shu, Chang; Wan, Yi

2016-04-01

This study aimed to determine the accuracy of the Microlife BP A200 Comfort and W2 Slim automated blood pressure monitors according to the European Society of Hypertension International Protocol revision 2010 and the ANSI/AAMI/ISO 81060-2:2013 protocols. The devices were assessed on 33 participants according to the European Society of Hypertension requirements and were then tested on 85 participants according to the ANSI/AAMI/ISO 81060-2:2013 criteria. Procedures and data analysis were carried out following protocol guidelines precisely. The Microlife BP A200 Comfort and W2 Slim devices passed the criteria of the European Society of Hypertension International Protocol revision 2010 for both systolic blood pressure and diastolic blood pressure. The devices also fulfilled the ANSI/AAMI/ISO 81060-2:2013 criteria, with mean differences of SBP and DPB between the devices and observers of 0.38±5.12 and 0.28±4.29 mmHg for the BP A200 Comfort and 1.01±6.80 and 0.34±5.62 mmHg for the W2 Slim, respectively. The Microlife BP A200 Comfort and W2 Slim automated blood pressure monitors fulfilled the European Society of Hypertension revision 2010 and the ANSI/AAMI/ISO 81060-2:2013 protocols, and can be recommended for self-measurement in the general population.

KETAMINE-MEDETOMIDINE AND KETAMINE-MEDETOMIDINE-MIDAZOLAM ANESTHESIA IN CAPTIVE CHEETAHS (ACINONYX JUBATUS)-COMPARISON OF BLOOD PRESSURE AND KIDNEY BLOOD FLOW.

PubMed

Stagegaard, Julia; Hørlyck, Arne; Hydeskov, Helle B; Bertelsen, Mads F

2017-06-01

Six clinically healthy captive cheetahs ( Acinonyx jubatus ) were anesthetized twice using two different drug combinations to investigate if blood pressure and kidney blood flow are affected by medetomidine dosage. Protocol KM (2.0 mg/kg ketamine and 0.05 mg/kg medetomidine) was compared with protocol KMM (2.0 mg/kg ketamine, 0.02 mg/kg medetomidine, and 0.1 mg/kg midazolam). Heart rate (HR), respiratory rate (RR), body temperature, end-tidal carbon dioxide pressure (ETCO 2 ), and anesthetic depth were monitored every 10 min. Noninvasive mean (MAP), systolic (SAP), and diastolic (DAP) arterial blood pressure were measured, and Duplex Doppler ultrasonography was performed on the kidneys. The mean arterial resistive index (RI) was determined and the pulse pressure index (PPI) was calculated, as indicators for kidney blood flow. There were no significant differences in induction and recovery times. MAP was significantly higher with KM than KMM at 35 min, and in both protocols decreased significantly after atipamezole administration. DAP was significantly higher at 25 and 35 min in animals anesthetized with KM; it also decreased significantly with both protocols after atipamezole administration. The PPI was significantly lower throughout the procedure with KM, and with both protocols increased significantly after atipamezole administration. Both the higher blood pressure and the reduced PPI with KM were likely a direct effect of the higher medetomidine dosage, and these findings indicate that lower medetomidine dosages might reduce hypertension and lead to a better PPI in cheetah immobilization.

Suspension properties of whole blood and its components under glucose influence studied in patients with acute coronary syndrome

NASA Astrophysics Data System (ADS)

Malinova, Lidia I.; Simonenko, Georgy V.; Denisova, Tatyana P.; Dovgalevsky, Pavel Y.; Tuchin, Valery V.

2004-05-01

The protocol of our study includes men with acute myocardial infarction, stable angina pectoris of II and III functional classes and unstable angina pectoris. Patients with arterial hypertension, disorders in carbohydrate metabolism were excluded from the study. Blood samples taken under standardized conditions, were stabilized with citrate sodium 3,8% (1:9). Erythrocytes and platelets aggregation activity under glucose influence (in vitro) was studied by means of computer aided microphotometer -- a visual analyzer. Erythrocyte and platelets were united in special subsystem of whole blood. Temporal and functional characteristics of their aggregation were analyzed by creation of phase patterns fragments. The received data testify to interrelation of erythrocytes and platelets processes of aggregation under conditions of increasing of glucose concentration of the incubatory environment, which temporal and functional characteristics may be used for diagnostics and the prognosis of destabilization coronary blood flow at an acute coronary syndrome.

Absolute counting of neutrophils in whole blood using flow cytometry.

PubMed

Brunck, Marion E G; Andersen, Stacey B; Timmins, Nicholas E; Osborne, Geoffrey W; Nielsen, Lars K

2014-12-01

Absolute neutrophil count (ANC) is used clinically to monitor physiological dysfunctions such as myelosuppression or infection. In the research laboratory, ANC is a valuable measure to monitor the evolution of a wide range of disease states in disease models. Flow cytometry (FCM) is a fast, widely used approach to confidently identify thousands of cells within minutes. FCM can be optimised for absolute counting using spiked-in beads or by measuring the sample volume analysed. Here we combine the 1A8 antibody, specific for the mouse granulocyte protein Ly6G, with flow cytometric counting in straightforward FCM assays for mouse ANC, easily implementable in the research laboratory. Volumetric and Trucount™ bead assays were optimized for mouse neutrophils, and ANC values obtained with these protocols were compared to ANC measured by a dual-platform assay using the Orphee Mythic 18 veterinary haematology analyser. The single platform assays were more precise with decreased intra-assay variability compared with ANC obtained using the dual protocol. Defining ANC based on Ly6G expression produces a 15% higher estimate than the dual protocol. Allowing for this difference in ANC definition, the flow cytometry counting assays using Ly6G can be used reliably in the research laboratory to quantify mouse ANC from a small volume of blood. We demonstrate the utility of the volumetric protocol in a time-course study of chemotherapy induced neutropenia using four drug regimens. © 2014 International Society for Advancement of Cytometry.

Comparison of butorphanol-detomidine versus butorphanol-azaperone for the standing sedation of captive greater one-horned rhinoceroses (Rhinoceros unicornis).

PubMed

Bapodra, Priya; Cracknell, Jonathan; Wolfe, Barbara A

2014-03-01

Three adult and two subadult greater one-horned rhinoceroses (Rhinoceros unicornis) were sedated a total of nine times using two different intramuscular sedative combinations in order to compare the effectiveness of these combinations in inducing consistent standing sedation in this species. The sedation protocols compared were butorphanol tartrate (50-60 mg) and detomidine hydrochloride (20-30 mg; BD) versus butorphanol tartrate (80-120 mg) and azaperone (80-120 mg; BA). Specific doses were adjusted according to age and sex class, and based on previous experience. Parameters compared included time to achieve defined levels of sedation, time to recovery following antagonism, physiological parameters including heart rate, respiratory rate, indirect arterial blood pressure, and venous blood gas values. A hydraulic restraint chute was utilized to mechanically restrain animals during the procedures, and blood collection and ophthalmic examinations were conducted on all animals. Both protocols resulted in standing sedation for > or = 22.3 +/- 2.9 min or until antagonists were administered. The BD protocol resulted in deeper and more consistent sedation, compared to the BA protocol. Naltrexone hydrochloride (250-300 mg) and tolazoline hydrochloride (1,500-2,000 mg) were administered intramuscularly to antagonize protocol BD, whereas naltrexone alone (200-500 mg) was used to antagonize BA. Time to full antagonism, defined as normal mentation and ambulation following administration of antagonists, was prolonged in the BD protocol (132.3 +/- 17.2 min) compared with the BA protocol (7.5 +/- 2.5 min). Venous blood gas analysis did not reveal any significant blood gas deviations during sedation when compared with either conscious equine or white rhinoceros (Ceratotherium simum) venous reference ranges. In summary, both combinations resulted in adequate standing sedation for minimally invasive procedures, although BD resulted in more profound and consistent sedation.

Self-aliquoting micro-grooves in combination with laser ablation-ICP-mass spectrometry for the analysis of challenging liquids: quantification of lead in whole blood.

PubMed

Nischkauer, Winfried; Vanhaecke, Frank; Limbeck, Andreas

2016-08-01

We present a technique for the fast screening of the lead concentration in whole blood samples using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The whole blood sample is deposited on a polymeric surface and wiped across a set of micro-grooves previously engraved into the surface. The engraving of the micro-grooves was accomplished with the same laser system used for LA-ICP-MS analysis. In each groove, a part of the liquid blood is trapped, and thus, the sample is divided into sub-aliquots. These aliquots dry quasi instantly and are then investigated by means of LA-ICP-MS. For quantification, external calibration against aqueous standard solutions was relied on, with iron as an internal standard to account for varying volumes of the sample aliquots. The (208)Pb/(57)Fe nuclide ratio used for quantification was obtained via a data treatment protocol so far only used in the context of isotope ratio determination involving transient signals. The method presented here was shown to provide reliable results for Recipe ClinChek® Whole Blood Control levels I-III (nos. 8840-8842), with a repeatability of typically 3 % relative standard deviation (n = 6, for Pb at 442 μg L(-1)). Spiked and non-spiked real whole blood was analysed as well, and the results were compared with those obtained via dilution and sectorfield ICP-MS. A good agreement between both methods was observed. The detection limit (3 s) for lead in whole blood was established to be 10 μg L(-1) for the laser ablation method presented here. Graphical Abstract Micro-grooves are filled with whole blood, dried, and analyzed by laser ablation ICP-mass spectrometry. Notice that the laser moves in perpendicular direction with regard to the micro-grooves.

Red Cell Properties after Different Modes of Blood Transportation

PubMed Central

Makhro, Asya; Huisjes, Rick; Verhagen, Liesbeth P.; Mañú-Pereira, María del Mar; Llaudet-Planas, Esther; Petkova-Kirova, Polina; Wang, Jue; Eichler, Hermann; Bogdanova, Anna; van Wijk, Richard; Vives-Corrons, Joan-Lluís; Kaestner, Lars

2016-01-01

Transportation of blood samples is unavoidable for assessment of specific parameters in blood of patients with rare anemias, blood doping testing, or for research purposes. Despite the awareness that shipment may substantially alter multiple parameters, no study of that extent has been performed to assess these changes and optimize shipment conditions to reduce transportation-related artifacts. Here we investigate the changes in multiple parameters in blood of healthy donors over 72 h of simulated shipment conditions. Three different anticoagulants (K3EDTA, Sodium Heparin, and citrate-based CPDA) for two temperatures (4°C and room temperature) were tested to define the optimal transportation conditions. Parameters measured cover common cytology and biochemistry parameters (complete blood count, hematocrit, morphological examination), red blood cell (RBC) volume, ion content and density, membrane properties and stability (hemolysis, osmotic fragility, membrane heat stability, patch-clamp investigations, and formation of micro vesicles), Ca2+ handling, RBC metabolism, activity of numerous enzymes, and O2 transport capacity. Our findings indicate that individual sets of parameters may require different shipment settings (anticoagulants, temperature). Most of the parameters except for ion (Na+, K+, Ca2+) handling and, possibly, reticulocytes counts, tend to favor transportation at 4°C. Whereas plasma and intraerythrocytic Ca2+ cannot be accurately measured in the presence of chelators such as citrate and EDTA, the majority of Ca2+-dependent parameters are stabilized in CPDA samples. Even in blood samples from healthy donors transported using an optimized shipment protocol, the majority of parameters were stable within 24 h, a condition that may not hold for the samples of patients with rare anemias. This implies for as short as possible shipping using fast courier services to the closest expert laboratory at reach. Mobile laboratories or the travel of the patients to the specialized laboratories may be the only option for some groups of patients with highly unstable RBCs. PMID:27471472

Using the emergency department as a screening site for high blood pressure. A method for improving hypertension detection and appropriate referral.

PubMed

Mamon, J; Green, L; Levine, D M; Gibson, G; Gurley, H T

1987-08-01

This study describes the development and testing of a high blood pressure protocol for use in emergency departments (ED) to enhance detection of those patients appropriate for subsequent referral. The protocol involves two serial blood pressure measurements and a patient interview to determine: 1) previous history of high blood pressure (HBP), 2) treatment in past year for HBP, and 3) usual source of medical care. The accuracy of patient reporting was validated by comparison with the patients' hospital record (reflecting outpatient and inpatient visits). Results indicate that these self-reports have high levels of sensitivity (range 90-100%) and specificity (range 79-96%). Use of the additional patient information increased the sensitivity of the screening protocol in identifying when and where a patient should be referred. Use of this methodology indicates that the protocol is a simple and effective method for HBP screening. The findings also suggest that the ED is an ideal site for screening the "hard-to-reach" hypertensive population.

Cryopreservation prevents iron-initiated highly unsaturated fatty acid loss during storage of human blood on chromatography paper at -20°C.

PubMed

Metherel, Adam H; Stark, Ken D

2015-03-01

Fingertip prick whole blood collection on chromatography paper is amenable to high-throughput fatty acid (FA) profiling for large clinical and field studies. However, sample storage is problematic because highly unsaturated FAs (HUFAs) in erythrocytes rapidly degrade in samples stored at -20°C. The aim of the current study was to determine the mechanism of HUFA degradation and to develop prevention protocols. Free fatty acid (FFA) standards and whole blood reference material from a single participant were used to examine sample storage at -20°C for up to 90 d in triplicate. Iron chelation with deferoxamine (0-5000 μg), antioxidant protection with butylated hydroxytoluene (50 μg), cryopreservation with glycerol, and blood drying were examined using whole blood on chromatography strips. Biological replicate blood samples from additional participants (n = 6) with a range of ω-3 (n-3) HUFA concentrations were similarly assessed. FFAs were relatively stable when stored on chromatography strips at -20°C. Glycerol treatment prevented HUFA degradation in whole blood reference material for 30 d (45 ± 0.4 to 46.8 ± 0.1, means ± SDs) compared to untreated saline controls (45.9 ± 1.0 to 6.8 ± 0.2). Pretreatment of paper for blood spots with deferoxamine and drying blood before storage slowed, but not entirely prevented, HUFA degradation over 30 d to 22% and 19% below baseline, respectively, compared to 86-92% in the controls. Protection against HUFA degradation with blood drying and glycerol treatment was confirmed in the biological replicate study and confirmed by prevention of cell lysis. HUFA degradation during storage at -20°C appears to be due to hemolysis and subsequent iron-initiated peroxidation. This degradation may be prevented by glycerol, iron chelation, and/or dried blood spotting. A more thorough understanding of methods to prevent degradation during storage is critical with increasing use of FA profiling in large clinical studies. © 2015 American Society for Nutrition.

In vivo evaluation of titanium-prepared platelet-rich fibrin (T-PRF): a new platelet concentrate.

PubMed

Tunalı, Mustafa; Özdemir, Hakan; Küçükodacı, Zafer; Akman, Serhan; Fıratlı, Erhan

2013-07-01

We have developed a new, titanium-prepared, platelet-rich fibrin (T-PRF) together with the protocol for forming it, which is based on the hypothesis that titanium tubes may be more effective at activating platelets than the glass tubes used by Chouckroun in his platelet-rich fibrin (PRF) method. The aim of this study was to find a suitable animal model in which to evaluate the method and to investigate the efficacy of T-PRF for wound healing. Blood samples from 6 rabbits were used to confirm the protocol for formation of T-PRF. We evaluated T-PRF or T-PRF-like clots morphologically using scanning electron microscopy (EM). Blood samples from 5 rabbits were used to develop an experiment in which to evaluate the effects of T-PRF on wound healing. The mucoperiosteal flaps were filled with autologous T-PRF membranes from the vestibule in the anterior mandibular regions. Samples collected from the surgical sites were stained with haematoxylin and eosin. We found a mature fibrin network in T-PRF clots that had been centrifuged for 15 min at 3500 rpm and, 15 days after placement of the membrane, we found newly-forming connective tissue and islets of bony tissue in the T-PRF membrane. These results show that T-PRF could induce the formation of new bone with new connective tissue in a rabbit model of wound healing within 30 days of treatment. Published by Elsevier Ltd.

Validation of the Pangao PG-800B11 blood pressure monitor according to the European Society of Hypertension and the British Hypertension Society protocols.

PubMed

Bing, Sen; Zhang, Cunhai; Wang, Liping; Li, Linyi; Wan, Yi

2014-12-01

The study aimed to determine the accuracy of the fully automated oscillometric upper-arm blood pressure (BP) monitor Pangao PG-800B11 according to the European Society of Hypertension-International Protocol (ESH-IP) revision 2010 and the British Hypertension Society (BHS) protocol 1993. Data from 33 participants were initially examined according to the ESH-IP revision 2010. Furthermore, 52 participants were then enrolled to fulfill the BHS protocol requirements. In all participants, sequential left arm measurements were performed by two trained observers using a mercury sphygmomanometer and one supervisor using the device. The protocols' requirements were followed precisely. The device passed all parts of the ESH protocol for systolic blood pressure (SBP) and diastolic blood pressure (DBP) and was graded A according to the criteria of the BHS protocol for both SBP and DBP. The A/A grade was achieved in low (<130/80 mmHg), medium (130-160/80-100 mmHg), and high (>160/100 mmHg) BP categories. The mean BP difference between PG-800B11 and observers in the 85 participants was -0.6±5.0 mmHg for SBP and -0.6±4.5 mmHg for DBP; thus, the device also fulfilled the requirements of the Association for the Advancement of Medical Instrumentation (AAMI). The Pangao PG-800B11 passed all requirements of the ESH-IP revision 2010 and achieved A/A grade of the BHS protocol across a wide range of BPs.

Performance of a quantitative fecal immunochemical test for detecting advanced colorectal neoplasia: a prospective cohort study.

PubMed

Liles, Elizabeth G; Perrin, Nancy; Rosales, Ana G; Smith, David H; Feldstein, Adrianne C; Mosen, David M; Levin, Theodore R

2018-05-02

The fecal immunochemical test (FIT) is easier to use and more sensitive than the guaiac fecal occult blood test, but it is unclear how to optimize FIT performance. We compared the sensitivity and specificity for detecting advanced colorectal neoplasia between single-sample (1-FIT) and two-sample (2-FIT) FIT protocols at a range of hemoglobin concentration cutoffs for a positive test. We recruited 2,761 average-risk men and women ages 49-75 referred for colonoscopy within a large nonprofit, group-model health maintenance organization (HMO), and asked them to complete two separate single-sample FITs. We generated receiver-operating characteristic (ROC) curves to compare sensitivity and specificity estimates for 1-FIT and 2-FIT protocols among those who completed both FIT kits and colonoscopy. We similarly compared sensitivity and specificity between hemoglobin concentration cutoffs for a single-sample FIT. Differences in sensitivity and specificity between the 1-FIT and 2-FIT protocols were not statistically significant at any of the pre-specified hemoglobin concentration cutoffs (10, 15, 20, 25, and 30 μg/g). There was a significant difference in test performance of the one-sample FIT between 50 ng/ml (10 μg/g) and each of the higher pre-specified cutoffs. Disease prevalence was low. A two-sample FIT is not superior to a one-sample FIT in detection of advanced adenomas; the one-sample FIT at a hemoglobin concentration cutoff of 50 ng/ml (10 μg/g) is significantly more sensitive for advanced adenomas than at higher cutoffs. These findings apply to a population of younger, average-risk patients in a U.S. integrated care system with high rates of prior screening.

Validation of the Pangao PG-800B5 for clinical use and self-measurement according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Chen, Wan; Zeng, Zhaolin; Li, Lizhi; Wan, Xiaofen; Wan, Yi

2014-10-01

This study aimed to validate the Pangao PG-800B5 upper arm blood pressure monitor according to the European Society of Hypertension International Protocol revision 2010. A total of 33 participants, 16 men and 17 women, were included in the device evaluation. The protocol requirements were followed precisely. The mean age of the participants was 56.4±21.0 years (range 22-84 years). The mean systolic blood pressure was 143.6±25.5 mmHg (range 98-188 mmHg), the mean diastolic blood pressure was 85.7±17.2 mmHg (range 49-125 mmHg), and the mean arm circumference was 26.1±2.2 cm (range 23-32 cm). On average, the device overestimated the systolic blood pressure by 0.9±4.2 mmHg and diastolic blood pressure by 0.7±4.5 mmHg. The device passed all requirements, fulfilling the standards of the protocol. Therefore, the Pangao PG-800B5 upper arm blood pressure monitor can be recommended for clinical use and self-measurement in an adult population.

Protein substitute dosage in PKU: how much do young patients need?

PubMed

MacDonald, A; Chakrapani, A; Hendriksz, C; Daly, A; Davies, P; Asplin, D; Hall, K; Booth, I W

2006-07-01

The optimal dose of protein substitute has not been determined in children with phenylketonuria (PKU). To determine if a lower dose of protein substitute could achieve the same or better degree of blood phenylalanine control when compared to the dosage recommended by the UK MRC.(1) In a six week randomised, crossover study, two doses of protein substitute (Protocol A: 2 g/kg/day of protein equivalent; Protocol B: 1.2 g/kg/day protein equivalent) were compared in 25 children with well controlled PKU aged 2-10 years (median 6 years). Each dose of protein substitute was taken for 14 days, with a 14 day washout period in between. Twice daily blood samples (fasting pre-breakfast and evening, at standard times) for plasma phenylalanine were taken on day 8-14 of each protocol. The median usual dose of protein substitute was 2.2 g/kg/day (range 1.5-3.1 g/kg/day). When compared with control values, median plasma phenylalanine on the low dose of protein substitute increased at pre-breakfast by 301 mumol/l (95% CI 215 to 386) and in the evening by 337 micromol/l (95% CI 248 to 431). On the high dose of protein substitute, plasma phenylalanine concentrations remained unchanged when compared to control values. However, wide variability was seen between subjects. A higher dosage of protein substitute appeared to contribute to lower blood phenylalanine concentrations in PKU, but it did have a variable and individual impact and may have been influenced by the carbohydrate (+/- fat) content of the protein substitute.

The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools.

PubMed

Singh, Satwinder Kaur; Meyering, Maaike; Ramwadhdoebe, Tamara H; Stynenbosch, Linda F M; Redeker, Anke; Kuppen, Peter J K; Melief, Cornelis J M; Welters, Marij J P; van der Burg, Sjoerd H

2012-11-01

The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.

Single-cell high resolution melting analysis: A novel, generic, pre-implantation genetic diagnosis (PGD) method applied to cystic fibrosis (HRMA CF-PGD).

PubMed

Destouni, A; Poulou, M; Kakourou, G; Vrettou, C; Tzetis, M; Traeger-Synodinos, J; Kitsiou-Tzeli, S

2016-03-01

Institutions offering CF-PGD face the challenge of developing and optimizing single cell genotyping protocols that should cover for the extremely heterogeneous CF mutation spectrum. Here we report the development and successful clinical application of a generic CF-PGD protocol to facilitate direct detection of any CFTR nucleotide variation(s) by HRMA and simultaneous confirmation of diagnosis through haplotype analysis. A multiplex PCR was optimized supporting co-amplification of any CFTR exon-region, along with 6 closely linked STRs. Single cell genotypes were established through HRM analysis following melting of the 2nd round PCR products and were confirmed by STR haplotype analysis of the 1st PCR products. The protocol was validated pre-clinically, by testing 208 single lymphocytes, isolated from whole blood samples from 4 validation family trios. Fifteen PGD cycles were performed and 103 embryos were biopsied. In 15 clinical PGD cycles, genotypes were achieved in 88/93 (94.6%) embryo biopsy samples, of which 57/88 (64.8%) were deemed genetically suitable for embryo transfer. Amplification failed at all loci for 10/103 blastomeres biopsied from poor quality embryos. Six clinical pregnancies were achieved (2 twin, 4 singletons). PGD genotypes were confirmed following conventional amniocentesis or chorionic villus sampling in all achieved pregnancies. The single cell HRMA CF-PGD protocol described herein is a flexible, generic, low cost and robust genotyping method, which facilitates the analysis of any CFTR genotype combination. Single-cell HRMA can be beneficial to other clinical settings, for example the detection of single nucleotide variants in single cells derived from clinical tumor samples. Copyright © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Multicenter evaluation of the Sepsityper™ extraction kit and MALDI-TOF MS for direct identification of positive blood culture isolates using the BD BACTEC™ FX and VersaTREK(®) diagnostic blood culture systems.

PubMed

Schieffer, K M; Tan, K E; Stamper, P D; Somogyi, A; Andrea, S B; Wakefield, T; Romagnoli, M; Chapin, K C; Wolk, D M; Carroll, K C

2014-04-01

(i) Evaluation of delayed time to blood culture extraction by the Sepsityper kit and impact of shipping pellets off-site for MALDI-TOF MS analysis. (ii) Comparison of Sepsityper and laboratory-developed extraction methods from a literature review. Using two blood culture systems (BD BACTEC and VersaTREK), we extracted 411 positive blood cultures using the Sepsityper kit to mimic a potential protocol for institutions without a MALDI-TOF MS. Extracted pellets were shipped and analysed on the Bruker UltraflexIII. Successful extraction of 358 (87·1%) samples was determined by the presence of detectable proteins. MALDI-TOF MS correctly identified 332 (80·8%) samples. Delayed time to extraction did not affect Sepsityper extraction or MALDI-TOF MS accuracy. The extracted pellets remain stable and provide accurate results by MALDI-TOF MS when shipped at room temperature to off-site reference laboratories. This is the first study to show that institutions without a MALDI-TOF MS can take advantage of this innovative technology by shipping a volume of blood to an off-site laboratory for extraction and MALDI-TOF MS analysis. We also performed a literature review to compare various extraction methods. © 2014 The Society for Applied Microbiology.

Feasibility of quarantine procedures for bison (Bison bison) calves from Yellowstone National Park for conservation of brucellosis-free bison.

PubMed

Ryan Clarke, P; Frey, Rebecca K; Rhyan, Jack C; McCollum, Matt P; Nol, Pauline; Aune, Keith

2014-03-01

OBJECTIVE--To determine the feasibility of qualifying individuals or groups of Yellowstone National Park bison as free from brucellosis. DESIGN--Cohort study. SAMPLE--Serum, blood, and various samples from live bison and tissues taken at necropsy from 214 bison over 7 years. PROCEDURES--Blood was collected from bison every 30 to 45 days for serologic tests and microbiological culture of blood for Brucella abortus. Seropositive bison were euthanized until all remaining bison had 2 consecutive negative test results. Half the seronegative bison were randomly euthanized, and tissues were collected for bacteriologic culture. The remaining seronegative bison were bred, and blood was tested at least twice per year. Cow-calf pairs were sampled immediately after calving and 6 months after calving for evidence of B abortus. RESULTS--Post-enrollment serial testing for B abortus antibodies revealed no bison that seroconverted after 205 days (first cohort) and 180 days (second cohort). During initial serial testing, 85% of bison seroconverted within 120 days after removal from the infected population. Brucella abortus was not cultured from any euthanized seronegative bison (0/88). After parturition, no cows or calves had a positive test result for B abortus antibodies, nor was B abortus cultured from any samples. CONCLUSIONS AND CLINICAL RELEVANCE--Results suggested it is feasible to qualify brucellosis-free bison from an infected herd following quarantine procedures as published in the USDA APHIS brucellosis eradication uniform methods and rules. Latent infection was not detected in this sample of bison when applying the USDA APHIS quarantine protocol.

Tooth Tissue Engineering: The Importance of Blood Products as a Supplement in Tissue Culture Medium for Human Pulp Dental Stem Cells.

PubMed

Pisciolaro, Ricardo Luiz; Duailibi, Monica Talarico; Novo, Neil Ferreira; Juliano, Yara; Pallos, Debora; Yelick, Pamela Crotty; Vacanti, Joseph Phillip; Ferreira, Lydia Masako; Duailibi, Silvio Eduardo

2015-11-01

One of the goals in using cells for tissue engineering (TE) and cell therapy consists of optimizing the medium for cell culture. The present study compares three different blood product supplements for improved cell proliferation and protection against DNA damage in cultured human dental pulp stem cells for tooth TE applications. Human cells from dental pulp were first characterized as adult stem cells (ectomesenchymal mixed origin) by flow cytometry. Next, four different cell culture conditions were tested: I, supplement-free; II, supplemented with fetal bovine serum; III, allogeneic human serum; and IV, autologous human serum. Cultured cells were then characterized for cell proliferation, mineralized nodule formation, and colony-forming units (CFU) capability. After 28 days in culture, the comet assay was performed to assess possible damage in cellular DNA. Our results revealed that Protocol IV achieved higher cell proliferation than Protocol I (p = 0.0112). Protocols II and III resulted in higher cell proliferation than Protocol I, but no statistical differences were found relative to Protocol IV. The comet assay revealed less cell damage in cells cultured using Protocol IV as compared to Protocols II and III. The damage percentage observed on Protocol II was significantly higher than all other protocols. CFUs capability was highest using Protocol IV (p = 0.0018) and III, respectively, and the highest degree of mineralization was observed using Protocol IV as compared to Protocols II and III. Protocol IV resulted in significantly improved cell proliferation, and no cell damage was observed. These results demonstrate that human blood product supplements can be used as feasible supplements for culturing adult human dental stem cells.

Does a resistance exercise session with continuous or intermittent blood flow restriction promote muscle damage and increase oxidative stress?

PubMed

Neto, Gabriel R; Novaes, Jefferson S; Salerno, Verônica P; Gonçalves, Michel M; Batista, Gilmário R; Cirilo-Sousa, Maria S

2018-01-01

The aim of this study was to compare the effect of low-load resistance exercise (LLRE) with continuous and intermittent blood flow restriction (BFR) on the creatine kinase (CK), lactate dehydrogenase (LDH), protein carbonyl (PC), thiobarbituric acid-reactive substance (TBARS) and uric acid (UA) levels in military men. The study included 10 recreationally trained men aged 19 ± 0.82 years who underwent the following experimental protocols in random order on separate days (72-96 h): 4 LLRE sessions at a 20% 1RM (one-repetition maximum [1RM]) with continuous BFR (LLRE + CBFR); 4 LLRE sessions at 20% 1RM with intermittent BFR (LLRE + IBFR) and 4 high-intensity resistance exercise (HIRE) sessions at 80% 1RM. The CK and LDH (markers of muscle damage) levels were measured before exercise (BE), 24 h post-exercise and 48 h post-exercise, and the PC, TBARS and UA (markers of oxidative stress) levels were measured BE and immediately after each exercise session. There was a significant increase in CK in the HIRE 24 post-exercise samples compared with the LLRE + CBFR and LLRE + IBFR (P = 0.035, P = 0.036, respectively), as well as between HIRE 48 post-exercise and LLRE + CBFR (P = 0.049). Additionally, there was a significant increase in CK in the LLRE + CBFR samples BE and immediately after each exercise (Δ = 21.9%) and in the HIRE samples BE and immediately after each exercise, BE and 24 post-exercise, and BE and 48 post-exercise (Δ values of 35%, 177.6%, and 177.6%, respectively). However, there were no significant changes in LDH, PC, TBARS, and UA between the protocols (P > 0.05). Therefore, a physical exercise session with continuous or intermittent BFR did not promote muscle damage; moreover, neither protocol seemed to affect the oxidative stress markers.

Potential misdiagnosis of von Willebrand disease and haemophilia caused by ineffective mixing of thawed plasma.

PubMed

Favaloro, E J; Oliver, S; Mohammed, S; Ahuja, M; Grzechnik, E; Azimulla, S; McDonald, J; Lima-Oliveira, G; Lippi, G

2017-09-01

von Willebrand disease (VWD) reflects a loss or dysfunction in von Willebrand factor (VWF), while haemophilia represents a loss or dysfunction of clotting factors such as factor VIII (FVIII) or FIX. Their diagnosis requires laboratory testing, with this potentially compromised by preanalytical events, including poor sample quality. This study assessed the effect of inadequate mixing as a potential cause of VWD and haemophilia misdiagnosis. After completion of requested testing, 48 consecutive patient samples comprising separate aliquots from single collections were individually pooled, appropriately mixed, then frozen in separate aliquots, either at -20°C or -80°C for 2-7 days. Each sample set was then thawed and the separate aliquots subjected to separate mixing protocols (several inversions, blood roller, vortex) vs a non-mix sample, and all aliquots then tested for various VWF and factor assays. Non-mixing led to substantial reduction in VWF and factors in about 25% of samples, that in some cases could lead to misdiagnosis of VWD or haemophilia. Interestingly, there were also some differences observed with respect to different mixing protocols. Our study identified ineffective or variable mixing of thawed plasma samples as potential causes of misdiagnosis of VWD or haemophilia. Further education regarding the importance of appropriate mixing appears warranted. © 2017 John Wiley & Sons Ltd.

High density FTA plates serve as efficient long-term sample storage for HLA genotyping.

PubMed

Lange, V; Arndt, K; Schwarzelt, C; Boehme, I; Giani, A S; Schmidt, A H; Ehninger, G; Wassmuth, R

2014-02-01

Storage of dried blood spots (DBS) on high-density FTA(®) plates could constitute an appealing alternative to frozen storage. However, it remains controversial whether DBS are suitable for high-resolution sequencing of human leukocyte antigen (HLA) alleles. Therefore, we extracted DNA from DBS that had been stored for up to 4 years, using six different methods. We identified those extraction methods that recovered sufficient high-quality DNA for reliable high-resolution HLA sequencing. Further, we confirmed that frozen whole blood samples that had been stored for several years can be transferred to filter paper without compromising HLA genotyping upon extraction. Concluding, DNA derived from high-density FTA(®) plates is suitable for high-resolution HLA sequencing, provided that appropriate extraction protocols are employed. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Detection of toxoplasmosis in experimentally infected goats by PCR.

PubMed

Sreekumar, C; Rao, J R; Mishra, A K; Ray, D; Joshi, P; Singh, R K

2004-05-15

PCR was used to diagnose toxoplasmosis in two pairs of Barbari goats infected by oral administration of doses of either 10(4) or 10(5) oocysts of Toxoplasma gondii. Blood and lymph node aspirates were collected from the infected goats and control goat at intervals, and tissues were also collected from a fetus that was aborted and a doe that died during the trial. Both processed and unprocessed samples were used for the PCR, using primers directed to the multicopy B1 gene. None of the blood samples was positive, but a specific signal was obtained from the lymph node aspirates after partial DNA extraction. Direct PCR of the lung, muscle and mesenteric lymph node of the doe and lung tissue of the aborted fetus yielded the target fragment. The simplified PCR protocols, including partial DNA extraction and direct assay of lung tissue, were effective for the diagnosis of toxoplasmosis.

Understanding biological mechanisms underlying adverse birth outcomes in developing countries: protocol for a prospective cohort (AMANHI bio–banking) study

PubMed Central

Baqui, Abdullah H; Khanam, Rasheda; Rahman, Mohammad Sayedur; Ahmed, Aziz; Rahman, Hasna Hena; Moin, Mamun Ibne; Ahmed, Salahuddin; Jehan, Fyezah; Nisar, Imran; Hussain, Atiya; Ilyas, Muhammad; Hotwani, Aneeta; Sajid, Muhammad; Qureshi, Shahida; Zaidi, Anita; Sazawal, Sunil; Ali, Said M; Deb, Saikat; Juma, Mohammed Hamad; Dhingra, Usha; Dutta, Arup; Ame, Shaali Makame; Hayward, Caroline; Rudan, Igor; Zangenberg, Mike; Russell, Donna; Yoshida, Sachiyo; Polašek, Ozren; Manu, Alexander; Bahl, Rajiv

2017-01-01

Objectives The AMANHI study aims to seek for biomarkers as predictors of important pregnancy–related outcomes, and establish a biobank in developing countries for future research as new methods and technologies become available. Methods AMANHI is using harmonised protocols to enrol 3000 women in early pregnancies (8–19 weeks of gestation) for population–based follow–up in pregnancy up to 42 days postpartum in Bangladesh, Pakistan and Tanzania, with collection taking place between August 2014 and June 2016. Urine pregnancy tests will be used to confirm reported or suspected pregnancies for screening ultrasound by trained sonographers to accurately date the pregnancy. Trained study field workers will collect very detailed phenotypic and epidemiological data from the pregnant woman and her family at scheduled home visits during pregnancy (enrolment, 24–28 weeks, 32–36 weeks & 38+ weeks) and postpartum (days 0–6 or 42–60). Trained phlebotomists will collect maternal and umbilical blood samples, centrifuge and obtain aliquots of serum, plasma and the buffy coat for storage. They will also measure HbA1C and collect a dried spot sample of whole blood. Maternal urine samples will also be collected and stored, alongside placenta, umbilical cord tissue and membrane samples, which will both be frozen and prepared for histology examination. Maternal and newborn stool (for microbiota) as well as paternal and newborn saliva samples (for DNA extraction) will also be collected. All samples will be stored at –80°C in the biobank in each of the three sites. These samples will be linked to numerous epidemiological and phenotypic data with unique study identification numbers. Importance of the study AMANHI biobank proves that biobanking is feasible to implement in LMICs, but recognises that biobank creation is only the first step in addressing current global challenges. PMID:29163938

An improved in-house lysis-filtration protocol for bacterial identification from positive blood culture bottles with high identification rates by MALDI-TOF MS.

PubMed

Tsuchida, Sachio; Murata, Syota; Miyabe, Akiko; Satoh, Mamoru; Takiwaki, Masaki; Matsushita, Kazuyuki; Nomura, Fumio

2018-05-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is now a well-established method for identification of microorganisms from positive blood cultures. Pretreatments to effectively remove non-bacterial proteins are a prerequisite for successful identification, and a variety of protocols have been reported. Although commercially available kits, mainly the Sepsityper Kit, are increasingly used, the identification rates reported often are not satisfactory, particularly for Gram-positive isolates. We developed a new, in-house lysis-filtration protocol and prospectively evaluated its performance compared to the Sepsityper kit. The in-house protocol consists of three simple steps: lysis by ammonium chloride, aspiration with a syringe fitted with a 0.45-μm membrane, and centrifugation to collect microbes. The novel protocol requires only 20 min. Performance of the in-house protocol was evaluated using a total of 117 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the in-house protocol or the commercial kit, and isolated cells were subjected to direct identification by mass spectrometry fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with score > 1.7 and > 2.0 obtained using the in-house protocol were 99.2% and 85.5%, respectively, whereas those obtained using the Sepsityper Kit were 85.4% and 61.5%, respectively. For Gram-positive cases, the in-house protocol yielded scores >1.7 and > 2.0 at 98.5% and 76.1%, respectively, whereas the commercial kit yielded these scores at 76.1% and 43.3%, respectively. Although these are preliminary results, these values suggest that this easy lysis-filtration protocol deserves assessment in a larger-scale test. Copyright © 2018 Elsevier B.V. All rights reserved.

Determination of proflavine in rat whole blood without sample pretreatment by laser desorption postionization mass spectrometry.

PubMed

Chen, Jiaxin; Hu, Yongjun; Lu, Qiao; Wang, Pengchao; Zhan, Huaqi

2017-04-01

A novel pretreatment-free method involving laser desorption postionization (LDPI) coupled with time-of-flight mass spectrometry (MS) was developed for the monitoring of proflavine level in rat whole blood. It comprises a protocol for dosing via intravenous administration and collection of whole blood, followed by direct LDPI-MS analysis without any sample pretreatment. An intense ion signal at m/z 209 was observed from whole blood without any interference signals, except some background signals below m/z 100. The calibration curve was established with use of 9-phenylacridine as the internal standard for proflavine determination from the plotting of the peak ratios of proflavine to the internal standard, with a correlation coefficient (R 2 ) greater than 0.99. The limit of detection was estimated to be 0.48 pmol/mm 2 and the quantification range was 0.5-16.5 μg/mL for proflavine. In addition, only a minimal matrix effect was observed, as expected from considerations of the desorption and ionization mechanism. Interday and intraday accuracy and precision were calculated to be within 13% and 82-114%, respectively. Estimated concentrations of proflavine residue in whole blood were also successfully obtained at selected time points after dosing. The proposed method is simple, low cost, and sensitive, and should be seen as a complementary method for monitoring drug levels in blood. Graphical Abstract Monitoring proflavine levels in rat whole blood at different time points using laser desorption postionization mass spectrometry (LDPI-MS).

HEART RATE AND INDIRECT BLOOD PRESSURE RESPONSES TO FOUR DIFFERENT FIELD ANESTHETIC PROTOCOLS IN WILD-BORN CAPTIVE CHIMPANZEES (PAN TROGLODYTES).

PubMed

Atencia, Rebeca; Stöhr, Eric J; Drane, Aimee L; Stembridge, Mike; Howatson, Glyn; Del Rio, Pablo Rodriguez Lopez; Feltrer, Yedra; Tafon, Babila; Redrobe, Sharon; Peck, Bruce; Eng, Jaclyn; Unwin, Steve; Sanchez, Carlos R; Shave, Rob E

2017-09-01

Limited data are available on hemodynamic responses to anesthetic protocols in wild-born chimpanzees (Pan troglodytes). Accordingly, this study characterized the heart rate (HR) and blood pressure responses to four anesthetic protocols in 176 clinically healthy, wild-born chimpanzees undergoing routine health assessments. Animals were anesthetized with medetomidine-ketamine (MK) (n = 101), tiletamine-zolazepam (TZ) (n = 30), tiletamine-zolazepam-medetomidine (TZM) (n = 24), or medetomidine-ketamine (maintained with isoflurane) (MKI) (n = 21). During each procedure, HR, systolic blood pressure (SBP), and diastolic blood pressure (DBP) were regularly recorded. Data were grouped according to anesthetic protocol, and mean HR, SBP, and DBP were calculated. Differences between mean HR, SBP, and DBP for each anesthetic protocol were assessed using the Kruskall-Wallis test and a Dunn multiple comparisons post hoc analysis. To assess the hemodynamic time course response to each anesthetic protocol, group mean data (±95% confidence interval [CI]) were plotted against time postanesthetic induction. Mean HR (beats/min [CI]) was significantly higher in TZ (86 [80-92]) compared to MKI (69 [61-78]) and MK (62 [60-64]) and in TZM (73 [68-78]) compared to MK. The average SBP and DBP values (mm Hg [CI]) were significantly higher in MK (130 [126-134] and 94 [91-97]) compared to TZ (104 [96-112] and 58 [53-93]) and MKI (113 [103-123] and 78 [69-87]) and in TZM (128 [120-135] and 88 [83-93]) compared to TZ. Time course data were markedly different between protocols, with MKI showing the greatest decline over time. Both the anesthetic protocol adopted and the timing of measurement after injection influence hemodynamic recordings in wild-born chimpanzees and need to be considered when monitoring or assessing cardiovascular health.

External Quality Control for Dried Blood Spot Based C-reactive Protein Assay: Experience from the Indonesia Family Life Survey and the Longitudinal Aging Study in India

PubMed Central

Hu, Peifeng; Herningtyas, Elizabeth H.; Kale, Varsha; Crimmins, Eileen M.; Risbud, Arun R.; McCreath, Heather; Lee, Jinkook; Strauss, John; O’Brien, Jennifer C.; Bloom, David E.; Seeman, Teresa E.

2015-01-01

Measurement of C-reactive protein, a marker of inflammation, in dried blood spots has been increasingly incorporated in community-based social surveys internationally. Although the dried blood spot based CRP assay protocol has been validated in the United States, it remains unclear whether laboratories in other less developed countries can generate C-reactive protein results of similar quality. We therefore conducted external quality monitoring for dried blood spot based C-reactive protein measurement for the Indonesia Family Life Survey and the Longitudinal Aging Study in India. Our results show that dried blood spot based C-reactive protein results in these two countries have excellent and consistent correlations with serum-based values and dried blood spot based results from the reference laboratory in the United States. Even though the results from duplicate samples may have fluctuations in absolute values over time, the relative order of C-reactive protein levels remains similar and the estimates are reasonably precise for population-based studies that investigate the association between socioeconomic factors and health. PMID:25879265

Comparative evaluation of PCR amplification of RLEP, 16S rRNA, rpoT and Sod A gene targets for detection of M. leprae DNA from clinical and environmental samples.

PubMed

Turankar, Ravindra P; Pandey, Shradha; Lavania, Mallika; Singh, Itu; Nigam, Astha; Darlong, Joydeepa; Darlong, Fam; Sengupta, Utpal

2015-03-01

PCR assay is a highly sensitive, specific and reliable diagnostic tool for the identification of pathogens in many infectious diseases. Genome sequencing Mycobacterium leprae revealed several gene targets that could be used for the detection of DNA from clinical and environmental samples. The PCR sensitivity of particular gene targets for specific clinical and environmental isolates has not yet been established. The present study was conducted to compare the sensitivity of RLEP, rpoT, Sod A and 16S rRNA gene targets in the detection of M. leprae in slit skin smear (SSS), blood, soil samples of leprosy patients and their surroundings. Leprosy patients were classified into Paucibacillary (PB) and Multibacillary (MB) types. Ziehl-Neelsen (ZN) staining method for all the SSS samples and Bacteriological Index (BI) was calculated for all patients. Standard laboratory protocol was used for DNA extraction from SSS, blood and soil samples. PCR technique was performed for the detection of M. leprae DNA from all the above-mentioned samples. RLEP gene target was able to detect the presence of M. leprae in 83% of SSS, 100% of blood samples and in 36% of soil samples and was noted to be the best out of all other gene targets (rpoT, Sod A and 16S rRNA). It was noted that the RLEP gene target was able to detect the highest number (53%) of BI-negative leprosy patients amongst all the gene targets used in this study. Amongst all the gene targets used in this study, PCR positivity using RLEP gene target was the highest in all the clinical and environmental samples. Further, the RLEP gene target was able to detect 53% of blood samples as positive in BI-negative leprosy cases indicating its future standardization and use for diagnostic purposes. Copyright © 2015 Asian African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

Rapid screening method for male DNA by using the loop-mediated isothermal amplification assay.

PubMed

Kitamura, Masashi; Kubo, Seiji; Tanaka, Jin; Adachi, Tatsushi

2017-08-12

Screening for male-derived biological material from collected samples plays an important role in criminal investigations, especially those involving sexual assaults. We have developed a loop-mediated isothermal amplification (LAMP) assay targeting multi-repeat sequences of the Y chromosome for detecting male DNA. Successful amplification occurred with 0.5 ng of male DNA under isothermal conditions of 61 to 67 °C, but no amplification occurred with up to 10 ng of female DNA. Under the optimized conditions, the LAMP reaction initiated amplification within 10 min and amplified for 20 min. The LAMP reaction was sensitive at levels as low as 1-pg male DNA, and a quantitative LAMP assay could be developed because of the strong correlation between the reaction time and the amount of template DNA in the range of 10 pg to 10 ng. Furthermore, to apply the LAMP assay to on-site screening for male-derived samples, we evaluated a protocol using a simple DNA extraction method and a colorimetric intercalating dye that allows detection of the LAMP reaction by evaluating the change in color of the solution. Using this protocol, samples of male-derived blood and saliva stains were processed in approximately 30 min from DNA extraction to detection. Because our protocol does not require much hands-on time or special equipment, this LAMP assay promises to become a rapid and simple screening method for male-derived samples in forensic investigations.

Platelet-Rich Plasma and Adipose-Derived Mesenchymal Stem Cells for Regenerative Medicine-Associated Treatments in Bottlenose Dolphins (Tursiops truncatus)

PubMed Central

Griffeth, Richard J.; García-Párraga, Daniel; Mellado-López, Maravillas; Crespo-Picazo, Jose Luis; Soriano-Navarro, Mario; Martinez-Romero, Alicia; Moreno-Manzano, Victoria

2014-01-01

Dolphins exhibit an extraordinary capacity to heal deep soft tissue injuries. Nevertheless, accelerated wound healing in wild or captive dolphins would minimize infection and other side effects associated with open wounds in marine animals. Here, we propose the use of a biological-based therapy for wound healing in dolphins by the application of platelet-rich plasma (PRP). Blood samples were collected from 9 different dolphins and a specific and simple protocol which concentrates platelets greater than two times that of whole blood was developed. As opposed to a commonly employed human protocol for PRP preparation, a single centrifugation for 3 minutes at 900 rpm resulted in the best condition for the concentration of dolphin platelets. By FACS analysis, dolphin platelets showed reactivity to platelet cell-surface marker CD41. Analysis by electron microscopy revealed that dolphin platelets were larger in size than human platelets. These findings may explain the need to reduce the duration and speed of centrifugation of whole blood from dolphins to obtain a 2-fold increase and maintain proper morphology of the platelets. For the first time, levels of several growth factors from activated dolphin platelets were quantified. Compared to humans, concentrations of PDGF-BB were not different, while TGFβ and VEGF-A were significantly lower in dolphins. Additionally, adipose tissue was obtained from cadaveric dolphins found along the Spanish Mediterranean coast, and adipose-derived mesenchymal stem cells (ASCs) were successfully isolated, amplified, and characterized. When dolphin ASCs were treated with 2.5 or 5% dolphin PRP they exhibited significant increased proliferation and improved phagocytotic activity, indicating that in culture, PRP may improve the regenerative capacity of ASCs. Taken together, we show an effective and well-defined protocol for efficient PRP isolation. This protocol alone or in combination with ASCs, may constitute the basis of a biological treatment for wound-healing and tissue regeneration in dolphins. PMID:25251412

Platelet-rich plasma and adipose-derived mesenchymal stem cells for regenerative medicine-associated treatments in bottlenose dolphins (Tursiops truncatus).

PubMed

Griffeth, Richard J; García-Párraga, Daniel; Mellado-López, Maravillas; Crespo-Picazo, Jose Luis; Soriano-Navarro, Mario; Martinez-Romero, Alicia; Moreno-Manzano, Victoria

2014-01-01

Dolphins exhibit an extraordinary capacity to heal deep soft tissue injuries. Nevertheless, accelerated wound healing in wild or captive dolphins would minimize infection and other side effects associated with open wounds in marine animals. Here, we propose the use of a biological-based therapy for wound healing in dolphins by the application of platelet-rich plasma (PRP). Blood samples were collected from 9 different dolphins and a specific and simple protocol which concentrates platelets greater than two times that of whole blood was developed. As opposed to a commonly employed human protocol for PRP preparation, a single centrifugation for 3 minutes at 900 rpm resulted in the best condition for the concentration of dolphin platelets. By FACS analysis, dolphin platelets showed reactivity to platelet cell-surface marker CD41. Analysis by electron microscopy revealed that dolphin platelets were larger in size than human platelets. These findings may explain the need to reduce the duration and speed of centrifugation of whole blood from dolphins to obtain a 2-fold increase and maintain proper morphology of the platelets. For the first time, levels of several growth factors from activated dolphin platelets were quantified. Compared to humans, concentrations of PDGF-BB were not different, while TGFβ and VEGF-A were significantly lower in dolphins. Additionally, adipose tissue was obtained from cadaveric dolphins found along the Spanish Mediterranean coast, and adipose-derived mesenchymal stem cells (ASCs) were successfully isolated, amplified, and characterized. When dolphin ASCs were treated with 2.5 or 5% dolphin PRP they exhibited significant increased proliferation and improved phagocytotic activity, indicating that in culture, PRP may improve the regenerative capacity of ASCs. Taken together, we show an effective and well-defined protocol for efficient PRP isolation. This protocol alone or in combination with ASCs, may constitute the basis of a biological treatment for wound-healing and tissue regeneration in dolphins.

Do clinical guidelines improve management of sepsis in critically ill elderly patients? A before-and-after study of the implementation of a sepsis protocol.

PubMed

Heppner, Hans Juergen; Singler, Katrin; Kwetkat, Anja; Popp, Steffen; Esslinger, Adelheid Susanne; Bahrmann, Philipp; Kaiser, Matthias; Bertsch, Thomas; Sieber, Cornel Christian; Christ, Michael

2012-10-01

Guidelines for the management of sepsis have been published but not validated for elderly patients, though a prompt work-up and initiation of appropriate therapy are crucial. This study assesses the impact of a sepsis protocol on timelines for therapy and mortality in standardized management. Consecutive patients aged 70 years and older who were diagnosed with sepsis and admitted during the observation periods were included in this before-and-after study at a medical intensive care unit (ICU). Age, sex, and process-of-care variables including timely administration of antibiotics, obtaining blood cultures before the start of antibiotics, documenting central venous pressure, evaluation of central venous blood oxygen saturation, fluid resuscitation, and patient outcome were recorded. A total of 122 patients were included. Sepsis was diagnosed in 22.9 % of patients prior to the introduction of the protocol and 57.4 % after introduction. Volume therapy was conducted in 63.9 % of the patients (11.5 % preprotocol). Blood culture samples were taken prior to the administration of antibiotics in 67.2 % of patients (4.9 % preprotocol), and antibiotics were applied early in 72.1 % of patients (32.8 % preprotocol). Lactate was set in 77.0 % of patients (11.5 % preprotocol). A central venous catheter was inserted in 88.5 % of patients (68.9 % preprotocol), and the target central venous pressure was achieved in 64.3 % of patients (47.2 % preprotocol). ICU mortality was reduced by 5.2 % and hospital mortality by 6.4 %. The use of standardized order sets for the management of sepsis in elderly patients should be strongly recommended for better performance in treatment. Compliance with the protocol was associated with reduced length of stay, reduced mortality, and improved initial appropriate therapy.

Development of new method and protocol for cryopreservation related to embryo and oocytes freezing in terms of fertilization rate: A comparative study including review of literature

PubMed Central

Barik, Mayadhar; Bajpai, Minu; Patnaik, Santosh; Mishra, Pravash; Behera, Priyamadhaba; Dwivedi, Sada Nanda

2016-01-01

Background: Cryopreservation is basically related to meritorious thin samples or small clumps of cells that are cooled quickly without loss. Our main objective is to establish and formulate an innovative method and protocol development for cryopreservation as a gold standard for clinical uses in laboratory practice and treatment. The knowledge regarding usefulness of cryopreservation in clinical practice is essential to carry forward the clinical practice and research. Materials and Methods: We are trying to compare different methods of cryopreservation (in two dozen of cells) at the same time we compare the embryo and oocyte freezing interms of fertilization rate according to the International standard protocol. Results: The combination of cryoprotectants and regimes of rapid cooling and rinsing during warming often allows successful cryopreservation of biological materials, particularly cell suspensions or thin tissue samples. Examples include semen, blood, tissue samples like tumors, histological cross-sections, human eggs and human embryos. Although presently many studies have reported that the children born from frozen embryos or “frosties,” show consistently positive results with no increase in birth defects or development abnormalities is quite good enough and similar to our study (50–85%). Conclusions: We ensure that cryopreservation technology provided useful cell survivability, tissue and organ preservation in a proper way. Although it varies according to different laboratory conditions, it is certainly beneficial for patient's treatment and research. Further studies are needed for standardization and development of new protocol. PMID:27512686

Rationale, design and methodology for the Navajo Health and Nutrition Survey.

PubMed

White, L L; Goldberg, H I; Gilbert, T J; Ballew, C; Mendlein, J M; Peter, D G; Percy, C A; Mokdad, A H

1997-10-01

As recently as 1990, there was no reservation-wide, population-based health status information about Navajo Indians. To remedy this shortcoming, the Navajo Health and Nutrition Survey was conducted from 1991 to 1992 to assess the health and nutritional status of Navajo Reservation residents using a population-based sample. Using a three-stage design, a representative sample of reservation households was selected for inclusion. All members of selected households 12 y of age and older were invited to participate. A total of 985 people in 459 households participated in the study. Survey protocols were modeled on those of previous national surveys and included a standard blood chemistry profile, complete blood count, oral glucose tolerance test, blood pressure, anthropometric measurements, a single 24-h dietary recall and a questionnaire on health behaviors. The findings from this survey, reported in the accompanying papers, inform efforts to prevent and control chronic disease among the Navajo. Lessons learned from this survey may be of interest to those conducting similar surveys in other American Indian and Alaska Native populations.

Novel, high-yield red blood cell production methods from CD34-positive cells derived from human embryonic stem, yolk sac, fetal liver, cord blood, and peripheral blood.

PubMed

Olivier, Emmanuel; Qiu, Caihong; Bouhassira, Eric E

2012-08-01

The current supply of red blood cells expressing rare blood groups is not sufficient to cover all the existing transfusion needs for chronically transfused patients, such as sickle cell disease homozygous carriers, because of alloimmunization. In vitro production of cultured red blood cells is slowly emerging as a possible complement to the existing collection-based red blood cell procurement system. The yield of cultured red blood cells can theoretically be maximized by amplifying the stem, progenitor, or precursor compartment. Here, we combined methods designed to expand these three compartments to optimize the yield of cultured red blood cells and found that exposing CD34(+) cells to a short pulse of cytokines favorable for erythroid differentiation prior to stem cell expansion followed by progenitor expansion produced the highest yield of erythroid cells. This novel serum-free red blood cell production protocol was efficient on CD34(+) cells derived from human embryonic stem cells, 6-8-week yolk sacs, 16-18-week fetal livers, cord blood, and peripheral blood. The yields of cells obtained with these new protocols were larger by an order of magnitude than the yields observed previously. Globin expression analysis by high-performance liquid chromatography revealed that these expansion protocols generally yielded red blood cells that expressed a globin profile similar to that expected for the developmental age of the CD34(+) cells.

Accuracy of LightCycler® SeptiFast for the detection and identification of pathogens in the blood of patients with suspected sepsis: a systematic review protocol

PubMed Central

Wilson, Claire; Blackwood, Bronagh; McAuley, Danny F; Perkins, Gavin D; McMullan, Ronan; Gates, Simon; Warhurst, Geoffrey

2012-01-01

Background There is growing interest in the potential utility of molecular diagnostics in improving the detection of life-threatening infection (sepsis). LightCycler® SeptiFast is a multipathogen probe-based real-time PCR system targeting DNA sequences of bacteria and fungi present in blood samples within a few hours. We report here the protocol of the first systematic review of published clinical diagnostic accuracy studies of this technology when compared with blood culture in the setting of suspected sepsis. Methods/design Data sources: the Cochrane Database of Systematic Reviews, the Database of Abstracts of Reviews of Effects (DARE), the Health Technology Assessment Database (HTA), the NHS Economic Evaluation Database (NHSEED), The Cochrane Library, MEDLINE, EMBASE, ISI Web of Science, BIOSIS Previews, MEDION and the Aggressive Research Intelligence Facility Database (ARIF). Study selection: diagnostic accuracy studies that compare the real-time PCR technology with standard culture results performed on a patient's blood sample during the management of sepsis. Data extraction: three reviewers, working independently, will determine the level of evidence, methodological quality and a standard data set relating to demographics and diagnostic accuracy metrics for each study. Statistical analysis/data synthesis: heterogeneity of studies will be investigated using a coupled forest plot of sensitivity and specificity and a scatter plot in Receiver Operator Characteristic (ROC) space. Bivariate model method will be used to estimate summary sensitivity and specificity. The authors will investigate reporting biases using funnel plots based on effective sample size and regression tests of asymmetry. Subgroup analyses are planned for adults, children and infection setting (hospital vs community) if sufficient data are uncovered. Dissemination Recommendations will be made to the Department of Health (as part of an open-access HTA report) as to whether the real-time PCR technology has sufficient clinical diagnostic accuracy potential to move forward to efficacy testing during the provision of routine clinical care. Registration PROSPERO—NIHR Prospective Register of Systematic Reviews (CRD42011001289). PMID:22240646

Validation of the Artsana CSI 610 automated blood pressure monitor in adults according to the International Protocol of the European Society of Hypertension.

PubMed

Pini, Claudio; Pastori, Marco; Baccheschi, Jordan; Omboni, Stefano; Parati, Gianfranco

2007-06-01

There is evidence that blood pressure measurement outside the doctor's office can provide valuable information for the diagnostic evaluation of hypertensive patients and for monitoring their response to treatment. Home blood pressure monitoring devices have a major role in this setting, provided that their accuracy in measuring blood pressure is demonstrated by validation studies. This study aimed at verifying whether the automatic electronic oscillometric blood pressure measuring device Artsana CSI 610 complied with the standard of accuracy indicated by the ESH International Protocol. Sequential measurements of systolic and diastolic blood pressure were obtained in 33 participants using the mercury sphygmomanometer (two observers) and the test device (one supervisor). A standard adult cuff was always employed during the study. According to the ESH validation protocol, 99 couples of test device and reference blood pressure measurements were obtained during the two phases of the study (three pairs for each of the 33 participants). The Artsana CSI 610 device successfully passed phase 1 of study validation with the number of absolute differences between test and reference device never <35 within 5 mmHg and never <40 within 10 and 15 mmHg. The test device also passed phase 2 of the validation study with a mean (+/-SD) device-observer difference of -1.4+/-4.8 mmHg for systolic and -0.9+/-3.5 mmHg for diastolic blood pressure. According to the results of the validation study on the basis of the ESH International Protocol, the Artsana CSI 610 can be recommended for clinical use in adults.

