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Sample records for human whole-blood mononuclear

  1. Human whole-blood oxygen affinity: effect of temperature.

    PubMed

    Zwart, A; Kwant, G; Oeseburg, B; Zijlstra, W G

    1984-08-01

    phe effect of temperature changes on human whole-blood O2 affinity was measured in the blood of six healthy donors over almost the entire O2 saturation (SO2) range (1-99%). The results showed that temperature has no influence on the shape of the O2 dissociation curve, implying that the temperature coefficient (delta log PO2/delta T) is independent of SO2. Simultaneous measurements of the total (proton) Haldane factor (delta[HbH]/[delta HbO2]) at the five temperatures under study (22, 27, 32, 37, and 42 degrees C) revealed that this factor depends on temperature. The liberation of protons from hemoglobin appeared to be linear with respect to changes in SO2. We therefore conclude that the (proton) Bohr factor (H+ factor) is dependent on temperature over the entire SO2 range in the same way as previously described for SO2 = 50%. The exothermic oxygenation reaction in whole blood was accompanied by a heat evolution (delta HO2) of 42.7 kJ/mol (monomeric) hemoglobin.

  2. A Systematic Heritability Analysis of the Human Whole Blood Transcriptome

    PubMed Central

    Huan, Tianxiao; Liu, Chunyu; Joehanes, Roby; Zhang, Xiaoling; Chen, Brian H.; Johnson, Andrew D.; Yao, Chen; Courchesne, Paul; O'Donnell, Christopher J.; Munson, Peter J.; Levy, Daniel

    2015-01-01

    Genome-wide expression quantitative trait locus (eQTL) mapping may reveal common genetic variants regulating gene expression. In addition to mapping eQTLs, we systematically evaluated the heritability of the whole blood transcriptome in 5626 participants from the Framingham Heart Study. Of all gene expression measurements, about 40% exhibit evidence of being heritable (hgeneExp2>0, (p<0.05]), the average heritability was estimated to be 0.13, and 10% display hgeneExp2>0.2. In order to identify the role of eQTLs in promoting phenotype differences and disease susceptibility, we investigated the proportion of cis/trans eQTLs in different heritability categories and discovered that genes with higher heritability are more likely to have cis eQTLs that explain large proportions of variance in the expression of the corresponding genes. Single cis eQTLs explain 0.33–0.53 of variance in transcripts on average, whereas single trans eQTLs only explain 0.02–0.07. The top cis eQTLs tend to explain more variance in the corresponding gene when its hgeneExp2 is greater. Taking body mass index (BMI) as a case study, we cross-linked cis/trans eQTLs with both GWAS SNPs and differentially expressed genes for BMI. We discovered that BMI GWAS SNPs in 16p11.2 (e.g., rs7359397) are associated with several BMI differentially expressed genes in a cis manner (e.g. SULT1A1, SPNS1, and TUFM). These BMI signature genes explain a much larger proportion of variance in BMI than do the GWAS SNPs. Our results shed light the impact of eQTLs on the heritability of the human whole blood transcriptome and its relations to phenotype differences. PMID:25585846

  3. Potent Innate Immune Response to Pathogenic Leptospira in Human Whole Blood

    PubMed Central

    Hartskeerl, Rudy A.; van Gorp, Eric C. M.; Schuller, Simone; Monahan, Avril M.; Nally, Jarlath E.; van der Poll, Tom; van 't Veer, Cornelis

    2011-01-01

    Background Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. The bacteria enter the human body via abraded skin or mucous membranes and may disseminate throughout. In general the clinical picture is mild but some patients develop rapidly progressive, severe disease with a high case fatality rate. Not much is known about the innate immune response to leptospires during haematogenous dissemination. Previous work showed that a human THP-1 cell line recognized heat-killed leptospires and leptospiral LPS through TLR2 instead of TLR4. The LPS of virulent leptospires displayed a lower potency to trigger TNF production by THP-1 cells compared to LPS of non-virulent leptospires. Methodology/Principal Findings We investigated the host response and killing of virulent and non-virulent Leptospira of different serovars by human THP-1 cells, human PBMC's and human whole blood. Virulence of each leptospiral strain was tested in a well accepted standard guinea pig model. Virulent leptospires displayed complement resistance in human serum and whole blood while in-vitro attenuated non-virulent leptospires were rapidly killed in a complement dependent manner. In vitro stimulation of THP-1 and PBMC's with heat-killed and living leptospires showed differential serovar and cell type dependence of cytokine induction. However, at low, physiological, leptospiral dose, living virulent complement resistant strains were consistently more potent in whole blood stimulations than the corresponding non-virulent complement sensitive strains. At higher dose living virulent and non-virulent leptospires were equipotent in whole blood. Inhibition of different TLRs indicated that both TLR2 and TLR4 as well as TLR5 play a role in the whole blood cytokine response to living leptospires. Conclusions/Significance Thus, in a minimally altered system as human whole blood, highly virulent Leptospira are potent inducers of the cytokine response. PMID:21483834

  4. Egg beater as centrifuge: isolating human blood plasma from whole blood in resource-poor settings.

    PubMed

    Wong, Amy P; Gupta, Malancha; Shevkoplyas, Sergey S; Whitesides, George M

    2008-12-01

    This paper demonstrates that a hand-powered egg beater can be modified to serve as a centrifuge for separating plasma from human whole blood. Immunoassays used to diagnose infectious diseases often require plasma from whole blood, and obtaining plasma typically requires electrically-powered centrifuges, which are not widely available in resource-limited settings. Human whole blood was loaded into polyethylene (PE) tubing, and the tubing was attached to the paddle of an egg beater. Spinning the paddle pelleted the blood cells to the distal end of the PE tubing; the plasma remained as the supernatant. A cholesterol assay (run on patterned paper) demonstrated the suitability of this plasma for use in diagnostic assays. The physics of the system was also analyzed as a guide for the selection of other rotating systems for use in centrifugation. Egg beaters, polyethylene tubing, and paper are readily available devices and supplies that can facilitate the use of point-of-care diagnostics at sites far from centralized laboratory facilities.

  5. Investigation of prothrombin time in human whole-blood samples with a quartz crystal biosensor.

    PubMed

    Müller, Lothar; Sinn, Stefan; Drechsel, Hartmut; Ziegler, Christiane; Wendel, Hans-Peter; Northoff, Hinnak; Gehring, Frank K

    2010-01-15

    Monitoring of blood coagulation and fibrinolysis is an important issue in treatment of patients with cardiovascular problems and in surgery when blood gets into contact with artificial surfaces. In this work a new method for measuring the coagulation time (prothrombin time, PT) of human whole-blood samples based on a quartz crystal microbalance (QCM) biosensor is presented. The 10 MHz sensors used in this work respond with a frequency shift to changes in viscosity during blood clot formation. For driving and for readout of the quartz, both a network analyzer and an oscillator circuit were utilized. The sensor surfaces were specifically coated with a thin polyethylene layer. We found that both frequency analysis methods are suitable to measure exact prothrombin times in a very good conformity with a mechanical coagulometer as a reference. The anticoagulant effect of heparin on the prothrombin time was exemplarily shown as well as the reverse effect of the heparin antagonist polybrene. The change of the viscoelastic properties during blood coagulation, reflected by the ratio of frequency and dissipation shifts, is discussed for different dilutions of the whole-blood samples. In conclusion, QCM is a distinguished biosensor technique to determine prothrombin time and to monitor heparin therapy in whole-blood samples. Due to the excellent potential of miniaturization and the availability of direct digital signals, the method is predestinated for incorporation and integration into other devices and is thus opening the field of application for inline coagulation diagnostic in extracorporeal blood circuits.

  6. Human whole-blood culture system for ex vivo characterization of designer-cell function.

    PubMed

    Schukur, Lina; Geering, Barbara; Fussenegger, Martin

    2016-03-01

    Encapsulated designer cells implanted into mice are currently used to validate the efficacy of therapeutic gene networks for the diagnosis and treatment of various human diseases in preclinical research. Because many human conditions cannot be adequately replicated by animal models, complementary and alternative procedures to test future treatment strategies are required. Here we describe a novel approach utilizing an ex vivo human whole-blood culture system to validate synthetic biology-inspired designer cell-based treatment strategies. The viability and functionality of transgenic mammalian designer cells co-cultured with primary human immune cells were characterized. We demonstrated that transgenic mammalian designer cells required adequate insulation from the human blood microenvironment to maintain viability and functionality. The biomaterial alginate-(poly-l-lysine)-alginate used to encapsulate the transgenic designer cells did neither affect the viability of primary granulocytes and lymphocytes nor the functionality of lymphocytes. Additionally, alginate-encapsulated transgenic designer cells remained responsive to the release of the pro-inflammatory cytokine tumor necrosis factor (TNF) from the whole-blood culture upon exposure to bacterial lipopolysaccharide (LPS). TNF diffused into the alginate capsules, bound to the specific TNF receptors on the transgenic designer cells' surface and triggered the expression of the reporter gene SEAP (human placental secreted alkaline phosphatase) that was rewired to the TNF-specific signaling cascade. Human whole-blood culture systems can therefore be considered as valuable complementary assays to animal models for the validation of synthetic circuits in genetically modified mammalian cells and may speed up preclinical research in a world of personalized medicine.

  7. 21 CFR 640.1 - Whole Blood.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Whole Blood. 640.1 Section 640.1 Food and Drugs... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.1 Whole Blood. The proper name of this product shall be Whole Blood. Whole Blood is defined as blood collected from human donors for...

  8. 21 CFR 640.1 - Whole Blood.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Whole Blood. 640.1 Section 640.1 Food and Drugs... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.1 Whole Blood. The proper name of this product shall be Whole Blood. Whole Blood is defined as blood collected from human donors for...

  9. 21 CFR 640.1 - Whole Blood.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Whole Blood. 640.1 Section 640.1 Food and Drugs... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.1 Whole Blood. The proper name of this product shall be Whole Blood. Whole Blood is defined as blood collected from human donors for...

  10. 21 CFR 640.1 - Whole Blood.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Whole Blood. 640.1 Section 640.1 Food and Drugs... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.1 Whole Blood. The proper name of this product shall be Whole Blood. Whole Blood is defined as blood collected from human donors for...

  11. 21 CFR 640.1 - Whole Blood.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Whole Blood. 640.1 Section 640.1 Food and Drugs... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.1 Whole Blood. The proper name of this product shall be Whole Blood. Whole Blood is defined as blood collected from human donors for...

  12. Extraction of human genomic DNA from whole blood using a magnetic microsphere method.

    PubMed

    Gong, Rui; Li, Shengying

    2014-01-01

    With the rapid development of molecular biology and the life sciences, magnetic extraction is a simple, automatic, and highly efficient method for separating biological molecules, performing immunoassays, and other applications. Human blood is an ideal source of human genomic DNA. Extracting genomic DNA by traditional methods is time-consuming, and phenol and chloroform are toxic reagents that endanger health. Therefore, it is necessary to find a more convenient and efficient method for obtaining human genomic DNA. In this study, we developed urea-formaldehyde resin magnetic microspheres and magnetic silica microspheres for extraction of human genomic DNA. First, a magnetic microsphere suspension was prepared and used to extract genomic DNA from fresh whole blood, frozen blood, dried blood, and trace blood. Second, DNA content and purity were measured by agarose electrophoresis and ultraviolet spectrophotometry. The human genomic DNA extracted from whole blood was then subjected to polymerase chain reaction analysis to further confirm its quality. The results of this study lay a good foundation for future research and development of a high-throughput and rapid extraction method for extracting genomic DNA from various types of blood samples.

  13. A new method of preparing monocyte suspensions from human whole blood.

    PubMed

    Stoll, H P; Krämer, S; Oberhausen, E

    1986-01-01

    A method of isolating monocytes from human whole blood is described. The technique is primarily based on simple centrifugation steps that follow Tylose-sedimentation as well as on the use of the new density gradient medium Nycodens. Counterflow centrifugation is not involved. The final monocyte suspension is free of platelets. The contaminating cells are predominantly lymphocytes. As a whole, the method is a modification of the Nycodens technique published by Boyum in 1983, which leads to a total elimination of platelet contamination in the final cell suspension.

  14. In vitro study of thimerosal reactions in human whole blood and plasma surrogate samples.

    PubMed

    Trümpler, Stefan; Meermann, Björn; Nowak, Sascha; Buscher, Wolfgang; Karst, Uwe; Sperling, Michael

    2014-04-01

    Because of its bactericidal and fungicidal properties, thimerosal is used as a preservative in drugs and vaccines and is thus deliberately injected into the human body. In aqueous environment, it decomposes into thiosalicylic acid and the ethylmercury cation. This organomercury fragment is a potent neurotoxin and is suspected to have similar toxicity and bioavailability like the methylmercury cation. In this work, human whole blood and physiological simulation solutions were incubated with thimerosal to investigate its behaviour and binding partners in the blood stream. Inductively coupled plasma with optical emission spectrometry (ICP-OES) was used for total mercury determination in different blood fractions, while liquid chromatography (LC) coupled to electrospray ionisation time-of-flight (ESI-TOF) and inductively coupled plasma-mass spectrometry (ICP-MS) provided information on the individual mercury species in plasma surrogate samples. Analogous behaviour of methylmercury and ethylmercury species in human blood was shown and an ethylmercury-glutathione adduct was identified.

  15. Stability of some atypical antipsychotics in human plasma, haemolysed whole blood, oral fluid, human serum and calf serum.

    PubMed

    Fisher, Danielle S; Partridge, Suzanne J; Handley, Simon A; Flanagan, Robert J

    2013-06-10

    Long-term stability data of atypical antipsychotics in different matrices are not widely available. The aim of this work was to assess the stability of amisulpride, aripiprazole and dehydroaripiprazole, clozapine and norclozapine, olanzapine, quetiapine, risperidone and 9-hydroxyrisperidone, and sulpiride in human EDTA plasma, heparinised haemolysed human whole blood, oral fluid, human serum, and newborn calf serum stored in tightly capped plastic containers under a range of conditions. Measurements were performed by LC-MS/MS. Analyte instability was defined as a deviation of 15% or greater from the expected concentration. All analytes were stable following 3 freeze-thaw cycles in human plasma, and were stable in this matrix for at least 5 days at ambient temperature (olanzapine, 3 days); 4 weeks at 2-8°C (olanzapine, 2 weeks), and 2 years at -20°C (except for dehydroaripiprazole, olanzapine, and quetiapine, 1 year). In human serum, aripiprazole, dehydroaripiprazole, norclozapine, olanzapine, quetiapine, risperidone, 9-hydroxyrisperidone, and sulpiride were unstable after 5 days at ambient temperature, 3 weeks at 2-8°C, and 9 months at -20°C. Olanzapine was unstable in whole blood and oral fluid under most conditions studied, although prior addition of ascorbic acid had a moderate stabilising effect. All other analytes were stable in whole blood and oral fluid for at least 2 days at ambient temperature, 1 week at 2-8°C, and 2 months at -20°C (clozapine and norclozapine, 1 month whole blood). These results confirm that plasma (EDTA anticoagulant) is the sample of choice for TDM of atypical antipsychotics. Delayed (more than 1 week) analysis of patient samples should be undertaken with caution, especially with serum and with haemolysed whole blood. With olanzapine, only plasma collected and stored appropriately is likely to give reliable quantitative results.

  16. Detoxification of mercury species--an in vitro study with antidotes in human whole blood.

    PubMed

    Trümpler, Stefan; Nowak, Sascha; Meermann, Björn; Wiesmüller, Gerhard A; Buscher, Wolfgang; Sperling, Michael; Karst, Uwe

    2009-11-01

    To investigate the effects of mercury species intoxication and to test the efficiency of different commonly applied antidotes, human whole blood and plasma surrogate samples were spiked with inorganic mercury (Hg2+) and methylmercury (MeHg+, CH3Hg+) prior to treatment with the antidotes 2,3-dimercaptopropan-1-ol (British Anti Lewisite), 2,3-dimercaptosuccinic acid (DMSA), and N-acetylcysteine (NAC). For mercury speciation analysis in these samples, liquid chromatography was coupled to either inductively coupled plasma mass spectrometry (ICP-MS) or electrospray ionisation time-of-flight mass spectrometry (ESI-TOF-MS). Adduct formation between mercury species and physiological thiols (cysteine and glutathione) was observed as well as the release of glutathione under treatment with the antidotes DMSA and NAC.

  17. The relationship between the iron isotopic composition of human whole blood and iron status parameters.

    PubMed

    Van Heghe, Lana; Delanghe, Joris; Van Vlierberghe, Hans; Vanhaecke, Frank

    2013-11-01

    As the iron status of an individual cannot be adequately assessed on the basis of the (total) Fe concentration in whole blood or serum, in medicine a number of parameters, such as the serum concentrations of ferritin, transferrin and soluble transferrin receptor and the transferrin saturation, are routinely determined instead. As previous research has shown that also the isotopic composition of Fe in blood and tissues is dependent on the metabolism, the present study assessed whether Fe isotopic composition in whole blood provides information as to an individual's iron status. Fe isotopic analysis of whole blood samples from a reference population (healthy volunteers) was carried out using multi-collector ICP-mass spectrometry (after chromatographic target element isolation) and the results obtained were investigated by statistical means as to their potential relation with the iron status parameters conventionally used in medicine. A low δ(56)Fe value was demonstrated to coincide with high iron status and a high δ(56)Fe value with low iron status, thus reflecting the response of the body to this iron status in terms of iron uptake, distribution between blood and stores and mobilization of storage iron. In a second phase, the iron isotopic composition in blood from patients treated for hemochromatosis type I and from patients with anemia of chronic disease (ACD) was determined. The results for hemochromatosis patients plotted with the values of low iron status, while those for ACD patients plotted with the values of high iron status. By taking a closer look at the aberrant iron metabolism that comes with these diseases, it can be seen that the patient samples confirm the conclusions drawn for the reference population. Patients with hemochromatosis type I have a strongly upregulated iron uptake, like healthy individuals with low iron status. The metabolism of patients suffering from ACD tries to remove iron from the circulation by downregulating the iron uptake

  18. Quantitative assessment of human whole blood RNA as a potential biomarker for infectious disease.

    PubMed

    Smith, Claire L; Dickinson, Paul; Forster, Thorsten; Khondoker, Mizanur; Craigon, Marie; Ross, Alan; Storm, Petter; Burgess, Stewart; Lacaze, Paul; Stenson, Benjamin J; Ghazal, Peter

    2007-12-01

    Infection remains a significant cause of morbidity and mortality especially in newborn infants. Analytical methods for diagnosing infection are severely limited in terms of sensitivity and specificity and require relatively large samples. It is proposed that stringent regulation of the human transcriptome affords a new molecular diagnostic approach based on measuring a highly specific systemic inflammatory response to infection, detectable at the RNA level. This proposition raises a number of as yet poorly characterised technical and biological variation issues that urgently need to be addressed. Here we report a quantitative assessment of methodological approaches for processing and extraction of RNA from small samples of infant whole blood and applying analysis of variation from biochip measurements. On the basis of testing and selection from a battery of assays we show that sufficient high quality RNA for analysis using multiplex array technology can be obtained from small neonatal samples. These findings formed the basis of implementing a set of robust clinical and experimental standard operating procedures for whole blood RNA samples from 58 infants. Modelling and analysis of variation between samples revealed significant sources of variation from the point of sample collection to processing and signal generation. These experiments further permitted power calculations to be run indicating the tractability and requirements of using changes in RNA expression profiles to detect different states between patient groups. Overall the results of our investigation provide an essential first step toward facilitating an alternative way for diagnosing infection from very small neonatal blood samples, providing methods and requirements for future chip-based studies.

  19. A Human In Vitro Whole Blood Assay to Predict the Systemic Cytokine Response to Therapeutic Oligonucleotides Including siRNA

    PubMed Central

    Schwickart, Anna; Putschli, Bastian; Renn, Marcel; Höller, Tobias; Barchet, Winfried

    2013-01-01

    Therapeutic oligonucleotides including siRNA and immunostimulatory ligands of Toll-like receptors (TLR) or RIG-I like helicases (RLH) are a promising novel class of drugs. They are in clinical development for a broad spectrum of applications, e.g. as adjuvants in vaccines and for the immunotherapy of cancer. Species-specific immune activation leading to cytokine release is characteristic for therapeutic oligonucleotides either as an unwanted side effect or intended pharmacology. Reliable in vitro tests designed for therapeutic oligonucleotides are therefore urgently needed in order to predict clinical efficacy and to prevent unexpected harmful effects in clinical development. To serve this purpose, we here established a human whole blood assay (WBA) that is fast and easy to perform. Its response to synthetic TLR ligands (R848: TLR7/8, LPS: TLR4) was on a comparable threshold to the more time consuming peripheral blood mononuclear cell (PBMC) based assay. By contrast, the type I IFN profile provoked by intravenous CpG-DNA (TLR9 ligand) in humans in vivo was more precisely replicated in the WBA than in stimulated PBMC. Since Heparin and EDTA, but not Hirudin, displaced oligonucleotides from their delivery agent, only Hirudin qualified as the anticoagulant to be used in the WBA. The Hirudin WBA exhibited a similar capacity as the PBMC assay to distinguish between TLR7-activating and modified non-stimulatory siRNA sequences. RNA-based immunoactivating TLR7/8- and RIG-I-ligands induced substantial amounts of IFN-α in the Hirudin-WBA dependent on delivery agent used. In conclusion, we present a human Hirudin WBA to determine therapeutic oligonucleotide-induced cytokine release during preclinical development that can readily be performed and offers a close reflection of human cytokine response in vivo. PMID:23940691

  20. A new method to measure air-borne pyrogens based on human whole blood cytokine response.

    PubMed

    Kindinger, Ilona; Daneshian, Mardas; Baur, Hans; Gabrio, Thomas; Hofmann, Andreas; Fennrich, Stefan; von Aulock, Sonja; Hartung, Thomas

    2005-03-01

    Air-borne microorganisms, as well as their fragments and components, are increasingly recognized to be associated with pulmonary diseases, e.g. organic dust toxic syndrome, humidifier lung, building-related illness, "Monday sickness." We have previously described and validated a new method for the detection of pyrogenic (fever-inducing) microbial contaminations in injectable drugs, based on the inflammatory reaction of human blood to pyrogens. We have now adapted this test to evaluate the total inflammatory capacity of air samples. Air was drawn onto PTFE membrane filters, which were incubated with human whole blood from healthy volunteers inside the collection device. Cytokine release was measured by ELISA. The test detects endotoxins and non-endotoxins, such as fungal spores, Gram-positive bacteria and their lipoteichoic acid moiety and pyrogenic dust particles with high sensitivity, thus reflecting the total inflammatory capacity of a sample. When air from different surroundings such as working environments and animal housing was assayed, the method yielded reproducible data which correlated with other parameters of microbial burden tested. We further developed a standard material for quantification and showed that this assay can be performed with cryopreserved as well as fresh blood. The method offers a test to measure the integral inflammatory capacity of air-borne microbial contaminations relevant to humans. It could thus be employed to assess air quality in different living and work environments.

  1. Whole Blood Human Neutrophil Trafficking in a Microfluidic Model of Infection and Inflammation

    PubMed Central

    Hamza, Bashar; Irimia, Daniel

    2015-01-01

    Appropriate inflammatory responses to wounds and infections require adequate numbers of neutrophils arriving at injury sites. Both insufficient and excessive neutrophil recruitment can be detrimental, favouring systemic spread of microbes or triggering severe tissue damage. Despite its importance in health and disease, the trafficking of neutrophils through tissues remains difficult to control and the mechanisms regulating it are insufficiently understood. These mechanisms are also complex and difficult to isolate using traditional in vivo models. Here we designed a microfluidic model of tissue infection/inflammation, in which human neutrophils emerge from a droplet-size samples of whole blood and display bi-directional traffic between this and micro-chambers containing chemoattractant and microbe-like particles. Two geometrical barriers restrict the entrance of red blood cells from the blood to the micro-chambers and simulate the mechanical function of the endothelial barrier separating the cells in blood from cells in tissues. We found that in the presence of chemoattractant, the number of neutrophils departing the chambers by retrotaxis is in dynamic equilibrium with the neutrophils recruited by chemotaxis. We also found that in the presence of microbe-like particles, the number of neutrophils trapped in the chambers is proportional to the number of particles. Together, the dynamic equilibrium between migration, reversed-migration and trapping processes determine the optimal number of neutrophils at a site. These neutrophils are continuously refreshed and responsive to the number of microbes. Further studies using this infection-inflammation-on-a-chip-model could help study the processes of inflammation resolution. The new in vitro experimental tools may also eventually help testing new therapeutic strategies to limit neutrophil accumulation in tissues during chronic inflammation, without increasing the risk for infections. PMID:25987163

  2. Comparison of human whole blood, plasma, and serum matrices for the determination of perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), and other fluorochemicals

    SciTech Connect

    Ehresman, David J.; Froehlich, John W.; Olsen, Geary W. . E-mail: gwolsen@mmm.com; Chang, Shu-Ching; Butenhoff, John L.

    2007-02-15

    Interest in human exposure to perfluorinated acids, including perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), perfluorooctanesulfonate (PFOS), and perfluorooctanoate (PFOA) has led to their measurement in whole blood, plasma and serum. Comparison of measurements in these different blood-based matrices, however, has not been rigorously investigated to allow for across-matrix comparisons. This research evaluated concentrations of PFBS, PFHS, PFOS, and PFOA in whole blood collected in heparin (lithium) and ethylenediamine tetraacetic acid (EDTA), plasma samples collected in heparin and EDTA, and serum (from whole blood allowed to clot). Blood samples were collected from 18 voluntary participants employed at 3M Company. Solid phase extraction methods were used for all analytical sample preparations, and analyses were completed using high-pressure liquid chromatography/tandem mass spectrometry methods. Serum concentrations ranged from: limit of quantitation (LOQ, 5 ng/mL) to 25 ng/mL for PFBS; LOQ (5 ng/mL) to 75 ng/mL for PFHS; LOQ (5 ng/mL) to 880 ng/mL for PFOS; and LOQ (5 or 10 ng/mL) to 7320 ng/mL for PFOA. Values less than the LOQ were not included in the statistical analyses of the mean of the ratios of individual values for the matrices. PFBS was not quantifiable in most samples. Serum to plasma ratios for PFHS, PFOS, and PFOA were 1:1 and this ratio was independent of the level of concentrations measured. Serum or plasma to whole blood ratios, regardless of the anticoagulant used, approximated 2:1. The difference between plasma and serum and whole blood corresponded to volume displacement by red blood cells, suggesting that the fluorochemicals are not found intracellularly or attached to the red blood cells.

  3. Comparison of human whole blood, plasma, and serum matrices for the determination of perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), and other fluorochemicals.

    PubMed

    Ehresman, David J; Froehlich, John W; Olsen, Geary W; Chang, Shu-Ching; Butenhoff, John L

    2007-02-01

    Interest in human exposure to perfluorinated acids, including perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), perfluorooctanesulfonate (PFOS), and perfluorooctanoate (PFOA) has led to their measurement in whole blood, plasma and serum. Comparison of measurements in these different blood-based matrices, however, has not been rigorously investigated to allow for across-matrix comparisons. This research evaluated concentrations of PFBS, PFHS, PFOS, and PFOA in whole blood collected in heparin (lithium) and ethylenediamine tetraacetic acid (EDTA), plasma samples collected in heparin and EDTA, and serum (from whole blood allowed to clot). Blood samples were collected from 18 voluntary participants employed at 3M Company. Solid phase extraction methods were used for all analytical sample preparations, and analyses were completed using high-pressure liquid chromatography/tandem mass spectrometry methods. Serum concentrations ranged from: limit of quantitation (LOQ, 5 ng/mL) to 25 ng/mL for PFBS; LOQ (5 ng/mL) to 75 ng/mL for PFHS; LOQ (5 ng/mL) to 880 ng/mL for PFOS; and LOQ (5 or 10 ng/mL) to 7320 ng/mL for PFOA. Values less than the LOQ were not included in the statistical analyses of the mean of the ratios of individual values for the matrices. PFBS was not quantifiable in most samples. Serum to plasma ratios for PFHS, PFOS, and PFOA were 1:1 and this ratio was independent of the level of concentrations measured. Serum or plasma to whole blood ratios, regardless of the anticoagulant used, approximated 2:1. The difference between plasma and serum and whole blood corresponded to volume displacement by red blood cells, suggesting that the fluorochemicals are not found intracellularly or attached to the red blood cells.

  4. Raman and SERS recognition of β-carotene and haemoglobin fingerprints in human whole blood.

    PubMed

    Casella, Michele; Lucotti, Andrea; Tommasini, Matteo; Bedoni, Marzia; Forvi, Elena; Gramatica, Furio; Zerbi, Giuseppe

    2011-09-01

    The present work reports on Raman and Surface Enhanced Raman Scattering (SERS) vibrational fingerprints of β-carotene and haemoglobin in fresh whole blood (i.e. right after blood test) with different laser excitations, i.e. visible (514 nm) and near-infrared (NIR, 785 nm). The use of colloidal silver nanoparticles significantly increases the Raman signal, thus providing a clear SERS spectrum of blood. The collected spectra have been examined and marker bands of β-carotene and of the haem prosthetic group of haemoglobin have been found. In particular, the fundamental features of β-carotene (514 nm excitation), blood proteins and haem molecules (785 nm excitation) were recognized and assigned. Moreover haemoglobin SERS signals can be identified and related with its oxygenation state (oxy-haemoglobin). The data reported show the prospects of Raman and SERS techniques to detect important bio-molecules in a whole blood sample with no pre-treatment.

  5. Continuous separation of serum from human whole blood within a microfluidic device

    NASA Astrophysics Data System (ADS)

    Davis, John; Inglis, David; Sturm, James; Austin, Robert

    2006-03-01

    We were able to demonstrate separation of red and white blood cells from their native blood plasma, using a technique known as deterministic lateral displacement. The device takes advantage of asymmetric bifurcation of laminar flow around obstacles. This asymmetry creates a size dependent deterministic path through the device. All components of a given size follow equivalent migration paths, leading to high resolution. A subsequent version of the device will focus on the removal of platelets from whole blood. Samples will be extracted from the microfluidic device and analyzed by conventional flow cytometry.