Evaluation of two different protocols for peripheral blood stem cell collection with the Fresenius AS 104 blood cell separator.

PubMed

Menichella, G; Lai, M; Pierelli, L; Vittori, M; Serafini, R; Ciarli, M; Foddai, M L; Salerno, G; Sica, S; Scambia, G; Leone, G; Bizzi, B

1997-01-01

Reconstitution of hematopoiesis by means of peripheral blood stem cells is a valid alternative to autologous bone marrow transplantation. The aim of this investigation was to increase the efficiency of collection of circulating blood progenitor cells and to obtain a purer product for transplant. We carried out leukapheresis procedures with the Fresenius AS 104 blood cell separator, using two different protocols, the previously used PBSC-LYM and a new mononuclear cell collection program. Both programs were highly effective in collecting mononuclear cells (MNC) and CD34+ cells. Some differences were found, especially regarding MNC yield and efficiencies. There are remarkable differences in the efficiency of collection of CD34+ cells (62.38% with the new program as opposed to 31.69% with the older one). Linear regression analysis showed a negative correlation between blood volume processed and MNC efficiency only for the PBSC-LYM program. Differences were also observed in the degree of inverse correlation existing in both programs between patients' white blood cell precount and MNC collection efficiency. The inverse correlation was stronger for the PBSC-LYM program. Seven patients with solid tumors and hematologic malignancies received high dose chemotherapy and were subsequently transplanted with peripheral blood stem cells collected using the new protocol. All patients obtained a complete and stable engraftment with the reinfusion product collected with one or two leukapheresis procedures. High efficiencies and yields were observed in the new protocol for MNC and CD34+ cells. These were able to effect rapid and complete bone marrow recovery after myeloablative chemotherapy.

Who's really hypertensive?--Quality control issues in the assessment of blood pressure for randomized trials.

PubMed

Reid, Christopher M; Ryan, Philip; Miles, Helen; Willson, Kristyn; Beilin, Laurence J; Brown, Mark A; Jennings, Garry L; Johnston, Colin I; Macdonald, Graham J; Marley, John E; McNeil, John J; Morgan, Trefor O; West, Malcolm J; Wing, Lindon M H

2005-01-01

The characterization of blood pressure in treatment trials assessing the benefits of blood pressure lowering regimens is a critical factor for the appropriate interpretation of study results. With numerous operators involved in the measurement of blood pressure in many thousands of patients being screened for entry into clinical trials, it is essential that operators follow pre-defined measurement protocols involving multiple measurements and standardized techniques. Blood pressure measurement protocols have been developed by international societies and emphasize the importance of appropriate choice of cuff size, identification of Korotkoff sounds, and digit preference. Training of operators and auditing of blood pressure measurement may assist in reducing the operator-related errors in measurement. This paper describes the quality control activities adopted for the screening stage of the 2nd Australian National Blood Pressure Study (ANBP2). ANBP2 is cardiovascular outcome trial of the treatment of hypertension in the elderly that was conducted entirely in general practices in Australia. A total of 54 288 subjects were screened; 3688 previously untreated subjects were identified as having blood pressure >140/90 mmHg at the initial screening visit, 898 (24%) were not eligible for study entry after two further visits due to the elevated reading not being sustained. For both systolic and diastolic blood pressure recording, observed digit preference fell within 7 percentage points of the expected frequency. Protocol adherence, in terms of the required minimum blood pressure difference between the last two successive recordings, was 99.8%. These data suggest that adherence to blood pressure recording protocols and elimination of digit preferences can be achieved through appropriate training programs and quality control activities in large multi-centre community-based trials in general practice. Repeated blood pressure measurement prior to initial diagnosis and study entry is essential to appropriately characterize hypertension in these elderly patients.

Regularly scheduled, day-time, slow-onset 60 Hz electric and magnetic field exposure does not depress serum melatonin concentration in nonhuman primates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rogers, W.R.; Smith, H.D.; Orr, J.L.

Experiments conducted with laboratory rodents indicate that exposure to 60 Hz electric fields or magnetic fields can suppress nocturnal melatonin concentrations in pineal gland and blood. In three experiments employing three field-exposed and three sham-exposed nonhuman primates, each implanted with an indwelling venous cannula to allow repeated blood sampling, the authors studied the effects of either 6 kV/m and 50 {micro}T (0.5 G) or 30 kV/m and 100 {micro}T (1.0 G) on serum melatonin patterns. The fields were ramped on and off slowly, so that no transients occurred. Extensive quality control for the melatonin assay, computerized control and monitoring ofmore » field intensities, and consistent exposure protocols were used. No changes in nocturnal serum melatonin concentration resulted from 6 weeks of day-time exposure with slow field onset/offset and a highly regular exposure protocol. These results indicate that, under the conditions tested, day-time exposure to 60 Hz electric and magnetic fields in combination does not result in melatonin suppression in primates.« less

Effect of two complex training protocols of back squats in blood indicators of muscular damage in military athletes

PubMed Central

Ojeda, Álvaro Huerta; Ríos, Luis Chirosa; Barrilao, Rafael Guisado; Ríos, Ignacio Chirosa; Serrano, Pablo Cáceres

2016-01-01

[Purpose] The aim of this study was to determine the variations in the blood muscular damage indicators post application of two complex training programs for back squats. [Subjects and Methods] Seven military athletes were the subjects of this study. The study had a quasi-experimental cross-over intra-subject design. Two complex training protocols were applied, and the variables to be measured were cortisol, metabolic creatine kinase, and total creatine kinase. For the statistical analysis, Student’s t-test was used. [Results] Twenty-four hours post effort, a significant decrease in cortisol level was shown for both protocols; however, the metabolic creatine kinase and total creatine kinase levels showed a significant increase. [Conclusion] Both protocols lowered the indicator of main muscular damage in the blood supply (cortisol). This proved that the work weight did not generate significant muscular damage in the 24-hour post-exercise period. PMID:27313356

Effect of two complex training protocols of back squats in blood indicators of muscular damage in military athletes.

PubMed

Ojeda, Álvaro Huerta; Ríos, Luis Chirosa; Barrilao, Rafael Guisado; Ríos, Ignacio Chirosa; Serrano, Pablo Cáceres

2016-05-01

[Purpose] The aim of this study was to determine the variations in the blood muscular damage indicators post application of two complex training programs for back squats. [Subjects and Methods] Seven military athletes were the subjects of this study. The study had a quasi-experimental cross-over intra-subject design. Two complex training protocols were applied, and the variables to be measured were cortisol, metabolic creatine kinase, and total creatine kinase. For the statistical analysis, Student's t-test was used. [Results] Twenty-four hours post effort, a significant decrease in cortisol level was shown for both protocols; however, the metabolic creatine kinase and total creatine kinase levels showed a significant increase. [Conclusion] Both protocols lowered the indicator of main muscular damage in the blood supply (cortisol). This proved that the work weight did not generate significant muscular damage in the 24-hour post-exercise period.

Validation of the Andon KD-5917 automatic upper arm blood pressure monitor, for clinic use and self-measurement, according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Guo, Wan-Gang; Li, Bing-Ling; He, Yong; Xue, Yu-Sheng; Wang, Hai-Yan; Zheng, Qiang-Sun; Xiang, Ding-Cheng

2014-08-01

To validate the Andon KD-5917 automatic upper arm blood pressure monitor according to the European Society of Hypertension International Protocol revision 2010. Sequential same-left-arm measurements of systolic blood pressure (SBP) and diastolic blood pressure (DBP) were obtained in 33 participants using the mercury sphygmomanometer and the test device. According to the validation protocol, 99 pairs of test device and reference blood pressure measurements (three pairs for each of the 33 participants) were obtained in the study. The device produced 73, 98, and 99 measurements within 5, 10, and 15 mmHg for SBP and 86, 98, and 99 for DBP, respectively. The mean ± SD device-observer difference was 3.07 ± 3.68 mmHg for SBP and -0.89 ± 3.72 mmHg for DBP. The number of patients with two or three of the device-observer difference within 5 mmHg was 26 for SBP and 29 for DBP, and no patient had a device-observer difference within 5 mmHg. The Andon KD-5917 automatic upper arm blood pressure monitor can be recommended for clinical use and self-measurement in an adult population on the basis of the European Society of Hypertension International Protocol revision 2010.

Blood flow dynamics in heart failure

NASA Technical Reports Server (NTRS)

Shoemaker, J. K.; Naylor, H. L.; Hogeman, C. S.; Sinoway, L. I.

1999-01-01

BACKGROUND: Exercise intolerance in heart failure (HF) may be due to inadequate vasodilation, augmented vasoconstriction, and/or altered muscle metabolic responses that lead to fatigue. METHODS AND RESULTS: Vascular and metabolic responses to rhythmic forearm exercise were tested in 9 HF patients and 9 control subjects (CTL) during 2 protocols designed to examine the effect of HF on the time course of oxygen delivery versus uptake (protocol 1) and on vasoconstriction during exercise with 50 mm Hg pressure about the forearm to evoke a metaboreflex (protocol 2). In protocol 1, venous lactate and H+ were greater at 4 minutes of exercise in HF versus CTL (P<0.05) despite similar blood flow and oxygen uptake responses. In protocol 2, mean arterial pressure increased similarly in each group during ischemic exercise. In CTL, forearm blood flow and vascular conductance were similar at the end of ischemic and ambient exercise. In HF, forearm blood flow and vascular conductance were reduced during ischemic exercise compared with the ambient trial. CONCLUSIONS: Intrinsic differences in skeletal muscle metabolism, not vasodilatory dynamics, must account for the augmented glycolytic metabolic responses to moderate-intensity exercise in class II and III HF. The inability to increase forearm vascular conductance during ischemic handgrip exercise, despite a normal pressor response, suggests that enhanced vasoconstriction of strenuously exercising skeletal muscle contributes to exertional fatigue in HF.

Validation of Transtek blood pressure monitor TMB-1491 for self-measurement according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Tian, Huiyong; Zeng, Sijian; Zhong, Xiaoyan; Gong, Wei; Liu, Wenjun

2015-10-01

Transtek blood pressure monitor TMB-1491 is an automatic upper arm device designed for self/home measurement in adult populations. This study aimed to evaluate its accuracy according to the European Society of Hypertension International Protocol revision 2010. The protocol requirements were followed precisely with the recruitment of 33 adult individuals on whom same-left-arm sequential systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured. According to the validation protocol, 99 pairs of test device and reference blood pressure measurements were obtained in this study (three pairs for each of the 33 participants). The device produced 74, 95 and 99 measurements within 5, 10, and 15 mmHg for SBP and 85, 97, and 99 for DBP, respectively. The mean±SD device-observer difference was -0.6±4.4 mmHg for SBP and -0.6±3.4 mmHg for DBP. The number of participants with two or three device-observer difference within 5 mmHg was 24 for SBP and 29 for DBP. In addition, none of the participants had a device-observer difference within 5 mmHg for SBP, and three of the participants had the same for DBP. Transtek TMB-1491 has passed all phases of European Society of Hypertension International Protocol revision 2010 and can be recommended for self/home measurement in adult populations.

Application of an industrial robot to nuclear pharmacy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Viola, J.

1994-12-31

Increased patient throughput and lengthened P.E.T. scan protocols have increased the radiation dose received by P.E.T. technologists. Automated methods of tracer infusion and blood sampling have been introduced to reduce direct contact with the radioisotopes, but significant radiation exposure still exists during the receipt and dispensing of the patient dose. To address this situation the authors have developed an automated robotic system which performs these tasks, thus limiting the physical contact between operator and radioisotope.

Validation of the SEJOY BP-1307 upper-arm blood pressure monitor for home blood pressure monitoring according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Lei, Lei; Chen, Yi; Chen, Qi; Li, Yan; Wang, Ji-Guang

2017-12-01

The present study aimed to evaluate the accuracy of the automated oscillometric upper-arm blood pressure monitor SEJOY BP-1307 (also called JOYTECH DBP-1307) for home blood pressure monitoring according to the International Protocol of the European Society of Hypertension revision 2010. Systolic and diastolic blood pressures were sequentially measured in 33 adult Chinese individuals (13 women, 45.1 years of mean age) using a mercury sphygmomanometer (two observers) and the SEJOY BP-1307 device (one supervisor). Ninety-nine pairs of comparisons were obtained from 33 participants for judgments in two parts with three grading phases. The average±SD of the device-observer differences was 0.2±4.1 and -1.7±4.7 mmHg for systolic and diastolic blood pressure, respectively. The SEJOY BP-1307 device achieved the criteria in both part 1 and part 2 of the validation study. The SEJOY upper-arm blood pressure monitor BP-1307 has passed the requirements of the International Protocol revision 2010, and hence can be recommended for home use in adults.

Is reducing variability of blood glucose the real but hidden target of intensive insulin therapy?

PubMed

Egi, Moritoki; Bellomo, Rinaldo; Reade, Michael C

2009-01-01

Since the first report that intensive insulin therapy reduced mortality in selected surgical critically ill patients, lowering of blood glucose levels has been recommended as a means of improving patient outcomes. In this initial Leuven trial, blood glucose control by protocol using insulin was applied to 98.7% of patients in the intensive group but to only 39.2% (P < 0.0001) of patients in the control group. If appropriately applied, such protocols should decrease both the mean blood glucose concentration and its variability (variation of blood glucose concentration). Thus, it is logically possible that the benefit of intensive insulin therapy in the first Leuven trial was due to a decrease in mean glucose levels, a decrease in their variability, or both. Several recent studies have confirmed significant associations between variability of blood glucose levels and patient outcomes. Decreasing the variability of blood glucose levels might be an important dimension of glucose management, a possible mechanism by which an intensive insulin protocol exerts its putative beneficial effects, and an important goal of glucose management in the intensive care unit. Clinicians need to be aware of this controversy when considering the application of intensive insulin therapy and interpreting future trials.

The influence of mechanical ventilation on physiological parameters in ball pythons (Python regius).

PubMed

Jakobsen, Sashia L; Williams, Catherine J A; Wang, Tobias; Bertelsen, Mads F

2017-05-01

Mechanical ventilation is widely recommended for reptiles during anesthesia, and while it is well-known that their low ectothermic metabolism requires much lower ventilation than in mammals, very little is known about the influence of ventilation protocol on the recovery from anesthesia. Here, 15 ball pythons (Python regius) were induced and maintained with isoflurane for 60min at one of three ventilation protocols (30, 125, or 250mlmin -1 kg -1 body mass) while an arterial catheter was inserted, and ventilation was then continued on 100% oxygen at the specified rate until voluntary extubation. Mean arterial blood pressure and heart rate (HR) were measured, and arterial blood samples collected at 60, 80, 180min and 12 and 24h after intubation. In all three groups, there was evidence of a metabolic acidosis, and snakes maintained at 30mlmin -1 kg -1 experienced an additional respiratory acidosis, while the two other ventilation protocols resulted in normal or low arterial PCO 2 . In general, normal acid-base status was restored within 12h in all three protocols. HR increased by 143±64% during anesthesia with high mechanical ventilation (250mlmin -1 kg -1 ) in comparison with recovered values. Recovery times after mechanical ventilation at 30, 125, or 250mlmin -1 kg -1 were 289±70, 126±16, and 68±7min, respectively. Mild overventilation may result in a faster recovery, and the associated lowering of arterial PCO 2 normalised arterial pH in the face of metabolic acidosis. Copyright © 2017 Elsevier Inc. All rights reserved.

Standardization of a Protocol for Obtaining Platelet Rich Plasma from blood Donors; a Tool for Tissue Regeneration Procedures.

PubMed

Gómez, Lina Andrea; Escobar, Magally; Peñuela, Oscar

2015-01-01

To develop a protocol for obtaining autologous platelet rich plasma in healthy individuals and to determine the concentration of five major growth factors before platelet activation. This protocol could be integrated into the guidelines of good clinical practice and research in regenerative medicine. Platelet rich plasma was isolated by centrifugation from 38 healthy men and 42 women ranging from 18 to 59 years old. The platelet count and quantification of growth factors were analyzed in eighty samples, stratified for age and gender of the donor. Analyses were performed using parametric the t-test or Pearson's analysis for non-parametric distribution. P < 0.05 was considered statistically significant. Our centrifugation protocol allowed us to concentrate basal platelet counts from 1.6 to 4.9 times (mean = 2.8). There was no correlation between platelet concentration and the level of the following growth factors: VEGF-D (r = 0.009, p = 0.4105), VEGF-A (r = 0.0068, p = 0.953), PDGF subunit AA (p = 0.3618; r = 0.1047), PDGF-BB (p = 0.5936; r = 0.6095). In the same way, there was no correlation between donor gender and growth factor concentrations. Only TGF-β concentration was correlated to platelet concentration (r = 0.3163, p = 0.0175). The procedure used allowed us to make preparations rich in platelets, low in leukocytes and red blood cells, and sterile. Our results showed biological variations in content of growth factors in PRP. The factors influencing these results should be further studied.

Shift in responsibilities in diabetes care: the Nurse-Driven Diabetes In-Hospital Treatment protocol (N-DIABIT).

PubMed

Manders, I G; Stoecklein, K; Lubach, C H C; Bijl-Oeldrich, J; Nanayakkara, P W B; Rauwerda, J A; Kramer, M H H; Eekhoff, E M W

2016-06-01

To investigate the feasibility, safety and efficacy of the Nurse-Driven Diabetes In-Hospital Treatment protocol (N-DIABIT), which consists of nurse-driven correctional therapy, in addition to physician-guided basal therapy, and is carried out by trained ward nurses. Data on 210 patients with diabetes consecutively admitted in the 5-month period after the introduction of N-DIABIT (intervention group) were compared with the retrospectively collected data on 200 consecutive patients with diabetes admitted in the 5-month period before N-DIABIT was introduced (control group). Additional per-protocol analyses were performed in patients in whom mean patient-based protocol adherence was ≥ 70% (intervention subgroup, n = 173 vs. control subgroup, n = 196). There was no difference between the intervention and the control group in mean blood glucose levels (8.9 ± 0.1 and 9.1 ± 0.2 mmol/l, respectively; P = 0.38), consecutive hyperglycaemic (blood glucose ≥ 10.0 mmol/l) episodes; P = 0.15), admission duration (P = 0.79), mean number of blood glucose measurements (P = 0.21) and incidence of severe hypoglycaemia (P = 0.29). Per-protocol analyses showed significant reductions in mean blood glucose levels and consecutive hypoglycaemia and hyperglycaemia in the intervention compared with the control group. Implementation of N-DIABIT by trained ward nurses in non-intensive care unit diabetes care is feasible, safe and non-inferior to physician-driven care alone. High protocol adherence was associated with improved glycaemic control. © 2015 Diabetes UK.

Clinical impact of reducing routine blood culture incubation time from 7 to 5 days.

PubMed

Marginson, Michael J; Daveson, Kathryn L; Kennedy, Karina J

2014-12-01

The aim of this study was to determine the clinical impact of reducing the blood culture incubation protocol from 7 to 5 days.A laboratory data extraction identified positive blood cultures occurring after 5 or more days of incubation at Canberra Hospital, Australia between 1 January 2001 and 31 August 2011. Isolates were identified as clinically significant using a pre-existing prospective bacteraemia database. Medical records review determined whether the positive result affected clinical management.Positive blood cultures after 5 or more days of incubation accounted for 2.65% (423/15979) of all positive blood cultures, although the majority were false positives or contaminants. Eighty-five were significant/indeterminate, representing an average of eight cases per year or 0.47% (85/15979) of all positive blood cultures sets. Forty-three were isolated for the first time, representing 1.1% of all significant/indeterminate blood culture episodes. Fungi and anaerobic bacteria compromised over 50% of isolates. In 26 cases (2.4 cases per year), the culture result led to a change in patient management.A 7 day incubation protocol is preferable due to late isolation of organisms; however, if space is required to accommodate increasing blood culture numbers, reducing to a 5 or 6 day protocol would miss only a small percentage of clinically significant isolates.

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood.

PubMed

Park, Yang-Seok; Sunkara, Vijaya; Kim, Yubin; Lee, Won Seok; Han, Ja-Ryoung; Cho, Yoon-Kyoung

2016-04-16

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

Blood Flow After Exercise-Induced Muscle Damage

PubMed Central

Selkow, Noelle M.; Herman, Daniel C.; Liu, Zhenqi; Hertel, Jay; Hart, Joseph M.; Saliba, Susan A.

2015-01-01

Context: The most common modality used to address acute inflammation is cryotherapy. Whereas pain decreases with cryotherapy, evidence that changes occur in perfusion of skeletal muscle is limited. We do not know whether ice attenuates the increases in perfusion associated with acute inflammation. Objective: To examine the effects of repeated applications of ice bags on perfusion of the gastrocnemius muscle after an eccentric exercise protocol. Design: Controlled laboratory study. Setting: Laboratory. Patients or Other Participants: Eighteen healthy participants (3 men, 15 women; age = 22.2 ± 2.2 years, height = 166.0 ± 11.9 cm, mass = 69.4 ± 25.0 kg). Intervention(s): To induce eccentric muscle damage, participants performed 100 unilateral heel-lowering exercises off a step to the beat of a metronome. A randomized intervention (cryotherapy, sham, control) was applied to the exercised lower extremity immediately after the protocol and again at 10, 24, and 34 hours after the protocol. Main Outcome Measure(s): Baseline perfusion measurements (blood volume, blood flow, and blood flow velocity) were taken using contrast-enhanced ultrasound of the exercised leg. Perfusion was reassessed after the first intervention and 48 hours after the protocol as percentage change scores. Pain was measured with a visual analog scale at baseline and at 10, 24, 34, and 48 hours after the protocol. Separate repeated-measures analyses of variance were used to assess each dependent variable. Results: We found no interactions among interventions for microvascular perfusion. Blood volume and blood flow, however, increased in all conditions at 48 hours after exercise (P < .001), and blood flow velocity decreased postintervention from baseline (P = .041). We found a time-by-intervention interaction for pain (P = .009). Visual analog scale scores were lower for the cryotherapy group than for the control group at 34 and 48 hours after exercise. Conclusions: Whereas eccentric muscle damage resulted in increased blood flow, ice did not decrease muscle perfusion 48 hours after exercise. Therefore, ice does not seem to decrease muscle perfusion when blood flow is elevated, as it would be during inflammation. PMID:25658816

Sample preparation method influences direct identification of anaerobic bacteria from positive blood culture bottles using MALDI-TOF MS.

PubMed

Jeverica, Samo; Nagy, Elisabeth; Mueller-Premru, Manica; Papst, Lea

2018-05-15

Rapid detection and identification of anaerobic bacteria from blood is important to adjust antimicrobial therapy by including antibiotics with activity against anaerobic bacteria. Limited data is available about direct identification of anaerobes from positive blood culture bottles using MALDI-TOF mass spectrometry (MS). In this study, we evaluated the performance of two sample preparation protocols for direct identification of anaerobes from positive blood culture bottles, the MALDI Sepsityper kit (Sepsityper) and the in-house saponin (saponin) method. Additionally, we compared two blood culture bottle types designed to support the growth of anaerobic bacteria, the BacT/ALERT-FN Plus (FN Plus) and the BACTEC-Lytic (Lytic), and their influence on direct identification. A selection of 30 anaerobe strains belonging to 22 different anaerobic species (11 reference strains and 19 clinical isolates) were inoculated to 2 blood culture bottle types in duplicate. In total, 120 bottles were inoculated and 99.2% (n = 119) signalled growth within 5 days of incubation. The Sepsityper method correctly identified 56.3% (n = 67) of anaerobes, while the saponin method correctly identified 84.9% (n = 101) of anaerobes with at least log(score) ≥1.6 (low confidence correct identification), (p < 0.001). Gram negative anaerobes were better identified with the saponin method (100% vs. 46.5%; p < 0.001), while Gram positive anaerobes were better identified with the Sepsityper method (70.8% vs. 62.5%; p = 0.454). Average log(score) values among only those isolates that were correctly identified simultaneously by both sample preparation methods were 2.119 and 2.029 in favour of the Sepsityper method, (p = 0.019). The inoculated bottle type didn't influence the performance of the two sample preparation methods. We confirmed that direct identification from positive blood culture bottles with MALDI-TOF MS is reliable for anaerobic bacteria. However, the results are influenced by the sample preparation method used. Copyright © 2018 Elsevier Ltd. All rights reserved.

Diagnostic accuracy of serological diagnosis of hepatitis C and B using dried blood spot samples (DBS): two systematic reviews and meta-analyses.

PubMed

Lange, Berit; Cohn, Jennifer; Roberts, Teri; Camp, Johannes; Chauffour, Jeanne; Gummadi, Nina; Ishizaki, Azumi; Nagarathnam, Anupriya; Tuaillon, Edouard; van de Perre, Philippe; Pichler, Christine; Easterbrook, Philippa; Denkinger, Claudia M

2017-11-01

Dried blood spots (DBS) are a convenient tool to enable diagnostic testing for viral diseases due to transport, handling and logistical advantages over conventional venous blood sampling. A better understanding of the performance of serological testing for hepatitis C (HCV) and hepatitis B virus (HBV) from DBS is important to enable more widespread use of this sampling approach in resource limited settings, and to inform the 2017 World Health Organization (WHO) guidance on testing for HBV/HCV. We conducted two systematic reviews and meta-analyses on the diagnostic accuracy of HCV antibody (HCV-Ab) and HBV surface antigen (HBsAg) from DBS samples compared to venous blood samples. MEDLINE, EMBASE, Global Health and Cochrane library were searched for studies that assessed diagnostic accuracy with DBS and agreement between DBS and venous sampling. Heterogeneity of results was assessed and where possible a pooled analysis of sensitivity and specificity was performed using a bivariate analysis with maximum likelihood estimate and 95% confidence intervals (95%CI). We conducted a narrative review on the impact of varying storage conditions or limits of detection in subsets of samples. The QUADAS-2 tool was used to assess risk of bias. For the diagnostic accuracy of HBsAg from DBS compared to venous blood, 19 studies were included in a quantitative meta-analysis, and 23 in a narrative review. Pooled sensitivity and specificity were 98% (95%CI:95%-99%) and 100% (95%CI:99-100%), respectively. For the diagnostic accuracy of HCV-Ab from DBS, 19 studies were included in a pooled quantitative meta-analysis, and 23 studies were included in a narrative review. Pooled estimates of sensitivity and specificity were 98% (CI95%:95-99) and 99% (CI95%:98-100), respectively. Overall quality of studies and heterogeneity were rated as moderate in both systematic reviews. HCV-Ab and HBsAg testing using DBS compared to venous blood sampling was associated with excellent diagnostic accuracy. However, generalizability is limited as no uniform protocol was applied and most studies did not use fresh samples. Future studies on diagnostic accuracy should include an assessment of impact of environmental conditions common in low resource field settings. Manufacturers also need to formally validate their assays for DBS for use with their commercial assays.

Preemptive Genotyping for Personalized Medicine: Design of the Right Drug, Right Dose, Right Time – Using Genomic Data to Individualize Treatment Protocol

PubMed Central

Bielinski, Suzette J.; Olson, Janet E.; Pathak, Jyotishman; Weinshilboum, Richard M.; Wang, Liewei; Lyke, Kelly J.; Ryu, Euijung; Targonski, Paul V.; Van Norstrand, Michael D.; Hathcock, Matthew A.; Takahashi, Paul Y.; McCormick, Jennifer B.; Johnson, Kiley J.; Maschke, Karen J.; Rohrer Vitek, Carolyn R.; Ellingson, Marissa S.; Wieben, Eric D.; Farrugia, Gianrico; Morrisette, Jody A.; Kruckeberg, Keri J.; Bruflat, Jamie K.; Peterson, Lisa M.; Blommel, Joseph H.; Skierka, Jennifer M.; Ferber, Matthew J.; Black, John L.; Baudhuin, Linnea M.; Klee, Eric W.; Ross, Jason L.; Veldhuizen, Tamra L.; Schultz, Cloann G.; Caraballo, Pedro J.; Freimuth, Robert R.; Chute, Christopher G.; Kullo, Iftikhar J.

2014-01-01

Objective To report the design and implementation of the Right Drug, Right Dose, Right Time: Using Genomic Data to Individualize Treatment Protocol that was developed to test the concept that prescribers can deliver genome guided therapy at the point-of-care by using preemptive pharmacogenomics (PGx) data and clinical decision support (CDS) integrated in the electronic medical record (EMR). Patients and Methods We used a multivariable prediction model to identify patients with a high risk of initiating statin therapy within 3 years. The model was used to target a study cohort most likely to benefit from preemptive PGx testing among Mayo Clinic Biobank participants with a recruitment goal of 1000 patients. Cox proportional hazards model was utilized using the variables selected through the Lasso shrinkage method. An operational CDS model was adapted to implement PGx rules within the EMR. Results The prediction model included age, sex, race, and 6 chronic diseases categorized by the Clinical Classifications Software for ICD-9 codes (dyslipidemia, diabetes, peripheral atherosclerosis, disease of the blood-forming organs, coronary atherosclerosis and other heart diseases, and hypertension). Of the 2000 Biobank participants invited, 50% provided blood samples, 13% refused, 28% did not respond, and 9% consented but did not provide a blood sample within the recruitment window (October 4, 2012 – March 20, 2013). Preemptive PGx testing included CYP2D6 genotyping and targeted sequencing of 84 PGx genes. Synchronous real-time CDS is integrated in the EMR and flags potential patient-specific drug-gene interactions and provides therapeutic guidance. Conclusion These interventions will improve understanding and implementation of genomic data in clinical practice. PMID:24388019

Postnatal gestational age estimation using newborn screening blood spots: a proposed validation protocol

PubMed Central

Murphy, Malia S Q; Hawken, Steven; Atkinson, Katherine M; Milburn, Jennifer; Pervin, Jesmin; Gravett, Courtney; Stringer, Jeffrey S A; Rahman, Anisur; Lackritz, Eve; Chakraborty, Pranesh; Wilson, Kumanan

2017-01-01

Background Knowledge of gestational age (GA) is critical for guiding neonatal care and quantifying regional burdens of preterm birth. In settings where access to ultrasound dating is limited, postnatal estimates are frequently used despite the issues of accuracy associated with postnatal approaches. Newborn metabolic profiles are known to vary by severity of preterm birth. Recent work by our group and others has highlighted the accuracy of postnatal GA estimation algorithms derived from routinely collected newborn screening profiles. This protocol outlines the validation of a GA model originally developed in a North American cohort among international newborn cohorts. Methods Our primary objective is to use blood spot samples collected from infants born in Zambia and Bangladesh to evaluate our algorithm’s capacity to correctly classify GA within 1, 2, 3 and 4 weeks. Secondary objectives are to 1) determine the algorithm's accuracy in small-for-gestational-age and large-for-gestational-age infants, 2) determine its ability to correctly discriminate GA of newborns across dichotomous thresholds of preterm birth (≤34 weeks, <37 weeks GA) and 3) compare the relative performance of algorithms derived from newborn screening panels including all available analytes and those restricted to analyte subsets. The study population will consist of infants born to mothers already enrolled in one of two preterm birth cohorts in Lusaka, Zambia, and Matlab, Bangladesh. Dried blood spot samples will be collected and sent for analysis in Ontario, Canada, for model validation. Discussion This study will determine the validity of a GA estimation algorithm across ethnically diverse infant populations and assess population specific variations in newborn metabolic profiles. PMID:29104765

Evaluation of a manual DNA extraction protocol and an isothermal amplification assay for detecting HIV-1 DNA from dried blood spots for use in resource-limited settings.

PubMed

Jordan, Jeanne A; Ibe, Christine O; Moore, Miranda S; Host, Christel; Simon, Gary L

2012-05-01

In resource-limited settings (RLS) dried blood spots (DBS) are collected on infants and transported through provincial laboratories to a central facility where HIV-1 DNA PCR testing is performed using specialized equipment. Implementing a simpler approach not requiring such equipment or skilled personnel could allow the more numerous provincial laboratories to offer testing, improving turn-around-time to identify and treat infected infants sooner. Assess performances of a manual DNA extraction method and helicase-dependent amplification (HDA) assay for detecting HIV-1 DNA from DBS. 60 HIV-1 infected adults were enrolled, blood samples taken and DBS made. DBS extracts were assessed for DNA concentration and beta globin amplification using PCR and melt-curve analysis. These same extracts were then tested for HIV-1 DNA using HDA and compared to results generated by PCR and pyrosequencing. Finally, HDA limit of detection (LOD) studies were performed using DBS extracts prepared with known numbers of 8E5 cells. The manual extraction protocol consistently yielded high concentrations of amplifiable DNA from DBS. LOD assessment demonstrated HDA detected ∼470 copies/ml of HIV-1 DNA extracts in 4/4 replicates. No statistical difference was found using the McNemar's test when comparing HDA to PCR for detecting HIV-1 DNA from DBS. Using just a magnet, heat block and pipettes, the manual extraction protocol and HDA assay detected HIV-1 DNA from DBS at levels that would be useful for early infant diagnosis. Next steps will include assessing HDA for non-B HIV-1 subtypes recognition and comparison to Roche HIV-1 DNA v1.5 PCR assay. Copyright © 2012 Elsevier B.V. All rights reserved.

Protein substitute dosage in PKU: how much do young patients need?

PubMed Central

MacDonald, A; Chakrapani, A; Hendriksz, C; Daly, A; Davies, P; Asplin, D; Hall, K; Booth, I W

2006-01-01

Background The optimal dose of protein substitute has not been determined in children with phenylketonuria (PKU). Aim To determine if a lower dose of protein substitute could achieve the same or better degree of blood phenylalanine control when compared to the dosage recommended by the UK MRC.1 Methods In a six week randomised, crossover study, two doses of protein substitute (Protocol A: 2 g/kg/day of protein equivalent; Protocol B: 1.2 g/kg/day protein equivalent) were compared in 25 children with well controlled PKU aged 2–10 years (median 6 years). Each dose of protein substitute was taken for 14 days, with a 14 day washout period in between. Twice daily blood samples (fasting pre‐breakfast and evening, at standard times) for plasma phenylalanine were taken on day 8–14 of each protocol. The median usual dose of protein substitute was 2.2 g/kg/day (range 1.5–3.1 g/kg/day). Results When compared with control values, median plasma phenylalanine on the low dose of protein substitute increased at pre‐breakfast by 301 μmol/l (95% CI 215 to 386) and in the evening by 337 μmol/l (95% CI 248 to 431). On the high dose of protein substitute, plasma phenylalanine concentrations remained unchanged when compared to control values. However, wide variability was seen between subjects. Conclusions A higher dosage of protein substitute appeared to contribute to lower blood phenylalanine concentrations in PKU, but it did have a variable and individual impact and may have been influenced by the carbohydrate (+/− fat) content of the protein substitute. PMID:16547085

Factors influencing European consumer uptake of personalised nutrition. Results of a qualitative analysis.

PubMed

Stewart-Knox, Barbara; Kuznesof, Sharron; Robinson, Jenny; Rankin, Audrey; Orr, Karen; Duffy, Maresa; Poínhos, Rui; de Almeida, Maria Daniel Vaz; Macready, Anna; Gallagher, Caroline; Berezowska, Aleksandra; Fischer, Arnout R H; Navas-Carretero, Santiago; Riemer, Martina; Traczyk, Iwona; Gjelstad, Ingrid M F; Mavrogianni, Christina; Frewer, Lynn J

2013-07-01

The aim of this research was to explore consumer perceptions of personalised nutrition and to compare these across three different levels of "medicalization": lifestyle assessment (no blood sampling); phenotypic assessment (blood sampling); genomic assessment (blood and buccal sampling). The protocol was developed from two pilot focus groups conducted in the UK. Two focus groups (one comprising only "older" individuals between 30 and 60 years old, the other of adults 18-65 yrs of age) were run in the UK, Spain, the Netherlands, Poland, Portugal, Ireland, Greece and Germany (N=16). The analysis (guided using grounded theory) suggested that personalised nutrition was perceived in terms of benefit to health and fitness and that convenience was an important driver of uptake. Negative attitudes were associated with internet delivery but not with personalised nutrition per se. Barriers to uptake were linked to broader technological issues associated with data protection, trust in regulator and service providers. Services that required a fee were expected to be of better quality and more secure. An efficacious, transparent and trustworthy regulatory framework for personalised nutrition is required to alleviate consumer concern. In addition, developing trust in service providers is important if such services to be successful. Copyright © 2013 Elsevier Ltd. All rights reserved.

Molt-associated changes in hematologic and plasma biochemical values and stress hormone levels in African penguins (Spheniscus demersus).

PubMed

Mazzaro, Lisa M; Meegan, Jenny; Sarran, Delphine; Romano, Tracy A; Bonato, Vinicius; Deng, Shibing; Dunn, J Lawrence

2013-12-01

Handling, including blood collection, has often been discouraged in molting penguins because it is considered an additional stress imposed on birds already experiencing major physiologic stress associated with molting. To evaluate the degree of physiologic stress posed by molting, we compared the hematologic and plasma biochemical values and hormone levels of molting and nonmolting African penguins, Spheniscus demersus. Five male and 5 female penguins randomly chosen were given complete physical examinations, were weighed, and blood samples were taken at 7 time points before, during, and after the molt. Data were analyzed by linear mixed-model analysis of variance. Throughout the study, behavior and appetite remained normal. Catecholamine levels were highly variable within and among subjects, whereas mean corticosterone levels were significantly different between baseline, molt, and postmolt values. Significant differences from baseline values were observed in many of the hematologic analytes; however, only decreases in hematocrit and red blood cell count values were considered clinically significant. Anemia due to experimentally induced blood loss as a possible cause of the significant hematologic changes was ruled out based on results of a follow-up control study during the nonmolt season, which showed no significant changes in hematocrit level or total red blood cell counts when using similar sampling protocols, which indicates that these changes were associated with molt.

Evaluation of a simple method for storage of blood samples that enables isolation of circulating tumor cells 96 h after sample collection.

PubMed

Apostolou, Panagiotis; Ntanovasilis, Dimitrios-Athanasios; Papasotiriou, Ioannis

2017-12-01

Minimizing the effects of transportation on the properties of biological material is a major challenge for the scientific community. The viability of cells is important in cases where their study is urgent for evaluation of treatment response or for the study of cancer progression. Circulating tumor cells (CTCs) constitute a cell subpopulation with great importance for oncologists, because of their prognostic value. Detection and isolation of CTCs from blood samples is a routine activity in many laboratories, but concerns exist with regard to the maintenance of the cells during transportation. In this study, experiments were conducted to determine the stability of gene and protein expression in CTCs over a period of 96 h. Blood samples collected from healthy individuals and patients with cancer were each divided into five aliquots, which were stored at 2-8 °C and analyzed after 0, 24, 48, 72 and 96 h of storage. CTCs from patients and CD45-negative cells from healthy individuals were isolated each day using enrichment protocols, and qPCR was performed to determine expression levels of genes encoding specific biological markers. In addition, cells from breast and colon cancer cell lines were spiked into blood samples from healthy individuals, and these samples were stored and analyzed over a period of 96 h by PCR and by flow cytometry. The markers that were studied included housekeeping genes and genes associated with the response to chemotherapy, as well as genes encoding transcription factors. The results demonstrated that the expression profiles of specific genes and proteins in CTCs were not significantly affected by 72 h of storage. After 96 h of storage, expression of some genes was altered. The transportation of blood at low temperature (2-8 °C) in the presence of the anticoagulant EDTA can protect CTCs from alteration of gene and protein expression for at least 72 h. Furthermore, under these conditions, CTCs can be detected and isolated 96 h after blood collection.

Functional characterization of neotropical snakes peripheral blood leukocytes subsets: Linking flow cytometry cell features, microscopy images and serum corticosterone levels.

PubMed

de Carvalho, Marcelo Pires Nogueira; Queiroz-Hazarbassanov, Nicolle Gilda Teixeira; de Oliveira Massoco, Cristina; Sant'Anna, Sávio Stefanini; Lourenço, Mariana Mathias; Levin, Gabriel; Sogayar, Mari Cleide; Grego, Kathleen Fernandes; Catão-Dias, José Luiz

2017-09-01

Reptiles are the unique ectothermic amniotes, providing the key link between ectothermic anamniotes fish and amphibians, and endothermic birds and mammals; becoming an important group to study with the aim of providing significant knowledge into the evolutionary history of vertebrate immunity. Classification systems for reptiles' leukocytes have been described by their appearance rather than function, being still inconsistent. With the advent of modern techniques and the establishment of analytical protocols for snakes' blood by flow cytometry, we bring a qualitative and quantitative assessment of innate activities presented by snakes' peripheral blood leukocytes, thereby linking flow cytometric features with fluorescent and light microscopy images. Moreover, since corticosterone is an important immunomodulator in reptiles, hormone levels of all blood samples were measured. We provide novel and additional information which should contribute to better understanding of the development of the immune system of reptiles and vertebrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparison of Performance Characteristics of Aspergillus PCR in Testing a Range of Blood-Based Samples in Accordance with International Methodological Recommendations.

PubMed

Springer, Jan; White, P Lewis; Hamilton, Shanna; Michel, Denise; Barnes, Rosemary A; Einsele, Hermann; Löffler, Juergen

2016-03-01

Standardized methodologies for the molecular detection of invasive aspergillosis (IA) have been established by the European Aspergillus PCR Initiative for the testing of whole blood, serum, and plasma. While some comparison of the performance of Aspergillus PCR when testing these different sample types has been performed, no single study has evaluated all three using the recommended protocols. Standardized Aspergillus PCR was performed on 423 whole-blood pellets (WBP), 583 plasma samples, and 419 serum samples obtained from hematology patients according to the recommendations. This analysis formed a bicenter retrospective anonymous case-control study, with diagnosis according to the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (11 probable cases and 36 controls). Values for clinical performance using individual and combined samples were calculated. For all samples, PCR positivity was significantly associated with cases of IA (for plasma, P = 0.0019; for serum, P = 0.0049; and for WBP, P = 0.0089). Plasma PCR generated the highest sensitivity (91%); the sensitivities for serum and WBP PCR were 80% and 55%, respectively. The highest specificity was achieved when testing WBP (96%), which was significantly superior to the specificities achieved when testing serum (69%, P = 0.0238) and plasma (53%, P = 0.0002). No cases were PCR negative in all specimen types, and no controls were PCR positive in all specimens. This study confirms that Aspergillus PCR testing of plasma provides robust performance while utilizing commercial automated DNA extraction processes. Combining PCR testing of different blood fractions allows IA to be both confidently diagnosed and excluded. A requirement for multiple PCR-positive plasma samples provides similar diagnostic utility and is technically less demanding. Time to diagnosis may be enhanced by testing multiple contemporaneously obtained sample types. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Optimizing direct amplification of forensic commercial kits for STR determination.

PubMed

Caputo, M; Bobillo, M C; Sala, A; Corach, D

2017-04-01

Direct DNA amplification in forensic genotyping reduces analytical time when large sample sets are being analyzed. The amplification success depends mainly upon two factors: on one hand, the PCR chemistry and, on the other, the type of solid substrate where the samples are deposited. We developed a workflow strategy aiming to optimize times and cost when starting from blood samples spotted onto diverse absorbent substrates. A set of 770 blood samples spotted onto Blood cards, Whatman ® 3 MM paper, FTA™ Classic cards, and Whatman ® Grade 1 was analyzed by a unified working strategy including a low-cost pre-treatment, a PCR amplification volume scale-down, and the use of the 3500 Genetic Analyzer as the analytical platform. Samples were analyzed using three different commercial multiplex STR direct amplification kits. The efficiency of the strategy was evidenced by a higher percentage of high-quality profiles obtained (over 94%), a reduced number of re-injections (average 3.2%), and a reduced amplification failure rate (lower than 5%). Average peak height ratio among different commercial kits was 0.91, and the intra-locus balance showed values ranging from 0.92 to 0.94. A comparison with previously reported results was performed demonstrating the efficiency of the proposed modifications. The protocol described herein showed high performance, producing optimal quality profiles, and being both time and cost effective. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Hypertension and related lifestyle factors among persons living in rural Nicaragua.

PubMed

Alicea-Planas, Jessica; Greiner, Lydia; Greiner, Philip A

2016-02-01

Globally about 40% of adults are diagnosed with hypertension, with high-income countries having a lower prevalence than low-income countries. However, there are limited data about adult hypertension prevalence in Nicaragua. The purpose of this study was to determine the prevalence of hypertension in rural coffee farm workers. A convenience sample of 229 adult coffee farm workers was used. Blood pressure was measured using an established protocol and the Joint National Committee on Prevention, Detection, Evaluation and Treatment of High Blood Pressure (JNC-7) guidelines. Nearly 60% of the sample reported at least one prior blood pressure measurement. Hypertension was detected in 16.7% of males and 26.3% of females (20.7% of the total). Prehypertension was detected in 59.3% of males and 27.7% of females (46.2% of the total). Of the men, 51.4% reported smoking at least some days and just over one third of the sample reported adding extra salt to their food. While the prevalence of hypertension in this sample is lower than global estimates, almost half of the sample had prehypertension, demonstrating an area where health promotion efforts could be focused. Given the limited funding and resources often available in these areas, increasing disease prevention efforts (including health promotion and wellness programs) and establishing settings that provide outreach and education, may improve chronic disease management and prevent comorbidities from occurring. Copyright © 2015 Elsevier Inc. All rights reserved.

TH-C-18A-09: Exam and Patient Parameters Affecting the DNA Damage Response Following CT Studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Elgart, S; Adibi, A; Bostani, M

Purpose: To identify exam and patient parameters affecting the biological response to CT studies using in vivo and ex vivo blood samples. Methods: Blood samples were collected under IRB approval from 16 patients undergoing clinically-indicated CT exams. Blood was procured prior to, immediately after and 30minutes following irradiation. A sample of preexam blood was placed on the patient within the exam region for ex vivo analysis. Whole blood samples were fixed immediately following collection and stained for γH2AX to assess DNA damage response (DDR). Median fluorescence of treated samples was compared to non-irradiated control samples for each patient. Patients weremore » characterized by observed biological kinetic response: (a) fast — phosphorylation increased by 2minutes and fell by 30minutes, (b) slow — phosphorylation continued to increase to 30minutes and (c) none — little change was observed or irradiated samples fell below controls. Total dose values were normalized to exam time for an averaged dose-rate in dose/sec for each exam. Relationships between patient biological responses and patient and exam parameters were investigated. Results: A clearer dose response at 30minutes is observed for young patients (<61yoa; R2>0.5) compared to old patients (>61yoa; R{sup 2}<0.11). Fast responding patients were significantly younger than slow responding patients (p<0.05). Unlike in vivo samples, age did not significantly affect the patient response ex vivo. Additionally, fast responding patients received exams with significantly smaller dose-rate than slow responding patients (p<0.05). Conclusion: Age is a significant factor in the biological response suggesting that DDR may be more rapid in a younger population and slower as the population ages. Lack of an agerelated response ex vivo suggests a systemic response to radiation not present when irradiated outside the body. Dose-rate affects the biological response suggesting that patient response may be related to scan timing and dose delivery within an exam protocol. All authors receive(d) funding from a Master Research Agreement from Siemens Healthcare with UCLA Radiological Sciences.« less

Evaluation of a rapid protocol for the assessment of salt sensitivity against the blood pressure response to dietary sodium chloride restriction.

PubMed

Galletti, F; Ferrara, I; Stinga, F; Iacone, R; Noviello, F; Strazzullo, P

1997-04-01

The "gold standard" for the assessment of salt sensitivity of hypertension is the blood pressure response to dietary NaCl restriction; nevertheless, for practical purposes, a more rapid test that would not depend on the patient's compliance to the dietary prescription would be very useful in clinical research and medical practice. The aim of this study was thus to evaluate the effectiveness and reliability of a rapid, easy-to-standardize protocol for the assessment of salt sensitivity against the blood pressure response to dietary salt restriction. A total of 108 hypertensive patients were screened for salt sensitivity by the modified protocol of Grim et al. Thereafter, nine patients identified by the test as salt sensitive and nine identified as salt resistant followed, for two consecutive periods of 1 week, a diet with normal (200 mmol/day) or low (50 mmol/day) NaCl content. Compliance to the diet was checked by repeated 24-h urine collections. The group as a whole experienced a significant fall in blood pressure during the low Na diet (mean pressure = 123 +/- 3 v 118 +/- 3 mm Hg; P < .05). However, whereas patients identified as salt sensitive by the Grim protocol had a marked and significant blood pressure decrease (systolic -12 mm Hg, diastolic -7 mm Hg), no change was observed in those classified as salt resistant (systolic -2 mm Hg, diastolic -2 mm Hg). A significant correlation between changes in urinary Na excretion and changes in blood pressure was found only in salt-sensitive hypertensive patients. In conclusion, the modified Grim protocol tested in this study was able to correctly predict a significant blood pressure response to dietary salt restriction in the majority of cases. A validation of this test in a larger patient population may be advisable.

Protocol for a randomised controlled trial of fetal scalp blood lactate measurement to reduce caesarean sections during labour: the Flamingo trial [ACTRN12611000172909].

PubMed

East, Christine E; Kane, Stefan C; Davey, Mary-Ann; Kamlin, C Omar; Brennecke, Shaun P

2015-11-03

The rate of caesarean sections around the world is rising each year, reaching epidemic proportions. Although many caesarean sections are performed for concerns about fetal welfare on the basis of abnormal cardiotocography, the majority of babies are shown to be well at birth, meaning that the operation, with its inherent short and long term risks, could have been avoided without compromising the baby's health. Previously, fetal scalp blood sampling for pH estimation was performed in the context of an abnormal cardiotocograph, to improve the identification of babies in need of expedited delivery. This test has largely been replaced by lactate measurement, although its validity is yet to be established through a randomised controlled trial. This study aims to test the hypothesis that the performance of fetal scalp blood lactate measurement for women in labour with an abnormal cardiotocograph will reduce the rate of birth by caesarean section from 38 % to 25 % (a 35 % relative reduction). Prospective unblinded randomised controlled trial conducted at a single tertiary perinatal centre. Women labouring with a singleton fetus in cephalic presentation at 37 or more weeks' gestation with ruptured membranes and with an abnormal cardiotocograph will be eligible. Participants will be randomised to one of two groups: fetal monitoring by cardiotocography alone, or cardiotocography augmented by fetal scalp blood lactate analysis. Decisions regarding the timing and mode of delivery will be made by the treating team, in accordance with hospital protocols. The primary study endpoint is caesarean section with secondary outcomes collected from maternal, fetal and neonatal clinical course and morbidities. A cost effectiveness analysis will also be performed. A sample size of 600 will provide 90 % power to detect the hypothesised difference in the proportion of women who give birth by caesarean section. This world-first trial is adequately powered to determine the impact of fetal scalp blood lactate measurement on rates of caesarean section. Preventing unnecessary caesarean sections will reduce the health and financial burdens associated with this operation, both in the index and any future pregnancies. Australian New Zealand Clinical Trials Registry ACTRN12611000172909.

A retrospective review of safety using a nursing driven protocol for autonomic dysreflexia in patients with spinal cord injuries.

PubMed

Solinsky, Ryan; Svircev, Jelena N; James, Jennifer J; Burns, Stephen P; Bunnell, Aaron E

2016-11-01

Autonomic dysreflexia is a potentially life-threatening condition which afflicts a significant proportion of individuals with spinal cord injuries (SCI). To date, the safety and efficacy of several commonly used interventions for this condition have not been studied. A retrospective chart review of the safety of a previously implemented nursing driven inpatient autonomic dysreflexia protocol. Seventy-eight male patients with SCI who experienced autonomic dysreflexia while inpatient at our Veterans Affairs SCI unit over a 3-1/2-year period were included. The safety of a nursing driven protocol utilizing conservative measures, nitroglycerin paste, and oral hydralazine was evaluated. Occurrence of adverse events and relative hypotensive events during all episodes treated with the protocol, and efficacy of attaining target blood pressure for all episodes with protocol adherence and for initial episode experienced by each patient. Four hundred forty-five episodes of autonomic dysreflexia were recorded in the study period, with 92% adherence to the protocol. When the protocol was followed, target blood pressure was achieved for 97.6% of all episodes. Twenty-three total adverse events occurred (5.2% of all episodes). All adverse events were due to hypotension and only 0.9% required interventions beyond clinical monitoring. Of each patient's initial autonomic dysreflexia episode, 97.3% resolved using the protocol without need for further escalation of care. This inpatient nursing driven-protocol for treating autonomic dysreflexia utilizing conservative measures, nitroglycerin paste and oral hydralazine achieved target blood pressure with a high success rate and a low incidence of adverse events.

Safety and efficacy of an intensive insulin protocol in a burn-trauma intensive care unit.

PubMed

Cochran, Amalia; Davis, Lynn; Morris, Stephen E; Saffle, Jeffrey R

2008-01-01

Aggressive glycemic management in critically ill patients with acute burn injury or life-threatening soft-tissue infections has not been thoroughly evaluated. An intensive insulin protocol with target glucose values of less than 120 mg/dl was implemented in October 2005 in our regional Burn-Trauma intensive care unit. We reviewed our initial experience with this protocol to evaluate the safety and efficacy of aggressive glycemic control in these patient groups. Patients were placed on the intensive insulin protocol based upon the need for glycemic management during their hospitalization for burn or soft-tissue disease. Patient information prospectively collected while on protocol included all measured blood glucose values, total daily insulin use, and incidence of hypoglycemic episodes, defined as serum glucose <60 mg/dl. Thirty patients (17 burns, 13 soft-tissue infections) were placed on the intensive insulin protocol during the first 16 months of use. The mean daily blood glucose level for burn patients was 115.9 mg/dl and for soft-tissue disease patients was 119.5 mg/dl. There was a 5% incidence of hypoglycemic episodes per protocol day. All hypoglycemic episodes were treated by holding the insulin infusion, and no episode had known adverse effects. Hyperglycemia in critically ill patients with burns and extensive soft-tissue disease can be effectively managed with an insulin protocol that targets blood glucose values of less than 120 mg/dl with minimal incidence of hypoglycemia. A multicenter prospective randomized trial would provide the ideal forum for evaluating clinical outcome benefits of using an intensive insulin protocol.

gDNA enrichment by a transposase-based technology for NGS analysis of the whole sequence of BRCA1, BRCA2, and 9 genes involved in DNA damage repair.

PubMed

Chevrier, Sandy; Boidot, Romain

2014-10-06

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3'-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

Total knee replacement induces peripheral blood lymphocytes apoptosis and it is not prevented by regional anesthesia - a randomized study.

PubMed

Kosel, Juliusz; Rusak, Małgorzata; Gołembiewski, Łukasz; Dąbrowska, Milena; Siemiątkowski, Andrzej

2016-01-01

Among the many changes caused by a surgical insult one of the least studied is postoperative immunosuppression. This phenomenon is an important cause of infectious complications of surgery such as surgical site infection or hospital acquired pneumonia. One of the mechanisms leading to postoperative immunosuppression is the apoptosis of immunological cells. Anesthesia during surgery is intended to minimize harmful changes and maintain perioperative homeostasis. The aim of the study was evaluation of the effect of the anesthetic technique used for total knee replacement on postoperative peripheral blood lymphocyte apoptosis. 34 patients undergoing primary total knee replacement were randomly assigned to two regional anesthetic protocols: spinal anesthesia and combined spinal-epidural anesthesia. 11 patients undergoing total knee replacement under general anesthesia served as control group. Before surgery, immediately after surgery, during first postoperative day and seven days after the surgery venous blood samples were taken and the immunological status of the patient was assessed with the use of flow cytometry, along with lymphocyte apoptosis using fluorescent microscopy. Peripheral blood lymphocyte apoptosis was seen immediately in the postoperative period and was accompanied by a decrease of the number of T cells and B cells. There were no significant differences in the number of apoptotic lymphocytes according to the anesthetic protocol. Changes in the number of T CD3/8 cells and the number of apoptotic lymphocytes were seen on the seventh day after surgery. Peripheral blood lymphocyte apoptosis is an early event in the postoperative period that lasts up to seven days and is not affected by the choice of the anesthetic technique. Copyright © 2014 Sociedade Brasileira de Anestesiologia. Published by Elsevier Editora Ltda. All rights reserved.

Reduced use of occult bacteremia blood screens by emergency medicine physicians using immunization registry for children presenting with fever without a source.

PubMed

Zeretzke, Cristina M; McIntosh, Mark S; Kalynych, Colleen J; Wylie, Todd; Lott, Michelle; Wood, David

2012-07-01

This study examined whether utilization of the Florida State Health Online Tracking System (SHOTS) immunization registry to determine Haemophilus influenzae type B and heptavalent pneumococcal conjugate (PCV7) vaccine status impacts the protocolized decision to perform a screening blood draw for occult bacteremia (OB) in young children. A convenience sample of children 6 to 24 months of age presenting to the pediatric emergency department with fever of greater than 39°C without a source was enrolled. Physicians were trained to use the SHOTS immunization registry and reviewed the emergency department's fever protocol. A "preregistry" workup plan was documented for each patient based on clinical history, immunization status before accessing SHOTS, and physical examination. A "postregistry" workup plan was then documented based on the SHOTS record. Demographic and registry data were recorded. Preregistry workup plans indicated OB screening blood draws for 100% (n = 91; 95% confidence interval [CI], 96-100) of patients with unconfirmed immunization status. Of those 91 children, 58% (n = 53; 95% CI, 55-61) were documented in SHOTS as having received their primary conjugate vaccine series at ages 2, 4, and 6 months. Registry access reduced the percentage of screening blood draws from 100% (n = 91) to 42% (n = 38; 95% CI, 37-53; P < 0.001). The state immunization registry is an adjunctive tool to caregiver recall, which can be used by emergency medicine practitioners to confirm completion of the primary conjugate vaccine series before making the decision to perform blood screens for OB in children aged 6 to 24 months who present with fever without a source.

Validation of the Rossmax CF175 upper-arm blood pressure monitor for home blood pressure monitoring according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Zhang, Lu; Kang, Yuan-Yuan; Zeng, Wei-Fang; Li, Yan; Wang, Ji-Guang

2015-04-01

The present study aimed to evaluate the accuracy of the Rossmax CF175 upper-arm blood pressure monitor for home blood pressure monitoring according to the International Protocol of the European Society of Hypertension revision 2010. Systolic and diastolic blood pressures were sequentially measured in 33 adult Chinese (17 women, mean age 46 years) using a mercury sphygmomanometer (two observers) and the Rossmax CF175 device (one supervisor). A total of 99 pairs of comparisons were obtained from 33 participants for judgments in two parts with three grading phases. All the blood pressure requirements were fulfilled. The Rossmax CF175 device achieved the targets in part 1 of the validation study. The number of absolute differences between the device and observers within 5, 10, and 15 mmHg was 78/99, 94/99, and 98/99, respectively, for systolic blood pressure, and 81/99, 96/99, and 97/99, respectively, for diastolic blood pressure. The device also achieved the criteria in part 2 of the validation study. Twenty-nine participants, for both of systolic and diastolic blood pressure, had at least two of the three device-observers differences within 5 mmHg (required ≥24). Only one participant for diastolic blood pressure had all three device-observers comparisons greater than 5 mmHg. The Rossmax automated oscillometric upper-arm blood pressure monitor CF175 fulfilled the requirements of the International Protocol revision 2010, and hence can be recommended for blood pressure measurement in adults.

Validation of the Andon KD-5965 upper-arm blood pressure monitor for home blood pressure monitoring according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Huang, Jinhua; Li, Zhijie; Li, Guimei; Liu, Zhaoying

2015-10-01

This study aimed to evaluate the accuracy of the Andon KD-5965 upper-arm blood pressure monitor according to the European Society of Hypertension International Protocol revision 2010. Systolic and diastolic blood pressures were sequentially measured in 33 adults, with 20 women using a mercury sphygmomanometer (two observers) and the Andon KD-5965 device (one supervisor). A total of 99 pairs of comparisons were obtained from 33 participants for judgments in two parts with three grading phases. The device achieved the targets in part 1 of the validation study. The number of absolute differences between the device and observers within 5, 10, and 15 mmHg was 70/99, 91/99, and 98/99, respectively, for systolic blood pressure and 81/99, 99/99, and 99/99, respectively, for diastolic blood pressure. The device also fulfilled the criteria in part 2 of the validation study. Twenty-five and 29 participants, for systolic and diastolic blood pressure, respectively, had at least two of the three device-observers differences within 5 mmHg (required≥24). Two and one participants for systolic and diastolic blood pressure, respectively, had all three device-observers comparisons greater than 5 mmHg. According to the validation results, with better performance for diastolic blood pressure than that for systolic blood pressure, the Andon automated oscillometric upper-arm blood pressure monitor KD-5965 fulfilled the requirements of the European Society of Hypertension International Protocol revision 2010, and hence can be recommended for blood pressure measurement in adults.