  6. Potential Epigenetic Biomarkers of Obesity Related Insulin Resistance in Human Whole-blood.

    PubMed

    Day, Samantha E; Coletta, Richard L; Kim, Joon Young; Garcia, Luis A; Campbell, Latoya E; Benjamin, Tonya R; Roust, Lori R; De Filippis, Elena A; Mandarino, Lawrence J; Coletta, Dawn K

    2017-01-20

    Obesity can increase the risk of complex metabolic diseases, including insulin resistance. Moreover, obesity can be caused by environmental and genetic factors. However, the epigenetic mechanisms of obesity are not well defined. Therefore, the identification of novel epigenetic biomarkers of obesity allows for a more complete understanding of the disease and its underlying insulin resistance. The aim of our study was to identify DNA methylation changes in whole-blood that were strongly associated with obesity and insulin resistance. Whole-blood was obtained from lean (n = 10; BMI = 23.6 ± 0.7 kg/m(2)) and obese (n = 10; BMI = 34.4 ± 1.3 kg/m(2)) participants in combination with euglycemic hyperinsulinemic clamps to assess insulin sensitivity. We performed reduced representation bisulfite sequencing on genomic DNA isolated from the blood. We identified 49 differentially methylated cytosines (DMCs; q<0.05) that were altered in obese compared to lean participants. We identified two sites (Chr.21:46,957,981 and Chr.21:46,957,915) in the 5' untranslated region of solute carrier family 19 member 1 (SLC19A1) with decreased in methylation in obese participants (lean 0.73 ± 0.11 vs. obese 0.09 ± 0.05; lean 0.68 ± 0.10 vs. obese 0.09 ± 0.05, respectively). These two DMCs identified by obesity were also significantly predicted by insulin sensitivity (r = 0.68, P = 0.003; r = 0.66; P = 0.004). In addition, we performed a differentially methylated region (DMR) analysis and demonstrated a decrease in methylation of Chr.21:46,957,915-46,958,001 in SLC19A1 of -34.9% (70.4% lean vs. 35.5% obese). The decrease in whole-blood SLC19A1 methylation in our obese participants was similar to the change observed in skeletal muscle (Chr.21:46,957,981, lean 0.70 ± 0.09 vs. obese 0.31 ± 0.11 and Chr.21:46,957,915, lean 0.72 ± 0.11 vs. obese 0.31 ± 0.13). Pyrosequencing analysis further demonstrated a decrease in methylation at Chr.21:46,957,915 in both whole-blood (lean 0.71 ± 0

  7. Comparison of Non-Human Primate and Human Whole Blood Tissue Gene Expression Profiles

    DTIC Science & Technology

    2005-03-01

    studies have used rhesus, chimpanzee, gorilla, or orangutan RNA, but to date no gene expression profiling studies are available that use AGM or cynomologus...previous work has been published using human genechips to study NHPs, particularly rhesus, chimpanzee, gorilla, and orangutan (Uddin et al., 2004; Kayo

  8. Leucocytes isolated from simply frozen whole blood can be used in human biomonitoring for DNA damage measurement with the comet assay.

    PubMed

    Akor-Dewu, Maryam B; El Yamani, Naouale; Bilyk, Olena; Holtung, Linda; Tjelle, Torunn E; Blomhoff, Rune; Collins, Andrew R

    2014-04-01

    Preservation of human blood cells for DNA damage analysis with the comet assay conventionally involves the isolation of mononuclear cells by centrifugation, suspension in freezing medium and slow freezing to -80 °C-a laborious process. A recent publication (Al-Salmani et al. Free Rad Biol Med 2011; 51: 719-725) describes a simple method in which small volumes of whole blood are frozen to -20 or -80 °C; on subsequent thawing, the comet assay is performed, with no indication of elevated DNA strand breakage resulting from the rapid freezing. However, leucocytes in whole blood (whether fresh or frozen) are abnormally resistant to damage by H2 O2 , and so a common test of antioxidant status (resistance to strand breakage by H2 O2 ) cannot be used. We have refined this method by separating the leucocytes from the thawed blood; we find that, after three washes, the cells respond normally to H2 O2 . In addition, we have measured specific endogenous base damage (oxidized purines) in the isolated leucocytes, using the enzyme formamidopyrimidine DNA glycosylase. In a study of blood samples from 10 subjects, H2 O2 sensitivity and endogenous damage-both reflecting the antioxidant status of the cells-correlated significantly. This modified approach to sample collection and storage is particularly applicable when the available volume of blood is limited and has great potential in biomonitoring and ecogenotoxicology studies where samples are obtained in the field or at sites remote from the testing laboratory.

  9. LC-MS/MS of some atypical antipsychotics in human plasma, serum, oral fluid and haemolysed whole blood.

    PubMed

    Fisher, Danielle S; Partridge, Suzanne J; Handley, Simon A; Couchman, Lewis; Morgan, Phillip E; Flanagan, Robert J

    2013-06-10

    Therapeutic drug monitoring (TDM) of atypical antipsychotics is common, but published methods often specify relatively complex sample preparation and analysis procedures. The aim of this work was to develop and validate a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of amisulpride, aripiprazole and dehydroaripiprazole, clozapine and norclozapine, olanzapine, quetiapine, risperidone and 9-hydroxyrisperidone, and sulpiride in small (200 μL) volumes of plasma or serum for TDM purposes. The applicability of the method as developed to haemolysed whole blood and to oral fluid was also investigated. Analytes and internal standards were extracted into butyl acetate:butanol (9+1, v/v) and a portion of the extract analysed by LC-MS/MS (100 mm × 2.1 mm i.d. Waters Spherisorb S5SCX; eluent: 50 mmol/L methanolic ammonium acetate, pH* 6.0; flow-rate 0.5 mL/min; positive ion APCI-SRM, two transitions per analyte). Assay calibration (human plasma, oral fluid, and haemolysed whole blood calibration solutions) was performed by plotting the ratio of the peak area of the analyte to that of the appropriate internal standard. Assay validation was as per FDA guidelines. Assay calibration was linear across the concentration ranges studied. Inter- and intra-assay precision and accuracy were within 10% for all analytes in human plasma. Similar results were obtained for oral fluid and haemolysed whole blood, except that aripiprazole and dehydroaripiprazole were within 15% accuracy at low concentration (15 μg/L) in oral fluid, and olanzapine inter-assay precision could not be assessed in these matrices due to day-by-day degradation of this analyte. Recoveries varied between 16% (sulpiride) and 107% (clozapine), and were reproducible as well as comparable between human plasma, human serum, calf serum and haemolysed whole blood. For oral fluid, recoveries were reproducible, but differed slightly from those in plasma suggesting the need for

  10. Hemocompatible control of sulfobetaine-grafted polypropylene fibrous membranes in human whole blood via plasma-induced surface zwitterionization.

    PubMed

    Chen, Sheng-Han; Chang, Yung; Lee, Kueir-Rarn; Wei, Ta-Chin; Higuchi, Akon; Ho, Feng-Ming; Tsou, Chia-Chun; Ho, Hsin-Tsung; Lai, Juin-Yih

    2012-12-21

    In this work, the hemocompatibility of zwitterionic polypropylene (PP) fibrous membranes with varying grafting coverage of poly(sulfobetaine methacrylate) (PSBMA) via plasma-induced surface polymerization was studied. Charge neutrality of PSBMA-grafted layers on PP membrane surfaces was controlled by the low-pressure and atmospheric plasma treatment in this study. The effects of grafting composition, surface hydrophilicity, and hydration capability on blood compatibility of the membranes were determined. Protein adsorption onto the different PSBMA-grafted PP membranes from human fibrinogen solutions was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Blood platelet adhesion and plasma clotting time measurements from a recalcified platelet-rich plasma solution were used to determine if platelet activation depends on the charge bias of the grafted PSBMA layer. The charge bias of PSBMA layer deviated from the electrical balance of positively and negatively charged moieties can be well-controlled via atmospheric plasma-induced interfacial zwitterionization and was further tested with human whole blood. The optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and keeps its original blood-inert property of antifouling, anticoagulant, and antithrmbogenic activities when it comes into contact with human blood. This work suggests that the hemocompatible nature of grafted PSBMA polymers by controlling grafting quality via atmospheric plasma treatment gives a great potential in the surface zwitterionization of hydrophobic membranes for use in human whole blood.

  11. Enhanced differentiation of human osteoblasts on Ti surfaces pre-treated with human whole blood.

    PubMed

    Kopf, Brigitte S; Schipanski, Angela; Rottmar, Markus; Berner, Simon; Maniura-Weber, Katharina

    2015-06-01

    Early and effective integration of a metal implant into bone tissue is of crucial importance for its long-term stability. While different material properties including surface roughness and wettability but also initial blood-implant surface interaction are known to influence this osseointegration, implications of the latter process are still poorly understood. In this study, early interaction between blood and the implant surface and how this affects the mechanism of osseointegration were investigated. For this, blood coagulation on a micro-roughened hydrophobic titanium (Ti) surface (SLA-H(phob)) and on a hydrophilic micro-roughened Ti surface with nanostructures (SLActive-H(phil)NS), as well as the effects of whole human blood pre-incubation of these two surfaces on the differentiation potential of primary human bone cells (HBC) was assessed. Interestingly, pre-incubation with blood resulted in a dense fibrin network over the entire surface on SLActive-H(phil)NS but only in single patches of fibrin and small isolated fibre complexes on SLA-H(phob). On SLActive-H(phil)NS, the number of HBCs attaching to the fibrin network was greatly increased and the cells displayed enhanced cell contact to the fibrin network. Notably, HBCs displayed increased expression of the osteogenic marker proteins alkaline phosphatase and collagen-I when cultivated on both surfaces upon blood pre-incubation. Additionally, blood pre-treatment promoted an earlier and enhanced mineralization of HBCs cultivated on SLActive-H(phil)NS compared to SLA-H(phob). The results presented in this study therefore suggest that blood pre-incubation of implant surfaces mimics a more physiological situation, eventually providing a more predictive in vitro model for the evaluation of novel bone implant surfaces.

  12. Multiple enzyme-doped thread-based microfluidic system for blood urea nitrogen and glucose detection in human whole blood

    PubMed Central

    Yang, Yu-An

    2015-01-01

    This research presents a multiple enzyme-doped thread-based microfluidic system for blood urea nitrogen (BUN) and glucose detection in human whole blood. A novel enzyme-doped thread coated with a thin polyvinylchloride (PVC) membrane is produced for on-site electrochemical detection of urea and glucose in whole blood. Multiple enzymes can be directly applied to the thread without delicate pretreatment or a surface modification process prior to sealing the thread with PVC membrane. Results indicate that the developed device exhibits a good linear dynamic range for detecting urea and glucose in concentrations from 0.1 mM–10.0 mM (R2 = 0.9850) and 0.1 mM–13.0 mM (R2 = 0.9668), which is suitable for adoption in detecting the concentrations of blood urea nitrogen (BUN, 1.78–7.12 mM) and glucose (3.89–6.11 mM) in serum. The detection result also shows that the developed thread-based microfluidic system can successfully separate and detect the ions, BUN, and glucose in blood. The calculated concentrations of BUN and glucose ante cibum (glucose before meal) in the whole blood sample are 3.98 mM and 4.94 mM, respectively. The developed thread-based microfluidic system provides a simple yet high performance for clinical diagnostics. PMID:25825613

  13. Development of a Modular Assay for Detailed Immunophenotyping of Peripheral Human Whole Blood Samples by Multicolor Flow Cytometry

    PubMed Central

    Rühle, Paul F.; Fietkau, Rainer; Gaipl, Udo S.; Frey, Benjamin

    2016-01-01

    The monitoring of immune cells gained great significance in prognosis and prediction of therapy responses. For analyzing blood samples, the multicolor flow cytometry has become the method of choice as it combines high specificity on single cell level with multiple parameters and high throughput. Here, we present a modular assay for the detailed immunophenotyping of blood (DIoB) that was optimized for an easy and direct application in whole blood samples. The DIoB assay characterizes 34 immune cell subsets that circulate the peripheral blood including all major immune cells such as T cells, B cells, natural killer (NK) cells, monocytes, dendritic cells (DCs), neutrophils, eosinophils, and basophils. In addition, it evaluates their functional state and a few non-leukocytes that also have been associated with the outcome of cancer therapy. This DIoB assay allows a longitudinal and close-meshed monitoring of a detailed immune status in patients requiring only 2.0 mL of peripheral blood and it is not restricted to peripheral blood mononuclear cells. It is currently applied for the immune monitoring of patients with glioblastoma multiforme (IMMO-GLIO-01 trial, NCT02022384), pancreatic cancer (CONKO-007 trial, NCT01827553), and head and neck cancer (DIREKHT trial, NCT02528955) and might pave the way for immune biomarker identification for prediction and prognosis of therapy outcome. PMID:27529227

  14. Human genomic DNA isolation from whole blood using a simple microfluidic system with silica- and polymer-based stationary phases.

    PubMed

    Günal, Gülçin; Kip, Çiğdem; Öğüt, Sevim Eda; Usta, Duygu Deniz; Şenlik, Erhan; Kibar, Güneş; Tuncel, Ali

    2017-05-01

    Monodisperse-porous silica microspheres 5.1μm in size with a bimodal pore-size distribution (including both mesoporous and macroporous compartments) were obtained using a newly developed staged-shape templated hydrolysis and condensation protocol. Synthesized silica microspheres and monodisperse-porous polymer-based microspheres with different functionalities, synthesized by staged-shape template polymerization, were comparatively tested as sorbents for human genomic DNA (hgDNA) isolation in a microfluidic system. Microcolumns with a permeability range of 1.8-8.5×10(-13)m(2) were fabricated by the slurry-packing of silica- or polymer-based microspheres. The monodisperse-porous silica microspheres showed the best performance in hgDNA isolation in an aqueous buffer medium; >2500ng of hgDNA was recovered with an isolation yield of about 50%, using an hgDNA feed concentration of 100ng/μL. Monodisperse-porous silica microspheres were also evaluated as a sorbent for genomic DNA isolation from human whole blood in the microfluidic system; 14ng of hgDNA was obtained from 10μL of whole blood lysate with an isolation yield of 64%. Based on these results, we conclude that monodisperse-porous silica microspheres with a bimodal pore size distribution are a promising sorbent for the isolation of hgDNA in larger amounts and with higher yields compared to the sorbents previously tried in similar microfluidic systems.

  15. Refractive index of human whole blood with different types in the visible and near-infrared ranges

    NASA Astrophysics Data System (ADS)

    Li, Hui; Lin, Lei; Xie, Shusen

    2000-06-01

    Knowledge of the optical properties of human whole blood has always been of great interest for medical applications. The aim of this study was to provide the dispersive relations of refractive index of human whole blood with different types in the visible and near-infrared ranges and other conditions. In order to overcome the scattering effect, we applied an unusual method based on total internal reflection. A focused light, a semicylindrical lens in contact with tissues and a linear CCD camera are used in the experimental apparatus. The critical angle and therefore the refractive index can be obtained from the spacial distribution of internal reflective light. A monochromator is chosen as the light source, the chromatic dispersion curve of materials can be determined directly and quickly. A set of values has been presented that relates the refractive index to wavelength and types of whole, undiluted blood. Our results suggest that the refractive dispersions be almost the same in the visible and near-infrared ranges no matter which blood type it belongs to. In addition, the relationship can be described by Cauchy's formula.

  16. Cellular Defense System Gene Expression Profiling of Human Whole Blood: Opportunities to Predict Health Benefits in Response to Diet12

    PubMed Central

    Drew, Janice E.

    2012-01-01

    Diet is a critical factor in the maintenance of human cellular defense systems, immunity, inflammation, redox regulation, metabolism, and DNA repair that ensure optimal health and reduce disease risk. Assessment of dietary modulation of cellular defense systems in humans has been limited due to difficulties in accessing target tissues. Notably, peripheral blood gene expression profiles associated with nonhematologic disease are detectable. Coupled with recent innovations in gene expression technologies, gene expression profiling of human blood to determine predictive markers associated with health status and dietary modulation is now a feasible prospect for nutrition scientists. This review focuses on cellular defense system gene expression profiling of human whole blood and the opportunities this presents, using recent technological advances, to predict health status and benefits conferred by diet. PMID:22797985

  17. A whole blood in vitro cytokine release assay with aqueous monoclonal antibody presentation for the prediction of therapeutic protein induced cytokine release syndrome in humans.

    PubMed

    Wolf, Babette; Morgan, Hannah; Krieg, Jennifer; Gani, Zaahira; Milicov, Adriana; Warncke, Max; Brennan, Frank; Jones, Stewart; Sims, Jennifer; Kiessling, Andrea

    2012-12-01

    The administration of several monoclonal antibodies (mAbs) to humans has been associated with acute adverse events characterized by clinically significant release of cytokines in the blood. The limited predictive value of toxicology species in this field has triggered intensive research to establish human in vitro assays using peripheral blood mononuclear cells or blood to predict cytokine release in humans. A thorough characterization of these assays is required to understand their predictive value for hazard identification and risk assessment in an optimal manner, and to highlight potential limitations of individual assay formats. We have characterized a whole human blood cytokine release assay with only minimal dilution by the test antibodies (95% v/v blood) in aqueous presentation format, an assay which has so far received less attention in the scientific world with respect to the evaluation of its suitability to predict cytokine release in humans. This format was compared with a human PBMC assay with immobilized mAbs presentation already well-characterized by others. Cytokine secretion into plasma or cell culture supernatants after 24h incubation with the test mAbs (anti-CD28 superagonist TGN1412-like material (TGN1412L), another anti-CD28 superagonistic mAb (ANC28.1), a T-cell depleting mAb (Orthoclone™), and a TGN1412 isotype-matched control (Tysabri™) not associated with clinically-relevant cytokine release) was detected by a multiplex assay based on electrochemiluminescent excitation. We provide proof that this whole blood assay is a suitable new method for hazard identification of safety-relevant cytokine release in the clinic based on its ability to detect the typical cytokine signatures found in humans for the tested mAbs and on a markedly lower assay background and cytokine release with the isotype-matched control mAb Tysabri™ - a clear advantage over the PBMC assay. Importantly, quantitative and qualitative differences in the relative cytokine

  18. Evaluating genomic DNA extraction methods from human whole blood using endpoint and real-time PCR assays.

    PubMed

    Koshy, Linda; Anju, A L; Harikrishnan, S; Kutty, V R; Jissa, V T; Kurikesu, Irin; Jayachandran, Parvathy; Jayakumaran Nair, A; Gangaprasad, A; Nair, G M; Sudhakaran, P R

    2017-02-01

    The extraction of genomic DNA is the crucial first step in large-scale epidemiological studies. Though there are many popular DNA isolation methods from human whole blood, only a few reports have compared their efficiencies using both end-point and real-time PCR assays. Genomic DNA was extracted from coronary artery disease patients using solution-based conventional protocols such as the phenol-chloroform/proteinase-K method and a non-phenolic non-enzymatic Rapid-Method, which were evaluated and compared vis-a-vis a commercially available silica column-based Blood DNA isolation kit. The appropriate method for efficiently extracting relatively pure DNA was assessed based on the total DNA yield, concentration, purity ratios (A260/A280 and A260/A230), spectral profile and agarose gel electrophoresis analysis. The quality of the isolated DNA was further analysed for PCR inhibition using a murine specific ATP1A3 qPCR assay and mtDNA/Y-chromosome ratio determination assay. The suitability of the extracted DNA for downstream applications such as end-point SNP genotyping, was tested using PCR-RFLP analysis of the AGTR1-1166A>C variant, a mirSNP having pharmacogenetic relevance in cardiovascular diseases. Compared to the traditional phenol-chloroform/proteinase-K method, our results indicated the Rapid-Method to be a more suitable protocol for genomic DNA extraction from human whole blood in terms of DNA quantity, quality, safety, processing time and cost. The Rapid-Method, which is based on a simple salting-out procedure, is not only safe and cost-effective, but also has the added advantage of being scaled up to process variable sample volumes, thus enabling it to be applied in large-scale epidemiological studies.

  19. EFFECTS OF STORAGE, RNA EXTRACTION, GENECHIP TYPE, AND DONOR SEX ON GENE EXPRESSION PROFILING OF HUMAN WHOLE BLOOD

    EPA Science Inventory

    Background: Gene expression profiling of whole blood may be useful for monitoring toxicological exposure and for diagnosis and monitoring of various diseases. Several methods are available that can be used to transport, store, and extract RNA from whole blood, but it is not clear...

  20. Whole blood coagulation analyzers.

    PubMed

    1997-08-01

    Whole blood Coagulation analyzers (WBCAs) are widely used point-of-care (POC) testing devices found primarily in cardiothoracic surgical suites and cardia catheterization laboratories. Most of these devices can perform a number of coagulation tests that provide information about a patient's blood clotting status. Clinicians use the results of the WBCA tests, which are available minutes after applying a blood sample, primarily to monitor the effectiveness of heparin therapy--an anticoagulation therapy used during cardiopulmonary bypass (CPB) surgery, angioplasty, hemodialysis, and other clinical procedures. In this study we evaluated five WBCAs from four suppliers. Our testing focused on the applications for which WBCAs are primarily used: Monitoring moderate to high heparin levels, as would be required, for example, during CPB are angioplasty. For this function, WCBAs are typically used to perform an activated clotting time (ACT) test or, as one supplier refers to its test, a heparin management test (HMT). All models included in this study offered an ACT test or an HMT. Monitoring low heparin levels, as would be required, for example,during hemodialysis. For this function, WBCAs would normally be used to perform either a low-range ACT (LACT) test or a whole blood activated partial thromboplastin time (WBAPTT) test. Most of the evaluated units could perform at least one of these tests; one unit did not offer either test and was therefore not rated for this application. We rated and ranked each evaluated model separately for each of these two applications. In addition, we provided a combined rating and ranking that considers the units' appropriateness for performing both application. We based our conclusions on a unit's performance and humans factor design, as determined by our testing, and on its five-year life-cycle cost, as determined by our net present value (NPV) analysis. While we rated all evaluated units acceptable for each appropriate category, we did

  1. Monitoring of glucose, salt and pure water in human whole blood: An in vitro study.

    PubMed

    Imran, Muhammad; Ullah, Hafeez; Akhtar, Munir; Sial, Muhammad Aslam; Ahmed, Ejaz; Durr-E-Sabeeh; Ahmad, Mukhtar; Hussain, Fayyaz

    2016-07-01

    Designing and implementation of non-invasive methods for glucose monitoring in blood is main focus of biomedical scientists to provide a relief from skin puncturing of diabete patient. The objective of this research work is to investigate the shape deformations and the aggregation of red blood cells (RBCs) in the human blood after addition of three different analytes i) (0mM-400mM: Range) of glucose (C(6)H(12)O(6)), ii) (0mM-400mM: range) of pure salt (NaCl) and iii) (0mM- 350mM: range) of pure water (H(2)O). We have observed that the changes in the shape of individual cells from biconcave discs to spherical shapes and eventually the lysis of the cells at optimum concentration of glucose, salts and pure water. This demonstration also provides a base line to facilitate diabetes during partial diagnosis and monitoring of the glucose levels qualitatively both in research laboratories and clinical environment.

  2. Human whole-blood O2 affinity: effect of CO2.

    PubMed

    Kwant, G; Oeseburg, B; Zwart, A; Zijlstra, W G

    1988-06-01

    The proton Bohr factor (phi H = alpha log PO2/alpha pH), the carbamate Bohr factor (phi C = alpha log PO2/alpha log PCO2), the total Bohr factor (phi HC = d log PO2/dpH[base excess) and the CO2 buffer factor (d log PCO2/dpH) were determined in the blood of 12 healthy donors over the whole O2 saturation (SO2) range. All three Bohr factors proved to be dependent on SO2, although to a lesser extent than reported in some of the recent literature. At SO2 = 50% and 37 degrees C, we found phi H = -0.428 +/- 0.010 (SE), phi C = 0.054 +/- 0.006, and phi HC = -0.488 +/- 0.007. The values obtained for phi H, phi C, and d log PCO2/dpH were used to calculate phi HC. Calculated and measured values of phi HC proved to be in good agreement. In an additional series of 12 specimens of human blood we determined the influence of PCO2 on phi H and the influence of pH on phi C. At SO2 = 50%, phi H varied from -0.49 +/- 0.009 at PCO2 = 15 Torr to -0.31 +/- 0.010 at PCO2 = 105 Torr and phi C from 0.157 +/- 0.015 at pH = 7.80 to 0.006 +/- 0.009 at pH = 7.00. When on the basis of these data a second-order term is taken into account, a still slightly better agreement between measured and calculated values of phi HC can be attained.

  3. Quantitation of the enantiomers of tramadol and its three main metabolites in human whole blood using LC-MS/MS.

    PubMed

    Haage, Pernilla; Kronstrand, Robert; Carlsson, Björn; Kugelberg, Fredrik C; Josefsson, Martin

    2016-02-05

    The analgesic drug tramadol and its metabolites are chiral compounds, with the (+)- and (-)-enantiomers showing different pharmacological and toxicological effects. This novel enantioselective method, based on LC-MS/MS in reversed phase mode, enabled measurement of the parent compound and its three main metabolites O-desmethyltramadol, N-desmethyltramadol and N,O-didesmethyltramadol simultaneously. Whole blood samples of 0.5g were fortified with internal standards (tramadol-(13)C-D3 and O-desmethyl-cis-tramadol-D6) and extracted under basic conditions (pH 11) by liquid-liquid extraction. Chromatography was performed on a chiral alpha-1-acid glycoprotein (AGP) column preceded by an AGP guard column. The mobile phase consisted of 0.8% acetonitrile and 99.2% ammonium acetate (20mM, pH 7.2). A post-column infusion with 0.05% formic acid in acetonitrile was used to enhance sensitivity. Quantitation as well as enantiomeric ratio measurements were covered by quality controls. Validation parameters for all eight enantiomers included selectivity (high), matrix effects (no ion suppression/enhancement), calibration model (linear, weight 1/X(2), in the range of 0.25-250ng/g), limit of quantitation (0.125-0.50ng/g), repeatability (2-6%) and intermediate precision (2-7%), accuracy (83-114%), dilution integrity (98-115%), carry over (not exceeding 0.07%) and stability (stable in blood and extract). The method was applied to blood samples from a healthy volunteer administrated a single 100mg dose and to a case sample concerning an impaired driver, which confirmed its applicability in human pharmacokinetic studies as well as in toxicological and forensic investigations.

  4. Comparison of DNA extraction kits for detection of Burkholderia pseudomallei in spiked human whole blood using real-time PCR.

    PubMed

    Podnecky, Nicole L; Elrod, Mindy G; Newton, Bruce R; Dauphin, Leslie A; Shi, Jianrong; Chawalchitiporn, Sutthinan; Baggett, Henry C; Hoffmaster, Alex R; Gee, Jay E

    2013-01-01

    Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (C T ) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×10(4) colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.

  5. Validation of a method to quantify chromium, cadmium, manganese, nickel and lead in human whole blood, urine, saliva and hair samples by electrothermal atomic absorption spectrometry.

    PubMed

    Olmedo, P; Pla, A; Hernández, A F; López-Guarnido, O; Rodrigo, L; Gil, F

    2010-02-05

    For biological monitoring of heavy metal exposure in occupational toxicology, usually whole blood and urine samples are the most widely used and accepted matrix to assess internal xenobiotic exposure. Hair samples and saliva are also of interest in occupational and environmental health surveys but procedures for the determination of metals in saliva and hair are very scarce and to our knowledge there is no validation of a method to quantify Cr, Cd, Mn, Ni and Pb in four different human biological materials (whole blood, urine, saliva and axilary hair) by electrothermal atomization atomic absorption spectrometry (ETAAS). In the present study, quantification methods for the determination of Cr, Cd, Mn, Ni and Pb in whole blood, urine, saliva and axilary hair were validated according to the EU common standards. Pyrolisis and atomization temperatures have been determined. The main parameters evaluated were: detection and quantification limits, linearity range, repeatability, reproducibility, recovery and uncertainty. Accuracy of the methods was tested with the whole blood, urine and hair certified reference materials and recoveries of the spiked samples were acceptable ranged from 96.3 to 107.8%.

  6. Optical and spectroscopic properties of human whole blood and plasma with and without Y₂O₃ and Nd³⁺:Y₂O₃ nanoparticles.

    PubMed

    Barrera, Frederick J; Yust, Brian; Mimun, Lawrence C; Nash, Kelly L; Tsin, Andrew T; Sardar, Dhiraj K

    2013-11-01

    The optical properties of human whole blood and blood plasma with and without Y₂O₃ and Nd³⁺:Y₂O₃ nanoparticles are characterized in the near infrared region at 808 nm using a double integrating sphere technique. Using experimentally measured quantities of diffuse reflectance and diffuse transmittance, a computational analysis was conducted utilizing the Kubelka-Munk, the Inverse Adding Doubling, and Magic Light Kubelka-Munk and Monte Carlo Methods to determine optical properties of the absorption and scattering coefficients. Room temperature absorption and emission spectra were also acquired of Nd³⁺:Y₂O₃ nanoparticles elucidating their utility as biological markers. The emission spectra of Nd³⁺:Y₂O₃ were taken by exciting the nanoparticles before and after entering the whole blood sample. The emission from the ⁴F(3/2) → ⁴I(11/2) manifold transition of Nd³⁺:Y₂O₃ nanoparticles readily propagates through the blood sample at excitation of 808 nm and exhibits a shift in relative intensities of the peaks due to differences in scattering. At 808 nm, in both whole blood and plasma samples, a direct relationship was found with absorption coefficient and Y₂O₃ nanoparticle concentration. Results for the whole blood indicate a small inverse relationship with Y₂O₃ nanoparticle concentration and scattering coefficient and in contrast a direct relation for the plasma.

  7. Transcriptome Analysis of Neisseria meningitidis in Human Whole Blood and Mutagenesis Studies Identify Virulence Factors Involved in Blood Survival

    PubMed Central

    Del Tordello, Elena; Seib, Kate L.; Francois, Patrice; Rappuoli, Rino; Pizza, Mariagrazia; Serruto, Davide

    2011-01-01

    During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by

  8. Label-free cancer cell separation from human whole blood using inertial microfluidics at low shear stress.

    PubMed

    Lee, Myung Gwon; Shin, Joong Ho; Bae, Chae Yun; Choi, Sungyoung; Park, Je-Kyun

    2013-07-02

    We report a contraction-expansion array (CEA) microchannel device that performs label-free high-throughput separation of cancer cells from whole blood at low Reynolds number (Re). The CEA microfluidic device utilizes hydrodynamic field effect for cancer cell separation, two kinds of inertial effects: (1) inertial lift force and (2) Dean flow, which results in label-free size-based separation with high throughput. To avoid cell damages potentially caused by high shear stress in conventional inertial separation techniques, the CEA microfluidic device isolates the cells with low operational Re, maintaining high-throughput separation, using nondiluted whole blood samples (hematocrit ~45%). We characterized inertial particle migration and investigated the migration of blood cells and various cancer cells (MCF-7, SK-BR-3, and HCC70) in the CEA microchannel. The separation of cancer cells from whole blood was demonstrated with a cancer cell recovery rate of 99.1%, a blood cell rejection ratio of 88.9%, and a throughput of 1.1 × 10(8) cells/min. In addition, the blood cell rejection ratio was further improved to 97.3% by a two-step filtration process with two devices connected in series.

  9. Antithrombin III, but not C1 esterase inhibitor reduces inflammatory response in lipopolysaccharide-stimulated human monocytes in an ex-vivo whole blood setting.

    PubMed

    Kellner, Patrick; Nestler, Frank; Leimert, Anja; Bucher, Michael; Czeslick, Elke; Sablotzki, Armin; Raspè, Christoph

    2014-12-01

    In order to examine the immunomodulatory effects of antithrombin III (AT-III) and C1 esterase inhibitor (C1-INH) in human monocytes, we investigated the intracellular expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in an ex-vivo laboratory study in a whole blood setting. Heparinized whole blood samples from 23 healthy male and female volunteers (mean age: 27±7years) were pre-incubated with clinically relevant concentrations of AT-III (n=11) and C1-INH (n=12), then stimulated with 0.2 ng/mL lipopolysaccharide (LPS) for 3h. After phenotyping CD14⁺ monocytes, intracellular expression of IL-6, IL-8, and TNF-α was assessed using flow cytometry. In addition, 12 whole blood samples (AT-III and C1-INH, n=6 each) were examined using hirudin for anticoagulation; all samples were processed in the same way. To exclude cytotoxicity effects, 7-amino-actinomycin D and Nonidet P40 staining were used to investigate probes. This study is the first to demonstrate the influence of C1-INH and AT-III on the monocytic inflammatory response in a whole blood setting, which mimics the optimal physiological setting. Cells treated with AT-III exhibited significant downregulation of the proportion of gated CD14⁺ monocytes for IL-6 and IL-8, in a dose-dependent manner; downregulation for TNF-α did not reach statistical significance. There were no significant effects on mean fluorescence intensity (MFI). In contrast, C1-INH did not significantly reduce the proportion of gated CD14⁺ monocytes or the MFI regarding IL-6, TNF-α, and IL-8. When using hirudin for anticoagulation, no difference in the anti-inflammatory properties of AT-III and C1-INH in monocytes occurs. Taken together, in contrast to TNF-α, IL-6 and IL-8 were significantly downregulated in monocytes in an ex-vivo setting of human whole blood when treated with AT-III. This finding implicates monocytes as an important point of action regarding the anti-inflammatory properties of AT-III in sepsis. C1

  10. Glutamine and alanine-induced differential expression of intracellular IL-6, IL-8, and TNF-α in LPS-stimulated monocytes in human whole-blood.

    PubMed

    Raspé, C; Czeslick, E; Weimann, A; Schinke, C; Leimert, A; Kellner, P; Simm, A; Bucher, M; Sablotzki, A

    2013-04-01

    To investigate the effects of the commonly-used immunomodulators l-glutamine, l-alanine, and the combination of both l-alanyl-l-glutamine (Dipeptamin(®)) on intracellular expression of IL-6, IL-8, and TNF-α during endotoxemia, lipopolysaccharide (LPS)-stimulated human monocytes in a whole blood system were investigated by flow cytometry. Whole blood of twenty-seven healthy volunteers was stimulated with LPS and incubated with three different amino acid solutions (1. l-glutamine, 2. l-alanine, 3. l-alanyl-l-glutamine, each concentration 2 mM, 5 mM, incubation time 3 h). CD14(+) monocytes were phenotyped in whole-blood and intracellular expression of cytokines was assessed by flow cytometry. Our investigations showed for the first time in whole blood probes, imitating best physiologically present cellular interactions, that l-glutamine caused a dose-independent inhibitory effect on IL-6 and TNF-α production in human monocytes stimulated with LPS. However, l-alanine had contrary effects on IL-6 expression, significantly upregulating expression of IL-6 in LPS-treated monocytes. The impact of l-alanine on the expression of TNF-α was comparable with glutamine. Neither amino acid was able to affect IL-8 production in LPS-stimulated monocytes. The combination of both did not influence significantly IL-6 and IL-8 expression in monocytes during endotoxemia, however strongly reduced TNF-α production. For the regulation of TNF-α, l-glutamine, l-alanine and the combination of both show a congruent and exponentiated downregulating effect during endotoxemia, for the modulation of IL-6, l-glutamine and l-alanine featured opposite regulation leading to a canceling impact of each other when recombining both amino acids.

  11. Pipette tip solid-phase extraction and gas chromatography - mass spectrometry for the determination of methamphetamine and amphetamine in human whole blood.

    PubMed

    Hasegawa, Chika; Kumazawa, Takeshi; Lee, Xiao-Pen; Marumo, Akemi; Shinmen, Natsuko; Seno, Hiroshi; Sato, Keizo

    2007-09-01

    Methamphetamine and amphetamine were extracted from human whole blood samples using pipette tip solid-phase extraction (SPE) with MonoTip C(18) tips, on which C(18)-bonded monolithic silica gel was fixed. Human whole blood (0.1 mL) containing methamphetamine and amphetamine, with N-methylbenzylamine as an internal standard, was mixed with 0.4 mL of distilled water and 50 microL of 5 M sodium hydroxide solution. After centrifugation, the supernatant was extracted to the C(18) phase of the tip (pipette tip volume, 200 microL) by 25 repeated aspirating/dispensing cycles using a manual micropipettor. Analytes retained in the C(18) phase were eluted with methanol by five repeated aspirating/dispensing cycles. After derivatization with trifluoroacetic anhydride, analytes were measured by gas chromatography - mass spectrometry with selected ion monitoring in the positive-ion electron impact mode. Recoveries of methamphetamine and amphetamine spiked into whole blood were more than 87.6 and 81.7%, respectively. Regression equations for methamphetamine and amphetamine showed excellent linearity in the range of 0.5-100 ng/0.1 mL. The limits of detection for methamphetamine and amphetamine were 0.15 and 0.11 ng/0.1 mL, respectively. Intra- and interday coefficients of variation for both stimulants were not greater than 9.6 and 13.8%, respectively. The determination of methamphetamine and amphetamine in autopsy whole blood samples is presented, and was shown to validate the present methodology.

  12. 21 CFR 640.6 - Modifications of Whole Blood.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Modifications of Whole Blood. 640.6 Section 640.6...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.6 Modifications of Whole Blood. Upon approval by the Director, Center for Biologics Evaluation and Research, of a supplement...

  13. 21 CFR 640.6 - Modifications of Whole Blood.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Modifications of Whole Blood. 640.6 Section 640.6...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.6 Modifications of Whole Blood. Upon approval by the Director, Center for Biologics Evaluation and Research, of a supplement...

  14. Microwave-Accelerated Surface Plasmon-Coupled Directional Luminescence: application to fast and sensitive assays in buffer, human serum and whole blood.

    PubMed

    Aslan, Kadir; Malyn, Stuart N; Geddes, Chris D

    2007-05-31

    The applicability of a new technique, Microwave-Accelerated Surface Plasmon-Coupled Luminescence (MA-SPCL) for fast and sensitive bioassays in buffer, serum and whole blood using quantum dots as luminescence reporters is demonstrated. In this regard, a model bioassay based on the well-known interactions of biotin and streptavidin is used. Using MA-SPCL, the bioassay was kinetically completed within 1 min with the use of low power microwave heating as compared to the identical bioassay which took in excess of 30 min to reach >95% completion at room temperature, a 30-fold increase in assay kinetics. The luminescence emission from the quantum dots was coupled to surface plasmons of the gold film, enabling the detection of the luminescence emission in a highly directional fashion as compared to the normal isotropic emission, for enhanced sensitivity and detection. The combined effect of microwaves for faster assay kinetics, with surface plasmon-coupled luminescence for sensitive luminescence measurements, has also made possible the demonstration of the use of the MA-SPCL technique for assays run in complex media, such as human serum and whole blood, where the same assay could not be performed at room temperature due to the coagulation of blood. In the MA-SPCL assay run in serum and whole blood, the luminescence intensity from 33 nM quantum dots was 75% and 20% that of the luminescence intensity from the assay run in buffer, with a signal to noise ratio of 12.5 and 3, respectively.

  15. Significance of thrombin-receptors of thrombocytes for the interaction of heparins and low-molecular-weight heparin in human whole blood clotting.

    PubMed

    Harenberg, J; Schuler, M; Zimmermann, R; Heptner, W

    1988-01-01

    We describe in the present paper the results of the influence of normal and low-molecular-weight heparin on the interaction of human fibrinogen and thrombocytes in human whole blood cotting ex vivo. During the coagulation process sequential measurements of fibrinopeptide A reflect fibrin formation and determination of platelet factor 4 indicate activation of thrombocytes. The data show that low-molecular-weight heparin inhibits plasma thrombin generation in vivo for longer than normal heparin and it affects the fibrinogen platelet binding less. There is good evidence that a lonely factor Xa inhibition mediates this anticoagulant mechanism. Therefore, these data favor the hypothesis that antifactor Xa activity prevents indeed blood clotting.

  16. Simple Real-Time PCR and Amplicon Sequencing Method for Identification of Plasmodium Species in Human Whole Blood.

    PubMed

    Lefterova, Martina I; Budvytiene, Indre; Sandlund, Johanna; Färnert, Anna; Banaei, Niaz

    2015-07-01

    Malaria is the leading identifiable cause of fever in returning travelers. Accurate Plasmodium species identification has therapy implications for P. vivax and P. ovale, which have dormant liver stages requiring primaquine. Compared to microscopy, nucleic acid tests have improved specificity for species identification and higher sensitivity for mixed infections. Here, we describe a SYBR green-based real-time PCR assay for Plasmodium species identification from whole blood, which uses a panel of reactions to detect species-specific non-18S rRNA gene targets. A pan-Plasmodium 18S rRNA target is also amplified to allow species identification or confirmation by sequencing if necessary. An evaluation of assay accuracy, performed on 76 clinical samples (56 positives using thin smear microscopy as the reference method and 20 negatives), demonstrated clinical sensitivities of 95.2% for P. falciparum (20/21 positives detected) and 100% for the Plasmodium genus (52/52), P. vivax (20/20), P. ovale (9/9), and P. malariae (6/6). The sensitivity of the P. knowlesi-specific PCR was evaluated using spiked whole blood samples (100% [10/10 detected]). The specificities of the real-time PCR primers were 94.2% for P. vivax (49/52) and 100% for P. falciparum (51/51), P. ovale (62/62), P. malariae (69/69), and P. knowlesi (52/52). Thirty-three specimens were used to test species identification by sequencing the pan-Plasmodium 18S rRNA PCR product, with correct identification in all cases. The real-time PCR assay also identified two samples with mixed P. falciparum and P. ovale infection, which was confirmed by sequencing. The assay described here can be integrated into a malaria testing algorithm in low-prevalence areas, allowing definitive Plasmodium species identification shortly after malaria diagnosis by microscopy.

  17. Micropallet arrays for the capture, isolation and culture of circulating tumor cells from whole blood of mice engrafted with primary human pancreatic adenocarcinoma.

    PubMed

    Gach, Philip C; Attayek, Peter J; Whittlesey, Rebecca L; Yeh, Jen Jen; Allbritton, Nancy L

    2014-04-15

    Circulating tumor cells (CTCs) are important biomarkers of cancer progression and metastatic potential. The rarity of CTCs in peripheral blood has driven the development of technologies to isolate these tumor cells with high specificity; however, there are limited techniques available for isolating target CTCs following enumeration. A strategy is described to capture and isolate viable tumor cells from whole blood using an array of releasable microstructures termed micropallets. Specific capture of nucleated cells or cells expressing epithelial cell adhesion molecules (EpCAM) was achieved by functionalizing micropallet surfaces with either fibronectin, Matrigel or anti-EpCAM antibody. Surface grafting of poly(acrylic acid) followed by covalent binding of protein A/G enabled efficient capture of EpCAM antibody on the micropallet surface. MCF-7 cells, a human breast adenocarcinoma, were retained on the array surface with 90±8% efficiency when using an anti-EpCAM-coated array. To demonstrate the efficiency of tumor cell retention on micropallet arrays in the presence of blood, MCF-7 cells were mixed into whole blood and added to small arrays (71 mm(2)) coated with fibronectin, Matrigel or anti-EpCAM. These approaches achieved MCF-7 cell capture from ≤10 µL of whole blood with efficiencies greater than 85%. Furthermore, MCF-7 cells intermixed with 1 mL blood and loaded onto large arrays (7171 mm(2)) were captured with high efficiencies (≥97%), could be isolated from the array by a laser-based approach and were demonstrated to yield a high rate of colony formation (≥85%) after removal from the array. Clinical utility of this technology was shown through the capture, isolation and successful culture of CTCs from the blood of mice engrafted with primary human pancreatic tumors. Direct capture and isolation of living tumor cells from blood followed by analysis or culture will be a valuable tool for cancer cell characterization.

  18. Detection of total and A1c-glycosylated hemoglobin in human whole blood using sandwich immunoassays on polydimethylsiloxane-based antibody microarrays.

    PubMed

    Chen, Huang-Han; Wu, Chih-Hsing; Tsai, Mei-Ling; Huang, Yi-Jing; Chen, Shu-Hui

    2012-10-16

    The percentage of glycosylated hemoglobin A1c (%GHbA1c) in human whole blood indicates the average plasma glucose concentration over a prolonged period of time and is used to diagnose diabetes. However, detecting GHbA1c in the whole blood using immunoassays has limited detection sensitivity due to its low percentage in total hemoglobin (tHb) and interference from various glycan moieties in the sample. We have developed a sandwich immunoassay using an antibody microarray on a polydimethylsiloxane (PDMS) substrate modified with fluorinated compounds to detect tHb and glycosylated hemoglobin A1c (GHbA1c) in human whole blood without sample pretreatment. A polyclonal antibody against hemoglobin (Hb) immobilized on PDMS is used as a common capture probe to enrich all forms of Hb followed by detection via monoclonal anti-Hb and specific monoclonal anti-GHbA1c antibodies for tHb and GHbA1c detection, respectively. This method prevents the use of glycan binding molecules and dramatically reduces the background interference, yielding a detection limit of 3.58 ng/mL for tHb and 0.20 ng/mL for GHbA1c. The fluorinated modification on PDMS is superior to the glass substrate and eliminates the need for the blocking step which is required in commercial enzyme linked immunosorbent assay (ELISA) kits. Moreover, the detection sensitivity for GHbA1c is 4-5 orders of magnitude higher, but the required sample amount is 25 times less than the commercial method. On the basis of patient sample data, a good linear correlation between %GHbA1c values determined by our method and the certified high performance liquid chromatography (HPLC) standard method is shown with R(2) > 0.98, indicating the great promise of the developed method for clinical applications.

  19. Gene expression profiling of Gram-negative bacteria-induced inflammation in human whole blood: The role of complement and CD14-mediated innate immune response.

    PubMed

    Lau, Corinna; Olstad, Ole Kristoffer; Holden, Marit; Nygård, Ståle; Fure, Hilde; Lappegård, Knut Tore; Brekke, Ole-Lars; Espevik, Terje; Hovig, Eivind; Mollnes, Tom Eirik

    2015-09-01

    Non-sterile pathogen-induced sepsis and sterile inflammation like in trauma or ischemia-reperfusion injury may both coincide with the life threatening systemic inflammatory response syndrome and multi-organ failure. Consequently, there is an urgent need for specific biomarkers in order to distinguish sepsis from sterile conditions. The overall aim of this study was to uncover putative sepsis biomarkers and biomarker pathways, as well as to test the efficacy of combined inhibition of innate immunity key players complement and Toll-like receptor co-receptor CD14 as a possible therapeutic regimen for sepsis. We performed whole blood gene expression analyses using microarray in order to profile Gram-negative bacteria-induced inflammatory responses in an ex vivo human whole blood model. The experiments were performed in the presence or absence of inhibitors of complement proteins (C3 and CD88 (C5a receptor 1)) and CD14, alone or in combination. In addition, we used blood from a C5-deficient donor. Anti-coagulated whole blood was challenged with heat-inactivated Escherichia coli for 2 h, total RNA was isolated and microarray analyses were performed on the Affymetrix GeneChip Gene 1.0 ST Array platform. The initial experiments were performed in duplicates using blood from two healthy donors. C5-deficiency is very rare, and only one donor could be recruited. In order to increase statistical power, a technical replicate of the C5-deficient samples was run. Subsequently, log2-transformed intensities were processed by robust multichip analysis and filtered using a threshold of four. In total, 73 microarray chips were run and analyzed. The normalized and filtered raw data have been deposited in NCBI's Gene Expression Omnibus (GEO) and are accessible with GEO Series accession number GSE55537. Linear models for microarray data were applied to estimate fold changes between data sets and the respective multiple testing adjusted p-values (FDR q-values). The interpretation of the

  20. Ultrasound-assisted dispersive liquid-liquid microextraction for the determination of seven recreational drugs in human whole blood using gas chromatography-mass spectrometry.

    PubMed

    Lin, Zebin; Li, Jiaolun; Zhang, Xinyu; Qiu, Meihong; Huang, Zhibin; Rao, Yulan

    2017-03-01

    Recreational drugs have large impact on public health and security, and to monitor them is of urgent demand. In the present study, ultrasound-assisted dispersive liquid-liquid microextraction combined with the detection of gas chromatography-mass spectrometry was applied to the determination of seven common recreational drugs, including amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, meperidine, methadone and ketamine in 200μL of human whole blood. A series of factors which would affect the extraction efficiency were systematically investigated, including the nature and the volume of extraction and dispersing solvents, ultrasonication time, salting-out effect and pH value. The method consumed small amount of sample. The limits of detection and limits of quantification for each analyte were 10 and 40ng/mL, respectively, and the linearity was in the range of 0.04-25μg/mL (R(2) higher than 0.99). Good specificity, precision (1.5-8.2% for the intra-day study and 2.6-12.8% for the inter-day study), satisfactory accuracy (85.0-117.1%) and extraction recovery (77.0-92.4%) were obtained, which makes it a high performance method for the determination of recreational drugs in human whole blood samples.

  1. Simultaneous online SPE-HPLC-MS/MS analysis of docetaxel, temsirolimus and sirolimus in whole blood and human plasma.

    PubMed

    Navarrete, Alicia; Martínez-Alcázar, M Paz; Durán, Ignacio; Calvo, Emiliano; Valenzuela, Belén; Barbas, Coral; García, Antonia

    2013-03-15

    Docetaxel and temsirolimus are some of the most used drugs in a wide range of solid tumors. In preclinical studies, mTOR inhibitors such as temsirolimus have demonstrated synergistic cytotoxic effects with taxanes providing the rationale for combination studies. These anticancer agents exhibit a narrow therapeutic concentration range and due to their high inter- and intra-individual pharmacokinetic variability, therapeutic dose monitoring by highly sensitive methods as LC-MS/MS are important for clinical research. Therefore, the aim of this study was to develop and validate a sensitive, fast and convenient method for the simultaneous identification and quantification of docetaxel, temsirolimus and its main metabolite, sirolimus, using paclitaxel, another anticancer drug, as the internal standard. These analytes were quantified by an integrated online solid phase extraction-high performance liquid chromatography-tandem mass spectrometry (SPE-HPLC-MS/MS) system. Separation was performed on a Zorbax eclipse XDB-C8 (150mm×4.6mm, 5μm) column. The mass spectrometer tandem quadruple detector was equipped with jet stream electrospray ionization, monitored in multiple reactions monitoring (MRM) and operated in positive mode. A combination of protein precipitation with methanol/zinc sulphate (70:30) (v/v) and online SPE using a Zorbax eclipse plus C8 (12.5mm×4.6mm, 5μm) cartridge was used to extract the compounds. This method allows the use of the same reagents, sample treatment and analytical technique independently of whether the samples are whole blood or plasma. The method has been successfully validated and applied to real samples. It is a suitable method for dose adjustment and for evaluating potential drug interactions during combined treatments.

  2. [A new application for the human whole blood test: development of an assay to assess the health risk of air-borne microbial contaminations].

    PubMed

    Fennrich, S; Zucker, B; Hartung, T

    2001-01-01

    The pathogenic properties of environmental microorganisms as well as pyrogens as fragments of those bacteria (especially endotoxins) for humans is increasingly recognised. Various clinical syndromes are described after contact with airborne microbial contaminants via the respiratory tract: Sick-building-syndrome, humidifier lung (a form of hypersensitive pneumonitis), "Monday sickness" etc. Air-conditioning and ventilation systems intensify this problem as well as storage of compost within the household which represents a considerable source of airborne pollutants. In 1995 a new method for the detection of pyrogenic (fever-inducing) hazardous substances was described by Hartung and Wendel. This whole blood assay utilises the natural reaction of the immune system in order to detect a broad spectrum of pyrogens very sensitively in the relevant species. Injectable drugs are the main area of application in which this innovative test has already proven effective and is currently validated for inclusion into European Pharmacopoeia. In co-operation with the FU Berlin we could demonstrate in ventilation systems in animal stables that the whole blood pyrogen test can also detect airborne environmental microorganisms very sensitively. The filtration technique for collection of these germs is an established method for air-conditioning and ventilation systems. In co-operation with the FU Berlin (Institut für Tier-und Umwelthygiene) and the filter producer Sartorius this method is currently developed for the detection of airborne contaminations.

  3. A capillary gas chromatographic assay with nitrogen phosphorus detection for the quantification of topiramate in human plasma, urine and whole blood.

    PubMed

    Riffitts, J M; Gisclon, L G; Stubbs, R J; Palmer, M E

    1999-03-01

    An accurate and robust method involving liquid liquid extraction and capillary gas chromatographic (GC) assay with nitrogen phosphorus detection (NPD) was developed and validated for the quantitative determination of topiramate [2,3:4,5-bis-O-(-1-methylethylidene)-beta-D-fructopyranose sulfamate], Topamax, an anticonvulsant drug, in human plasma, urine, and whole blood. The galactopyranose analog of topiramate was used as the internal standard. A DB-5, fused silica capillary column (J&W Scientific, Folsom, CA) was used, yielding typical retention times of 4.95 min for topiramate and 5.32 min for the internal standard in human plasma. The assay involved organic extraction with methyl t-butyl ether (MTBE) from base, a back extraction into acid and a second extraction in MTBE. The organic solvent was evaporated, and the residue was redissolved and injected for analysis. The standard curve was validated from 0.5 to 50 microg/ml(-1) for human plasma and whole blood, and from 1.0 to 50 microg/ml(-1) for urine. Peak area ratios of drug to internal standard were determined and used to construct a standard curve. The resulting chromatograms showed no endogenous interfering peaks with the respective blank human fluids. Chromatograms corresponding to topiramate and the internal standard produced sharp peaks that were well resolved. This assay showed precision and accuracy of < or = 5%. Two minor human metabolites of topiramate did not interfere with the assay. This assay was successfully applied to determine the pharmacokinetics of topiramate during the development of this drug.

  4. Market study: Whole blood analyzer

    NASA Technical Reports Server (NTRS)

    1977-01-01

    A market survey was conducted to develop findings relative to the commercialization potential and key market factors of the whole blood analyzer which is being developed in conjunction with NASA's Space Shuttle Medical System.

  5. Chronic exposure to aluminum, nickel, thallium and uranium and their relationship with essential elements in human whole blood and blood serum.

    PubMed

    Zeneli, Lulzim; Sekovanić, Ankica; Daci, Nexhat

    2015-01-01

    This study aimed to evaluate the influence of exposure to aluminum, nickel, thallium and uranium on the metabolism of essential elements in humans, as well as the relationship between uranium, thallium, nickel, and aluminum and essential elements (Ca, Mg, Zn, Se, Mn, Co, Cr, and Mo) in the whole blood and blood serum of healthy men who were occupationally exposed. This study included 97 healthy men, 31-64 years age, including 70 workers in a thermo power plant and 27 control subjects. The results showed that chronic, moderate exposure of trace elements (Al, Ni, Tl, and U) lead to decreased serum chromium (SCr) and blood molybdenum levels (BMo), whereas by the results achieved in terms of correlations between non-essential and essential elements, non-essential elements such as uranium, thallium, nickel, and aluminum, despite their concentration within the reference values, are strongly competitive with essential elements in biochemical processes.

  6. Human transcriptome response to immunization with live-attenuated Venezuelan equine encephalitis virus vaccine (TC-83): Analysis of whole blood

    PubMed Central

    Erwin-Cohen, Rebecca A.; Porter, Aimee I.; Pittman, Phillip R.; Rossi, Cynthia A.; DaSilva, Luis

    2017-01-01

    ABSTRACT Venezuelan equine encephalitis virus (VEEV) is an important human and animal alphavirus pathogen transmitted by mosquitoes. The virus is endemic in Central and South America, but has also caused equine outbreaks in southwestern areas of the United States. In an effort to better understand the molecular mechanisms of the development of immunity to this important pathogen, we performed transcriptional analysis from whole, unfractionated human blood of patients who had been immunized with the live-attenuated vaccine strain of VEEV, TC-83. We compared changes in the transcriptome between naïve individuals who were mock vaccinated with saline to responses of individuals who received TC-83. Significant transcriptional changes were noted at days 2, 7, and 14 following vaccination. The top canonical pathways revealed at early and intermediate time points (days 2 and 7) included the involvement of the classic interferon response, interferon-response factors, activation of pattern recognition receptors, and engagement of the inflammasome. By day 14, the top canonical pathways included oxidative phosphorylation, the protein ubiquitination pathway, natural killer cell signaling, and B-cell development. Biomarkers were identified that differentiate between vaccinees and control subjects, at early, intermediate, and late stages of the development of immunity as well as markers which were common to all 3 stages following vaccination but distinct from the sham-vaccinated control subjects. The study represents a novel examination of molecular processes that lead to the development of immunity against VEEV in humans and which may be of value as diagnostic targets, to enhance modern vaccine design, or molecular correlates of protection. PMID:27870591

  7. [Mutagen influence with different mechanisms of action on DNA global methylation in human whole-blood lymphocytes in vitro].

    PubMed

    Smirnikhina, S A; Voronina, E S; Strelnikov, V V; Tanas, A S; Lavrov, A V

    2013-07-01

    Data that support the evidence of mutagens known to cause epigenetic abnormalities that could potentially result in genomic instability and the development of cancer rather than to modifications in the human genome at the gene and chromosomal levels only. The level of global methylation in human lymphocytes in vitro caused by exposure to two mutagens with different mechanisms of action, i.e., dioxidine and methyl methanesulphonate (MMS), was demonstrated in the present study. Global methylation was assessed by methyl-sensitive comet assay. An increase in the level of global methylation to 45.64% was revealed during culturing with dioxidine in a concentration of 0.01 mg/mL (p < 0.001), while the addition of dioxidine in a concentration of 0.1 mg/mL resulted in a decreased level of methylation up to 42.31% (p < 0.001). The addition of M MS in concentrations of 0.0025 and 0.01 mg/mL resulted in minor but significant modifications (p < 0.05) of the global methylation level ranged within natural variations in global methylation. Accordingly, the addition ofdioxidine in the concentration of 0.1 mg/mL might cause genomic instability and might be considered a potential carcinogen.

  8. A novel fully validated LC-MS/MS method for quantification of pyridoxal-5'-phosphate concentrations in samples of human whole blood.

    PubMed

    Ghassabian, Sussan; Griffiths, Lyn; Smith, Maree T

    2015-09-01

    Quantification of pyridoxal-5'-phosphate (PLP) in biological samples is challenging due to the presence of endogenous PLP in matrices used for preparation of calibrators and quality control samples (QCs). Hence, we have developed an LC-MS/MS method for accurate and precise measurement of the concentrations of PLP in samples (20μL) of human whole blood that addresses this issue by using a surrogate matrix and minimizing the matrix effect. We used a surrogate matrix comprising 2% bovine serum albumin (BSA) in phosphate buffer saline (PBS) for making calibrators, QCs and the concentrations were adjusted to include the endogenous PLP concentrations in the surrogate matrix according to the method of standard addition. PLP was separated from the other components of the sample matrix using protein precipitation with trichloroacetic acid 10% w/v. After centrifugation, supernatant were injected directly into the LC-MS/MS system. Calibration curves were linear and recovery was >92%. QCs were accurate, precise, stable for four freeze-thaw cycles, and following storage at room temperature for 17h or at -80°C for 3 months. There was no significant matrix effect using 9 different individual human blood samples. Our novel LC-MS/MS method has satisfied all of the criteria specified in the 2012 EMEA guideline on bioanalytical method validation.

  9. CD14 and Complement Crosstalk and Largely Mediate the Transcriptional Response to Escherichia coli in Human Whole Blood as Revealed by DNA Microarray

    PubMed Central

    Lau, Corinna; Nygård, Ståle; Fure, Hilde; Olstad, Ole Kristoffer; Holden, Marit; Lappegård, Knut Tore; Brekke, Ole-Lars; Espevik, Terje; Hovig, Eivind; Mollnes, Tom Eirik

    2015-01-01

    Systemic inflammation like in sepsis is still lacking specific diagnostic markers and effective therapeutics. The first line of defense against intruding pathogens and endogenous damage signals is pattern recognition by e.g., complement and Toll-like receptors (TLR). Combined inhibition of a key complement component (C3 and C5) and TLR-co-receptor CD14 has been shown to attenuate certain systemic inflammatory responses. Using DNA microarray and gene annotation analyses, we aimed to decipher the effect of combined inhibition of C3 and CD14 on the transcriptional response to bacterial challenge in human whole blood. Importantly, combined inhibition reversed the transcriptional changes of 70% of the 2335 genes which significantly responded to heat-inactivated Escherichia coli by on average 80%. Single inhibition was less efficient (p<0.001) but revealed a suppressive effect of C3 on 21% of the responding genes which was partially counteracted by CD14. Furthermore, CD14 dependency of the Escherichia coli-induced response was increased in C5-deficient compared to C5-sufficient blood. The observed crucial distinct and synergistic roles for complement and CD14 on the transcriptional level correspond to their broad impact on the inflammatory response in human blood, and their combined inhibition may become inevitable in the early treatment of acute systemic inflammation. PMID:25706641

  10. Factor H-binding protein is important for meningococcal survival in human whole blood and serum and in the presence of the antimicrobial peptide LL-37.

    PubMed

    Seib, K L; Serruto, D; Oriente, F; Delany, I; Adu-Bobie, J; Veggi, D; Aricò, B; Rappuoli, R; Pizza, M

    2009-01-01

    Factor H-binding protein (fHBP; GNA1870) is one of the antigens of the recombinant vaccine against serogroup B Neisseria meningitidis, which has been developed using reverse vaccinology and is the basis of a meningococcal B vaccine entering phase III clinical trials. Binding of factor H (fH), an inhibitor of the complement alternative pathway, to fHBP enables N. meningitidis to evade killing by the innate immune system. All fHBP null mutant strains analyzed were sensitive to killing in ex vivo human whole blood and serum models of meningococcal bacteremia with respect to the isogenic wild-type strains. The fHBP mutant strains of MC58 and BZ83 (high fHBP expressors) survived in human blood and serum for less than 60 min (decrease of >2 log(10) CFU), while NZ98/254 (intermediate fHBP expressor) and 67/00 (low fHBP expressor) showed decreases of >1 log(10) CFU after 60 to 120 min of incubation. In addition, fHBP is important for survival in the presence of the antimicrobial peptide LL-37 (decrease of >3 log(10) CFU after 2 h of incubation), most likely due to electrostatic interactions between fHBP and the cationic LL-37 molecule. Hence, the expression of fHBP by N. meningitidis strains is important for survival in human blood and human serum and in the presence of LL-37, even at low levels. The functional significance of fHBP in mediating resistance to the human immune response, in addition to its widespread distribution and its ability to induce bactericidal antibodies, indicates that it is an important component of the serogroup B meningococcal vaccine.

  11. Development and validation of a sensitive assay for the quantification of imatinib using LC/LC-MS/MS in human whole blood and cell culture

    PubMed Central

    Klawitter, Jelena; Zhang, Yan Ling; Klawitter, Jost; Anderson, Nora; Serkova, Natalie J.; Christians, Uwe

    2011-01-01

    We developed and validated a semi-automated LC/LC-MS/MS assay for the quantification of imatinib in human whole blood and leukemia cells. After protein precipitation, samples were injected into the HPLC system and trapped onto the enrichment column (flow 5 mL/min); extracts were back-flushed onto the analytical column. Ion transitions [M + H]+ of imatinib (m/z = 494.3 → 394.3) and its internal standard trazodone (372.5 → 176.3) were monitored. The range of reliable response was 0.03–75 ng/mL. The inter-day precisions were: 8.4% (0.03 ng/mL), 7.2% (0.1 ng/mL), 6.5% (1 ng/mL), 8.2% (10 ng/mL) and 4.3% (75 ng/mL) with no interference from ion suppression. Autosampler stability was 24 hs and samples were stable over three freeze–thaw cycles. This semi-automated method is simple with only one manual step, uses a commercially available internal standard, and has proven to be robust in larger studies. PMID:19517424

  12. Development, validation and clinical application of a LC-MS/MS method for the simultaneous quantification of hydroxychloroquine and its active metabolites in human whole blood.