Influence of Fasting Status and Sample Preparation on Metabolic Biomarker Measurements in Postmenopausal Women.

PubMed

Murphy, Neil; Falk, Roni T; Messinger, Diana B; Pollak, Michael; Xue, Xiaonan; Lin, Juan; Sgueglia, Robin; Strickler, Howard D; Gaudet, Mia M; Gunter, Marc J

2016-01-01

Epidemiologic data linking metabolic markers-such as insulin, insulin-like growth factors (IGFs)-and adipose tissue-derived factors with cancer are inconsistent. Between-study differences in blood collection protocols, in particular participant's fasting status, may influence measurements. We investigated the impact of fasting status and blood sample processing time on components of the insulin/IGF axis and in adipokines in a controlled feeding study of 45 healthy postmenopausal-women aged 50-75 years. Fasting blood samples were drawn (T0), after which subjects ate a standardized breakfast; subsequent blood draws were made at 1 hour (T1), 3 hours (T3), and 6 hours (T6) after breakfast. Serum samples were assayed for insulin, C-peptide, total- and free-IGF-I, IGF-binding protein [BP]-1 and -3, total and high molecular weight (HMW)-adiponectin, retinol binding protein-4, plasminogen activator inhibitor (PAI)-1, and resistin. Insulin and C-peptide levels followed similar postprandial trajectories; intra-class correlation coefficients [ICC] for insulin = 0.75, (95%CI:0.64-0.97) and C-peptide (ICC = 0.66, 95%CI:0.54-0.77) were similarly correlated in fasting (Spearman correlation, r = 0.78, 95%CI:0.64-0.88) and postprandial states (T1, r = 0.77 (95%CI: 0.62-0.87); T3,r = 0.78 (95%CI: 0.63-0.87); T6,r = 0.77 (95%CI: 0.61-0.87)). Free-IGF-I and IGFBP-1 levels were also affected by fasting status, whereas total-IGF-I and IGFBP-3 levels remained unchanged. Levels of adipokines were largely insensitive to fasting status and blood sample processing delays. Several components of the insulin/IGF axis were significantly impacted by fasting state and in particular, C-peptide levels were substantially altered postprandially and in a similar manner to insulin.

Broth versus solid agar culture of swab samples of cadaveric allograft musculoskeletal tissue.

PubMed

Varettas, Kerry

2013-12-01

As part of the donor assessment protocol, bioburden assessment must be performed on allograft musculoskeletal tissue samples collected at the time of tissue retrieval. Swab samples of musculoskeletal tissue allografts from cadaveric donors are received at the microbiology department of the South Eastern Area Laboratory Services (Australia) to determine the presence of bacteria and fungi. This study will review the isolation rate of organisms from solid agar and broth culture of swab samples of cadaveric allograft musculoskeletal tissue over a 6-year period, 2006-2011. Swabs were inoculated onto horse blood agar (anaerobic, 35 °C) and chocolate agar (CO2, 35 °C) and then placed into a cooked meat broth (aerobic, 35 °C). A total of 1,912 swabs from 389 donors were received during the study period. 557 (29.1 %) swabs were culture positive with the isolation of 713 organisms, 249 (34.9 %) from solid agar culture and an additional 464 (65.1 %) from broth culture only. This study has shown that the broth culture of cadaveric allograft musculoskeletal swab samples recovered a greater amount of organisms than solid agar culture. Isolates such as Clostridium species and Staphylococcus aureus would not have been isolated from solid agar culture alone. Broth culture is an essential part of the bioburden assessment protocol of swab samples of cadaveric allograft musculoskeletal tissue in this laboratory.

Determination of Δ9-tetrahydrocannabinolic acid A (Δ9-THCA-A) in whole blood and plasma by LC-MS/MS and application in authentic samples from drivers suspected of driving under the influence of cannabis.

PubMed

Raikos, Nikolaos; Schmid, Helene; Nussbaumer, Susanne; Ambach, Lars; Lanz, Stephan; Längin, Andreas; König, Stefan; Roth, Nadine; Auwärter, Volker; Weinmann, Wolfgang

2014-10-01

Delta-9-tetrahydrocannabinolic acid A (THCA-A) is the biosynthetic precursor of delta-9-tetrahydrocannabinol (THC) in cannabis plants, and has no psychotropic effects. THCA-A can be detected in blood and urine, and several metabolites have been identified. THCA-A was also shown to be incorporated in hair by side stream smoke to a minor extent, but incorporation via blood stream or sweat seems unlikely. The detection of THCA-A in biological fluids may serve as a marker for differentiating between the intake of prescribed THC medication - containing only pure THC - and cannabis products containing THC besides THC-acid A and other cannabinoids. However, the knowledge about its usefulness in forensic cases is very limited. The aim of the present work was the development of a reliable method for THCA-A determination in human blood or plasma using LC-MS/MS and application to cases of driving under the influence of drugs. Fifty eight (58) authentic whole blood and the respective plasma samples were collected from drivers suspected of driving under the influence of cannabis from the region of Bern (Switzerland). Samples were first tested for THC, 11-OH-THC and THC-COOH, and then additionally for THCA-A. For this purpose, the existing LC-MS/MS method was modified and validated, and found to be selective and linear over a range of 1.0 to 200ng/mL (the correlation coefficients were above 0.9980 in all validation runs). Limit of detection (LOD) and limit of quantification (LOQ) were 0.3ng/mL and 1.0ng/mL respectively. Intra- and inter-assay accuracy were equal or better than 90% and intra- and inter-assay precision were equal or better than 11.1%. The mean extraction efficiencies were satisfactory being equal or higher than 85.4%. THCA-A was stable in whole blood samples after 3 freeze/thaw cycles and storage at 4°C for 7 days. Re-injection (autosampler) stability was also satisfactory. THC was present in all blood samples with levels ranging from 0.7 to 51ng/mL. THCA-A concentrations ranged from 1.0 to 496ng/mL in blood samples and from 1.4 to 824ng/mL in plasma samples. The plasma:blood partition coefficient had a mean value of 1.7 (±0.21, SD). No correlation was found between the degree of intoxication or impairment stated in the police protocols or reports of medical examinations and the detected THCA-A-concentration in blood. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Dried venous blood samples for the detection and quantification of measles IgG using a commercial enzyme immunoassay.

PubMed Central

Riddell, Michaela A.; Byrnes, Graham B.; Leydon, Jennie A.; Kelly, Heath A.

2003-01-01

OBJECTIVES: To determine whether samples of dried venous blood (DVB) were an acceptable alternative to serum for detecting measles-specific IgG in a commercial enzyme immunoassay. METHODS: Paired samples of serum and DVB were collected from 98 suspected cases of measles and 1153 schoolchildren in Victoria, Australia. All samples were tested using the Dade Behring Enzygnost Anti-Measles-Virus/IgG immunoassay. DVB samples were eluted using either the sample buffer provided with the kit or 5% dry milk powder in phosphate-buffered saline-Tween 20. FINDINGS: DVB samples eluted by sample buffer showed significantly better linear correlation to the serum samples than did DVB samples eluted in 5% dry milk in phosphate-buffered saline-Tween 20. To improve the comparability of serum and DVB samples an adjustment factor of 1.28 was applied to the optical density (OD) values of DVB. This adjustment also enabled quantification of the titre of measles IgG in mIU/ml directly from the OD value using the alpha calculation as specified by the kit protocol. For DVB samples stored for less than six months at 4 degrees C, the assay showed an overall sensitivity of 98.4% and a specificity of 97.2% compared with the results of serum testing. CONCLUSION: These results illustrate the potential for DVB samples to be widely used with the Dade Behring enzyme immunoassay system for determining the immunity of the individual and the population to the measles virus. PMID:14758429

Cytomegalovirus pp65 antigenemia-guided pre-emptive treatment with ganciclovir after allogeneic stem transplantation: a single-center experience.

PubMed

Manteiga, R; Martino, R; Sureda, A; Labeaga, R; Brunet, S; Sierra, J; Rabella, N

1998-11-01

The optimal prophylactic strategy for cytomegalovirus (CMV) disease after allogeneic hematopoietic stem cell transplantation has not yet been established. The aim of this study was to analyze our single-center experience with a uniform protocol of CMV antigenemia-guided pre-emptive treatment with ganciclovir (GCV) after allografting. Fifty-two consecutive adult patients, 48 of them transplanted from HLA-identical matched related donors were included. T cell-depleted marrow or peripheral blood were used in 21 cases. After engraftment, weekly blood samples were tested for CMV pp65 antigenemia and viremia (conventional cultures) until day +100. GCV was started if CMV antigenemia and/or CMV viremia were detected. CMV infection (CMV-I) was found in 19 patients (37%). Seven patients suffered from CMV disease (CMV-D), three colitis and four pneumonias. There was one death directly related to CMV-D and three further cases died from refractory GVHD with CMV-D. Only one patient developed CMV pneumonia without any previous positive antigenemia and/or viremia. Multivariate analysis identified grades II-IV acute GVHD (P = 0.02) and peripheral blood stem cell transplantation (P = 0.03) to be risk factors for developing CMV-I. In conclusion, this monitoring protocol allowed early treatment of CMV-I without progression to CMV-D. Pre-emptive therapy had the additional advantage of avoiding GCV administration in most of our allograft recipients.

Comparative Analytical Utility of DNA Derived from Alternative Human Specimens for Molecular Autopsy and Diagnostics

PubMed Central

Klassen, Tara L.; von Rüden, Eva-Lotta; Drabek, Janice; Noebels, Jeffrey L.; Goldman, Alica M.

2013-01-01

Genetic testing and research have increased the demand for high-quality DNA that has traditionally been obtained by venipuncture. However, venous blood collection may prove difficult in special populations and when large-scale specimen collection or exchange is prerequisite for international collaborative investigations. Guthrie/FTA card–based blood spots, buccal scrapes, and finger nail clippings are DNA-containing specimens that are uniquely accessible and thus attractive as alternative tissue sources (ATS). The literature details a variety of protocols for extraction of nucleic acids from a singular ATS type, but their utility has not been systematically analyzed in comparison with conventional sources such as venous blood. Additionally, the efficacy of each protocol is often equated with the overall nucleic acid yield but not with the analytical performance of the DNA during mutation detection. Together with a critical in-depth literature review of published extraction methods, we developed and evaluated an all-inclusive approach for serial, systematic, and direct comparison of DNA utility from multiple biological samples. Our results point to the often underappreciated value of these alternative tissue sources and highlight ways to maximize the ATS-derived DNA for optimal quantity, quality, and utility as a function of extraction method. Our comparative analysis clarifies the value of ATS in genomic analysis projects for population-based screening, diagnostics, molecular autopsy, medico-legal investigations, or multi-organ surveys of suspected mosaicisms. PMID:22796560

Direct identification from positive blood broth culture by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS).

PubMed

Barberino, Maria Goreth; Silva, Marcio de Oliveira; Arraes, Ana Carolina Palmeiras; Correia, Luís Cláudio; Mendes, Ana Verena

Bloodstream infections (BSIs) are among the most concerning bacterial infections. They are one of the leading causes of morbidity and mortality, and occur in 30-70% of critical care patients. The prompt identification of the causative microorganism can help choosing the appropriate antimicrobial therapy that will lead to better clinical outcomes. Blood culture is one of the most relevant tests for microbiological diagnosis of bacterial infections. The introduction of the MALDI-TOF microbiological diagnosis significantly decreased the time of identifying microorganisms. However, it depends on the growth on solid culture medium. In this study, 538 bottles of positive blood cultures were evaluated to test the accuracy of an in house modified protocol. The study sample consisted of 198 Gram-negative and 350 Gram-positive bacteria. In all, 460 (83.94%) species were identified based on the direct plate findings. The protocol allowed the identification of 185/198 (93.43%) of the Gram-negative bacteria, including aerobes, anaerobes, and non-fermenters, and 275/350 (78.85%) of the Gram-positive bacteria. The proposed method has the potential to provide accurate results in comparison to the traditional method with the potential to reduce the turnaround time for the results and optimize antimicrobial therapy in BSI. Copyright © 2017 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

The Karlsruhe Metabolomics and Nutrition (KarMeN) Study: Protocol and Methods of a Cross-Sectional Study to Characterize the Metabolome of Healthy Men and Women

PubMed Central

Kriebel, Anita; Dörr, Claudia; Bandt, Susanne; Rist, Manuela; Roth, Alexander; Hummel, Eva; Kulling, Sabine; Hoffmann, Ingrid; Watzl, Bernhard

2016-01-01

Background The human metabolome is influenced by various intrinsic and extrinsic factors. A precondition to identify such biomarkers is the comprehensive understanding of the composition and variability of the metabolome of healthy humans. Sample handling aspects have an important impact on the composition of the metabolome; therefore, it is crucial for any metabolomics study to standardize protocols on sample collection, preanalytical sample handling, storage, and analytics to keep the nonbiological variability as low as possible. Objective The main objective of the KarMeN study is to analyze the human metabolome in blood and urine by targeted and untargeted metabolite profiling (gas chromatography-mass spectrometry [GC-MS], GC×GC-MS, liquid chromatography-mass spectrometry [LC-MS/MS], and1H nuclear magnetic resonance [NMR] spectroscopy) and to determine the impact of sex, age, body composition, diet, and physical activity on metabolite profiles of healthy women and men. Here, we report the outline of the study protocol with special regard to all aspects that should be considered in studies applying metabolomics. Methods Healthy men and women, aged 18 years or older, were recruited. In addition to a number of anthropometric (height, weight, body mass index, waist circumference, body composition), clinical (blood pressure, electrocardiogram, blood and urine clinical chemistry) and functional parameters (lung function, arterial stiffness), resting metabolic rate, physical activity, fitness, and dietary intake were assessed, and 24-hour urine, fasting spot urine, and plasma samples were collected. Standard operating procedures were established for all steps of the study design. Using different analytical techniques (LC-MS, GC×GC-MS,1H NMR spectroscopy), metabolite profiles of urine and plasma were determined. Data will be analyzed using univariate and multivariate as well as predictive modeling methods. Results The project was funded in 2011 and enrollment was carried out between March 2012 and July 2013. A total of 301 volunteers were eligible to participate in the study. Metabolite profiling of plasma and urine samples has been completed and data analysis is currently underway. Conclusions We established the KarMeN study applying a broad set of clinical and physiological examinations with a high degree of standardization. Our experimental approach of combining scheduled timing of examinations and sampling with the multiplatform approach (GC×GC-MS, GC-MS, LC-MS/MS, and1H NMR spectroscopy) will enable us to differentiate between current and long-term effects of diet and physical activity on metabolite profiles, while enabling us at the same time to consider confounders such as age and sex in the KarMeN study. Trial Registration German Clinical Trials Register DRKS00004890; https://drks-neu.uniklinik-freiburg.de/drks_web/navigate.do? navigationId=trial.HTML&TRIAL_ID=DRKS00004890 (Archived by WebCite at http://www.webcitation.org/6iyM8dMtx) PMID:27421387

Continuous haematic pH monitoring in extracorporeal circulation using a disposable florescence sensing element

NASA Astrophysics Data System (ADS)

Ferrari, Luca; Rovati, Luigi; Fabbri, Paola; Pilati, Francesco

2013-02-01

During extracorporeal circulation (ECC), blood is periodically sampled and analyzed to maintain the blood-gas status of the patient within acceptable limits. This protocol has well-known drawbacks that may be overcome by continuous monitoring. We present the characterization of a new pH sensor for continuous monitoring in ECC. This monitoring device includes a disposable fluorescence-sensing element directly in contact with the blood, whose fluorescence intensity is strictly related to the pH of the blood. In vitro experiments show no significant difference between the blood gas analyzer values and the sensor readings; after proper calibration, it gives a correlation of R>0.9887, and measuring errors were lower than the 3% of the pH range of interest (RoI) with respect to a commercial blood gas analyzer. This performance has been confirmed also by simulating a moderate ipothermia condition, i.e., blood temperature 32°C, frequently used in cardiac surgery. In ex vivo experiments, performed with animal models, the sensor is continuously operated in an extracorporeal undiluted blood stream for a maximum of 11 h. It gives a correlation of R>0.9431, and a measuring error lower than the 3% of the pH RoI with respect to laboratory techniques.

Continuous haematic pH monitoring in extracorporeal circulation using a disposable florescence sensing element.

PubMed

Ferrari, Luca; Rovati, Luigi; Fabbri, Paola; Pilati, Francesco

2013-02-01

During extracorporeal circulation (ECC), blood is periodically sampled and analyzed to maintain the blood-gas status of the patient within acceptable limits. This protocol has well-known drawbacks that may be overcome by continuous monitoring. We present the characterization of a new pH sensor for continuous monitoring in ECC. This monitoring device includes a disposable fluorescence-sensing element directly in contact with the blood, whose fluorescence intensity is strictly related to the pH of the blood. In vitro experiments show no significant difference between the blood gas analyzer values and the sensor readings; after proper calibration, it gives a correlation of R>0.9887, and measuring errors were lower than the 3% of the pH range of interest (RoI) with respect to a commercial blood gas analyzer. This performance has been confirmed also by simulating a moderate ipothermia condition, i.e., blood temperature 32°C, frequently used in cardiac surgery. In ex vivo experiments, performed with animal models, the sensor is continuously operated in an extracorporeal undiluted blood stream for a maximum of 11 h. It gives a correlation of R>0.9431, and a measuring error lower than the 3% of the pH RoI with respect to laboratory techniques.

Raman spectroscopy of coloured resins used in antiquity: dragon's blood and related substances.

PubMed

Edward, H G; de Oliveira, L F; Quye, A

2001-12-01

Dragon's blood is a deep red resin which has been used for centuries by many cultures and much prized for it's rarity, depth of colour and alchemical associations. The original source of dragon's blood resin is believed to be Dracaena cinnabari from Socotra in Africa, but since mediaeval times there have been several alternatives from different geographical locations from the Canary Islands to the East Indies. Here, the Raman spectra of dragon's blood resins from Dracaena draco Liliacae trees growing in several different locations bordering the Mediterranean and Middle East are compared with the resins from alternative botanical sources such as Daemonorops draco, Dracaena cinnabari and Eucalyptus terminalis, which all generically come under the description of dragon's blood. Key vibrational spectroscopic marker bands are identified in the Raman spectra of the resins, which are suggested for adoption as a protocol for the identification of the botanical and possible geographical sources of modern dragon's blood resins. The Raman spectra of materials, which are falsely attributed to dragon's blood resin are also shown for comparison and identification purposes. Changes in the Raman spectra of genuine dragon's blood resin specimens arising from simple processing treatment during the preparation of the resins for sale are also identified, which suggests a possible attribution characteristic for unknown samples.

Raman spectroscopy of coloured resins used in antiquity: dragon's blood and related substances

NASA Astrophysics Data System (ADS)

Edward, Howell G. M.; de Oliveira, Luiz Fernando C.; Quye, Anita

2001-12-01

Dragon's blood is a deep red resin which has been used for centuries by many cultures and much prized for it's rarity, depth of colour and alchemical associations. The original source of dragon's blood resin is believed to be Dracaena cinnabari from Socotra in Africa, but since mediaeval times there have been several alternatives from different geographical locations from the Canary Islands to the East Indies. Here, the Raman spectra of dragon's blood resins from Dracaena draco Liliacae trees growing in several different locations bordering the Mediterranean and Middle East are compared with the resins from alternative botanical sources such as Daemonorops draco, Dracaena cinnabari and Eucalyptus terminalis, which all generically come under the description of dragon's blood. Key vibrational spectroscopic marker bands are identified in the Raman spectra of the resins, which are suggested for adoption as a protocol for the identification of the botanical and possible geographical sources of modern dragon's blood resins. The Raman spectra of materials, which are falsely attributed to dragon's blood resin are also shown for comparison and identification purposes. Changes in the Raman spectra of genuine dragon's blood resin specimens arising from simple processing treatment during the preparation of the resins for sale are also identified, which suggests a possible attribution characteristic for unknown samples.

Follicle vascularity coordinates corpus luteum blood flow and progesterone production.

PubMed

de Tarso, S G S; Gastal, G D A; Bashir, S T; Gastal, M O; Apgar, G A; Gastal, E L

2017-03-01

Colour Doppler ultrasonography was used to compare the ability of preovulatory follicle (POF) blood flow and its dimensions to predict the size, blood flow and progesterone production capability of the subsequent corpus luteum (CL). Cows (n=30) were submitted to a synchronisation protocol. Follicles ≥7mm were measured and follicular wall blood flow evaluated every 12h for approximately 3.5 days until ovulation. After ovulation, cows were scanned daily for 8 days and similar parameters were evaluated for the CL. Blood samples were collected and plasma progesterone concentrations quantified. All parameters were positively correlated. Correlation values ranged from 0.26 to 0.74 on data normalised to ovulation and from 0.31 to 0.74 on data normalised to maximum values. Correlations between calculated ratios of both POF and CL in data normalised to ovulation and to maximum values ranged from moderate (0.57) to strong (0.87). Significant (P<0.0001) linear regression analyses were seen in all comparisons. In conclusion, higher correlations were observed between the dimensions of POF and/or CL and blood flow of both structures, as well as POF and/or CL blood flow with plasma progesterone concentrations of the resultant CL. These findings indicate that follicle vascularity coordinates CL blood flow and progesterone production in synchronised beef cows.

Genotoxic evaluation of pirfenidone using erythrocyte rodent micronucleus assay.

PubMed

Alcántar-Díaz, Blanca E; Gómez-Meda, Belinda C; Zúñiga-González, Guillermo M; Zamora-Perez, Ana L; González-Cuevas, Jaime; Alvarez-Rodríguez, Bertha A; Sánchez-Parada, María Guadalupe; García-Bañuelos, Jesús J; Armendáriz-Borunda, Juan

2012-08-01

Pirfenidone is a non-steroidal antifibrotic compound that has been proposed in clinical protocols and experimental studies as a pharmacological treatment for fibroproliferative diseases. The objective of this study was to determine the genotoxicity or cytotoxicity of three doses of pirfenidone using the micronuclei test in peripheral blood erythrocytes of rodent models. Pirfenidone was administered orally to Balb-C mice for 3 days, and also was administered topically to hairless Sprague Dawley rats during the final stage of gestation. Mice were sampled every 24 h over the course of 6 days; pregnant rats were sampled every 24 h during the last 6 days of gestation, and pups were sampled at birth. Blood smears were analyzed and the frequencies of micronucleated erythrocytes (MNEs), micronucleated polychromatic erythrocytes (MNPCEs), and the proportion of polychromatic erythrocytes (PCEs), were recorded in samples from mice, pregnant rats and rat neonates. Increases in MN frequencies (p<0.03) were noted only in the positive control groups. No genotoxic effects or decreased PCE values were observed neither in newborn rats transplacentally exposed to pirfenidone, or in two adult rodent models when pirfenidone was administered orally or topically. Copyright © 2012 Elsevier Ltd. All rights reserved.

Ultra-fast, label-free isolation of circulating tumor cells from blood using spiral microfluidics.

PubMed

Warkiani, Majid Ebrahimi; Khoo, Bee Luan; Wu, Lidan; Tay, Andy Kah Ping; Bhagat, Ali Asgar S; Han, Jongyoon; Lim, Chwee Teck

2016-01-01

Circulating tumor cells (CTCs) are rare cancer cells that are shed from primary or metastatic tumors into the peripheral blood circulation. Phenotypic and genetic characterization of these rare cells can provide important information to guide cancer staging and treatment, and thus further research into their characteristics and properties is an area of considerable interest. In this protocol, we describe detailed procedures for the production and use of a label-free spiral microfluidic device to allow size-based isolation of viable CTCs using hydrodynamic forces that are present in curvilinear microchannels. This spiral system enables us to achieve ≥ 85% recovery of spiked cells across multiple cancer cell lines and 99.99% depletion of white blood cells in whole blood. The described spiral microfluidic devices can be produced at an extremely low cost using standard microfabrication and soft lithography techniques (2-3 d), and they can be operated using two syringe pumps for lysed blood samples (7.5 ml in 12.5 min for a three-layered multiplexed chip). The fast processing time and the ability to collect CTCs from a large patient blood volume allows this technique to be used experimentally in a broad range of potential genomic and transcriptomic applications.

Estimating the ratio of multivariate recurrent event rates with application to a blood transfusion study.

PubMed

Ning, Jing; Rahbar, Mohammad H; Choi, Sangbum; Piao, Jin; Hong, Chuan; Del Junco, Deborah J; Rahbar, Elaheh; Fox, Erin E; Holcomb, John B; Wang, Mei-Cheng

2017-08-01

In comparative effectiveness studies of multicomponent, sequential interventions like blood product transfusion (plasma, platelets, red blood cells) for trauma and critical care patients, the timing and dynamics of treatment relative to the fragility of a patient's condition is often overlooked and underappreciated. While many hospitals have established massive transfusion protocols to ensure that physiologically optimal combinations of blood products are rapidly available, the period of time required to achieve a specified massive transfusion standard (e.g. a 1:1 or 1:2 ratio of plasma or platelets:red blood cells) has been ignored. To account for the time-varying characteristics of transfusions, we use semiparametric rate models for multivariate recurrent events to estimate blood product ratios. We use latent variables to account for multiple sources of informative censoring (early surgical or endovascular hemorrhage control procedures or death). The major advantage is that the distributions of latent variables and the dependence structure between the multivariate recurrent events and informative censoring need not be specified. Thus, our approach is robust to complex model assumptions. We establish asymptotic properties and evaluate finite sample performance through simulations, and apply the method to data from the PRospective Observational Multicenter Major Trauma Transfusion study.

The Clinical Urine Culture: Enhanced Techniques Improve Detection of Clinically Relevant Microorganisms

PubMed Central

Price, Travis K.; Dune, Tanaka; Hilt, Evann E.; Thomas-White, Krystal J.; Kliethermes, Stephanie; Brincat, Cynthia; Brubaker, Linda; Wolfe, Alan J.

2016-01-01

Enhanced quantitative urine culture (EQUC) detects live microorganisms in the vast majority of urine specimens reported as “no growth” by the standard urine culture protocol. Here, we evaluated an expanded set of EQUC conditions (expanded-spectrum EQUC) to identify an optimal version that provides a more complete description of uropathogens in women experiencing urinary tract infection (UTI)-like symptoms. One hundred fifty adult urogynecology patient-participants were characterized using a self-completed validated UTI symptom assessment (UTISA) questionnaire and asked “Do you feel you have a UTI?” Women responding negatively were recruited into the no-UTI cohort, while women responding affirmatively were recruited into the UTI cohort; the latter cohort was reassessed with the UTISA questionnaire 3 to 7 days later. Baseline catheterized urine samples were plated using both standard urine culture and expanded-spectrum EQUC protocols: standard urine culture inoculated at 1 μl onto 2 agars incubated aerobically; expanded-spectrum EQUC inoculated at three different volumes of urine onto 7 combinations of agars and environments. Compared to expanded-spectrum EQUC, standard urine culture missed 67% of uropathogens overall and 50% in participants with severe urinary symptoms. Thirty-six percent of participants with missed uropathogens reported no symptom resolution after treatment by standard urine culture results. Optimal detection of uropathogens could be achieved using the following: 100 μl of urine plated onto blood (blood agar plate [BAP]), colistin-nalidixic acid (CNA), and MacConkey agars in 5% CO2 for 48 h. This streamlined EQUC protocol achieved 84% uropathogen detection relative to 33% detection by standard urine culture. The streamlined EQUC protocol improves detection of uropathogens that are likely relevant for symptomatic women, giving clinicians the opportunity to receive additional information not currently reported using standard urine culture techniques. PMID:26962083

Moderate glycemic control safe in critically ill adult burn patients: A 15 year cohort study.

PubMed

Stoecklin, Patricia; Delodder, Frederik; Pantet, Olivier; Berger, Mette M

2016-02-01

Hyperglycemia is a metabolic alteration in major burn patients associated with complications. The study aimed at evaluating the safety of general ICU glucose control protocols applied in major burns receiving prolonged ICU treatment. 15 year retrospective analysis of consecutive, adult burn patients admitted to a single specialized centre. death or length of stay <10 days, age <16 years. demographic variables, burned surface (TBSA), severity scores, infections, ICU stay, outcome. Metabolic variables: total energy, carbohydrate and insulin delivery/24h, arterial blood glucose and CRP values. Analysis of 4 periods: 1, before protocol; 2, tight doctor driven; 3, tight nurse driven; 4, moderate nurse driven. 229 patients, aged 45 ± 20 years (mean ± SD), burned 32 ± 20% TBSA were analyzed. SAPSII was 35 ± 13. TBSA, Ryan and ABSI remained stable. Inhalation injury increased. A total of 28,690 blood glucose samples were analyzed: the median value remained unchanged with a narrower distribution over time. After the protocol initiation, the normoglycemic values increased from 34.7% to 65.9%, with a reduction of hypoglycaemic events (no extreme hypoglycemia in period 4). Severe hyperglycemia persisted throughout with a decrease in period 4 (9.25% in period 4). Energy and glucose deliveries decreased in periods 3 and 4 (p<0.0001). Infectious complications increased during the last 2 periods (p=0.01). A standardized ICU glucose control protocol improved the glycemic control in adult burn patients, reducing glucose variability. Moderate glycemic control in burns was safe specifically related to hypoglycemia, reducing the incidence of hypoglycaemic events compared to the period before. Hyperglycemia persisted at a lower level. Copyright © 2015 Elsevier Ltd and ISBI. All rights reserved.

Use of portable blood physiology point-of-care devices for basic and applied research on vertebrates: a review

PubMed Central

Stoot, Lauren J.; Cairns, Nicholas A.; Cull, Felicia; Taylor, Jessica J.; Jeffrey, Jennifer D.; Morin, Félix; Mandelman, John W.; Clark, Timothy D.; Cooke, Steven J.

2014-01-01

Non-human vertebrate blood is commonly collected and assayed for a variety of applications, including veterinary diagnostics and physiological research. Small, often non-lethal samples enable the assessment and monitoring of the physiological state and health of the individual. Traditionally, studies that rely on blood physiology have focused on captive animals or, in studies conducted in remote settings, have required the preservation and transport of samples for later analysis. In either situation, large, laboratory-bound equipment and traditional assays and analytical protocols are required. The use of point-of-care (POC) devices to measure various secondary blood physiological parameters, such as metabolites, blood gases and ions, has become increasingly popular recently, due to immediate results and their portability, which allows the freedom to study organisms in the wild. Here, we review the current uses of POC devices and their applicability to basic and applied studies on a variety of non-domesticated species. We located 79 individual studies that focused on non-domesticated vertebrates, including validation and application of POC tools. Studies focused on a wide spectrum of taxa, including mammals, birds and herptiles, although the majority of studies focused on fish, and typical variables measured included blood glucose, lactate and pH. We found that calibrations for species-specific blood physiology values are necessary, because ranges can vary within and among taxa and are sometimes outside the measurable range of the devices. In addition, although POC devices are portable and robust, most require durable cases, they are seldom waterproof/water-resistant, and factors such as humidity and temperature can affect the performance of the device. Overall, most studies concluded that POC devices are suitable alternatives to traditional laboratory devices and eliminate the need for transport of samples; however, there is a need for greater emphasis on rigorous calibration and validation of these units and appreciation of their limitations. PMID:27293632

Validation of the AVITA BPM63S upper arm blood pressure monitor for home blood pressure monitoring according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Kang, Yuan-Yuan; Zeng, Wei-Fang; Liu, Ming; Li, Yan; Wang, Ji-Guang

2014-02-01

The present study aimed to evaluate the accuracy of the AVITA BPM63S upper arm blood pressure monitor for home blood pressure monitoring according to the International Protocol of the European Society of Hypertension revision 2010. Systolic and diastolic blood pressures were sequentially measured in 33 adult Chinese (14 women, mean age of 47 years) using a mercury sphygmomanometer (two observers) and the AVITA BPM63S device (one supervisor). Ninety-nine pairs of comparisons were obtained from 33 participants for judgments in two parts with three grading phases. All the blood pressure requirements were fulfilled. The AVITA BPM63S device achieved the targets in part 1 of the validation study. The number of absolute differences between device and observers within 5, 10, and 15 mmHg was 68/99, 89/99, and 96/99, respectively, for systolic blood pressure, and 75/99, 95/99, and 97/99, respectively, for diastolic blood pressure. The device also achieved the criteria in part 2 of the validation study. Twenty-four and 25 participants for systolic and diastolic blood pressure, respectively, had at least two of the three device-observers differences within 5 mmHg (required ≥24). One and two participants for systolic and diastolic blood pressure, respectively, had all three device-observers differences greater than 5 mmHg. The AVITA BPM63S automated oscillometric upper arm blood pressure monitor has passed the requirements of the International Protocol revision 2010, and hence can be recommended for blood pressure measurement at home in adults.

Hospital protocols for targeted glycemic control: Development, implementation, and models for cost justification.

PubMed

Magee, Michelle F

2007-05-15

Evolving elements of best practices for providing targeted glycemic control in the hospital setting, clinical performance measurement, basal-bolus plus correction-dose insulin regimens, components of standardized subcutaneous (s.c.) insulin order sets, and strategies for implementation and cost justification of glycemic control initiatives are discussed. Best practices for targeted glycemic control should address accurate documentation of hyperglycemia, initial patient assessment, management plan, target blood glucose range, blood glucose monitoring frequency, maintenance of glycemic control, criteria for glucose management consultations, and standardized insulin order sets and protocols. Establishing clinical performance measures, including desirable processes and outcomes, can help ensure the success of targeted hospital glycemic control initiatives. The basal-bolus plus correction-dose regimen for insulin administration will be used to mimic the normal physiologic pattern of endogenous insulin secretion. Standardized insulin order sets and protocols are being used to minimize the risk of error in insulin therapy. Components of standardized s.c. insulin order sets include specification of the hyperglycemia diagnosis, finger stick blood glucose monitoring frequency and timing, target blood glucose concentration range, cutoff values for excessively high or low blood glucose concentrations that warrant alerting the physician, basal and prandial or nutritional (i.e., bolus) insulin, correction doses, hypoglycemia treatment, and perioperative or procedural dosage adjustments. The endorsement of hospital administrators and key physician and nursing leaders is needed for glycemic control initiatives. Initiatives may be cost justified on the basis of the billings for clinical diabetes management services and/or the return- on-investment accrued to reductions in hospital length of stay, readmissions, and accurate documentation and coding of unrecognized or uncontrolled diabetes, and diabetes complications. Standardized insulin order sets and protocols may minimize risk of insulin errors. The endorsement of these protocols by administrators, physicians, nurses, and pharmacists is also needed for success.

In vivo decontamination of the nerve agent VX using the domestic swine model.

PubMed

Misik, Jan; Pavlik, Michal; Novotny, Ladislav; Pavlikova, Ruzena; Chilcott, Robert P; Cabal, Jiri; Kuca, Kamil

2012-11-01

The purpose of this in vivo study was to assess a new, putatively optimised method for mass casualty decontamination ("ORCHIDS protocol") for effectiveness in removing the chemical warfare agent VX from the skin of anaesthetised, domestic white pigs. ORCHIDS protocol consists of a 1.5-minute shower with a mild detergent (Argos™) supplemented by physical removal. A standard method of wet decontamination was used for comparison. Experimental animals were divided into four groups (A-D). Two groups were exposed to a supra-lethal percutaneous dose (5 × LD(50); 300 μg kg(-1)) of VX for 1 h prior to decontamination with either the ORCHIDS (C) or standard protocol (D). A third (B, positive control) group was exposed but not subject to decontamination. Blank controls (A) received anaesthesia and the corresponding dose of normal saline instead of VX. Observations of the clinical signs of intoxication were supplemented by measurements of whole blood cholinesterase (ChE) performed on samples of arterial blood acquired at 30-minute intervals for the duration of the study (up to 6 h). Untreated (B) animals displayed typical cholinergic signs consistent with VX intoxication (local fasciculation, mastication, salivation, pilo-erection and motor convulsions) and died 165-240 min post exposure. All animals in both decontamination treatment groups (C, D) survived the duration of the study and exhibited less severe signs of cholinergic poisoning. Thus, both the standard and ORCHIDS protocol were demonstrably effective against exposure to the potent nerve agent VX, even after a delay of 1 h. A critical advantage of the ORCHIDS protocol is the relatively short shower duration (1½ min compared to 3 min). In practice, this could substantially improve the rate at which individuals could be decontaminated by emergency responders following exposure to toxic materials such as chemical warfare agents.

Technical pre-analytical effects on the clinical biochemistry of Atlantic salmon (Salmo salar L.).

PubMed

Braceland, M; Houston, K; Ashby, A; Matthews, C; Haining, H; Rodger, H; Eckersall, P D

2017-01-01

Clinical biochemistry has long been utilized in human and veterinary medicine as a vital diagnostic tool, but despite occasional studies showing its usefulness in monitoring health status in Atlantic salmon (Salmo salar L.), it has not yet been widely utilized within the aquaculture industry. This is due, in part, to a lack of an agreed protocol for collection and processing of blood prior to analysis. Moreover, while the analytical phase of clinical biochemistry is well controlled, there is a growing understanding that technical pre-analytical variables can influence analyte concentrations or activities. In addition, post-analytical interpretation of treatment effects is variable in the literature, thus making the true effect of sample treatment hard to evaluate. Therefore, a number of pre-analytical treatments have been investigated to examine their effect on analyte concentrations and activities. In addition, reference ranges for salmon plasma biochemical analytes have been established to inform veterinary practitioners and the aquaculture industry of the importance of clinical biochemistry in health and disease monitoring. Furthermore, a standardized protocol for blood collection has been proposed. © 2016 The Authors Journal of Fish Diseases Published by John Wiley & Sons Ltd.

Validation of the Kingyield BP210 wrist blood pressure monitor for home blood pressure monitoring according to the European Society of Hypertension-International Protocol.

PubMed

Zeng, Wei-Fang; Huang, Qi-Fang; Sheng, Chang-Sheng; Li, Yan; Wang, Ji-Guang

2012-02-01

The present study aimed to evaluate the accuracy of the automated oscillometric wrist blood pressure monitor BP210 for home blood pressure monitoring according to the International Protocol of the European Society of Hypertension. Systolic and diastolic blood pressures were sequentially measured in 33 adult Chinese participants (21 women, 51 years of mean age) using a mercury sphygmomanometer (two observers) and the BP210 device (one supervisor). Ninety-nine pairs of comparisons were obtained from 15 participants in phase 1 and a further 18 participants in phase 2 of the validation study. Data analysis was conducted using the ESHIP analyzer. The BP210 device successfully passed phase 1 of the validation study with a number of absolute differences between device and observers within 5, 10, and 15 mmHg for at least 33/45, 44/45, and 44/45 measurements, respectively. The device also achieved the targets for phase 2.1, with 77/99, 95/99, and 97/99 differences within 5, 10, and 15 mmHg, respectively for systolic blood pressure, and with 78/99, 97/99, and 99/99 within 5, 10, and 15 mmHg, respectively for diastolic blood pressure. In phase 2.2, 29 and 25 participants had at least two of the three device-observers differences within 5 mmHg (required≥22) for systolic blood pressure and diastolic blood pressure, respectively. The Kingyield wrist blood pressure monitor BP210 has passed the International Protocol requirements, and hence can be recommended for home use in adults.

Validation of the AVITA BPM15S wrist blood pressure monitor for home blood pressure monitoring according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Kang, Yuan-Yuan; Zeng, Wei-Fang; Zhang, Lu; Li, Yan; Wang, Ji-Guang

2014-06-01

The present study aimed to evaluate the accuracy of the automated oscillometric wrist blood pressure monitor AVITA BPM15S for home blood pressure monitoring according to the International Protocol revision 2010 of the European Society of Hypertension. Systolic and diastolic blood pressures were sequentially measured in 33 Chinese adults (15 women, mean age 51 years) using a mercury sphygmomanometer (two observers) and the AVITA BPM15S device (one supervisor). Ninety-nine pairs of comparisons were obtained from 33 participants for judgments in two parts with three grading phases. The AVITA BPM15S device achieved the targets in part 1 of the validation study. The number of absolute differences between the device and observers within 5, 10, and 15 mmHg were 85/99, 94/99, and 98/99, respectively, for systolic blood pressure, and 82/99, 96/99, and 98/99, respectively, for diastolic blood pressure. The device also achieved the criteria in part 2 of the validation study. Thirty-two and 28 participants for systolic and diastolic blood pressure, respectively, had at least two of the three device-observer differences within 5 mmHg (required ≥ 24). No participant had all of the three device-observer comparisons greater than 5 mmHg for systolic or diastolic blood pressure. The AVITA wrist blood pressure monitor BPM15S fulfilled the requirements of the International Protocol revision 2010 and hence can be recommended for home use in an adult population.

Validation of the SCIAN LD-735 wrist blood pressure monitor for home blood pressure monitoring according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Kang, Yuan-Yuan; Chen, Qi; Li, Yan; Wang, Ji-Guang

2016-08-01

This study aimed to evaluate the accuracy of the automated oscillometric wrist blood pressure monitor SCIAN LD-735 for home blood pressure monitoring according to the International Protocol of the European Society of Hypertension revision 2010. Systolic and diastolic blood pressures were measured sequentially in 33 adult Chinese participants (10 women, mean age 44.8 years) using a mercury sphygmomanometer (two observers) and the SCIAN LD-735 device (one supervisor). A total of 99 pairs of comparisons were obtained from 33 participants for judgments in two parts with three grading phases. The SCIAN LD-735 device achieved the targets in part 1 of the validation study. The number of absolute differences between device and observers within 5, 10, and 15 mmHg was 86/99, 97/99, and 98/99, respectively, for systolic blood pressure and 85/99, 98/99, and 99/99, respectively, for diastolic blood pressure. The device also fulfilled the criteria in part 2 of the validation study. In total, 30 and 33 participants for systolic and diastolic blood pressure, respectively, had at least two of the three device-observer differences within 5 mmHg (required ≥24). No participant had all of the three device-observer comparisons greater than 5 mmHg for systolic or diastolic blood pressure. The SCIAN wrist blood pressure monitor LD-735 has passed the requirements of the International Protocol revision 2010, and hence can be recommended for home use in adults.

Validation of the AVITA BPM17 wrist blood pressure monitor for home blood pressure monitoring according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Kang, Yuan-Yuan; Chen, Qi; Liu, Chang-Yuan; Li, Yan; Wang, Ji-Guang

2017-08-01

The aim of the present study was to evaluate the accuracy of the automated oscillometric wrist blood pressure monitor AVITA BPM17 for home blood pressure monitoring according to the International Protocol of the European Society of Hypertension revision 2010. Systolic and diastolic blood pressures were sequentially measured in 33 adult Chinese (19 men, 45.7 years of mean age) using a mercury sphygmomanometer (two observers) and the AVITA BPM17 device (one supervisor). Ninety-nine pairs of comparisons were obtained from 33 participants for judgments in two parts with three grading phases. The AVITA BPM17 device achieved the targets in part 1 of the validation study. The number of absolute differences between device and observers within 5, 10, and 15 mmHg was 94/99, 98/99, and 98/99, respectively, for systolic blood pressure and 92/99, 99/99, and 99/99, respectively, for diastolic blood pressure. The device also fulfilled the criteria in part 2 of the validation study. Overall, 32 participants for both systolic and diastolic blood pressure, respectively, had at least two of the three device-observerss differences within 5 mmHg (required ≥24). None had all the three device-observers comparisons greater than 5 mmHg for systolic and diastolic blood pressure. The AVITA wrist blood pressure monitor BPM17 has passed the requirements of the International Protocol revision 2010, and hence can be recommended for home use in adults.

Cryopreservation of Circulating Tumor Cells for Enumeration and Characterization.

PubMed

Nejlund, Sarah; Smith, Julie; Kraan, Jaco; Stender, Henrik; Van, Mai N; Langkjer, Sven T; Nielsen, Mikkel T; Sölétormos, György; Hillig, Thore

2016-08-01

A blood sample containing circulating tumor cells (CTCs) may serve as a surrogate for metastasis in invasive cancer. Cryopreservation will provide new opportunities in management of clinical samples in the laboratory and allow collection of samples over time for future analysis of existing and upcoming cancer biomarkers. Blood samples from healthy volunteers were spiked with high (∼500) and low (∼50) number of tumor cells from culture. The samples were stored at -80C with cryopreservative dimethyl sulfoxide mixed with Roswell Park Memorial Institute 1640 medium. Flow cytometry tested if cryopreservation affected specific biomarkers regularly used to detect CTCs, i.e. cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM) and white blood cell specific lymphocyte common antigen (CD45). After various time intervals (up to 6 months), samples were thawed and tumor cell recovery (enumeration) was examined. Clinical samples may differ from cell line studies, so the cryopreservation protocol was tested on 17 patients with invasive breast cancer and tumor cell recovery was examined. Two blood samples were drawn from each patient. Biomarkers, CK, CD45, and EpCAM, were not affected by the freezing and thawing procedures. Cryopreserved samples (n = 2) spiked with a high number of tumor cells (∼500) had a ∼90% recovery compared with the spiked fresh samples. In samples spiked with lower numbers of tumor cells (median = 43 in n = 5 samples), the recovery was 63% after cryopreservation (median 27 tumor cells), p = 0.03. With an even lower number of spiked tumor cells (median = 3 in n = 8 samples), the recovery rate of tumor cells after cryopreservation did not seem to be affected (median = 8), p = 0.09. Time of cryopreservation did not affect recovery. When testing the effect of cryopreservation on enumeration in clinical samples, no difference was observed in the number of CTCs between the fresh and the cryopreserved samples based on n = 17 pairs, p = 0.83; however, the variation was large. This large variation was confirmed by clinically paired fresh samples (n = 64 pairs), where 95% of the samples (<30 CTCs) vary in number up to ±15 CTCs, p = 0.18. A small loss of CTCs after cryopreservation may be expected; however, cryopreservation of CTCs for biomarker characterization for clinical applications seems promising.

Small RNA profiling of low biomass samples: identification and removal of contaminants.

PubMed

Heintz-Buschart, Anna; Yusuf, Dilmurat; Kaysen, Anne; Etheridge, Alton; Fritz, Joëlle V; May, Patrick; de Beaufort, Carine; Upadhyaya, Bimal B; Ghosal, Anubrata; Galas, David J; Wilmes, Paul

2018-05-14

Sequencing-based analyses of low-biomass samples are known to be prone to misinterpretation due to the potential presence of contaminating molecules derived from laboratory reagents and environments. DNA contamination has been previously reported, yet contamination with RNA is usually considered to be very unlikely due to its inherent instability. Small RNAs (sRNAs) identified in tissues and bodily fluids, such as blood plasma, have implications for physiology and pathology, and therefore the potential to act as disease biomarkers. Thus, the possibility for RNA contaminants demands careful evaluation. Herein, we report on the presence of small RNA (sRNA) contaminants in widely used microRNA extraction kits and propose an approach for their depletion. We sequenced sRNAs extracted from human plasma samples and detected important levels of non-human (exogenous) sequences whose source could be traced to the microRNA extraction columns through a careful qPCR-based analysis of several laboratory reagents. Furthermore, we also detected the presence of artefactual sequences related to these contaminants in a range of published datasets, thereby arguing in particular for a re-evaluation of reports suggesting the presence of exogenous RNAs of microbial and dietary origin in blood plasma. To avoid artefacts in future experiments, we also devise several protocols for the removal of contaminant RNAs, define minimal amounts of starting material for artefact-free analyses, and confirm the reduction of contaminant levels for identification of bona fide sequences using 'ultra-clean' extraction kits. This is the first report on the presence of RNA molecules as contaminants in RNA extraction kits. The described protocols should be applied in the future to avoid confounding sRNA studies.

A pilot evaluation of whole blood finger-prick sampling for point-of-care HIV viral load measurement: the UNICORN study.

PubMed

Fidler, Sarah; Lewis, Heather; Meyerowitz, Jodi; Kuldanek, Kristin; Thornhill, John; Muir, David; Bonnissent, Alice; Timson, Georgina; Frater, John

2017-10-20

There is a global need for HIV viral load point-of-care (PoC) assays to monitor patients receiving antiretroviral therapy. UNICORN was the first study of an off-label protocol using whole blood finger-prick samples tested with and without a simple three minute spin using a clinic-room microcentrifuge. Two PoC assays were evaluated in 40 HIV-positive participants, 20 with detectable and 20 with undetectable plasma viral load (pVL) (<20 copies/ml). Using 100 µl finger-prick blood samples, the Cepheid Xpert HIV-1 Viral Load and HIV-1 Qual cartridges were compared with laboratory pVL assessment (TaqMan, Roche). For participants with undetectable viraemia by TaqMan, there was poor concordance without centrifugation with the TaqMan platform with only 40% 'undetectable' using Xpert VL and 25% 'not detected' using the Qual assay. After a 3 minute spin, 100% of samples were undetectable using either assay, showing full concordance with the TaqMan assay. Defining a lower limit of detection of 1000 copies/ml when including a spin, there was 100% concordance with the TaqMan platform with strong correlation (rho 0.95 and 0.94; p < 0.0001 for both assays). When including a simple microcentrifugation step, finger-prick PoC testing was a quick and accurate approach for assessing HIV viraemia, with excellent concordance with validated laboratory approaches.

Quality assessment program for EuroFlow protocols: summary results of four-year (2010-2013) quality assurance rounds.

PubMed

Kalina, Tomas; Flores-Montero, Juan; Lecrevisse, Quentin; Pedreira, Carlos E; van der Velden, Vincent H J; Novakova, Michaela; Mejstrikova, Ester; Hrusak, Ondrej; Böttcher, Sebastian; Karsch, Dennis; Sędek, Łukasz; Trinquand, Amelie; Boeckx, Nancy; Caetano, Joana; Asnafi, Vahid; Lucio, Paulo; Lima, Margarida; Helena Santos, Ana; Bonaccorso, Paola; van der Sluijs-Gelling, Alita J; Langerak, Anton W; Martin-Ayuso, Marta; Szczepański, Tomasz; van Dongen, Jacques J M; Orfao, Alberto

2015-02-01

Flow cytometric immunophenotyping has become essential for accurate diagnosis, classification, and disease monitoring in hemato-oncology. The EuroFlow Consortium has established a fully standardized "all-in-one" pipeline consisting of standardized instrument settings, reagent panels, and sample preparation protocols and software for data analysis and disease classification. For its reproducible implementation, parallel development of a quality assurance (QA) program was required. Here, we report on the results of four consecutive annual rounds of the novel external QA EuroFlow program. The novel QA scheme aimed at monitoring the whole flow cytometric analysis process (cytometer setting, sample preparation, acquisition and analysis) by reading the median fluorescence intensities (MedFI) of defined lymphocytes' subsets. Each QA participant applied the predefined reagents' panel on blood cells of local healthy donors. A uniform gating strategy was applied to define lymphocyte subsets and to read MedFI values per marker. The MedFI values were compared with reference data and deviations from reference values were quantified using performance score metrics. In four annual QA rounds, we analyzed 123 blood samples from local healthy donors on 14 different instruments in 11 laboratories from nine European countries. The immunophenotype of defined cellular subsets appeared sufficiently standardized to permit unified (software) data analysis. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%, average MedFI in each QA round ranged from 86 to 125% from overall median. Calculation of performance scores was instrumental to pinpoint standardization failures and their causes. Overall, the new EuroFlow QA system for the first time allowed to quantify the technical variation that is introduced in the measurement of fluorescence intensities in a multicentric setting over an extended period of time. EuroFlow QA is a proficiency test specific for laboratories that use standardized EuroFlow protocols. It may be used to complement, but not replace, established proficiency tests. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

Molecular screening for hemotropic mycoplasmas in captive Barbary sheep (Ammotragus lervia) in southern Brazil.

PubMed

Santos, Leonilda C; Vidotto, Odilon; Morikawa, Vivien M; Santos, Nelson J R; Vieira, Thállitha S W J; Barros Filho, Ivan R; Vieira, Rafael F C; Biondo, Alexander W

2017-08-01

This study is part of an active surveillance program for monitoring animal health status in endangered species, and was conducted to screen captive Barbary sheep ( Ammotragus lervia ) for hemoplasma infection. A total of 12 blood samples were collected, DNA extracted and further tested by a pan-hemoplasma polymerase chain reaction protocol. Animals were clinically healthy and not infested by ectoparasites. Although housekeeping gene DNA was successfully amplified, all the Barbary sheep samples tested negative for Mycoplasma sp. Notwithstanding the negative results, molecular pathogen surveys on Barbary sheep and other exotic wild mammals may provide insights regarding infection of endangered species caused by captivity stress in association with exposure to new pathogens worldwide.

Design of a multi-center immunophenotyping analysis of peripheral blood, sputum and bronchoalveolar lavage fluid in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS).

PubMed

Freeman, Christine M; Crudgington, Sean; Stolberg, Valerie R; Brown, Jeanette P; Sonstein, Joanne; Alexis, Neil E; Doerschuk, Claire M; Basta, Patricia V; Carretta, Elizabeth E; Couper, David J; Hastie, Annette T; Kaner, Robert J; O'Neal, Wanda K; Paine, Robert; Rennard, Stephen I; Shimbo, Daichi; Woodruff, Prescott G; Zeidler, Michelle; Curtis, Jeffrey L

2015-01-27

Subpopulations and Intermediate Outcomes in COPD Study (SPIROMICS) is a multi-center longitudinal, observational study to identify novel phenotypes and biomarkers of chronic obstructive pulmonary disease (COPD). In a subset of 300 subjects enrolled at six clinical centers, we are performing flow cytometric analyses of leukocytes from induced sputum, bronchoalveolar lavage (BAL) and peripheral blood. To minimize several sources of variability, we use a "just-in-time" design that permits immediate staining without pre-fixation of samples, followed by centralized analysis on a single instrument. The Immunophenotyping Core prepares 12-color antibody panels, which are shipped to the six Clinical Centers shortly before study visits. Sputum induction occurs at least two weeks before a bronchoscopy visit, at which time peripheral blood and bronchoalveolar lavage are collected. Immunostaining is performed at each clinical site on the day that the samples are collected. Samples are fixed and express shipped to the Immunophenotyping Core for data acquisition on a single modified LSR II flow cytometer. Results are analyzed using FACS Diva and FloJo software and cross-checked by Core scientists who are blinded to subject data. Thus far, a total of 152 sputum samples and 117 samples of blood and BAL have been returned to the Immunophenotyping Core. Initial quality checks indicate useable data from 126 sputum samples (83%), 106 blood samples (91%) and 91 BAL samples (78%). In all three sample types, we are able to identify and characterize the activation state or subset of multiple leukocyte cell populations (including CD4+ and CD8+ T cells, B cells, monocytes, macrophages, neutrophils and eosinophils), thereby demonstrating the validity of the antibody panel. Our study design, which relies on bi-directional communication between clinical centers and the Core according to a pre-specified protocol, appears to reduce several sources of variability often seen in flow cytometric studies involving multiple clinical sites. Because leukocytes contribute to lung pathology in COPD, these analyses will help achieve SPIROMICS aims of identifying subgroups of patients with specific COPD phenotypes. Future analyses will correlate cell-surface markers on a given cell type with smoking history, spirometry, airway measurements, and other parameters. This study was registered with ClinicalTrials.gov as NCT01969344 .

The HoMedics BPA-200 and BPA-300 home blood pressure devices fail the European Society of Hypertension International Protocol and cannot be recommended for patient use.

PubMed

Grim, Clarence E; Grim, Carlene M

2008-08-01

We have tested two home blood pressure monitors made by the HoMedics, Inc., 300 Pontiac Trail, Commerce Township, Michigan 48390, USA, using the European Society of Hypertension International Protocol. Both failed and we believe it is important to get this into the literature quickly to protect patients and practitioners.

A method for reducing the sloughing of thick blood films for malaria diagnosis.

PubMed

Norgan, Andrew P; Arguello, Heather E; Sloan, Lynne M; Fernholz, Emily C; Pritt, Bobbi S

2013-07-08

The gold standard for malaria diagnosis is the examination of thick and thin blood films. Thick films contain 10 to 20 times more blood than thin films, correspondingly providing increased sensitivity for malaria screening. A potential complication of thick film preparations is sloughing of the blood droplet from the slide during staining or rinsing, resulting in the loss of sample. In this work, two methods for improving thick film slide adherence ('scratch' (SCM) and 'acetone dip' (ADM) methods) were compared to the 'standard method' (SM) of thick film preparation. Standardized blood droplets from 26 previously examined EDTA whole blood specimens (22 positive and four negative) were concurrently spread on glass slides using the SM, ADM, and SCM. For the SM and ADM prepared slides, the droplet was gently spread to an approximate 22 millimeters in diameter spot on the slide using the edge of a second glass slide. For the SCM, the droplet was spread by carefully grinding (or scratching) it into the slide with the point of a second glass slide. Slides were dried for one hour in a laminar flow hood. For the ADM, slides were dipped once in an acetone filled Coplin jar and allowed to air dry. All slides were then Giemsa-stained and examined in a blinded manner. Adherence was assessed by blinded reviewers. No significant or severe defects were observed for slides prepared with the SCM. In contrast, 8 slides prepared by the ADM and 3 prepared using the SM displayed significant or severe defects. Thick films prepared by the three methods were microscopically indistinguishable and concordant results (positive or negative) were obtained for the three methods. Estimated parasitaemia of the blood samples ranged from 25 to 429,169 parasites/μL of blood. The SCM is an inexpensive, rapid, and simple method that improves the adherence of thick blood films to standard glass slides without altering general slide preparation, microscopic appearance or interpretability. Using the SCM, thick films can be reliably examined less than two hours after sample receipt. This represents a significant diagnostic improvement over protocols requiring extended drying periods.

Validation of the YuWell YE690A upper-arm blood pressure monitor, for clinic use and self-measurement, according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Chen, Qi; Lei, Lei; Li, Yan; Wang, Ji-Guang

2017-10-01

The present study aimed to evaluate the accuracy of the automated oscillometric upper-arm blood pressure monitor YuWell YE690A for blood pressure measurement according to the International Protocol of the European Society of Hypertension revision 2010. Systolic and diastolic blood pressures were measured sequentially in 33 adult Chinese (12 women, 44.2 years of mean age) using a mercury sphygmomanometer (two observers) and the YE690A device (one supervisor). A total of 99 pairs of comparisons were obtained from 33 participants for judgments in two parts with three grading phases. All the blood pressure requirements were fulfilled. The YuWell YE690A device achieved the targets in part 1 of the validation study. The number of absolute differences between device and observers within 5, 10, and 15 mmHg was 79/99, 96/99, and 97/99, respectively, for systolic blood pressure and 72/99, 95/99, and 98/99, respectively, for diastolic blood pressure. The device also fulfilled the criteria in part 2 of the validation study. Thirty-one and 25 participants for systolic and diastolic blood pressure, respectively, had at least two of the three device-observer differences within 5 mmHg (required ≥24). No participant for systolic and two participants for diastolic blood pressure had all the three device-observer comparisons greater than 5 mmHg. The YuWell blood pressure monitor YE690A has passed the requirements of the International Protocol revision 2010 and hence can be recommended for blood pressure measurement in adults.

Alcohol and drugs use among drivers injured in road accidents in Campania (Italy): A 8-years retrospective analysis.