    PubMed

    Soichot, Marion; Mégarbane, Bruno; Houzé, Pascal; Chevillard, Lucie; Fonsart, Julien; Baud, Frédéric J; Laprévote, Olivier; Bourgogne, Emmanuel

    2014-11-01

    A rapid, sensitive and specific method using liquid chromatography coupled to tandem mass spectrometry was developed for the simultaneous quantification of hydroxychloroquine (HCQ) and its three major metabolites in human whole blood. The assay, using a sample volume of 100μL, was linear in a dynamic 25-2000ng/mL range (R(2)>0.99) for all four compounds and suitable for the determination of elevated HCQ concentrations up to 20,000ng/mL, after appropriate sample dilution. Inter- and intra-assay precisions were <18.2% and accuracies were between 84% and 113% for any analyte. No matrix effects were observed. The assay was successfully applied to a blood sample obtained from one poisoned patient following a massive HCQ self-ingestion resulting in an estimated concentration of 19,500ng/mL on hospital admission. In this patient, HCQ metabolites were identified and quantified at 1123, 465 and 91ng/mL for monodesethylhydroxychloroquine, desethylchloroquine and bisdesethylchloroquine, respectively. Further investigations are still required to assess the usefulness of the simultaneous measurement of blood concentrations of HCQ and its three active metabolites for monitoring HCQ treatment and managing HCQ poisoning.

  13. Application of a new nanocarbonaceous sorbent in electromembrane surrounded solid phase microextraction for analysis of amphetamine and methamphetamine in human urine and whole blood.

    PubMed

    Rezazadeh, Maryam; Yamini, Yadollah; Seidi, Shahram

    2015-05-29

    Application of a new carbon-based sorbent was studied for the first time for extraction and quantification of amphetamine and methamphetamine as model analytes by means of electromembrane surrounded solid phase microextraction (EM-SPME). Since the basis of this microextraction method is adsorption of target analytes on the sorbent surface (after transferring across a supported liquid membrane) in an electrical field, the sorbent, which also performs the electrical potential, should have a conductive nature. On the other hand, using a synthesized fiber is a suitable solution to eliminate the interfering compounds existing in the fiber. To extract the model analytes from acidic sample solution through a thin layer of organic phase and into the aqueous acceptor phase and their final adsorption, 150V electrical potential was applied for 15min. Regardless of the high sample cleanup ability of the proposed method, which makes the analysis of complicated biological fluids possible, admissible extraction recoveries (9.0-18.8%) and suitable detection limits (less than 2.0ngmL(-1)) were obtained. Repeatability and reproducibility of the method were studied and intra- and inter-assay precisions were in the ranges of 2.0-7.3% and 7.5-12.5%, respectively. Coefficients of determination larger than 0.9964 were achieved by scrutinizing of the linearity up to 500ngmL(-1) and calibration curves were utilized for quantification of analytes of interest in human urine and whole blood samples.

  14. Simultaneous determination of ethanol's four types of non-oxidative metabolites in human whole blood by liquid chromatography tandem mass spectrometry.

    PubMed

    Zhang, Xinyu; Zheng, Feng; Lin, Zebin; Johansen, Sys Stybe; Yu, Tianfang; Liu, Yuming; Huang, Zhibin; Li, Jiaolun; Yan, Jie; Rao, Yulan

    2017-04-22

    The importance of ethanol non-oxidative metabolites as the specific biomarkers of alcohol consumption in clinical and forensic settings is increasingly acknowledged. Simultaneous determination of these metabolites can provide a wealth of information like drinking habit and history, but it was difficult to achieve because of their wide range of polarity. This work describes development and validation of a simple liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for 4 types of ethanol non-oxidative metabolites (ethyl glucuronide, ethyl sulfate, fatty acid ethyl esters and phosphatidylethanols) in 50 μL of human whole blood. Pretreatment method, column and MS conditions were optimized. For the first time, the four types of ethanol non-oxidative metabolites with enormous discrepancies of property were simultaneously extracted and analyzed in one run within 40 min. The limits of detections (LODs) were among 0.1-10 ng/mL, and good linearity was obtained. Deviations in precision and accuracy were all lower than 15% at three QC levels. This method was then applied to two forensic samples, resulting in information on drinking habits and drinking time which were very useful for the interpretation of the blood alcohol results.

  15. An integrated direct loop-mediated isothermal amplification microdevice incorporated with an immunochromatographic strip for bacteria detection in human whole blood and milk without a sample preparation step.

    PubMed

    Lee, Dohwan; Kim, Yong Tae; Lee, Jee Won; Kim, Do Hyun; Seo, Tae Seok

    2016-05-15

    We have developed an integrated direct loop-mediated isothermal amplification (Direct LAMP) microdevice incorporated with an immunochromatographic strip (ICS) to identify bacteria contaminated in real samples. The Direct LAMP is a novel isothermal DNA amplification technique which does not require thermal cycling steps as well as any sample preparation steps such as cell lysis and DNA extraction for amplifying specific target genes. In addition, the resultant amplicons were colorimetrically detected on the ICS, thereby enabling the entire genetic analysis process to be simplified. The two functional units (Direct LAMP and ICS) were integrated on a single device without use of the tedious and complicated microvalve and tubing systems. The utilization of a slidable plate allows us to manipulate the fluidic control in the microchannels manually and the sequential operation of the Direct LAMP and ICS detection could be performed by switching the slidable plate to each functional unit. Thus, the combination of the direct isothermal amplification without any sample preparation and thermal cycling steps, the ICS based amplicon detection by naked eyes, and the slidable plate to eliminate the microvalves in the integrated microdevice would be an ideal platform for point-of-care DNA diaganotics. On the integrated Direct LAMP-ICS microdevice, we could analyze Staphylococcus aureus (S. aureus) and Escherichia coli O157:H7 (E. coli O157:H7) contaminated in human whole blood or milk at a single-cell level within 1h.

  16. Blood cell oxidative stress precedes hemolysis in whole blood-liver slice co-cultures of rat, dog, and human tissues

    SciTech Connect

    Vickers, Alison E.M.; Sinclair, John R.; Fisher, Robyn L.; Morris, Stephen R.; Way, William

    2010-05-01

    A novel in vitro model to investigate time-dependent and concentration-dependent responses in blood cells and hemolytic events is studied for rat, dog, and human tissues. Whole blood is co-cultured with a precision-cut liver slice. Methimazole (MMI) was selected as a reference compound, since metabolism of its imidazole thione moiety is linked with hematologic disorders and hepatotoxicity. An oxidative stress response occurred in all three species, marked by a decline in blood GSH levels by 24 h that progressed, and preceded hemolysis, which occurred at high MMI concentrations in the presence of a liver slice with rat (>= 1000 muM at 48 h) and human tissues (>= 1000 muM at 48 h, >= 750 muM at 72 h) but not dog. Human blood-only cultures exhibited a decline of GSH levels but minimal to no hemolysis. The up-regulation of liver genes for heme degradation (Hmox1 and Prdx1), iron cellular transport (Slc40a1), and GSH synthesis and utilization (mGST1 and Gclc) were early markers of the oxidative stress response. The up-regulation of the Kupffer cell lectin Lgals3 gene expression indicated a response to damaged red blood cells, and Hp (haptoglobin) up-regulation is indicative of increased hemoglobin uptake. Up-regulation of liver IL-6 and IL-8 gene expression suggested an activation of an inflammatory response by liver endothelial cells. In summary, MMI exposure led to an oxidative stress response in blood cells, and an up-regulation of liver genes involved with oxidative stress and heme homeostasis, which was clearly separate and preceded frank hemolysis.

  17. Methanethiol metabolism in whole blood

    SciTech Connect

    Blom, H.J.; Tangerman, A.

    1988-06-01

    The metabolism of methanethiol in whole blood has been described. Incubation of carbon 14-labeled or sulfur 35-labeled gaseous methanethiol resulted in complete trapping of methanethiol by whole blood within 30 minutes. After trapping, both labels were found to be equally distributed over plasma and erythrocytes. Eighty to ninety percent of both labels could be extracted from erythrocytes incubated in saline solution. The chemical properties of the /sup 14/C and /sup 35/S labels in saline solution differed completely. The /sup 14/C label was not precipitated by BaCl/sub 2/, was moderately volatile, and could be extracted by either (pH 1). In contrast, the 35S label was precipitated by BaCl/sub 2/, was not volatile, and was not extracted by ether. It is concluded that the central carbon-sulfur bond of methanethiol is split by incubation with whole blood. Plasma components are not involved in this process. Most likely, methanethiol becomes largely oxidized by erythrocytes to formic acid and sulfite or sulfate. Only 10% of methanethiol became firmly bound to erythrocytes. One to two percent was transformed to protein--S--S--CH/sub 3/ and 1% to dimethyl sulfide by the enzyme thiol methyltransferase.

  18. A dried blood spots technique based LC-MS/MS method for the analysis of posaconazole in human whole blood samples.

    PubMed

    Reddy, Todime M; Tama, Cristina I; Hayes, Roger N

    2011-11-15

    A rugged and robust liquid chromatographic tandem mass spectrometric (LC-MS/MS) method utilizing dried blood spots (DBS) was developed and validated for the analysis of posaconazole in human whole blood. Posaconazole fortified blood samples were spotted (15 μL) onto Ahlstrom Alh-226 DBS cards and dried for at least 2h. Punched spots were then extracted by using a mixture of acetonitrile and water containing stable labeled internal standard (IS). Posaconazole and its IS were separated from endogenous matrix components on a Kinetex™ C18 column under gradient conditions with a mobile phase A consisting of 0.1% formic acid and a mobile phase B consisting of 0.1% formic acid in acetonitrile/methanol (70/30, v/v). The analyte and IS were detected using a Sciex API 4000 triple quadrupole LC-MS/MS system equipped with a TurboIonSpray™ source operated in the positive ion mode. The assay was linear over the concentration range of 5-5000 ng/mL. The inter-run accuracy and precision of the assay were -1.8% to 0.8% and 4.0% to 10.4%, respectively. Additional assessments unique to DBS were investigated including sample spot homogeneity, spot volume, and hematocrit. Blood spot homogeneity was maintained and accurate and precise quantitation results were obtained when using a blood spot volume of between 15 and 35 μL. Human blood samples with hematocrit values ranging between 25% and 41% gave acceptable quantitation results. The validation results indicate that the method is accurate, precise, sensitive, selective and reproducible.

  19. Separation of uncompromised whole blood mixtures for single source STR profiling using fluorescently-labeled human leukocyte antigen (HLA) probes and fluorescence activated cell sorting (FACS).

    PubMed

    Dean, Lee; Kwon, Ye Jin; Philpott, M Katherine; Stanciu, Cristina E; Seashols-Williams, Sarah J; Dawson Cruz, Tracey; Sturgill, Jamie; Ehrhardt, Christopher J

    2015-07-01

    Analysis of biological mixtures is a significant problem for forensic laboratories, particularly when the mixture contains only one cell type. Contributions from multiple individuals to biologic evidence can complicate DNA profile interpretation and often lead to a reduction in the probative value of DNA evidence or worse, its total loss. To address this, we have utilized an analytical technique that exploits the intrinsic immunological variation among individuals to physically separate cells from different sources in a mixture prior to DNA profiling. Specifically, we applied a fluorescently labeled antibody probe to selectively bind to one contributor in a mixture through allele-specific interactions with human leukocyte antigen (HLA) proteins that are expressed on the surfaces of most nucleated cells. Once the contributor's cells were bound to the probe, they were isolated from the mixture using fluorescence activated cell sorting (FACS)-a high throughput technique for separating cell populations based on their optical properties-and then subjected to STR analysis. We tested this approach on two-person and four-person whole blood mixtures where one contributor possessed an HLA allele (A*02) that was not shared by other contributors to the mixture. Results showed that hybridization of the mixture with a fluorescently-labeled antibody probe complimentary to the A*02 allele's protein product created a cell population with a distinct optical profile that could be easily differentiated from other cells in the mixture. After sorting the cells with FACS, genetic analysis showed that the STR profile of this cell population was consistent with that of the contributor who possessed the A*02 allele. Minor peaks from the A*02 negative contributor(s) were observed but could be easily distinguished from the profile generated from A*02 positive cells. Overall, this indicates that HLA antibody probes coupled to FACS may be an effective approach for generating STR profiles of

  20. A Single Meal Containing Raw, Crushed Garlic Influences Expression of Immunity- and Cancer-Related Genes in Whole Blood of Humans1234

    PubMed Central

    Charron, Craig S; Dawson, Harry D; Albaugh, George P; Solverson, Patrick M; Vinyard, Bryan T; Solano-Aguilar, Gloria I; Molokin, Aleksey; Novotny, Janet A

    2015-01-01

    Background: Preclinical and epidemiologic studies suggest that garlic intake is inversely associated with the progression of cancer and cardiovascular disease. Objective: We designed a study to probe the mechanisms of garlic action in humans. Methods: We conducted a randomized crossover feeding trial in which 17 volunteers consumed a garlic-containing meal (100 g white bread, 15 g butter, and 5 g raw, crushed garlic) or a garlic-free control meal (100 g white bread and 15 g butter) after 10 d of consuming a controlled, garlic-free diet. Blood was collected before and 3 h after test meal consumption for gene expression analysis in whole blood. Illumina BeadArray was used to screen for genes of interest, followed by real-time quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) on selected genes. To augment human study findings, Mono Mac 6 cells were treated with a purified garlic extract (0.5 μL/mL), and mRNA was measured by qRT-PCR at 0, 3, 6, and 24 h. Results: The following 7 genes were found to be upregulated by garlic intake: aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT), hypoxia-inducible factor 1α (HIF1A), proto-oncogene c-Jun (JUN), nuclear factor of activated T cells (NFAT) activating protein with immunoreceptor tyrosine-based activation motif 1 (NFAM1), oncostatin M (OSM), and V-rel avian reticuloendotheliosis viral oncogene homolog (REL). Fold-increases in mRNA transcripts ranged from 1.6 (HIF1A) to 3.0 (NFAM1) (P < 0.05). The mRNA levels of 5 of the 7 genes that were upregulated in the human trial were also upregulated in cell culture at 3 and 6 h: AHR, HIF1A, JUN, OSM, and REL. Fold-increases in mRNA transcripts in cell culture ranged from 1.7 (HIF1A) to 12.1 (JUN) (P < 0.01). OSM protein was measured by ELISA and was significantly higher than the control at 3, 6, and 24 h (24 h: 19.5 ± 1.4 and 74.8 ± 1.4 pg/mL for control and garlic, respectively). OSM is a pleiotropic cytokine that

  1. Carbon isotopes profiles of human whole blood, plasma, red blood cells, urine and feces for biological/biomedical 14C-accelerator mass spectrometry applications.

    PubMed

    Kim, Seung-Hyun; Chuang, Jennifer C; Kelly, Peter B; Clifford, Andrew J

    2011-05-01

    Radiocarbon ((14)C) is an ideal tracer for in vivo human ADME (absorption, distribution, metabolism, elimination) and PBPK (physiological-based pharmacokinetic) studies. Living plants peferentially incorporate atmospheric (14)CO(2) versus (13)CO(2) versus (12)CO(2), which result in unique signature. Furthermore, plants and the food chains they support also have unique carbon isotope signatures. Humans, at the top of the food chain, consequently acquire isotopic concentrations in the tissues and body fluids depending on their dietary habits. In preparation of ADME and PBPK studies, 12 healthy subjects were recruited. The human baseline (specific to each individual and their diet) total carbon (TC) and carbon isotope (13)C (δ(13)C) and (14)C (F(m)) were quantified in whole blood (WB), plasma, washed red blood cell (RBC), urine, and feces. TC (mg of C/100 μL) in WB, plasma, RBC, urine, and feces were 11.0, 4.37, 7.57, 0.53, and 1.90, respectively. TC in WB, RBC, and feces was higher in men over women, P < 0.05. Mean δ(13)C were ranked low to high as follows: feces < WB = plasma = RBC = urine, P < 0.0001. δ(13)C was not affected by gender. Our analytic method shifted δ(13)C by only ±1.0 ‰ ensuring our F(m) measurements were accurate and precise. Mean F(m) were ranked low to high as follows: plasma = urine < WB = RBC = feces, P < 0.05. F(m) in feces was higher for men over women, P < 0.05. Only in WB, (14)C levels (F(m)) and TC were correlated with one another (r = 0.746, P < 0.01). Considering the lag time to incorporate atmospheric (14)C into plant foods (vegetarian) and or then into animal foods (nonvegetarian), the measured F(m) of WB in our population (recruited April 2009) was 1.0468 ± 0.0022 (mean ± SD), and the F(m) of WB matched the (extrapolated) atmospheric F(m) of 1.0477 in 2008. This study is important in presenting a procedure to determine a baseline for a study group for human ADME and PBPK studies using (14)C as a tracer.

  2. Direct polymerase chain reaction (PCR) from human whole blood and filter-paper-dried blood by using a PCR buffer with a higher pH.

    PubMed

    Bu, Ying; Huang, Huan; Zhou, Guohua

    2008-04-15

    We described a novel approach to directly amplify genomic DNA from whole blood and dried blood spotted on filter paper without any DNA isolation by using the PCR buffer with a higher pH, which was optimized as pH 9.1-9.6. Direct PCR on blood treated with various anticoagulants showed that the buffer worked well with the blood treated by citrate, EDTA, or heparinate. DNA fragments with different lengths could be efficiently amplified directly from various forms of blood samples. By coupling the buffer with tetra-PCR, a "true" single-tube genotyping was realized by using whole blood or paper-dried blood as starting material.

  3. Simultaneous LC-MS/MS determination of JWH-210, RCS-4, ∆(9)-tetrahydrocannabinol, and their main metabolites in pig and human serum, whole blood, and urine for comparing pharmacokinetic data.

    PubMed

    Schaefer, Nadine; Kettner, Mattias; Laschke, Matthias W; Schlote, Julia; Peters, Benjamin; Bregel, Dietmar; Menger, Michael D; Maurer, Hans H; Ewald, Andreas H; Schmidt, Peter H

    2015-05-01

    A series of new synthetic cannabinoids (SC) has been consumed without any toxicological testing. For example, pharmacokinetic data have to be collected from forensic toxicological case work and/or animal studies. To develop a corresponding model for assessing such data, samples of controlled pig studies with two selected SC (JWH-210, RCS-4) and, as reference, ∆(9)-tetrahydrocannabinol (THC) should be analyzed as well as those of human cases. Therefore, a method for determination of JWH-210, RCS-4, THC, and their main metabolites in pig and human serum, whole blood, and urine samples is presented. Specimens were analyzed by liquid-chromatography tandem mass spectrometry and multiple-reaction monitoring with three transitions per compound. Full validation was carried out for the pig specimens and cross-validation for the human specimens concerning precision and bias. For the pig studies, the limits of detection were between 0.05 and 0.50 ng/mL in serum and whole blood and between 0.05 and 1.0 ng/mL in urine, the lower limits of quantification between 0.25 and 1.0 ng/mL in serum and 0.50 and 2.0 ng/mL in whole blood and urine, and the intra- and interday precision values lower than 15% and bias values within ±15%. The applicability was tested with samples taken from a pharmacokinetic pilot study with pigs following intravenous administration of a mixture of 200 μg/kg body mass dose each of JWH-210, RCS-4, and THC. The cross-validation data for human serum, whole blood, and urine showed that this approach should also be suitable for human specimens, e.g., of clinical or forensic cases.

  4. Analysis of human serum and whole blood for mineral content by ICP-MS and ICP-OES: development of a mineralomics method.

    PubMed

    Harrington, James M; Young, Daniel J; Essader, Amal S; Sumner, Susan J; Levine, Keith E

    2014-07-01

    Minerals are inorganic compounds that are essential to the support of a variety of biological functions. Understanding the range and variability of the content of these minerals in biological samples can provide insight into the relationships between mineral content and the health of individuals. In particular, abnormal mineral content may serve as an indicator of illness. The development of robust, reliable analytical methods for the determination of the mineral content of biological samples is essential to developing biological models for understanding the relationship between minerals and illnesses. This paper describes a method for the analysis of the mineral content of small volumes of serum and whole blood samples from healthy individuals. Interday and intraday precision for the mineral content of the blood (250 μL) and serum (250 μL) samples was measured for eight essential minerals--sodium (Na), calcium (Ca), magnesium (Mg), potassium (K), iron (Fe), zinc (Zn), copper (Cu), and selenium (Se)--by plasma spectrometric methods and ranged from 0.635 to 10.1% relative standard deviation (RSD) for serum and 0.348-5.98% for whole blood. A comparison of the determined ranges for ten serum samples and six whole blood samples provided good agreement with literature reference ranges. The results demonstrate that the digestion and analysis methods can be used to reliably measure the content of these minerals and potentially of other minerals.

  5. Analysis of Human Serum and Whole Blood for Mineral Content by ICP-MS and ICP-OES: Development of a Mineralomics Method

    PubMed Central

    Harrington, James M.; Young, Daniel J.; Essader, Amal S.; Sumner, Susan J.; Levine, Keith E.

    2014-01-01

    Minerals are inorganic compounds that are essential to the support of a variety of biological functions. Understanding the range and variability of the content of these minerals in biological samples can provide insight into the relationships between mineral content and the health of individuals. In particular, abnormal mineral content may serve as an indicator of illness. The development of robust, reliable analytical methods for the determination of the mineral content of biological samples is essential to developing biological models for understanding the relationship between minerals and illnesses. This manuscript describes a method for the analysis of the mineral content of small volumes of serum and whole blood samples from healthy individuals. Interday and intraday precision for the mineral content of the blood (250 μl) and serum (250 μl) samples was measured for eight essential minerals, sodium (Na), calcium (Ca), magnesium (Mg), potassium (K), iron (Fe), zinc (Zn), copper (Cu), and selenium (Se) by plasma spectrometric methods and ranged from 0.635 – 10.1% relative standard deviation (RSD) for serum and 0.348 – 5.98% for whole blood. A comparison of the determined ranges for ten serum samples and six whole blood samples provided good agreement with literature reference ranges. The results demonstrate that the digestion and analysis methods can be used to reliably measure the content of these minerals, and potentially to add other minerals. PMID:24917052

  6. Detection and quantification of 12 anabolic steroids and analogs in human whole blood and 20 in hair using LC-HRMS/MS: application to real cases.

    PubMed

    Fabresse, Nicolas; Grassin-Delyle, Stanislas; Etting, Isabelle; Alvarez, Jean-Claude

    2017-02-24

    We developed and validated a method to detect and quantify 12 anabolic steroids in blood (androstenedione, dihydrotestosterone, boldenone, epitestosterone, mesterolone, methandienone, nandrolone, stanozolol, norandrostenedione, tamoxifene, testosterone, trenbolone) and eight more in hair samples (nandrolone phenylpropionate, nandrolone decanoate, testosterone propionate, testosterone benzoate, testosterone cypionate, testosterone decanoate, testosterone phenylpropionate, testosterone undecanoate) using liquid chromatography coupled to high-resolution mass spectrometry. This method used a benchtop Orbitrap mass spectrometer operating with an APCI probe under positive ionization mode. Analysis was realized in full scan experiment with a nominal resolving power of 140,000. After addition of the internal standard (testosterone-D3) and incubation in phosphate buffer pH = 5 for hair, 200 μL of blood and 30 mg of hair samples were extracted with heptane. LOQ and LOD were determined at 5 and 1 ng mL(-1) in whole blood and 10 to 100 pg mg(-1) and 2 to 20 pg mg(-1) in hair according to the compounds, respectively. The method was linear in the 5-1000 ng mL(-1) range in whole blood and between 10 or 100 pg mg(-1) and 1000 pg mg(-1) in hair with correlation coefficients >0.99, and intra- and inter-day accuracy and precision were <14.8% for all compounds except for some esters in hairs (<19.9%) probably due to an important matrix effect for these compounds. This sensitive and specific method to detect anabolic steroids has been successfully applied to two real cases, for which various anabolic steroids in whole blood, urine, and hair were identified and quantified.

  7. Whole blood pumping with a microthrottle pump

    PubMed Central

    Davies, M. J.; Johnston, I. D.; Tan, C. K. L.; Tracey, M. C.

    2010-01-01

    We have previously reported that microthrottle pumps (MTPs) display the capacity to pump solid phase suspensions such as polystyrene beads which prove challenging to most microfluidic pumps. In this paper we report employing a linear microthrottle pump (LMTP) to pump whole, undiluted, anticoagulated, human venous blood at 200 μl min−1 with minimal erythrocyte lysis and no observed pump blockage. LMTPs are particularly well suited to particle suspension transport by virtue of their relatively unimpeded internal flow-path. Micropumping of whole blood represents a rigorous real-world test of cell suspension transport given blood’s high cell content by volume and erythrocytes’ relative fragility. A modification of the standard Drabkin method and its validation to spectrophotometrically quantify low levels of erythrocyte lysis by hemoglobin release is also reported. Erythrocyte lysis rates resulting from transport via LMTP are determined to be below one cell in 500 at a pumping rate of 102 μl min−1. PMID:21264059

  8. Enantioselective analysis of citalopram and demethylcitalopram in human whole blood by chiral LC-MS/MS and application in forensic cases.

    PubMed

    Johansen, Sys Stybe

    2017-02-08

    Citalopram is one of the most frequently used antidepressants in Denmark. Citalopram is marketed as a racemic mixture (50:50) of S- and R-enantiomers as well as of the S-enantiomer alone, which is the active enantiomer named escitalopram that processes the inhibitory effects. In this study, a chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is developed for the measurement of citalopram and demethylcitalopram enantiomers in whole blood and is applied to forensic cases. Whole blood samples (0.10 g) were extracted with butyl acetate after adjusting the pH with 2 M NaOH. The analytes were separated on a 250 x 4.6 mm Chirobiotic V, 5 µm column by isocratic elution with methanol:ammonia:acetic acid (1000:1:1) using an ultra-high-pressure liquid chromatography (UHPLC) system. Quantification was performed by tandem mass spectrometry (MS/MS) using multiple reaction monitoring in the positive mode. The total chromatographic run time was 20 min. The limit of detection (LOD) and quantification (LOQ) were 0.001 and 0.005 mg/kg of all four enantiomers, respectively. Linear behaviour was obtained for all four enantiomers from LOQ to 0.50 mg/kg blood with absolute recoveries from 71 to 80%. The method showed an acceptable precision and accuracy as the obtained coefficient of variation, and bias values were ≤ 16% for all enantiomers. After the validation of the method, a correlation with the racemic method was assessed and found to be acceptable. Then, the method was successfully applied to authentic blood samples from forensic investigations demonstrating that escitalopram was less frequent than citalopram among all forensic cases.

  9. Microfluidic immunomagnetic cell separation from whole blood.

    PubMed

    Bhuvanendran Nair Gourikutty, Sajay; Chang, Chia-Pin; Puiu, Poenar Daniel

    2016-02-01

    Immunomagnetic-based separation has become a viable technique for the separation of cells and biomolecules. Here we report on the design and analysis of a simple and efficient microfluidic device for high throughput and high efficiency capture of cells tagged with magnetic particles. This is made possible by using a microfluidic chip integrated with customized arrays of permanent magnets capable of creating large magnetic field gradients, which determine the effective capturing of the tagged cells. This method is based on manipulating the cells which are under the influence of a combination of magnetic and fluid dynamic forces in a fluid under laminar flow through a microfluidic chip. A finite element analysis (FEA) model is developed to analyze the cell separation process and predict its behavior, which is validated subsequently by the experimental results. The magnetic field gradients created by various arrangements of magnetic arrays have been simulated using FEA and the influence of these field gradients on cell separation has been studied with the design of our microfluidic chip. The proof-of-concept for the proposed technique is demonstrated by capturing white blood cells (WBCs) from whole human blood. CD45-conjugated magnetic particles were added into whole blood samples to label WBCs and the mixture was flown through our microfluidic device to separate the labeled cells. After the separation process, the remaining WBCs in the elute were counted to determine the capture efficiency, and it was found that more than 99.9% WBCs have been successfully separated from whole blood. The proposed design can be used for positive selection as well as for negative enrichment of rare cells.

  10. Evaluation of whole blood coagulation process by optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Xu, Xiangqun; Lin, Jia

    2010-11-01

    This study was to investigate the feasibility of using optical coherence tomography (OCT) to evaluate whole blood coagulation process. Attenuation coefficients and 1/e light penetration depth (D1/e) against time of human whole blood during in vitro clot formation under static were measured from the OCT profiles of reflectance vs depth. The results obtained clearly showed that the optical parameters are able to identify three stages during the in vitro blood clotting process. It is concluded that D1/e measured by OCT is a potential parameter to quantify and follow the liquid-gel transition of blood during clotting.

  11. High level of IFN-γ released from whole blood of human tuberculosis infections following stimulation with Rv2073c of Mycobacterium tuberculosis.

    PubMed

    Tan, Kun; Zhang, Jingyan; Teng, Xindong; Liang, Jinping; Wang, Xiaochun; Yuan, Xuefeng; Tian, Maopeng; Fan, Xionglin

    2015-07-01

    More efficacious and specific biomarkers are urgently needed for better control of tuberculosis (TB), the second leading infectious cause of mortality worldwide. The region of difference 9 (RD9) presents the genome of the causative pathogen Mycobacterium tuberculosis rather than other species of the genus Mycobacterium, which might be promising targets for specific diagnosis, vaccine development and pathogenesis. In this study, two proteins Rv2073c and Rv2074, encoded by the RD9 were expressed and purified from Escherichia coli system. Following stimulation with both proteins, the levels of IFN-γ secreted by T cells from a total of 49 whole blood samples obtained from clinically diagnosed active TB patients, patients with latent TB infections (LTBIs), and healthy donors, were compared with those of the incubation with recombinant fusion protein of CFP21 and MPT64 (rCM). Our results demonstrated that only Rv2073c could induce a higher level of IFN-γ in TB infections than healthy controls and there was a positive correlation between Rv2073c- and rCM-specific IFN-γ levels in TB infections and healthy donors, respectively. These findings indicate that Rv2073c might be a promising antigen for specific diagnostic reagents and vaccine candidates of TB.

  12. Analysis of MT-45, a Novel Synthetic Opioid, in Human Whole Blood by LC-MS-MS and its Identification in a Drug-Related Death.

    PubMed

    Papsun, Donna; Krywanczyk, Alison; Vose, James C; Bundock, Elizabeth A; Logan, Barry K

    2016-05-01

    MT-45 (1-cyclohexyl-4-(1,2-diphenylethyl)piperazine) is just one of the many novel psychoactive substances (NPS) to have reached the recreational drug market in the twenty-first century; it is however, one of the first designer opioids to achieve some degree of popularity, in a market currently dominated by synthetic cannabinoids and designer stimulants. A single fatality involving MT-45 and etizolam is described. A method for the quantitation of MT-45 in whole blood using liquid chromatography-tandem mass spectrometry was developed and validated. The linear range was determined to be 1.0-100 ng/mL with a detection limit of 1.0 ng/mL, and the method met the requirements for acceptable linearity, precision and accuracy. After analyzing the sample on dilution and by standard addition, the concentration of MT-45 in the decedent's blood was determined to be 520 ng/mL, consistent with other concentrations of MT-45 reported in drug-related fatalities. Etizolam was present at a concentration of 35 ng/mL. This case illustrates the importance of considering non-traditional drugs in unexplained apparent drug-related deaths.

  13. Determination of clozapine, and five antidepressants in human plasma, serum and whole blood by gas chromatography-mass spectrometry: A simple tool for clinical and postmortem toxicological analysis.

    PubMed

    Boumba, Vassiliki A; Rallis, George; Petrikis, Petros; Vougiouklakis, Theodore; Mavreas, Venetsanos

    2016-12-01

    In this study, we describe a simple and rapid method for the determination of the antipsychotic drug clozapine and five commonly co-administered antidepressants - bupropion, mirtazapine, sertraline, clomipramine and citalopram - in serum, plasma and whole blood. Sample preparation includes solid phase extraction of analytes and determination of drug concentrations by gas chromatography-mass spectrometry without any derivatization steps. The method was fully validated according to international criteria and can be successfully applied for routine analyses. Correlation coefficients of calibration curves for the tested drugs in the three specimens were in the range 0.9977-0.9999. Intra-day and inter-day precisions ranged from 0.81-7.85% and 3.60-12.91% respectively for the studied analytes and matrices. Recoveries were satisfactory for different concentrations of each drug in each specimen allowing accurate determinations in the range from sub-therapeutic to toxic levels. The presented method shows acceptable sensitivity, linearity in wide concentration ranges (sub-therapeutic, therapeutic, supra-therapeutic/toxic levels), it is simple and rapid and it is applicable for qualitative and quantitative routine toxicological analyses of clinical and postmortem cases.

  14. Flow Cytometric Analysis of Mononuclear Phagocytes in Nondiseased Human Lung and Lung-Draining Lymph Nodes

    PubMed Central

    Desch, A. Nicole; Gibbings, Sophie L.; Goyal, Rajni; Kolde, Raivo; Bednarek, Joe; Bruno, Tullia; Slansky, Jill E.; Jacobelli, Jordan; Mason, Robert; Ito, Yoko; Messier, Elise; Randolph, Gwendalyn J.; Prabagar, Miglena; Atif, Shaikh M.; Segura, Elodie; Xavier, Ramnik J.; Bratton, Donna L.; Janssen, William J.; Henson, Peter M.

    2016-01-01

    Rationale: The pulmonary mononuclear phagocyte system is a critical host defense mechanism composed of macrophages, monocytes, monocyte-derived cells, and dendritic cells. However, our current characterization of these cells is limited because it is derived largely from animal studies and analysis of human mononuclear phagocytes from blood and small tissue resections around tumors. Objectives: Phenotypic and morphologic characterization of mononuclear phagocytes that potentially access inhaled antigens in human lungs. Methods: We acquired and analyzed pulmonary mononuclear phagocytes from fully intact nondiseased human lungs (including the major blood vessels and draining lymph nodes) obtained en bloc from 72 individual donors. Differential labeling of hematopoietic cells via intrabronchial and intravenous administration of antibodies within the same lobe was used to identify extravascular tissue-resident mononuclear phagocytes and exclude cells within the vascular lumen. Multiparameter flow cytometry was used to identify mononuclear phagocyte populations among cells labeled by each route of antibody delivery. Measurements and Main Results: We performed a phenotypic analysis of pulmonary mononuclear phagocytes isolated from whole nondiseased human lungs and lung-draining lymph nodes. Five pulmonary mononuclear phagocytes were observed, including macrophages, monocyte-derived cells, and dendritic cells that were phenotypically distinct from cell populations found in blood. Conclusions: Different mononuclear phagocytes, particularly dendritic cells, were labeled by intravascular and intrabronchial antibody delivery, countering the notion that tissue and blood mononuclear phagocytes are equivalent systems. Phenotypic descriptions of the mononuclear phagocytes in nondiseased lungs provide a precedent for comparative studies in diseased lungs and potential targets for therapeutics. PMID:26551758

  15. Modulation of fibronectin gene expression in human mononuclear phagocytes.

    PubMed Central

    Yamauchi, K; Martinet, Y; Crystal, R G

    1987-01-01

    Under some conditions, mononuclear phagocytes spontaneously synthesize and release fibronectin, an extracellular matrix glycoprotein with versatile effects on cell-matrix interactions. To gain insight into the processes that modulate the level of fibronectin secretion by these cells, we used monocytes, in vitro matured monocytes and alveolar macrophages as models to compare fibronectin mRNA levels and fibronectin secretion in a variety of circumstances. Using Northern analysis and dot-blot analysis with a 32P-labeled human fibronectin cDNA probe, we evaluated steady-state mRNA levels and a human fibronectin-specific ELISA was used to evaluate fibronectin secretion. In all cases the amounts of fibronectin secreted paralleled fibronectin mRNA levels. Specifically (a) when fibronectin mRNA was undetectable, as in the case of normal blood monocytes, no fibronectin was secreted, but whenever fibronectin mRNA was present, as in normal alveolar macrophages, fibronectin was secreted by the cells; (b) as monocytes matured into macrophages in vitro, the cells began to express fibronectin mRNA and the cells secreted fibronectin; (c) when alveolar macrophages were activated with surface stimuli such as lipopolysaccharide (LPS) or immune complexes, fibronectin mRNA levels decreased and in parallel, the cells secreted less fibronectin; (d) in idiopathic pulmonary fibrosis (IPF), alveolar macrophages contained severalfold more fibronectin mRNA transcripts that normal and the cells spontaneously secreted severalfold more fibronectin than normal; and (e) when IPF alveolar macrophages were placed in culture the fibronectin mRNA levels in the cells decreased with time, and concurrently the amounts of fibronectin produced per unit time continually decreased. The observation of a strict concordance of fibronectin mRNA levels and fibronectin release by mononuclear phagocytes suggests that, at least in many circumstances, fibronectin secretion by mononuclear phagocytes is controlled by

  16. High throughput online solid phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry method for polyfluoroalkyl phosphate esters, perfluoroalkyl phosphonates, and other perfluoroalkyl substances in human serum, plasma, and whole blood.

    PubMed

    Poothong, Somrutai; Lundanes, Elsa; Thomsen, Cathrine; Haug, Line Småstuen

    2017-03-08

    A rapid, sensitive and reliable method was developed for the determination of a broad range of poly- and perfluoroalkyl substances (PFASs) in various blood matrices (serum, plasma, and whole blood), and uses only 50 μL of sample material. The method consists of a rapid protein precipitation by methanol followed by high throughput online solid phase extraction (SPE), ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), and negative electrospray ionization detection. The method was developed for simultaneous determination of twenty-five PFASs, including polyfluoroalkyl phosphate esters (PAPs; 6:2, 8:2, 6:2/6:2, and 8:2/8:2), perfluoroalkyl phosphonates (PFPAs; C6, C8, and C10), perfluoroalkyl sulfonates (PFSAs; C4, C6, C7, C8, and C10), perfluoroalkyl carboxylates (PFCAs; C5C14), and perfluoroalkyl sulfonamides (FOSAs; C8, N-methyl, and N-ethyl). High linearity of matrix-matched calibration standards (correlation coefficients, R = 0.99-0.999) were obtained in the range of 0.006-45 ng mL(-1) blood. Excellent sensitivity was achieved with method detection limits (MDLs) between 0.0018 and 0.09 ng mL(-1), depending on the compound and matrix. The method was validated for serum, plasma, and whole blood (n = 5 + 5) at six levels in the range 0.0180-30 ng mL(-1). The accuracy (n = 5) was on average 102± 12%. The intermediate precision (n = 10) ranged from 2 to 40% with an average between-batch of analyses difference of 10± 10%. Two human serum samples from a former interlaboratory comparison were analyzed and the differences between the applied method and the consensus values were below ≤22% (n = 5). The method was also successfully applied to samples of human plasma and whole blood with coefficients of variation in the range 0.8-15.2% (n = 5).

  17. Differences in iron concentration in whole blood of animal models using NAA

    NASA Astrophysics Data System (ADS)

    Bahovschi, V.; Zamboni, C. B.; Lopes Silva, L. F. F.; Metairon, S.; Medeiros, I. M. M. A.

    2015-07-01

    In this study Neutron Activation Analysis technique (NAA) was applied to determine Fe concentration in whole blood samples of several animal models such as: mice (Mus musculus), Golden Hamster (Mesocricetus auratus), Wistar rats, Albinic Rabbits of New Zealand, Golden Retriever dogs and Crioulabreed horses. These results were compared with human whole blood estimation to check their similarities.

  18. Ex-vivo in-vitro inhibition of lipopolysaccharide stimulated tumor necrosis factor-alpha and interleukin-1 beta secretion in human whole blood by extractum urticae dioicae foliorum.

    PubMed

    Obertreis, B; Ruttkowski, T; Teucher, T; Behnke, B; Schmitz, H

    1996-04-01

    An extract of Urtica dioica folium (IDS 23, Rheuma-Hek), monographed positively for adjuvant therapy of rheumatic diseases and with known effects in partial inhibition of prostaglandin and leukotriene synthesis in vitro, was investigated with respect to effects of the extract on the lipopolysaccharide (LPS) stimulated secretion of proinflammatory cytokines in human whole blood of healthy volunteers. In the assay system used, LPS stimulated human whole blood showed a straight increase of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) secretion reaching maximum concentrations within 24 h following a plateau and slight decrease up to 65 h, respectively. The concentrations of these cytokines was strongly positively correlated with the number of monocytes/macrophages of each volunteer. TNF-alpha and IL-1 beta concentration after LPS stimulation was significantly reduced by simultaneously given IDS 23 in a strictly dose dependent manner. At time 24 h these cytokine concentrations were reduced by 50.8% and 99.7%, respectively, using the highest test IDS 23 assay concentration of 5 mg/ml (p < 0.001). After 65 h the corresponding inhibition was 38.9% and 99.9%, respectively (p < 0.001). On the other hand IDS 23 showed no inhibition but stimulated IL-6 secretion in absence of LPS alone. Simultaneously given LPS and IDS 23 resulted in no further increase. In contrast to described effects on arachidonic acid cascade in vitro, tested Urtica dioica phenol carbon acid derivates and flavonoides such as caffeic malic acid, caffeic acid, chlorogenic acid, quercetin and rutin did not influence LPS stimulated TNF-alpha, IL-1 beta and IL-6 secretion in tested concentrations up to 5 x 10(-5) mol/l. These further findings on the pharmacological mechanism of action of Urticae dioica folia may explain the positive effects of this extract in the treatment of rheumatic diseases.

  19. Whole blood stimulation with Toll-like receptor (TLR)-7/8 and TLR-9 agonists induces interleukin-12p40 expression in plasmacytoid dendritic cells in rhesus macaques but not in humans.

    PubMed

    Koopman, G; Beenhakker, N; Burm, S; Bouwhuis, O; Bajramovic, J; Sommandas, V; Mudde, G; Mooij, P; 't Hart, B A; Bogers, W M J M

    2013-10-01

    Macaques provide important animal models in biomedical research into infectious and chronic inflammatory disease. Therefore, a proper understanding of the similarities and differences in immune function between macaques and humans is needed for adequate interpretation of the data and translation to the human situation. Dendritic cells are important as key regulators of innate and adaptive immune responses. Using a new whole blood assay we investigated functional characteristics of blood plasmacytoid dendritic cells (pDC), myeloid dendritic cells (mDC) and monocytes in rhesus macaques by studying induction of activation markers and cytokine expression upon Toll-like receptor (TLR) stimulation. In a head-to-head comparison we observed that rhesus macaque venous blood contained relatively lower numbers of pDC than human venous blood, while mDC and monocytes were present at similar percentages. In contrast to humans, pDC in rhesus macaques expressed the interleukin (IL)-12p40 subunit in response to TLR-7/8 as well as TLR-9 stimulation. Expression of IL-12p40 was confirmed by using different monoclonal antibodies and by reverse transcription-polymerase chain reaction (RT-PCR). Both in humans and rhesus macaques, TLR-4 stimulation induced IL-12p40 expression in mDC and monocytes, but not in pDC. The data show that, in contrast to humans, pDC in macaques are able to express IL-12p40, which could have consequences for evaluation of human vaccine candidates and viral infection.

  20. A single meal containing raw, crushed garlic influences expression of immunity- and cancer-related genes in whole blood of humans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Preclinical and epidemiological studies suggest that garlic intake is inversely associated with the progression of cancer and cardiovascular disease. To probe mechanisms of garlic action in humans, we conducted a randomized crossover feeding trial in which 17 volunteers consumed a garlic-containing...

  1. Short report: Seroprevalence of human leptospirosis in Reunion Island (Indian Ocean) assessed by microscopic agglutination test on paper disc-absorbed whole blood.

    PubMed

    Desvars, Amélie; Gigan, Jimmy; Hoarau, Géraldine; Gérardin, Patrick; Favier, François; Michault, Alain

    2011-12-01

    In the last decade, leptospirosis has emerged as a globally important infectious disease. Humans most commonly become infected through occupational, recreational, or domestic contact with the urine of carrier animals, either directly or through contaminated water or soil. The disease occurs in urban areas of industrialized and developing countries as well as rural regions worldwide. We present a retrospective study conducted in 2006 on 2,269 randomly selected Reunion Island inhabitants. Blood sampling was performed on individual blotting papers, and microscopic agglutination test (MAT) was conducted on paper disc-absorbed (PDA) blood. We showed that seroprevalence of leptospirosis was 0.66% ± 0.34 in the global population of Reunion Island, which is 1.78 lower than the seroprevalence estimated 20 years before. The serological method is described, and the results discussion focuses on methodology and socio-economic factors.

  2. Detection of progesterone in whole blood samples.

    PubMed

    Ehrentreich-Förster, Eva; Scheller, Frieder W; Bier, Frank F

    2003-04-01

    The progesterone concentration in blood samples can be utilised as a marker for the diagnosis of early pregnancy, endocrinopathy and virilism. Here, we describe a method for progesterone detection and measurement in whole blood samples by a surface sensitive biosensor used in conjunction with an integrated optical grating coupler. This device determines refractive index changes near the biosensor's surface. Hence, biological species bound to a surface layer can be measured in real-time without any label. For the measurements, we have modified the indirect competitive immunoassay principle. The concentration of the progesterone antibody was kept at 1 microg/ml. Progesterone concentration was determined in buffer solution and whole blood in a range between 0.005 and 10 ng/ml. The detection limit was determined to be 3 pM. The relative standard deviation was calculated to be 3.5%.

  3. Ingestion of Giardia lamblia trophozoites by human mononuclear phagocytes.

    PubMed Central

    Hill, D R; Pearson, R D

    1987-01-01

    Mononuclear phagocytes may be important effector cells against Giardia lamblia. Human monocyte-derived macrophages were incubated with G. lamblia trophozoites in 13% heat-inactivated autologous serum. At a G. lamblia/macrophage ratio of 1:1, the number of trophozoites ingested per 100 macrophages ranged from 1 to 12 at 0.5 h and increased for all donors (n = 6) to 10 to 92 at 8 h. Ingestion was confirmed by electron microscopy. Increasing the parasite/phagocyte ratio to 5:1 increased the percentage of macrophages with adherent but not ingested trophozoites. Incubating Giardia cells and macrophages with 20% immune serum increased ingestion of parasites eightfold, indicating that anti-G. lamblia antibody can enhance ingestion. Both phase-contrast microscopy and electron microscopy documented trophozoite destruction within macrophages. Ingestion of parasites elicited an oxidative burst as measured by luminol-enhanced chemiluminescence. In vitro, Giardia trophozoites were killed by greater than or equal to 5 X 10(-5) M H2O2. Fusion of lysosomes with parasite-containing phagosomes was suggested by acridine orange-stained preparations. Human macrophages have the capacity to ingest Giardia trophozoites and to kill intracellular parasites, possibly by oxidative microbicidal mechanisms. Images PMID:3679547

  4. Activity of microemulsion-based nanoparticles at the human bio-nano interface: concentration-dependent effects on thrombosis and hemolysis in whole blood

    NASA Astrophysics Data System (ADS)

    Morey, Timothy E.; Varshney, Manoj; Flint, Jason A.; Seubert, Christoph N.; Smith, W. Brit; Bjoraker, David G.; Shah, Dinesh O.; Dennis, Donn M.

    2004-06-01

    Background: Although microemulsion-based nanoparticles (MEs) may be useful for drug delivery or scavenging, these benefits must be balanced against potential nanotoxicological effects in biological tissue (bio-nano interface). We investigated the actions of assembled MEs and their individual components at the bio-nano interface of thrombosis and hemolysis in human blood. Methods: Oil-in-water MEs were synthesized using ethylbutyrate, sodium caprylate, and pluronic F-68 (ME4) or F-127 (ME6) in 0.9% NaClw/v. The effects of MEs or components on thrombosis were determined using thrombo-elastography, platelet contractile force, clot elastic modulus, and platelet counting. For hemolysis, ME or components were incubated with erythrocytes, centrifuged, and washed for measurement of free hemoglobin by spectroscopy. Results and conclusions: The mean particle diameters (polydispersity index) for ME6 and ME4 were 23.6 ± 2.5 nm (0.362) and 14.0 ± 1.0 nm (0.008), respectively. MEs (0, 0.03, 0.3, 3 mM) markedly reduced the thromboelastograph maximal amplitude in a concentration-dependent manner (49.0 ± 4.2, 39.0 ± 5.6, 15.0 ± 8.7, 3.8 ± 1.3 mm, respectively), an effect highly correlated ( r2 = 0.94) with similar changes caused by pluronic surfactants (48.7 ± 10.9, 30.7 ± 15.8, 20.0 ± 11.3, 2.0 ± 0.5) alone. Neither oil nor sodium caprylate alone affected the thromboelastograph. The clot contractile force was reduced by ME (27.3 ± 11.1-6.7 ± 3.4 kdynes/cm2, P = 0.02, n = 5) whereas the platelet population not affected (175 ± 28-182 ± 23 106/ml, P = 0.12, n = 6). This data suggests that MEs reduced platelet activity due to associated pluronic surfactants, but caused minimal changes in protein function necessary for coagulation. Although pharmacological concentrations of sodium caprylate caused hemolysis (EC50 = 213 mM), MEs and pluronic surfactants did not disrupt erythrocytes. Knowledge of nanoparticle activity and potential associated nanotoxicity at this bio

  5. Mechanism and role of MCP-1 upregulation upon chikungunya virus infection in human peripheral blood mononuclear cells.

    PubMed

    Ruiz Silva, Mariana; van der Ende-Metselaar, Heidi; Mulder, H Lie; Smit, Jolanda M; Rodenhuis-Zybert, Izabela A

    2016-08-25

    Monocyte chemoattractant protein-1 (MCP-1/CCL2)-mediated migration of monocytes is essential for immunological surveillance of tissues. During chikungunya virus (CHIKV) infection however, excessive production of MCP-1 has been linked to disease pathogenesis. High MCP-1 serum levels are detected during the viremic phase of CHIKV infection and correlate with the virus titre. In vitro CHIKV infection was also shown to stimulate MCP-1 production in whole blood; yet the role and the mechanism of MCP-1 production upon infection of human peripheral blood mononuclear cells remain unknown. Here we found that active CHIKV infection stimulated production of MCP-1 in monocytes. Importantly however, we found that communication with other leukocytes is crucial to yield MCP-1 by monocytes upon CHIKV infection. Indeed, blocking interferon-α/β receptor or the JAK1/JAK2 signalling downstream of the receptor abolished CHIKV-mediated MCP-1 production. Additionally, we show that despite the apparent correlation between IFN type I, CHIKV replication and MCP-1, modulating the levels of the chemokine did not influence CHIKV infection. In summary, our data disclose the complexity of MCP-1 regulation upon CHIKV infection and point to a crucial role of IFNβ in the chemokine secretion. We propose that balance between these soluble factors is imperative for an appropriate host response to CHIKV infection.

  6. Mechanism and role of MCP-1 upregulation upon chikungunya virus infection in human peripheral blood mononuclear cells

    PubMed Central

    Ruiz Silva, Mariana; van der Ende-Metselaar, Heidi; Mulder, H. Lie; Smit, Jolanda M.; Rodenhuis-Zybert, Izabela A.

    2016-01-01

    Monocyte chemoattractant protein-1 (MCP-1/CCL2)-mediated migration of monocytes is essential for immunological surveillance of tissues. During chikungunya virus (CHIKV) infection however, excessive production of MCP-1 has been linked to disease pathogenesis. High MCP-1 serum levels are detected during the viremic phase of CHIKV infection and correlate with the virus titre. In vitro CHIKV infection was also shown to stimulate MCP-1 production in whole blood; yet the role and the mechanism of MCP-1 production upon infection of human peripheral blood mononuclear cells remain unknown. Here we found that active CHIKV infection stimulated production of MCP-1 in monocytes. Importantly however, we found that communication with other leukocytes is crucial to yield MCP-1 by monocytes upon CHIKV infection. Indeed, blocking interferon-α/β receptor or the JAK1/JAK2 signalling downstream of the receptor abolished CHIKV-mediated MCP-1 production. Additionally, we show that despite the apparent correlation between IFN type I, CHIKV replication and MCP-1, modulating the levels of the chemokine did not influence CHIKV infection. In summary, our data disclose the complexity of MCP-1 regulation upon CHIKV infection and point to a crucial role of IFNβ in the chemokine secretion. We propose that balance between these soluble factors is imperative for an appropriate host response to CHIKV infection. PMID:27558873

  7. Zebrafish thrombocyte aggregation by whole blood aggregometry and flow cytometry.

    PubMed

    Sundaramoorthi, Hemalatha; Panapakam, Rekha; Jagadeeswaran, Pudur

    2015-01-01

    Zebrafish has become an excellent model system to study mammalian hemostasis. Despite our extensive efforts to develop technologies to measure zebrafish hemostasis and even with previously established thrombocyte qualitative and quantitative functional assays, quantifying thrombocyte function for high throughput applications has been a challenge. In this paper, we have developed two quantitative methods to estimate thrombocyte aggregation: one by whole blood aggregometry and the other by flow cytometry. We found that it is possible to conduct whole blood aggregometry using only 2 µl of blood and the currently available aggregometer. Each of three agonists, arachidonic acid, ADP, and collagen yielded impedance curves similar to those obtained with human blood. We were also able to use flow cytometry to indirectly quantify the extent of thrombocyte aggregation by labeling whole blood with mepacrine, aggregating in the presence of each of the above agonists, separating the aggregates from the white blood cells by centrifugation, and then sorting the resulting white cell fraction for thrombocyte numbers. These methods have high throughput capabilities and have the potential to be used in large scale screens to detect and characterize mutants with thrombocyte functional defects or to identify genes involved in thrombocyte function by large scale knockdowns.

  8. Improved Whole-Blood-Staining Device

    NASA Technical Reports Server (NTRS)

    Sams, Clarence F.; Crucian, Brian; Paul, Bonnie; Melton, Shannon; Guess, Terry

    2012-01-01

    Dramatic improvements have been made in NASA s Whole Blood Staining Device (WBSD) since it was last described in "Whole-Blood-Staining Device," NASA Tech Briefs, Vol. 23, No. 10 (October 1999), page 64. The new system has a longer shelf life, a simpler and more effective operational procedure, improved interface with instrumentation, and shorter processing time. More specifically, the improvements have targeted bag and locking clip materials, sampling ports, and air pocket prevention. The WBSD stains whole blood collected during spaceflight for subsequent flow cytometric analysis. In short, the main device stains white blood cells by use of monoclonal antibodies conjugated to various fluorochromes, followed by lysing and fixing of the cells by use of a commercial reagent that has been diluted according to NASA safety standards. This system is compact, robust, and does not require electric power, precise mixing, or precise incubation times. Figure 1 depicts the present improved version for staining applications, which is a poly(tetrafluoroethylene) bag with a Luer-lock port and plastic locking clips. An InterLink (or equivalent) intravenous- injection port screws into the Luer-lock port. The inflatable/collapsible nature of the bag facilitates loading and helps to minimize the amount of air trapped in the fully loaded bag. Some additional uses have been identified for the device beyond whole blood staining. The WBSD has been configured for functional assays that require culture of live cells by housing sterile culture media, mitogens, and fixatives prior to use [Figure 2(a)]. Simple injection of whole blood allows cell-stimulation culture to be performed in reduced gravity conditions, and product stabilization prior to storage, while protecting astronauts from liquid biohazardous materials. Also, the improved WBSD has reconstituted powdered injectable antibiotics by mixing them with diluent liquids [Figure 2(b)]. Although such mixing can readily be performed on

  9. Determination of ecgonine and seven other cocaine metabolites in human urine and whole blood by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry.

    PubMed

    Xiong, Lingjuan; Wang, Rong; Liang, Chen; Cao, Fangqi; Rao, Yulan; Wang, Xin; Zeng, Libo; Ni, Chunfang; Ye, Haiying; Zhang, Yurong

    2013-12-01

    Ecgonine is suggested to be a promising marker of cocaine (COC) ingestion. A combined mass spectrometry (MS) and tandem MS (MS/MS) method was developed to simultaneously determine ecgonine and seven other metabolites of cocaine in human urine and whole blood with ultra-high-pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The compounds were extracted from as little as 100 μL of sample by solid-phase extraction with a 96-well μElution solid-phase extraction plate. The protonated molecules or fragment ions at accurate mass acquired in MS mode were used to quantify specific analytes, following by dedicated MS/MS identification. The assay was linear in the range from 5 to 50-100 ng/mL for urine samples, except for ecgonine methyl ester (10-200 ng/mL) and ecgonine (40-400 ng/mL), and was linear from 1-2 to 50 ng/mL for whole blood samples, except for ecgonine methyl ester (20-1,000 ng/mL) and ecgonine (40-2,000 ng/mL). The correlation coefficients were all greater than 0.99. The limits of detection ranged from 0.2 to 16 ng/mL, and the lower limits of quantification ranged from 1 to 40 ng/mL. The repeatability and intermediate precision were 18.1% or less. The accuracy was in the range from 80.0 to 122.9%, process efficiencies were in the range from 8.6 to 177.4%, matrix effects were in the range from 28.7 to 171.0%, and extraction recoveries were in the range from 41.0 to 114.3%, except for ecgonine (12.8% and 9.3% at low and high concentrations, respectively). This method was highly sensitive in comparison with previously published methods. The validated method was successfully applied to the analysis of real samples derived from forensic cases, and the results verified that, on the basis of data from four positive samples, ecgonine is a promising marker of cocaine ingestion.

  10. 21 CFR 864.7500 - Whole blood hemoglobin assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  11. 21 CFR 864.7500 - Whole blood hemoglobin assays.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  12. 21 CFR 864.7500 - Whole blood hemoglobin assays.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  13. 21 CFR 864.7500 - Whole blood hemoglobin assays.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  14. 21 CFR 864.7500 - Whole blood hemoglobin assays.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  15. Combined Inhibition of Complement and CD14 Attenuates Bacteria-Induced Inflammation in Human Whole Blood More Efficiently Than Antagonizing the Toll-like Receptor 4–MD2 Complex

    PubMed Central

    Gustavsen, Alice; Nymo, Stig; Landsem, Anne; Christiansen, Dorte; Ryan, Liv; Husebye, Harald; Lau, Corinna; Pischke, Søren E.; Lambris, John D.; Espevik, Terje; Mollnes, Tom E.

    2016-01-01

    Background. Single inhibition of the Toll-like receptor 4 (TLR4)–MD2 complex failed in treatment of sepsis. CD14 is a coreceptor for several TLRs, including TLR4 and TLR2. The aim of this study was to investigate the effect of single TLR4-MD2 inhibition by using eritoran, compared with the effect of CD14 inhibition alone and combined with the C3 complement inhibitor compstatin (Cp40), on the bacteria-induced inflammatory response in human whole blood. Methods. Cytokines were measured by multiplex technology, and leukocyte activation markers CD11b and CD35 were measured by flow cytometry. Results. Lipopolysaccharide (LPS)–induced inflammatory markers were efficiently abolished by both anti-CD14 and eritoran. Anti-CD14 was significantly more effective than eritoran in inhibiting LPS-binding to HEK-293E cells transfected with CD14 and Escherichia coli–induced upregulation of monocyte activation markers (P < .01). Combining Cp40 with anti-CD14 was significantly more effective than combining Cp40 with eritoran in reducing E. coli–induced interleukin 6 (P < .05) and monocyte activation markers induced by both E. coli (P < .001) and Staphylococcus aureus (P < .01). Combining CP40 with anti-CD14 was more efficient than eritoran alone for 18 of 20 bacteria-induced inflammatory responses (mean P < .0001). Conclusions. Whole bacteria–induced inflammation was inhibited more efficiently by anti-CD14 than by eritoran, particularly when combined with complement inhibition. Combined CD14 and complement inhibition may prove a promising treatment strategy for bacterial sepsis. PMID:26977050

  16. Detection and quantification of benzodiazepines and Z-drugs in human whole blood, plasma, and serum samples as part of a comprehensive multi-analyte LC-MS/MS approach.

    PubMed

    Montenarh, Deborah; Hopf, Markus; Maurer, Hans H; Schmidt, Peter; Ewald, Andreas H

    2014-01-01

    For the first time, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) multi-analyte approach based on a simple liquid-liquid extraction was developed and validated for fast target screening and quantification of benzodiazepines and Z-drugs in case of driving ability and crime responsibility in the three most important biosamples whole blood, plasma, and serum. Whole blood, plasma, and serum (500 μL each) were extracted twice at pH 7.4 and at pH 10 with ether/ethyl acetate (1:1). Separation, detection, and quantification were performed using LC-MS/MS with electrospray ionization in positive mode. The method was validated with respect to selectivity, ion suppression/enhancement of co-eluting analytes, matrix effects, recovery, process efficiency, accuracy and precision, stabilities, and limits of detection and quantification. For accuracy and precision, full calibration was performed with ranges from subtherapeutic to toxic concentrations. The presented LC-MS/MS approach as part of a universal multi-analyte concept for over 100 drugs was applicable for selective detection as well as accurate and precise quantification in whole blood, plasma, and serum. The approach was selective, sensitive, accurate, and precise for 16 of the 19 tested drugs in whole blood, 18 in plasma, and 17 in serum. Only semiquantitative results could be obtained for zopiclone because of its instability in all tested biosamples.

  17. Whole Blood RNA as a Source of Transcript-Based Nutrition- and Metabolic Health-Related Biomarkers

    PubMed Central

    Petrov, Petar D.; Bonet, M. Luisa; Reynés, Bárbara; Oliver, Paula; Palou, Andreu; Ribot, Joan

    2016-01-01

    Blood cells are receiving an increasing attention as an easily accessible source of transcript-based biomarkers. We studied the feasibility of using mouse whole blood RNA in this context. Several paradigms were studied: (i) metabolism-related transcripts known to be affected in rat tissues and peripheral blood mononuclear cells (PBMC) by fasting and upon the development of high fat diet (HFD)-induced overweight were assessed in whole blood RNA of fasted rats and mice and of HFD-fed mice; (ii) retinoic acid (RA)-responsive genes in tissues were assessed in whole blood RNA of control and RA-treated mice; (iii) lipid metabolism-related transcripts previously identified in PBMC as potential biomarkers of metabolic health in a rat model were assessed in whole blood in an independent model, namely retinoblastoma haploinsufficient (Rb+/-) mice. Blood was collected and stored in RNAlater® at -80°C until analysis of selected transcripts by real-time RT-PCR. Comparable changes with fasting were detected in the expression of lipid metabolism-related genes when RNA from either PBMC or whole blood of rats or mice was used. HFD-induced excess body weight and fat mass associated with expected changes in the expression of metabolism-related genes in whole blood of mice. Changes in gene expression in whole blood of RA-treated mice reproduced known transcriptional actions of RA in hepatocytes and adipocytes. Reduced expression of Fasn, Lrp1, Rxrb and Sorl1 could be validated as early biomarkers of metabolic health in young Rb+/- mice using whole blood RNA. Altogether, these results support the use of whole blood RNA in studies aimed at identifying blood transcript-based biomarkers of nutritional/metabolic status or metabolic health. Results also support reduced expression of Fasn, Lrp1, Rxrb and Sorl1 in blood cells at young age as potential biomarkers of metabolic robustness. PMID:27163124

  18. Ultrastructural characterization of macrophage-like mononuclear leukocytes in human astrocytic tumors.

    PubMed

    Arismendi-Morillo, Gabriel; Castellano-Ramírez, Alan; Medina, Zulamita

    2010-12-01

    The aim of this study was to describe the ultrastructural features of macrophage-like mononuclear leukocytes associated with human astrocytic tumors. Tumoral biopsies of 10 patients with a pathological diagnosis of astrocytic tumor by means of transmission electron microscopy were examined. The macrophage-like mononuclear leukocyte shows ultrastructural characteristics related with the physiologic phenotype of the alternatively activated macrophage (M2), localized principally around of tumoral vasculature and tumor milieu; classically activated macrophages (M1) in surrounding necrosis areas were observed. The presence of these two ultrastructural kinds of macrophage-like mononuclear leukocytes into different areas of the tumor denotes that cellular response of TAMs is dependent of microenvironment stimuli in different parts of a tumor. The process of transvascular emigration of monocyte/macrophage-like mononuclear leukocytes into tumor is presented. The preponderance of alternatively activated macrophage-like mononuclear leukocytes suggests disequilibrium between pro-tumoral leukocytes and anti-tumoral leukocytes. Therefore, macrophage polarization toward anti-tumoral macrophage-like mononuclear leukocytes would be a potential target for therapeutic manipulation in human astrocytic tumors.