PubMed

Carfora, Anna; Campobasso, Carlo Pietro; Cassandro, Paola; Petrella, Raffaella; Borriello, Renata

2018-05-09

A recent update of the Italian Road Traffic Law (RTL 41/2016), established severe penal sanctions when drivers, driving under the influence of alcohol (DUI) or drugs (DUID), are involved in road accident that results in death or injuries. A study was carried out to assess the trends of consumption of alcohol, illicit drugs or pharmaceutical among injured drivers suspected for DUI or DUID from 2009 to 2016 in the region of Campania (Italy). Confirmation toxicological analyses were performed on 780 blood samples and 1017 urine samples collected from 1797 injured drivers. These drivers all tested positive for alcohol or drug use through immunoassay screening applied at hospital emergency units and their biological samples transferred to the Forensic Reference Laboratory (FRL) for confirmation analysis. The GC/HS-FID methodology was used to test Blood Alcohol Concentration (BAC). Qualitative and quantitative analyses for drugs were performed using the GC/MS or LC-MS/MS methodology. The BAC >0.5g/L was confirmed in 91.5% of drivers suspected for DUI cases and in 93% of DUID respectively. In DUI cases, results show an increasing incidence of road accidents involving drivers with BAC above 1.5g/L while at concentrations above 0.8g/L alcohol and drugs are both used. Among the suspected DUID cases, the intake of alcohol in association with drugs has consistently increased over time and positive results on blood samples was confirmed for multiple drugs (20%) or cannabis and cocaine alone (18%) followed by benzodiazepines (6%) and methadone (3.5%) respectively. The majority of injured drivers suspected for DUID (1017 cases) did not authorize blood sampling, therefore only urine was analyzed showing the prevalent use of cannabis, followed by multiple drug>cocaine>benzodiazepines>opiates. Among 1797 drivers, suspected at screening for DUI or DUID, 15.4% of cases (64 blood and 213 urine samples) were not confirmed by GC/HS, GC/MS or LC-MS/MS analysis. In forensic toxicological investigations, it is mandatory to satisfy the best quality standards, which is not achievable if immunochemical screening is only performed on urine. Therefore, only confirmed positive results of alcohol or drugs on blood samples can represent conclusive evidence to demonstrate the DUI or DUID related offences. An improvement of the protocols currently applied in Italy for the assessment of DUI or DUID crimes is needed and the confirmation analysis on blood should be considered mandatory. Copyright © 2018 Elsevier B.V. All rights reserved.

Trypanosoma cruzi infection in captive Neotropical primates in the Brazilian Amazon.

PubMed

Bahia, Michele; de Nazaré Leite Barros, Flávia; Magalhães-Matos, Paulo Cesar; de Souza Gonçalves, Thamirys; Chiesorin Neto, Laerzio; Oliveira Faria, Diogo Cesar Lagroteria; Aparecida Romeiro, Sandra; Barros Monteiro, Frederico Ozanan; Góes-Cavalcante, Gustavo; Scofield, Alessandra

2017-02-01

The aim of this study was to detect the infection by Trypanosoma cruzi in captive Neotropical primates in the Brazilian Amazon. From February 2013 to July 2014, 112 blood samples were collected from Neotropical primates from the Amazonas, Amapá, and Pará States, north of Brazil. The subjects belonged to the families Cebidae (N = 59), Atelidae (N = 41), Callitrichidae (N = 5), Pitheciidae (N = 4), and Aotidae (N = 3). Blood smears also were examined for the presence of trypomastigotes by optical microscopy. For the detection of T. cruzi DNA, a Nested-PCR with primers TCZ1/TCZ2 and TCZ3/TCZ4 was performed. T. cruzi DNA was detected in 12.5% (14/112) of Neotropical primates examined. Positive samples were detected in 16%, 12.5%, and 11.11% of the different species of primates sampled from the Amapá, Pará, and Amazonas states, respectively. The analysis of the blood smears did not reveal trypomastigote forms of T. cruzi. In conclusion, Neotropical primates kept in captivity were infected by T. cruzi in the studied areas. We recommend that a health management protocol be put into place to prevent the transmission of infectious agents among captive populations, captive and wild populations, and between NHPs and the technicians who handle these animals. © 2016 Wiley Periodicals, Inc.

Validation of the custo screen pediatric blood pressure monitor according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Beime, Beate; Deutsch, Cornelia; Krüger, Ralf; Wolf, Andreas; Müller, Peter; Hammel, Gertrud; Bramlage, Peter

2017-05-01

The purpose of the study was to validate the ambulatory blood pressure monitoring (ABPM) device custo screen pediatric in children aged 3 to 12 years according to the International Protocol of the European Society of Hypertension (ESH-IP revision 2010). Thirty-three children were included and systolic and diastolic blood pressure measurements were performed according to the ESH-IP. The protocol was modified for children considering data from the German Health Interview and Examination Survey for Children and Adolescents (KIGGS). The custo screen pediatric met all the requirements of the ESH-IP. The mean difference between the test device and the reference was -1.4 ± 3.0 mmHg for systolic blood pressure (SBP) and -0.7 ± 3.2 mmHg for diastolic blood pressure (DBP). For SBP and DBP, all 99 measurements were within the absolute difference of 10 mmHg between the test device and the reference. As to part 2 of the protocol, for DBP in all subjects, two out of three measurements were within 5 mmHg between the device and the standard, whereas for SBP in 32 of 33 subjects, two out of three measurements were within this range. The custo screen pediatric met all criteria of the ESH-IP review 2010, modified for children from 3 to about 12 years, and can be recommended for ABPM in children. What is Known: • Validation of blood pressure measuring devices is essential to provide patients with an accurate blood pressure measuring device. • The majority of devices has not been validated in children. What is New: • Prior to the present validation, study protocol adjustments of ESH-IP review 2010 for children were defined according to German Health Interview and Examination Survey for Children and Adolescents 2013 (KIGGS). • The custo screen pediatric test device met all criteria of ESH-IP revision 2010, modified for children, and can be recommended for ABPM in children aged 3 to about 12 years.

Tiered protocol implementation improves treatment of hypoglycaemia in a neurosciences critical care and surgical intensive care unit.

PubMed

Van Berkel, Megan A; MacDermott, Jennifer; Dungan, Kathleen M; Cook, Charles H; Murphy, Claire V

2017-12-01

Although studies demonstrate techniques to limit hypoglycaemia in critically ill patients, there are limited data supporting methods to improve management of existing hypoglycaemia. Assess the impact and sustainability of a computerised, three tiered, nurse driven protocol for hypoglycaemia treatment. Retrospective pre and post protocol study. Neurosciences and surgical intensive care units at a tertiary academic medical centre. Patients with a hypoglycaemic episode were included during a pre-protocol or post-protocol implementation period. An additional six-month cohort was evaluated to assess sustainability. Fifty-four patients were included for evaluation (35 pre- and 19 post-protocol); 122 patients were included in the sustainability cohort. Hypoglycaemia treatment significantly improved in the post-protocol cohort (20% vs. 52.6%, p=0.014); with additional improvement to 79.5% in the sustainability cohort. Time to follow-up blood glucose was decreased after treatment from 122 [Q1-Q3: 46-242] minutes pre-protocol to 25 [Q1-Q3: 9-48] minutes post protocol (p<0.0001). This reduction was maintained in the sustainability cohort [median of 29min (Q1-Q3: 20-51)]. Implementation of a nurse-driven, three-tiered protocol for treatment of hypoglyacemia significantly improved treatment rates, as well as reduced time to recheck blood glucose measurement. These benefits were sustained during a six-month period after protocol implementation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Newborn screening for cystic fibrosis in Wisconsin: comparison of biochemical and molecular methods.

PubMed

Gregg, R G; Simantel, A; Farrell, P M; Koscik, R; Kosorok, M R; Laxova, A; Laessig, R; Hoffman, G; Hassemer, D; Mischler, E H; Splaingard, M

1997-06-01

To evaluate neonatal screening for cystic fibrosis (CF), including study of the screening procedures and characteristics of false-positive infants, over the past 10 years in Wisconsin. An important objective evolving from the original design has been to compare use of a single-tier immunoreactive trypsinogen (IRT) screening method with that of a two-tier method using IRT and analyses of samples for the most common cystic fibrosis transmembrane regulator (CFTR) (DeltaF508) mutation. We also examined the benefit of including up to 10 additional CFTR mutations in the screening protocol. From 1985 to 1994, using either the IRT or IRT/DNA protocol, 220 862 and 104 308 neonates, respectively, were screened for CF. For the IRT protocol, neonates with an IRT >/=180 ng/mL were considered positive, and the standard sweat chloride test was administered to determine CF status. For the IRT/DNA protocol, samples from the original dried-blood specimen on the Guthrie card of neonates with an IRT >/=110 ng/mL were tested for the presence of the DeltaF508 CFTR allele, and if the DNA test revealed one or two DeltaF508 alleles, a sweat test was obtained. Both screening procedures had very high specificity. The sensitivity tended to be higher with the IRT/DNA protocol, but the differences were not statistically significant. The positive predictive value of the IRT/DNA screening protocol was 15.2% compared with 6.4% if the same samples had been screened by the IRT method. Assessment of the false-positive IRT/DNA population revealed that the two-tier method eliminates the disproportionate number of infants with low Apgar scores and also the high prevalence of African-Americans identified previously in our study of newborns with high IRT levels. We found that 55% of DNA-positive CF infants were homozygous for DeltaF508 and 40% had one DeltaF508 allele. Adding analyses for 10 more CFTR mutations has only a small effect on the sensitivity but is likely to add significantly to the cost of screening. Advantages of the IRT/DNA protocol over IRT analysis include improved positive predictive value, reduction of false-positive infants, and more rapid diagnosis with elimination of recall specimens.

A randomized trial of protocol-based care for early septic shock.

PubMed

Yealy, Donald M; Kellum, John A; Huang, David T; Barnato, Amber E; Weissfeld, Lisa A; Pike, Francis; Terndrup, Thomas; Wang, Henry E; Hou, Peter C; LoVecchio, Frank; Filbin, Michael R; Shapiro, Nathan I; Angus, Derek C

2014-05-01

In a single-center study published more than a decade ago involving patients presenting to the emergency department with severe sepsis and septic shock, mortality was markedly lower among those who were treated according to a 6-hour protocol of early goal-directed therapy (EGDT), in which intravenous fluids, vasopressors, inotropes, and blood transfusions were adjusted to reach central hemodynamic targets, than among those receiving usual care. We conducted a trial to determine whether these findings were generalizable and whether all aspects of the protocol were necessary. In 31 emergency departments in the United States, we randomly assigned patients with septic shock to one of three groups for 6 hours of resuscitation: protocol-based EGDT; protocol-based standard therapy that did not require the placement of a central venous catheter, administration of inotropes, or blood transfusions; or usual care. The primary end point was 60-day in-hospital mortality. We tested sequentially whether protocol-based care (EGDT and standard-therapy groups combined) was superior to usual care and whether protocol-based EGDT was superior to protocol-based standard therapy. Secondary outcomes included longer-term mortality and the need for organ support. We enrolled 1341 patients, of whom 439 were randomly assigned to protocol-based EGDT, 446 to protocol-based standard therapy, and 456 to usual care. Resuscitation strategies differed significantly with respect to the monitoring of central venous pressure and oxygen and the use of intravenous fluids, vasopressors, inotropes, and blood transfusions. By 60 days, there were 92 deaths in the protocol-based EGDT group (21.0%), 81 in the protocol-based standard-therapy group (18.2%), and 86 in the usual-care group (18.9%) (relative risk with protocol-based therapy vs. usual care, 1.04; 95% confidence interval [CI], 0.82 to 1.31; P=0.83; relative risk with protocol-based EGDT vs. protocol-based standard therapy, 1.15; 95% CI, 0.88 to 1.51; P=0.31). There were no significant differences in 90-day mortality, 1-year mortality, or the need for organ support. In a multicenter trial conducted in the tertiary care setting, protocol-based resuscitation of patients in whom septic shock was diagnosed in the emergency department did not improve outcomes. (Funded by the National Institute of General Medical Sciences; ProCESS ClinicalTrials.gov number, NCT00510835.).

Skin blood flow and local temperature independently modify sweat rate during passive heat stress in humans.

PubMed

Wingo, Jonathan E; Low, David A; Keller, David M; Brothers, R Matthew; Shibasaki, Manabu; Crandall, Craig G

2010-11-01

Sweat rate (SR) is reduced in locally cooled skin, which may result from decreased temperature and/or parallel reductions in skin blood flow. The purpose of this study was to test the hypotheses that decreased skin blood flow and decreased local temperature each independently attenuate sweating. In protocols I and II, eight subjects rested supine while wearing a water-perfused suit for the control of whole body skin and internal temperatures. While 34°C water perfused the suit, four microdialysis membranes were placed in posterior forearm skin not covered by the suit to manipulate skin blood flow using vasoactive agents. Each site was instrumented for control of local temperature and measurement of local SR (capacitance hygrometry) and skin blood flow (laser-Doppler flowmetry). In protocol I, two sites received norepinephrine to reduce skin blood flow, while two sites received Ringer solution (control). All sites were maintained at 34°C. In protocol II, all sites received 28 mM sodium nitroprusside to equalize skin blood flow between sites before local cooling to 20°C (2 sites) or maintenance at 34°C (2 sites). In both protocols, individuals were then passively heated to increase core temperature ~1°C. Both decreased skin blood flow and decreased local temperature attenuated the slope of the SR to mean body temperature relationship (2.0 ± 1.2 vs. 1.0 ± 0.7 mg·cm(-2)·min(-1)·°C(-1) for the effect of decreased skin blood flow, P = 0.01; 1.2 ± 0.9 vs. 0.07 ± 0.05 mg·cm(-2)·min(-1)·°C(-1) for the effect of decreased local temperature, P = 0.02). Furthermore, local cooling delayed the onset of sweating (mean body temperature of 37.5 ± 0.4 vs. 37.6 ± 0.4°C, P = 0.03). These data demonstrate that local cooling attenuates sweating by independent effects of decreased skin blood flow and decreased local skin temperature.

Blood serotonin levels in autism spectrum disorder: a systematic review and meta-analysis.

PubMed

Gabriele, Stefano; Sacco, Roberto; Persico, Antonio M

2014-06-01

Elevated blood serotonin (5-HT) levels were the first biomarker identified in autism research. Many studies have contrasted blood 5-HT levels in autistic patients and controls, but different measurement protocols, technologies, and biomaterials have been used through the years. We performed a systematic review and meta-analysis to provide an overall estimate of effect size and between-study heterogeneity, while verifying whether and to what extent different methodological approaches influence the strength of this association. Our literature search strategy identified 551 papers, from which 22 studies providing patient and control blood 5-HT values were selected for meta-analysis. Significantly higher 5-HT levels in autistic patients compared to controls were recorded both in whole blood (WB) [O.R.=4.6; (3.1-5.2); P=1.0×10(-12]), and in platelet-rich plasma (PRP) [O.R.=2.6 (1.8-3.9); P=2.7×10(-7)]. Predictably, studies measuring 5-HT levels in platelet-poor plasma (PPP) yielded no significant group difference [O.R.=0.54 (0.2-2-0); P=0.36]. Altogether, elevated 5-HT blood levels were recorded in 28.3% in WB and 22.5% in PRP samples of autistic individuals, as reported in 15 and 4 studies, respectively. Studies employing HPLC vs fluorometric assays yield similar cumulative effect sizes, but the former display much lower variability. In summary, despite some limitations mainly due to small study sample sizes, our results significantly reinforce the reliability of elevated 5-HT blood levels as a biomarker in ASD, providing practical indications potentially useful for its inclusion in multi-marker diagnostic panels for clinical use. Copyright © 2014 Elsevier B.V. and ECNP. All rights reserved.

Individualized Molecular Analyses Guide Efforts (IMAGE): A Prospective Study of Molecular Profiling of Tissue and Blood in Metastatic Triple-Negative Breast Cancer.

PubMed

Parsons, Heather A; Beaver, Julia A; Cimino-Mathews, Ashley; Ali, Siraj M; Axilbund, Jennifer; Chu, David; Connolly, Roisin M; Cochran, Rory L; Croessmann, Sarah; Clark, Travis A; Gocke, Christopher D; Jeter, Stacie C; Kennedy, Mark R; Lauring, Josh; Lee, Justin; Lipson, Doron; Miller, Vincent A; Otto, Geoff A; Rosner, Gary L; Ross, Jeffrey S; Slater, Shannon; Stephens, Philip J; VanDenBerg, Dustin A; Wolff, Antonio C; Young, Lauren E; Zabransky, Daniel J; Zhang, Zhe; Zorzi, Jane; Stearns, Vered; Park, Ben H

2017-01-15

The clinical utility of next-generation sequencing (NGS) in breast cancer has not been demonstrated. We hypothesized that we could perform NGS of a new biopsy from patients with metastatic triple-negative breast cancer (TNBC) in a clinically actionable timeframe. We planned to enroll 40 patients onto a prospective study, Individualized Molecular Analyses Guide Efforts (IMAGE), to evaluate the feasibility of obtaining a new biopsy of a metastatic site, perform NGS (FoundationOne), and convene a molecular tumor board to formulate treatment recommendations within 28 days. We collected blood at baseline and at time of restaging to assess cell-free circulating plasma tumor DNA (ptDNA). We enrolled 26 women with metastatic TNBC who had received ≥1 line of prior chemotherapy, and 20 (77%) underwent NGS of a metastatic site biopsy. Twelve (60%) evaluable patients received treatment recommendations within 28 days of consent. The study closed after 20 patients underwent NGS, based on protocol-specified interim futility analysis. Three patients went on to receive genomically directed therapies. Twenty-four of 26 patients had genetic alterations successfully detected in ptDNA. Among 5 patients, 4 mutations found in tumor tissues were not identified in blood, and 4 mutations found in blood were not found in corresponding tumors. In 9 patients, NGS of follow-up blood samples showed 100% concordance with baseline blood samples. This study demonstrates challenges of performing NGS on prospective tissue biopsies in patients with metastatic TNBC within 28 days, while also highlighting the potential use of blood as a more time-efficient and less invasive method of mutational assessment. Clin Cancer Res; 23(2); 379-86. ©2016 AACR. ©2016 American Association for Cancer Research.

The association of estimated salt intake with blood pressure in a Viet Nam national survey.

PubMed

Jensen, Paul N; Bao, Tran Quoc; Huong, Tran Thi Thanh; Heckbert, Susan R; Fitzpatrick, Annette L; LoGerfo, James P; Ngoc, Truong Le Van; Mokdad, Ali H

2018-01-01

To evaluate the association of salt consumption with blood pressure in Viet Nam, a developing country with a high level of salt consumption. Analysis of a nationally representative sample of Vietnamese adults 25-65 years of age who were surveyed using the World Health Organization STEPwise approach to Surveillance protocol. Participants who reported acute illness, pregnancy, or current use of antihypertensive medications were excluded. Daily salt consumption was estimated from fasting mid-morning spot urine samples. Associations of salt consumption with systolic blood pressure and prevalent hypertension were assessed using adjusted linear and generalized linear models. Interaction terms were tested to assess differences by age, smoking, alcohol consumption, and rural/urban status. The analysis included 2,333 participants (mean age: 37 years, 46% male, 33% urban). The average estimated salt consumption was 10g/day. No associations of salt consumption with blood pressure or prevalent hypertension were observed at a national scale in men or women. The associations did not differ in subgroups defined by age, smoking, or alcohol consumption; however, associations differed between urban and rural participants (p-value for interaction of urban/rural status with salt consumption, p = 0.02), suggesting that higher salt consumption may be associated with higher systolic blood pressure in urban residents but lower systolic blood pressure in rural residents. Although there was no evidence of an association at a national level, associations of salt consumption with blood pressure differed between urban and rural residents in Viet Nam. The reasons for this differential association are not clear, and given the large rate of rural to urban migration experienced in Viet Nam, this topic warrants further investigation.

The association of estimated salt intake with blood pressure in a Viet Nam national survey

PubMed Central

Bao, Tran Quoc; Huong, Tran Thi Thanh; Heckbert, Susan R.; Fitzpatrick, Annette L.; LoGerfo, James P.; Ngoc, Truong Le Van; Mokdad, Ali H.

2018-01-01

Objective To evaluate the association of salt consumption with blood pressure in Viet Nam, a developing country with a high level of salt consumption. Design and setting Analysis of a nationally representative sample of Vietnamese adults 25–65 years of age who were surveyed using the World Health Organization STEPwise approach to Surveillance protocol. Participants who reported acute illness, pregnancy, or current use of antihypertensive medications were excluded. Daily salt consumption was estimated from fasting mid-morning spot urine samples. Associations of salt consumption with systolic blood pressure and prevalent hypertension were assessed using adjusted linear and generalized linear models. Interaction terms were tested to assess differences by age, smoking, alcohol consumption, and rural/urban status. Results The analysis included 2,333 participants (mean age: 37 years, 46% male, 33% urban). The average estimated salt consumption was 10g/day. No associations of salt consumption with blood pressure or prevalent hypertension were observed at a national scale in men or women. The associations did not differ in subgroups defined by age, smoking, or alcohol consumption; however, associations differed between urban and rural participants (p-value for interaction of urban/rural status with salt consumption, p = 0.02), suggesting that higher salt consumption may be associated with higher systolic blood pressure in urban residents but lower systolic blood pressure in rural residents. Conclusions Although there was no evidence of an association at a national level, associations of salt consumption with blood pressure differed between urban and rural residents in Viet Nam. The reasons for this differential association are not clear, and given the large rate of rural to urban migration experienced in Viet Nam, this topic warrants further investigation. PMID:29346423

Adrenal Venous Sampling: Where Is the Aldosterone Disappearing to?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Solar, Miroslav; Ceral, Jiri, E-mail: ceral.jiri@fnhk.c; Krajina, Antonin

Adrenal venous sampling (AVS) is generally considered to be the gold standard in distinguishing unilateral and bilateral aldosterone hypersecretion in primary hyperaldosteronism. However, during AVS, we noticed a considerable variability in aldosterone concentrations among samples thought to have come from the right adrenal glands. Some aldosterone concentrations in these samples were even lower than in samples from the inferior vena cava. We hypothesized that the samples with low aldosterone levels were unintentionally taken not from the right adrenal gland, but from hepatic veins. Therefore, we sought to analyze the impact of unintentional cannulation of hepatic veins on AVS. Thirty consecutivemore » patients referred for AVS were enrolled. Hepatic vein sampling was implemented in our standardized AVS protocol. The data were collected and analyzed prospectively. AVS was successful in 27 patients (90%), and hepatic vein cannulation was successful in all procedures performed. Cortisol concentrations were not significantly different between the hepatic vein and inferior vena cava samples, but aldosterone concentrations from hepatic venous blood (median, 17 pmol/l; range, 40-860 pmol/l) were markedly lower than in samples from the inferior vena cava (median, 860 pmol/l; range, 460-4510 pmol/l). The observed difference was statistically significant (P < 0.001). Aldosterone concentrations in the hepatic veins are significantly lower than in venous blood taken from the inferior vena cava. This finding is important for AVS because hepatic veins can easily be mistaken for adrenal veins as a result of their close anatomic proximity.« less

Normalization of mass cytometry data with bead standards

PubMed Central

Finck, Rachel; Simonds, Erin F.; Jager, Astraea; Krishnaswamy, Smita; Sachs, Karen; Fantl, Wendy; Pe’er, Dana; Nolan, Garry P.; Bendall, Sean C.

2013-01-01

Mass cytometry uses atomic mass spectrometry combined with isotopically pure reporter elements to currently measure as many as 40 parameters per single cell. As with any quantitative technology, there is a fundamental need for quality assurance and normalization protocols. In the case of mass cytometry, the signal variation over time due to changes in instrument performance combined with intervals between scheduled maintenance must be accounted for and then normalized. Here, samples were mixed with polystyrene beads embedded with metal lanthanides, allowing monitoring of mass cytometry instrument performance over multiple days of data acquisition. The protocol described here includes simultaneous measurements of beads and cells on the mass cytometer, subsequent extraction of the bead-based signature, and the application of an algorithm enabling correction of both short- and long-term signal fluctuations. The variation in the intensity of the beads that remains after normalization may also be used to determine data quality. Application of the algorithm to a one-month longitudinal analysis of a human peripheral blood sample reduced the range of median signal fluctuation from 4.9-fold to 1.3-fold. PMID:23512433

A Method for Identification and Analysis of Non-Overlapping Myeloid Immunophenotypes in Humans

PubMed Central

Gustafson, Michael P.; Lin, Yi; Maas, Mary L.; Van Keulen, Virginia P.; Johnston, Patrick B.; Peikert, Tobias; Gastineau, Dennis A.; Dietz, Allan B.

2015-01-01

The development of flow cytometric biomarkers in human studies and clinical trials has been slowed by inconsistent sample processing, use of cell surface markers, and reporting of immunophenotypes. Additionally, the function(s) of distinct cell types as biomarkers cannot be accurately defined without the proper identification of homogeneous populations. As such, we developed a method for the identification and analysis of human leukocyte populations by the use of eight 10-color flow cytometric protocols in combination with novel software analyses. This method utilizes un-manipulated biological sample preparation that allows for the direct quantitation of leukocytes and non-overlapping immunophenotypes. We specifically designed myeloid protocols that enable us to define distinct phenotypes that include mature monocytes, granulocytes, circulating dendritic cells, immature myeloid cells, and myeloid derived suppressor cells (MDSCs). We also identified CD123 as an additional distinguishing marker for the phenotypic characterization of immature LIN-CD33+HLA-DR- MDSCs. Our approach permits the comprehensive analysis of all peripheral blood leukocytes and yields data that is highly amenable for standardization across inter-laboratory comparisons for human studies. PMID:25799053

Traumatic Hemothorax Blood Contains Elevated Levels of Microparticles that are Prothrombotic but Inhibit Platelet Aggregation.

PubMed

Mitchell, Thomas A; Herzig, Maryanne C; Fedyk, Chriselda G; Salhanick, Marc A; Henderson, Aaron T; Parida, Bijaya K; Prat, Nicolas J; Dent, Daniel L; Schwacha, Martin G; Cap, Andrew P

2017-06-01

Autotransfusion of shed blood from traumatic hemothorax is an attractive option for resuscitation of trauma patients in austere environments. However, previous analyses revealed that shed hemothorax (HX) blood is defibrinated, thrombocytopenic, and contains elevated levels of D-dimer. Mixing studies with normal pooled plasma demonstrated hypercoagulability, evoking concern for potentiation of acute traumatic coagulopathy. We hypothesized that induction of coagulopathic changes by shed HX blood may be due to increases in cellular microparticles (MP) and that these may also affect recipient platelet function. Shed HX blood was obtained from 17 adult trauma patients under an Institutional Review Board approved prospective observational protocol. Blood samples were collected every hour up to 4 h after thoracostomy tube placement. The corresponding plasma was isolated and frozen for analysis. The effects of shed HX frozen plasma (HFP) and isolated HX microparticles (HMP) on coagulation and platelet function were assessed through mixing studies with platelet-rich plasma at various dilutions followed by analysis with thromboelastometry (ROTEM), platelet aggregometry (Multiplate), enzyme-linked immunosorbent assays, and flow cytometry. Furthermore, HFP was assessed for von Willebrand factor antigen levels and multimer content, and plasma-free hemoglobin. ROTEM analysis demonstrated that diluted HFP and isolated HMP samples decreased clotting time, clotting formation time, and increased α angle, irrespective of sample concentrations, when compared with diluted control plasma. Isolated HMP inhibited platelet aggregation in response to adenosine diphosphate, arachidonic acid, and collagen. HFP contained elevated levels of fibrin-degradation products and tissue factor compared with control fresh frozen plasma samples. MP concentrations in HFP were significantly increased and enriched in events positive for phosphatidylserine, tissue factor, CD235, CD45, CD41a, and CD14. von Willebrand factor (vWF) multimer analysis revealed significant loss of high molecular weight multimers in HFP samples. Plasma-free hemoglobin levels were 8-fold higher in HFP compared with fresh frozen plasma. HFP induces plasma hypercoagulability that is likely related to increased tissue factor and phosphatidylserine expression originating from cell-derived MP. In contrast, platelet dysfunction is induced by HMP, potentially aggravated by depletion of high molecular weight multimers of vWF. Thus, autologous transfusion of shed traumatic hemothorax blood may induce a range of undesirable effects in patients with acute traumatic coagulopathy.

Assessment of carboxyhemoglobin, hydrogen cyanide and methemoglobin in fire victims: a novel approach.

PubMed

Ferrari, Luis A; Giannuzzi, Leda

2015-11-01

To establish the cause of death, carboxyhemoglobin (COHb), total hemoglobin (tHb), methemoglobin (MetHb), and hydrogen cyanide (HCN) were quantified in the blood of fire victims. We analyzed 32 out of 33 blood samples from forensic autopsy cases in a disastrous polyurethane mattress fire, which caused the deaths of 33 inmates at a prison in Argentina in 2006. The cadaveric blood samples were collected by femoral vein puncture. These samples were analyzed using the IL80 CO-oximeter system for tHb, MetHb, and COHb levels and by microdiffusion for HCN and COHb levels. Blood alcohol (ethanol) and drugs were examined by headspace gas chromatography-flame ionization detection (HS-GC-FID) and GC-mass spectrometry (MS), respectively. Polyurethane mattress samples were analyzed according to the California 117 protocol. The saturation of COHb ranged from 10% to 43%, tHb from 2% to 19.7%, MetHb from 0.10% to 35.7%, and HCN from 0.24 to 15mg/L. These HCN values are higher than the lethal levels reported in the literature. Other toxic components routinely measured (ethanol, methanol, aldehydes, and other volatile compounds) gave negative results in the 32 cases. Neither drugs of abuse nor psychotropic drugs were detected. The results indicate that death in the 32 fire victims was probably caused in part by HCN, generated during the extensive polyurethane decomposition stimulated by a rapid increase in temperature. We also considered the influence of oxygen depletion and the formation of other volatile compounds such as NOx in this disaster, as well as pathological evidence demonstrating that heat was not the cause of death in all victims. Furthermore, statistical analysis showed that the percentage values of COHb and MetHb in the blood were not independent variables, with χ(2)=11.12 (theoretical χ(2)=4.09, degrees of freedom=12, and α=0.05). However, no correlation was found between HCN and MetHb in the blood of the victims. This is the first report to assess the relationship between COHb and MetHb in forensic blood samples. We further discuss other factors that could lead to a lethal atmosphere generated by the fire and compare the data from this disaster with that of other published fire episodes. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

An Evaluation of the Accuracy of the Subtraction Method Used for Determining Platelet Counts in Advanced Platelet-Rich Fibrin and Concentrated Growth Factor Preparations

PubMed Central

Watanabe, Taisuke; Isobe, Kazushige; Suzuki, Taiji; Kawabata, Hideo; Nakamura, Masayuki; Tsukioka, Tsuneyuki; Okudera, Toshimitsu; Okudera, Hajime; Uematsu, Kohya; Okuda, Kazuhiro; Nakata, Koh; Kawase, Tomoyuki

2017-01-01

Platelet concentrates should be quality-assured of purity and identity prior to clinical use. Unlike for the liquid form of platelet-rich plasma, platelet counts cannot be directly determined in solid fibrin clots and are instead calculated by subtracting the counts in other liquid or semi-clotted fractions from those in whole blood samples. Having long suspected the validity of this method, we herein examined the possible loss of platelets in the preparation process. Blood samples collected from healthy male donors were immediately centrifuged for advanced platelet-rich fibrin (A-PRF) and concentrated growth factors (CGF) according to recommended centrifugal protocols. Blood cells in liquid and semi-clotted fractions were directly counted. Platelets aggregated on clot surfaces were observed by scanning electron microscopy. A higher centrifugal force increased the numbers of platelets and platelet aggregates in the liquid red blood cell fraction and the semi-clotted red thrombus in the presence and absence of the anticoagulant, respectively. Nevertheless, the calculated platelet counts in A-PRF/CGF preparations were much higher than expected, rendering the currently accepted subtraction method inaccurate for determining platelet counts in fibrin clots. To ensure the quality of solid types of platelet concentrates chairside in a timely manner, a simple and accurate platelet-counting method should be developed immediately. PMID:29563413

[Assessment of two DNA extraction methods to amplify the pneumolysin gene (PLY) from blood culture samples of Streptococcus pneumoniae].

PubMed

Hernández, Carolina; Durán, Claudia; Ulloa, María Teresa; Prado, Valeria

2004-05-01

Streptococcus pneumoniae is a common etiologic agent of invasive respiratory infections among children under 5 years of age and older adults. Isolation rates of S. pneumoniae by traditional culture techniques are low. To study the sensitivity and specificity of two different DNA extraction methods to amplify the ply gene, applied to three different types of blood culture broths, experimentally inoculated with S. pneumoniae. DNA was extracted from the cultures using an organic method or a technique that consists in dilution, washing with NaOH and concentration of the sample. This was followed by PCR amplification of a 355 pb fragment of the pneumolysin gene (ply). The organic DNA extraction method inhibited the PCR reaction at all concentrations studied (0.6 to 10(6) colony forming units/mL). Using the NaOH extraction, ply gene amplification was positive in all three blood culture broths, but only at concentrations of 10(3) colony forming units/mL, or higher. Using the same DNA extraction method, PCR was negative when the broths were inoculated with seven other related bacterial species, which results in a 100% specificity. Detection of S. pneumoniae by amplification of ply gene from blood cultures using the protocol of NaOH for DNA extraction is specific and provides results in a short lapse. However, the diagnostic sensitivity is not optimal, which limits its clinical use.

[Usefulness of the initial medical examination on matters relating to persons suspected of driving under the influence of amphetamine and its analogs or delta9-tetrahydrocannabinol based on the materials the Department of Forensic Medicine, Pomeranian Medical University in Szczecin].

PubMed

Wolski, Stanisław; Lewandowska, Ewa; Kurzejamska-Parafiniuk, Małgorzata

2013-01-01

Polish law forbids persons to drive under the influence of intoxicating substances, and those after the use of substances producing effects similar to alcohol. Therefore, there is a need to give an opinion based on a blood test, to establish whether or not the person from whom the blood was taken was under the influence of an intoxicating substance or after use of the drug while driving. Some authors reported that the final opinion should take into account chemical and toxicology test results identifying the parent compound and/or the metabolite only, but also the sampling time of the material to be analyzed in relation to the driving time, the result of the medical examination conducted prior to the collection of material for analysis, and the results of screening tests executed at the scene. Circumstances relating to the event, the findings and observations of third parties, and the testimony of the suspect are also relevant. The aim of this study was to evaluate the results of the medical examination in the evaluation of cases concerning driving by persons who were potentially under the influence of amphetamine and its analogs, or delta9-tetrahydrocannabinol, (delta9-THC) and conformity assessment of these results with the results of blood tests. An additional aim was to determine the factors considered by doctors when making their evaluation of patient's condition. The study group consisted of 350 persons suspected of driving while under the influence of amphetamine and its analogs, and/or delta9-THC, from whom blood samples were taken to test amphetamine content or its analogues and/or delta9-tetrahydrocannabinol. Blood tests were carried out according to the existing procedure developed by the department. Blood samples were initially analyzed with immunochemical methods. Positive preliminary results were verified by gas chromatography-mass spectrometry. Statistical analysis was conducted with independent tests for multi-way tables, i.e. the Pearson chi2 test and the 2 x 2 tables Yates' correction was used for the low numbers. Comparison of mean concentrations of amphetamine and delta9-THC in the blood was performed using the U Mann-Whitney test. The analysis revealed a significant correlation between the symptoms and the results of the chemical-toxicological blood tests for mood only. There was no significant correlation between the results of toxicological and physical elements contained in the protocol of blood collection as the skin's appearance, speech, heart rate, pupil, pupil reaction to light, walking, lifting objects off the ground, the Romberg test, the finger-to-nose test and orientation in the space time environment. In the analysis of the relationship between the medical assessment and physical elements significant relationships with the assessment of mood, pupils, pupil reaction to light and gait were found. A significant correlation between the prevalence of symptoms in terms of any/none and medical evaluation was found. 1. Preliminary medical examination based on the blood sampling protocol has no practical importance for determining whether a person is tested under the influence of amphetamines or its analogues and/or delta9-THC. 2. The confirmation of the state "under the influence of narcotics or psychotropics" should only be based on blood test results. 3. Doctors, when completing the blood collection report, are often guided by factors other than the test results when making assessment. 4. The low utility of the medical examination is affected by lack of standardization of test items. Doctors often find symptoms in a subjective manner.

Development and testing of a new disposable sterile device for labelling white blood cells.

PubMed

Signore, A; Glaudemans, A W J M; Malviya, G; Lazzeri, E; Prandini, N; Viglietti, A L; De Vries, E F J; Dierckx, R A J O

2012-08-01

White blood cell (WBC) labelling requires isolation of cells from patient's blood under sterile conditions using sterile materials, buffers and disposables under good manufacturing practice (GMP) conditions. Till now, this limited the use of white blood cell scintigraphy (WBC-S) only to well equipped laboratories with trained personnel. We invented, developed and tested a disposable, sterile, closed device for blood manipulation, WBC purification and radionuclide labelling without exposing patient's blood and the operator to contamination risks. This device prototype and a final industrialized device (Leukokit®) were tested for WBC labelling and compared to standard procedure. Leukokit® was also tested in an international multi-centre study for easiness of WBC purification and labelling. On the device prototype we tested in parallel, with blood samples from 7 volunteers, the labelling procedure compared to the standard procedure of the International Society of Radiolabeled Blood Elements (ISORBE) consensus protocol with respect to cell recovery, labelling efficiency (LE), cell viability (Trypan Blue test) and sterility (haemoculture). On the final Leukokit® we tested the biocompatibility of all components, and again the LE, erythro-sedimentation rate, cell viability, sterility and apyrogenicity. ACD-A, HES and PBS provided by Leukokit® were also compared to Heparin, Dextran and autologous plasma, respectively. In 4 samples, we tested the chemotactic activity of purified WBC against 1 mg/ml of lipopolysaccharide (LPS) and chemotaxis of 99mTc-HMPAO-labelled WBC (925 MBq) was compared to that of unlabelled cells. For the multi-centre study, 70 labellings were performed with the Leukokit® by 9 expert operators and 3 beginners from five centers using blood from both patients and volunteers. Finally, Media-Fill tests were performed by 3 operators on two different days (11 procedures) by replacing blood and kit reagents with bacterial culture media (Tryptic Soy Broth) and testing sterility of aliquots of the medium at the end of procedure. Tests performed with the prototype showed no significant differences with the standard procedure but a faster and safer approach. Tests performed with the final Leukokit® confirmed full biocompatibility, sterility and apyrogenicity of all reagents and plastic ware. Average WBC recovery with Leukokit® was comparable to that of the ISORBE protocol (117x106±24x106 vs. 132x106±29x106 cells, P=not significant). No differences in red blood cells and platelet content were observed. LE was 82% ± 3% for Leukokit® and 65±5% for control (P=0.0003) being PBS vs autologous plasma the main reason of such difference. Cell viability was always >99.9% in both conditions. Chemotactic tests showed no differences between all Leukokit® samples and controls. Haemocultures and Media-Fill tests were always sterile. The procedure was well accepted by expert operators and beginners, with a very fast learning curve (confidence after 2±2 labellings). The invented device offers high level of protection to operators and patients. The derived Leukokit® is safe and easy to use, and gives a high LE of WBC without affecting cell viability and function. Being a registered closed, sterile medical device, it may allow easier and faster WBC labelling that is not limited to only well equipped laboratories. Also simultaneously labelling of multiple patients is possible.

Validation of the Pangao PG-800A11 wrist device assessed according to the European Society of Hypertension and the British Hypertension Society protocols.

PubMed

Xie, Peigen; Wang, Yanling; Xu, Xiaoying; Huang, Fei; Pan, Jingru

2015-04-01

The objective of this study was to determine the accuracy of the Pangao PG-800A11 wrist blood pressure monitor according to the European Society of Hypertension International Protocol (ESH-IP) revision 2010 and the protocol of the British Hypertension Society (BHS). The device evaluations were performed in 85 participants, 33 of whom were included according to the ESH-IP revision 2010 and 52 of whom were included on the basis of the requirements of the BHS protocol. The validation procedure and data analysis followed the protocols precisely. The device achieved an A/A grading for the BHS protocol and passed all phases of the ESH-IP revision 2010 protocol. The mean difference ±SD for the ESH and BHS protocols, respectively, was -0.6±4.5 and -0.8±6.2 mmHg for systolic pressure and 1.2±4.6 and -0.5±5.1 mmHg for diastolic pressure. The device maintained its A/A grading throughout the low, medium, and high-pressure ranges. The Pangao PG-800A11 wrist blood pressure monitor passed all requirements of the ESH-IP revision 2010 and achieved A/A grade of the BHS protocol in an adult population.

A comparison of DNA extraction protocols from blood spotted on FTA cards for the detection of tick-borne pathogens by Reverse Line Blot hybridization.

PubMed

Hailemariam, Zerihun; Ahmed, Jabbar Sabir; Clausen, Peter-Henning; Nijhof, Ard Menzo

2017-01-01

An essential step in the molecular detection of tick-borne pathogens (TBPs) in blood is the extraction of DNA. When cooled storage of blood under field conditions prior to DNA extraction in a dedicated laboratory is not possible, the storage of blood on filter paper forms a promising alternative. We evaluated six DNA extraction methods from blood spotted on FTA Classic ® cards (FTA cards), to determine the optimal protocol for the subsequent molecular detection of TBPs by PCR and the Reverse Line Blot hybridization assay (RLB). Ten-fold serial dilutions of bovine blood infected with Babesia bovis, Theileria mutans, Anaplasma marginale or Ehrlichia ruminantium were made by dilution with uninfected blood and spotted on FTA cards. Subsequently, DNA was extracted from FTA cards using six different DNA extraction protocols. DNA was also isolated from whole blood dilutions using a commercial kit. PCR/RLB results showed that washing of 3mm discs punched from FTA cards with FTA purification reagent followed by DNA extraction using Chelex ® resin was the most sensitive procedure. The detection limit could be improved when more discs were used as starting material for the DNA extraction, whereby the use of sixteen 3mm discs proved to be most practical. The presented best practice method for the extraction of DNA from blood spotted on FTA cards will facilitate epidemiological studies on TBPs. It may be particularly useful for field studies where a cold chain is absent. Copyright © 2016 Elsevier GmbH. All rights reserved.

Validation of the Somnotouch-NIBP noninvasive continuous blood pressure monitor according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Bilo, Grzegorz; Zorzi, Cristina; Ochoa Munera, Juan E; Torlasco, Camilla; Giuli, Valentina; Parati, Gianfranco

2015-10-01

The present study aimed to evaluate the accuracy of the Somnotouch-NIBP noninvasive continuous blood pressure monitor according to the European Society of Hypertension International Protocol revision 2010. Systolic and diastolic blood pressures were sequentially measured in 33 adults (11 women, mean age 63.5±11.9 years) using a mercury sphygmomanometer (two observers) and the Somnotouch-NIBP device (one supervisor). A total of 99 pairs of comparisons were obtained from 33 participants for judgments in two parts with three grading phases. All the validation requirements were fulfilled. The Somnotouch-NIBP device fulfilled the requirements of the part 1 of the validation study. The number of absolute differences between device and observers within 5, 10, and 15 mmHg was 75/99, 90/99, and 96/99, respectively, for systolic blood pressure and 90/99, 99/99, and 99/99, respectively, for diastolic blood pressure. The device also fulfilled the criteria in part 2 of the validation study. Twenty-seven and 31 participants had at least two of the three device-observers differences less than or equal to 5 mmHg for systolic and diastolic blood pressure, respectively. All three device-observer differences were greater than 5 mmHg in two participants for systolic and in one participant for diastolic blood pressure. The Somnotouch-NIBP noninvasive continuous blood pressure monitor has passed the requirements of the International Protocol revision 2010, and hence can be recommended for blood pressure monitoring in adults, at least under conditions corresponding to those investigated in our study.

Sample acceptance time criteria, electronic issue and alloimmunisation in thalassaemia.

PubMed

Trompeter, S; Baxter, L; McBrearty, M; Zatkya, E; Porter, J

2015-12-01

To determine the safety of a 1-week acceptance criteria of sample receipt in laboratory to transfusion commencement in transfusion dependent thalassaemia with respect to alloimmunisation. To determine the safety of electronic issue of blood components in such a setting. Retrospective audit of alloimmunisation (1999-2012) and blood exposure in registered thalassaemia patients at a central London thalassaemia centre where the acceptance criteria for the group and save sample from arrival in the laboratory to the time of issue of blood for transfusion for someone who has been transfused in the last 28 days was 1 week, and there was electronic issue protocol for patients who have always had a negative antibody screen (other than temporary positivity in pregnant women receiving prophylactic anti-D or anti Le-a, Anti Le-b and Anti P1 that are no longer detectable). There were 133 patients with thalassemia variants regularly attending UCLH for review. A total of 105 patients had transfusion dependent thalassaemia (TDT) (7 E-beta thalassaemia, 98 beta thalassaemia major). Ten of the 84 patients who received their transfusions at UCLH were alloimmunised. Seven of them had been alloimmunised prior to arrival at UCLH. Only two patients developed antibodies at UCLH during this period. The prevalence of alloantibody formation of 2% in UCLH transfused patients, with presumptive incidence of 0.01 alloantibodies per 100 units or 0·001 immunisations per person per year compares favourably with other reported series and suggests that 1 week interval with appropriate electronic issue is acceptable practice. © 2015 British Blood Transfusion Society.

Citizens in the commons: blood and genetics in the making of the civic

PubMed Central

Reddy, Deepa S.

2013-01-01

This essay is based on ethnographic fieldwork conducted with the Indian community in Houston, as part of a NIH/NHGRI-sponsored ethics study and sample collection initiative entitled ‘Indian and Hindu Perspectives on Genetic Variation Research.’ Taking a cue from my Indian interlocutors who largely support and readily respond to such initiatives on the grounds that they will undoubtedly serve ‘humanity’ and the common good, I explore notions of the commons that are created in the process of soliciting blood for genetic research. How does blood become the stuff of which a civic discourse is made? How do idealistic individual appeals to donate blood, ethics research protocols, open-source databases, debates on approaches to genetic research, patents and Intellectual Property regulations, markets and the nation-state itself variously engage, limit or further ideas of the common good? Moving much as my interlocutors do, between India and the United States, I explore the nature of the commons that is both imagined and pragmatically reckoned in both local and global diasporic contexts. PMID:24478538

Examining Radiation-Induced In Vivo and In Vitro Gene Expression Changes of the Peripheral Blood in Different Laboratories for Biodosimetry Purposes: First RENEB Gene Expression Study.

PubMed

Abend, M; Badie, C; Quintens, R; Kriehuber, R; Manning, G; Macaeva, E; Njima, M; Oskamp, D; Strunz, S; Moertl, S; Doucha-Senf, S; Dahlke, S; Menzel, J; Port, M

2016-02-01

The risk of a large-scale event leading to acute radiation exposure necessitates the development of high-throughput methods for providing rapid individual dose estimates. Our work addresses three goals, which align with the directive of the European Union's Realizing the European Network of Biodosimetry project (EU-RENB): 1. To examine the suitability of different gene expression platforms for biodosimetry purposes; 2. To perform this examination using blood samples collected from prostate cancer patients (in vivo) and from healthy donors (in vitro); and 3. To compare radiation-induced gene expression changes of the in vivo with in vitro blood samples. For the in vitro part of this study, EDTA-treated whole blood was irradiated immediately after venipuncture using single X-ray doses (1 Gy/min(-1) dose rate, 100 keV). Blood samples used to generate calibration curves as well as 10 coded (blinded) samples (0-4 Gy dose range) were incubated for 24 h in vitro, lysed and shipped on wet ice. For the in vivo part of the study PAXgene tubes were used and peripheral blood (2.5 ml) was collected from prostate cancer patients before and 24 h after the first fractionated 2 Gy dose of localized radiotherapy to the pelvis [linear accelerator (LINAC), 580 MU/min, exposure 1-1.5 min]. Assays were run in each laboratory according to locally established protocols using either microarray platforms (2 laboratories) or qRT-PCR (2 laboratories). Report times on dose estimates were documented. The mean absolute difference of estimated doses relative to the true doses (Gy) were calculated. Doses were also merged into binary categories reflecting aspects of clinical/diagnostic relevance. For the in vitro part of the study, the earliest report time on dose estimates was 7 h for qRT-PCR and 35 h for microarrays. Methodological variance of gene expression measurements (CV ≤10% for technical replicates) and interindividual variance (≤twofold for all genes) were low. Dose estimates based on one gene, ferredoxin reductase (FDXR), using qRT-PCR were as precise as dose estimates based on multiple genes using microarrays, but the precision decreased at doses ≥2 Gy. Binary dose categories comprising, for example, unexposed compared with exposed samples, could be completely discriminated with most of our methods. Exposed prostate cancer blood samples (n = 4) could be completely discriminated from unexposed blood samples (n = 4, P < 0.03, two-sided Fisher's exact test) without individual controls. This could be performed by introducing an in vitro-to-in vivo correction factor of FDXR, which varied among the laboratories. After that the in vitro-constructed calibration curves could be used for dose estimation of the in vivo exposed prostate cancer blood samples within an accuracy window of ±0.5 Gy in both contributing qRT-PCR laboratories. In conclusion, early and precise dose estimates can be performed, in particular at doses ≤2 Gy in vitro. Blood samples of prostate cancer patients exposed to 0.09-0.017 Gy could be completely discriminated from pre-exposure blood samples with the doses successfully estimated using adjusted in vitro-constructed calibration curves.

An evidence-based approach to breastfeeding neonates at risk for hypoglycemia.

PubMed

Csont, Georgia Lowmaster; Groth, Susan; Hopkins, Patrick; Guillet, Ronnie

2014-01-01

The revised standard of care for breastfeeding infants at risk of developing hypoglycemia during transitioning to extrauterine life was developed using the American Academy of Pediatrics (AAP) 2011 hypoglycemia guidelines, the Academy of Breastfeeding Medicine protocol, and staff input. A pre/postimplementation chart audit indicated support of infant safety by glucose stabilization, breastfeeding within the first hour of life, and breastfeeding frequency without an increase in blood sampling, formula use, or admissions to the special care nursery. © 2014 AWHONN, the Association of Women's Health, Obstetric and Neonatal Nurses.

Evaluation of a xeno-free protocol for long-term cryopreservation of cord blood cells.

PubMed

Mairhofer, M; Schulz, J C; Parth, M; Beer, U; Zimmermann, H; Kolbus, A

2013-01-01

Cord blood is regarded as a powerful source for adult stem cells. Cord blood transplants have been used successfully to treat children and adults in autologous and allogeneic settings. Nevertheless, in many cases, the clinically relevant cell number (CD34+ cells and total leukocytes) is a limiting factor. To enable standardized cell banking and future in vitro expansion of adult stem/progenitor cells, elimination of serum, which inevitably differs from lot to lot and donor to donor, is highly desirable. Here, we demonstrate the feasibility of a xeno-free, chemically defined cryopreservation procedure for cord blood-derived cells over a period of 1 year. Cell recoveries with respect to retrieval of clinically relevant CD34+ cells, colony-forming units, and in vitro cultures of erythroid progenitor cells under standardized conditions were analyzed after 1 week or 1 year of cryopreservation and found to be very high and similar to the samples before freezing. The established xeno-free procedure is an important step toward using the full potential of adult stem cells from cord blood, enabling the elimination of serum-derived factors negatively influencing proliferation, differentiation, and survival of hematopoietic stem cells.

Automated selected reaction monitoring data analysis workflow for large-scale targeted proteomic studies.

PubMed

Surinova, Silvia; Hüttenhain, Ruth; Chang, Ching-Yun; Espona, Lucia; Vitek, Olga; Aebersold, Ruedi

2013-08-01

Targeted proteomics based on selected reaction monitoring (SRM) mass spectrometry is commonly used for accurate and reproducible quantification of protein analytes in complex biological mixtures. Strictly hypothesis-driven, SRM assays quantify each targeted protein by collecting measurements on its peptide fragment ions, called transitions. To achieve sensitive and accurate quantitative results, experimental design and data analysis must consistently account for the variability of the quantified transitions. This consistency is especially important in large experiments, which increasingly require profiling up to hundreds of proteins over hundreds of samples. Here we describe a robust and automated workflow for the analysis of large quantitative SRM data sets that integrates data processing, statistical protein identification and quantification, and dissemination of the results. The integrated workflow combines three software tools: mProphet for peptide identification via probabilistic scoring; SRMstats for protein significance analysis with linear mixed-effect models; and PASSEL, a public repository for storage, retrieval and query of SRM data. The input requirements for the protocol are files with SRM traces in mzXML format, and a file with a list of transitions in a text tab-separated format. The protocol is especially suited for data with heavy isotope-labeled peptide internal standards. We demonstrate the protocol on a clinical data set in which the abundances of 35 biomarker candidates were profiled in 83 blood plasma samples of subjects with ovarian cancer or benign ovarian tumors. The time frame to realize the protocol is 1-2 weeks, depending on the number of replicates used in the experiment.

Thoracic Impedance as a Potential Indicator of Gz-induced Presyncope

NASA Technical Reports Server (NTRS)

Howarth, M. S.; Moore, F. B.; Hinghofer-Szalkay, H.; Jezova, D.; Diedrich, A.; Ferris, M. B.; Patwardhan, A. R.; Knapp, C. F.; Evans, J. M.

2008-01-01

We investigated fluid shifts and regulatory responses to variations of posture, exercise, Gz level and radius of rotation in subjects riding NASA Ames 20G centrifuge. Results are from 4 protocols that address radius and exercise effects only. Protocol A: After 10 min supine control, 12 healthy men (35 9 yr, 82.8 7.9 kg) were exposed to rotational 1 Gz (2.5 m radius) for 2 min followed by 20 min alternating between 1 and 1.25 Gz. Blood samples were taken pre and post spin. Protocol B: Same as A, but lower limb exercise (70% V02max) preceded ramps to 1.25 Gz. Protocol C: Same as A but radius of rotation 8.3 m. Protocol D: Same as B but at 8.3 m. The 8 subjects who completed all protocols, increased heart rate (HR) from control by: A: 5, B: 39, C: 11, D: 44 bpm; and the 4 who did not: A: 6, B: 35, C: 20, D: 50 bpm. For thoracic fluid volume, (bioimpedance), the 8 subjects changed from control: A: -394, B: -548, C: -537, D: -708 mL; and the 4: A: -516, B: -652, C: -583, D: -1263 mL. The 4 subjects lost more thoracic fluid volume than the 8, especially in protocol D. A slightly greater increase in HR for the 4 compared to the 8 was not adequate to maintain cardiac output during D. Our data support the concept that thoracic impedance can detect inability to return adequate fluid to the heart, thereby predicting presyncope.

Lack of effect of drinking water barium on cardiovascular risk factors.

PubMed Central

Wones, R G; Stadler, B L; Frohman, L A

1990-01-01

Higher cardiovascular mortality has been associated in a single epidemiological study with higher levels of barium in drinking water. The purpose of this study was to determine whether drinking water barium at levels found in some U.S. communities alters the known risk factors for cardiovascular disease. Eleven healthy men completed a 10-week dose-response protocol in which diet was controlled (600 mg cholesterol; 40% fat, 40% carbohydrate, 20% protein; sodium and potassium controlled at the subject's pre-protocol estimated intake). Other aspects of the subjects' lifestyles known to affect cardiac risk factors were controlled, and the barium content (as barium chloride) of the drinking water (1.5 L/day) was varied from 0 (first 2 weeks), to 5 ppm (next 4 weeks), to 10 ppm (last 4 weeks). Multiple blood and urine samples, morning and evening blood pressure measurements, and 48-hr electrocardiographic monitoring were performed at each dose of barium. There were no changes in morning or evening systolic or diastolic blood pressures, plasma cholesterol or lipoprotein or apolipoprotein levels, serum potassium or glucose levels, or urine catecholamine levels. There were no arrhythmias related to barium exposure detected on continuous electrocardiographic monitoring. A trend was seen toward increased total serum calcium levels with exposure to barium, which was of borderline statistical significance and of doubtful clinical significance. In summary, drinking water barium at levels of 5 and 10 ppm did not appear to affect any of the known modifiable cardiovascular risk factors. PMID:2384067

Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays.

PubMed

Abras, Alba; Ballart, Cristina; Llovet, Teresa; Roig, Carme; Gutiérrez, Cristina; Tebar, Silvia; Berenguer, Pere; Pinazo, María-Jesús; Posada, Elizabeth; Gascón, Joaquim; Schijman, Alejandro G; Gállego, Montserrat; Muñoz, Carmen

2018-01-01

Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process. We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately. This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.

Field Immune Assessment during Simulated Planetary Exploration in the Canadian Arctic

NASA Technical Reports Server (NTRS)

Crucian, Brian; Lee, Pascal; Stowe, Raymond; Jones, Jeff; Effenhauser, Rainer; Widen, Raymond; Sams, Clarence

2006-01-01

Dysregulation of the immune system has been shown to occur during space flight, although the detailed nature of the phenomenon and the clinical risks for exploration class missions has yet to be established. In addition, the growing clinical significance of immune system evaluation combined with epidemic infectious disease rates in third world countries provides a strong rationale for the development of field-compatible clinical immunology techniques and equipment. In July 2002 NASA performed a comprehensive field immunology assessment on crewmembers participating in the Haughton-Mars Project (HMP) on Devon Island in the high Canadian Arctic. The purpose of the study was to evaluate mission-associated effects on the human immune system, as well as to evaluate techniques developed for processing immune samples in remote field locations. Ten HMP-2002 participants volunteered for the study. A field protocol was developed at NASA-JSC for performing sample collection, blood staining/processing for immunophenotype analysis, wholeblood mitogenic culture for functional assessments and cell-sample preservation on-location at Devon Island. Specific assays included peripheral leukocyte distribution; constitutively activated T cells, intracellular cytokine profiles and plasma EBV viral antibody levels. Study timepoints were L-30, midmission and R+60. The protocol developed for immune sample processing in remote field locations functioned properly. Samples were processed in the field location, and stabilized for subsequent analysis at the Johnson Space Center in Houston. The data indicated that some phenotype, immune function and stress hormone changes occurred in the HMP field participants that were largely distinct from pre-mission baseline and post-mission recovery data. These immune changes appear similar to those observed in Astronauts following spaceflight. The sample processing protocol developed for this study may have applications for immune assessment during exploration-class space missions or in remote terrestrial field locations. The data validate the use of the HMP as a ground-based spaceflight/planetary exploration analog for some aspects of human physiology.

The UAE healthy future study: a pilot for a prospective cohort study of 20,000 United Arab Emirates nationals.

PubMed

Abdulle, Abdishakur; Alnaeemi, Abdullah; Aljunaibi, Abdullah; Al Ali, Abdulrahman; Al Saedi, Khaled; Al Zaabi, Eiman; Oumeziane, Naima; Al Bastaki, Marina; Al-Houqani, Mohammed; Al Maskari, Fatma; Al Dhaheri, Ayesha; Shah, Syed M; Loney, Tom; El-Sadig, Mohamed; Oulhaj, Abderrahim; Wareth, Leila Abdel; Al Mahmeed, Wael; Alsafar, Habiba; Hirsch, Benjamin; Al Anouti, Fatme; Yaaqoub, Jamila; Inman, Claire K; Al Hamiz, Aisha; Al Hosani, Ayesha; Haji, Muna; Alsharid, Teeb; Al Zaabi, Thekra; Al Maisary, Fatima; Galani, Divya; Sprosen, Tim; El Shahawy, Omar; Ahn, Jiyoung; Kirchhoff, Tomas; Ramasamy, Ravichandran; Schmidt, Ann Marie; Hayes, Richard; Sherman, Scott; Ali, Raghib

2018-01-05

The United Arab Emirates (UAE) is faced with a rapidly increasing burden of non-communicable diseases including obesity, diabetes, and cardiovascular disease. The UAE Healthy Future study is a prospective cohort designed to identify associations between risk factors and these diseases amongst Emiratis. The study will enroll 20,000 UAE nationals aged ≥18 years. Environmental and genetic risk factors will be characterized and participants will be followed for future disease events. As this was the first time a prospective cohort study was being planned in the UAE, a pilot study was conducted in 2015 with the primary aim of establishing the feasibility of conducting the study. Other objectives were to evaluate the implementation of the main study protocols, and to build adequate capacity to conduct advanced clinical laboratory analyses. Seven hundred sixty nine UAE nationals aged ≥18 years were invited to participate voluntarily in the pilot study. Participants signed an informed consent, completed a detailed questionnaire, provided random blood, urine, and mouthwash samples and were assessed for a series of clinical measures. All specimens were transported to the New York University Abu Dhabi laboratories where samples were processed and analyzed for routine chemistry and hematology. Plasma, serum, and a small whole blood sample for DNA extraction were aliquoted and stored at -80 °C for future analyses. Overall, 517 Emirati men and women agreed to participate (68% response rate). Of the total participants, 495 (95.0%), 430 (82.2%), and 492 (94.4%), completed the questionnaire, physical measurements, and provided biological samples, respectively. The pilot study demonstrated the feasibility of recruitment and completion of the study protocols for the first large-scale cohort study designed to identify emerging risk factors for the major non-communicable diseases in the region.

A clinical pathway for the postoperative management of hypocalcemia after pediatric thyroidectomy reduces blood draws.