  19. Simple and Inexpensive Quantification of Ammonia in Whole Blood

    PubMed Central

    Ayyub, Omar B.; Behrens, Adam M.; Heligman, Brian T.; Natoli, Mary E.; Ayoub, Joseph J.; Cunningham, Gary; Summar, Marshall; Kofinas, Peter

    2015-01-01

    Quantification of ammonia in whole blood has applications in the diagnosis and management of many hepatic diseases, including cirrhosis and rare urea cycle disorders, amounting to more than 5 million patients in the United States. Current techniques for ammonia measurement suffer from limited range, poor resolution, false positives or large, complex sensor set-ups. Here we demonstrate a technique utilizing inexpensive reagents and simple methods for quantifying ammonia in 100 μl of whole blood. The sensor comprises a modified form of the indophenol reaction, which resists sources of destructive interference in blood, in conjunction with a cation-exchange membrane. The presented sensing scheme is selective against other amine containing molecules such as amino acids and has a shelf life of at least 50 days. Additionally, the resulting system has high sensitivity and allows for the accurate reliable quantification of ammonia in whole human blood samples at a minimum range of 25 to 500 μM, which is clinically for rare hyperammonemic disorders and liver disease. Furthermore, concentrations of 50 and 100 μM ammonia could be reliably discerned with p=0.0001. PMID:25936660

  20. Simple and inexpensive quantification of ammonia in whole blood.

    PubMed

    Ayyub, Omar B; Behrens, Adam M; Heligman, Brian T; Natoli, Mary E; Ayoub, Joseph J; Cunningham, Gary; Summar, Marshall; Kofinas, Peter

    2015-01-01

    Quantification of ammonia in whole blood has applications in the diagnosis and management of many hepatic diseases, including cirrhosis and rare urea cycle disorders, amounting to more than 5 million patients in the United States. Current techniques for ammonia measurement suffer from limited range, poor resolution, false positives or large, complex sensor set-ups. Here we demonstrate a technique utilizing inexpensive reagents and simple methods for quantifying ammonia in 100 μL of whole blood. The sensor comprises a modified form of the indophenol reaction, which resists sources of destructive interference in blood, in conjunction with a cation-exchange membrane. The presented sensing scheme is selective against other amine containing molecules such as amino acids and has a shelf life of at least 50 days. Additionally, the resulting system has high sensitivity and allows for the accurate reliable quantification of ammonia in whole human blood samples at a minimum range of 25 to 500 μM, which is clinically for rare hyperammonemic disorders and liver disease. Furthermore, concentrations of 50 and 100 μM ammonia could be reliably discerned with p = 0.0001.

  1. Evaluation of a standardised real-time PCR based DNA-detection method (Realstar®) in whole blood for the diagnosis of primary human cytomegalovirus (CMV) infections in immunocompetent patients.

    PubMed

    Berth, M; Benoy, I; Christensen, N

    2016-02-01

    Cytomegalovirus (CMV) DNA detection in blood could, as a supplementary test to serology, improve the accuracy and speed of diagnosis of an acute CMV infection. In this study we evaluated the performance of a commercially available and standardised CMV PCR assay in whole blood for the diagnosis of a primary infection in immunocompetent adults. Moreover, the kinetics of viral DNA was evaluated in order to provide a time frame in which viral DNA could be detected during an acute primary infection. Whole blood samples were collected from 66 patients with an acute CMV infection, 65 patients with an acute Epstein-Barr virus infection, 27 patients with various other acute infections (parvovirus B19, HIV, Toxoplasma gondii), 20 patients with past CMV infections (>1 year) and 20 apparently healthy persons. For CMV DNA detection and quantification a commercially available real-time PCR was applied (RealStar®, altona Diagnostics). The clinical sensitivity of CMV PCR in whole blood for the diagnosis of a recent primary CMV infection was 93.9 % and the diagnostic specificity 99.2 %. In the majority of the patients CMV DNA was not detectable anymore approximately within 4 weeks after the first blood sample was taken. From these data we concluded that, together with a suggestive serological profile, a positive CMV PCR result in whole blood can be regarded as a diagnostic confirmation of a recent CMV infection on a single blood sample in an immunocompetent patient. However, a negative CMV PCR result does not exclude a recent CMV infection.

  2. Whole blood: the future of traumatic hemorrhagic shock resuscitation.

    PubMed

    Murdock, Alan D; Berséus, Olle; Hervig, Tor; Strandenes, Geir; Lunde, Turid Helen

    2014-05-01

    Toward the end of World War I and during World War II, whole-blood transfusions were the primary agent in the treatment of military traumatic hemorrhage. However, after World War II, the fractionation of whole blood into its components became widely accepted and replaced whole-blood transfusion to better accommodate specific blood deficiencies, logistics, and financial reasons. This transition occurred with very few clinical trials to determine which patient populations or scenarios would or would not benefit from the change. A smaller population of patients with trauma hemorrhage will require massive transfusion (>10 U packed red blood cells in 24 h) occurring in 3% to 5% of civilian and 10% of military traumas. Advocates for hemostatic resuscitation have turned toward a ratio-balanced component therapy using packed red blood cells-fresh frozen plasma-platelet concentration in a 1:1:1 ratio due to whole-blood limited availability. However, this "reconstituted" whole blood is associated with a significantly anemic, thrombocytopenic, and coagulopathic product compared with whole blood. In addition, several recent military studies suggest a survival advantage of early use of whole blood, but the safety concerns have limited is widespread civilian use. Based on extensive military experience as well as recent published literature, low-titer leukocyte reduced cold-store type O whole blood carries low adverse risks and maintains its hemostatic properties for up to 21 days. A prospective randomized trial comparing whole blood versus ratio balanced component therapy is proposed with rationale provided.

  3. Separation of granulocytes from whole blood by leukoadhesion, phase 1

    NASA Technical Reports Server (NTRS)

    1976-01-01

    Capillary glass tubes are investigated for the separation and retrieval of large quantities of viable granulocytes and monocytes from whole blood on a continuous basis from a single donor. This effort represented the feasibility demonstration of a three phase program for development of a capillary tube cell separation device. The activity included the analysis and parametric laboratory testing with subscale models required to design a prototype device. Capillary tubes 40 cm long with a nominal 0.030 cm internal diameter yielded the highest total process efficiency. Recovery efficiencies as high as 89% of the adhering cell population were obtained. Granulocyte phagocytosis of latex particles indicated approximately 90% viability. Monocytes recovered from the separation column retained their capability to stimulate human bone marrow colony growth, as demonstrated in an in vitro cell culture assay.

  4. Paper‐Origami‐Based Multiplexed Malaria Diagnostics from Whole Blood

    PubMed Central

    Xu, Gaolian; Nolder, Debbie; Reboud, Julien; Oguike, Mary C.; van Schalkwyk, Donelly A.; Sutherland, Colin J.

    2016-01-01

    Abstract We demonstrate, for the first time, the multiplexed determination of microbial species from whole blood using the paper‐folding technique of origami to enable the sequential steps of DNA extraction, loop‐mediated isothermal amplification (LAMP), and array‐based fluorescence detection. A low‐cost handheld flashlight reveals the presence of the final DNA amplicon to the naked eye, providing a “sample‐to‐answer” diagnosis from a finger‐prick volume of human blood, within 45 min, with minimal user intervention. To demonstrate the method, we showed the identification of three species of Plasmodium, analyzing 80 patient samples benchmarked against the gold‐standard polymerase chain reaction (PCR) assay in an operator‐blinded study. We also show that the test retains its diagnostic accuracy when using stored or fixed reference samples. PMID:27554333

  5. Paper-Origami-Based Multiplexed Malaria Diagnostics from Whole Blood.

    PubMed

    Xu, Gaolian; Nolder, Debbie; Reboud, Julien; Oguike, Mary C; van Schalkwyk, Donelly A; Sutherland, Colin J; Cooper, Jonathan M

    2016-12-05

    We demonstrate, for the first time, the multiplexed determination of microbial species from whole blood using the paper-folding technique of origami to enable the sequential steps of DNA extraction, loop-mediated isothermal amplification (LAMP), and array-based fluorescence detection. A low-cost handheld flashlight reveals the presence of the final DNA amplicon to the naked eye, providing a "sample-to-answer" diagnosis from a finger-prick volume of human blood, within 45 min, with minimal user intervention. To demonstrate the method, we showed the identification of three species of Plasmodium, analyzing 80 patient samples benchmarked against the gold-standard polymerase chain reaction (PCR) assay in an operator-blinded study. We also show that the test retains its diagnostic accuracy when using stored or fixed reference samples.

  6. The synthesis of Rantes, G-CSF, IL-4, IL-5, IL-6, IL-12 and IL-13 in human whole-blood cultures is modulated by an extract from Eleutherococcus senticosus L. roots.

    PubMed

    Schmolz, M W; Sacher, F; Aicher, B

    2001-05-01

    An ethanol extract derived from the roots of Eleutherococcus senticosus was found to influence markedly the cytokine synthesis of activated whole blood cultures of ten healthy volunteers. Whereas the synthesis of Rantes was increased over a wide range of concentrations, the release of IL-4, IL-5 and IL-12 was significantly inhibited. An inhibition at higher concentrations, switching to a stimulation at lower doses of the extract was seen with G-CSF, IL-6 and IL-13. From these particular immuno-pharmacological effects of Eleutherococcus senticosus we suggest this herbal preparation possesses immuno-modulatory potency, rather than just being immuno-suppressive or -stimulating.

  7. Flow cytometric lymphocyte subset analysis using material from frozen whole blood.

    PubMed

    Alam, Iftikhar; Goldeck, David; Larbi, Anis; Pawelec, Graham

    2012-01-01

    Multicenter immune monitoring programs commonly rely on storing and shipping cryopreserved peripheral blood mononuclear cells (PBMC), isolated from whole blood before freezing. However, under many conditions in the field, facilities to separate PBMC are absent. Here, we investigate the feasibility of using whole blood (WB) frozen at -80°C as a source of viable lymphocytes for use in immunological studies. We compare the percentage of CD4 and CD8 T lymphocytes and their subsets from frozen WB with results from cryopreserved PBMC in five random healthy blood donors (three female, two male). We report that CD4 and CD8 values in lymphocytes from WB frozen up to 120 days were very similar to those of PBMC frozen up to 10 days. These data suggest that within the limits of parameters investigated in this study, contrary to our original assumptions, whole blood frozen at -80°C may in fact be an appropriate source of viable lymphocytes for T cell enumeration assays in immunological and epidemiological studies.

  8. Adjusting MtDNA Quantification in Whole Blood for Peripheral Blood Platelet and Leukocyte Counts

    PubMed Central

    Gonzalez-Lazaro, Monica; Moreno-Loshuertos, Raquel; Fernandez-Silva, Patricio; Enriquez, Jose Antonio; Laclaustra, Martin

    2016-01-01

    Alterations of mitochondrial DNA copy number (mtDNAcn) in the blood (mitochondrial to nuclear DNA ratio) appear associated with several systemic diseases, including primary mitochondrial disorders, carcinogenesis, and hematologic diseases. Measuring mtDNAcn in DNA extracted from whole blood (WB) instead of from peripheral blood mononuclear cells or buffy coat may yield different results due to mitochondrial DNA present in platelets. The aim of this work is to quantify the contribution of platelets to mtDNAcn in whole blood [mtDNAcn(WB)] and to propose a correction formula to estimate leukocytes' mtDNAcn [mtDNAcn(L)] from mtDNAcn(WB). Blood samples from 10 healthy adults were combined with platelet-enriched plasma and saline solution to produce artificial blood preparations. Aliquots of each sample were combined with five different platelet concentrations. In 46 of these blood preparations, mtDNAcn was measured by qPCR. MtDNAcn(WB) increased 1.07 (95%CI 0.86, 1.29; p<0.001) per 1000 platelets present in the preparation. We proved that leukocyte count should also be taken into account as mtDNAcn(WB) was inversely associated with leukocyte count; it increased 1.10 (95%CI 0.95, 1.25, p<0.001) per unit increase of the ratio between platelet and leukocyte counts. If hematological measurements are available, subtracting 1.10 the platelets/leukocyte ratio from mtDNAcn(WB) may serve as an estimation for mtDNAcn(L). Both platelet and leukocyte counts in the sample are important sources of variation if comparing mtDNAcn among groups of patients when mtDNAcn is measured in DNA extracted from whole blood. Not taking the platelet/leukocyte ratio into account in whole blood measurements, may lead to overestimation and misclassification if interpreted as leukocytes' mtDNAcn. PMID:27736919

  9. Whole blood tissue factor procoagulant activity is elevated in patients with sickle cell disease.

    PubMed

    Key, N S; Slungaard, A; Dandelet, L; Nelson, S C; Moertel, C; Styles, L A; Kuypers, F A; Bach, R R

    1998-06-01

    We developed a simple assay for the measurement of tissue factor procoagulant activity (TF PCA) in whole blood samples that avoids the need for mononuclear cell isolation. This method combines convenience of sample collection and processing with a high degree of sensitivity and specificity for TF. Using this method, we have determined that TF PCA is detectable in whole blood samples from normal individuals, which is itself a novel observation. Essentially all PCA could be shown to be localized in the mononuclear cell fraction of blood. Compared with controls, whole blood TF levels were significantly (P < .000001) elevated in patients with sickle cell disease (SCD), regardless of the subtype of hemoglobinopathy (SS or SC disease). No significant difference in TF PCA was observed between patients in pain crisis compared with those in steady-state disease. Because TF functions as cofactor in the proteolytic conversion of FVII to FVIIa in vitro, it was expected that an increase in circulating TF PCA would lead to an increased in vivo generation of FVIIa. On the contrary, FVIIa levels were actually decreased in the plasma of patients with SCD. Plasma TF pathway inhibitor (TFPI) antigen levels were normal in SCD patients, suggesting that accelerated clearance of FVIIa by the TFPI pathway was not responsible for the reduced FVIIa levels. We propose that elevated levels of circulating TF PCA may play an important role in triggering the activation of coagulation known to occur in patients with SCD. Because TF is the principal cellular ligand for FVIIa, it is possible that increased binding to TF accounts for the diminished plasma FVIIa levels.

  10. Whole blood is the sample matrix of choice for monitoring systemic triclocarban levels.

    PubMed

    Schebb, Nils Helge; Ahn, Ki Chang; Dong, Hua; Gee, Shirley J; Hammock, Bruce D

    2012-05-01

    The antibacterial triclocarban (TCC) concentrates in the cellular fraction of blood. Consequently, plasma levels are at least two-fold lower than the TCC amount present in blood. Utilizing whole blood sampling, a low but significant absorption of TCC from soap during showering is demonstrated for a small group of human subjects.

  11. Relationships of Whole Blood Serotonin and Plasma Norepinephrine within Families.

    ERIC Educational Resources Information Center

    Leventhal, Bennett L.; And Others

    1990-01-01

    This study of 47 families of autistic probands found that whole blood serotonin was positively correlated between autistic children and their mothers, fathers, and siblings, but plasma norepinephrine levels were not. (Author/JDD)

  12. Whole blood cyanide levels in patients with tobacco amblyopia.

    PubMed

    Jestico, J V; O'Brien, M D; Teoh, R; Toseland, P A; Wong, H C

    1984-06-01

    Three patients presented with painless bilateral visual failure due to tobacco amblyopia. The whole blood cyanide levels were raised above those predicted from their high tobacco consumption, approaching lethal levels reported from acute inhalation of cyanide. Each patient had an excessive alcohol intake with biochemical evidence of hepatic dysfunction, the elevated whole blood cyanide levels being attributed to the associated impairment of cyanide detoxification. In each case the improvement in visual acuities following abstinence and hydroxycobalamin therapy was accompanied by a reduction in the whole blood cyanide level to within the normal range. Serial measurements of whole blood cyanide, serum alcohol, and the detection of urinary nicotine provided valuable indices of the patient's subsequent compliance and clinical progress.

  13. Different responses of human mononuclear phagocyte populations to Mycobacterium tuberculosis.

    PubMed

    Duque, Camilo; Arroyo, Leonar; Ortega, Héctor; Montúfar, Franco; Ortíz, Blanca; Rojas, Mauricio; Barrera, Luis F

    2014-03-01

    Mycobacterium tuberculosis (Mtb) infects different populations of macrophages. Alveolar macrophages (AMs) are initially infected, and their response may contribute to controlling Mtb infection and dissemination. However, Mtb infection may disseminate to other tissues, infecting a wide variety of macrophages. Given the difficulty in obtaining AMs, monocyte-derived macrophages (MDMs) are used to model macrophage-mycobacteria interactions in humans. However, the response of other tissue macrophages to Mtb infection has been poorly explored. We have compared MDMs, AMs and splenic human macrophages (SMs) for their in vitro capacity to control Mtb growth, cytokine production, and induction of cell death in response to Mtb H37Rv, and the Colombian isolate UT205, and to the virulence factor ESAT-6. Significant differences in the magnitude of cell death and cytokine production depending mainly on the Mtb strain were observed; however, no major differences in the mycobacteriostatic/mycobacteriocidal activity were detected among the macrophage populations. Infection with the clinical isolate UT205 was associated with an increased cell death with membrane damage, particularly in IFNγ-treated SMs and H37Rv induced a higher production of cytokines compared to UT205. These results are concordant with the interpretation of a differential response to Mtb infection mainly depending upon the strain of Mtb.

  14. Enhanced production of prostaglandins and plasminogen activator during activation of human articular chondrocytes by products of mononuclear cells.

    PubMed

    Meats, J E; McGuire, M K; Ebsworth, N M; Englis, D J; Russell, R G

    1984-01-01

    We have examined the way in which products of cultured human blood mononuclear cells activate human articular chondrocytes. Conditioned medium from mononuclear cells enhanced the production of prostaglandin E by cultured human chondrocytes and also stimulated fibrinolytic activity in these cultures. These two effects may be interrelated, since the increased fibrinolysis in response to products of mononuclear cells was partially inhibited by indomethacin, an inhibitor of prostaglandin biosynthesis. The increased fibrinolysis is probably attributable to plasminogen activator, since it was strongly dependent on the presence of plasminogen. Increased amounts of PGE and chondroitin sulphate were also released from intact fragments of cartilage exposed to medium from cultured mononuclear cells. The time course and dose dependence of these effects were studied. The addition of exogenous arachidonic acid markedly enhanced production of PGE2. Ultrogel AcA54 was used to fractionate medium from cultured mononuclear cells and the chondrocyte-stimulating activity eluted with an apparent molecular weight between 12 000 and 25 000 daltons. Adherent and non-adherent mononuclear blood cells were also partially separated and conditioned medium from each was assayed for chondrocyte-stimulating factors. Both populations released factor(s) which increased the production of prostaglandin E by chondrocytes, but more activity came from the adherent mononuclear cells. The possible interrelationship between the chondrocyte activating factor studied here and others described in the literature is discussed.

  15. Rapid, label-free, electrical whole blood bioassay based on nanobiosensor systems.

    PubMed

    Chang, Hsiao-Kang; Ishikawa, Fumiaki N; Zhang, Rui; Datar, Ram; Cote, Richard J; Thompson, Mark E; Zhou, Chongwu

    2011-12-27

    Biomarker detection based on nanowire biosensors has attracted a significant amount of research effort in recent years. However, only very limited research work has been directed toward biomarker detection directly from physiological fluids mainly because of challenges caused by the complexity of media. This limitation significantly reduces the practical impact generated by the aforementioned nanobiosensors. In this study, we demonstrate an In(2)O(3) nanowire-based biosensing system that is capable of performing rapid, label-free, electrical detection of cancer biomarkers directly from human whole blood collected by a finger prick. Passivating the nanowire surface successfully blocked the signal induced by nonspecific binding when performing active measurement in whole blood. Passivated devices showed markedly smaller signals induced by nonspecific binding of proteins and other biomaterials in serum and higher sensitivity to target biomarkers than bare devices. The detection limit of passivated sensors for biomarkers in whole blood was similar to the detection limit for the same analyte in purified buffer solutions at the same ionic strength, suggesting minimal decrease in device performance in the complex media. We then demonstrated detection of multiple cancer biomarkers with high reliability at clinically meaningful concentrations from whole blood collected by a finger prick using this sensing system.

  16. Metals in air pollution particles decrease whole-blood coagulation time.

    PubMed

    Sangani, Rahul G; Soukup, Joleen M; Ghio, Andrew J

    2010-07-01

    The mechanism underlying procoagulative effects of air pollution particle exposure is not known. The authors tested the postulate that (1) the water-soluble components of an air pollution particle could affect whole-blood coagulation time and (2) metals included in this fraction were responsible for this effect. Exposure to the water-soluble fraction of particulate matter (PM), at doses as low as 50 ng/ml original particle, significantly diminished the whole-blood coagulation time. Inclusion of deferoxamine prolonged coagulation time following the exposures to the water-soluble fraction, whereas equivalent doses of ferroxamine had no effect. Except for nickel, all metal sulfates shortened the whole-blood coagulation time. Iron and zinc were two metals with the greatest capacity to reduce the coagulation time, with an effect observed at 10 ng/ml. Finally, in contrast to the anticoagulants citrate and EDTA, their iron complexes were found to be procoagulative. The authors conclude that metals in the water-soluble fraction of air pollution particles decrease whole-blood coagulation time. These metals can potentially contribute to procoagulative effects observed following human exposures to air pollution particles.

  17. Sensitive determination of xylenes in whole blood by capillary gas chromatography with cryogenic trapping.

    PubMed

    Hattori, H; Iwai, M; Kurono, S; Yamada, T; Watanabe-Suzuki, K; Ishii, A; Seno, H; Suzuki, O

    1998-11-06

    A new and sensitive method for measurement of o-, m- and p-xylenes in human whole blood by capillary gas chromatography (GC) with cryogenic trapping is presented. After heating 0.5 ml of whole blood and 0.5 ml of distilled water containing the xylenes and aniline (internal standard, I.S.) in a 4.0-ml vial at 100 degrees C for 30 min, 2 ml of the headspace vapor was drawn into a glass syringe. All vapor was introduced through the GC port into an AT-Wax middle-bore capillary column in the splitless mode at an oven temperature of 5 degrees C to trap the entire analytes, and the oven temperature was then programmed up to 180 degrees C. The present conditions gave sharp peaks for xylenes and aniline (I.S.), and low background noises for whole blood samples; the peaks of p- and m-xylenes showed about 90% separation with the AT-Wax column. As much as 41.0-46.3% of xylenes, which had been spiked to whole blood could be recovered. The calibration curves showed linearity in the range of 0.1-0.5 microg/0.5 ml of whole blood. The detection limit was estimated to be about 10 ng/0.5 ml. The coefficients of intra-day and inter-day variations for xylenes were not greater than 9.38%. The data for actual detection of xylenes in post-mortem blood of self-ignition suicide cases by the present method were also presented.

  18. Measurement and Comparison of Organic Compound Concentrations in Plasma, Whole Blood, and Dried Blood Spot Samples.

    PubMed

    Batterman, Stuart A; Chernyak, Sergey; Su, Feng-Chiao

    2016-01-01

    The preferred sampling medium for measuring human exposures of persistent organic compounds (POPs) is blood, and relevant sample types include whole blood, plasma, and dried blood spots (DBS). Because information regarding the performance and comparability of measurements across these sample types is limited, it is difficult to compare across studies. This study evaluates the performance of POP measurements in plasma, whole blood and DBS, and presents the distribution coefficients needed to convert concentrations among the three sample types. Blood samples were collected from adult volunteers, along with demographic and smoking information, and analyzed by GC/MS for organochlorine pesticides (OCPs), chlorinated hydrocarbons (CHCs), polychlorinated biphenyls (PCBs), and brominated diphenyl ethers (PBDEs). Regression models were used to evaluate the relationships between the sample types and possible effects of personal covariates. Distribution coefficients also were calculated using physically-based models. Across all compounds, concentrations in plasma were consistently the highest; concentrations in whole blood and DBS samples were comparable. Distribution coefficients for plasma to whole blood concentrations ranged from 1.74 to 2.26 for pesticides/CHCs, averaged 1.69 ± 0.06 for the PCBs, and averaged 1.65 ± 0.03 for the PBDEs. Regression models closely fit most chemicals (R (2) > 0.80), and whole blood and DBS samples generally showed very good agreement. Distribution coefficients estimated using biologically-based models were near one and did not explain the observed distribution. Among the study population, median concentrations of several pesticides/CHCs and PBDEs exceeded levels reported in the 2007-2008 National Health and Nutrition Examination Survey, while levels of other OCPs and PBDEs were comparable or lower. Race and smoking status appeared to slightly affect plasma/blood concentration ratios for several POPs. The experimentally

  19. A high-throughput assay of NK cell activity in whole blood and its clinical application

    SciTech Connect

    Lee, Saet-byul; Cha, Junhoe; Kim, Im-kyung; Yoon, Joo Chun; Lee, Hyo Joon; Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan; Lee, Jae Myun; Lee, Kang Young; Kim, Jongsun

    2014-03-14

    Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.

  20. Measurement of peripheral B cell subpopulations in common variable immunodeficiency (CVID) using a whole blood method

    PubMed Central

    Ferry, B L; Jones, J; Bateman, E A; Woodham, N; Warnatz, K; Schlesier, M; Misbah, S A; Peter, H H; Chapel, H M

    2005-01-01

    Recent reports have described reduced populations of CD27+ memory B cells and increased percentages of undifferentiated B cells in peripheral blood of patients with common variable immunodeficiency (CVID). This work has prompted two attempts to classify CVID based on rapid flow cytometric quantification of peripheral blood memory B cells and immature B cells. Evidence to support the hypothesis that such in vitro B cell classification systems correlate with clinical subtypes of CVID is being sought. For the classification to be useful in routine diagnosis, it is important that the flow cytometric method can be used without prior separation of peripheral blood mononuclear cells (PBMC). We have examined 23 CVID patients and 24 controls, using both PBMC and whole blood, and find an excellent correlation between these methods. The reproducibility of the method was excellent. We classified the CVID patients by all three of the existing classifications, including secretion of immunoglobulin by B cells in vitro as described by Bryant, as well as the more recent flow cytometric classification methods. Only one patient changed classification as a result of using whole blood. PMID:15932516

  1. Does whole blood coagulation analysis reflect developmental haemostasis?

    PubMed

    Ravn, Hanne Berg; Andreasen, Jo Bnding; Hvas, Anne-Mette

    2016-07-27

    Developmental haemostasis has been well documented over the last 3 decades and age-dependent reference ranges have been reported for a number of plasmatic coagulation parameters. With the increasing use of whole blood point-of-care tests like rotational thromboelastometry (ROTEM) and platelet function tests, an evaluation of age-dependent changes is warranted for these tests as well. We obtained blood samples from 149 children, aged 1 day to 5.9 years, and analysed conventional plasmatic coagulation tests, including activated partial prothrombin time, prothrombin time, and fibrinogen (functional). Whole blood samples were analysed using ROTEM to assess overall coagulation capacity and Multiplate analyzer to evaluate platelet aggregation. Age-dependent changes were analysed for all variables. We found age-dependent differences in all conventional coagulation tests (all P values < 0.05), but there was no sign of developmental changes in whole blood coagulation assessment when applying ROTEM, apart from clotting time in the EXTEM assay (P < 0.03). Despite marked differences in mean platelet aggregation between age groups, data did not reach statistical significance. Citrate-anticoagulated blood showed significantly reduced platelet aggregation compared with blood anticoagulated with heparin or hirudin (all P values < 0.003). We confirmed previous developmental changes in conventional plasmatic coagulation test. However, these age-dependent changes were not displayed in whole blood monitoring using ROTEM or Multiplate analyzer. Type of anticoagulant had a significant influence on platelet aggregation across all age groups.

  2. Metals in airpollution particles decrease whole blood coagulation time

    EPA Science Inventory

    The mechanism underlying the pro-coagulative effect of air pollution particle exposure is not known. We tested the postulate that 1) the soluble fraction ofan air pollution particle can affect whole blood coagulation time and 2) metals included in the soluble fraction are respons...

  3. Homogeneous assay for whole blood folate using photon upconversion.

    PubMed

    Arppe, Riikka; Mattsson, Leena; Korpi, Krista; Blom, Sami; Wang, Qi; Riuttamäki, Terhi; Soukka, Tero

    2015-02-03

    Red blood cell folate is measured for folate deficiency diagnosis, because it reflects the long-term folate level in tissues, whereas serum folate only represents the dietary intake. Direct homogeneous assay from whole blood would be ideal but conventional fluorescence techniques in blood suffer from high background and strong absorption of light at ultraviolet and visible wavelengths. In this study, a new photon upconversion-based homogeneous assay for whole blood folate is introduced based on resonance energy transfer from upconverting nanophosphor donor coated with folate binding protein to a near-infrared fluorescent acceptor dye conjugated to folate analogue. The sensitized acceptor emission is measured at 740 nm upon 980 nm excitation. Thus, optically transparent wavelengths are utilized for both donor excitation and sensitized acceptor emission to minimize the sample absorption, and anti-Stokes detection completely eliminates the Stokes-shifted autofluorescence. The IC50 value of the assay was 6.0 nM and the limit of detection (LOD) was 1 nM. The measurable concentration range was 2 orders of magnitude between 1.0-100 nM, corresponding to 40-4000 nM folate in the whole blood sample. Recoveries of added folic acid were 112%-114%. A good correlation was found when compared to a competitive heterogeneous assay based on the DELFIA-technology. The introduced assay provides a simple and fast method for whole blood folate measurement.

  4. Antibody conjugated magnetic iron oxide nanoparticles for cancer cell separation in fresh whole blood.

    PubMed

    Xu, Hengyi; Aguilar, Zoraida P; Yang, Lily; Kuang, Min; Duan, Hongwei; Xiong, Yonghua; Wei, Hua; Wang, Andrew

    2011-12-01

    A highly efficient process using iron oxide magnetic nanoparticles (IO)-based immunomagnetic separation of tumor cells from fresh whole blood has been developed. The process involved polymer coated 30 nm IO that was modified with antibodies (Ab) against human epithelial growth factor receptor 2 (anti-HER2 or anti-HER2/neu) forming IO-Ab. HER2 is a cell membrane protein that is overexpressed in several types of human cancer cells. Using a HER2/neu overexpressing human breast cancer cell line, SK-BR3, as a model cell, the IO-Ab was used to separate 73.6% (with a maximum capture of 84%) of SK-BR3 cells that were spiked in 1 mL of fresh human whole blood. The IO-Ab preferentially bound to SK-BR3 cells over normal cells found in blood due to the high level of HER2/neu receptor on the cancer cells unlike the normal cell surfaces. The results showed that the nanosized magnetic nanoparticles exhibited an enrichment factor (cancer cells over normal cells) of 1:10,000,000 in a magnetic field (with gradient of 100 T/m) through the binding of IO-Ab on the cell surface that resulted in the preferential capture of the cancer cells. This research holds promise for efficient separation of circulating cancer cells in fresh whole blood.

  5. 21 CFR 864.7140 - Activated whole blood clotting time tests.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Activated whole blood clotting time tests. 864....7140 Activated whole blood clotting time tests. (a) Identification. An activated whole blood clotting... pulmonary embolism by measuring the coagulation time of whole blood. (b) Classification. Class...

  6. 21 CFR 864.7140 - Activated whole blood clotting time tests.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Activated whole blood clotting time tests. 864....7140 Activated whole blood clotting time tests. (a) Identification. An activated whole blood clotting... pulmonary embolism by measuring the coagulation time of whole blood. (b) Classification. Class...

  7. 21 CFR 864.7140 - Activated whole blood clotting time tests.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Activated whole blood clotting time tests. 864....7140 Activated whole blood clotting time tests. (a) Identification. An activated whole blood clotting... pulmonary embolism by measuring the coagulation time of whole blood. (b) Classification. Class...

  8. 21 CFR 864.7140 - Activated whole blood clotting time tests.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Activated whole blood clotting time tests. 864....7140 Activated whole blood clotting time tests. (a) Identification. An activated whole blood clotting... pulmonary embolism by measuring the coagulation time of whole blood. (b) Classification. Class...

  9. 21 CFR 864.7140 - Activated whole blood clotting time tests.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Activated whole blood clotting time tests. 864....7140 Activated whole blood clotting time tests. (a) Identification. An activated whole blood clotting... pulmonary embolism by measuring the coagulation time of whole blood. (b) Classification. Class...

  10. Carvedilol differentially regulates cytokine production from activated human peripheral blood mononuclear cells.

    PubMed

    Yang, Shih-Ping; Ho, Ling-Jun; Cheng, Shu-Meng; Hsu, Yu-Lin; Tsao, Tien-Ping; Chang, Deh-Ming; Lai, Jenn-Haung

    2004-05-01

    Chronic inflammation is one of the important mechanisms involved in atherosclerosis formation. The activated monocytes and their secreted cytokines contribute significantly to this inflammatory process. Here we examined the effects of carvedilol, a recently introduced cardio-protective alpha-1- and beta-receptor blocker, on cytokine production from various stimuli-activated human immune effector cells. By ELISA analysis, we showed that carvedilol inhibited interferon-gamma (IFN-gamma), but enhanced interleukin (IL)-12 production in phytohemagglutinin (PHA)- and concanavalin A (ConA)-stimulated human peripheral blood mononuclear cells (PBMCs). The production of tumor necrosis factor-alpha (TNF-alpha) was marginally affected. When purified monocytes were examined, we observed the consistent up-regulation of IL-12 production while both IL-10 and TNF-alpha were unaffected or marginally down-regulated, respectively, by carvedilol. In agreement with the observation in monocytes, the production of IL-12 from activated macrophages was also up-regulated by carvedilol. We concluded that carvedilol might mediate its therapeutic effects through differentially regulating cytokine production from activated mononuclear cells, including at least monocytes and macrophages.

  11. Metabolic effects of microwave radiation and convection heating on human mononuclear leukocytes

    SciTech Connect

    Kiel, J.L.; Wong, L.S.; Erwin, D.N.

    1986-01-01

    The effects of microwave radiation (2450 MHz, continuous wave, mean specific absorption rate of 103.5 +/- 4.2 W/kg) and convection heating on the nonphosphorylating oxidative metabolism of human peripheral mononuclear leukocytes (96% lymphocytes, 4% monocytes) at 37 degrees C were investigated. Metabolic activity, determined by chemiluminescence (CL) of cells challenged with luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) linked to bovine serum albumin, was detected with a brightness photometer. A significant stimulation after microwave exposure (p less than 0.005) over total CL of matched 37 degrees C incubator controls was observed. A similar degree of stimulation compared to incubator controls was also detected after sham treatment. There was no significant difference between changes in total CL or stimulation indices of the microwave and sham exposed groups. It appears that exposure to microwave radiation, under normothermic (37 +/- 0.03 degrees C) conditions, has no effect on the oxidative metabolic activity of human peripheral mononuclear leukocytes. However, the significant differences between microwave or sham exposed cells and their respective incubator controls occurred because the temperature of the incubator controls did not exceed 35.9 degrees C and this temperature required 39 minutes to reach from 22 degrees C. Slow heating of incubator controls must be accounted for in thermal and radiofrequency radiation studies in vitro.

  12. High insulin and leptin increase resistin and inflammatory cytokine production from human mononuclear cells.

    PubMed

    Tsiotra, Panayoula C; Boutati, Eleni; Dimitriadis, George; Raptis, Sotirios A

    2013-01-01

    Resistin and the proinflammatory cytokines, such as TNF- α , IL-6, and IL-1 β , produced by adipocytes, and macrophages, are considered to be important modulators of chronic inflammation contributing to the development of obesity and atherosclerosis. Human monocyte-enriched mononuclear cells, from ten healthy individuals, were exposed to high concentrations of insulin, leptin, and glucose (alone or in combination) for 24 hours in vitro. Resistin, TNF- α , IL-6, and IL-1 β production was examined and compared to that in untreated cells. High insulin and leptin concentrations significantly upregulated resistin and the cytokines. The subsequent addition of high glucose significantly upregulated resistin and TNF- α mRNA and protein secretion, while it did not have any effect on IL-6 or IL-1 β production. By comparison, exposure to dexamethasone reduced TNF- α , IL-6, and IL-1 β production, while at this time point it increased resistin protein secretion. These data suggest that the expression of resistin, TNF- α , IL-6, and IL-1 β from human mononuclear cells, might be enhanced by the hyperinsulinemia and hyperleptinemia and possibly by the hyperglycemia in metabolic diseases as obesity, type 2 diabetes, and atherosclerosis. Therefore, the above increased production may contribute to detrimental effects of their increased adipocyte-derived circulating levels on systemic inflammation, insulin sensitivity, and endothelial function of these patients.

  13. DJ-1 isoforms in whole blood as potential biomarkers of Parkinson disease

    NASA Astrophysics Data System (ADS)

    Lin, Xiangmin; Cook, Travis J.; Zabetian, Cyrus P.; Leverenz, James B.; Peskind, Elaine R.; Hu, Shu-Ching; Cain, Kevin C.; Pan, Catherine; Edgar, John Scott; Goodlett, David R.; Racette, Brad A.; Checkoway, Harvey; Montine, Thomas J.; Shi, Min; Zhang, Jing

    2012-12-01

    DJ-1 is a multifunctional protein that plays an important role in oxidative stress, cell death, and synucleinopathies, including Parkinson disease. Previous studies have demonstrated that total DJ-1 levels decrease in the cerebrospinal fluid, but do not change significantly in human plasma from patients with Parkinson disease when compared with controls. In this study, we measured total DJ-1 and its isoforms in whole blood of patients with Parkinson disease at various stages, Alzheimer disease, and healthy controls to identify potential peripheral biomarkers of PD. In an initial discovery study of 119 subjects, 7 DJ-1 isoforms were reliably detected, and blood levels of those with 4-hydroxy-2-nonenal modifications were discovered to be altered in late-stage Parkinson disease. This result was further confirmed in a validation study of another 114 participants, suggesting that, unlike total DJ-1 levels, post-translationally modified isoforms of DJ-1 from whole blood are candidate biomarkers of late-stage Parkinson disease.

  14. Globin mRNA reduction for whole-blood transcriptome sequencing

    PubMed Central

    Krjutškov, Kaarel; Koel, Mariann; Roost, Anne Mari; Katayama, Shintaro; Einarsdottir, Elisabet; Jouhilahti, Eeva-Mari; Söderhäll, Cilla; Jaakma, Ülle; Plaas, Mario; Vesterlund, Liselotte; Lohi, Hannes; Salumets, Andres; Kere, Juha

    2016-01-01

    The transcriptome analysis of whole-blood RNA by sequencing holds promise for the identification and tracking of biomarkers; however, the high globin mRNA (gmRNA) content of erythrocytes hampers whole-blood and buffy coat analyses. We introduce a novel gmRNA locking assay (GlobinLock, GL) as a robust and simple gmRNA reduction tool to preserve RNA quality, save time and cost. GL consists of a pair of gmRNA-specific oligonucleotides in RNA initial denaturation buffer that is effective immediately after RNA denaturation and adds only ten minutes of incubation to the whole cDNA synthesis procedure when compared to non-blood RNA analysis. We show that GL is fully effective not only for human samples but also for mouse and rat, and so far incompletely studied cow, dog and zebrafish. PMID:27515369

  15. Integration of Genome-Wide SNP Data and Gene-Expression Profiles Reveals Six Novel Loci and Regulatory Mechanisms for Amino Acids and Acylcarnitines in Whole Blood

    PubMed Central

    Beutner, Frank; Holdt, Lesca M.; Gross, Arnd; Teren, Andrej; Tönjes, Anke; Becker, Susen; Krohn, Knut; Kovacs, Peter; Stumvoll, Michael; Teupser, Daniel; Thiery, Joachim; Ceglarek, Uta; Scholz, Markus

    2015-01-01

    Profiling amino acids and acylcarnitines in whole blood spots is a powerful tool in the laboratory diagnosis of several inborn errors of metabolism. Emerging data suggests that altered blood levels of amino acids and acylcarnitines are also associated with common metabolic diseases in adults. Thus, the identification of common genetic determinants for blood metabolites might shed light on pathways contributing to human physiology and common diseases. We applied a targeted mass-spectrometry-based method to analyze whole blood concentrations of 96 amino acids, acylcarnitines and pathway associated metabolite ratios in a Central European cohort of 2,107 adults and performed genome-wide association (GWA) to identify genetic modifiers of metabolite concentrations. We discovered and replicated six novel loci associated with blood levels of total acylcarnitine, arginine (both on chromosome 6; rs12210538, rs17657775), propionylcarnitine (chromosome 10; rs12779637), 2-hydroxyisovalerylcarnitine (chromosome 21; rs1571700), stearoylcarnitine (chromosome 1; rs3811444), and aspartic acid traits (chromosome 8; rs750472). Based on an integrative analysis of expression quantitative trait loci in blood mononuclear cells and correlations between gene expressions and metabolite levels, we provide evidence for putative causative genes: SLC22A16 for total acylcarnitines, ARG1 for arginine, HLCS for 2-hydroxyisovalerylcarnitine, JAM3 for stearoylcarnitine via a trans-effect at chromosome 1, and PPP1R16A for aspartic acid traits. Further, we report replication and provide additional functional evidence for ten loci that have previously been published for metabolites measured in plasma, serum or urine. In conclusion, our integrative analysis of SNP, gene-expression and metabolite data points to novel genetic factors that may be involved in the regulation of human metabolism. At several loci, we provide evidence for metabolite regulation via gene-expression and observed overlaps with GWAS

  16. Receptor expression and responsiveness of human peripheral blood mononuclear cells to a human cytomegalovirus encoded CC chemokine.

    PubMed

    Zheng, Qi; Xu, Jun; Gao, Huihui; Tao, Ran; Li, Wei; Shang, Shiqiang; Gu, Weizhong

    2015-01-01

    Human cytomegalovirus is a ubiquitous pathogen that infects the majority of the world's population. After long period of time co-evolving with human being, this pathogen has developed several strategies to evade host immune surveillance. One of the major trick is encoding homologous to those of the host organism or stealing host cellular genes that have significant functions in immune system. To date, we have found several viral immune analogous which include G protein coupled receptor, class I major histocompatibility complex and chemokine. Chemokine is a small group of molecules which is defined by the presence of four cysteines in highly conserved region. The four kinds of chemokines (C, CC, CXC, and CX3C) are classified based on the arrangement of 1 or 2 N-terminal cysteine residues. UL128 protein is one of the analogous that encoded by human cytomegalovirus that has similar amino acid sequences to the human CC chemokine. It has been proved to be one of the essential particles that involved in human cytomegalovirus entry into epithelial/endothelial cells as well as macrophages. It is also the target of potent neutralizing antibodies in human cytomegalovirus-seropositive individuals. We had demonstrated the chemotactic trait of UL128 protein in our previous study. Recombinant UL128 in vitro has the ability to attract monocytes to the infection region and enhances peripheral blood mononuclear cell proliferation by activating the MAPK/ERK signaling pathway. However, the way that this viral encoded chemokine interacting with peripheral blood mononuclear cells and the detailed mechanism that involving the virus entry into host cells keeps unknown. Here we performed in vitro investigation into the effects of UL128 protein on peripheral blood mononuclear cell's activation and receptor binding, which may help us further understand the immunomodulatory function of UL128 protein as well as human cytomegalovirus diffusion mechanism.

  17. Platelet concentrates, from whole blood or collected by apheresis?

    PubMed

    van der Meer, Pieter F

    2013-04-01

    Platelet concentrates can be isolated from donated whole blood with the platelet-rich plasma-method or the buffy coat-method. Alternatively, platelets can be obtained by apheresis, harvesting the platelets but returning all other cells to the donor. The quality and characteristics of platelets during storage are affected by a number of factors, such as anticoagulant, centrifugation and processing after collection, and pre- or post storage pooling, but when comparing literature on the various methods, most differences balance out. To have sufficient platelets to treat an adult patient, whole-blood-derived platelet concentrates need pooling of multiple donations, thereby increasing the risk of infectious agent transmission at least two-fold as compared with apheresis units. Allo immunization rates, acute reaction rates, and transfusion related acute lung injury rates are not different. Apheresis donation procedures have fewer adverse events. All these factors need to be considered and weighed when selecting a method of platelet collection for a blood center.

  18. Velocity measurements in whole blood using acoustic resolution photoacoustic Doppler

    PubMed Central

    Brunker, Joanna; Beard, Paul

    2016-01-01

    Acoustic resolution photoacoustic Doppler velocimetry promises to overcome the spatial resolution and depth penetration limitations of current blood flow measuring methods. Despite successful implementation using blood-mimicking fluids, measurements in blood have proved challenging, thus preventing in vivo application. A common explanation for this difficulty is that whole blood is insufficiently heterogeneous relative to detector frequencies of tens of MHz compatible with deep tissue photoacoustic measurements. Through rigorous experimental measurements we provide new insight that refutes this assertion. We show for the first time that, by careful choice of the detector frequency and field-of-view, and by employing novel signal processing methods, it is possible to make velocity measurements in whole blood using transducers with frequencies in the tens of MHz range. These findings have important implications for the prospects of making deep tissue measurements of blood flow relevant to the study of microcirculatory abnormalities associated with cancer, diabetes, atherosclerosis and other conditions. PMID:27446707

  19. Whole blood analysis rotor assembly having removable cellular sedimentation bowl

    DOEpatents

    Burtis, C.A.; Johnson, W.F.

    1975-08-26

    A rotor assembly for performing photometric analyses using whole blood samples is described. Following static loading of a gross blood sample within a centrally located, removable, cell sedimentation bowl, the red blood cells in the gross sample are centrifugally separated from the plasma, the plasm displaced from the sedimentation bowl, and measured subvolumes of plasma distributed to respective sample analysis cuvettes positioned in an annular array about the rotor periphery. Means for adding reagents to the respective cuvettes are also described. (auth)

  20. Analysis of cyanide in whole blood of dosed cathartids

    USGS Publications Warehouse

    Krynitsky, A.J.; Wiemeyer, Stanley N.; Hill, E.F.; Carpenter, J.W.

    1986-01-01

    A gas-liquid chromatographic method was modified to quantify both unmetabolized ('free') and metabolized ('bound', i.e., thiocyanates) cyanides. The methods for both are efficient and sensitive to 0.05 ppm. Repeated freezing and thawing of whole blood from treated cathartids caused an initial increase in free cyanide concentrations, followed by a gradual decline to a plateau. Bound cyanide concentrations declined after repeated freezing and thawing.

  1. Particulate matter induces prothrombotic microparticle shedding by human mononuclear and endothelial cells.

    PubMed

    Neri, Tommaso; Pergoli, Laura; Petrini, Silvia; Gravendonk, Lotte; Balia, Cristina; Scalise, Valentina; Amoruso, Angela; Pedrinelli, Roberto; Paggiaro, Pierluigi; Bollati, Valentina; Celi, Alessandro

    2016-04-01

    Particulate airborne pollution is associated with increased cardiopulmonary morbidity. Microparticles are extracellular vesicles shed by cells upon activation or apoptosis involved in physiological processes such as coagulation and inflammation, including airway inflammation. We investigated the hypothesis that particulate matter causes the shedding of microparticles by human mononuclear and endothelial cells. Cells, isolated from the blood and the umbilical cords of normal donors, were cultured in the presence of particulate from a standard reference. Microparticles were assessed in the supernatant as phosphatidylserine concentration. Microparticle-associated tissue factor was assessed by an one-stage clotting assay. Nanosight technology was used to evaluate microparticle size distribution. Particulate matter induces a dose- and time- dependent, rapid (1h) increase in microparticle generation in both cells. These microparticles express functional tissue factor. Particulate matter increases intracellular calcium concentration and phospholipase C inhibition reduces microparticle generation. Nanosight analysis confirmed that upon exposure to particulate matter both cells express particles with a size range consistent with the definition of microparticles (50-1000 nm). Exposure of mononuclear and endothelial cells to particulate matter upregulates the generation of microparticles at least partially mediated by calcium mobilization. This observation might provide a further link between airborne pollution and cardiopulmonary morbidity.

  2. Retinoid modulation of collagenase production by adherent human mononuclear cells in culture.

    PubMed Central

    Ohta, A; Louie, J S; Uitto, J

    1987-01-01

    Previous observations have suggested that retinoids might be useful for the treatment of rheumatoid arthritis. In this study we examined the effects of various retinoids on collagenase production by adherent human peripheral blood mononuclear cells in culture. We have previously shown that these cells, consisting predominantly of monocyte-macrophages, actively synthesize and secrete collagenase upon stimulation with concanavalin A. The cells were incubated in serum free medium with all-trans-retinoic acid, 13-cis-retinoic acid, all-trans-retinal, or Ro 10-9359 (trimethylmethoxyphenyl retinoic acid ethyl ester) for up to 72 hours, and the collagenase activity was determined with [3H]proline labelled type I collagen as substrate. The incubation of mononuclear cells with all-trans-retinoic acid in the concentration range 10(-7)-10(-5) mol/l resulted in a dose dependent inhibition of the collagenase production. All-trans-retinal was also a potent inhibitor, whereas 13-cis-retinoic acid and Ro 10-9359 in a concentration of 10(-5) mol/l had a lesser effect. Control experiments indicated that the inhibition of collagenase production by all-trans-retinoic acid did not result from inhibition of total protein synthesis nor could it be explained by induction of an inhibitory molecule. These results indicate that retinoids with distinct structural features can inhibit collagenase production by monocyte-macrophages, and suggest a role for retinoids in the treatment of rheumatoid arthritis. PMID:3036026

  3. Molecular recognition of HER-1 in whole-blood samples.

    PubMed

    Moldoveanu, Iuliana; Stanciu Gavan, Camelia; Stefan-van Staden, Raluca-Ioana

    2014-11-01

    Multimode sensing was proposed for molecular screening and recognition of HER-1 in whole blood. The tools used for molecular recognition were platforms based on nanostructured materials such as the complex of Mn(III) with meso-tetra (4-carboxyphenyl) porphyrin, and maltodextrin (dextrose equivalence between 4 and 7), immobilized in diamond paste, graphite paste or C60 fullerene paste. The identification of HER-1 in whole-blood samples, at molecular level, is performed using stochastic mode and is followed by the quantification of it using stochastic and differential pulse voltammetry modes. HER-1 can be identified in the concentration range between 280 fg/ml and 4.86 ng/ml using stochastic mode, this making possible the early detection of cancers such as gastrointestinal, pancreatic and lung cancers. The recovery tests performed using whole-blood samples proved that the platforms can be used for identification and quantification of HER-1 with high sensitivity and reliability in such samples, these making them good molecular screening tools.

  4. Low titer group O whole blood in emergency situations.

    PubMed

    Strandenes, Geir; Berséus, Olle; Cap, Andrew P; Hervig, Tor; Reade, Michael; Prat, Nicolas; Sailliol, Anne; Gonzales, Richard; Simon, Clayton D; Ness, Paul; Doughty, Heidi A; Spinella, Philip C; Kristoffersen, Einar K

    2014-05-01

    In past and ongoing military conflicts, the use of whole blood (WB) as a resuscitative product to treat trauma-induced shock and coagulopathy has been widely accepted as an alternative when availability of a balanced component-based transfusion strategy is restricted or lacking. In previous military conflicts, ABO group O blood from donors with low titers of anti-A/B blood group antibodies was favored. Now, several policies demand the exclusive use of ABO group-specific WB. In this short review, we argue that the overall risks, dangers, and consequences of "the ABO group-specific approach," in emergencies, make the use of universal group O WB from donors with low titers of anti-A/B safer. Generally, risks with ABO group-specific transfusions are associated with in vivo destruction of the red blood cells transfused. The risk with group O WB is from the plasma transfused to ABO-incompatible patients. In the civilian setting, the risk of clinical hemolytic transfusion reactions (HTRs) due to ABO group-specific red blood cell transfusions is relatively low (approximately 1:80,000), but the consequences are frequently severe. Civilian risk of HTRs due to plasma incompatible transfusions, using titered donors, is approximately 1:120,000 but usually of mild to moderate severity. Emergency settings are often chaotic and resource limited, factors well known to increase the potential for human errors. Using ABO group-specific WB in emergencies may delay treatment because of needed ABO typing, increase the risk of clinical HTRs, and increase the severity of these reactions as well as increase the danger of underresuscitation due to lack of some ABO groups. When the clinical decision has been made to transfuse WB in patients with life-threatening hemorrhagic shock, we recommend the use of group O WB from donors with low anti-A/B titers when logistical constraints preclude the rapid availability of ABO group-specific WB and reliable group matching between donor and recipient is

  5. Enhanced Detection of Rift Valley Fever Virus using Molecular Assays on Whole Blood Samples

    PubMed Central

    Grolla, Allen; Mehedi, Masfique; Lindsay, Robbin; Bosio, Catharine; Duse, Adriano; Feldmann, Heinz

    2012-01-01

    Background Rift Valley fever (RVF) is an emerging arthropod-borne zoonoses of global agricultural and public health importance. In December 2006, an RVF outbreak was recognized in Kenya which led to the deployment of international response laboratory teams to the area. Objectives A field laboratory was operated in Malindi, Kenya to provide safe sample handling and molecular testing for RVF virus (RVFV) as well as selected other pathogens for differential diagnosis. Study Design Safe sample handling was carried out using a negative pressure flexible film isolator (glovebox) and commercial reagents to inactivate clinical specimens and purify nucleic acid. Whole blood was routinely used for diagnostic testing although paired plasma samples were also tested in select cases. Subsequently, human macrophages were tested in vitro for their susceptibility to RVFV. Results The field laboratory received samples from 33 individuals and a definite laboratory diagnosis was provided in 16 of these cases. Using molecular diagnostic techniques, RVFV was more consistently detected in whole blood than in plasma samples most likely due to association of RVFV with blood cells. Subsequent in vitro studies identified macrophages as a target cell for RVFV replication. Conclusions RVFV appears to replicate in blood cells such as macrophages. Thus, the sensitivity of molecular diagnostic testing is improved if whole blood is used as the clinical specimen rather than plasma or serum. PMID:22632901

  6. Influence of testosterone and a novel SARM on gene expression in whole blood of Macaca fascicularis.

    PubMed

    Riedmaier, Irmgard; Tichopad, Ales; Reiter, Martina; Pfaffl, Michael W; Meyer, Heinrich H D

    2009-04-01

    Anabolic hormones, including testosterone, have been suggested as a therapy for aging-related conditions, such as osteoporosis and sarcopenia. These therapies are sometimes associated with severe androgenic side effects. A promising alternative to testosterone replacement therapy are selective androgen receptor modulators (SARMs). SARMs have the potential to mimic the desirable central and peripheral androgenic anabolic effects of testosterone without having its side effects. In this study we evaluated the effects of LGD2941, in comparison to testosterone, on mRNA expression of selected target genes in whole blood in an non-human model. The regulated genes can act as potential blood biomarker candidates in future studies with AR ligands. Cynomolgus monkeys (Macaca fascicularis) were treated either with testosterone or LGD2941 for 90 days in order to compare their effects on mRNA expression in blood. Blood samples were taken before SARM application, on day 16 and on day 90 of treatment. Gene expression of 37 candidate genes was measured using quantitative real-time RT-PCR (qRT-PCR) technology. Our study shows that both testosterone and LGD2941 influence mRNA expression of 6 selected genes out of 37 in whole blood. The apoptosis regulators CD30L, Fas, TNFR1 and TNFR2 and the interleukins IL-12B and IL-15 showed significant changes in gene expression between control and the treatment groups and represent potential biomarkers for androgen receptor ligands in whole blood.

  7. I am the 9%: Making the case for whole-blood platelets.

    PubMed

    Seheult, J N; Triulzi, D J; Yazer, M H

    2016-06-01

    Over the last 15 years, there has been a trend in the United States towards the increasing use of apheresis platelet (AP) concentrates over whole-blood-derived platelets (WBP). Although 1-h- and 24-h-corrected count increments tend to be higher with AP, this does not translate into improved haemostatic efficiency when used to prevent bleeding in haematology/oncology patients. WBP expose the recipient to more donors than apheresis products. However, recent studies have shown no significant differences in the rates of bacterial contamination, human leukocyte antigen alloimmunisation, RhD alloimmunisation, transfusion-related acute lung injury or febrile non-haemolytic transfusion reactions between these two products. Given the overall low rates of virally contaminated units in the era of nucleic acid testing and rigorous donor screening, the difference in donor exposures of 4-6 vs 1 has minimal clinical relevance. Although studies point to a marginally increased risk of donor adverse events associated with WBP, the absolute risk is too miniscule to act as a deterrent to making whole-blood donations. Both types of platelet concentrates should therefore be considered clinically equivalent; in this light, the most responsible use of the community donor resource pool, which both optimises the utility of a whole-blood donation and meets the clinical needs of thrombocytopenic recipients, is to have a mix of both types of platelet products so as to mitigate the risk of shortages.

  8. A field-deployable device for the rapid detection of cyanide poisoning in whole blood

    NASA Astrophysics Data System (ADS)

    Boehringer, Hans; Tong, Winnie; Chung, Roy; Boss, Gerry; O'Farrell, Brendan

    2012-06-01

    Feasibility of a field-deployable device for the rapid and early diagnosis of cyanide poisoning in whole blood using the spectral shift of the vitamin B12 precursor cobinamide upon binding with cyanide as an indicator is being assessed. Cyanide is an extremely potent and rapid acting poison with as little as 50 mg fatal to humans. Cyanide poisoning has been recognized as a threat from smoke inhalation and potentially through weapons of mass destruction. Currently, no portable rapid tests for the detection of cyanide in whole blood are available. Cobinamide has an extremely high affinity for cyanide and captures hemoglobin associated cyanide from red blood cells. Upon binding of cyanide, cobinamide undergoes a spectral shift that can be measured with a spectrophotometer. We have combined the unique cyanide-binding properties of cobinamide with blood separation technology, sample transport and a detection system, and are developing a rapid, field deployable, disposable device which will deliver an intuitive result to a first responder, allowing for rapid response to exposure events. Feasibility of the cobinamide-Cyanide chemistry in a rapid test using a whole blood sample from a finger-stick has been demonstrated with an assay time from sample collection to a valid result of under 5 minutes. Data showing the efficacy of the diagnostic method and initial device design concepts will be shown.

  9. Whole blood assay for trypsin activity using polyanionic focusing gel electrophoresis.

    PubMed

    Lefkowitz, Roy B; Schmid-Schönbein, Geert W; Heller, Michael J

    2010-07-01

    The measurement of trypsin activity directly in blood is important for the development of novel diagnostics and for biomedical research. Presently, most degradative enzyme assays require sample preparation, making them time consuming, costly, and less accurate. We recently demonstrated a simple and rapid electrophoretic assay for the measurement of trypsin activity directly in whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target enzyme. This fragment is then rapidly separated from whole blood by electrophoresis and quantified with a fluorescent detector. In this study, we demonstrate that polyanionic poly-L-glutamic acid-doped polyacrylamide gels can focus the fluorescent cleavage product and markedly improve the LODs of the assay. A LOD of 2 pg in 6 microL (0.3 ng/mL) in whole human blood was achieved after a 1-h reaction of enzyme and substrate followed by 10 min of electrophoresis. This is 50- to 200-fold better than the estimated reference levels for trypsin (15-60 ng/mL) in blood. This straightforward technique now allows for the rapid measurement of clinically relevant levels of trypsin activity in microliter volumes of whole blood, providing a useful tool for the development of novel point-of-care diagnostics.

  10. Stimulation of production of prostaglandin E in gingival cells exposed to products of human blood mononuclear cells.

    PubMed Central

    D'Souza, S M; Englis, D J; Clark, A; Russell, R G

    1981-01-01

    1. Supernatant media from cultures of unstimulated human peripheral blood mononuclear cells contained one or more factors that increased by several hundred-fold the production of prostaglandin E by fibroblast-like cells derived from both inflamed and normal human gingival tissue. 2. This stimulation occurred in a dose-dependent manner and was completely inhibited by 14 microM-indomethacin. 3. Responsiveness to the factor declined as the age of the cell culture increased. 4. An increase in prostaglandin E production was first observed after a 2h exposure to the mononuclear cell factor(s) and could be prevented by cycloheximide. 5. Brief exposure (0.5 and 1.0 h) to mononuclear cell factor did not increase prostaglandin E production by the cells in a subsequent 72 h incubation in the absence of mononuclear cell factor. 6. Addition of arachidonate (10 microM and 15 microM) further enhanced stimulation of prostaglandin E production in response to mononuclear cell factor. 7. The stimulatory activity was resistant to digestion by trypsin, but was heat-labile, so that only 17% remained after treatment at 56 degrees C for 30 min. PMID:6798975

  11. Stimulation of production of prostaglandin E in gingival cells exposed to products of human blood mononuclear cells.

    PubMed

    D'Souza, S M; Englis, D J; Clark, A; Russell, R G

    1981-08-15

    1. Supernatant media from cultures of unstimulated human peripheral blood mononuclear cells contained one or more factors that increased by several hundred-fold the production of prostaglandin E by fibroblast-like cells derived from both inflamed and normal human gingival tissue. 2. This stimulation occurred in a dose-dependent manner and was completely inhibited by 14 microM-indomethacin. 3. Responsiveness to the factor declined as the age of the cell culture increased. 4. An increase in prostaglandin E production was first observed after a 2h exposure to the mononuclear cell factor(s) and could be prevented by cycloheximide. 5. Brief exposure (0.5 and 1.0 h) to mononuclear cell factor did not increase prostaglandin E production by the cells in a subsequent 72 h incubation in the absence of mononuclear cell factor. 6. Addition of arachidonate (10 microM and 15 microM) further enhanced stimulation of prostaglandin E production in response to mononuclear cell factor. 7. The stimulatory activity was resistant to digestion by trypsin, but was heat-labile, so that only 17% remained after treatment at 56 degrees C for 30 min.