PubMed

Patel, Neha A; Bly, Randall A; Adams, Seth; Carlin, Kristen; Parikh, Sanjay R; Dahl, John P; Manning, Scott

2018-02-01

Postoperative calcium management is challenging following pediatric thyroidectomy given potential limitations in self-reporting symptoms and compliance with phlebotomy. A protocol was created at our tertiary children's institution utilizing intraoperative parathyroid hormone (PTH) levels to guide electrolyte management during hospitalization. The objective of this study was to determine the effect of a new thyroidectomy postoperative management protocol on two primary outcomes: (1) the number of postoperative calcium blood draws and (2) the length of hospital stay. Institutional review board approved retrospective study (2010-2016). Consecutive pediatric total thyroidectomy and completion thyroidectomy ± neck dissection cases from 1/1/2010 through 8/5/2016 at a single tertiary children's institution were retrospectively reviewed before and after initiation of a new management protocol. All cases after 2/1/2014 comprised the experimental group (post-protocol implementation). The pre-protocol control group consisted of cases prior to 2/1/2014. Multivariable linear and Poisson regression models were used to compare the control and experimental groups for outcome measure of number of calcium lab draws and hospital length of stay. 53 patients were included (n = 23, control group; n = 30 experimental group). The median age was 15 years. 41 patients (77.4%) were female. Postoperative calcium draws decreased from a mean of 5.2 to 3.6 per day post-protocol implementation (Rate Ratio = 0.70, p < .001), adjusting for covariates. The mean number of total inpatient calcium draws before protocol initiation was 13.3 (±13.20) compared to 7.2 (±4.25) in the post-protocol implementation group. Length of stay was 2.1 days in the control group and 1.8 days post-protocol implementation (p = .29). Patients who underwent concurrent neck dissection had a longer mean length of stay of 2.32 days compared to 1.66 days in those patients who did not undergo a neck dissection (p = .02). Hypocalcemia was also associated with a longer mean length of stay of 2.41 days compared to 1.60 days in patients who did not develop hypocalcemia (p < .01). The number of calcium blood draws was significantly reduced after introduction of a standardized protocol based on intraoperative PTH levels. The hospital length of stay did not change. Adoption of a standardized postoperative protocol based on intraoperative PTH levels may reduce the number of blood draws in children undergoing thyroidectomy. Copyright © 2017 Elsevier B.V. All rights reserved.

Providing Hemostatic and Blood Conservation Options for Jehovah's Witness Patients in a Large Medical System.

PubMed

Bai, Yu; Castillo, Brian S; Tchakarov, Amanda; Escobar, Miguel A; Cotton, Bryan A; Holcomb, John B; Brown, Robert E

2016-12-01

People of the Jehovah's Witness faith believe that they shall "abstain from blood." Because of this belief, we encounter the challenges from Jehovah's Witness patients who actively seek medical care for themselves and their children, but refuse the transfusion of blood products, which may result in increased morbidity and mortality in this patient population. With the development/availability of new hemostatic/coagulation products and the advances in medical technology, we, in collaboration with our clinical colleagues and our local Jehovah's Witness leadership, have developed a clinical guideline comprising medical protocol and surgical strategy for patients refusing blood products. Included in the medical protocol is an informative handout on related details to help treating physicians and patients make informed decisions about transfusion alternatives. Together, we have entered the medical protocol into the entire Memorial Hermann Hospital's electronic system. We report the detailed development and implementation process in order to share our experience and encourage others to develop their own management plan for this patient population. © 2016 by the Association of Clinical Scientists, Inc.

Preventing blood transfusion failures: FMEA, an effective assessment method.

PubMed

Najafpour, Zhila; Hasoumi, Mojtaba; Behzadi, Faranak; Mohamadi, Efat; Jafary, Mohamadreza; Saeedi, Morteza

2017-06-30

Failure Mode and Effect Analysis (FMEA) is a method used to assess the risk of failures and harms to patients during the medical process and to identify the associated clinical issues. The aim of this study was to conduct an assessment of blood transfusion process in a teaching general hospital, using FMEA as the method. A structured FMEA was recruited in our study performed in 2014, and corrective actions were implemented and re-evaluated after 6 months. Sixteen 2-h sessions were held to perform FMEA in the blood transfusion process, including five steps: establishing the context, selecting team members, analysis of the processes, hazard analysis, and developing a risk reduction protocol for blood transfusion. Failure modes with the highest risk priority numbers (RPNs) were identified. The overall RPN scores ranged from 5 to 100 among which, four failure modes were associated with RPNs over 75. The data analysis indicated that failures with the highest RPNs were: labelling (RPN: 100), transfusion of blood or the component (RPN: 100), patient identification (RPN: 80) and sampling (RPN: 75). The results demonstrated that mis-transfusion of blood or blood component is the most important error, which can lead to serious morbidity or mortality. Provision of training to the personnel on blood transfusion, knowledge raising on hazards and appropriate preventative measures, as well as developing standard safety guidelines are essential, and must be implemented during all steps of blood and blood component transfusion.

[Effectivity of screening, concepts and attitudes towards metabolic síndrome: a study in bipolar patients followed in Hospital Santa Maria psychiatric consultation].

PubMed

Guerreiro, Diogo Frasquilho; Navarro, Rita; Telles-Correia, Diogo; Martins, Paulo; Trigo, Elsa; Silva, Manuela; Neves, António; Góis, Carlos; Figueira, Maria Luísa

2010-01-01

The Metabolic Syndrome (MS) is constituted by a set of specific metabolic alterations being postulated that the main dysfunction is insulin resistance, associated with abdominal type obesity, hypertension, hyperglycemia and dyslipidemia. Epidemiological data indicates prevalence of MS of about 25%. Estimates point to higher prevalence of MS in bipolar (BP) patients, between 30 to 35%. Cost-effective screening methods, not recurring to blood test, have been researched. Test the viability of MS screening without using blood tests. Analyse knowledge and importance given to the issue of MS in Bipolar patients. Observational, cross-sectional, exploratory study. Random sample of 15 BP patients, in euthymic phase, between 18 and 65 years. Semi-structured interview, YMRS, HAMD were applied. MS diagnosis was investigated according to the International Diabetes Federation (IDF), including blood tests. Screening of MS was defined positive if blood pressure > or = to 130/85 or on anti-hypertensive medication and Abdominal Perimeter > 90 cm in males or > 80 cm in females. Afterwards a questionnaire about knowledge, attitudes and concerns on MS was applied. 14 patients completed the investigation protocol, 1 patient didn't do blood testing for unknown reasons. Five patients (36%) met IDF criteria for MS. Screening sensitivity was 80% and specificity 78% on our sample (1 false positive and 2 false negative). Twelve patients (80%) were overweight or obese. Mean IMC in patients that met IDF criteria for MS was 30 while in the other group mean IMC was 26, showing statistical significance. Only 3 (20%) have ever heard about MS, but the majority of the patients were concerned, in decreasing order, about weight gain, blood pressure cholesterol and hyperglycemia control. Although limited by small sample size, this study strengthens the idea that MS screening can be effective in clinical practice, it also indicates the need to educate BP patients about MS and to prevent overweight.

Indirect antiglobulin test-crossmatch using low-ionic-strength saline-albumin enhancement medium and reduced incubation time: effectiveness in the detection of most clinically significant antibodies and impact on blood utilization.

PubMed

Dinardo, C L; Bonifácio, S L; Mendrone, A

2014-01-01

Indirect antiglobulin test-crossmatch (IAT-XM) using enhancement media such as low-ionic-strength saline (LISS) and polyethylene glycol (PEG) usually requires 15 minutes of incubation. These methods are necessary when testing samples from blood recipients who have a higher risk of alloimmunization. In emergency situations, IAT-XM can be time-consuming and can influence presurgery routine, resulting in more red blood cell (RBC) units being tested and stored to avoid the transfusion of uncrossmatched ones. The objective of this study was to evaluate the performance of a LISS-albumin enhancer to intensify antigen-antibody reaction after 5 minutes of 37oC incubation and compare this performance with that of other enhancers, gel, and conventional tube testing. Second, the study evaluated the impact of this method's implementation in the C:T ratio (crossmatched to transfused RBC units) of a transfusion laboratory. Ninety serum samples containing alloantibodies of potential clinical significance were tested against phenotyped RBCs using four different methods: (1) tube with LISS-albumin enhancer (5 minutes of incubation), (2) tube with LISS-albumin and PEG (15 minutes of incubation), (3) gel, and (4) conventional tube method (60 minutes of incubation). In parallel, the study compared the C:T ratio of a tertiary-hospital transfusion laboratory in two different periods: 3 months before and 3 months after the implementation of the 5-minute IAT-XM protocol. The use of LISS-albumin with 5 minutes of incubation exhibited the same performance as LISS-albumin, PEG, and gel with 15 minutes of incubation. Conventional tube method results were equally comparable, but reactions were significantly less intense, except for anti-c (p = 0.406). Accuracy was 100 percent for all selected methods. After the implementation of the 5-minute IAT-XM protocol, the C:T ratio fell from 2.74 to 1.29 (p < 0.001). IAT-XM can have its incubation time reduced to 5 minutes with the use of LISS-albumin enhancement. We suggest this strategy should be used to quickly prepare RBC units for surgical patients, keeping transfusion safety without compromising blood supplies.

Using droplet digital PCR to analyze MYCN and ALK copy number in plasma from patients with neuroblastoma.

PubMed

Lodrini, Marco; Sprüssel, Annika; Astrahantseff, Kathy; Tiburtius, Daniela; Konschak, Robert; Lode, Holger N; Fischer, Matthias; Keilholz, Ulrich; Eggert, Angelika; Deubzer, Hedwig E

2017-10-17

The invasive nature of surgical biopsies deters sequential application, and single biopsies often fail to reflect tumor dynamics, intratumor heterogeneity and drug sensitivities likely to change during tumor evolution and treatment. Implementing molecular characterization of cell-free neuroblastoma-derived DNA isolated from blood plasma could improve disease assessment for treatment selection and monitoring of patients with high-risk neuroblastoma. We established droplet digital PCR (ddPCR) protocols for MYCN and ALK copy number status in plasma from neuroblastoma patients. Our ddPCR protocol accurately discriminated between MYCN and ALK amplification, gain and normal diploid status in a large panel of neuroblastoma cell lines, and discrepancies with reported MYCN and ALK status were detected, including a high-level MYCN amplification in NB-1, a MYCN gain in SH-SY5Y, a high-level ALK amplification in IMR-32 and ALK gains in BE(2)-C, Kelly, SH-SY5Y and LAN-6. MYCN and ALK status were also reliably determined from cell-free DNA derived from medium conditioned by the cell lines. MYCN and ALK copy numbers of subcutaneous neuroblastoma xenograft tumors were accurately determined from cell-free DNA in the mouse blood plasma. In a final validation step, we accurately distinguished MYCN and ALK copy numbers of the corresponding primary tumors using retrospectively collected blood plasma samples from 10 neuroblastoma patients. Our data justify the further development of molecular disease characterization using cell-free DNA in blood plasma from patients with neuroblastoma. This expanded molecular diagnostic palette may improve monitoring of disease progression including relapse and metastatic events as well as therapy success or failure in high-risk neuroblastoma patients.

Using droplet digital PCR to analyze MYCN and ALK copy number in plasma from patients with neuroblastoma

PubMed Central

Lodrini, Marco; Sprüssel, Annika; Astrahantseff, Kathy; Tiburtius, Daniela; Konschak, Robert; Lode, Holger N.; Fischer, Matthias; Keilholz, Ulrich; Eggert, Angelika; Deubzer, Hedwig E.

2017-01-01

The invasive nature of surgical biopsies deters sequential application, and single biopsies often fail to reflect tumor dynamics, intratumor heterogeneity and drug sensitivities likely to change during tumor evolution and treatment. Implementing molecular characterization of cell-free neuroblastoma-derived DNA isolated from blood plasma could improve disease assessment for treatment selection and monitoring of patients with high-risk neuroblastoma. We established droplet digital PCR (ddPCR) protocols for MYCN and ALK copy number status in plasma from neuroblastoma patients. Our ddPCR protocol accurately discriminated between MYCN and ALK amplification, gain and normal diploid status in a large panel of neuroblastoma cell lines, and discrepancies with reported MYCN and ALK status were detected, including a high-level MYCN amplification in NB-1, a MYCN gain in SH-SY5Y, a high-level ALK amplification in IMR-32 and ALK gains in BE(2)-C, Kelly, SH-SY5Y and LAN-6. MYCN and ALK status were also reliably determined from cell-free DNA derived from medium conditioned by the cell lines. MYCN and ALK copy numbers of subcutaneous neuroblastoma xenograft tumors were accurately determined from cell-free DNA in the mouse blood plasma. In a final validation step, we accurately distinguished MYCN and ALK copy numbers of the corresponding primary tumors using retrospectively collected blood plasma samples from 10 neuroblastoma patients. Our data justify the further development of molecular disease characterization using cell-free DNA in blood plasma from patients with neuroblastoma. This expanded molecular diagnostic palette may improve monitoring of disease progression including relapse and metastatic events as well as therapy success or failure in high-risk neuroblastoma patients. PMID:29156716

Modified salting-out method: high-yield, high-quality genomic DNA extraction from whole blood using laundry detergent.

PubMed

Nasiri, H; Forouzandeh, M; Rasaee, M J; Rahbarizadeh, F

2005-01-01

Different approaches have been used to extract DNA from whole blood. In most of these methods enzymes (such as proteinase K and RNAse A) or toxic organic solvents (such as phenol or guanidine isothiocyanate) are used. Since these enzymes are expensive, and most of the materials that are used routinely are toxic, it is desirable to apply an efficient DNA extraction procedure that does not require the use of such materials. In this study, genomic DNA was extracted by the salting-out method, but instead of using an analytical-grade enzyme and chemical detergents, as normally used for DNA isolation, a common laundry powder was used. Different concentrations of the powder were tested, and proteins were precipitated by NaCl-saturated distilled water. Finally, DNA precipitation was performed with the use of 96% ethanol. From the results, we conclude that the optimum concentration of laundry powder for the highest yield and purity of isolated DNA is 30 mg/mL. The procedure was optimized, and a final protocol is suggested. Following the same protocol, DNA was extracted from 100 blood samples, and their amounts were found to be >50 microg/mL of whole blood. The integrity of the DNA fragments was confirmed by agarose gel electrophoresis. Furthermore, the extracted DNA was used as a template for PCR reaction. The results obtained from PCR showed that the final solutions of extracted DNA did not contain any inhibitory material for the enzyme used in the PCR reaction, and indicated that the isolated DNA was of good quality. These results show that this method is simple, fast, safe, and cost-effective, and can be used in medical laboratories and research centers. Copyright 2005 Wiley-Liss, Inc.

Peak oxygen uptake in a sprint interval testing protocol vs. maximal oxygen uptake in an incremental testing protocol and their relationship with cross-country mountain biking performance.

PubMed

Hebisz, Rafał; Hebisz, Paulina; Zatoń, Marek; Michalik, Kamil

2017-04-01

In the literature, the exercise capacity of cyclists is typically assessed using incremental and endurance exercise tests. The aim of the present study was to confirm whether peak oxygen uptake (V̇O 2peak ) attained in a sprint interval testing protocol correlates with cycling performance, and whether it corresponds to maximal oxygen uptake (V̇O 2max ) determined by an incremental testing protocol. A sample of 28 trained mountain bike cyclists executed 3 performance tests: (i) incremental testing protocol (ITP) in which the participant cycled to volitional exhaustion, (ii) sprint interval testing protocol (SITP) composed of four 30 s maximal intensity cycling bouts interspersed with 90 s recovery periods, (iii) competition in a simulated mountain biking race. Oxygen uptake, pulmonary ventilation, work, and power output were measured during the ITP and SITP with postexercise blood lactate and hydrogen ion concentrations collected. Race times were recorded. No significant inter-individual differences were observed in regards to any of the ITP-associated variables. However, 9 individuals presented significantly increased oxygen uptake, pulmonary ventilation, and work output in the SITP compared with the remaining cyclists. In addition, in this group of 9 cyclists, oxygen uptake in SITP was significantly higher than in ITP. After the simulated race, this group of 9 cyclists achieved significantly better competition times (99.5 ± 5.2 min) than the other cyclists (110.5 ± 6.7 min). We conclude that mountain bike cyclists who demonstrate higher peak oxygen uptake in a sprint interval testing protocol than maximal oxygen uptake attained in an incremental testing protocol demonstrate superior competitive performance.

Performance of the Colson MAM BP 3AA1-2 automatic blood pressure monitor according to the European Society of Hypertension validation protocol.

PubMed

Pereira, Telmo; Maldonado, João

2005-11-01

To evaluate the performance of the Colson MAM BP 3AA1-2 oscillometric automatic blood pressure monitor according to the validation protocol of the European Society of Hypertension, testing its suitability for self-measurement of blood pressure. The performance of the device was assessed in relation to various clinical variables, including age, gender, body mass index, arm circumference and arterial stiffness. 33 subjects (15 men and 18 women), with a mean age of 47 +/- 10 years, were studied according to the procedures laid down in the European Society of Hypertension validation protocol. Sequential same-arm blood pressure measurements were made, alternating between a mercury standard and the automatic device. The differences among the test-control measurements were assessed and divided into categorization zones of 5, 10 and 15 mmHg discrepancy. Aortic pulse wave velocity was assessed in all subjects with a Complior device (Colson, Paris). The Colson MAM BP 3AA1-2 passed all three phases of the protocol for both systolic and diastolic blood pressure. The mean differences between the test and control measurements were -1.0 +/- 5.0 mmHg for systolic blood pressure and -1.1 +/- 4.1 mmHg for diastolic blood pressure. Both standard deviations are well below the 8 mmHg limit proposed by the Association for the Advancement of Medical Instrumentation. The predictive value of various clinical variables for the discrepancies was assessed by a regression model analysis, with no variable being found that independently undermined the performance of the monitor. In another regression analysis, we found a similar relation between test and control blood pressures and aortic pulse wave velocity, a widely recognized and validated index of target organ damage. These data show that the Colson MAM BP 3AA1-2 satisfies the quality requirements proposed by the European Society of Hypertension, demonstrating its suitability for inclusion in integrated programs of clinical surveillance based on self-measurement of blood pressure. The uniformity of its performance over a wide spectrum of clinical characteristics and the relation found with pulse wave velocity further reinforce its clinical validity.

Automated genomic DNA purification options in agricultural applications using MagneSil paramagnetic particles

NASA Astrophysics Data System (ADS)

Bitner, Rex M.; Koller, Susan C.

2002-06-01

The automated high throughput purification of genomic DNA form plant materials can be performed using MagneSil paramagnetic particles on the Beckman-Coulter FX, BioMek 2000, and the Tecan Genesis robot. Similar automated methods are available for DNA purifications from animal blood. These methods eliminate organic extractions, lengthy incubations and cumbersome filter plates. The DNA is suitable for applications such as PCR and RAPD analysis. Methods are described for processing traditionally difficult samples such as those containing large amounts of polyphenolics or oils, while still maintaining a high level of DNA purity. The robotic protocols have ben optimized for agricultural applications such as marker assisted breeding, seed-quality testing, and SNP discovery and scoring. In addition to high yield purification of DNA from plant samples or animal blood, the use of Promega's DNA-IQ purification system is also described. This method allows for the purification of a narrow range of DNA regardless of the amount of additional DNA that is present in the initial sample. This simultaneous Isolation and Quantification of DNA allows the DNA to be used directly in applications such as PCR, SNP analysis, and RAPD, without the need for separate quantitation of the DNA.

Characterization of blood lipoproteins and validation of cholesterol and triacylglycerol assays for free-ranging polar bears (Ursus maritimus).

PubMed

Whiteman, John P; Frank, Nicholas; Greller, Katie A; Harlow, Henry J; Ben-David, Merav

2013-05-01

Blood triacylglycerol (TG) and lipoproteins are important variables for evaluating nutritional status of wildlife, but measurements are often expensive and difficult. Performance of a small, portable blood analyzer intended for human medical diagnostics was evaluated in measuring these variables in plasma and serum from free-ranging polar bears (Ursus maritimus), which are experiencing nutritional stress related to sea ice loss. The analyzer accurately tracked changes in concentration of total cholesterol (Ctotal), cholesterol associated with high-density lipoprotein (CHDL), and TG during a validation protocol of diluting samples and spiking them with exogenous cholesterol and glycerol. Values of Ctotal and TG agreed well with values obtained by other methods (ultracentrifugation followed by colorimetric assays); agreement was variable for values of cholesterol associated with specific lipoproteins. Similar to a study of captive polar bears, ultracentrifugation methods revealed greater TG in very low-density lipoproteins than in low-density lipoprotein, which is unusual and merits additional study.

Chromosome Aberrations in Astronauts

NASA Technical Reports Server (NTRS)

George, Kerry A.; Durante, M.; Cucinotta, Francis A.

2007-01-01

A review of currently available data on in vivo induced chromosome damage in the blood lymphocytes of astronauts proves that, after protracted exposure of a few months or more to space radiation, cytogenetic biodosimetry analyses of blood collected within a week or two of return from space provides a reliable estimate of equivalent radiation dose and risk. Recent studies indicate that biodosimetry estimates from single spaceflights lie within the range expected from physical dosimetry and biophysical models, but very large uncertainties are associated with single individual measurements and the total sample population remains low. Retrospective doses may be more difficult to estimate because of the fairly rapid time-dependent loss of "stable" aberrations in blood lymphocytes. Also, biodosimetry estimates from individuals who participate in multiple missions, or very long (interplanetary) missions, may be complicated by an adaptive response to space radiation and/or changes in lymphocyte survival and repopulation. A discussion of published data is presented and specific issues related to space radiation biodosimetry protocols are discussed.

Physiological and Performance Measures for Baseline Concussion Assessment.

PubMed

Dobney, Danielle M; Thomas, Scott G; Taha, Tim; Keightley, Michelle

2017-05-17

Baseline testing is a common strategy for concussion assessment and management. Research continues to evaluate novel measures for potential to improve baseline testing methods. The primary objective was to; 1) determine the feasibility of including physiological, neuromuscular and mood measures as part of baseline concussion testing protocol, 2) describe typical values in a varsity athlete sample, and 3) estimate the influence of concussion history on these baseline measures. Prospective observational study. University Athletic Therapy Clinic. 100 varsity athletes. Frequency and domain measures of heart rate variability (HRV), blood pressure (BP), grip strength, Profile of Mood States and the Sport Concussion Assessment Tool-2. Physiological, neuromuscular performance and mood measures were feasible at baseline. Participants with a history of two or more previous concussions displayed significantly higher diastolic blood pressure. Females reported higher total mood disturbance compared to males. Physiological and neuromuscular performance measures are safe and feasible as baseline concussion assessment outcomes. History of concussion may have an influence on diastolic blood pressure.

Direct identification of microorganisms from positive blood cultures using the lysis-filtration technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): a multicentre study.

PubMed

Farina, Claudio; Arena, Fabio; Casprini, Patrizia; Cichero, Paola; Clementi, Massimo; Cosentino, Marina; Degl'Innocenti, Roberto; Giani, Tommaso; Luzzaro, Francesco; Mattei, Romano; Mauri, Carola; Nardone, Maria; Rossolini, Gian Maria; Serna Ortega, Paula Andrea; Vailati, Francesca

2015-04-01

Microbial identification from blood cultures is essential to institute optimal antibiotic therapy and improve survival possibilities. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been successfully applied to identify bacteria and yeasts from positive blood cultures broths. The aim of this multicentre study was to evaluate the reliability of the lysis-filtration technique associated with MALDI-TOF MS to directly identify microorganisms from 765 positive blood cultures collected in six Italian hospitals. Overall, 675/765 (78.1%) blood isolates were correctly identified at the species level, with significant differences between Gram-negative and Gram-positive bacteria (92.6%, and 69.8%, respectively). Some difficulties arise in identifying Streptococcus pneumoniae, Staphylococcus aureus, yeasts and anaerobes. The lysis-filtration protocol is a suitable procedure in terms of performance in identifying microorganisms, but it is quite expensive and technically time-consuming since the time of filtration is not regular for all the samples. The application of the MALDI-TOF MS technique to the direct microbial identification from positive blood cultures is a very promising approach, even if more experience must be gained to minimize errors and costs.

Validation of the Omron HEM-7201 upper arm blood pressure monitor, for self-measurement in a high-altitude environment, according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Cho, K; Tian, M; Lan, Y; Zhao, X; Yan, L L

2013-08-01

Few studies have been conducted on blood pressure monitors and their use at high altitude. This study is the first to evaluate the accuracy of an automatic blood pressure monitor in a high-altitude environment following a standard validation protocol. The Omron HEM-7201 upper arm blood pressure monitor was tested for accuracy in Lhasa, Tibet, China (3650 m above sea level) according to the European Society of Hypertension International Protocol revision 2010 (ESH-IP2). Thirty-three participants received 9-10 sequential blood pressure measurements alternating between a mercury sphygmomanometer and the device. The mean device-observer measurement difference was 1.0±5.9 mm Hg for systolic blood pressure (SBP) and -3.1±4.6 mm Hg for diastolic blood pressure (DBP). Of the 99 measurement pairs analyzed, 72, 90 and 97 device readings were within 5, 10 and 15 mm Hg, respectively, of the observer measurements for SBP, and 68, 92 and 99 readings for DBP. The number of participants with at least two out of three measurements within 5 mm Hg was 27 for SBP and 25 for DBP. Three participants had no measurements within 5 mm Hg for either SBP or DBP. As a result, the Omron HEM-7201 passes the ESH-IP2 validation criteria and can therefore be recommended for use in adults in this setting.

Validation of the iHealth BP3 upper-arm blood pressure monitor, for clinic use and self-measurement, according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Chen, Can; Shang, Fujun; Wang, Jiepin; Chen, Jianghong; Ji, Na; Wan, Yi

2012-12-01

This study aimed to evaluate the performance of the iHealth BP3 upper-arm blood pressure monitor, which is designed for clinic use and self-measurement of blood pressure using Apple touch devices as an interface. The European Society of Hypertension International Protocol (ESH-IP) revision 2010 for the validation of blood pressure measuring devices in adults was followed precisely. Ninty-nine couples of test device and reference blood pressure measurements were obtained during the study (three pairs for each of the 33 participants). The 33 participants, age 47.1±12.3 years (age range 27-69 years) and arm circumference 30.0±4.4 cm, had a mean systolic blood pressure (SBP) of 143.9±27.4 mmHg and a mean diastolic blood pressure (DBP) of 90.1±18.3 mmHg. The device passed all of the requirements fulfilling the standards of the protocol, and the mean±SD device-observer difference was 2.8±4.2 mmHg for SBP and -0.4±3.5 mmHg for DBP. According to the results of the validation study on the basis of the ESH-IP revision 2010, the iHealth BP3 can be recommended for clinic use and self-measurement in an adult population. © 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques

PubMed Central

Grüner, Nico; Stambouli, Oumaima; Ross, R. Stefan

2015-01-01

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since then, DBS testing for decades has remained predominantly focused on the diagnosis of infectious diseases especially in resource-limited settings or the systematic screening of newborns for inherited metabolic disorders and only recently have a variety of new and innovative DBS applications begun to emerge. For many years, pre-analytical variables were only inappropriately considered in the field of DBS testing and even today, with the exception of newborn screening, the entire pre-analytical phase, which comprises the preparation and processing of DBS for their final analysis has not been standardized. Given this background, a comprehensive step-by-step protocol, which covers al the essential phases, is proposed, i.e., collection of blood; preparation of blood spots; drying of blood spots; storage and transportation of DBS; elution of DBS, and finally analyses of DBS eluates. The effectiveness of this protocol was first evaluated with 1,762 coupled serum/DBS pairs for detecting markers of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus infections on an automated analytical platform. In a second step, the protocol was utilized during a pilot study, which was conducted on active drug users in the German cities of Berlin and Essen. PMID:25867233

A review of blood transfusions in a trauma unit for young children.

PubMed

Salverda, M; Ketharanathan, N; Van Dijk, M; Beltchev, E; Buys, H; Numanoglu, A; Van As, A B

2017-02-27

Trauma is the leading cause of mortality and morbidity worldwide. Blood transfusions play an incremental role in the acute phase, yet practice varies owing to variations in transfusion thresholds and concerns about potential complications, especially in children. To evaluate protocol adherence to blood transfusion thresholds in paediatric trauma patients and determine the degree of blood product wastage, as defined by discarded units. A retrospective, descriptive study of trauma patients (age 0 - 13 years) who received a blood transfusion in the trauma unit at Red Cross War Memorial Children's Hospital, Cape Town, South Africa, over a 5.5-year period (1 January 2009 - 1 July 2014). Haemoglobin (Hb) transfusion thresholds were defined as 10 g/dL for neurotrauma patients and patients requiring skin grafting or a musculocutaneous flap (group 1). All other trauma patients had an Hb transfusion threshold of 7 g/dL (group 2). A total of 144 patients were included (mean age 5.2 years (standard deviation (SD) 3.3), 68.1% male). The mean Hb increase after transfusion was 3.5 g/dL (SD 1.7). Adherence to the transfusion Hb threshold protocol was 96.7% for group 1 v. 34.0% for group 2. No complications were reported. Average blood wastage was 3.5 units per year during the study period. Adherence to paediatric blood transfusion protocol was low in the Hb threshold group <7 g/dL. However, transfusion-related complications and wastage were minimal. Further prospective research is required to determine optimal blood transfusion guidelines for paediatric trauma patients.

Evaluation of whole genome amplified DNA to decrease material expenditure and increase quality.

PubMed

Bækvad-Hansen, Marie; Bybjerg-Grauholm, Jonas; Poulsen, Jesper B; Hansen, Christine S; Hougaard, David M; Hollegaard, Mads V

2017-06-01

The overall aim of this study is to evaluate whole genome amplification of DNA extracted from dried blood spot samples. We wish to explore ways of optimizing the amplification process, while decreasing the amount of input material and inherently the cost. Our primary focus of optimization is on the amount of input material, the amplification reaction volume, the number of replicates and amplification time and temperature. Increasing the quality of the amplified DNA and the subsequent results of array genotyping is a secondary aim of this project. This study is based on DNA extracted from dried blood spot samples. The extracted DNA was subsequently whole genome amplified using the REPLIg kit and genotyped on the PsychArray BeadChip (assessing > 570,000 SNPs genome wide). We used Genome Studio to evaluate the quality of the genotype data by call rates and log R ratios. The whole genome amplification process is robust and does not vary between replicates. Altering amplification time, temperature or number of replicates did not affect our results. We found that spot size i.e. amount of input material could be reduced without compromising the quality of the array genotyping data. We also showed that whole genome amplification reaction volumes can be reduced by a factor of 4, without compromising the DNA quality. Whole genome amplified DNA samples from dried blood spots is well suited for array genotyping and produces robust and reliable genotype data. However, the amplification process introduces additional noise to the data, making detection of structural variants such as copy number variants difficult. With this study, we explore ways of optimizing the amplification protocol in order to reduce noise and increase data quality. We found, that the amplification process was very robust, and that changes in amplification time or temperature did not alter the genotyping calls or quality of the array data. Adding additional replicates of each sample also lead to insignificant changes in the array data. Thus, the amount of noise introduced by the amplification process was consistent regardless of changes made to the amplification protocol. We also explored ways of decreasing material expenditure by reducing the spot size or the amplification reaction volume. The reduction did not affect the quality of the genotyping data.

Determinants of blood-lead levels in children in Callao and Lima metropolitan area.

PubMed

Espinoza, Rocío; Hernández-Avila, Mauricio; Narciso, Juan; Castañaga, Carmen; Moscoso, Shirley; Ortiz, Georgina; Carbajal, Luz; Wegner, Steve; Noonan, Gary

2003-01-01

To determine blood lead levels in urban populations of children (n = 2,510) and women (n = 874) in the early postpartum in certain districts of Lima and Callao, and to correlate those levels with particular exposures. Between July 1998 and January 1999 cross sectional study was conducted. The study population was selected using three sampling strategies in the government operated school system and from public pediatric and maternity hospitals at Lima and Callao, Peru. Study personnel were trained to collect finger stick blood samples with a protocol that minimizes external lead contamination. Lead determinations in blood and environmental samples were performed at the study site using portable anodic striping voltamenters. To determine the simultaneous effects of different predictors on blood lead levels, multivariate regression models were used to estimate adjusted mean differences. The mean blood lead level in the children studied was 9.9 micrograms/dl ranging from 1 microgram/dl to 64 micrograms/dl with 29% of the children displaying values greater than 10 micrograms/dl and 9.4% at levels greater than 20 micrograms/dl. Among the women, the mean was 3.5 micrograms/dl (SD = 2.4 micrograms/dl), and 2.4% (n = 21) displayed levels greater than 10 micrograms/dl. Important differences were observed between the sample locations, and the highest levels were documented in the port region near Callao. The mean level of blood lead in this group was 25.6 micrograms/dl (SD = 4.6 micrograms/dl), while among the rest of the sample it was 7.1 micrograms/dl (SD = 5.1 micrograms/dl). The presence of a mineral storage area signified a difference in exposure in excess of 13 micrograms/dl for children living near the port area in contrast to the other children who were not as close to such fixed sources of lead exposure. For the participants in Lima, the risk of showing levels above 10 micrograms/dl was associated with exposure to high vehicular traffic. In metropolitan Lima, we conclude that the mean blood lead levels of the populations studied were not alarming and that a positive health impact can be made by a reduction of lead in gasoline. With regard to the port area, the study demonstrates that the presence of mineral storage areas pose a detrimental risk factor for the health of the children living in this area. The English version of this paper is available too at: http://www.insp.mx/salud/index.html.

World Health Organization Generic Protocol to Assess Drug-Resistant HIV Among Children <18 Months of Age and Newly Diagnosed With HIV in Resource-Limited Countries

PubMed Central

Penazzato, Martina; Jordan, Michael R.; Persaud, Deborah; Mofenson, Lynne M.; Bennett, Diane E.

2012-01-01

Increased use of nonnucleoside reverse transcriptase inhibitors (NNRTIs) in pregnant and breastfeeding women will result in fewer children infected with human immunodeficiency virus (HIV). However, among children infected despite prevention of mother-to-child transmission (PMTCT), a substantial proportion will acquire NNRTI-resistant HIV, potentially compromising response to NNRTI-based antiretroviral therapy (ART). In countries scaling up PMTCT and pediatric ART programs, it is crucial to assess the proportion of young children with drug-resistant HIV to improve health outcomes and support national and global decision making on optimal selection of pediatric first-line ART. This article summarizes a new World Health Organization surveillance protocol to assess resistance using remnant dried blood spot specimens from a representative sample of children aged <18 months being tested for early infant diagnosis. PMID:22544184

Novel Combined Training Approach Improves Sleep Quality but Does Not Change Body Composition in Healthy Elderly Women: A Preliminary Study

PubMed Central

Sousa, Thiago Matheus da Silva; Ruberti, Olivia de Moraes; Novais, Tânia Maria Gaspar; Oliveira, Paula de Lourdes Lauande; Almeida, Fabiano de Jesus Furtado; Pulcherio, Janaína Oliveira Bentivi

2017-01-01

The aim of this study was to investigate the effects of a novel combined training protocol on sleep quality and body composition of healthy elderly women. The study sample consisted of 8 sedentary elderly individuals with mean (±SD) of 67 (±8) years of age, 96.0 (±7.8) mg/dL fasting blood glucose, 94.4 (±36.1) mg/dL triglycerides, 179.1 (±22.4) mg/dL total cholesterol, 57.2 (±15.7) mg/dL high-density lipoprotein (HDL), 103.1 (±25.2) mg/dL low-density lipoprotein (LDL), 125.3 (±8.4) mmHg systolic blood pressure, and 72.6 (±10.1) mmHg diastolic blood pressure. The training protocol consisted of resistance training exercises (approximately 18-minute duration) combined with aerobic exercises (approximately 26-minute duration), performed interspersed in the same session, for 8 weeks (3 times a week), with a 24-hour interval rest between each session. Continuous variables were expressed as the mean (±standard deviation) and the paired sample t-test compares baseline with final measurement. The results showed a significant improvement (p = 0.01) in quality of sleep (4.9 ± 1.5 versus 3.8 ± 1.8 for total PSQI index) without body significant improvements in the fat-free mass (59.9 ± 4.0 versus 60.5 ± 4.4; p = 0.20) and fat mass (40.1 ± 4.0 versus 39.5 ± 4.4; p = 0.20) in healthy elderly women. In this sense, the novel combined training proposed may be an effective alternative or adjunct to present therapies aimed at improving the sleep quality in this population. PMID:29062575

Preemptive genotyping for personalized medicine: design of the right drug, right dose, right time-using genomic data to individualize treatment protocol.

PubMed

Bielinski, Suzette J; Olson, Janet E; Pathak, Jyotishman; Weinshilboum, Richard M; Wang, Liewei; Lyke, Kelly J; Ryu, Euijung; Targonski, Paul V; Van Norstrand, Michael D; Hathcock, Matthew A; Takahashi, Paul Y; McCormick, Jennifer B; Johnson, Kiley J; Maschke, Karen J; Rohrer Vitek, Carolyn R; Ellingson, Marissa S; Wieben, Eric D; Farrugia, Gianrico; Morrisette, Jody A; Kruckeberg, Keri J; Bruflat, Jamie K; Peterson, Lisa M; Blommel, Joseph H; Skierka, Jennifer M; Ferber, Matthew J; Black, John L; Baudhuin, Linnea M; Klee, Eric W; Ross, Jason L; Veldhuizen, Tamra L; Schultz, Cloann G; Caraballo, Pedro J; Freimuth, Robert R; Chute, Christopher G; Kullo, Iftikhar J

2014-01-01

To report the design and implementation of the Right Drug, Right Dose, Right Time-Using Genomic Data to Individualize Treatment protocol that was developed to test the concept that prescribers can deliver genome-guided therapy at the point of care by using preemptive pharmacogenomics (PGx) data and clinical decision support (CDS) integrated into the electronic medical record (EMR). We used a multivariate prediction model to identify patients with a high risk of initiating statin therapy within 3 years. The model was used to target a study cohort most likely to benefit from preemptive PGx testing among the Mayo Clinic Biobank participants, with a recruitment goal of 1000 patients. We used a Cox proportional hazards model with variables selected through the Lasso shrinkage method. An operational CDS model was adapted to implement PGx rules within the EMR. The prediction model included age, sex, race, and 6 chronic diseases categorized by the Clinical Classifications Software for International Classification of Diseases, Ninth Revision codes (dyslipidemia, diabetes, peripheral atherosclerosis, disease of the blood-forming organs, coronary atherosclerosis and other heart diseases, and hypertension). Of the 2000 Biobank participants invited, 1013 (51%) provided blood samples, 256 (13%) declined participation, 555 (28%) did not respond, and 176 (9%) consented but did not provide a blood sample within the recruitment window (October 4, 2012, through March 20, 2013). Preemptive PGx testing included CYP2D6 genotyping and targeted sequencing of 84 PGx genes. Synchronous real-time CDS was integrated into the EMR and flagged potential patient-specific drug-gene interactions and provided therapeutic guidance. This translational project provides an opportunity to begin to evaluate the impact of preemptive sequencing and EMR-driven genome-guided therapy. These interventions will improve understanding and implementation of genomic data in clinical practice. Copyright © 2014 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.

A major haemorrhage protocol improves the delivery of blood component therapy and reduces waste in trauma massive transfusion.

PubMed

Khan, Sirat; Allard, Shubha; Weaver, Anne; Barber, Colin; Davenport, Ross; Brohi, Karim

2013-05-01

Major haemorrhage protocols (MHP) are required as part of damage control resuscitation regimens in modern trauma care. The primary objectives of this study were to ascertain whether a MHP improved blood product administration and reduced waste compared to traditional massive transfusion protocols (MTP). Datasets on adult trauma admissions 1 year prior and 1 year post implementation of a MHP at a Level 1 trauma centre were obtained from the trauma registry. Demographic and clinical data were collected prospectively including mechanism of injury, physiological observations, ICU admission and length of stay. The volume of blood components (packed red blood cells, platelets, cryoprecipitate and fresh frozen plasma) issued, transfused, returned to stock and wasted within the first 24h was gathered retrospectively. Over the 2-year study period 2986 patient records were available for analysis. 40 patients required a 10+ Units of packed red blood ells transfusion in the MTP group vs. 56 patients post MHP implementation. The administration of blood component therapy improved significantly post MHP implementation. FFP:PRBC transfusion improved from 1:3 to 1:2 (p<0.01) and CRYO:PRBC improved from 1:10 to 1:7 (p<0.05). We reported a significant reduction in the waste of platelets from 14% to 2% (p<0.01). Outcomes had improved: Median hospital length of stay was reduced from 54 days to 26 days (p<0.05). Implementation of a MHP results in improved delivery of blood components and a reduction in the waste of blood products compared to the older model of MTP. In combination with educational programmes MHP can significantly improve blood product administration and patient outcomes in trauma haemorrhage. Level III diagnostic test study. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

Non-invasive prenatal diagnosis of paternally inherited disorders from maternal plasma: detection of NF1 and CFTR mutations using droplet digital PCR.

PubMed

Gruber, Aurélia; Pacault, Mathilde; El Khattabi, Laila Allach; Vaucouleur, Nicolas; Orhant, Lucie; Bienvenu, Thierry; Girodon, Emmanuelle; Vidaud, Dominique; Leturcq, France; Costa, Catherine; Letourneur, Franck; Anselem, Olivia; Tsatsaris, Vassilis; Goffinet, François; Viot, Géraldine; Vidaud, Michel; Nectoux, Juliette

2018-04-25

To limit risks of miscarriages associated with invasive procedures of current prenatal diagnosis practice, we aim to develop a personalized medicine-based protocol for non-invasive prenatal diagnosis (NIPD) of monogenic disorders relying on the detection of paternally inherited mutations in maternal blood using droplet digital PCR (ddPCR). This study included four couples at risk of transmitting paternal neurofibromatosis type 1 (NF1) mutations and four couples at risk of transmitting compound heterozygous CFTR mutations. NIPD was performed between 8 and 15 weeks of gestation, in parallel to conventional invasive diagnosis. We designed specific hydrolysis probes to detect the paternal mutation and to assess the presence of cell-free fetal DNA by ddPCR. Analytical performances of each assay were determined from paternal sample, an then fetal genotype was inferred from maternal plasma sample. Presence or absence of the paternal mutant allele was correctly determined in all the studied plasma DNA samples. We report an NIPD protocol suitable for implementation in an experienced laboratory of molecular genetics. Our proof-of-principle results point out a high accuracy for early detection of paternal NF1 and CFTR mutations in cell-free DNA, and open new perspectives for extending the technology to NIPD of many other monogenic diseases.

Training Status as a Marker of the Relationship between Nitric Oxide, Oxidative Stress, and Blood Pressure in Older Adult Women

PubMed Central

Mourão Jacomini, André; Celso Dutra de Souza, Hugo; da Silva Dias, Danielle; de Oliveira Brito, Janaina; Cezar Pinheiro, Lucas; Bernardino da Silva, Anderson; Fernanda da Silva, Roberta; Alexandre Trapé, Atila; De Angelis, Kátia; Tanus-Santos, José Eduardo; Lia do Amaral, Sandra; Saranz Zago, Anderson

2016-01-01

The purpose of this study was to evaluate the influence of functional fitness and oxidative capacity on the nitric oxide concentration associated with hemodynamic control in older adult women. The sample consisted of 134 women (65.73 ± 6.14 years old). All subjects underwent a physical examination to assess body mass index, waist-hip ratio, body fat measurement by dual energy X-ray absorptiometry, and blood pressure (BP). Training status (TS) was evaluated by indirect determination of maximal oxygen uptake by a treadmill test using Balke protocol modified for older adults. Functional fitness was also evaluated through a “Functional Fitness Battery Test” to determine the general fitness functional index (GFFI). All participants were separated according to the functional fitness (TS1, very weak and weak; TS2, regular; TS3, good and very good). Plasma blood samples were used to evaluate prooxidant and antioxidant activity and nitrite and nitrate concentrations. The general results of this study showed that good levels of TS were related to lower levels of lipoperoxidation and protein damage, higher levels of antioxidant, and higher concentration of nitrite and nitrate. This combination may be responsible for the lower levels of BP in subjects with better TS. PMID:26697141

Training Status as a Marker of the Relationship between Nitric Oxide, Oxidative Stress, and Blood Pressure in Older Adult Women.

PubMed

Jacomini, André Mourão; de Souza, Hugo Celso Dutra; Dias, Danielle da Silva; Brito, Janaina de Oliveira; Pinheiro, Lucas Cezar; da Silva, Anderson Bernardino; da Silva, Roberta Fernanda; Trapé, Atila Alexandre; De Angelis, Kátia; Tanus-Santos, José Eduardo; do Amaral, Sandra Lia; Zago, Anderson Saranz

2015-01-01

The purpose of this study was to evaluate the influence of functional fitness and oxidative capacity on the nitric oxide concentration associated with hemodynamic control in older adult women. The sample consisted of 134 women (65.73 ± 6.14 years old). All subjects underwent a physical examination to assess body mass index, waist-hip ratio, body fat measurement by dual energy X-ray absorptiometry, and blood pressure (BP). Training status (TS) was evaluated by indirect determination of maximal oxygen uptake by a treadmill test using Balke protocol modified for older adults. Functional fitness was also evaluated through a "Functional Fitness Battery Test" to determine the general fitness functional index (GFFI). All participants were separated according to the functional fitness (TS1, very weak and weak; TS2, regular; TS3, good and very good). Plasma blood samples were used to evaluate prooxidant and antioxidant activity and nitrite and nitrate concentrations. The general results of this study showed that good levels of TS were related to lower levels of lipoperoxidation and protein damage, higher levels of antioxidant, and higher concentration of nitrite and nitrate. This combination may be responsible for the lower levels of BP in subjects with better TS.

Molecular screening for hemotropic mycoplasmas in captive Barbary sheep (Ammotragus lervia) in southern Brazil

PubMed Central

Santos, Leonilda C.; Vidotto, Odilon; Morikawa, Vivien M.; Santos, Nelson J. R.; Vieira, Thállitha S. W. J.; Barros Filho, Ivan R.; Vieira, Rafael F. C.; Biondo, Alexander W.

2017-01-01

Aim: This study is part of an active surveillance program for monitoring animal health status in endangered species, and was conducted to screen captive Barbary sheep (Ammotragus lervia) for hemoplasma infection. Materials and Methods: A total of 12 blood samples were collected, DNA extracted and further tested by a pan-hemoplasma polymerase chain reaction protocol. Results: Animals were clinically healthy and not infested by ectoparasites. Although housekeeping gene DNA was successfully amplified, all the Barbary sheep samples tested negative for Mycoplasma sp. Conclusion: Notwithstanding the negative results, molecular pathogen surveys on Barbary sheep and other exotic wild mammals may provide insights regarding infection of endangered species caused by captivity stress in association with exposure to new pathogens worldwide. PMID:28919684

Prophylactic use of intravascular balloon catheters in women with placenta accreta, increta and percreta.

PubMed

Cali, Giuseppe; Forlani, Francesco; Giambanco, Laura; Amico, Maria Luisa; Vallone, Mario; Puccio, Giuseppe; Alio, Luigi

2014-08-01

To evaluate outcomes of women undergoing cesarean hysterectomy for abnormal invasive placenta (AIP) with and without preoperative balloon catheter placement in internal iliac arteries. A prospective observational study of women with ultrasound diagnosis of AIP and a planned delivery at our institution. From January 2004 to June 2009, all AIP cases were treated with planned multidisciplinary cesarean hysterectomy alone (CHa group). From July 2009 to September 2013 a pre-operative balloon catheter protocol was introduced (BC group). Statistical analysis considered the entire sample (placenta accreta/increta and percreta) and the individual subgroups (accreta/increta vs percreta). Twenty-three cases of AIP (10 accreta/increta and 13 percreta) were treated with cesarean hysterectomy alone, and 30 cases of AIP (12 accreta/increta and 18 percreta) were treated with cesarean hysterectomy and pre-operative balloon catheters. For the entire sample, a significant difference in estimated blood loss and transfused blood products units was observed between CHa group and BC group. When women with placenta accreta/increta and women with placenta percreta were analysed separately, no difference in estimated blood loss and transfused blood products units was found between the BC and the CHa groups in women with placenta accreta/increta. However, in women with placenta percreta, mean estimated blood loss and transfused blood products units were higher in the CHa group compared with BC group (1507ml vs 933.33ml; 3.31 units vs 0.67 units). Postoperative recovery differed between the two groups, but no differences were observed in any other outcomes. Pre-operative placement of intravascular balloon catheters is a feasible treatment for AIP, and is particularly useful in cases of placenta percreta. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Competition for DNA binding sites using Promega DNA IQ™ paramagnetic beads.

PubMed

Frégeau, Chantal J; De Moors, Anick

2012-09-01

The Promega DNA IQ™ system is easily amenable to automation and has been an integral part of standard operating procedures for many forensic laboratories including those of the Royal Canadian Mounted Police (RCMP) since 2004. Due to some failure to extract DNA from samples that should have produced DNA using our validated automated DNA IQ™-based protocol, the competition for binding sites on the DNA IQ™ magnetic beads was more closely examined. Heme from heavily blooded samples interfered slightly with DNA binding. Increasing the concentration of Proteinase K during lysis of these samples did not enhance DNA recovery. However, diluting the sample lysate following lysis prior to DNA extraction overcame the reduction in DNA yield and preserved portions of the lysates for subsequent manual or automated extraction. Dye/chemicals from black denim lysates competed for binding sites on the DNA IQ™ beads and significantly reduced DNA recovery. Increasing the size or number of black denim cuttings during lysis had a direct adverse effect on DNA yield from various blood volumes. The dilution approach was successful on these samples and permitted the extraction of high DNA yields. Alternatively, shortening the incubation time for cell lysis to 30 min instead of the usual overnight at 56 °C prevented competition from black denim dye/chemicals and increased DNA yields. Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.

Protocol deviations before and after IV tPA in community hospitals

PubMed Central

Adelman, Eric E.; Scott, Phillip A.; Skolarus, Lesli E.; Fox, Allison K.; Frederiksen, Shirley M.; Meurer, William J.

2015-01-01

Background Protocol deviations before and after tPA treatment for ischemic stroke are common. It is unclear if patient or hospital factors predict protocol deviations. We examined predictors of protocol deviations and the effects of protocol violations on symptomatic intracerebral hemorrhage. Methods We used data from the INSTINCT trial, a cluster-randomized, controlled trial evaluating the efficacy of a barrier assessment and educational intervention to increase appropriate tPA use in 24 Michigan community hospitals, to review tPA treatments between 2007 and 2010. Protocol violations were defined as deviations from the standard tPA protocol, both before and after treatment. Multi-level logistic regression models were fitted to determine if patient and hospital variables were associated with pre-treatment or post-treatment protocol deviations. Results During the study, 557 patients (mean age 70; 52% male; median NIHSS 12) were treated with tPA. Protocol deviations occurred in 233 (42%) patients: 16% had pre-treatment deviations, 35% had post-treatment deviations, and 9% had both. The most common protocol deviations included elevated post-treatment blood pressure, antithrombotic agent use within 24 hours of treatment, and elevated pre-treatment blood pressure. Protocol deviations were not associated with symptomatic intracerebral hemorrhage, stroke severity, or hospital factors. Older age was associated with pre-treatment protocol deviations (adjusted OR 0.52; 95% confidence interval 0.30-0.92). Pre-treatment deviations were associated with post-treatment deviations (adjusted OR 3.20; 95% confidence interval 1.91-5.35). Conclusions Protocol deviations were not associated with symptomatic intracerebral hemorrhage. Aside from age, patient and hospital factors were not associated with protocol deviations. PMID:26419527

Heart rate, blood pressure and repolarization effects of an energy drink as compared to coffee.

PubMed

Brothers, R Matthew; Christmas, Kevin M; Patik, Jordan C; Bhella, Paul S

2017-11-01

The goal of this study was to investigate the impact of energy drinks on haemodynamic and cardiac physiology. Comparisons were made to coffee as well as water consumption. In Protocol #1 the caffeine content was normalized to body weight to represent a controlled environment. Heart rate, blood pressure and cardiac QTc interval were assessed in 15 participants, on 4 days, prior to and for 6·5 h postconsumption of (i) energy drink (2 mg caffeine per kg body weight; low dose), (ii) energy drink (3 mg caffeine per kg body weight; medium dose), (iii) coffee (2 mg caffeine per kg body weight) and (iv) 250 ml water. In Protocol #2, the beverages were consumed in volumes that they are purchased to represent real-life conditions. The aforementioned measurements were repeated in 15 participants following (i) 1 16 oz can of energy drink (16 oz Monster), (ii) 1 24 oz can of energy drink (24 oz Monster), (iii) 1 packet of Keurig K-Cup Starbucks coffee (coffee) and (iv) 250 ml water. The order of the beverages was performed in a randomized double-blinded fashion. For both protocols, QTc interval, heart rate and systolic blood pressure were unchanged in any condition (P>0·05). Diastolic blood pressure and mean blood pressure were slightly elevated in Protocol #1 (P<0·05, main effect of time) with no difference between beverages (P<0·05, interaction of beverage × time); however, they were unaffected in Protocol #2 (P>0·05). These findings suggest that acute consumption of these commonly consumed beverages has no negative effect on cardiac QTc interval. © 2016 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd.

Experimentally induced hyperchloremic and DL-lactic acidosis in calves: an attempt to study the effects of oral rehydration on acid-base status.

PubMed

Schwedhelm, L; Kirchner, D; Klaus, B; Bachmann, L

2013-04-01

Many diarrheic calves suffer from metabolic acidosis, which is commonly treated by oral rehydration therapy. Oral rehydration solutions can be prepared in water, milk, or milk replacer. Therefore, the aim of the study was to verify dietary effects of water- or milk replacer-based oral rehydration solutions on parameters of acid-base balance in calves with experimentally induced hyperchloremic and dl-lactate acidosis. In 12 calves, hyperchloremic or dl-lactate acidosis was induced by HCl or dl-lactic acid infusions according to protocols outlined in previous literature. Immediately after induction, the calves were fed with milk replacer or water- or milk replacer-based oral rehydration solutions, or remained fasting, respectively. Blood samples were taken to monitor acid-base status over an experimental period of 4h. Using the protocols, all calves revealed a manifest hyperchloremic or dl-lactate acidosis. Because of high infusion volumes, plasma volume was expanded and effects of feeding regimens on blood parameters were rare. Unexpected clinical aberrations occurred after repeated induction of dl-lactate acidosis: all calves developed a thrombophlebitis of the jugular vein, whereas HCl infusion had no effect on endothelium. Induction of acidosis via infusion is not suitable to study dietary effects. A protocol to induce acidosis and dehydration simultaneously is required to duplicate the metabolic conditions of diarrheic calves. In further investigations, attention should be focused on effects of d-lactate or its metabolites on endothelial tissue. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A simplified protocol employing elacridar in rodents: a screening model in drug discovery to assess P-gp mediated efflux at the blood brain barrier.

PubMed

Kallem, Rajareddy; Kulkarni, Chetan P; Patel, Dakshay; Thakur, Megha; Sinz, Michael; Singh, Sheelendra P; Mahammad, S Shahe; Mandlekar, Sandhya

2012-06-01

In the present study we have developed a simple, time, and cost effective in vivo rodent protocol to screen the susceptibility of a test compound for P-glycoprotein (P-gp) mediated efflux at the blood brain barrier (BBB) during early drug discovery. We used known P-gp substrates as test compounds (quinidine, digoxin, and talinolol) and elacridar (GF120918) as a chemical inhibitor to establish the model. The studies were carried out in both mice and rats. Elacridar was dosed intravenously at 5 mg/kg, 0.5 h prior to probe substrate administration. Plasma and brain samples were collected and analyzed using UPLC-MS/MS. In the presence of elacridar, the ratio of brain to plasma area under the curve (B/P) in mouse increased 2, 4, and 38-fold, respectively, for talinolol, digoxin, and quinidine; whereas in rat, a 70-fold increase was observed for quinidine. Atenolol, a non P-gp substrate, exhibited poor brain penetration in the presence or absence of elacridar in both species (B/P ratio ~ 0.1). Elacridar had no significant effect on the systemic clearance of digoxin or quinidine; however, a trend towards increasing volume of distribution and half life was observed. Our results support the utility of elacridar in evaluation of the influence of P-gp mediated efflux on drug distribution to the brain. Our protocol employing a single intravenous dose of elacridar and test compound provides a cost effective alternative to expensive P-gp knockout mice models during early drug discovery.

Design, rationale and feasibility of a multidimensional experimental protocol to study early life stress.

PubMed

Bartholomeusz, M Dillwyn; Bolton, Philip S; Callister, Robin; Skinner, Virginia; Hodgson, Deborah

2017-09-01

There is a rapidly accumulating body of evidence regarding the influential role of early life stress (ELS) upon medical and psychiatric conditions. While self-report instruments, with their intrinsic limitations of recall, remain the primary means of detecting ELS in humans, biological measures are generally limited to a single biological system. This paper describes the design, rationale and feasibility of a study to simultaneously measure neuroendocrine, immune and autonomic nervous system (ANS) responses to psychological and physiological stressors in relation to ELS. Five healthy university students were recruited by advertisement. Exclusion criteria included chronic medical conditions, psychotic disorders, needle phobia, inability to tolerate pain, and those using anti-inflammatory medications. They were clinically interviewed and physiological recordings made over a two-hour period pre, during and post two acute stressors: the cold pressor test and recalling a distressing memory. The Childhood Trauma Questionnaire and the Parental Bonding Index were utilised to measure ELS. Other psychological measures of mood and personality were also administered. Measurements of heart rate, blood pressure, respiratory rate, skin conductance, skin blood flow and temporal plasma samples were successfully obtained before, during and after acute stress. Participants reported the extensive psychological and multisystem physiological data collection and stress provocations were tolerable. Most (4/5) participants indicated a willingness to return to repeat the protocol, indicating acceptability. Our protocol is viable and safe in young physically healthy adults and allows us to assess simultaneously neuroendocrine, immune and autonomic nervous system responses to stressors in persons assessed for ELS.

Validation of four automatic devices for self-measurement of blood pressure according to the international protocol of the European Society of Hypertension.

PubMed

Topouchian, Jirar; Agnoletti, Davide; Blacher, Jacques; Youssef, Ahmed; Ibanez, Isabel; Khabouth, Jose; Khawaja, Salwa; Beaino, Layale; Asmar, Roland

2011-01-01

Four oscillometric devices for self-measurement of blood pressure (SBPM) were evaluated according to the European Society of Hypertension (ESH) international protocol and its 2010 revision in four separate studies. The Omron® M2, Omron M3, and Omron M6 measure blood pressure (BP) at the brachial level, while the Omron R2 measures BP at the wrist level. The international protocol requires a total number of 33 subjects in which the validation is performed. The Omron M2 and Omron R2 were validated in 2009 according to the ESH international protocol, while the Omron M3 and Omron M6 were validated in 2010-2011 according to the 2010 ESH international protocol revision. The protocol procedures were followed precisely. All four tested devices passed the validation process. The mean differences between the device and mercury readings were 2.7 ± 5.0 and -1.4 ± 3.2 mmHg for systolic and diastolic BP, respectively, using the Omron M2 device, and 1.7 ± 3.2 and -0.9 ± 2.6 mmHg using the Omron M3, 1.6 ± 2.9 and -0.9 ± 2.5 mmHg using the Omron M6, and -1.1 ± 4.8 and -0.9 ± 4.3 mmHg using the Omron R2. Readings from the Omron M2, Omron M3, Omron M6, and Omron R2, differing by less than 5, 10, and 15 mmHg, fulfill the ESH international protocol and its 2010 revision requirements. Therefore, each of these four devices can be used by patients for SBPM.

Does mental arithmetic before head up tilt have an effect on the orthostatic cardiovascular and hormonal responses?

NASA Astrophysics Data System (ADS)

Goswami, Nandu; Lackner, Helmut Karl; Papousek, Ilona; Montani, Jean-Pierre; Jezova, Daniela; Hinghofer-Szalkay, Helmut G.

2011-05-01

Passive head up tilt (HUT) and mental arithmetic (MA) are commonly used for providing mental and orthostatic challenges, respectively. In animal experiments, even a single exposure to a stressor has been shown to modify the response to subsequent stress stimulus. We investigated whether MA applied before HUT elicits synergistic responses in orthostatic heart rate (HR), cardiac output (CO), heart rate variability and arterial blood pressure. The 15 healthy young males were subjected to two randomized protocols: (a) HUT and (b) HUT preceded by MA, with sessions randomized and ≥2 weeks apart. Beat to beat continuous hemodynamic variables were measured and saliva samples taken for hormonal assay. HUT alone increased HR from 59±7 (baseline) to 80±10 bpm (mean±SD) and mean blood pressure (MBP) from 88±10 to 91±14 mmHg. HUT results after MA were not different from those with HUT alone. The activity of alpha amylase showed differences during the experiments irrespective of the protocols. We conclude that mental challenge does not affect orthostatic cardiovascular responses when applied before; the timing of mental loading seems to be critical if it is intended to alter cardiovascular responses to upright standing.

The effects of a walking program on older Chinese American immigrants with hypertension: a pretest and posttest quasi-experimental design.

PubMed

Chiang, Chun-Ying; Sun, Fan-Ko

2009-01-01

Hypertension is known to have high rates among Chinese Americans. Identifying culturally specific interventions to reduce sedentary behavior may be effective in reducing hypertension. This study examines the effects of an 8-week walking program with and without cultural modification. The study used a 2-group, pretest and posttest, quasi-experimental design. A total sample of 128 Chinese American immigrants with hypertension were assigned to walking groups. The results showed that the walking program had no significant effects upon participant blood pressure or walking endurance. The results also revealed that individuals in the maintenance stage walked longer than those in the preparation stage. A comparison of demographic data showed that subjects with a lower level of education walked more minutes per week, which contributed to lower systolic blood pressures among this group as compared with those with a higher level of education. These results suggest that this walking protocol, when translated into Chinese and when accompanied by a weekly telephone reminder and other interactions with a Chinese-speaking nurse, is appropriate to use without additional cultural modification. Future research should examine other components of Chinese culture or should apply this protocol for a longer period of time.

A priori collaboration in population imaging: The Uniform Neuro-Imaging of Virchow-Robin Spaces Enlargement consortium.

PubMed

Adams, Hieab H H; Hilal, Saima; Schwingenschuh, Petra; Wittfeld, Katharina; van der Lee, Sven J; DeCarli, Charles; Vernooij, Meike W; Katschnig-Winter, Petra; Habes, Mohamad; Chen, Christopher; Seshadri, Sudha; van Duijn, Cornelia M; Ikram, M Kamran; Grabe, Hans J; Schmidt, Reinhold; Ikram, M Arfan

2015-12-01

Virchow-Robin spaces (VRS), or perivascular spaces, are compartments of interstitial fluid enclosing cerebral blood vessels and are potential imaging markers of various underlying brain pathologies. Despite a growing interest in the study of enlarged VRS, the heterogeneity in rating and quantification methods combined with small sample sizes have so far hampered advancement in the field. The Uniform Neuro-Imaging of Virchow-Robin Spaces Enlargement (UNIVRSE) consortium was established with primary aims to harmonize rating and analysis (www.uconsortium.org). The UNIVRSE consortium brings together 13 (sub)cohorts from five countries, totaling 16,000 subjects and over 25,000 scans. Eight different magnetic resonance imaging protocols were used in the consortium. VRS rating was harmonized using a validated protocol that was developed by the two founding members, with high reliability independent of scanner type, rater experience, or concomitant brain pathology. Initial analyses revealed risk factors for enlarged VRS including increased age, sex, high blood pressure, brain infarcts, and white matter lesions, but this varied by brain region. Early collaborative efforts between cohort studies with respect to data harmonization and joint analyses can advance the field of population (neuro)imaging. The UNIVRSE consortium will focus efforts on other potential correlates of enlarged VRS, including genetics, cognition, stroke, and dementia.

Multicenter Evaluation of a New Shortened Peptide Nucleic Acid Fluorescence In Situ Hybridization Procedure for Species Identification of Select Gram-Negative Bacilli from Blood Cultures▿

PubMed Central

Morgan, Margie; Marlowe, Elizabeth; Della-Latta, Phyllis; Salimnia, Hossein; Novak-Weekley, Susan; Wu, Fann; Crystal, Benjamin S.

2010-01-01

A shortened protocol for two peptide nucleic acid fluorescence in situ hybridization (PNA FISH) assays for the detection of Gram-negative bacilli from positive blood cultures was evaluated in a multicenter trial. There was 100% concordance between the two protocols for each assay (368 of 368 and 370 of 370 results) and 99.7% (367 of 368 and 369 of 370 results) agreement with routine laboratory techniques. PMID:20357212

Thiolation mediated pegylation platform to generate functional universal red blood cells.

PubMed

Nacharaju, Parimala; Manjula, Belur N; Acharya, Seetharama A

2007-01-01

The PEGylation that adds an extension arm on protein amino groups with the conservation of their positive charge masks the A and D antigens of erythrocytes efficiently. In the present study, the efficiency of masking the antigens of RBC by PEGylation protocols that do not conserve the charge with and without adding extension arms is compared. The conjugation of PEG-5000 to RBCs through the addition of extension arms masked the D antigen more efficiently than the other protocol. A combination of PEG-5 K and PEG-20 K is needed to mask the A antigen, irrespective of the PEGylation approach. The oxygen affinity of the PEGylated RBCs increased by the extension arm facilitated PEGylation. The protocol involving the conjugation of PEG-chains without adding extension arm did not alter the oxygen affinity of RBCs. A combination of PEGylation protocols is an alternate strategy to generate universal red blood cells with good levels of oxygen affinity.

Coagulation changes during lower body negative pressure and blood loss in humans.

PubMed

van Helmond, Noud; Johnson, Blair D; Curry, Timothy B; Cap, Andrew P; Convertino, Victor A; Joyner, Michael J

2015-11-01

We tested the hypothesis that markers of coagulation activation are greater during lower body negative pressure (LBNP) than those obtained during blood loss (BL). We assessed coagulation using both standard clinical tests and thrombelastography (TEG) in 12 men who performed a LBNP and BL protocol in a randomized order. LBNP consisted of 5-min stages at 0, -15, -30, and -45 mmHg of suction. BL included 5 min at baseline and following three stages of 333 ml of blood removal (up to 1,000 ml total). Arterial blood draws were performed at baseline and after the last stage of each protocol. We found that LBNP to -45 mmHg is a greater central hypovolemic stimulus versus BL; therefore, the coagulation markers were plotted against central venous pressure (CVP) to obtain stimulus-response relationships using the linear regression line slopes for both protocols. Paired t-tests were used to determine whether the slopes of these regression lines fell on similar trajectories for each protocol. Mean regression line slopes for coagulation markers versus CVP fell on similar trajectories during both protocols, except for TEG α° angle (-0.42 ± 0.96 during LBNP vs. -2.41 ± 1.13°/mmHg during BL; P < 0.05). During both LBNP and BL, coagulation was accelerated as evidenced by shortened R-times (LBNP, 9.9 ± 2.4 to 6.2 ± 1.1; BL, 8.7 ± 1.3 to 6.4 ± 0.4 min; both P < 0.05). Our results indicate that LBNP models the general changes in coagulation markers observed during BL. Copyright © 2015 the American Physiological Society.

Identification of anaerobic threshold by analysis of heart rate variability during discontinuous dynamic and resistance exercise protocols in healthy older men.

PubMed

Simões, Rodrigo Polaquini; Castello-Simões, Viviane; Mendes, Renata Gonçalves; Archiza, Bruno; Dos Santos, Daniel Augusto; Bonjorno, José Carlos; de Oliveira, Claudio Ricardo; Catai, Aparecida Maria; Arena, Ross; Borghi-Silva, Audrey

2014-03-01

The purposes of this study were to determine anaerobic threshold (AT) during discontinuous dynamic and resistive exercise protocols by analysing of heart rate variability (HRV) and blood lactate (BL) in healthy elderly subjects and compare the cardiovascular, metabolic and autonomic variables obtained from these two forms of exercise. Fourteen elderly (70 ± 4 years) apparently healthy males underwent the following tests: (i) incremental ramp test on cycle ergometer, (ii) one repetition maximum (1RM) leg press at 45°, (iii) a discontinuous exercise test on a cycle ergometer (DET-C) protocol and (iv) a resistance exercise leg press (DET-L) protocol. Heart rate, blood pressure and BL were obtained during each increment of exercise intensity. No significant differences (P>0·05) were found between methods of AT determination (BL and HRV) nor the relative intensity corresponding to AT (30% of maximum intensity) between the types of exercise (DET-C and DET-L). Furthermore, no significant differences (P>0·05) were found between the DET-C and DET-L in relation to HRV, however, the DET-L provided higher values of systolic blood pressure and BL (P<0·05) from the intensity corresponding to AT. We conclude that HRV was effective in determination of AT, and the parasympathetic modulation responses obtained during dynamic and resistive exercise protocols were similar when compared at the same relative intensity. However, DET-L resulted in higher values of blood pressure and BL at workloads beyond AT. © 2013 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd.

Use of filter papers to determine seroprevalence of Toxoplasma gondii among hunted ungulates in remote Peruvian Amazon.

PubMed

Aston, Emily J; Mayor, Pedro; Bowman, Dwight D; Mohammed, Hussni O; Liotta, Janice L; Kwok, Oliver; Dubey, J P

2014-04-01

Toxoplasmosis is a zoonosis caused by the protozoan Toxoplasma gondii, and it is found worldwide. To determine whether ungulates are reservoirs of T. gondii in an isolated and remote region of the northeastern Peruvian Amazon, antibodies to T. gondii were determined in 5 species of ungulates by the modified agglutination test (MAT). These animals were hunted by subsistence hunters along the Yavarí-Mirín River, in the northeastern Peruvian Amazon. Blood samples were collected by hunters on filter papers. For determination of T. gondii antibodies, blood was eluted from filter papers, and a titer of 1:25 was considered indicative of exposure to T. gondii. Antibodies to T. gondii were found in 26 (31.0%) peccaries (Pecari tajacu, Tayassu pecari), six (17.1%) brocket deer (Mazama americana, Mazama gouazoubira), and four (40.0%) lowland tapir (Tapirus terrestris). We also introduced a modification to the MAT protocol that allows the extraction of fluid samples from several types of laboratory-grade filter paper, thus enabling researchers to easily adapt their approaches to the materials presented to them.

Use of filter papers to determine seroprevalence of Toxoplasma gondii among hunted ungulates in remote Peruvian Amazon☆

PubMed Central

Aston, Emily J.; Mayor, Pedro; Bowman, Dwight D.; Mohammed, Hussni O.; Liotta, Janice L.; Kwok, Oliver; Dubey, J.P.

2013-01-01

Toxoplasmosis is a zoonosis caused by the protozoan Toxoplasma gondii, and it is found worldwide. To determine whether ungulates are reservoirs of T. gondii in an isolated and remote region of the northeastern Peruvian Amazon, antibodies to T. gondii were determined in 5 species of ungulates by the modified agglutination test (MAT). These animals were hunted by subsistence hunters along the Yavarí-Mirín River, in the northeastern Peruvian Amazon. Blood samples were collected by hunters on filter papers. For determination of T. gondii antibodies, blood was eluted from filter papers, and a titer of 1:25 was considered indicative of exposure to T. gondii. Antibodies to T. gondii were found in 26 (31.0%) peccaries (Pecari tajacu, Tayassu pecari), six (17.1%) brocket deer (Mazama americana, Mazama gouazoubira), and four (40.0%) lowland tapir (Tapirus terrestris). We also introduced a modification to the MAT protocol that allows the extraction of fluid samples from several types of laboratory-grade filter paper, thus enabling researchers to easily adapt their approaches to the materials presented to them. PMID:24918073

Detection of Plasmodium sp. in capybara.

PubMed

dos Santos, Leonilda Correia; Curotto, Sandra Mara Rotter; de Moraes, Wanderlei; Cubas, Zalmir Silvino; Costa-Nascimento, Maria de Jesus; de Barros Filho, Ivan Roque; Biondo, Alexander Welker; Kirchgatter, Karin

2009-07-07

In the present study, we have microscopically and molecularly surveyed blood samples from 11 captive capybaras (Hydrochaeris hydrochaeris) from the Sanctuary Zoo for Plasmodium sp. infection. One animal presented positive on blood smear by light microscopy. Polymerase chain reaction was carried out accordingly using a nested genus-specific protocol, which uses oligonucleotides from conserved sequences flanking a variable sequence region in the small subunit ribosomal RNA (ssrRNA) of all Plasmodium organisms. This revealed three positive animals. Products from two samples were purified and sequenced. The results showed less than 1% divergence between the two capybara sequences. When compared with GenBank sequences, a 55% similarity was obtained to Toxoplasma gondii and a higher similarity (73-77.2%) was found to ssrRNAs from Plasmodium species that infect reptile, avian, rodents, and human beings. The most similar Plasmodium sequence was from Plasmodium mexicanum that infects lizards of North America, where around 78% identity was found. This work is the first report of Plasmodium in capybaras, and due to the low similarity with other Plasmodium species, we suggest it is a new species, which, in the future could be denominated "Plasmodium hydrochaeri".

Validation of the Samsung SBM-100A and Microlife BP 3BU1-5 wrist blood pressure measuring devices in adults according to the International Protocol.

PubMed

Altunkan, Sekip; Ilman, Nevzat; Altunkan, Erkan

2007-04-01

A variety of automatic blood measurement devices with diverse features have been introduced to the medical markets recently. Among these devices, models that measure at the wrist have become increasingly popular in self measurements. The objective of this study was to evaluate the accuracy of the Samsung SBM-100A and Microlife BP 3BU1-5 wrist blood pressure devices against the mercury sphygmomanometer in adults according to the International Protocol criteria. Fifty-four patients over 30 years of age were studied and classified based on the International Protocol range. Blood pressure measurements at the wrist with the Samsung SBM-100A and Microlife BP 3BU1-5 were compared with the results obtained by two trained observers using a mercury sphygmomanometer. Nine sequential blood pressure measurements were taken. A total of 33 participants with randomly distributed arm circumferences were selected for both of the validation studies. During each validation study, 99 measurements were obtained for comparison from 33 participants. The first phase was performed on 15 participants and if the device passed this phase, 18 more participants were selected. Mean discrepancies and standard deviations of the device-sphygmomanometer were 0.9+/-9.2 and -2.7+/-9.3 mmHg for systolic blood pressure and -1.4+/-8.0 mmHg and 1.4+/-5.7 for diastolic blood pressure in the Samsung and Microlife study groups, respectively. The Samsung SBM-100A passed Phase 1 in 15 participants. Despite the fact that Microlife BP 3BU1-5 passed Phase 1 for diastolic pressure, it failed according to the systolic pressure criteria. Eighteen patients were added and Phase 2 was continued, in which Samsung SBM-100A failed to meet the criteria of Phases 2.1 and 2.2 for adults in systolic and diastolic blood pressure. It was found that the Microlife BP 3BU1-5 does not meet the criteria of either of Phases 2.1 and 2.2 for systolic blood pressure and Phase 2.2 for diastolic blood pressure. In this study, Samsung SBM-100A and Microlife blood pressure 3BU1-5 wrist blood pressure monitoring devices were found to be incompetent to meet the criteria of the International Protocol and it has not been possible to suggest any one of them for clinical use in adults.

Guidelines and sample protocol for sampling forest gaps.

Treesearch

J.R. Runkle

1992-01-01

A protocol for sampling forest canopy gaps is presented. Methods used in published gap studies are reviewed. The sample protocol will be useful in developing a broader understanding of forest structure and dynamics through comparative studies across different forest ecosystems.

It's Time to Develop a New "Draft Test Protocol" for a Mars Sample Return Mission (or Two…).

PubMed

Rummel, John D; Kminek, Gerhard

2018-04-01

The last time NASA envisioned a sample return mission from Mars, the development of a protocol to support the analysis of the samples in a containment facility resulted in a "Draft Test Protocol" that outlined required preparations "for the safe receiving, handling, testing, distributing, and archiving of martian materials here on Earth" (Rummel et al., 2002 ). This document comprised a specific protocol to be used to conduct a biohazard test for a returned martian sample, following the recommendations of the Space Studies Board of the US National Academy of Sciences. Given the planned launch of a sample-collecting and sample-caching rover (Mars 2020) in 2 years' time, and with a sample return planned for the end of the next decade, it is time to revisit the Draft Test Protocol to develop a sample analysis and biohazard test plan to meet the needs of these future missions. Key Words: Biohazard detection-Mars sample analysis-Sample receiving facility-Protocol-New analytical techniques-Robotic sample handling. Astrobiology 18, 377-380.

Epsilon Aminocaproic Acid to Reduce Blood Loss and Transfusion After Total Hip and Total Knee Arthroplasty.

PubMed

Hobbs, Juliann C; Welsby, Ian J; Green, Cynthia L; Dhakal, Ishwori B; Wellman, Samuel S

2018-01-01

Total hip and knee arthroplasty (THA and TKA) are associated with significant blood loss and some patients require postoperative blood transfusion. While tranexamic acid has been studied extensively among this population, we tested the hypothesis that epsilon aminocaproic acid (EACA) can reduce blood loss and transfusion after joint arthroplasty. In April 2014, our Veterans Affairs Medical Center introduced a protocol to administer EACA during THA and TKA. No antifibrinolytics were used previously. We retrospectively compared blood loss and incidence of transfusion among patients who underwent primary arthroplasty in the year before standardized administration of EACA with patients having the same procedures the following year. Blood loss was measured as delta hemoglobin (preoperative hemoglobin - hemoglobin on postoperative day 1). All patients undergoing primary THA or TKA were included. Patients having revision surgery were excluded. We identified 185 primary arthroplasty patients from the year before and 184 from the year after introducing the EACA protocol. There were no changes in surgical technique or attending surgeons during this period. Delta hemoglobin was significantly lower in the EACA group (2.7 ± 0.8 mg/dL) compared to the control group (3.4 ± 1.1 mg/dL) (P < .0001). The incidence of blood transfusion was also significantly lower in the EACA group (2.7%) compared to the control group (25.4%) (P < .0001). There was no difference in venous thromboembolic complications between groups. We demonstrated reductions in hemoglobin loss and transfusion following introduction of the EACA protocol in patients undergoing primary arthroplasty. EACA offers a lower cost alternative to TXA for reducing blood loss and transfusion in this population. Published by Elsevier Inc.

Tranexamic Acid versus Placebo to Prevent Blood Transfusion during Radical Cystectomy for Bladder Cancer (TACT): Study Protocol for a Randomized Controlled Trial.

PubMed

Breau, Rodney H; Lavallée, Luke T; Cnossen, Sonya; Witiuk, Kelsey; Cagiannos, Ilias; Momoli, Franco; Bryson, Gregory; Kanji, Salmaan; Morash, Christopher; Turgeon, Alexis; Zarychanski, Ryan; Mallick, Ranjeeta; Knoll, Greg; Fergusson, Dean A

2018-05-02

Radical cystectomy for bladder cancer is associated with a high risk of needing red blood cell transfusion. Tranexamic acid reduces blood loss during cardiac and orthopedic surgery, but no study has yet evaluated tranexamic acid use during cystectomy. A randomized, double-blind (surgeon-, anesthesiologist-, patient-, data-monitor-blinded), placebo-controlled trial of tranexamic acid during cystectomy was initiated in June 2013. Prior to incision, the intervention arm participants receive a 10 mg/kg loading dose of intravenously administered tranexamic acid, followed by a 5 mg/kg/h maintenance infusion. In the control arm, the patient receives an identical volume of normal saline that is indistinguishable from the intervention. The primary outcome is any blood transfusion from the start of surgery up to 30 days post operative. There are no strict criteria to mandate the transfusion of blood products. The decision to transfuse is entirely at the discretion of the treating physicians who are blinded to patient allocation. Physicians are allowed to utilize all resources to make transfusion decisions, including serum hemoglobin concentration and vital signs. To date, 147 patients of a planned 354 have been randomized to the study. This protocol reviews pertinent data relating to blood transfusion during radical cystectomy, highlighting the need to identify methods for reducing blood loss and preventing transfusion in patients receiving radical cystectomy. It explains the clinical rationale for using tranexamic acid to reduce blood loss during cystectomy, and outlines the study methods of our ongoing randomized controlled trial. Canadian Institute for Health Research (CIHR) Protocol: MOP-342559; ClinicalTrials.gov, ID: NCT01869413. Registered on 5 June 2013.

Rapid Identification of Microorganisms from Positive Blood Culture by MALDI-TOF MS After Short-Term Incubation on Solid Medium.

PubMed

Curtoni, Antonio; Cipriani, Raffaella; Marra, Elisa Simona; Barbui, Anna Maria; Cavallo, Rossana; Costa, Cristina

2017-01-01

Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a useful tool for rapid identification of microorganisms. Unfortunately, its direct application to positive blood culture is still lacking standardized procedures. In this study, we evaluated an easy- and rapid-to-perform protocol for MALDI-TOF MS direct identification of microorganisms from positive blood culture after a short-term incubation on solid medium. This protocol was used to evaluate direct identification of microorganisms from 162 positive monomicrobial blood cultures; at different incubation times (3, 5, 24 h), MALDI-TOF MS assay was performed from the growing microorganism patina. Overall, MALDI-TOF MS concordance with conventional methods at species level was 60.5, 80.2, and 93.8% at 3, 5, and 24 h, respectively. Considering only bacteria, the identification performances at species level were 64.1, 85.0, and 94.1% at 3, 5, and 24 h, respectively. This protocol applied to a commercially available MS typing system may represent, a fast and powerful diagnostic tool for pathogen direct identification and for a promptly and pathogen-driven antimicrobial therapy in selected cases.

Physiological response of wild dugongs (Dugong dugon) to out-of-water sampling for health assessment

USGS Publications Warehouse

Lanyon, Janet M.; Sneath, Helen L.; Long, Trevor; Bonde, Robert K.

2010-01-01

The dugong (Dugong dugon) is a vulnerable marine mammal with large populations living in urban Queensland waters. A mark-recapture program for wild dugongs has been ongoing in southern Queensland since 2001. This program has involved capture and in-water sampling of more than 700 dugongs where animals have been held at the water surface for 5 min to be gene-tagged, measured, and biopsied. In 2008, this program expanded to examine more comprehensively body condition, reproductive status, and the health of wild dugongs in Moreton Bay. Using Sea World's research vessel, captured dugongs were lifted onto a boat and sampled out-of-water to obtain accurate body weights and morphometrics, collect blood and urine samples for baseline health parameters and hormone profiles, and ultrasound females for pregnancy status. In all, 30 dugongs, including two pregnant females, were sampled over 10 d and restrained on deck for up to 55 min each while biological data were collected. Each of the dugongs had their basic temperature-heart rate-respiration (THR) monitored throughout their period of handling, following protocols developed for the West Indian manatee (Trichechus manatus). This paper reports on the physiological response of captured dugongs during this out-of-water operation as indicated by their vital signs and the suitability of the manatee monitoring protocols to this related sirenian species. A recommendation is made that the range of vital signs of these wild dugongs be used as benchmark criteria of normal parameters for other studies that intend to sample dugongs out-of-water.

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels.

PubMed

Brzica, Hrvoje; Abdullahi, Wazir; Reilly, Bianca G; Ronaldson, Patrick T

2018-05-07

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of central nervous system (CNS) diseases. Many BBB changes that affect pharmacotherapy, involve proteins that are localized and expressed at the level of endothelial cells. Indeed, such knowledge on BBB physiology in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for a simple but robust and reproducible method for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in the presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to prepare samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this manuscript, details regarding a method that is utilized for isolation of rat brain microvessels and preparation of membrane samples are provided. Microvessel enrichment, from samples derived, is achieved by using four centrifugation steps where dextran is included in the sample buffer. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from protein analysis experiments that can greatly aid the understanding of BBB responses to physiological, pathophysiological, and pharmacological stimuli.

Relevance to Home Blood Pressure Monitoring Protocol of Blood Pressure Measurements Taken Before First-Morning Micturition and in the Afternoon

PubMed Central

de Almeida, Antonio Eduardo Monteiro; Stein, Ricardo; Gus, Miguel; Nascimento, João Agnaldo; Belli, Karlyse Claudino; Arévalo, Jorge Rene Garcia; Fuchs, Flávio Dani

2014-01-01

Background The importance of measuring blood pressure before morning micturition and in the afternoon, while working, is yet to be established in relation to the accuracy of home blood pressure monitoring (HBPM). Objective: To compare two HBPM protocols, considering 24-hour ambulatory blood pressure monitoring (wakefulness ABPM) as gold-standard and measurements taken before morning micturition (BM) and in the afternoon (AM), for the best diagnosis of systemic arterial hypertension (SAH), and their association with prognostic markers. Methods After undergoing 24-hour wakefulness ABPM, 158 participants (84 women) were randomized for 3- or 5-day HBPM. Two variations of the 3-day protocol were considered: with measurements taken before morning micturition and in the afternoon (BM+AM); and with post-morning-micturition and evening measurements (PM+EM). All patients underwent echocardiography (for left ventricular hypertrophy - LVH) and urinary albumin measurement (for microalbuminuria - MAU). Result Kappa statistic for the diagnosis of SAH between wakefulness-ABPM and standard 3-day HBPM, 3-day HBPM (BM+AM) and (PM+EM), and 5-day HBPM were 0.660, 0.638, 0.348 and 0.387, respectively. The values of sensitivity of (BM+AM) versus (PM+EM) were 82.6% × 71%, respectively, and of specificity, 84.8% × 74%, respectively. The positive and negative predictive values were 69.1% × 40% and 92.2% × 91.2%, respectively. The comparisons of intraclass correlations for the diagnosis of LVH and MAU between (BM+AM) and (PM+EM) were 0.782 × 0.474 and 0.511 × 0.276, respectively. Conclusions The 3 day-HBPM protocol including measurements taken before morning micturition and during work in the afternoon showed the best agreement with SAH diagnosis and the best association with prognostic markers. PMID:25352508

Simulated night shift work induces circadian misalignment of the human peripheral blood mononuclear cell transcriptome.

PubMed

Kervezee, Laura; Cuesta, Marc; Cermakian, Nicolas; Boivin, Diane B

2018-05-22

Misalignment of the endogenous circadian timing system leads to disruption of physiological rhythms and may contribute to the development of the deleterious health effects associated with night shift work. However, the molecular underpinnings remain to be elucidated. Here, we investigated the effect of a 4-day simulated night shift work protocol on the circadian regulation of the human transcriptome. Repeated blood samples were collected over two 24-hour measurement periods from eight healthy subjects under highly controlled laboratory conditions before and 4 days after a 10-hour delay of their habitual sleep period. RNA was extracted from peripheral blood mononuclear cells to obtain transcriptomic data. Cosinor analysis revealed a marked reduction of significantly rhythmic transcripts in the night shift condition compared with baseline at group and individual levels. Subsequent analysis using a mixed-effects model selection approach indicated that this decrease is mainly due to dampened rhythms rather than to a complete loss of rhythmicity: 73% of transcripts rhythmically expressed at baseline remained rhythmic during the night shift condition with a similar phase relative to habitual bedtimes, but with lower amplitudes. Functional analysis revealed that key biological processes are affected by the night shift protocol, most notably the natural killer cell-mediated immune response and Jun/AP1 and STAT pathways. These results show that 4 days of simulated night shifts leads to a loss in temporal coordination between the human circadian transcriptome and the external environment and impacts biological processes related to the adverse health effects associated to night shift work.

[Effectiveness of a nursing intervention on patient anxiety before transfusion of packed red blood cells].

PubMed

Martín Díaz, Jesús Fernando; Hidalgo Gutiérrez, M Jesús; Cerezo Solana, M Fátima; Martín Morcillo, Jaime

2013-01-01

To evaluate the effectiveness of an educational intervention regarding anxiety and satisfaction in patients requiring a red blood cell transfusion. Randomised, controlled, single-blind clinical trial in patients requiring a packed red blood cell transfusion. alpha=.05, beta=.10, to detect a 10% difference, 70 subjects in each group. The sampling recruitment was randomised to the intervention group (IG) and the control group (CG). an intervention protocol with oral and written information using a published guide on the safety, risks and benefits of haemotherapy for the IG, and an equivalent one on general health topics for the CG. pre- and post-anxiety state; Spielberger's validated questionnaire: STAI. Satisfaction, by an ad hoc questionnaire. Sociodemographic and clinical variables: description, reason for transfusion, prescription knowledge, incidents, records. There was a total of 144 subjects, 73 (50.69%) in the IG, and 71 (49.31%) in the CG. The mean age was 55.80 years, with 56.94% males, and a first transfusion in 52.08%. Comparability between the IG and the CG was tested and confirmed. The decrease in anxiety after the intervention for the IG was 19.99, compared to 25.48 in CG. The difference was greater than the proposed 10%, and was statistically significant. The preference for information was 98.60% in IG, compared to 43.70% in CG. The hypothesis was confirmed; a protocolised nursing educational intervention protocol increased patient satisfaction with nursing care, and decreased patient anxiety, thus preventing complications and providing greater safety to the users. Copyright © 2013 Elsevier España, S.L. All rights reserved.

Clinical investigation of the protective effects of palm vitamin E tocotrienols on brain white matter.

PubMed

Gopalan, Yogheswaran; Shuaib, Ibrahim Lutfi; Magosso, Enrico; Ansari, Mukhtar Alam; Abu Bakar, Mohd Rizal; Wong, Jia Woei; Khan, Nurzalina Abdul Karim; Liong, Wei Chuen; Sundram, Kalyana; Ng, Bee Hong; Karuthan, Chinna; Yuen, Kah Hay

2014-05-01

Previous cell-based and animal studies showed mixed tocotrienols are neuroprotective, but the effect is yet to be proven in humans. Thus, the present study aimed to evaluate the protective activity of mixed tocotrienols in humans with white matter lesions (WMLs). WMLs are regarded as manifestations of cerebral small vessel disease, reflecting varying degrees of neurodegeneration and tissue damage with potential as a surrogate end point in clinical trials. A total of 121 volunteers aged ≥35 years with cardiovascular risk factors and MRI-confirmed WMLs were randomized to receive 200 mg mixed tocotrienols or placebo twice a day for 2 years. The WML volumes were measured from MRI images taken at baseline, 1 year, and 2 years using a validated software and were compared. Fasting blood samples were collected for full blood chemistry investigation. According to per-protocol (88 volunteers) and intention-to-treat (121 volunteers) analyses, the mean WML volume of the placebo group increased after 2 years, whereas that of the tocotrienol-supplemented group remained essentially unchanged. The mean WML volume change between the 2 groups was not significantly different (P=0.150) at the end of 1 year but was significant at the end of 2 years for both per-protocol and intention-to-treat analyses (P=0.019 and P=0.018). No significant difference was observed in the blood chemistry parameters between the 2 groups. Mixed tocotrienols were found to attenuate the progression of WMLs. http://www.clinicaltrials.gov. Unique identifier: NCT00753532.

Mapping of Schistosomiasis and Soil-Transmitted Helminths in Namibia: The First Large-Scale Protocol to Formally Include Rapid Diagnostic Tests.

PubMed

Sousa-Figueiredo, José Carlos; Stanton, Michelle C; Katokele, Stark; Arinaitwe, Moses; Adriko, Moses; Balfour, Lexi; Reiff, Mark; Lancaster, Warren; Noden, Bruce H; Bock, Ronnie; Stothard, J Russell

2015-01-01

Namibia is now ready to begin mass drug administration of praziquantel and albendazole against schistosomiasis and soil-transmitted helminths, respectively. Although historical data identifies areas of transmission of these neglected tropical diseases (NTDs), there is a need to update epidemiological data. For this reason, Namibia adopted a new protocol for mapping of schistosomiasis and geohelminths, formally integrating rapid diagnostic tests (RDTs) for infections and morbidity. In this article, we explain the protocol in detail, and introduce the concept of 'mapping resolution', as well as present results and treatment recommendations for northern Namibia. This new protocol allowed a large sample to be surveyed (N = 17,896 children from 299 schools) at relatively low cost (7 USD per person mapped) and very quickly (28 working days). All children were analysed by RDTs, but only a sub-sample was also diagnosed by light microscopy. Overall prevalence of schistosomiasis in the surveyed areas was 9.0%, highly associated with poorer access to potable water (OR = 1.5, P<0.001) and defective (OR = 1.2, P<0.001) or absent sanitation infrastructure (OR = 2.0, P<0.001). Overall prevalence of geohelminths, more particularly hookworm infection, was 12.2%, highly associated with presence of faecal occult blood (OR = 1.9, P<0.001). Prevalence maps were produced and hot spots identified to better guide the national programme in drug administration, as well as targeted improvements in water, sanitation and hygiene. The RDTs employed (circulating cathodic antigen and microhaematuria for Schistosoma mansoni and S. haematobium, respectively) performed well, with sensitivities above 80% and specificities above 95%. This protocol is cost-effective and sensitive to budget limitations and the potential economic and logistical strains placed on the national Ministries of Health. Here we present a high resolution map of disease prevalence levels, and treatment regimens are recommended.

Mapping of Schistosomiasis and Soil-Transmitted Helminths in Namibia: The First Large-Scale Protocol to Formally Include Rapid Diagnostic Tests

PubMed Central

Sousa-Figueiredo, José Carlos; Stanton, Michelle C.; Katokele, Stark; Arinaitwe, Moses; Adriko, Moses; Balfour, Lexi; Reiff, Mark; Lancaster, Warren; Noden, Bruce H.; Bock, Ronnie; Stothard, J. Russell

2015-01-01

Background Namibia is now ready to begin mass drug administration of praziquantel and albendazole against schistosomiasis and soil-transmitted helminths, respectively. Although historical data identifies areas of transmission of these neglected tropical diseases (NTDs), there is a need to update epidemiological data. For this reason, Namibia adopted a new protocol for mapping of schistosomiasis and geohelminths, formally integrating rapid diagnostic tests (RDTs) for infections and morbidity. In this article, we explain the protocol in detail, and introduce the concept of ‘mapping resolution’, as well as present results and treatment recommendations for northern Namibia. Methods/Findings/Interpretation This new protocol allowed a large sample to be surveyed (N = 17 896 children from 299 schools) at relatively low cost (7 USD per person mapped) and very quickly (28 working days). All children were analysed by RDTs, but only a sub-sample was also diagnosed by light microscopy. Overall prevalence of schistosomiasis in the surveyed areas was 9.0%, highly associated with poorer access to potable water (OR = 1.5, P<0.001) and defective (OR = 1.2, P<0.001) or absent sanitation infrastructure (OR = 2.0, P<0.001). Overall prevalence of geohelminths, more particularly hookworm infection, was 12.2%, highly associated with presence of faecal occult blood (OR = 1.9, P<0.001). Prevalence maps were produced and hot spots identified to better guide the national programme in drug administration, as well as targeted improvements in water, sanitation and hygiene. The RDTs employed (circulating cathodic antigen and microhaematuria for Schistosoma mansoni and S. haematobium, respectively) performed well, with sensitivities above 80% and specificities above 95%. Conclusion/Significance This protocol is cost-effective and sensitive to budget limitations and the potential economic and logistical strains placed on the national Ministries of Health. Here we present a high resolution map of disease prevalence levels, and treatment regimens are recommended. PMID:26196386

Protocol for obtaining platelet-rich plasma (PRP), platelet-poor plasma (PPP), and thrombin for autologous use.

PubMed

Franco, Diogo; Franco, Talita; Schettino, Angélica Maria; Filho, João Medeiros Tavares; Vendramin, Fabiel Spani

2012-10-01

Plasma has been widely studied and used in many different situations to speed up healing with better tissue adherence and hemostasis. Research projects are now attempting to isolate platelet-rich plasma (PRP) and platelet-poor plasma (PPP), making better use of their properties, particularly during operations and for wounds that are slow to heal. In view of the wide diversity of industrial machines and extraction protocols, together with the variety of industrially produced biologic glues, this article suggests an option for obtaining PRP, PPP, and human thrombin for autologous use. A way of obtaining PRP, PPP, and thrombin is reproduced through a protocol defined and established by the authors. Autologous thrombin and plasma were obtained through the collection and successive centrifugation of ten whole blood samples, until the desired hemocomponents were isolated, followed by quantitative and qualitative analyses of the elements obtained. The mean platelet concentration obtained was 6.03 × 10(8) platelets/ml, with a mean thrombin concentration of 33.54 nM, both values compatible with reports in the literature when different protocols are applied. The protocol described is a good option for the preparation and application of PRP, PPP, and autologous thrombin, particularly as they can be obtained simultaneously, eliminating the possibilities of viral contamination and allergic reactions. Moreover, the cost of this procedure is low, it is easy to perform, and replicable. This journal requires that authors assign a level of evidence to each article.

EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols

PubMed Central

Kalina, T; Flores-Montero, J; van der Velden, V H J; Martin-Ayuso, M; Böttcher, S; Ritgen, M; Almeida, J; Lhermitte, L; Asnafi, V; Mendonça, A; de Tute, R; Cullen, M; Sedek, L; Vidriales, M B; Pérez, J J; te Marvelde, J G; Mejstrikova, E; Hrusak, O; Szczepański, T; van Dongen, J J M; Orfao, A

2012-01-01

The EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis and classification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory procedures and antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection of optimal combinations of compatible fluorochromes and the design and evaluation of adequate standard operating procedures (SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for the evaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentally versus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility based on strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments and the analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first time laboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal and malignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a single database. PMID:22948490

Emergency Whole Blood Use in the Field: A Simplified Protocol for Collection and Transfusion

DTIC Science & Technology

2014-01-01

renewed interest in the reintroduction of fresh whole blood and cold- stored whole blood to patient care in austere environments. There is scant...blood use in the treat- ment of exsanguinating hemorrhage and have renewed interest in the reintroduction of warm whole blood (WWB) stored at 22-C for...data pro- vide a biological rationale for whole-blood use in the treatment of exsanguinating hemorrhage and have renewed interest in the reintroduction

Sampling protocol for post-landfall Deepwater Horizon oil release, Gulf of Mexico, 2010

USGS Publications Warehouse

Wilde, F.D.; Skrobialowski, S.C.; Hart, J.S.

2010-01-01

The protocols and procedures described in this report are designed to be used by U.S. Geological Survey (USGS) field teams for the collection of environmental data and samples in coastal areas affected by the 2010 Deepwater Horizon oil spill in the Gulf of Mexico. This sampling protocol focuses specifically on sampling for water, sediments, benthic invertebrates, and microorganisms (ambient bacterial populations) after shoreline arrival of petroleum-associated product on beach, barrier island, and wetland environments of the Gulf of Mexico coastal states. Deployment to sampling sites, site setup, and sample collection in these environments necessitates modifications to standard USGS sampling procedures in order to address the regulatory, logistical, and legal requirements associated with samples collected in oil-impacted coastal areas. This document, therefore, has been written as an addendum to the USGS National Field Manual for the Collection of Water-Quality Data (NFM) (http://pubs.water.usgs.gov/twri9A/), which provides the basis for training personnel in the use of standard USGS sampling protocols. The topics covered in this Gulf of Mexico oil-spill sampling protocol augment NFM protocols for field-deployment preparations, health and safety precautions, sampling and quality-assurance procedures, and decontamination requirements under potentially hazardous environmental conditions. Documentation procedures and maintenance of sample integrity by use of chain-of-custody procedures also are described in this protocol.

Pre-cut Filter Paper for Detecting Anti-Japanese Encephalitis Virus IgM from Dried Cerebrospinal Fluid Spots

PubMed Central

Bharucha, Tehmina; Chanthongthip, Anisone; Phuangpanom, Soumphou; Phonemixay, Ooyanong; Sengvilaipaseuth, Onanong; Vongsouvath, Manivanh; Lee, Sue; Newton, Paul N.; Dubot-Pérès, Audrey

2016-01-01

Background The use of filter paper as a simple, inexpensive tool for storage and transportation of blood, ‘Dried Blood Spots’ or Guthrie cards, for diagnostic assays is well-established. In contrast, there are a paucity of diagnostic evaluations of dried cerebrospinal fluid (CSF) spots. These have potential applications in low-resource settings, such as Laos, where laboratory facilities for central nervous system (CNS) diagnostics are only available in Vientiane. In Laos, a major cause of CNS infection is Japanese encephalitis virus (JEV). We aimed to develop a dried CSF spot protocol and to evaluate its diagnostic performance using the World Health Organisation recommended anti-JEV IgM antibody capture enzyme-linked immunosorbent assay (JEV MAC-ELISA). Methodology and Principal Findings Sample volumes, spotting techniques and filter paper type were evaluated using a CSF-substitute of anti-JEV IgM positive serum diluted in Phosphate Buffer Solution (PBS) to end-limits of detection by JEV MAC-ELISA. A conventional protocol, involving eluting one paper punch in 200μl PBS, did not detect the end-dilution, nor did multiple punches utilising diverse spotting techniques. However, pre-cut filter paper enabled saturation with five times the volume of CSF-substitute, sufficiently improving sensitivity to detect the end-dilution. The diagnostic accuracy of this optimised protocol was compared with routine, neat CSF in a pilot, retrospective study of JEV MAC-ELISA on consecutive CSF samples, collected 2009–15, from three Lao hospitals. In comparison to neat CSF, 132 CSF samples stored as dried CSF spots for one month at 25–30°C showed 81.6% (65.7–92.3 95%CI) positive agreement, 96.8% (91.0–99.3 95%CI) negative agreement, with a kappa coefficient of 0.81 (0.70–0.92 95%CI). Conclusions/Significance The novel design of pre-cut filter paper saturated with CSF could provide a useful tool for JEV diagnostics in settings with limited laboratory access. It has the potential to improve national JEV surveillance and inform vaccination policies. The saturation of filter paper has potential use in the wider context of pathogen detection, including dried spots for detecting other analytes in CSF, and other body fluids. PMID:26986061

Performance of strip-based glucose meters and cassette-based blood gas analyzer for monitoring glucose levels in a surgical intensive care setting.

PubMed

Claerhout, Helena; De Prins, Martine; Mesotten, Dieter; Van den Berghe, Greet; Mathieu, Chantal; Van Eldere, Johan; Vanstapel, Florent

2016-01-01

We verified the analytical performance of strip-based handheld glucose meters (GM) for prescription use, in a comparative split-sample protocol using blood gas samples from a surgical intensive care unit (ICU). Freestyle Precision Pro (Abbott), StatStrip Connectivity Meter (Nova), ACCU-CHEK Inform II (Roche) were evaluated for recovery/linearity, imprecision/repeatability. The GMs and the ABL90 (Radiometer) blood gas analyzer (BGA) were tested for relative accuracy vs. the comparator hexokinase glucose-6-phosphate-dehydrogenase (HK/G6PDH) assay on a Cobas c702 analyzer (Roche). Recovery of spiked glucose was linear up to 19.3 mmol/L (347 mg/dL) with a slope of 0.91-0.94 for all GMs. Repeatability estimated by pooling duplicate measurements on samples below (n=9), in (n=51) or above (n=80) the 4.2-5.9 mM (74-106 mg/dL) range were for Freestyle Precision Pro: 4.2%, 4.0%, 3.6%; StatStrip Connectivity Meter: 4.0%, 4.3%, 4.5%; and ACCU-CHEK Inform II: 1.4%, 2.5%, 3.5%. GMs were in agreement with the comparator method. The BGA outperformed the GMs, with a MARD of 3.9% compared to 6.5%, 5.8% and 4.4% for the FreeStyle, StatStrip and ACCU-CHEK, respectively. Zero % of the BGA results deviated more than the FDA 10% criterion as compared to 9.4%, 3.7% and 2.2% for the FreeStyle, StatStrip and ACCU-CHEK, respectively. For all GMs, icodextrin did not interfere. Variation in the putative influence factors hematocrit and O2 tension could not explain observed differences with the comparator method. GMs quantified blood glucose in whole blood at about the 10% total error criterion, proposed by the FDA for prescription use.

Mars Sample Handling Protocol Workshop Series: Workshop 4

NASA Technical Reports Server (NTRS)

Race Margaret S. (Editor); DeVincenzi, Donald L. (Editor); Rummel, John D. (Editor); Acevedo, Sara E. (Editor)

2001-01-01

In preparation for missions to Mars that will involve the return of samples to Earth, it will be necessary to prepare for the receiving, handling, testing, distributing, and archiving of martian materials here on Earth. Previous groups and committees have studied selected aspects of sample return activities, but specific detailed protocols for the handling and testing of returned samples must still be developed. To further refine the requirements for sample hazard testing and to develop the criteria for subsequent release of sample materials from quarantine, the NASA Planetary Protection Officer convened a series of workshops in 2000-2001. The overall objective of the Workshop Series was to produce a Draft Protocol by which returned martian sample materials can be assessed for biological hazards and examined for evidence of life (extant or extinct) while safeguarding the purity of the samples from possible terrestrial contamination. This report also provides a record of the proceedings of Workshop 4, the final Workshop of the Series, which was held in Arlington, Virginia, June 5-7, 2001. During Workshop 4, the sub-groups were provided with a draft of the protocol compiled in May 2001 from the work done at prior Workshops in the Series. Then eight sub-groups were formed to discuss the following assigned topics: Review and Assess the Draft Protocol for Physical/Chemical Testing Review and Assess the Draft Protocol for Life Detection Testing Review and Assess the Draft Protocol for Biohazard Testing Environmental and Health/Monitoring and Safety Issues Requirements of the Draft Protocol for Facilities and Equipment Contingency Planning for Different Outcomes of the Draft Protocol Personnel Management Considerations in Implementation of the Draft Protocol Draft Protocol Implementation Process and Update Concepts This report provides the first complete presentation of the Draft Protocol for Mars Sample Handling to meet planetary protection needs. This Draft Protocol, which was compiled from deliberations and recommendations from earlier Workshops in the Series, represents a consensus that emerged from the discussions of all the sub-groups assembled over the course of the five Workshops of the Series. These discussions converged on a conceptual approach to sample handling, as well as on specific analytical requirements. Discussions also identified important issues requiring attention, as well as research and development needed for protocol implementation.

Validation of the Andon KD595 for clinical use and self-measurement according to the European Society of Hypertension International Protocol.

PubMed

Wu, Ning; Zhang, Xuezhong; Wang, Wen; Zhang, Hongye

2016-04-01

This study aimed to evaluate the accuracy of the automated oscillometric upper arm blood pressure monitor Andon KD595 for home blood pressure monitoring according to the European Society of Hypertension International Protocol revision 2010. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were sequentially measured in 33 participants using the standard mercury sphygmomanometer and the Andon KD595 device. Ninety-nine pairs of comparisons were obtained from 33 participants for analysis. The KD595 device achieved the targets in part 1 of the validation study. The number of absolute differences between the device and the observers within a range of 5, 10, and 15 mmHg was 72/99, 93/99, and 96/99, respectively, for SBP and 72/99, 96/99, and 99/99, respectively, for DBP. The device also achieved the targets in part 2 of the validation study. A total of 28 and 25 participants had at least two of the three device-observer differences within 5 mmHg (required≥24) for SBP and DBP, respectively. The number of participants without device-observer difference within 5 mmHg was two for SBP and two for DBP (required≤3). The Andon upper arm blood pressure monitor KD595 has passed the International Protocol requirements and it can be recommended for clinical use and self-measurement in adults.

Outcomes in cardiac surgery in 500 consecutive Jehovah's Witness patients: 21 year Experience

PubMed Central

2012-01-01

Background Refusal of heterogenic blood products can be for religious reasons as in Jehovah's Witnesses or otherwise or as requested by an increasing number of patients. Furthermore blood reserves are under continuous demand with increasing costs. Therefore, transfusion avoidance strategies are desirable. We describe a historic comparison and current results of blood saving protocols in Jehovah's Witnesses patients. Methods Data on 250 Jehovah's Witness patients operated upon between 1991 and 2003 (group A) were reviewed and compared with a second population of 250 patients treated from 2003 to 2012 (group B). Results In group A, mean age was 51 years of age compared to 68 years in group B. An iterative procedure was performed in 13% of patients in group B. Thirty days mortality was 3% in group A and 1% in group B despite greater operative risk factors, with more redo, and lower ejection fraction in group B. Several factors contributed to the low morbidity-mortality in group B, namely: preoperative erythropoietin to attain a minimal hemoglobin value of 14 g/dl, warm blood cardioplegia, the implementation of the Cornell University protocol and fast track extubation. Conclusions Cardiac surgery without transfusion in high-risk patients such as Jehovah Witnesses can be carried out with results equivalent to those of low risk patients. Recent advances in surgical techniques and blood conservation protocols are main contributing factors. PMID:23013647

Real-time PCR strategy for the identification of Trypanosoma cruzi discrete typing units directly in chronically infected human blood.

PubMed

Muñoz-San Martín, Catalina; Apt, Werner; Zulantay, Inés

2017-04-01

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a major public health problem in Latin America. This parasite has a complex population structure comprised by six or seven major evolutionary lineages (discrete typing units or DTUs) TcI-TcVI and TcBat, some of which have apparently resulted from ancient hybridization events. Because of the existence of significant biological differences between these lineages, strain characterization methods have been essential to study T. cruzi in its different vectors and hosts. However, available methods can be laborious and costly, limited in resolution or sensitivity. In this study, a new genotyping strategy by real-time PCR to identify each of the six DTUs in clinical blood samples have been developed and evaluated. Two nuclear (SL-IR and 18S rDNA) and two mitochondrial genes (COII and ND1) were selected to develop original primers. The method was evaluated with eight genomic DNA of T. cruzi populations belonging to the six DTUs, one genomic DNA of Trypanosoma rangeli, and 53 blood samples from individuals with chronic Chagas disease. The assays had an analytical sensitivity of 1-25fg of DNA per reaction tube depending on the DTU analyzed. The selectivity of trials with 20fg/μL of genomic DNA identified each DTU, excluding non-targets DTUs in every test. The method was able to characterize 67.9% of the chronically infected clinical samples with high detection of TcII followed by TcI. With the proposed original genotyping methodology, each DTU was established with high sensitivity after a single real-time PCR assay. This novel protocol reduces carryover contamination, enables detection of each DTU independently and in the future, the quantification of each DTU in clinical blood samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Solid-phase extraction of the alcohol abuse biomarker phosphatidylethanol using newly synthesized polymeric sorbent materials containing quaternary heterocyclic groups.

PubMed

Duarte, Mariana; Jagadeesan, Kishore Kumar; Billing, Johan; Yilmaz, Ecevit; Laurell, Thomas; Ekström, Simon

2017-10-13

Phosphatidylethanol (PEth) is an interesting biomarker finding increased use for detecting long term alcohol abuse with high specificity and sensitivity. Prior to detection, sample preparation is an unavoidable step in the work-flow of PEth analysis and new protocols may facilitate it. Solid-phase extraction (SPE) is a versatile sample preparation method widely spread in biomedical laboratories due to its simplicity of use and the possibility of automation. In this work, SPE was used for the first time to directly extract PEth from spiked human plasma and spiked human blood. A library of polymeric SPE materials with different surface functionalities was screened for PEth extraction in order to identify the surface characteristics that control PEth retention and recovery. The plasma samples were diluted 1:10 (v/v) in water and spiked at different concentrations ranging from 0.3 to 5μM. The library of SPE materials was then evaluated using the proposed SPE method and detection was done by LC-MS/MS. One SPE material efficiently retained and recovered PEth from spiked human plasma. With this insight, four new SPE materials were formulated and synthesized based on the surface characteristics of the best SPE material found in the first screening. These new materials were tested with spiked human blood, to better mimic a real clinical sample. All the newly synthetized materials outperformed the pre-existing commercially available materials. Recovery values for the new SPE materials were found between 29.5% and 48.6% for the extraction of PEth in spiked blood. A material based on quaternized 1-vinylimidazole with a poly(trimethylolpropane trimethacrylate) backbone was found suitable for PEth extraction in spiked blood showing the highest analyte recovery in this experiment, 48.6%±6.4%. Copyright © 2017 Elsevier B.V. All rights reserved.

Validation of the Tensoval Duo Control II blood pressure monitor for clinic use and self-measurement according to the British Hypertension Society protocol and the European Society of Hypertension International Protocol Revision 2010.

PubMed

de Greeff, Annemarie; Shennan, Andrew H

2013-06-01

The Tensoval Duo Control II is an automated upper arm device that uses a combination of oscillometric and auscultatory technology to determine blood pressure noninvasively. The accuracy of this device was assessed according to the British Hypertension Society (BHS) protocol and the European Society of Hypertension International Protocol revision 2010 (ESH-IP2) in an adult population. Ethical approval was obtained. Eighty-five and 33 adult individuals, respectively, were recruited to fulfil the requirements of each protocol. Trained observers took nine sequential same-arm measurements alternating between a mercury sphygmomanometer and the device. The device had to achieve at least a B grade for both systolic and diastolic pressures to pass the BHS protocol and had to fulfil the criteria of all three phases of the ESH-IP2 protocol to receive recommendation. The device achieved an A/A grading for the BHS protocol and passed all three phases of the ESH-IP2 protocol. The mean difference±SD for the BHS/ESH protocols, respectively, was -1.8±6.5/-0.7±5.7 mmHg for systolic pressure and 1.9±5.1/2.4±4.5 mmHg for diastolic pressure. The device maintained its A/A grading throughout the low-pressure, medium-pressure and high-pressure ranges. The Tensoval Duo Control II device is recommended for clinical and home use according to both the BHS and the ESH-IP2 standard.

Frecuency of antigens and alloantibodies of Diego system in blood

PubMed

Góngora, Fernando B; Chiriboga-Ponce, Rosa F

2018-01-01

The Diego blood group is an irregular blood system which has been involved in cases of hemolytic disease of the newborn and post transfusion reactions, in this system have been identified 22 erythrocyte antigens, among which the pair Di a /Di b is the most important because those have the most immunogenic potential. This research aims to determine the frequency of antigen Dia and their respective alloantibody in the Ecuadorian population. Methods: It was performed a simple random sampling in the donor population, being later tested tube agglutination by the presence or absence of the antigen and its alloantibody Di a applying gel agglutination technique. It was observed an antigen prevalence of 25% against a 6.09% of percentage alloimmunization due to Di a antigen, without significant differences between men and women and these being independent of the age and origin of the donor, showing that there are some Diego positive cases in Ecuadorian population as probably cases of transfusional alloinmunization or due to fetal-maternal alloinmunization. The frequency distribution of antigens and alloantibodies from the Diego blood group is almost uniform in the population, due presumably to the high incidence of miscegenation in our country. Therefore it becomes vitally important the implementation of this blood system inside the protocols of irregular antibodies identification in Ecuadorian blood banks. Copyright: © 2018 SecretarÍa de Salud

Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma.

PubMed

Li, Congfen; Takahashi, Chikara; Zhang, Liangxuan; Huseni, Mahrukh; Stankovich, Basha; Mashhedi, Haider; Lee, Joanna; French, Dorothy; Anderson, Jeff Eastham; Kim, Doris; Howell, Kathy; Brauer, Matthew J; Kowanetz, Marcin; Yan, Yibing; Humke, Eric; Ebens, Allen; Hampton, Garret; Lackner, Mark R; Hegde, Priti; Jia, Shidong

2013-03-23

The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.

Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma

PubMed Central

2013-01-01

Background The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. Methods We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. Results The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. Conclusions We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials. PMID:23522020

Evaluation of storage and filtration protocols for alpine/subalpine lake water quality samples

Treesearch

John L. Korfmacher; Robert C. Musselman

2007-01-01

Many government agencies and other organizations sample natural alpine and subalpine surface waters using varying protocols for sample storage and filtration. Simplification of protocols would be beneficial if it could be shown that sample quality is unaffected. In this study, samples collected from low ionic strength waters in alpine and subalpine lake inlets...

Pathology in Continuous Infusion Studies in Rodents and Non-Rodents and ITO (Infusion Technology Organisation)-Recommended Protocol for Tissue Sampling and Terminology for Procedure-Related Lesions

PubMed Central

Weber, Klaus; Mowat, Vasanthi; Hartmann, Elke; Razinger, Tanja; Chevalier, Hans-Jörg; Blumbach, Kai; Green, Owen P.; Kaiser, Stefan; Corney, Stephen; Jackson, Ailsa; Casadesus, Agustin

2011-01-01

Many variables may affect the outcome of continuous infusion studies. The results largely depend on the experience of the laboratory performing these studies, the technical equipment used, the choice of blood vessels and hence the surgical technique as well the quality of pathological evaluation. The latter is of major interest due to the fact that the pathologist is not involved until necropsy in most cases, i.e. not dealing with the complicated surgical or in-life procedures of this study type. The technique of tissue sampling during necropsy and the histology processing procedures may influence the tissues presented for evaluation, hence the pathologist may be a source of misinterpretation. Therefore, ITO proposes a tissue sampling procedure and a standard nomenclature for pathological lesions for all sites and tissues in contact with the port-access and/or catheter system. PMID:22272050

7 CFR 301.92-11 - Inspection and sampling protocols.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 5 2010-01-01 2010-01-01 false Inspection and sampling protocols. 301.92-11 Section... Inspection and sampling protocols. Type(s) of plants in the nursery Type(s) of plants shipped interstate... interstate. (1) Annual inspection, sampling, and testing—(i) Inspection. The nursery must be inspected...

7 CFR 301.92-11 - Inspection and sampling protocols.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 5 2011-01-01 2011-01-01 false Inspection and sampling protocols. 301.92-11 Section... Inspection and sampling protocols. Type(s) of plants in the nursery Type(s) of plants shipped interstate... interstate. (1) Annual inspection, sampling, and testing—(i) Inspection. The nursery must be inspected...

A pilot study to understand feasibility and acceptability of stool and cord blood sample collection for a large-scale longitudinal birth cohort.

PubMed

Bailey, S R; Townsend, C L; Dent, H; Mallet, C; Tsaliki, E; Riley, E M; Noursadeghi, M; Lawley, T D; Rodger, A J; Brocklehurst, P; Field, N

2017-12-28

Few data are available to guide biological sample collection around the time of birth for large-scale birth cohorts. We are designing a large UK birth cohort to investigate the role of infection and the developing immune system in determining future health and disease. We undertook a pilot to develop methodology for the main study, gain practical experience of collecting samples, and understand the acceptability of sample collection to women in late pregnancy. Between February-July 2014, we piloted the feasibility and acceptability of collecting maternal stool, baby stool and cord blood samples from participants recruited at prolonged pregnancy and planned pre-labour caesarean section clinics at University College London Hospital. Participating women were asked to complete acceptability questionnaires. Overall, 265 women were approached and 171 (65%) participated, with ≥1 sample collected from 113 women or their baby (66%). Women had a mean age of 34 years, were primarily of white ethnicity (130/166, 78%), and half were nulliparous (86/169, 51%). Women undergoing planned pre-labour caesarean section were more likely than those who delivered vaginally to provide ≥1 sample (98% vs 54%), but less likely to provide maternal stool (10% vs 43%). Pre-sample questionnaires were completed by 110/171 women (64%). Most women reported feeling comfortable with samples being collected from their baby (<10% uncomfortable), but were less comfortable about their own stool (19% uncomfortable) or a vaginal swab (24% uncomfortable). It is possible to collect a range of biological samples from women around the time of delivery, and this was acceptable for most women. These data inform study design and protocol development for large-scale birth cohorts.

Diagnosis of brucellosis by using blood cultures.

PubMed Central

Ruiz, J; Lorente, I; Pérez, J; Simarro, E; Martínez-Campos, L

1997-01-01

The performances of three blood culture systems, Hemoline performance diphasic medium (bioMérieux, Marcy l'Etoile, France), Bactec Plus Aerobic/F* (Becton Dickinson, Paramus, N.J.), and Vital Aer (bioMérieux), were compared for the diagnosis of 17 cases of brucellosis. By using a 5-day incubation protocol, positive results were 52.9, 82.4, and 11.8%, respectively. When the protocol was extended to 7 days, the results were 76.5, 94.1, and 47.1%, respectively. Bactec was the fastest system (P < 0.05). PMID:9276429

Impairment of liver synthetic function and the production of plasma proteins in primary breast cancer patients on doxorubicincyclophosphamide (AC) protocol.

PubMed

Saleem, Zikria; Ahmad, Mobasher; Hashmi, Furqan Khurshid; Saeed, Hamid; Aziz, Muhammad Tahir

2016-09-01

Doxorubicin and Cyclophosphamide (AC protocol) combination is usually considered as a first line therapy in newly diagnosed breast cancer patients. Thus, a retrospective observational study was conducted to monitor the effect of AC protocol on liver synthetic functions and production of plasma proteins in breast cancer patients, reporting to specialized cancer care hospital of Lahore, Pakistan. A total of 75 patients (n=75) on AC protocol with breast cancer were observed in this study. The patient data including age, gender, body surface area, dosage, disease status and laboratory biochemical values were recorded by reviewing historical treatment records. Pre-treatment values were taken as baseline values for albumin, globulin, blood urea nitrogen (BUN), albumin/globulin (A/G) ratio and total proteins. The baseline values were compared after each cycle of by applying ANOVA using statistical tool SPSS® version 21. The plasma levels of blood urea nitrogen (BUN), total protein and globulin dropped significantly (p<0.05) in patients of all age groups. However, the albumin levels were not significantly changed (p>0.05). The A/G ratio level increased (p<0.05) as a result of reduction in globulin levels. Significant changes in plasma protein levels were observed in the elderly patients (50 to 65 years) than patients between 20 to 50 years of age. AC protocol impairs liver synthetic functions as observed by decreased blood urea nitrogen (BUN) and plasma protein levels.

A Versatile Microarray Platform for Capturing Rare Cells

NASA Astrophysics Data System (ADS)

Brinkmann, Falko; Hirtz, Michael; Haller, Anna; Gorges, Tobias M.; Vellekoop, Michael J.; Riethdorf, Sabine; Müller, Volkmar; Pantel, Klaus; Fuchs, Harald

2015-10-01

Analyses of rare events occurring at extremely low frequencies in body fluids are still challenging. We established a versatile microarray-based platform able to capture single target cells from large background populations. As use case we chose the challenging application of detecting circulating tumor cells (CTCs) - about one cell in a billion normal blood cells. After incubation with an antibody cocktail, targeted cells are extracted on a microarray in a microfluidic chip. The accessibility of our platform allows for subsequent recovery of targets for further analysis. The microarray facilitates exclusion of false positive capture events by co-localization allowing for detection without fluorescent labelling. Analyzing blood samples from cancer patients with our platform reached and partly outreached gold standard performance, demonstrating feasibility for clinical application. Clinical researchers free choice of antibody cocktail without need for altered chip manufacturing or incubation protocol, allows virtual arbitrary targeting of capture species and therefore wide spread applications in biomedical sciences.

[Identification and management of intra-operative suspicious tissues in 20 transsphenoidal surgery cases].

PubMed

Liu, Jun-Feng; Ke, Chang-Shu; Chen, Xi; Xu, Yu; Zhang, Hua-Qiu; Chen, Juan; Gan, Chao; Li, Chao-Xi; Lei, Ting

2013-05-01

To determine appropriate protocols for the identification and management of intra operative suspicious tissues during transsphenoidal surgery. Clinical data and pathological reports of 20 patients with intra-operative suspicious tissues during transsphenoidal surgeries were analyzed retrospectively. The methods for discriminating between adenoma and normal pituitary tissues were reviewed. The postoperative pathological reports revealed that adenoma and normal pituitary tissues coexisted in 9 samples, while 5 samples were identified as normal pituitary tissues, 2 as adenoma tissues, and 4 as other tissues. Adenomas were distinguished from normal pituitary tissues on the basis of intra-operative appearance, texture, blood supply and possible existence of boundary. If decisions are difficult to made during surgeries from the appearance of the suspicious tissues, pathological examinations are advised as a guidance for the next steps.

Effects of exercise with or without light exposure on sleep quality and hormone reponses

PubMed Central

Lee, Hayan; Kim, Sunho; Kim, Donghee

2014-01-01

[Purpose] The objectives of the present study were to determine the effect of sun exposure and aerobic exercise on quality of sleep and investigate sleep-related hormonal responses in college-aged males. [Methods] In this study, the cross-over design was utilized. The subjects (N = 10) without any physical problems or sleep disorders participated in the experimental performed 4 protocols in only sun exposure (for 30 minutes, EG1) protocol, only aerobic exercise (walking and jogging for 30 minutes, EG2) protocol, aerobic exercise with sun exposure (EG3) protocol, and control (no exercise and no sun exposure, EG4) protocol. Each protocol was 5 times per week with one-week break (wash-out period) between protocols to prevent the effects of the previous protocol. Total test period was should be 7 weeks (one week of protocol and one week of break). Before and after each aerobic exercise session, the subjects completed stretching to warm up for 5 to 10 minutes. Surveys consisting of (bedtime, wake-up time, sleep onset latency, and (Pittsburgh Sleep Quality Index (PSQI) were obtained before the test and after each protocol. After each protocol, the following sleep-related hormonal responses were measured: blood concentrations of melatonin, cortisol, epinephrine, and norepinephrine. One-way ANOVA was used to determine differences between protocols. Statistical significance was set at p < 0.05. [Results] Bedtime of EG4 was significantly later than that of the EG1 or EG3. Wake-up time in the EG4 was significantly later than that of the EG1 or the EG3. Sleep onset latency in the EG4 was longer than that of the EG3. The quality of sleep in the EG4 was lower than that of the EG3. Sleep cycle in the EG4 was significantly shorter than that of the EG1. Blood melatonin concentrations of the EG3 was significantly higher than that of the EG4. There were no significant differences in blood concentrations of cortisol, epinephrine, or norepinephrine among protocols, with the order from the lowest to the highest values of EG1 < EG2 < EG3 < EG4. [Conclusion] The present data found that EG1 and EG3 showed positive sleep-related hormonal responses, sleep habits, and quality of sleep, indicating that sun exposure or exercise with sun exposure may improve the physical status and quality of life. PMID:25566466

Prevalence and Risk Factors of Elevated Blood Lead in Children in Gold Ore Processing Communities, Zamfara, Nigeria, 2012.

PubMed

Kaufman, John A; Brown, Mary Jean; Umar-Tsafe, Nasir T; Adbullahi, Muhammad Bashir; Getso, Kabiru I; Kaita, Ibrahim M; Sule, Binta Bako; Ba'aba, Ahmed; Davis, Lora; Nguku, Patrick M; Sani-Gwarzo, Nasir

2016-09-01

In March 2010, Medecins Sans Frontieres/Doctors Without Borders detected an outbreak of acute lead poisoning in Zamfara State, northwestern Nigeria, linked to low-technology gold ore processing. The outbreak killed more than 400 children ≤5 years of age in the first half of 2010 and has left more than 2,000 children with permanent disabilities. The aims of this study were to estimate the statewide prevalence of children ≤5 years old with elevated blood lead levels (BLLs) in gold ore processing and non-ore-processing communities, and to identify factors associated with elevated blood lead levels in children. A representative, population-based study of ore processing and non-ore-processing villages was conducted throughout Zamfara in 2012. Blood samples from children, outdoor soil samples, indoor dust samples, and survey data on ore processing activities and other lead sources were collected from 383 children ≤5 years old in 383 family compounds across 56 villages. 17.2% of compounds reported that at least one member had processed ore in the preceding 12 months (95% confidence intervals (CI): 9.7, 24.7). The prevalence of BLLs ≥10 µg/dL in children ≤5 years old was 38.2% (95% CI: 26.5, 51.4) in compounds with members who processed ore and 22.3% (95% CI: 17.8, 27.7) in compounds where no one processed ore. Ore processing activities were associated with higher lead concentrations in soil, dust, and blood samples. Other factors associated with elevated BLL were a child's age and sex, breastfeeding, drinking water from a piped tap, and exposure to eye cosmetics. Childhood lead poisoning is widespread in Zamfara State in both ore processing and non-ore-processing settings, although it is more prevalent in ore processing areas. Although most children's BLLs were below the recommended level for chelation therapy, environmental remediation and use of safer ore processing practices are needed to prevent further exposures. Obtained. The study protocol was approved by the US Centers for Disease Control Institutional Review Board-A and the National Health Research Ethics Committee of Nigeria. The authors declare no competing financial interests.

Variability in Usual Care Mechanical Ventilation for Pediatric Acute Respiratory Distress Syndrome: Time for a Decision Support Protocol?

PubMed

Newth, Christopher J L; Sward, Katherine A; Khemani, Robinder G; Page, Kent; Meert, Kathleen L; Carcillo, Joseph A; Shanley, Thomas P; Moler, Frank W; Pollack, Murray M; Dalton, Heidi J; Wessel, David L; Berger, John T; Berg, Robert A; Harrison, Rick E; Holubkov, Richard; Doctor, Allan; Dean, J Michael; Jenkins, Tammara L; Nicholson, Carol E

2017-11-01

Although pediatric intensivists philosophically embrace lung protective ventilation for acute lung injury and acute respiratory distress syndrome, we hypothesized that ventilator management varies. We assessed ventilator management by evaluating changes to ventilator settings in response to blood gases, pulse oximetry, or end-tidal CO2. We also assessed the potential impact that a pediatric mechanical ventilation protocol adapted from National Heart Lung and Blood Institute acute respiratory distress syndrome network protocols could have on reducing variability by comparing actual changes in ventilator settings to those recommended by the protocol. Prospective observational study. Eight tertiary care U.S. PICUs, October 2011 to April 2012. One hundred twenty patients (age range 17 d to 18 yr) with acute lung injury/acute respiratory distress syndrome. Two thousand hundred arterial and capillary blood gases, 3,964 oxygen saturation by pulse oximetry, and 2,757 end-tidal CO2 values were associated with 3,983 ventilator settings. Ventilation mode at study onset was pressure control 60%, volume control 19%, pressure-regulated volume control 18%, and high-frequency oscillatory ventilation 3%. Clinicians changed FIO2 by ±5 or ±10% increments every 8 hours. Positive end-expiratory pressure was limited at ~10 cm H2O as oxygenation worsened, lower than would have been recommended by the protocol. In the first 72 hours of mechanical ventilation, maximum tidal volume/kg using predicted versus actual body weight was 10.3 (8.5-12.9) (median [interquartile range]) versus 9.2 mL/kg (7.6-12.0) (p < 0.001). Intensivists made changes similar to protocol recommendations 29% of the time, opposite to the protocol's recommendation 12% of the time and no changes 56% of the time. Ventilator management varies substantially in children with acute respiratory distress syndrome. Opportunities exist to minimize variability and potentially injurious ventilator settings by using a pediatric mechanical ventilation protocol offering adequately explicit instructions for given clinical situations. An accepted protocol could also reduce confounding by mechanical ventilation management in a clinical trial.

Successful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis

PubMed Central

Carrol, Enitan D; Salway, Fiona; Pepper, Stuart D; Saunders, Emma; Mankhambo, Limangeni A; Ollier, William E; Hart, C Anthony; Day, Phillip

2007-01-01

Background The challenge of gene expression studies is to reliably quantify levels of transcripts, but this is hindered by a number of factors including sample availability, handling and storage. The PAXgene™ Blood RNA System includes a stabilizing additive in a plastic evacuated tube, but requires 2.5 mL blood, which makes routine implementation impractical for paediatric use. The aim of this study was to modify the PAXgene™ Blood RNA System kit protocol for application to small, sick chidren, without compromising RNA integrity, and subsequently to perform quantitative analysis of ICAM and interleukin-6 gene expression. Aliquots of 0.86 mL PAXgene™ reagent were put into microtubes and 0.3 mL whole blood added to maintain the same recommended proportions as in the PAXgene™ evacuated tube system. RNA quality was assessed using the Agilent BioAnalyser 2100 and an in-house TaqMan™ assay which measures GAPDH transcript integrity by determining 3' to 5' ratios. qPCR analysis was performed on an additional panel of 7 housekeeping genes. Three reference genes (HPRT1, YWHAZ and GAPDH) were identified using the GeNORM algorithm, which were subsequently used to normalising target gene expression levels. ICAM-1 and IL-6 gene expression were measured in 87 Malawian children with invasive pneumococcal disease. Results Total RNA yield was between 1,114 and 2,950 ng and the BioAnalyser 2100 demonstrated discernible 18s and 28s bands. The cycle threshold values obtained for the seven housekeeping genes were between 15 and 30 and showed good consistency. Median relative ICAM and IL-6 gene expression were significantly reduced in non-survivors compared to survivors (ICAM: 3.56 vs 4.41, p = 0.04, and IL-6: 2.16 vs 6.73, p = 0.02). Conclusion We have successfully modified the PAXgene™ blood collection system for use in small children and demonstrated preservation of RNA integrity and successful quantitative real-time PCR analysis. PMID:17850649

T cell activation and proliferation following acute exercise in human subjects is altered by storage conditions and mitogen selection.

PubMed

Siedlik, Jacob A; Deckert, Jake A; Benedict, Stephen H; Bhatta, Anuja; Dunbar, Amanda J; Vardiman, John P; Gallagher, Philip M

2017-07-01

Recent work investigating exercise induced changes in immunocompetence suggests that some of the ambiguity in the literature is resultant from different cell isolation protocols and mitogen selection. To understand this effect, we compared post-exercise measures of T cell activation and proliferation using two different stimulation methods (costimulation through CD28 or stimulation with phytohaemagglutinin [PHA]). Further, we investigated whether exercise induced changes are maintained when T cell isolation from whole blood is delayed overnight in either a room temperature or chilled (4°C) environment. As expected, an increased proliferation response was observed post-exercise in T cells isolated from whole blood of previously trained individuals immediately after blood collection. Also, cells stimulated with PHA after resting overnight in whole blood were not adversely impacted by the storage conditions. In contrast, allowing cells to rest overnight in whole blood prior to stimulation through CD28, lessened the proliferation observed by cells following exercise rendering both the room temperature and chilled samples closer to the results seen in the control condition. Changes in early markers of activation (CD25), followed a similar pattern, with activation in PHA stimulated cells remaining fairly robust after overnight storage; whereas cell activation following stimulation through CD3+CD28 was disproportionately decreased by the influence of overnight storage. These findings indicate that decisions regarding cell stimulation methods need to be paired with the timeline for T cell isolation from whole blood. These considerations will be especially important for field based studies of immunocompetence where there is a delay in getting whole blood samples to a lab for processing as well as clinical applications where a failure to isolate T cells in a timely manner may result in loss of the response of interest. Copyright © 2017 Elsevier B.V. All rights reserved.

A Comparison of Hamster Anesthetics and Their Effect on Mosquito Blood Feeding

USDA-ARS?s Scientific Manuscript database

Hamsters or mice are often anesthetized when they are used as the hosts for insect feeding experiments. An experiment was done to determine if there was a difference in mosquito blood feeding success when fed on hamsters anesthetized using two commonly used protocols. The number of blood-fed females...

Prevention of Infection in Patients With Hematologic Cancer and Persistent Fever Caused by a Low White Blood Cell Count

ClinicalTrials.gov

2012-09-20

Bone Marrow Suppression; Fever, Sweats, and Hot Flashes; Infection; Leukemia; Lymphoma; Multiple Myeloma and Plasma Cell Neoplasm; Myelodysplastic Syndromes; Unspecified Adult Solid Tumor, Protocol Specific; Unspecified Childhood Solid Tumor, Protocol Specific

The detection of blood on dental surgery surfaces and equipment following dental hygiene treatment.

PubMed

McColl, E; Bagg, J; Winning, S

1994-01-22

The Kastle-Meyer technique, a forensic test for blood, has been employed to assess the frequency and potential routes of contamination by blood between patients, staff and equipment during routine dental hygiene treatment. Fifty treatment sessions were studied and units were cleaned between patients according to the current hospital protocol. The surfaces most frequently contaminated after treatment were the 3-in-1 syringe buttons (40%), protective bibs (22%), tap handles (20%), light handles (18%) and operating cart handles (16%). Following cleaning of the units, the surfaces remaining contaminated were the 3-in-1 syringes (10%), tap handles (4%) and cart handles (2%). Modifications to the cross-infection control protocol have been made to eliminate these sources of contamination.

The Gulf Long-Term Follow-Up Study (GuLF STUDY): Biospecimen collection at enrollment.

PubMed

Engel, Lawrence S; Kwok, Richard K; Miller, Aubrey K; Blair, Aaron; Curry, Matthew D; McGrath, John A; Sandler, Dale P

2017-01-01

The 2010 Deepwater Horizon (DWH) explosion in the Gulf of Mexico led to the largest ever marine oil spill by volume. The GuLF STUDY is investigating possible adverse human health effects associated with oil spill activities. One objective of the study was to utilize biological specimens from study participants to examine spill-related adverse health effects. This study describes the methods for collecting, processing, shipping, and storing specimens during the enrollment phase of the study. GuLF STUDY participants living in Gulf States (Alabama, Florida, Louisiana, Mississippi, and eastern Texas) were eligible to complete a home visit at enrollment, one to three years after the DWH explosion. During this visit, blood, urine, toenail and hair clippings, and house dust samples were collected. Specimens were shipped overnight to a central processing laboratory in containers with cold and ambient temperature compartments. Most blood and urine specimens were then aliquoted and stored in liquid nitrogen vapor or at -80°C, with some samples stored at -20°C. A total of 11,193 participants completed a home visit, and over 99% provided at least one biospecimen. Most participants provided blood (93%), urine (99%), and toenail clippings (89%), and 40% provided hair. Nearly all participants (95%) provided house-dust samples. Most samples were received by the laboratory one (58%) or two (25%) days after collection. These biospecimens enable investigation of a range of biomarkers of spill-related adverse health effects, and possibly some biomarkers of spill-related exposures. The biospecimen collection, handling, and storage protocols were designed to maximize current and future scientific value within logistical and budgetary constraints and might serve as a template for future studies conducted in similar time-critical and geographically dispersed settings.

A Draft Test Protocol for Detecting Possible Biohazards in Martian Samples Returned to Earth

NASA Technical Reports Server (NTRS)

Rummel, John D. (Editor); Race, Margaret S.; DeVincenzi, Donald L.; Schad, P. Jackson; Stabekis, Pericles D.; Viso, Michel; Acevedo, Sara E.

2002-01-01

This document presents the first complete draft of a protocol for detecting possible biohazards in Mars samples returned to Earth: it is the final product of the Mars Sample Handling Protocol Workshop Series. convened in 2000-2001 by NASA's Planetary Protection Officer. The goal of the five-workshop Series vas to develop a comprehensive protocol by which returned martian sample materials could be assessed k r the presence of any biological hazard(s) while safeguarding the purity of the samples from possible terrestrial contamination.

A Simple Method to Measure Renal Function in Swine by the Plasma Clearance of Iohexol

PubMed Central

Luis-Lima, Sergio; García-Contreras, Consolación; Carrara, Fabiola; Negrín-Mena, Natalia; Jiménez-Sosa, Alejandro; Jiménez-Hernández, Hugo; Porrini, Esteban

2018-01-01

There is no simple method to measure glomerular filtration rate (GFR) in swine, an established model for studying renal disease. We developed a protocol to measure GFR in conscious swine by using the plasma clearance of iohexol. We used two groups, test and validation, with eight animals each. Ten milliliters of iohexol (6.47 g) was injected into the marginal auricular vein and blood samples (3 mL) were collected from the orbital sinus at different points after injection. GFR was determined using two models: two-compartment (CL2: all samples) and one-compartment (CL1: the last six samples). In the test group, CL1 overestimated CL2 by ~30%: CL2 = 245 ± 93 and CL1 = 308 ± 123 mL/min. This error was corrected by a first-order polynomial quadratic equation to CL1, which was considered the simplified method: SM = −47.909 + (1.176xCL1) − (0.00063968xCL12). The SM showed narrow limits of agreement with CL2, a concordance correlation of 0.97, and a total deviation index of 14.73%. Similar results were obtained for the validation group. This protocol is reliable, reproducible, can be performed in conscious animals, uses a single dose of the marker, and requires a reduced number of samples, and avoids urine collection. Finally, it presents a significant improvement in animal welfare conditions and handling necessities in experimental trials. PMID:29329247

Individual, contextual and network characteristics of blood donors and non-donors: a systematic review of recent literature

PubMed Central

Piersma, Tjeerd W.; Bekkers, René; Klinkenberg, Elisabeth F.; de Kort, Wim L.A.M.; Merz, Eva-Maria

2017-01-01

Background The ageing population and recent migration flows may negatively affect the blood supply in the long term, increasing the importance of targeted recruitment and retention strategies to address donors. This review sought to identify individual, network and contextual characteristics related to blood donor status and behaviour, to systematically discuss differences between study results, and to identify possible factors to target in recruitment and retention efforts. Methods The systematic review was conducted in accordance with a predefined PROSPERO protocol (CRD42016039591). After quality assessments by multiple independent raters, a final set of 66 peer-reviewed papers, published between October 2009 and January 2017, were included for review. Results Individual and contextual characteristics of blood donor status and behaviour were categorised into five main lines of research: donor demographics, motivations and barriers, adverse reactions and deferral, contextual factors, and blood centre factors. Results on donor demographics, motivations and barriers, and contextual factors were inconclusive, differing between studies, countries, and sample characteristics. Adverse reactions and deferral were negatively related to blood donor behaviour. Blood centre factors play an important role in donor management, e.g., providing information, reminders, and (non-)monetary rewards. No studies were found on network characteristics of (non-)donors. Discussion Although individual and contextual characteristics strongly relate to blood donor status and behaviour, mechanisms underlying these relations have not been studied sufficiently. We want to stress the importance of longitudinal studies in donor behaviour, exploring the role of life events and network characteristics within blood donor careers. Increased understanding of donor behaviour will assist policy makers of blood collection agencies, with the ultimate goal of safeguarding a sufficient and matching blood supply. PMID:28686151

Individual, contextual and network characteristics of blood donors and non-donors: a systematic review of recent literature.

PubMed

Piersma, Tjeerd W; Bekkers, René; Klinkenberg, Elisabeth F; De Kort, Wim L A M; Merz, Eva-Maria

2017-09-01

The ageing population and recent migration flows may negatively affect the blood supply in the long term, increasing the importance of targeted recruitment and retention strategies to address donors. This review sought to identify individual, network and contextual characteristics related to blood donor status and behaviour, to systematically discuss differences between study results, and to identify possible factors to target in recruitment and retention efforts. The systematic review was conducted in accordance with a predefined PROSPERO protocol (CRD42016039591). After quality assessments by multiple independent raters, a final set of 66 peer-reviewed papers, published between October 2009 and January 2017, were included for review. Individual and contextual characteristics of blood donor status and behaviour were categorised into five main lines of research: donor demographics, motivations and barriers, adverse reactions and deferral, contextual factors, and blood centre factors. Results on donor demographics, motivations and barriers, and contextual factors were inconclusive, differing between studies, countries, and sample characteristics. Adverse reactions and deferral were negatively related to blood donor behaviour. Blood centre factors play an important role in donor management, e.g., providing information, reminders, and (non-)monetary rewards. No studies were found on network characteristics of (non-)donors. Although individual and contextual characteristics strongly relate to blood donor status and behaviour, mechanisms underlying these relations have not been studied sufficiently. We want to stress the importance of longitudinal studies in donor behaviour, exploring the role of life events and network characteristics within blood donor careers. Increased understanding of donor behaviour will assist policy makers of blood collection agencies, with the ultimate goal of safeguarding a sufficient and matching blood supply.

Individual and environmental risk factors for high blood lead concentrations in Danish indoor shooters.

PubMed

Grandahl, Kasper; Suadicani, Poul; Jacobsen, Peter

2012-08-01

International studies have shown blood lead at levels causing health concern in recreational indoor shooters. We hypothesized that Danish recreational indoor shooters would also have a high level of blood lead, and that this could be explained by shooting characteristics and the physical environment at the shooting range. This was an environmental case study of 58 male and female shooters from two indoor shooting ranges with assumed different ventilation and cleaning conditions. Information was obtained on general conditions including age, gender, tobacco and alcohol use, and shooting conditions: weapon type, number of shots fired, frequency of stays at the shooting range and hygiene habits. A venous blood sample was drawn to determine blood lead concentrations; 14 non-shooters were included as controls. Almost 60% of the shooters, hereof five out of 14 women, had a blood lead concentration above 0.48 micromol/l, a level causing long-term health concern. All controls had blood lead values below 0.17 micromol/l. Independent significant associations with blood lead concentrations above 0.48 micromol/l were found for shooting at a poorly ventilated range, use of heavy calibre weapons, number of shots and frequency of stays at the shooting range. A large proportion of Danish recreational indoor shooters had potentially harmful blood lead concentrations. Ventilation, amounts of shooting, use of heavy calibre weapons and stays at the shooting ranges were independently associated with increased blood lead. The technical check at the two ranges was performed by the Danish Technological Institute and costs were defrayed by the Danish Rifle Association. To pay for the analyses of blood lead, the study was supported by the The Else & Mogens Wedell-Wedellsborg Foundation. The Danish Regional Capital Scientific Ethics Committee approved the study, protocol number H-4-2010-130.

Improving the accuracy of blood pressure measurement: the influence of the European Society of Hypertension International Protocol (ESH-IP) for the validation of blood pressure measuring devices and future perspectives.

PubMed

Stergiou, George S; Asmar, Roland; Myers, Martin; Palatini, Paolo; Parati, Gianfranco; Shennan, Andrew; Wang, Jiguang; O'Brien, Eoin

2018-03-01

The European Society of Hypertension (ESH) International Protocol (ESH-IP) for the validation of blood pressure (BP) measuring devices was published in 2002, with the main objective of simplifying the validation procedures, so that more BP monitors would be subjected to independent validation. This article provides an overview of the international impact of the ESH-IP and of the lessons learned from its use, to be able to justify further developments in validation protocols. A review of published (PubMed) validation studies from 2002 to 2017 was performed. One hundred and seventy-seven validation studies using the ESH-IP, 59 using the British Hypertension Society protocol, 46 using the Association for the Advancement of Medical Instrumentation (AAMI) standard and 23 using the International Organization for Standardization (ISO) standard were identified. Lists of validated office-clinic, home and ambulatory BP monitors are provided. Of the ESH-IP studies, 93% tested oscillometric devices, 80% upper arm, 71% home, 25% office and 7% ambulatory monitors (some had more than one function). The original goal of the ESH-IP has been fulfilled in that in the last decade the number of published validation studies has more than doubled. It is now recognized that the provision of accurate devices would be best served by having a universal protocol. An international initiative has been put in place by AAMI, ESH and ISO experts aiming to reach consensus for a universal validation protocol to be accepted worldwide, which will allow a more thorough evaluation of the accuracy and performance of future BP monitors.

77 FR 5492 - Magnuson-Stevens Act Provisions; General Provisions for Domestic Fisheries; Application for...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-03

... assess the performance of an approved sampling protocol and to allow for continued sample collection and... developmental sampling protocol. While this application was being reviewed and was available for public comment, the sampling protocol being tested was adopted into the National Shellfish Sanitation Program by the...

Effect of cholesterol and triglycerides levels on the rheological behavior of human blood

NASA Astrophysics Data System (ADS)

Moreno, Leonardo; Calderas, Fausto; Sanchez-Olivares, Guadalupe; Medina-Torres, Luis; Sanchez-Solis, Antonio; Manero, Octavio

2015-02-01

Important public health problems worldwide such as obesity, diabetes, hyperlipidemia and coronary diseases are quite common. These problems arise from numerous factors, such as hyper-caloric diets, sedentary habits and other epigenetic factors. With respect to Mexico, the population reference values of total cholesterol in plasma are around 200 mg/dL. However, a large proportion has higher levels than this reference value. In this work, we analyze the rheological properties of human blood obtained from 20 donors, as a function of cholesterol and triglyceride levels, upon a protocol previously approved by the health authorities. Samples with high and low cholesterol and triglyceride levels were selected and analyzed by simple-continuous and linear-oscillatory shear flow. Rheometric properties were measured and related to the structure and composition of human blood. In addition, rheometric data were modeled by using several constitutive equations: Bautista-Manero-Puig (BMP) and the multimodal Maxwell equations to predict the flow behavior of human blood. Finally, a comparison was made among various models, namely, the BMP, Carreau and Quemada equations for simple shear rate flow. An important relationship was found between cholesterol, triglycerides and the structure of human blood. Results show that blood with high cholesterol levels (400 mg/dL) has flow properties fully different (higher viscosity and a more pseudo-plastic behavior) than blood with lower levels of cholesterol (tendency to Newtonian behavior or viscosity plateau at low shear rates).

Single Sodium Pyruvate Ingestion Modifies Blood Acid-Base Status and Post-Exercise Lactate Concentration in Humans

PubMed Central

Olek, Robert A.; Kujach, Sylwester; Wnuk, Damian; Laskowski, Radoslaw

2014-01-01

This study examined the effect of a single sodium pyruvate ingestion on a blood acid-base status and exercise metabolism markers. Nine active, but non-specifically trained, male subjects participated in the double-blind, placebo-controlled, crossover study. One hour prior to the exercise, subjects ingested either 0.1 g·kg−1 of body mass of a sodium pyruvate or placebo. The capillary blood samples were obtained at rest, 60 min after ingestion, and then three and 15 min after completing the workout protocol to analyze acid-base status and lactate, pyruvate, alanine, glucose concentrations. The pulmonary gas exchange, minute ventilation and the heart rate were measured during the exercise at a constant power output, corresponding to ~90% O2max. The blood pH, bicarbonate and the base excess were significantly higher after sodium pyruvate ingestion than in the placebo trial. The blood lactate concentration was not different after the ingestion, but the post-exercise was significantly higher in the pyruvate trial (12.9 ± 0.9 mM) than in the placebo trial (10.6 ± 0.3 mM, p < 0.05) and remained elevated (nonsignificant) after 15 min of recovery. The blood pyruvate, alanine and glucose concentrations, as well as the overall pulmonary gas exchange during the exercise were not affected by the pyruvate ingestion. In conclusion, the sodium pyruvate ingestion one hour before workout modified the blood acid-base status and the lactate production during the exercise. PMID:24841105

A streamlined workflow for single-cells genome-wide copy-number profiling by low-pass sequencing of LM-PCR whole-genome amplification products.

PubMed

Ferrarini, Alberto; Forcato, Claudio; Buson, Genny; Tononi, Paola; Del Monaco, Valentina; Terracciano, Mario; Bolognesi, Chiara; Fontana, Francesca; Medoro, Gianni; Neves, Rui; Möhlendick, Birte; Rihawi, Karim; Ardizzoni, Andrea; Sumanasuriya, Semini; Flohr, Penny; Lambros, Maryou; de Bono, Johann; Stoecklein, Nikolas H; Manaresi, Nicolò

2018-01-01

Chromosomal instability and associated chromosomal aberrations are hallmarks of cancer and play a critical role in disease progression and development of resistance to drugs. Single-cell genome analysis has gained interest in latest years as a source of biomarkers for targeted-therapy selection and drug resistance, and several methods have been developed to amplify the genomic DNA and to produce libraries suitable for Whole Genome Sequencing (WGS). However, most protocols require several enzymatic and cleanup steps, thus increasing the complexity and length of protocols, while robustness and speed are key factors for clinical applications. To tackle this issue, we developed a single-tube, single-step, streamlined protocol, exploiting ligation mediated PCR (LM-PCR) Whole Genome Amplification (WGA) method, for low-pass genome sequencing with the Ion Torrent™ platform and copy number alterations (CNAs) calling from single cells. The method was evaluated on single cells isolated from 6 aberrant cell lines of the NCI-H series. In addition, to demonstrate the feasibility of the workflow on clinical samples, we analyzed single circulating tumor cells (CTCs) and white blood cells (WBCs) isolated from the blood of patients affected by prostate cancer or lung adenocarcinoma. The results obtained show that the developed workflow generates data accurately representing whole genome absolute copy number profiles of single cell and allows alterations calling at resolutions down to 100 Kbp with as few as 200,000 reads. The presented data demonstrate the feasibility of the Ampli1™ WGA-based low-pass workflow for detection of CNAs in single tumor cells which would be of particular interest for genome-driven targeted therapy selection and for monitoring of disease progression.

Recommended protocols for sampling macrofungi

Treesearch

Gregory M. Mueller; John Paul Schmit; Sabine M. Hubndorf Leif Ryvarden; Thomas E. O' Dell; D. Jean Lodge; Patrick R. Leacock; Milagro Mata; Loengrin Umania; Qiuxin (Florence) Wu; Daniel L. Czederpiltz

2004-01-01

This chapter discusses several issues regarding reommended protocols for sampling macrofungi: Opportunistic sampling of macrofungi, sampling conspicuous macrofungi using fixed-size, sampling small Ascomycetes using microplots, and sampling a fixed number of downed logs.

National Sample Assessment Protocols

ERIC Educational Resources Information Center

Ministerial Council on Education, Employment, Training and Youth Affairs (NJ1), 2012

2012-01-01

These protocols represent a working guide for planning and implementing national sample assessments in connection with the national Key Performance Measures (KPMs). The protocols are intended for agencies involved in planning or conducting national sample assessments and personnel responsible for administering associated tenders or contracts,…

Validation of the G.LAB MD2200 wrist blood pressure monitor according to the European Society of Hypertension, the British Hypertension Society, and the International Organization for Standardization Protocols.

PubMed

Liu, Ze-Yu; Zhang, Qing-Han; Ye, Xiao-Lei; Liu, Da-Peng; Cheng, Kang; Zhang, Chun-Hai; Wan, Yi

2017-04-01

To validate the G.LAB MD2200 automated wrist blood pressure (BP) monitors according to the European Society of Hypertension International Protocol (ESH-IP) revision 2010, the British Hypertension Society (BHS), and the International Organization for Standardization (ISO) 81060-2:2013 protocols. The device was assessed on 33 participants according to the ESH requirements and was then tested on 85 participants according to the BHS and ISO 81060-2:2013 criteria. The validation procedures and data analysis followed the protocols precisely. The G.LAB MD2200 devices passed all parts of ESH-IP revision 2010 for both systolic and diastolic BP, with a device-observer difference of 2.15±5.51 and 1.51±5.16 mmHg, respectively. The device achieved A/A grading for the BHS protocol and it also fulfilled the criteria of ISO 81060-2:2013, with mean differences of systolic and diastolic BP between the device and the observer of 2.19±5.21 and 2.11±4.70 mmHg, respectively. The G.LAB MD2200 automated wrist BP monitor passed the ESH-IP revision 2010 and the ISO 81060-2:2013 protocol, and achieved the A/A grade of the BHS protocol, which can be recommended for self-measurement in the general population.

Cardiovascular responses to plyometric exercise are affected by workload in athletes.

PubMed

Arazi, Hamid; Asadi, Abbas; Mahdavi, Seyed Amir; Nasiri, Seyed Omid Mirfalah

2014-01-01

With regard to blood pressure responses to plyometric exercise and decreasing blood pressure after exercise (post-exercise hypotension), the influence of different workloads of plyometric exercise on blood pressure is not clear. The purpose of this investigation was to examine the effects of a low, moderate and high workload of plyometric exercise on the post-exercise systolic (SBP) and diastolic blood pressure (DBP), heart rate (HR) and rate-pressure product (RPP) responses in athletes. TEN MALE ATHLETES (AGE: 22.6 ±0.5 years; height: 178.2 ±3.3 cm; and body mass: 75.2 ±2.8 kg) underwent PE protocols involving 5 × 10 reps (Low Workload - LW), 10 × 10 reps (Moderate Workload - MW), and 15 × 10 reps (High Workload - HW) depth jump exercise from a 50-cm box in 3 non-consecutive days. After each exercise session, SBP, DBP and HR were measured every 10 min for a period of 70 min. No significant differences were observed among post-exercise SBP and DBP when the protocols (LW, MW and HW) were compared. The MW and HW protocols showed greater increases in HR compared with LW. Also the HW indicated greater increases than LW in RPP at post-exercise (p < 0.05). All protocols increased SBP, HR and RPP responses at the 10(th) and 20(th) min of post-exercise. With regard to different workloads of plyometric exercise, HW condition indicated greater increases in HR and RPP and strength and conditioning professionals and athletes must keep in their mind that HW of plyometric exercise induces greater cardiovascular responses.

Validation of the BPLab(®) 24-hour blood pressure monitoring system according to the European standard BS EN 1060-4:2004 and British Hypertension Society protocol.

PubMed

Koudryavtcev, Sergey A; Lazarev, Vyacheslav M

2011-01-01

Automatic blood pressure (BP) measuring devices are more and more often used in BP self-checks and in 24-hour BP monitoring. Nowadays, 24-hour BP monitoring is a necessary procedure in arterial hypertension treatment. The aim of this study was to validate the BPLab(®) ambulatory blood pressure monitor according to the European standard BS EN 1060-4:2004 and the British Hypertension Society (BHS) protocol, as well as to work out solutions regarding the suitability of using this device in clinical practice. A group of 85 patients of both sexes and different ages, who voluntarily agreed to take part in the tests and were given detailed instructions on the measurement technique were recruited for this study. The results of the BP measurement obtained by a qualified operator using the BPLab(®) device were compared with the BP values measured using the Korotkov auscultatory method. Data were obtained simultaneously by two experts with experience of over 10 years and had completed a noninvasive BP measurement standardization training course. Discrepancies in the systolic and diastolic BP measurements (N = 510; 255 for each expert) were analyzed according to the criteria specified in the BHS-93 protocol. The device passed the requirements of the European Standard BS EN 1060-4:2004 and was graded 'A' according to the criteria of the BHS protocol for both systolic BP and diastolic BP. The BPLab(®) 24-hour ambulatory blood pressure monitoring device may be recommended for extensive clinical use.

Lack of reliability of nanotechnology in the of free plasma DNA in samples of patients with prostate cancer

PubMed Central

2013-01-01

Background Several studies seek biological markers that give diagnostic and degree of tumor development. The aim of this study was to validate the determination of plasma DNA using nanotechnology (Nanovue™-NV) in samples of 80 patients with prostate cancer. Methods Blood samples of 80 patients of the Urology Ambulatory of Faculdade de Medicina do ABC with prostate cancer confirmed by anatomical-pathology criteria were analyzed. DNA extraction was performed using a GFX TM kit (Amersham Pharmacia Biotech, Inc, USA) following the adapted protocol. Plasma was subjected to centrifugation. Results There was a big difference between the first and the second value obtained by NanoVue Only two samples had no differences between duplicates. Maximum difference between duplicates was 38 μg/mL. Average variation between 51 samples was 10.29 μg/mL, although 21 samples had differences above this average. No correlation was observed between pDNA obtained by traditional spectrophotometry and by nanotechnology. Conclusion Determination of plasma DNA by nanotechnology was not reproducible. PMID:23311763

Enhancing the Detection of Dysmorphic Red Blood Cells and Renal Tubular Epithelial Cells with a Modified Urinalysis Protocol.

PubMed

Chu-Su, Yu; Shukuya, Kenichi; Yokoyama, Takashi; Lin, Wei-Chou; Chiang, Chih-Kang; Lin, Chii-Wann

2017-01-11

Urinary sediment is used to evaluate patients with possible urinary tract diseases. Currently, numerous protocols are applied to detect dysmorphic red blood cells (RBCs) and renal tubular epithelial cells (RTECs) in urinary sediment. However, distinct protocols are used by nephrologists and medical technologists for specimen concentration and observation, which leads to major discrepancies in the differential counts of formed elements such as dysmorphic RBCs and RTECs and might interfere with an accurate clinical diagnosis. To resolve these problems, we first tested a modified urinalysis protocol with an increased relative centrifuge force and concentration factor in 20 biopsy-confirmed glomerulonephritis patients with haematuria. We successfully improved the recovery ratio of dysmorphic RBCs in clinical specimens from 34.7% to 42.0% (P < 0.001). Furthermore, we confirmed the correlation between counts by the modified urinary protocol and Sysmex UF-1000i urinary flow cytometer (r ≥ 0.898, P < 0.001). A total of 28 types of isomorphic and dysmorphic RBCs were detected using a bright field microscope, with results comparable to those using a standard phase contrast microscope. Finally, we applied Sternheimer stain to enhance the contrast of RTECs in the urinary sediments. We concluded that this modified urinalysis protocol significantly enhanced the quality of urinalysis.

Enhancing the Detection of Dysmorphic Red Blood Cells and Renal Tubular Epithelial Cells with a Modified Urinalysis Protocol

PubMed Central

Chu-Su, Yu; Shukuya, Kenichi; Yokoyama, Takashi; Lin, Wei-Chou; Chiang, Chih-Kang; Lin, Chii-Wann

2017-01-01

Urinary sediment is used to evaluate patients with possible urinary tract diseases. Currently, numerous protocols are applied to detect dysmorphic red blood cells (RBCs) and renal tubular epithelial cells (RTECs) in urinary sediment. However, distinct protocols are used by nephrologists and medical technologists for specimen concentration and observation, which leads to major discrepancies in the differential counts of formed elements such as dysmorphic RBCs and RTECs and might interfere with an accurate clinical diagnosis. To resolve these problems, we first tested a modified urinalysis protocol with an increased relative centrifuge force and concentration factor in 20 biopsy-confirmed glomerulonephritis patients with haematuria. We successfully improved the recovery ratio of dysmorphic RBCs in clinical specimens from 34.7% to 42.0% (P < 0.001). Furthermore, we confirmed the correlation between counts by the modified urinary protocol and Sysmex UF-1000i urinary flow cytometer (r ≥ 0.898, P < 0.001). A total of 28 types of isomorphic and dysmorphic RBCs were detected using a bright field microscope, with results comparable to those using a standard phase contrast microscope. Finally, we applied Sternheimer stain to enhance the contrast of RTECs in the urinary sediments. We concluded that this modified urinalysis protocol significantly enhanced the quality of urinalysis. PMID:28074941

Duration of serological response to canine parvovirus-type 2, canine distemper virus, canine adenovirus type 1 and canine parainfluenza virus in client-owned dogs in Australia.

PubMed

Mitchell, S A; Zwijnenberg, R J; Huang, J; Hodge, A; Day, M J

2012-12-01

To determine whether client-owned dogs in Australia, last vaccinated with Canvac(®) vaccines containing canine parvovirus-type 2 (CPV-2), canine distemper virus (CDV), canine adenovirus type 2 (CAV-2) ± canine parainfluenza virus (CPiV) at least 18 months ago, were seropositive or responded serologically to revaccination. A total of 235 dogs were recruited from 23 veterinary clinics, representing a variety of breeds, ages and time since last vaccination (TSLV: range 1.5-9 years, mean 2.8 years). Dogs had a blood sample taken and were revaccinated on day 0. A second blood sample was taken 7-14 days later. Blood samples were assessed for antibody titres to CPV-2 (by haemagglutination inhibition) and CDV, CAV type 1 (CAV-1) and CPiV (by virus neutralisation). Dogs with a day 0 titre >10 or a four-fold increase in titre following revaccination were considered to be serological responders. The overall percentage of dogs classified as serological responders was 98.7% for CPV-2, 96.6% for CDV, 99.6% for CAV-1 and 90.3% for CPiV. These results suggest that the duration of serological response induced by modified-live vaccines against CPV-2, CDV, CAV-1 and CPiV, including Canvac(®) vaccines, is beyond 18 months and may extend up to 9 years. Accordingly, these vaccines may be considered for use in extended revaccination interval protocols as recommended by current canine vaccine guidelines. © 2012 The Authors. Australian Veterinary Journal © 2012 Australian Veterinary Association.

Adapting the γ-H2AX assay for automated processing in human lymphocytes. 1. Technological aspects.

PubMed

Turner, Helen C; Brenner, David J; Chen, Youhua; Bertucci, Antonella; Zhang, Jian; Wang, Hongliang; Lyulko, Oleksandra V; Xu, Yanping; Shuryak, Igor; Schaefer, Julia; Simaan, Nabil; Randers-Pehrson, Gerhard; Yao, Y Lawrence; Amundson, Sally A; Garty, Guy

2011-03-01

The immunofluorescence-based detection of γ-H2AX is a reliable and sensitive method for quantitatively measuring DNA double-strand breaks (DSBs) in irradiated samples. Since H2AX phosphorylation is highly linear with radiation dose, this well-established biomarker is in current use in radiation biodosimetry. At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a fully automated high-throughput system, the RABIT (Rapid Automated Biodosimetry Tool), that can be used to measure γ-H2AX yields from fingerstick-derived samples of blood. The RABIT workstation has been designed to fully automate the γ-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes in heparin-coated PVC capillaries to the immunolabeling of γ-H2AX protein and image acquisition to determine fluorescence yield. High throughput is achieved through the use of purpose-built robotics, lymphocyte handling in 96-well filter-bottomed plates, and high-speed imaging. The goal of the present study was to optimize and validate the performance of the RABIT system for the reproducible and quantitative detection of γ-H2AX total fluorescence in lymphocytes in a multiwell format. Validation of our biodosimetry platform was achieved by the linear detection of a dose-dependent increase in γ-H2AX fluorescence in peripheral blood samples irradiated ex vivo with γ rays over the range 0 to 8 Gy. This study demonstrates for the first time the optimization and use of our robotically based biodosimetry workstation to successfully quantify γ-H2AX total fluorescence in irradiated peripheral lymphocytes.

The Renin-Angiotensin System, Not the Kinin-Kallikrein System, Affects Post-Exercise Proteinuria.

PubMed

Koçer, Günnur; Basralı, Filiz; Kuru, Oktay; Şentürk, Ümit Kemal

2018-05-17

Temporary proteinuria post-exercise is common and is caused predominantly by renal haemodynamic alterations. One reason is up-regulation of angiotensin II (Ang II) due to the reducing effect of angiotensin-converting enzyme (ACE) inhibitors. However, another, ignored, reason could be the kininase effect of ACE inhibition. This study investigated how ACE inhibition reduces post-exercise proteinuria: by either Ang II up-regulation inhibition or bradykinin elevation due to kininase activity inhibition. Our study included 10 volunteers, who completed 3 high-intensity exercise protocols involving cycling at 1-week intervals. The first protocol was a control arm, the second evaluated the effect of ACE inhibition and the third examined the effect of angiotensin type 1 receptor blockade. Upon application, both agents reduced systolic and diastolic blood pressure; however, there were no statistically significant -differences. In addition, total protein, microalbumin and -β2-microglobulin excretion levels in urine specimens were analysed before, 30 min after and 120 min after the exercise protocols. Total protein levels in urine samples were elevated in all 3 protocols after 30 min of high-intensity exercise, compared to baseline levels. However, both ACE inhibition and angiotensin type 1 receptor blockade suppressed total protein in the 30th min. In each protocol, total protein levels returned to the baseline after 120 min. Urinary microalbumin and β2-microglobulin levels during the control protocol were significantly higher 30 min post-exercise; however, only angiotensin type 1 receptor blockade suppressed microalbumin levels. The results indicated Ang II up-regulation, not bradykinin elevation, plays a role in post-exercise proteinuria. © 2018 S. Karger AG, Basel.

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution.

PubMed

Yamazaki, Tomoko; Li, Wenling; Mukouyama, Yoh-Suke

2018-03-29

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching morphogenesis and patterning, as well as immune cell distribution at a cellular level. The analysis of peripheral nerve and blood vessel anatomical structures in adult tissues provides some insights into the understanding of functional neuro-vascular wiring and neuro-vascular degeneration in pathological conditions such as wound healing. As a highly informative model system, we have focused our studies on adult ear skin, which is readily accessible for dissection. Our simple and reproducible protocol provides an accurate depiction of the cellular components in the entire skin, such as peripheral nerves (sensory axons, sympathetic axons, and Schwann cells), blood vessels (endothelial cells and vascular smooth muscle cells), and inflammatory cells. We believe this protocol will pave the way to investigate morphological abnormalities in peripheral nerves and blood vessels as well as the inflammation in the adult ear skin under different pathological conditions.

A Draft Test Protocol for Detecting Possible Biohazards in Martian Samples Returned to Earth

NASA Technical Reports Server (NTRS)

Rummel, John D.; Race, Margaret S.; DeVinenzi, Donald L.; Schad, P. Jackson; Stabekis, Pericles D.; Viso, Michel; Acevedo, Sara E.

2002-01-01

This document presents the first complete draft of a protocol for detecting possible biohazards in Mars samples returned to Earth; it is the final product of the Mars Sample Handling Protocol Workshop Series, convened in 2000-2001 by NASA's Planetary Protection Officer. The goal of the five-workshop Series vas to develop a comprehensive protocol by which returned martian sample materials could be assessed for the presence of any biological hazard(s) while safeguarding the purity of the samples from possible terrestrial contamination The reference numbers for the proceedings from the five individual Workshops.

Detection and quantification of extracellular microRNAs in murine biofluids

PubMed Central

2014-01-01

Background MicroRNAs (miRNAs) are short RNA molecules which regulate gene expression in eukaryotic cells, and are abundant and stable in biofluids such as blood serum and plasma. As such, there has been heightened interest in the utility of extracellular miRNAs as minimally invasive biomarkers for diagnosis and monitoring of a wide range of human pathologies. However, quantification of extracellular miRNAs is subject to a number of specific challenges, including the relatively low RNA content of biofluids, the possibility of contamination with serum proteins (including RNases and PCR inhibitors), hemolysis, platelet contamination/activation, a lack of well-established reference miRNAs and the biochemical properties of miRNAs themselves. Protocols for the detection and quantification of miRNAs in biofluids are therefore of high interest. Results The following protocol was validated by quantifying miRNA abundance in C57 (wild-type) and dystrophin-deficient (mdx) mice. Important differences in miRNA abundance were observed depending on whether blood was taken from the jugular or tail vein. Furthermore, efficiency of miRNA recovery was reduced when sample volumes greater than 50 μl were used. Conclusions Here we describe robust and novel procedures to harvest murine serum/plasma, extract biofluid RNA, amplify specific miRNAs by RT-qPCR and analyze the resulting data, enabling the determination of relative and absolute miRNA abundance in extracellular biofluids with high accuracy, specificity and sensitivity. PMID:24629058

Use of an Intravascular Fluorescent Continuous Glucose Sensor in ICU Patients.

PubMed

Strasma, Paul J; Finfer, Simon; Flower, Oliver; Hipszer, Brian; Kosiborod, Mikhail; Macken, Lewis; Sechterberger, Marjolein; van der Voort, Peter H J; DeVries, J Hans; Joseph, Jeffrey I

2015-07-01

Hyperglycemia and hypoglycemia are associated with adverse clinical outcomes in intensive care patients. In product development studies at 4 ICUs, the safety and performance of an intravascular continuous glucose monitoring (IV-CGM) system was evaluated in 70 postsurgical patients. The GluCath System (GluMetrics, Inc) used a quenched chemical fluorescence mechanism to optically measure blood glucose when deployed via a radial artery catheter or directly into a peripheral vein. Periodic ultrasound assessed blood flow and thrombus formation. Patient glucose levels were managed according to the standard of care and existing protocols at each site. Reference blood samples were acquired hourly and compared against prospectively calibrated sensor results. In all, 63 arterial sensors and 9 venous sensors were deployed in 70 patients. Arterial sensors did not interfere with invasive blood pressure monitoring, sampling or other aspects of patient care. A majority of venous sensors (66%) exhibited thrombus on ultrasound. In all, 89.4% (1383/1547) of arterial and 72.2% (182/252) of venous measurements met ISO15197:2003 criteria (within 20%), and 72.7% (1124/1547) of arterial and 56.3% (142/252) of venous measurements met CLSI POCT 12-A3 criteria (within 12.5%). The aggregate mean absolute relative difference (MARD) between the sensors and the reference was 9.6% for arterial and 14.2% for venous sensors. The GluCath System exhibited acceptable accuracy when deployed in a radial artery for up to 48 hours in ICU patients after elective cardiac surgery. Accuracy of venous deployment was substantially lower with significant rates of intravascular thrombus observed using ultrasound. © 2015 Diabetes Technology Society.

A single-platform approach using flow cytometry and microbeads to evaluate immune reconstitution in mice after bone marrow transplantation.

PubMed

Perruche, Sylvain; Kleinclauss, François; Lienard, Agnès; Robinet, Eric; Tiberghien, Pierre; Saas, Philippe

2004-11-01

The monitoring of immune reconstitution in murine models of HC transplantation, using accurate and automated methods, is necessary in view of the recent developments of hematopoietic cell (HC) transplantation (including reduced intensity conditioning regimens) as well as emerging immunological concepts (such as the involvement of dendritic cells or regulatory T cells). Here, we describe the use of a single-platform approach based on flow cytometry and tubes that contain a defined number of microbeads to evaluate absolute blood cell counts in mice. This method, previously used in humans to quantify CD34+ stem cells or CD4+ T cells in HIV infected patients, was adapted for mouse blood samples. A CD45 gating strategy in this "lyse no wash" protocol makes it possible to discriminate erythroblasts or red blood cell debris from CD45+ leukocytes, thus avoiding cell loss. Tubes contain a lyophilized brightly fluorescent microbead pellet permitting the acquisition of absolute counts of leukocytes after flow cytometric analysis. We compared this method to determine absolute counts of circulating cells with another method combining Unopette reservoir diluted blood samples, hemocytometer, microscopic examination and flow cytometry. The sensitivity of this single-platform approach was evaluated in different situations encountered in allogeneic HC transplantation, including immune cell depletion after different conditioning regimens, activation status of circulating cells after transplantation, evaluation of in vivo cell depletion and hematopoietic progenitor mobilization in the periphery. This single-platform flow cytometric assay can also be proposed to standardize murine (or other mammalian species) leukocyte count determination for physiological, pharmacological/toxicological and diagnostic applications in veterinary practice.

Cardiovascular risk factors in two Ecuadorian urban and rural populations. The Ecuadorian-Japan Cooperative CARDIAC Study Group.

PubMed

Del Pozo, G; Davalos, P; Yamori, Y

1990-01-01

We examined the specific hypotheses linking the intake of sodium, potassium, calcium, magnesium, and protein to blood pressure (BP) and the relationship between dietary factors and mortality from the major cardiovascular diseases (CVD) in the Ecuadorian populations. Two Ecuadorian populations, the urban and the rural, were selected from Quito and Vilcabamba, respectively. From Quito: 87 men and 83 women; from Vilcabamba: 71 men and 91 women aged 50-54 were randomly selected for BP measurement, 24-h urine collection, and blood sampling according to the Cardiovascular Disease and Alimentary Comparison (CARDIAC) Study protocol. Samples were analyzed at CARDIAC center in Izumo, Japan. Mean systolic blood pressure (SBP) was not much different in the two populations, but mean diastolic blood pressure (DBP) and body mass index (BMI) were significantly lower in Vilcabamba (p less than 0.001). Mortality from stroke was higher in Vilcabamba, whereas coronary death rate was higher in Quito. Both sodium intake and sodium/potassium ratio were higher in Vilcabamba (p less than 0.001). Protein intake and serum cholesterol were higher in Quito (p less than 0.001). Urinary taurine excretion was higher in Quito. There was no difference in W3/W6 fatty acids ratio between the two populations. Multiple regression analyses of intracommunity correlation indicated that both SBP and DBP were highly significantly related with BMI in Quito and that urinary excretions were inversely related to SBP. Serum cholesterol was positively related to coronary death rate. Mortality from stroke was inversely related to both serum cholesterol and protein and was positively related to salt consumption.

Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry.

PubMed

Keshishian, Hasmik; Burgess, Michael W; Specht, Harrison; Wallace, Luke; Clauser, Karl R; Gillette, Michael A; Carr, Steven A

2017-08-01

Proteomic characterization of blood plasma is of central importance to clinical proteomics and particularly to biomarker discovery studies. The vast dynamic range and high complexity of the plasma proteome have, however, proven to be serious challenges and have often led to unacceptable tradeoffs between depth of coverage and sample throughput. We present an optimized sample-processing pipeline for analysis of the human plasma proteome that provides greatly increased depth of detection, improved quantitative precision and much higher sample analysis throughput as compared with prior methods. The process includes abundant protein depletion, isobaric labeling at the peptide level for multiplexed relative quantification and ultra-high-performance liquid chromatography coupled to accurate-mass, high-resolution tandem mass spectrometry analysis of peptides fractionated off-line by basic pH reversed-phase (bRP) chromatography. The overall reproducibility of the process, including immunoaffinity depletion, is high, with a process replicate coefficient of variation (CV) of <12%. Using isobaric tags for relative and absolute quantitation (iTRAQ) 4-plex, >4,500 proteins are detected and quantified per patient sample on average, with two or more peptides per protein and starting from as little as 200 μl of plasma. The approach can be multiplexed up to 10-plex using tandem mass tags (TMT) reagents, further increasing throughput, albeit with some decrease in the number of proteins quantified. In addition, we provide a rapid protocol for analysis of nonfractionated depleted plasma samples analyzed in 10-plex. This provides ∼600 quantified proteins for each of the ten samples in ∼5 h of instrument time.

Effect of nutritional status and sampling intensity on recovery after dorsal aorta cannulation in free-swimming Atlantic salmon (Salmo salar L.).

PubMed

Djordjevic, B; Kristensen, T; Øverli, Ø; Rosseland, B O; Kiessling, A

2012-02-01

Recovery from implantation of a cannula in the dorsal aorta (DA) of Atlantic salmon (Salmo salar) was studied in relation to nutritional status and sampling intensity. The incentive for the study was the inconsistency between published reports and our own experience of recovery and longevity of fish exposed to this protocol. In two studies using starved fish, blood (0.3 ml) was sampled 0, 1 and 24 h after DA-cannulation, and thereafter at 48 and 72 h and thereafter once weekly for four weeks. In a third study using fed fish, four consecutive samples (0, 3, 6 and 12 h after a meal) were obtained twice a week over a four-week period. All fish displayed a sharp increase in pCO(2) and haematocrit (Hct) during surgery, followed by a marked raise in cortisol, glucose, sodium and potassium (1 h). pCO(2), pH and Hct approached baseline levels as early as the 1 h post-surgery sample, while this was not the case for cortisol and electrolytes before the 24 h post-surgery sample. Glucose did not display any significant changes post surgery. From then on, all variables displayed minor but non-significant (P > 0.05) changes indicating a steady state close to baseline values for unstressed fish. This pattern was independent of sampling procedure, i.e. repeated single or multiple samples and thus volume of blood removed. Nutritional status (fed vs. starved) did not affect post-surgical recovery pattern. Only K(+) and Hct displayed consistent and significant post-prandial patterns. We found marked differences between baseline level of cannulated fish and uncannulated control fish, in pH, K(+) and Hct indicating that cannulation may be the preferred method to obtain representative resting values in fish.

Analytical and pre-analytical performance characteristics of a novel cartridge-type blood gas analyzer for point-of-care and laboratory testing.

PubMed

Oyaert, Matthijs; Van Maerken, Tom; Bridts, Silke; Van Loon, Silvi; Laverge, Heleen; Stove, Veronique

2018-03-01

Point-of-care blood gas test results may benefit therapeutic decision making by their immediate impact on patient care. We evaluated the (pre-)analytical performance of a novel cartridge-type blood gas analyzer, the GEM Premier 5000 (Werfen), for the determination of pH, partial carbon dioxide pressure (pCO 2 ), partial oxygen pressure (pO 2 ), sodium (Na + ), potassium (K + ), chloride (Cl - ), ionized calcium ( i Ca 2+ ), glucose, lactate, and total hemoglobin (tHb). Total imprecision was estimated according to the CLSI EP5-A2 protocol. The estimated total error was calculated based on the mean of the range claimed by the manufacturer. Based on the CLSI EP9-A2 evaluation protocol, a method comparison with the Siemens RapidPoint 500 and Abbott i-STAT CG8+ was performed. Obtained data were compared against preset quality specifications. Interference of potential pre-analytical confounders on co-oximetry and electrolyte concentrations were studied. The analytical performance was acceptable for all parameters tested. Method comparison demonstrated good agreement to the RapidPoint 500 and i-STAT CG8+, except for some parameters (RapidPoint 500: pCO 2 , K + , lactate and tHb; i-STAT CG8+: pO 2 , Na + , i Ca 2+ and tHb) for which significant differences between analyzers were recorded. No interference of lipemia or methylene blue on CO-oximetry results was found. On the contrary, significant interference for benzalkonium and hemolysis on electrolyte measurements were found, for which the user is notified by an interferent specific flag. Identification of sample errors from pre-analytical sources, such as interferences and automatic corrective actions, along with the analytical performance, ease of use and low maintenance time of the instrument, makes the evaluated instrument a suitable blood gas analyzer for both POCT and laboratory use. Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Validation of the A&D BP UA-651 device for home blood pressure measurement according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Benetti, Elisabetta; Fania, Claudio; Palatini, Paolo

2014-02-01

The objective of this study was to determine the accuracy of the A&D BP UA-651 device for home blood pressure (BP) measurement according to the International Protocol of the European Society of Hypertension. Device evaluation was carried out in 33 patients. The mean age of the patients was 48.3±15.5 years, the mean systolic BP was 138.3±24.9 mmHg (range 90-180), the mean diastolic BP was 88.3±13.8 mmHg (range 60-108), and the mean arm circumference was 28.6±3.4 cm (range 23-36). The protocol requirements were followed precisely. The device passed all requirements, fulfilling the standards of the protocol. On average, the device underestimated the systolic BP by 0.4±4.4 mmHg and diastolic BP by 1.3±3.5 mmHg. The device-observer discrepancies were unrelated to patients' clinical characteristics. These data show that the A&D BP UA-651 device fulfilled the requirements for validation by the International Protocol and can be recommended for clinical use in the adult population.

Development of the Diabetes Technology Society Blood Glucose Monitor System Surveillance Protocol

PubMed Central

Klonoff, David C.; Lias, Courtney; Beck, Stayce; Parkes, Joan Lee; Kovatchev, Boris; Vigersky, Robert A.; Arreaza-Rubin, Guillermo; Burk, Robert D.; Kowalski, Aaron; Little, Randie; Nichols, James; Petersen, Matt; Rawlings, Kelly; Sacks, David B.; Sampson, Eric; Scott, Steve; Seley, Jane Jeffrie; Slingerland, Robbert; Vesper, Hubert W.

2015-01-01

Background: Inaccurate blood glucsoe monitoring systems (BGMSs) can lead to adverse health effects. The Diabetes Technology Society (DTS) Surveillance Program for cleared BGMSs is intended to protect people with diabetes from inaccurate, unreliable BGMS products that are currently on the market in the United States. The Surveillance Program will provide an independent assessment of the analytical performance of cleared BGMSs. Methods: The DTS BGMS Surveillance Program Steering Committee included experts in glucose monitoring, surveillance testing, and regulatory science. Over one year, the committee engaged in meetings and teleconferences aiming to describe how to conduct BGMS surveillance studies in a scientifically sound manner that is in compliance with good clinical practice and all relevant regulations. Results: A clinical surveillance protocol was created that contains performance targets and analytical accuracy-testing studies with marketed BGMS products conducted by qualified clinical and laboratory sites. This protocol entitled “Protocol for the Diabetes Technology Society Blood Glucose Monitor System Surveillance Program” is attached as supplementary material. Conclusion: This program is needed because currently once a BGMS product has been cleared for use by the FDA, no systematic postmarket Surveillance Program exists that can monitor analytical performance and detect potential problems. This protocol will allow identification of inaccurate and unreliable BGMSs currently available on the US market. The DTS Surveillance Program will provide BGMS manufacturers a benchmark to understand the postmarket analytical performance of their products. Furthermore, patients, health care professionals, payers, and regulatory agencies will be able to use the results of the study to make informed decisions to, respectively, select, prescribe, finance, and regulate BGMSs on the market. PMID:26481642

Protocol for Identifying Natural Agents That Selectively Affect Adhesion, Thickness, Architecture, Cellular Phenotypes, Extracellular Matrix, and Human White Blood Cell Impenetrability of Candida albicans Biofilms

PubMed Central

Park, Yang-Nim; Srikantha, Thyagarajan; Daniels, Karla J.; Jacob, Melissa R.; Agarwal, Ameeta K.; Li, Xing-Cong

2017-01-01

ABSTRACT In the screening of natural plant extracts for antifungal activity, assessment of their effects on the growth of cells in suspension or in the wells of microtiter plates is expedient. However, microorganisms, including Candida albicans, grow in nature as biofilms, which are organized cellular communities with a complex architecture capable of conditioning their microenvironment, communicating, and excluding low- and high-molecular-weight molecules and white blood cells. Here, a confocal laser scanning microscopy (CLSM) protocol for testing the effects of large numbers of agents on biofilm development is described. The protocol assessed nine parameters from a single z-stack series of CLSM scans for each individual biofilm analyzed. The parameters included adhesion, thickness, formation of a basal yeast cell polylayer, hypha formation, the vertical orientation of hyphae, the hyphal bend point, pseudohypha formation, calcofluor white staining of the extracellular matrix (ECM), and human white blood cell impenetrability. The protocol was applied first to five plant extracts and derivative compounds and then to a collection of 88 previously untested plant extracts. They were found to cause a variety of phenotypic profiles, as was the case for 64 of the 88 extracts (73%). Half of the 46 extracts that did not affect biofilm thickness affected other biofilm parameters. Correlations between specific effects were revealed. The protocol will be useful not only in the screening of chemical libraries but also in the analysis of compounds with known effects and mutations. PMID:28893778

Development of the Diabetes Technology Society Blood Glucose Monitor System Surveillance Protocol.

PubMed

Klonoff, David C; Lias, Courtney; Beck, Stayce; Parkes, Joan Lee; Kovatchev, Boris; Vigersky, Robert A; Arreaza-Rubin, Guillermo; Burk, Robert D; Kowalski, Aaron; Little, Randie; Nichols, James; Petersen, Matt; Rawlings, Kelly; Sacks, David B; Sampson, Eric; Scott, Steve; Seley, Jane Jeffrie; Slingerland, Robbert; Vesper, Hubert W

2016-05-01

Inaccurate blood glucsoe monitoring systems (BGMSs) can lead to adverse health effects. The Diabetes Technology Society (DTS) Surveillance Program for cleared BGMSs is intended to protect people with diabetes from inaccurate, unreliable BGMS products that are currently on the market in the United States. The Surveillance Program will provide an independent assessment of the analytical performance of cleared BGMSs. The DTS BGMS Surveillance Program Steering Committee included experts in glucose monitoring, surveillance testing, and regulatory science. Over one year, the committee engaged in meetings and teleconferences aiming to describe how to conduct BGMS surveillance studies in a scientifically sound manner that is in compliance with good clinical practice and all relevant regulations. A clinical surveillance protocol was created that contains performance targets and analytical accuracy-testing studies with marketed BGMS products conducted by qualified clinical and laboratory sites. This protocol entitled "Protocol for the Diabetes Technology Society Blood Glucose Monitor System Surveillance Program" is attached as supplementary material. This program is needed because currently once a BGMS product has been cleared for use by the FDA, no systematic postmarket Surveillance Program exists that can monitor analytical performance and detect potential problems. This protocol will allow identification of inaccurate and unreliable BGMSs currently available on the US market. The DTS Surveillance Program will provide BGMS manufacturers a benchmark to understand the postmarket analytical performance of their products. Furthermore, patients, health care professionals, payers, and regulatory agencies will be able to use the results of the study to make informed decisions to, respectively, select, prescribe, finance, and regulate BGMSs on the market. © 2015 Diabetes Technology Society.

Cardiovascular Risk and Its Associated Factors in Health Care Workers in Colombia: A Study Protocol.

PubMed

Gamboa Delgado, Edna M; Rojas Sánchez, Lyda Z; Bermon Angarita, Anderson; Rangel Díaz, Yully Andrea; Jaraba Suárez, Silvia J; Serrano Díaz, Norma C; Vega Fernández, Evaristo

2015-07-30

Cardiovascular diseases are the leading cause of mortality worldwide, for this reason, they are a public health problem. In Colombia, cardiovascular diseases are the main cause of mortality, having a death rate of 152 deaths per 100,000 population. There are 80% of these cardiovascular events that are considered avoidable. The objective of the study is to determine the prevalence of the cardiovascular risk and its associated factors among the institution's workers in order to design and implement interventions in the work environment which may achieve a decrease in such risk. An analytical cross-sectional study was designed to determine the cardiovascular risk and its associated factors among workers of a high complexity health care institution. A self-applied survey will be conducted considering sociodemographic aspects, physical activity, diet, alcohol consumption, smoking, level of perceived stress, and personal and family history. In a second appointment, a physical examination will be made, as well as anthropometric measurements and blood pressure determination. Also, blood samples for evaluating total and high density lipoprotein cholesterol, triglycerides, and fasting blood sugar will be taken. A ten-year global risk for cardiovascular disease will be determined using the Framingham score. A descriptive analysis of the population's characteristics and a stratified analysis by sex, age, and occupation will be made. Bivariate and multivariate analysis will be made using logistic regression models to evaluate the association between cardiovascular risk and the independent variables. The research protocol was approved by the Scientific and Technical Committee and the Ethics Committee on Research of the Fundación Cardiovascular de Colombia. The protocol has already received funding and the enrollment phase will begin in the coming months. The results of this study will give the foundation for the design, implementation, and evaluation of a program based on promoting healthy lifestyles, such as performing regular physical activity and healthy food intake in order to avoid and/or control the cardiovascular risk in the workers of a high complexity health care institution.

The optimal protocol to reduce blood loss and blood transfusion after unilateral total knee replacement: Low-dose IA-TXA plus 30-min drain clamping versus drainage clamping for the first 3 h without IA-TXA.

PubMed

Park, Joo Hyun; Choi, Sung Wook; Shin, Eun Ho; Park, Myung Hoon; Kim, Myung Ku

2017-01-01

Although intraarticular tranexamic acid (IA-TXA) administration or drainage clamping are popular methods used to reduce blood loss after total knee replacement (TKR), the protocol remains controversial. We aimed (1) to establish new protocols through investigating whether two methods, that is, low-dose (500 mg) IA-TXA plus 30-min drain clamping and drainage clamping for the first 3 h without IA-TXA, can reduce blood loss and blood transfusion after unilateral TKR and (2) to make recommendations related to clinical application. This study, conducted from September 2014 to June 2016 related to enrolled 95 patients with primary osteoarthritis who were to have a unilateral cemented TKR, was nonrandomized and retrospective. In group A, the drain was released following tourniquet deflation. In group B, 500-mg TXA was injected into the knee joint via a drain tube after fascia closure and the drain was clamped for the first 30 min to prevent leakage. In group C, the drain was clamped for the first 3-h postoperation. Demographic characteristics and clinical data were collected, including the levels of hematocrit (Hct), the total blood loss (TBL), drained blood volume (BV), the amount of blood transfused, and any complications that developed. We found a significantly lower postoperative TBL, drained BV, decreasing Hct level, and less transfused BV in the IA-TXA injection group (group B) and the 3-h drainage clamping group (group C) compared to the conventional negative drainage group (group A; p < 0.001). There was no significant difference between groups B and C ( p = 0.99). The drainage clamping method can be safer than IA-TXA administration in patients with risk factor of venous thromboembolic complication. Furthermore, the IA-TXA administration can be more optimal than drainage clamping in patients with high bleeding tendency or lateral retinacular release during TKR, who would be concerned about postoperative wound complication.

Diagnostic accuracy of the ROCHE Septifast PCR system for the rapid detection of blood pathogens in neonatal sepsis-A prospective clinical trial.

PubMed

Straub, Julia; Paula, Helga; Mayr, Michaela; Kasper, David; Assadian, Ojan; Berger, Angelika; Rittenschober-Böhm, Judith

2017-01-01

Diagnosis of neonatal sepsis remains a major challenge in neonatology. Most molecular-based methods are not customized for neonatal requirements. The aim of the present study was to assess the diagnostic accuracy of a modified multiplex PCR protocol for the detection of neonatal sepsis using small blood volumes. 212 episodes of suspected neonatal late onset sepsis were analyzed prospectively using the Roche SeptiFast® MGRADE PCR with a modified DNA extraction protocol and software-handling tool. Results were compared to blood culture, laboratory biomarkers and clinical signs of sepsis. Of 212 episodes, 85 (40.1%) were categorized as "not infected". Among these episodes, 1 was false positive by blood culture (1.2%) and 23 were false positive by PCR (27.1%). Of 51 (24.1%) episodes diagnosed as "culture proven sepsis", the same pathogen was detected by blood culture and PCR in 39 episodes (76.5%). In 8 episodes, more pathogens were detected by PCR compared to blood culture, and in 4 episodes the pathogen detected by blood culture was not found by PCR. One of these episodes was caused by Bacillus cereus, a pathogen not included in the PCR panel. In 76/212 (35.8%) episodes, clinical sepsis was diagnosed. Among these, PCR yielded positive results in 39.5% of episodes (30/76 episodes). For culture-positive sepsis, PCR showed a sensitivity of 90.2% (95%CI 86.2-94.2%) and a specificity of 72.9% (95%CI 67.0-79.0%). The Roche SeptiFast® MGRADE PCR using a modified DNA extraction protocol showed acceptable results for rapid detection of neonatal sepsis in addition to conventional blood culture. The benefit of rapid pathogen detection has to be balanced against the considerable risk of contamination, loss of information on antibiotic sensitivity pattern and increased costs.

Identification of Ixodes ricinus blood meals using an automated protocol with high resolution melting analysis (HRMA) reveals the importance of domestic dogs as larval tick hosts in Italian alpine forests.

PubMed

Collini, Margherita; Albonico, Francesca; Rosà, Roberto; Tagliapietra, Valentina; Arnoldi, Daniele; Conterno, Lorenza; Rossi, Chiara; Mortarino, Michele; Rizzoli, Annapaola; Hauffe, Heidi Christine

2016-12-12

In Europe, Ixodes ricinus L. is the main vector of a variety of zoonotic pathogens, acquired through blood meals taken once per stage from a vertebrate host. Defining the main tick hosts in a given area is important for planning public health interventions; however, until recently, no robust molecular methods existed for blood meal identification from questing ticks. Here we improved the time- and cost-effectiveness of an HRMA protocol for blood meal analysis and used it to identify blood meal sources of sheep tick larvae from Italian alpine forests. Nine hundred questing nymphs were collected using blanket-dragging in 18 extensive forests and 12 forest patches close to rural villages in the Province of Trento. Total DNA was either extracted manually, with the QIAamp DNA Investigator kit, or automatically using the KingFisher™ Flex Magnetic Particle Processors (KingFisher Cell and Tissue DNA Kit). Host DNA was amplified with six independent host group real-time PCR reactions and identified by means of HRMA. Statistical analyses were performed in R to assess the variables important for achieving successful identification and to compare host use in the two types of forest. Automating DNA extraction improved time- and cost-effectiveness of the HRMA protocol, but identification success fell to 22.4% (KingFisher™) from 55.1% (QIAamp), with larval hosts identified in 215 of 848 questing nymphs; 23 mixed blood meals were noted. However, the list of hosts targeted by our primer sets was extended, improving the potential of the method. Host identification to species or genus level was possible for 137 and 102 blood meals, respectively. The most common hosts were Rodentia (28.9%) and, unexpectedly, Carnivora (28.4%), with domestic dogs accounting for 21.3% of all larval blood meals. Overall, Cetartiodactyla species fed 17.2% of larvae. Passeriformes (14.6%) fed a significantly higher proportion of larvae in forest patches (22.3%) than in extensive forest (9.6%), while Soricomorpha (10.9%) were more important hosts in extensive forest (15.2%) than in forest patches (4.3%). The HRMA protocol for blood meal analysis is a valuable tool in the study of feeding ecology of sheep ticks, especially with the cost- and time- reductions introduced here. To our knowledge, we show for the first time that domestic dogs are important larval hosts in the Alps, which may have possible implications for tick-borne disease cycles in urbanized areas.

Interleukin (IL)-1A and IL-6: Applications to the predictive diagnostic testing of radiation pneumonitis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen Yuhchyau; Hyrien, Ollivier; Williams, Jacqueline

2005-05-01

Purpose: To explore the application of interleukin (IL)-1{alpha} and IL-6 measurements in the predictive diagnostic testing for symptomatic radiation pneumonitis (RP). Methods and materials: In a prospective protocol investigating RP and cytokines, IL-1{alpha} and IL-6 values were analyzed by enzyme-linked immunosorbent assay from serial weekly blood samples of patients receiving chest radiation. We analyzed sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) over selected threshold values for both cytokines in the application to diagnostic testing. Results: The average coefficient of variation was 51% of the weekly mean IL-1{alpha} level and 39% of the weekly mean IL-6 value.more » Interleukin 1{alpha} and IL-6 became positively correlated with time. Specificity for both cytokines was better than sensitivity. IL-6 globally outperformed IL-1{alpha} in predicting RP, with higher PPV and NPV. Conclusions: Our data demonstrate the feasibility of applying IL-1{alpha} and IL-6 measurements of blood specimens to predict RP. Interleukin-6 measurements offer stronger positive predictive value than IL-1{alpha}. This application might be further explored in a larger sample of patients.« less

Rationale and Design of the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) Study. Sarcoidosis Protocol.

PubMed

Moller, David R; Koth, Laura L; Maier, Lisa A; Morris, Alison; Drake, Wonder; Rossman, Milton; Leader, Joseph K; Collman, Ronald G; Hamzeh, Nabeel; Sweiss, Nadera J; Zhang, Yingze; O'Neal, Scott; Senior, Robert M; Becich, Michael; Hochheiser, Harry S; Kaminski, Naftali; Wisniewski, Stephen R; Gibson, Kevin F

2015-10-01

Sarcoidosis is a systemic disease characterized by noncaseating granulomatous inflammation with tremendous clinical heterogeneity and uncertain pathobiology and lacking in clinically useful biomarkers. The Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study is an observational cohort study designed to explore the role of the lung microbiome and genome in these two diseases. This article describes the design and rationale for the GRADS study sarcoidosis protocol. The study addresses the hypothesis that distinct patterns in the lung microbiome are characteristic of sarcoidosis phenotypes and are reflected in changes in systemic inflammatory responses as measured by peripheral blood changes in gene transcription. The goal is to enroll 400 participants, with a minimum of 35 in each of 9 clinical phenotype subgroups prioritized by their clinical relevance to understanding of the pathobiology and clinical heterogeneity of sarcoidosis. Participants with a confirmed diagnosis of sarcoidosis undergo a baseline visit with self-administered questionnaires, chest computed tomography, pulmonary function tests, and blood and urine testing. A research or clinical bronchoscopy with a research bronchoalveolar lavage will be performed to obtain samples for genomic and microbiome analyses. Comparisons will be made by blood genomic analysis and with clinical phenotypic variables. A 6-month follow-up visit is planned to assess each participant's clinical course. By the use of an integrative approach to the analysis of the microbiome and genome in selected clinical phenotypes, the GRADS study is powerfully positioned to inform and direct studies on the pathobiology of sarcoidosis, identify diagnostic or prognostic biomarkers, and provide novel molecular phenotypes that could lead to improved personalized approaches to therapy for sarcoidosis.

Application of the IAP cardiovascular fitness test protocol for Austromars candidate screening

NASA Astrophysics Data System (ADS)

Grasser, E. K.; Goswami, N.; Jantscher, A.; Roessler, A.; Vrecko, K.; Groemer, G.; Hinghofer-Szalkay, H.

2007-10-01

Rationale: The purpose of our research was to test for cardiovascular stability within 16 Austromars candidates, and to determine hemodynamic variables and hormones in a presyncopal state as evoked by a specific test protocol. Procedures and methods: We used a graded orthostatic stress (GOS) paradigm consisting of head-up tilt (HUT) combined with lower body negative pressure (LBNP) up to a presyncopal end-point on 15 males and one healthy female. Hemodynamic parameters were monitored and venous blood samples taken. Results: From supine control to presyncope, mean standing time was 12.3 ± 1.2 min, heart rate (HR) increased by 68 ± 12% (p < 0.0001) and thoracic impedance (TI) rose by 12 ± 1% (p < 0.0001), whereas following parameters decreased: stroke volume index (SI) 44 ± 4% (p < 0.0001), systolic pressure (SBP) 26 ± 3% (p < 0.0001), diastolic pressure (DBP) 16 ± 5% (p = 0.004), mean arterial blood pressure (MAP) 19 ± 3% (p < 0.0001), pulse pressure 41 ± 8% (p = 0.0003) and total peripheral resistance index (TPRI) 11 ± 5% (p = 0.03). Heart rate and blood pressure variabilities decreased together with pulse pressure. Plasma volume decreased by 11 ± 2% (p = 0.0004). Plasma norepinephrine (NE) increased by 86 ± 16% (p = 0.0001), epinephrine (E) by 460 ± 266% (p = 0.06), cortisol by 10 ± 6% (p = 0.02), plasma renin activity by 147 ± 26% (p = 0.002) and aldosterone by 24 ± 21% (p = 0.2). Conclusion: Our combined HUT- graded LBNP paradigm is useful to study CV regulation and hormonal responses under severe stress conditions.

Rationale and Design of the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) Study. Sarcoidosis Protocol

PubMed Central

Koth, Laura L.; Maier, Lisa A.; Morris, Alison; Drake, Wonder; Rossman, Milton; Leader, Joseph K.; Collman, Ronald G.; Hamzeh, Nabeel; Sweiss, Nadera J.; Zhang, Yingze; O’Neal, Scott; Senior, Robert M.; Becich, Michael; Hochheiser, Harry S.; Kaminski, Naftali; Wisniewski, Stephen R.; Gibson, Kevin F.

2015-01-01

Sarcoidosis is a systemic disease characterized by noncaseating granulomatous inflammation with tremendous clinical heterogeneity and uncertain pathobiology and lacking in clinically useful biomarkers. The Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study is an observational cohort study designed to explore the role of the lung microbiome and genome in these two diseases. This article describes the design and rationale for the GRADS study sarcoidosis protocol. The study addresses the hypothesis that distinct patterns in the lung microbiome are characteristic of sarcoidosis phenotypes and are reflected in changes in systemic inflammatory responses as measured by peripheral blood changes in gene transcription. The goal is to enroll 400 participants, with a minimum of 35 in each of 9 clinical phenotype subgroups prioritized by their clinical relevance to understanding of the pathobiology and clinical heterogeneity of sarcoidosis. Participants with a confirmed diagnosis of sarcoidosis undergo a baseline visit with self-administered questionnaires, chest computed tomography, pulmonary function tests, and blood and urine testing. A research or clinical bronchoscopy with a research bronchoalveolar lavage will be performed to obtain samples for genomic and microbiome analyses. Comparisons will be made by blood genomic analysis and with clinical phenotypic variables. A 6-month follow-up visit is planned to assess each participant’s clinical course. By the use of an integrative approach to the analysis of the microbiome and genome in selected clinical phenotypes, the GRADS study is powerfully positioned to inform and direct studies on the pathobiology of sarcoidosis, identify diagnostic or prognostic biomarkers, and provide novel molecular phenotypes that could lead to improved personalized approaches to therapy for sarcoidosis. PMID:26193069

Caffeine affects autonomic control of heart rate and blood pressure recovery after aerobic exercise in young adults: a crossover study.

PubMed

Gonzaga, Luana Almeida; Vanderlei, Luiz Carlos Marques; Gomes, Rayana Loch; Valenti, Vitor Engrácia

2017-10-26

The post-exercise recovery period is associated with changes in autonomic modulation, which can promote an intercurrent-favorable environment. Caffeine has the ability to release catecholamines, but its effects after exercises is little explored. The present study aims to evaluate the acute effects of caffeine on the autonomic control and cardiorespiratory parameters after moderate intensity aerobic exercise. 32 young males (23,59 ± 3,45 years) were submitted to two protocols: Placebo and Caffeine, consisting of 15 minutes of rest, 30 minutes of exercise on a treadmill to 60% on VO2peak, followed by 60 minutes of recovery. Heart rate variability indices and cardiorespiratory parameters were determined at different times during the protocols. The RMSSD and SD1 indices recovered faster in placebo (p < 0.05). The systolic blood pressure differences were found from the 1st to the 5th minute of recovery with the caffeine protocol and from the 1st and 3rd minute with the placebo, whereas, for diastolic blood pressure, significant differences (p < 0.0001) were observed only for the caffeine protocol at the 1st and 3rd minutes of recovery. Caffeine was shown to be capable of delaying parasympathetic recovery but did not influence the behavior of the respiratory rate, oxygen saturation or frequency-domain HRV indices.

Introduction of sodium pentosan polysulfate and avoidance of urethral catheterisation: improved outcomes in children with haemorrhagic cystitis post stem cell transplant/chemotherapy.

PubMed

Duthie, Gillian; Whyte, Lisa; Chandran, Harish; Lawson, Sarah; Velangi, Mark; McCarthy, Liam

2012-02-01

Haemorrhagic cystitis (HC) is an uncommon but potentially devastating complication of chemotherapy and bone marrow transplantation in children. We aimed to test the hypothesis that early recognition, sodium pentosan polysulfate (SPP), and avoidance of urethral catheterisation improve outcomes in children with HC. A retrospective case note review was performed of all patients treated for HC in our hospital from 2002 to 2010. A protocol for the management of HC was introduced in 2007 advocating early detection, use of SPP, and avoidance of urethral catheterisation. Data collected on each patient included primary condition, medications at onset, blood transfusions, duration of symptoms, catheter usage, and outcome. Statistical analysis was performed using the Mann-Whitney U test, and Fisher's Exact test as appropriate, P < .05 being significant. Five patients were treated using protocol with 5 historical controls. There was no significant difference between the ages of the group, diagnosis, and treatment at onset of HC. In the historical group, 4 of 5 died with HC, but all recovered in the protocol group (P < .05). Blood transfusion requirements were also significantly reduced after protocol introduction (P < .05). Early identification, avoidance of urethral catheterisation, and use of SPP significantly reduces blood transfusion requirements and mortality from HC. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

Serum Dried Samples to Detect Dengue Antibodies: A Field Study.

PubMed

Maldonado-Rodríguez, Angelica; Rojas-Montes, Othon; Vazquez-Rosales, Guillermo; Chavez-Negrete, Adolfo; Rojas-Uribe, Magdalena; Posadas-Mondragon, Araceli; Aguilar-Faisal, Leopoldo; Cevallos, Ana Maria; Xoconostle-Cazares, Beatriz; Lira, Rosalia

2017-01-01

Dried blood and serum samples are useful resources for detecting antiviral antibodies. The conditions for elution of the sample need to be optimized for each disease. Dengue is a widespread disease in Mexico which requires continuous surveillance. In this study, we standardized and validated a protocol for the specific detection of dengue antibodies from dried serum spots (DSSs). Paired serum and DSS samples from 66 suspected cases of dengue were collected in a clinic in Veracruz, Mexico. Samples were sent to our laboratory, where the conditions for optimal elution of DSSs were established. The presence of anti-dengue antibodies was determined in the paired samples. DSS elution conditions were standardized as follows: 1 h at 4°C in 200  µ l of DNase-, RNase-, and protease-free PBS (1x). The optimal volume of DSS eluate to be used in the IgG assay was 40  µ l. Sensitivity of 94%, specificity of 93.3%, and kappa concordance of 0.87 were obtained when comparing the antidengue reactivity between DSSs and serum samples. DSS samples are useful for detecting anti-dengue IgG antibodies in the field.

Serum Dried Samples to Detect Dengue Antibodies: A Field Study

PubMed Central

Maldonado-Rodríguez, Angelica; Rojas-Montes, Othon; Chavez-Negrete, Adolfo; Rojas-Uribe, Magdalena; Posadas-Mondragon, Araceli; Aguilar-Faisal, Leopoldo; Xoconostle-Cazares, Beatriz

2017-01-01

Background Dried blood and serum samples are useful resources for detecting antiviral antibodies. The conditions for elution of the sample need to be optimized for each disease. Dengue is a widespread disease in Mexico which requires continuous surveillance. In this study, we standardized and validated a protocol for the specific detection of dengue antibodies from dried serum spots (DSSs). Methods Paired serum and DSS samples from 66 suspected cases of dengue were collected in a clinic in Veracruz, Mexico. Samples were sent to our laboratory, where the conditions for optimal elution of DSSs were established. The presence of anti-dengue antibodies was determined in the paired samples. Results DSS elution conditions were standardized as follows: 1 h at 4°C in 200 µl of DNase-, RNase-, and protease-free PBS (1x). The optimal volume of DSS eluate to be used in the IgG assay was 40 µl. Sensitivity of 94%, specificity of 93.3%, and kappa concordance of 0.87 were obtained when comparing the antidengue reactivity between DSSs and serum samples. Conclusion DSS samples are useful for detecting anti-dengue IgG antibodies in the field. PMID:28630868

Curcumin and long-chain Omega-3 polyunsaturated fatty acids for Prevention of type 2 Diabetes (COP-D): study protocol for a randomised controlled trial.

PubMed

Thota, Rohith N; Acharya, Shamasunder H; Abbott, Kylie A; Garg, Manohar L

2016-11-29

Lifestyle interventions, including increase in physical activity and dietary counselling, have shown the ability to prevent type 2 diabetes (T2D) in high-risk state individuals, but the prevalence is still skyrocketing in Australia, in line with global prevalence. Currently, no medicines are approved by the Therapeutic Goods Administration in Australia for the management of prediabetes. Therefore, there is a need of developing a safer, biologically efficacious and cost-effective alternative for delaying the transition of individual health state from prediabetes into T2D. In the current trial we propose to evaluate the effects of curcumin and/or long-chain omega-3 polyunsaturated fatty acids on improving glycosylated haemoglobin as a primary outcome, along with secondary outcomes of glycaemic indices, lipid profile and inflammatory parameters. Eighty individuals diagnosed with prediabetes, aged between 30 and 70 years, will be randomly assigned to double placebo, curcumin alone, fish oil alone or double active groups according to a computer-generated randomisation sequence for 12 weeks. At baseline and post-intervention visits participants will be asked to provide blood samples and undergo body composition measurements. A blood sample is used for estimating glycaemic profiles, lipid profiles and inflammatory parameters (C-reactive protein, whole blood cell count, adiponectin, leptin, interleukin-6). The interim visit includes review on compliance with supplements based on capsule log and capsule count, adverse events and anthropometric measurements. In addition to these procedures, participants provide self-reported questionnaires on dietary intake (using a 3-day food record), a physical activity questionnaire and medical history. This trial aims to determine whether curcumin and/or long-chain omega-3 polyunsaturated fatty acids affect surrogate markers of glycaemic control which is relevant to delaying T2D. To date 38 participants completed the trial. No changes have been made to the clinical protocol post recruitment. If successful, this trial will provide considerable evidence for performing a larger trial to investigate whether this combination can be administered for preventing or delaying the onset of T2D in high-risk individuals. ACTRN12615000559516 , registered on 29 May 2015).

Dried blood spots as a source of anti-malarial antibodies for epidemiological studies

PubMed Central

Corran, Patrick H; Cook, Jackie; Lynch, Caroline; Leendertse, Heleen; Manjurano, Alphaxard; Griffin, Jamie; Cox, Jonathan; Abeku, Tarekegn; Bousema, Teun; Ghani, Azra C; Drakeley, Chris; Riley, Eleanor

2008-01-01

Background Blood spots collected onto filter paper are an established and convenient source of antibodies for serological diagnosis and epidemiological surveys. Although recommendations for the storage and analysis of small molecule analytes in blood spots exist, there are no published systematic studies of the stability of antibodies under different storage conditions. Methods Blood spots, on filter paper or glass fibre mats and containing malaria-endemic plasma, were desiccated and stored at various temperatures for different times. Eluates of these spots were assayed for antibodies against two Plasmodium falciparum antigens, MSP-119 and MSP2, and calculated titres used to fit an exponential (first order kinetic) decay model. The first order rate constants (k) for each spot storage temperature were used to fit an Arrhenius equation, in order to estimate the thermal and temporal stability of antibodies in dried blood spots. The utility of blood spots for serological assays was confirmed by comparing antibodies eluted from blood spots with the equivalent plasma values in a series of samples from North Eastern Tanzania and by using blood spot-derived antibodies to estimate malaria transmission intensity in this site and for two localities in Uganda. Results Antibodies in spots on filter paper and glass fibre paper had similar stabilities but blood was more easily absorbed onto filter papers than glass fibre, spots were more regular and spot size was more closely correlated with blood volume for filter paper spots. Desiccated spots could be stored at or below 4°C for extended periods, but were stable for only very limited periods at ambient temperature. When desiccated, recoveries of antibodies that are predominantly of IgG1 or IgG3 subclasses were similar. Recoveries of antibodies from paired samples of serum and of blood spots from Tanzania which had been suitably stored showed similar recoveries of antibodies, but spots which had been stored for extended periods at ambient humidity and temperature showed severe loss of recoveries. Estimates of malaria transmission intensity obtained from serum and from blood spots were similar, and values obtained using blood spots agreed well with entomologically determined values. Conclusion This study has demonstrated the suitability of filter paper blood spots paper for collection of serum antibodies, and provided clear guidelines for the treatment and storage of filter papers which emphasize the importance of desiccation and minimisation of time spent at ambient temperatures. A recommended protocol for collecting, storing and assaying blood spots is provided. PMID:18826573

Waiver of consent in noninterventional, observational emergency research: the PROMMTT experience.

PubMed

Fox, Erin E; Bulger, Eileen M; Dickerson, Aisha S; del Junco, Deborah J; Klotz, Patricia; Podbielski, Jeanette; Matijevic, Nena; Brasel, Karen J; Holcomb, John B; Schreiber, Martin A; Cotton, Bryan A; Phelan, Herb A; Cohen, Mitchell J; Myers, John G; Alarcon, Louis H; Muskat, Peter; Wade, Charles E; Rahbar, Mohammad H

2013-07-01

In the PRospective Observational Multicenter Major Trauma Transfusion (PROMMTT) study, waiver of consent was used because previous literature reported low response rates and subsequent bias. The goal of this article was to examine the rationale and tradeoffs of using waiver of consent in PROMMTT. PROMMTT enrolled trauma patients receiving at least 1 U of red blood cells within 6 hours after admission at 10 US Level 1 trauma centers. Local institutional review boards (IRBs) from all sites approved the study. Site 8 was required by their IRB to attempt consent but was allowed to retain data on patients unable to be consented. Of 121 subjects enrolled at Site 8, 55 consents were obtained (46%), and no patient or legally authorized representative refused to give consent. Of the patients, 36 (30%) died, and 6 (5%) were discharged before consent could be attempted. Consent was attempted but not possible among 24 patients (20%). Of the 10 clinical sites, 6 of the local IRBs approved collection of residual blood samples, 1 had previous approval to collect timed blood samples under a separate protocol, and 3 reported that their local IRBs would not approve collection of residual blood under a waiver of consent. Waiver of consent was used in PROMMTT because of the potential adverse impact of consent refusals; however, there were no refusals. If the IRB for Site 8 had required withdrawal of patients unable to consent and destruction of their data, a serious bias would likely have been introduced. Other tradeoffs included a reduction in sites participating in residual blood collection and a smaller than expected amount of residual blood collected among sites operating under a waiver of consent. Noninterventional emergency research studies should consider these potential tradeoffs carefully before deciding whether waiver of consent would best achieve the goals of a study.

Waiver of consent in non-interventional, observational emergency research: the PROMMTT experience

PubMed Central

Fox, Erin E.; Bulger, Eileen M.; Dickerson, Aisha S.; del Junco, Deborah J.; Klotz, Patricia; Podbielski, Jeanette; Matijevic, Nena; Brasel, Karen J.; Holcomb, John B.; Schreiber, Martin A.; Cotton, Bryan A.; Phelan, Herb A.; Cohen, Mitchell J.; Myers, John G.; Alarcon, Louis H.; Muskat, Peter; Wade, Charles E.; Rahbar, Mohammad H.

2013-01-01

Background In the PRospective Observational Multi-center Major Trauma Transfusion (PROMMTT) study, waiver of consent was utilized because previous literature reported low response rates and subsequent bias. The goal of this manuscript is to examine the rationale and tradeoffs of using waiver of consent in PROMMTT. Methods PROMMTT enrolled trauma patients receiving at least one unit of red blood cells within 6 hours after admission at ten US Level 1 Trauma Centers. Local Institutional Review Boards (IRBs) from all sites approved the study. Site 8 was required by their IRB to attempt consent, but was allowed to retain data on patients unable to be consented. Results Of 121 subjects enrolled at Site 8, 55 consents were obtained (46%) and no patient or legally-authorized representative refused to give consent. Thirty-six (30%) patients died and 6 (5%) were discharged before consent could be attempted. Consent was attempted but not possible among 24 patients (20%). Of the 10 clinical sites, six of the local IRBs approved collection of residual blood samples, one had prior approval to collect timed blood samples under a separate protocol, and three reported that their local IRBs would not approve collection of residual blood under a waiver of consent. Conclusions Waiver of consent was used in PROMMTT because of the potential adverse impact of consent refusals; however, there were no refusals. If Site 8’s IRB had required withdrawal of patients unable to consent and destruction of their data, a serious bias would likely have been introduced. Other tradeoffs included a reduction in sites participating in residual blood collection, and a smaller than expected amount of residual blood collected among sites operating under a waiver of consent. Non-interventional emergency research studies should consider these potential tradeoffs carefully before deciding whether waiver of consent would best achieve the goals of a study. Level of Evidence Prospective, Level II PMID:23778508

A comparison of blood loss during the Halal slaughter of lambs following Traditional Religious Slaughter without stunning, Electric Head-Only Stunning and Post-Cut Electric Head-Only Stunning.

PubMed

Khalid, Rizvan; Knowles, Toby G; Wotton, Steve B

2015-12-01

Blood lost at exsanguination during the Halal slaughter of lambs was compared between the slaughter methods of Traditional Religious Slaughter without stunning (TRS), Electric Head-Only Stunning (EHOS) and Post-Cut Electric Head-Only Stunning (PCEHOS). Two protocols were examined, Experimental (80 lambs) and Commercial (360 lambs), assessing varying periods of animal orientation during the 4 min bleeding process (upright orientation before vertical hanging). Live-weight, blood weight (Experimental only), carcass weights and by-product weights were recorded. The Experimental protocol highlighted an increase in blood loss at 60s in EHOS and PCEHOS compared to TRS (P<0.001) but by 90 s there was no significant difference. A post-slaughter change in animal orientation from an upright to a vertical hanging position aided the amount of blood loss. The bleeding of lambs is largely completed by 2 min. There were no significant differences (P>0.05) in final blood loss between treatments. This research was undertaken to inform discussion on the merits of different slaughter methods compatible with Halal requirements. Copyright © 2015. Published by Elsevier Ltd.

Validation of the Andon KD-5851 upper arm blood pressure monitor, for self-measurement according to the European Society of Hypertension International Protocol revision 2010.

PubMed

Wu, Lili; Jiao, Yinghui; Wang, Chengdong; Chen, Lei; Di, Dalin; Zhang, Haiyan

2015-08-01

This study aimed to validate the Andon KD-5851 upper arm blood pressure (BP) monitor according to the European Society of Hypertension International Protocol (ESH-IP) revision 2010. A total of 33 eligible participants were included in the study. Sequential measurements of BPs were performed using a mercury sphygmomanometer and the device, and the data analysis was carried out following precisely the ESH-IP revision 2010. The device had 82, 98, and 99 measurements within 5, 10, and 15 mmHg for systolic blood pressure and 85, 95, and 99 measurements for diastolic blood pressure, respectively. The average device-observer difference was -0.53±4.00 mmHg for systolic blood pressure and -1.15±4.06 mmHg for diastolic blood pressure. The device passed all the criteria according to the ESH-IP revision 2010. According to the validation results on the basis of the ESH-IP revision 2010, the Andon KD-5851 upper arm BP monitor can be recommended for self/home measurement in adults. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

Validation of the Grandway MD2301 digital automatic blood pressure monitor according to the European Society of Hypertension International Protocol.

PubMed

Chen, Wan; Zeng, Zhao-Lin; Bing, Sen; Li, Lin-Yi; Wang, Rui; Wan, Yi

2016-08-01

The aim of the present study was to validate the Grandway MD2301 digital automatic blood pressure monitor according to the European Society of Hypertension International Protocol (ESH-IP) revision 2010. The ESH-IP revision 2010 for the validation of blood pressure-measuring devices in adults was followed precisely. Systolic and diastolic blood pressure (SBP and DBP, respectively) were measured sequentially in 33 adult patients and compared with a standard mercury sphygmomanometer (two observers). A total of 99 comparison pairs were obtained. The device produced 78, 95 and 99 measurements within 5, 10, and 15 mmHg for SBP and 83, 96, and 99 for DBP, respectively. The average device-observer difference was -1.81±4.22 mmHg for SBP and -0.15±3.93 mmHg for DBP. All of the data were within the standards requirements to pass the testing. The Grandway MD2301 digital automatic blood pressure monitor meets the standards of the ESH-IP revision 2010 and can be recommended for self/home measurement in the general population.

A multigear protocol for sampling crayfish assemblages in Gulf of Mexico coastal streams

Treesearch

William R. Budnick; William E. Kelso; Susan B. Adams; Michael D. Kaller

2018-01-01

Identifying an effective protocol for sampling crayfish in streams that vary in habitat and physical/chemical characteristics has proven problematic. We evaluated an active, combined-gear (backpack electrofishing and dipnetting) sampling protocol in 20 Coastal Plain streams in Louisiana. Using generalized linear models and rarefaction curves, we evaluated environmental...

An overview of human biomonitoring of environmental chemicals in the Canadian Health Measures Survey: 2007-2019.

PubMed

Haines, Douglas A; Saravanabhavan, Gurusankar; Werry, Kate; Khoury, Cheryl

2017-03-01

Human biomonitoring (HBM) is used to indicate and quantify exposure by measuring environmental chemicals, their metabolites or reaction products in biological specimens. The biomonitoring component of the Canadian Health Measures Survey (CHMS) is the most comprehensive initiative providing general population HBM data in Canada. The CHMS is an ongoing cross-sectional direct measures survey implemented in 2-year cycles. It provides nationally-representative data on health, nutritional status, environmental exposures, and related risks and protective characteristics. The survey follows a robust planning, design and sampling protocol as well as a comprehensive quality assurance and quality control regime implemented for all aspect of the survey to ensure the validity of the HBM results. HBM blood and urine data are available for CHMS cycles 1 (2007-2009), 2 (2009-2011) and 3 (2012-2013). Field collection has been completed for cycle 4 (2014-2015), with cycle 5 (2016-2017) in progress and cycle 6 planning (2018-2019) being finalized. Biomonitoring results for 279 chemicals are expected over the six cycles of the CHMS (220 in individual blood, urine or hair samples, and 59 in pooled serum samples). The chemicals include metals and trace elements, polychlorinated biphenyls (PCBs), organochlorines, flame retardants, perfluoroalkyl substances, volatile organic compounds (VOCs) and metabolites, environmental phenols, triclocarban, acrylamide, pesticides (e.g., triazines, carbamates, organophosphates, phenoxy, pyrethroids) and/or their metabolites, chlorophenols, polycyclic aromatic hydrocarbon (PAH) metabolites, phthalates and alternate plasticizer metabolites, and tobacco biomarkers. Approximately one half of the chemicals measured in individual blood and urine samples over the first three cycles were detected in more than 60% of samples. CHMS biomonitoring data have been used to establish baseline HBM concentrations in Canadians; inform public health, regulatory risk assessment and management decisions; and fulfil national and international reporting requirements. Concurrent efforts are underway in Canada to develop statistically- and risk-based concepts and tools to interpret biomonitoring data. Crown Copyright © 2016. Published by Elsevier GmbH. All rights reserved.

Cardiovascular responses to plyometric exercise are affected by workload in athletes

PubMed Central

Arazi, Hamid; Mahdavi, Seyed Amir; Nasiri, Seyed Omid Mirfalah

2014-01-01

Introduction With regard to blood pressure responses to plyometric exercise and decreasing blood pressure after exercise (post-exercise hypotension), the influence of different workloads of plyometric exercise on blood pressure is not clear. Aim The purpose of this investigation was to examine the effects of a low, moderate and high workload of plyometric exercise on the post-exercise systolic (SBP) and diastolic blood pressure (DBP), heart rate (HR) and rate-pressure product (RPP) responses in athletes. Material and methods Ten male athletes (age: 22.6 ±0.5 years; height: 178.2 ±3.3 cm; and body mass: 75.2 ±2.8 kg) underwent PE protocols involving 5 × 10 reps (Low Workload – LW), 10 × 10 reps (Moderate Workload – MW), and 15 × 10 reps (High Workload – HW) depth jump exercise from a 50-cm box in 3 non-consecutive days. After each exercise session, SBP, DBP and HR were measured every 10 min for a period of 70 min. Results No significant differences were observed among post-exercise SBP and DBP when the protocols (LW, MW and HW) were compared. The MW and HW protocols showed greater increases in HR compared with LW. Also the HW indicated greater increases than LW in RPP at post-exercise (p < 0.05). Conclusions All protocols increased SBP, HR and RPP responses at the 10th and 20th min of post-exercise. With regard to different workloads of plyometric exercise, HW condition indicated greater increases in HR and RPP and strength and conditioning professionals and athletes must keep in their mind that HW of plyometric exercise induces greater cardiovascular responses. PMID:24799919

Three Dimensional Imaging of Paraffin Embedded Human Lung Tissue Samples by Micro-Computed Tomography

PubMed Central

Scott, Anna E.; Vasilescu, Dragos M.; Seal, Katherine A. D.; Keyes, Samuel D.; Mavrogordato, Mark N.; Hogg, James C.; Sinclair, Ian; Warner, Jane A.; Hackett, Tillie-Louise; Lackie, Peter M.

2015-01-01

Background Understanding the three-dimensional (3-D) micro-architecture of lung tissue can provide insights into the pathology of lung disease. Micro computed tomography (µCT) has previously been used to elucidate lung 3D histology and morphometry in fixed samples that have been stained with contrast agents or air inflated and dried. However, non-destructive microstructural 3D imaging of formalin-fixed paraffin embedded (FFPE) tissues would facilitate retrospective analysis of extensive tissue archives of lung FFPE lung samples with linked clinical data. Methods FFPE human lung tissue samples (n = 4) were scanned using a Nikon metrology µCT scanner. Semi-automatic techniques were used to segment the 3D structure of airways and blood vessels. Airspace size (mean linear intercept, Lm) was measured on µCT images and on matched histological sections from the same FFPE samples imaged by light microscopy to validate µCT imaging. Results The µCT imaging protocol provided contrast between tissue and paraffin in FFPE samples (15mm x 7mm). Resolution (voxel size 6.7 µm) in the reconstructed images was sufficient for semi-automatic image segmentation of airways and blood vessels as well as quantitative airspace analysis. The scans were also used to scout for regions of interest, enabling time-efficient preparation of conventional histological sections. The Lm measurements from µCT images were not significantly different to those from matched histological sections. Conclusion We demonstrated how non-destructive imaging of routinely prepared FFPE samples by laboratory µCT can be used to visualize and assess the 3D morphology of the lung including by morphometric analysis. PMID:26030902

Evaluating blood perfusion of the corpus luteum in beef cows during fescue toxicosis.

PubMed

Cline, G F; Muth-Spurlock, A M; Voelz, B E; Lemley, C O; Larson, J E

2016-01-01

The aim of this study was to determine if fescue toxicosis altered blood perfusion in the corpus luteum (CL) and peripheral concentrations of progesterone in cattle. The estrous cycles of 36 nonpregnant Angus or Charolais cows were synchronized in 2 replicates using the CO-Synch+CIDR protocol. Seven days after initiation of the protocol, cows were assigned (d 0) to 1 of 2 dietary treatments: 2.5 kg of 1) Kentucky-31 endophyte-infected (KY31; = 14) or 2) MaxQ novel endophyte (MaxQ; = 12) tall fescue seed. On d 7, ovaries were examined using ultrasonography, and only cows that had 1 CL present remained on the study ( = 26). Images of blood perfusion of CL, blood samples, rectal temperatures, and blood pressure of tails were collected on d 10, 13, 15, and 18. Images of CL blood perfusion were analyzed using ImageJ software for pixel density, and scored visually (0 to 9 with 0 = no perfusion, 9 = complete perfusion) by 2 independent technicians. The MIXED procedure of SAS was used with day as a repeated measure. Least squares means and SEM are reported. Cows receiving KY31 had greater rectal temperatures ( 0.003; 38.76 ± 0.08°C) than those receiving MaxQ (38.44 ± 0.08°C), providing evidence that the cows treated with KY31 were influenced by fescue toxicosis. Pulse pressure and mean arterial pressure were decreased ( < 0.01) in cows receiving KY31 (55.26 ± 2.81 and 80.06 ± 2.72 mmHg, respectively) than MaxQ (66.58 ± 3.03 and 91.38 ± 2.93 mmHg, respectively). Concentrations of progesterone were similar ( = 0.54) between cows receiving KY31 (6.04 ± 0.53 ng/mL) or MaxQ (6.36 ± 0.63 ng/mL). Pixel densities ( = 0.14) and visual perfusion scores were similar ( = 0.11) between cows receiving KY31 (1477.20 ± 655.62 pixels and 2.23 ± 0.34, respectively) or MaxQ (2934.70 ± 718.20 pixels and 3.00 ± 0.36, respectively). Mean CL volume was similar ( 0.95) between treatments. In conclusion, blood perfusion of CL or peripheral concentrations of progesterone were not altered at the onset of fescue toxicosis in this short-term study, indicating that a decrease in blood perfusion of the CL may not be a primary mechanism involved in decreased reproductive efficiency of cattle during fescue toxicosis.

Evaluation of four automated protocols for extraction of DNA from FTA cards.

PubMed

Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura; Frank-Hansen, Rune; Poulsen, Lena; Hansen, Anders J; Morling, Niels

2013-10-01

Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore, we demonstrated that it was possible to successfully extract sufficient DNA for STR profiling from previously processed FTA card pieces that had been stored at 4 °C for up to 1 year. This showed that rare or precious FTA card samples may be saved for future analyses even though some DNA was already extracted from the FTA cards.

Application of a comprehensive protocol for the identification of Gaucher disease in Brazil.

PubMed

Michelin, Kristiane; Wajner, Alessandro; de Souza, Fernanda T S; de Mello, Alexandre S; Burin, Maira G; Pereira, Maria Luiza S; Pires, Ricardo F; Giugliani, Roberto; Coelho, Janice C

2005-07-01

Gaucher disease (GD) is a sphingolipidosis caused by a genetic defect that leads to glucocerebrosidase (beta-glucosidase) deficiency. Between January 1982 and October 2003, 1,081 blood samples from patients suspected of having GD were referred for biochemical analysis. The activities of the enzymes beta-glucosidase (beta-glu) and chitotriosidase (CT) were measured in these samples. Among the 412 diagnosed cases of GD (38.1%), the great majority were GD type 1. The Brazilian regions with the greatest concentration of these patients were the Southeast, South, and Northeast. The mean age of patients at diagnosis was 19 years. The activity of beta-glu in patients with GD was, on average, 10.7% of that of normal individuals. CT was, on average, 269 times more elevated in this group of patients. Among the 669 cases with no confirmation of GD, there were patients with Niemann-Pick disease types A, B, or C (44 cases), possible heterozygotes for GD (59 cases), patients with other lysosomal storage diseases (LSDs) (19 cases) or with other inborn errors of metabolism (3 cases). In 508 cases, no metabolic disorder was found. This study shows that the biochemical protocol employed was effective for the detection of GD, a disease that is reasonably frequent in Brazil. Copyright (c) 2005 Wiley-Liss, Inc.

Manual versus automated blood sampling: impact of repeated blood sampling on stress parameters and behavior in male NMRI mice

PubMed Central

Kalliokoski, Otto; Sørensen, Dorte B; Hau, Jann; Abelson, Klas S P

2014-01-01

Facial vein (cheek blood) and caudal vein (tail blood) phlebotomy are two commonly used techniques for obtaining blood samples from laboratory mice, while automated blood sampling through a permanent catheter is a relatively new technique in mice. The present study compared physiological parameters, glucocorticoid dynamics as well as the behavior of mice sampled repeatedly for 24 h by cheek blood, tail blood or automated blood sampling from the carotid artery. Mice subjected to cheek blood sampling lost significantly more body weight, had elevated levels of plasma corticosterone, excreted more fecal corticosterone metabolites, and expressed more anxious behavior than did the mice of the other groups. Plasma corticosterone levels of mice subjected to tail blood sampling were also elevated, although less significantly. Mice subjected to automated blood sampling were less affected with regard to the parameters measured, and expressed less anxious behavior. We conclude that repeated blood sampling by automated blood sampling and from the tail vein is less stressful than cheek blood sampling. The choice between automated blood sampling and tail blood sampling should be based on the study requirements, the resources of the laboratory and skills of the staff. PMID:24958546

Effectiveness of droxidopa compared to midodrine in standing blood pressure and orthostatic tolerance in adults with neurogenic orthostatic hypotension: a systematic review protocol.

PubMed

Patrick, Kelli; Martin, Tina

2017-09-01

The question of this review is: what is the effectiveness of droxidopa compared to midodrine on standing blood pressure and orthostatic intolerance symptoms in adults with neurogenic orthostatic hypotension?

Validation of the Omron MIT Elite blood pressure device in a pregnant population with large arm circumference.

PubMed

James, Lauren; Nzelu, Diane; Hay, Anna; Shennan, Andrew; Kametas, Nikos A

2017-04-01

The aim of this study was to evaluate the accuracy of the Omron MIT Elite automated device in pregnant women with an arm circumference of or above 32 cm, using the British Hypertension Society validation protocol. Blood pressure was measured sequentially in 46 women of any gestation requiring the use of a large cuff (arm circumference ≥32 cm) alternating between the mercury sphygmomanometer and the Omron MIT Elite device. The Omron MIT Elite achieved an overall D/D grade with a mean of the device-observer difference being 7.17±6.67 and 9.31±6.59 for systolic and diastolic blood pressure respectively. Interobserver accuracy was 94.6% for systolic and 95% for diastolic readings within 5 mmHg. The Omron MIT Elite overestimates blood pressure and has failed the British Hypertension Society protocol requirements. Therefore, it cannot be recommended for use in pregnant women with an arm circumference of or above 32 cm.

An ultra-high-throughput spiral microfluidic biochip for the enrichment of circulating tumor cells.

PubMed

Warkiani, Majid Ebrahimi; Khoo, Bee Luan; Tan, Daniel Shao-Weng; Bhagat, Ali Asgar S; Lim, Wan-Teck; Yap, Yoon Sim; Lee, Soo Chin; Soo, Ross A; Han, Jongyoon; Lim, Chwee Teck

2014-07-07

The detection and characterization of rare circulating tumor cells (CTCs) from the blood of cancer patients can potentially provide critical insights into tumor biology and hold great promise for cancer management. The ability to collect a large number of viable CTCs for various downstream assays such as quantitative measurements of specific biomarkers or targeted somatic mutation analysis is increasingly important in medical oncology. Here, we present a simple yet reliable microfluidic device for the ultra-high-throughput, label-free, size-based isolation of CTCs from clinically relevant blood volumes. The fast processing time of the technique (7.5 mL blood in less than 10 min) and the ability to collect more CTCs from larger blood volumes lends itself to a broad range of potential genomic and transcriptomic applications. A critical advantage of this protocol is the ability to return all fractions of blood (i.e., plasma (centrifugation), CTCs and white blood cells (WBCs) (size-based sorting)) that can be utilized for diverse biomarker studies or time-sensitive molecular assays such as RT-PCR. The clinical use of this biochip was demonstrated by detecting CTCs from 100% (10/10) of blood samples collected from patients with advanced-stage metastatic breast and lung cancers. The CTC recovery rate ranged from 20 to 135 CTCs mL(-1) and obtained under high purity (of 1 CTC out of every 30-100 WBCs which gives ∼4 log depletion of WBCs). They were identified with immunofluorescence assays (pan-cytokeratin+/CD45-) and molecular probes such as HER2/neu.

Altered Mental Status: Current Evidence-based Recommendations for Prehospital Care.

PubMed

Sanello, Ashley; Gausche-Hill, Marianne; Mulkerin, William; Sporer, Karl A; Brown, John F; Koenig, Kristi L; Rudnick, Eric M; Salvucci, Angelo A; Gilbert, Gregory H

2018-05-01

In the United States emergency medical services (EMS) protocols vary widely across jurisdictions. We sought to develop evidence-based recommendations for the prehospital evaluation and treatment of a patient with an acute change in mental status and to compare these recommendations against the current protocols used by the 33 EMS agencies in the State of California. We performed a literature review of the current evidence in the prehospital treatment of a patient with altered mental status (AMS) and augmented this review with guidelines from various national and international societies to create our evidence-based recommendations. We then compared the AMS protocols of each of the 33 EMS agencies for consistency with these recommendations. The specific protocol components that we analyzed were patient assessment, point-of-care tests, supplemental oxygen, use of standardized scoring, evaluating for causes of AMS, blood glucose evaluation, toxicological treatment, and pediatric evaluation and management. Protocols across 33 EMS agencies in California varied widely. All protocols call for a blood glucose check, 21 (64%) suggest treating adults at <60mg/dL, and half allow for the use of dextrose 10%. All the protocols recommend naloxone for signs of opioid overdose, but only 13 (39%) give specific parameters. Half the agencies (52%) recommend considering other toxicological causes of AMS, often by using the mnemonic AEIOU TIPS. Eight (24%) recommend a 12-lead electrocardiogram; others simply suggest cardiac monitoring. Fourteen (42%) advise supplemental oxygen as needed; only seven (21%) give specific parameters. In terms of considering various etiologies of AMS, 25 (76%) give instructions to consider trauma, 20 (61%) to consider stroke, and 18 (55%) to consider seizure. Twenty-three (70%) of the agencies have separate pediatric AMS protocols; others include pediatric considerations within the adult protocol. Protocols for patients with AMS vary widely across the State of California. The evidence-based recommendations that we present for the prehospital diagnosis and treatment of this condition may be useful for EMS medical directors tasked with creating and revising these protocols.

High-throughput, automated extraction of DNA and RNA from clinical samples using TruTip technology on common liquid handling robots.

PubMed

Holmberg, Rebecca C; Gindlesperger, Alissa; Stokes, Tinsley; Brady, Dane; Thakore, Nitu; Belgrader, Philip; Cooney, Christopher G; Chandler, Darrell P

2013-06-11

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

A distance limited method for sampling downed coarse woody debris

Treesearch

Jeffrey H. Gove; Mark J. Ducey; Harry T. Valentine; Michael S. Williams

2012-01-01

A new sampling method for down coarse woody debris is proposed based on limiting the perpendicular distance from individual pieces to a randomly chosen sample point. Two approaches are presented that allow different protocols to be used to determine field measurements; estimators for each protocol are also developed. Both protocols are compared via simulation against...

Validation of Non-Invasive Tracer Kinetic Analysis of 18F-Florbetaben PET Using a Dual Time-Window Acquisition Protocol.

PubMed

Bullich, Santiago; Barthel, Henryk; Koglin, Norman; Becker, Georg A; De Santi, Susan; Jovalekic, Aleksandar; Stephens, Andrew W; Sabri, Osama

2017-11-24

Accurate amyloid PET quantification is necessary for monitoring amyloid-beta accumulation and response to therapy. Currently, most of the studies are analyzed using the static standardized uptake value ratio (SUVR) approach because of its simplicity. However, this approach may be influenced by changes in cerebral blood flow (CBF) or radiotracer clearance. Full tracer kinetic models require arterial blood sampling and dynamic image acquisition. The objectives of this work were: (1) to validate a non-invasive kinetic modeling approach for 18 F-florbetaben PET using an acquisition protocol with the best compromise between quantification accuracy and simplicity and (2) to assess the impact of CBF changes and radiotracer clearance on SUVRs and non-invasive kinetic modeling data in 18 F-florbetaben PET. Methods: Data from twenty subjects (10 patients with probable Alzheimer's dementia/ 10 healthy volunteers) were used to compare the binding potential (BP ND ) obtained from the full kinetic analysis to the SUVR and to non-invasive tracer kinetic methods (simplified reference tissue model (SRTM), and multilinear reference tissue model 2 (MRTM2)). Different approaches using shortened or interrupted acquisitions were compared to the results of the full acquisition (0-140 min). Simulations were carried out to assess the effect of CBF and radiotracer clearance changes on SUVRs and non-invasive kinetic modeling outputs. Results: A 0-30 and 120-140 min dual time-window acquisition protocol using appropriate interpolation of the missing time points provided the best compromise between patient comfort and quantification accuracy. Excellent agreement was found between BP ND obtained using full and dual time-window (2TW) acquisition protocols (BP ND,2TW =0.01+ 1.00 BP ND,FULL , R2=0.97 (MRTM2); BP ND,2TW = 0.05+ 0.92·BP ND,FULL , R2=0.93 (SRTM)). Simulations showed a limited impact of CBF and radiotracer clearance changes on MRTM parameters and SUVRs. Conclusion: This study demonstrates accurate non-invasive kinetic modeling of 18 F-florbetaben PET data using a dual time-window acquisition protocol, thus providing a good compromise between quantification accuracy, scan duration and patient burden. The influence of CBF and radiotracer clearance changes on amyloid-beta load estimates was small. For most clinical research applications, the SUVR approach is appropriate. However, for longitudinal studies in which a maximum quantification accuracy is desired, this non-invasive dual time-window acquisition protocol and kinetic analysis is recommended. Copyright © 2017 by the Society of Nuclear Medicine and Molecular Imaging, Inc.