  12. In vitro expansion of Lin+ and Lin- mononuclear cells from human peripheral blood

    NASA Astrophysics Data System (ADS)

    Norhaiza, H. Siti; Rohaya, M. A. W.; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul

    2013-11-01

    Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin-) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin+) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin- cell population. The ability of Lin+ and Lin- to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin+ and Lin- were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin+ mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin- stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation medium support Lin

  13. Human mononuclear cell function after 4 C storage during 1-G and microgravity conditions of spaceflight

    NASA Technical Reports Server (NTRS)

    Meehan, Richard; Taylor, Gerald; Lionetti, Fabian; Neale, Laurie; Curren, Tim

    1989-01-01

    To investigate the possibility of restoring immune competence of crewmembers during a prolonged spaceflight by infusions of autologous blood components, the effect of storage at 4 C aboard Space Shuttle Columbia (Mission 61-c) on the activity of human peripheral blood mononuclear cells (PBMNCs), stored as leukocyte concentrates in autologous plasa, was investigated. The results of preflight storage at 4 C demonstrated a progressive daily loss in mitogen-stimulated protein synthesis, and thymidine uptake, as well as a progressive reduction in the percentage of PBMNCs expressing cell-surface phenotype markers. The ability of PBMNCs stored at 4 C for 8 d in Columbia's middeck, to become activated and proliferate in vitro was similar to that of cells that remained for 7 d on ground.

  14. Grass immunotherapy induces inhibition of allergen-specific human peripheral blood mononuclear cell proliferation.

    PubMed

    Baskar, S; Hamilton, R G; Norman, P S; Ansari, A A

    1997-02-01

    The peripheral blood mononuclear cells (PBMC) from humans allergic to grass pollens (GR+ subjects) show strong in vitro proliferative responses to purified allergens from Lolium perenne pollen Lol p 1, and to a lesser extent to Lol p 2 and Lol p 3. By contrast, PBMC from grass allergic patients undergoing immunotherapy (GR + IT subjects) exhibit a very poor Lol p-specific proliferative response, similar to that observed in nongrass allergic subjects (GR-subjects). Unlike GR-subjects, both GR+ and GR + IT subjects have high levels of antigen-specific serum IgG and IgE antibodies to Lol p 1, Lol p 2 and Lol p 3. While GR+ subjects exhibit a significant correlation between antigen-specific serum antibody and PBMC responses, GR + IT subjects do not show a correlation between the two responses. The possible mechanisms by which immunotherapy may modulate allergen-specific T cell proliferative response are discussed.

  15. The effect of catechol on human peripheral blood mononuclear cells (in vitro study).

    PubMed

    Bukowska, Bożena; Michałowicz, Jaromir; Marczak, Agnieszka

    2015-01-01

    Catechol also known as pyrocatechol or 1,2-dihydroxybenzene is formed endogenously in the organism from neurotransmitters including adrenaline, noradrenaline, and dopamine. It is also a metabolite of many drugs like DOPA, isoproterenol or aspirin and it is also formed in the environment during transformation of various xenobiotics. We evaluated in vitro the effect of catechol on the structure and function of human peripheral blood mononuclear cells (PBMCs). The cells were incubated with xenobiotic at concentration range from 2 to 500μg/mL for 1h. Human blood mononuclear cells were obtained from leucocyte-platelet buffy coat taken from healthy donors in the Blood Bank of Łódź, Poland. Using flow cytometry we have evaluated necrotic, apoptotic and morphological changes in PBMCs incubated with catechol. Moreover, we have estimated changes in reactive oxygen species (ROS) formation, protein carbonylation and lipid peroxidation in the cells studied. The compound studied provoked necrotic (from 250μg/mL), apoptotic (from 100μg/mL), and morphological changes (from 250μg/mL) in the incubated cells. We have also noted that catechol decreased H2DCF oxidation at 2 and 10μg/mL but at higher concentrations of 250 and 500μg/mL it caused statistically significant increase in the oxidation of this probe. We also observed an increase in lipid peroxidation (from 250μg/mL) and protein carbonylation (from 50μg/mL) of PBMCs. It was observed that catechol only at high concentrations was capable of inducing changes in PBMCs. The obtained results clearly showed that catechol may induce change in PBMCs only in the caste of poisoning with this compound.

  16. MHC-unrestricted lysis of MUC1-expressing cells by human peripheral blood mononuclear cells.

    PubMed

    Wright, Stephen E; Rewers-Felkins, Kathleen A; Quinlin, Imelda S; Fogler, William E; Phillips, Catherine A; Townsend, Mary; Robinson, William; Philip, Ramila

    2008-01-01

    Many human adenocarcinomas can be killed in vitro by targeted cytotoxic T-lymphocytes (CTL); however, major histocompatibility complex (MHC)-restrictions are typically required. The MUC1 antigen is common in many human adenocarcinomas, and is associated with a variable number of tandem repeats. It has been proposed that antigens with such repeated epitopes may be vulnerable to cytotoxic T-lymphocyte killing without MHC-restriction. Therefore, it is possible that MUC1-expressing malignant cells may be killed by targeted cytotoxic T-lymphocyte in the absence of MHC-restriction. In this study, a human MUC1-expressing murine mammary carcinoma cell line was used to determine if cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells requires MHC-restriction. Specifically, MUC1-stimulated human mononuclear cells (M1SMC) were observed to kill human MUC1-transfected, MUC1-expressing murine mammary carcinoma cells, but not the mock-transfected, non-MUC1-expressing murine mammary carcinoma cells. Furthermore, the killing was blocked by antibody to MUC1, indicating MUC1-specific killing. In conclusion, cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells can be MHC-unrestricted.

  17. Whole blood tissue factor procoagulant activity remains detectable during severe aplasia following bone marrow and peripheral blood stem cell transplantation.

    PubMed

    Ozcan, M; Morton, C T; Solovey, A; Dandelet, L; Bach, R R; Hebbel, R P; Slungaard, A; Key, N S

    2001-02-01

    Using a novel whole blood assay, we recently demonstrated that tissue factor procoagulant activity (TF PCA) is present in normal individuals. Preliminary experiments suggested that this activity is localized in the mononuclear cell fraction. Postulating that whole blood TF PCA would therefore be undetectable when monocytes and neutrophils are absent from peripheral blood, we assayed TF PCA during the peri-transplant period in 15 consecutive patients undergoing allogeneic (n = 12) or autologous (n = 3) bone marrow transplantation (BMT) or peripheral blood stem cell transplantation (PBSCT). Baseline (pre-transplant) mean TF PCA was higher in patients compared to normal controls (P <0.005). Unexpectedly, although TF PCA during the period of profound aplasia was significantly reduced compared to baseline (p <0.05), fully 55% of the initial activity remained detectable. During the engraftment phase, TF PCA returned to pre-transplant levels, with a linear correlation between monocyte counts and TF PCA (r = 0.63). In contrast to normal whole blood, incubation of aplastic samples with E. Coli lipopolysaccharide ex vivo failed to induce TF PCA. Throughout the period of study--but especially during the aplastic phase--the absolute number of circulating endothelial cells (CECs) that were TF antigen-positive was increased compared to normals (P <0.001). However, removal of these cells from whole blood samples failed to significantly diminish total TF PCA indicating that CECs alone could not account for the detectable TF PCA during aplasia. We conclude that neither circulating mature myelo-monocytic cells nor endothelial cells can account for all the functionally intact TF in peripheral blood. Further studies are needed to identify the other source(s) of TF PCA.

  18. Platelet dynamics in three-dimensional simulation of whole blood.

    PubMed

    Vahidkhah, Koohyar; Diamond, Scott L; Bagchi, Prosenjit

    2014-06-03

    A high-fidelity computational model using a 3D immersed boundary method is used to study platelet dynamics in whole blood. We focus on the 3D effects of the platelet-red blood cell (RBC) interaction on platelet margination and near-wall dynamics in a shear flow. We find that the RBC distribution in whole blood becomes naturally anisotropic and creates local clusters and cavities. A platelet can enter a cavity and use it as an express lane for a fast margination toward the wall. Once near the wall, the 3D nature of the platelet-RBC interaction results in a significant platelet movement in the transverse (vorticity) direction and leads to anisotropic platelet diffusion within the RBC-depleted zone or cell-free layer (CFL). We find that the anisotropy in platelet motion further leads to the formation of platelet clusters, even in the absence of any platelet-platelet adhesion. The transverse motion, and the size and number of the platelet clusters are observed to increase with decreasing CFL thickness. The 3D nature of the platelet-RBC collision also induces fluctuations in off-shear plane orientation and, hence, a rotational diffusion of the platelets. Although most marginated platelets are observed to tumble just outside the RBC-rich zone, platelets further inside the CFL are observed to flow with an intermittent dynamics that alters between sliding and tumbling, as a result of the off-shear plane rotational diffusion, bringing them even closer to the wall. To our knowledge, these new findings are based on the fundamentally 3D nature of the platelet-RBC interaction, and they underscore the importance of using cellular-scale 3D models of whole blood to understand platelet margination and near-wall platelet dynamics.

  19. 77 FR 59000 - Guidance for Industry: Pre-Storage Leukocyte Reduction of Whole Blood and Blood Components...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-25

    ... HUMAN SERVICES Food and Drug Administration Guidance for Industry: Pre-Storage Leukocyte Reduction of Whole Blood and Blood Components Intended for Transfusion; Availability AGENCY: Food and Drug Administration, HHS. ACTION: Notice. SUMMARY: The Food and Drug Administration (FDA) is announcing...

  20. Identification of Recent Cannabis Use: Whole-Blood and Plasma Free and Glucuronidated Cannabinoid Pharmacokinetics following Controlled Smoked Cannabis Administration

    PubMed Central

    Schwope, David M.; Karschner, Erin L.; Gorelick, David A.; Huestis, Marilyn A.

    2013-01-01

    BACKGROUND Δ9-Tetrahydrocannabinol (THC) is the most frequently observed illicit drug in investigations of accidents and driving under the influence of drugs. THC-glucuronide has been suggested as a marker of recent cannabis use, but there are no blood data following controlled THC administration to test this hypothesis. Furthermore, there are no studies directly examining whole-blood cannabinoid pharmacokinetics, although this matrix is often the only available specimen. METHODS Participants (9 men, 1 woman) resided on a closed research unit and smoked one 6.8% THC cannabis cigarette ad libitum. We quantified THC, 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol (CBD), cannabinol (CBN), THC-glucuronide and THCCOOH-glucuronide directly in whole blood and plasma by liquid chromatography/ tandem mass spectrometry within 24 h of collection to obviate stability issues. RESULTS Median whole blood (plasma) observed maximum concentrations (Cmax) were 50 (76), 6.4 (10), 41 (67), 1.3 (2.0), 2.4 (3.6), 89 (190), and 0.7 (1.4) μg/L 0.25 h after starting smoking for THC, 11-OH-THC, THCCOOH, CBD, CBN, and THCCOOH-glucuronide, respectively, and 0.5 h for THC-glucuronide. At observed Cmax, whole-blood (plasma) detection rates were 60% (80%), 80% (90%), and 50% (80%) for CBD, CBN, and THC-glucuronide, respectively. CBD and CBN were not detectable after 1 h in either matrix (LOQ 1.0 μg/L). CONCLUSIONS Human whole-blood cannabinoid data following cannabis smoking will assist whole blood and plasma cannabinoid interpretation, while furthering identification of recent cannabis intake. PMID:21836075

  1. Validated Method for the Screening and Quantification of Baclofen, Gabapentin and Pregabalin in Human Post-Mortem Whole Blood Using Protein Precipitation and Liquid Chromatography-Tandem Mass Spectrometry.

    PubMed

    Nahar, Limon; Smith, Amy; Patel, Rajan; Andrews, Rebecca; Paterson, Sue

    2017-03-10

    There has been a rapid increase in the number of prescriptions for baclofen (BLF), gabapentin (GBP) and pregabalin (PGL) in the UK since their introduction to therapy. Recent studies across the European Union and USA have shown the illicit abuse potential of these drugs and deaths have been observed. A simple, reliable and fully validated method was developed for the screening and quantification of BLF, GBP and PGL in human post-mortem (PM) blood. The analytes and their deuterated analogs as internal standard were extracted from blood using a single addition acetonitrile protein precipitation reaction followed by analysis using liquid chromatography-tandem mass spectrometry (LC-MS-MS) with triggered dynamic multiple reaction monitoring mode for simultaneous confirmation and quantification. The assay was linear from 0.05 to 1.00 µg/mL for BLF and 0.5 to 50.0 µg/mL for GBP and PGL, respectively with r2 > 0.999 (n = 9) for all analytes. Intra-day and inter-day imprecisions (n = 80) were calculated using one-way ANOVA; no significant difference (P > 0.99) was observed for all analytes over 8 non-consecutive days. The average recovery for all analytes was >98.9%. The limits of detection and quantification were both 0.05 µg/mL for BLF, and 0.5 µg/mL for GBP and PGL. The method was highly selective with no interference from endogenous compounds or from 54 drugs commonly encountered in PM toxicology. To prove method applicability, 17 PM blood samples submitted for analysis were successfully analyzed. The concentration range observed in PM blood for BLF was 0.08-102.00 µg/mL (median = 0.25 µg/mL), for GBP 1.0-134.0 µg/mL (median = 49.0 µg/mL) and 2.0-540.0 µg/mL (median = 42.0 µg/mL) for PGL.

  2. Influence of calorie reduction on DNA repair capacity of human peripheral blood mononuclear cells.

    PubMed

    Matt, Katja; Burger, Katharina; Gebhard, Daniel; Bergemann, Jörg

    2016-03-01

    Caloric restrictive feeding prolongs the lifespan of a variety of model organisms like rodents and invertebrates. It has been shown that caloric restriction reduces age-related as well as overall-mortality, reduces oxidative stress and influences DNA repair ability positively. There are numerous studies underlining this, but fewer studies involving humans exist. To contribute to a better understanding of the correlation of calorie reduction and DNA repair in humans, we adapted the host cell reactivation assay to an application with human peripheral blood mononuclear cells. Furthermore, we used this reliable and reproducible assay to research the influence of a special kind of calorie reduction, namely F. X. Mayr therapy, on DNA repair capacity. We found a positive effect in all persons with low pre-existing DNA repair capacity. In individuals with normal pre-existing DNA repair capacity, no effect on DNA repair capacity was detectable. Decline of DNA repair, accumulation of oxidative DNA damages, mitochondrial dysfunction, telomere shortening as well as caloric intake are widely thought to contribute to aging. With regard to that, our results can be considered as a strong indication that calorie reduction may support DNA repair processes and thus contribute to a healthier aging.

  3. Nanostructured Substrates for Capturing Circulating Tumor Cells in Whole Blood

    NASA Astrophysics Data System (ADS)

    Tseng, Hsian-Rong

    2009-03-01

    Over the past decade, circulating tumor cells (CTCs) has become an emerging ``biomarker'' for detecting early-stage cancer metastasis, predicting patient prognosis, as well as monitoring disease progression and therapeutic outcomes. However, isolation of CTCs has been technically challenging due to the extremely low abundance (a few to hundreds per ml) of CTCs among a high number of hematologic cells (109 per mL) in the blood. Our joint research team at UCLA has developed a new cell capture technology for quantification of CTCs in whole blood samples. Similar to most of the existing approaches, epithelial cell adhesion molecule antibody (anti-EpCAM) was grafted onto the surfaces to distinguish CTCs from the surrounding hematologic cells. The uniqueness of our technology is the use of nanostructured surfaces, which facilitates local topographical interactions between CTCs and substrates at the very first cell/substrate contacting time point. We demonstrated the ability of these nanostructured substrates to capture CTCs in whole blood samples with significantly improved efficiency and selectivity. The successful demonstration of this cell capture technology using brain, breast and prostate cancer cell lines encouraged us to test this approach in clinical setting. We have been able to bond our first validation study with a commercialized technology based on the use of immunomagnetic nanoparticles. A group of clinically well-characterized prostate cancer patients at UCLA hospital have been recruited and tested in parallel by these two technologies.

  4. [Distribution of chemical elements in whole blood and plasma].

    PubMed

    Barashkov, G K; Zaĭtseva, L I; Kondakhchan, M A; Konstantinova, E A

    2003-01-01

    The distribution factor (Fd) of 35 elements of plasma and whole blood in 26 healthy men and women was detected by ICP-OES. Usilig this parameter the elements were subdivided in 3 pools. 9 of them have Fd higher than 1.5 ("elements of plasma"-Ag, Ca, Cu, In, Li, Na, Se, Si, Sr); 6 have lower than 0.5 ("elements of blood cells"-Fe, K, Mn, Ni, V, Zn), other 20-about 1 ("blood elements"). Fd of all elements depends on ionic radius. Elements of 2nd sub-groups of all groups of Mendeleev's periodic table ("heavy metals") depend on the similar law: "with growing of ionic radius the concentration of elements in plasma enhances". In alkaline metals Fd depends on the opposite law:" with growing of ionic radius of alkaline metal the quantity of elements in blood cells enhance". Dependence of Fd on the value of atomic mass in periods or in exterior electronic cloud (s-, p-, d-, f-) was not established. The table of distribution of all detected elements in whole blood in relation to 8 macroelements (Ca, Mg, K, Na, S, P, Fe, Zn,) is presented, as a basic diagnostic criteria in metal-ligand homeostasis disturbance.

  5. Ultrasonic monitoring of droplets' evaporation: Application to human whole blood.

    PubMed

    Laux, D; Ferrandis, J Y; Brutin, D

    2016-09-01

    During a colloidal droplet evaporation, a sol-gel transition can be observed and is described by the desiccation time τD and the gelation time τG. These characteristic times, which can be linked to viscoelastic properties of the droplet and to its composition, are classically rated by analysis of mass droplet evolution during evaporation. Even if monitoring mass evolution versus time seems straightforward, this approach is very sensitive to environmental conditions (vibrations, air flow…) as mass has to be evaluated very accurately using ultra-sensitive weighing scales. In this study we investigated the potentialities of ultrasonic shear reflectometry to assess τD and τG in a simple and reliable manner. In order to validate this approach, our study has focused on blood droplets evaporation on which a great deal of work has recently been published. Desiccation and gelation times measured with shear ultrasonic reflectometry have been perfectly correlated to values obtained from mass versus time analysis. This ultrasonic method which is not very sensitive to environmental perturbations is therefore very interesting to monitor the drying of blood droplets in a simple manner and is more generally suitable for complex fluid droplets evaporation investigation.

  6. Human whole-blood oxygen affinity: effect of carbon monoxide.

    PubMed

    Zwart, A; Kwant, G; Oeseburg, B; Zijlstra, W G

    1984-07-01

    Oxygen dissociation curves (ODC) were recorded in the presence of carboxyhemoglobin fractions (FHbCO) up to 60%. The gradual shift to the left of the ODC at increasing amounts of HbCO was reflected in a gradual fall in the half-saturation pressure of the remaining Hb and was accompanied by a gradual change in the shape of the ODC to a hyperbolic one. The H+ factor (delta log PO2/delta pH) was determined over the entire oxygen saturation (SO2) range at three different FHbCO levels (14, 30, and 52%). At FHbCO = 14 and 30% and for the SO2 range 20-90%, the H+ factor vs. SO2 curve was not significantly different from that in the absence of HbCO. At FHbCO = 52%, however, the value found for the H+ factor (-0.55) was appreciably more negative than in the case of blood containing less than 1% HbCO (-0.44), and there was no dependence on SO2. Comparison of measured and calculated ODCs at varying HbCO fractions showed, for FHbCO less than or equal to 50%, that measured and calculated ODCs coincide over the greater part of the SO2 range. For FHbCO greater than 50%, the measured ODC was situated to the left of the calculated one over the entire SO2 range. We conclude that the heme-heme interaction for CO is appreciably larger than for O2 only for FHbCO greater than 50%, whereas for FHbCO less than 50% there is virtually no difference.

  7. In vitro transdifferentiation of human peripheral blood mononuclear cells to photoreceptor-like cells.

    PubMed

    Komuta, Yukari; Ishii, Toshiyuki; Kaneda, Makoto; Ueda, Yasuji; Miyamoto, Kiyoko; Toyoda, Masashi; Umezawa, Akihiro; Seko, Yuko

    2016-06-15

    Direct reprogramming is a promising, simple and low-cost approach to generate target cells from somatic cells without using induced pluripotent stem cells. Recently, peripheral blood mononuclear cells (PBMCs) have attracted considerable attention as a somatic cell source for reprogramming. As a cell source, PBMCs have an advantage over dermal fibroblasts with respect to the ease of collecting tissues. Based on our studies involving generation of photosensitive photoreceptor cells from human iris cells and human dermal fibroblasts by transduction of photoreceptor-related transcription factors via retrovirus vectors, we transduced these transcription factors into PBMCs via Sendai virus vectors. We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions. In functional studies, a light-induced inward current was detected in some CRX-transduced cells. Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs. These data suggest that CRX acts as a master control gene for reprogramming PBMCs into photoreceptor-like cells and that our induced photoreceptor-like cells might contribute to individualized drug screening and disease modeling of inherited retinal degeneration.

  8. Differences in vanadocene dichloride and cisplatin effect on MOLT-4 leukemia and human peripheral blood mononuclear cells.

    PubMed

    Havelek, Radim; Siman, Pavel; Cmielova, Jana; Stoklasova, Alena; Vavrova, Jirina; Vinklarek, Jaromir; Knizek, Jiri; Rezacova, Martina

    2012-07-01

    Modern chemotherapy is interested in developing new agents with high efficiency of treatment in low-dose medication strategies, lower side toxicity and stronger specificity to the tumor cells. Vanadocene dichloride (VDC) belongs to the group of the most promising metallocene antitumor agents; however, its mechanism of action and cytotoxicity profile are not fully understood. In this paper we assess cytotoxic effects of VDC in comparison to cisplatin using opposite prototype of cells; human peripheral blood mononuclear (PBMCs) cells and human acute lymphoblastic leukemia cell line (MOLT-4). Our findings showed cytotoxic effect of VDC on leukemia cells, but unfortunately on human peripheral blood mononuclear cells as well. VDC induces apoptosis in leukemia cells; the induction is, however, lower than that of cisplatin, and in contrary to cisplatin, VDC does not induce p53 up-regulation. Cytotoxic effect of VDC on leukemia cells is less pronounced than that of cisplatin and more pronounced in PBMCs than in MOLT-4 cells.

  9. The lost art of whole blood transfusion in austere environments.

    PubMed

    Strandenes, Geir; Hervig, Tor A; Bjerkvig, Christopher K; Williams, Steve; Eliassen, Håkon S; Fosse, Theodor K; Torvanger, Hans; Cap, Andrew P

    2015-01-01

    The optimal resuscitation fluid for uncontrolled bleeding and hemorrhagic shock in both pre- and in-hospital settings has been an ongoing controversy for decades. Hemorrhage continues to be a major cause of death in both the civilian and military trauma population, and survival depends on adequacy of hemorrhage control and resuscitation between onset of bleeding and arrival at a medical treatment facility. The terms far-forward and austere are defined, respectively, as the environment where professional health care providers normally do not operate and a setting in which basic equipment and capabilities necessary for resuscitation are often not available. The relative austerity of a treatment setting may be a function of timing rather than just location, as life-saving interventions must be performed quickly before hemorrhagic shock becomes irreversible. Fresh whole blood transfusions in the field may be a feasible life-saving procedure when facing significant hemorrhage.

  10. Fluorometric determination of whole blood with various excitation wavelengths

    NASA Astrophysics Data System (ADS)

    Lan, Xiufeng; Gao, Shumei; Peng, Changde; Liu, Ying; Ni, Xiao-Wu

    2003-12-01

    Autofluorescence spectra from whole blood of laboratory rat are measured in this paper. The excitation lights are light emitting diode (LED), Ar+ laser, and He-Ne laser with the wavelength located at 457nm, 457.9nm, and 632.8nm respectively. The three spectral profiles are found to be substantially different, each displaying its own characteristic fluorescence bands. Ar+ laser-induced spectrum has very rich and sharp peaks. The LED-induced one has the strongest and widest fluorescence bands. And the intensity of the spectrum induced by He-Ne laser is much lower than the former two. Comparisons of those three fluorescence spectra indicate that Ar+ laser induced spectrum can show partly fine structure of blood cells. Based on the theoretical analysis, it is presented that the absorption of the fluorophores in blood cells to the wavelength of exciting light has definite selectivity, which depend on energy level structure and state of the fluorophores.

  11. Filter characteristics influencing circulating tumor cell enrichment from whole blood.

    PubMed

    Coumans, Frank A W; van Dalum, Guus; Beck, Markus; Terstappen, Leon W M M

    2013-01-01

    A variety of filters assays have been described to enrich circulating tumor cells (CTC) based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of whole blood spiked with cells from tumor cell lines were passed through silicon nitride microsieves, polymer track-etched filters and metal TEM grids with various pore sizes. The recovery and size of 9 different culture cell lines was determined and compared to the size of EpCAM+CK+CD45-DNA+ CTC from patients with metastatic breast, colorectal and prostate cancer. The 8 µm track-etched filter and the 5 µm microsieve had the best performance on MDA-231, PC3-9 and SKBR-3 cells, enriching >80% of cells from whole blood. TEM grids had poor recovery of ∼25%. Median diameter of cell lines ranged from 10.9-19.0 µm, compared to 13.1, 10.7, and 11.0 µm for breast, prostate and colorectal CTC, respectively. The 11.4 µm COLO-320 cell line had the lowest recovery of 17%. The ideal filter for CTC enrichment is constructed of a stiff, flat material, is inert to blood cells, has at least 100,000 regularly spaced 5 µm pores for 1 ml of blood with a ≤10% porosity. While cell size is an important factor in determining recovery, other factors must be involved as well. To evaluate a filtration procedure, cell lines with a median size of 11-13 µm should be used to challenge the system.

  12. Pharmacokinetic properties of γ-hydroxybutyrate (GHB) in whole blood, serum, and urine.

    PubMed

    Brailsford, Alan D; Cowan, David A; Kicman, Andrew T

    2012-03-01

    Over the last 10-15 years, γ-hydroxybutyrate (GHB) and γ-butyrolactone have become increasingly popular "club drugs", but they have also gained attention as potential agents of drug-facilitated sexual assault (DFSA). Several studies have attempted to characterize GHB's pharmacokinetic properties in humans, and the aim of this paper is to build on this research with an emphasis on DFSA cases. A 25 mg/kg dose of GHB was given to 12 GHB-naïve volunteers (6 men and 6 women). Urine and blood samples (serum and whole blood) were collected and analyzed by gas chromatography-mass spectrometry following liquid-liquid extraction. The urinary T(max) was 1 h in 11 volunteers with a mean C(max) of 67.6 mg/L (32.6-161.3 mg/L). Urinary concentrations rapidly decreased to < 10 mg/L (interpretive limit) for 11 volunteers after just 4 h. Data derived from whole blood (mean C(max) = 48.0 mg/L, T(max) = 24.6 min) closely matched that from serum (mean C(max) = 59.4 mg/L, T(max) = 23.3 min), suggesting GHB is distributed into erythrocytes. All 12 volunteers had GHB concentrations of less than 5 mg/L in both whole blood and serum after 3 h. Results verify the rapid elimination of GHB and the limited retrospective power of a concentration-based approach to prove GHB administration in blood and urine and confirm that, in DFSA cases, samples should be collected as soon as possible.

  13. Allograft inflammatory factor-1 stimulates chemokine production and induces chemotaxis in human peripheral blood mononuclear cells.

    PubMed

    Kadoya, Masatoshi; Yamamoto, Aihiro; Hamaguchi, Masahide; Obayashi, Hiroshi; Mizushima, Katsura; Ohta, Mitsuhiro; Seno, Takahiro; Oda, Ryo; Fujiwara, Hiroyoshi; Kohno, Masataka; Kawahito, Yutaka

    2014-06-06

    Allograft inflammatory factor-1 (AIF-1) is expressed by macrophages, fibroblasts, endothelial cells and smooth muscle cells in immune-inflammatory disorders such as systemic sclerosis, rheumatoid arthritis and several vasculopathies. However, its molecular function is not fully understood. In this study, we examined gene expression profiles and induction of chemokines in monocytes treated with recombinant human AIF (rhAIF-1). Using the high-density oligonucleotide microarray technique, we compared mRNA expression profiles of rhAIF-1-stimulated CD14(+) peripheral blood mononuclear cells (CD14(+) PBMCs) derived from healthy volunteers. We demonstrated upregulation of genes for several CC chemokines such as CCL1, CCL2, CCL3, CCL7, and CCL20. Next, using ELISAs, we confirmed that rhAIF-1 promoted the secretion of CCL3/MIP-1α and IL-6 by CD14(+) PBMCs, whereas only small amounts of CCL1, CCL2/MCP-1, CCL7/MCP-3 and CCL20/MIP-3α were secreted. Conditioned media from rhAIF-1stimulated CD14(+) PBMCs resulted in migration of PBMCs. These findings suggest that AIF-1, which induced chemokines and enhanced chemotaxis of monocytes, may represent a molecular target for the therapy of immune-inflammatory disorders.

  14. Human cord blood mononuclear cell transplantation for the treatment of premature ovarian failure in nude mice

    PubMed Central

    Dang, Jianhong; Jin, Zhijun; Liu, Xiaojun; Hu, Dian; Wang, Zhifeng

    2015-01-01

    Objective: This study explored the potential of human cord blood mononuclear cell (HCMNC) transplantation as a treatment for premature ovarian failure (POF) in a nude mouse model. Methods: Female nude mice were randomly divided into three groups; a normal control group (n = 35), a POF group (POF plus vehicle, n = 35) and a POF plus cell transplantation group (HCMNCs were implanted into the ovaries, n = 35). HCMNCs were isolated by Ficoll density gradient centrifugation and labeled with BrdU. Four weeks after transplantation, the nude mice were sacrificed to determine serum levels of E2, FSH and LH as indicators of ovarian function, and the ovaries were examined both histologically and immunochemically. Results: The transplanted HCMNCs survived in the transplantation group and were detected by BrdU. In the transplantation group, serum levels of E2 significantly increased while serum levels of FSH and LH significantly decreased compared to the POF control group. Additionally, the transplantation group had a recovery in follicle number. Conclusion: HCMNCs can be successfully transplanted into the ovaries of nude mice and can improve ovarian function in POF. PMID:26064319

  15. Analysis of cytotoxic effects of silver nanoclusters on human peripheral blood mononuclear cells 'in vitro'.

    PubMed

    Orta-García, Sandra Teresa; Plascencia-Villa, Germán; Ochoa-Martínez, Angeles Catalina; Ruiz-Vera, Tania; Pérez-Vázquez, Francisco Javier; Velázquez-Salazar, J Jesús; Yacamán, Miguel José; Navarro-Contreras, Hugo Ricardo; Pérez-Maldonado, Iván N

    2015-10-01

    The antimicrobial properties of silver nanoparticles (AgNPs) have made these particles one of the most used nanomaterials in consumer products. Therefore, an understanding of the interactions (unwanted toxicity) between nanoparticles and human cells is of significant interest. The aim of this study was to assess the in vitro cytotoxicity effects of silver nanoclusters (AgNC, < 2 nm diameter) on peripheral blood mononuclear cells (PBMC). Using flow cytometry and comet assay methods, we demonstrate that exposure of PBMC to AgNC induced intracellular reactive oxygen species (ROS) generation, DNA damage and apoptosis at 3, 6 and 12 h, with a dose-dependent response (0.1, 1, 3, 5 and 30 µg ml(-1)). Advanced electron microscopy imaging of complete and ultrathin-sections of PBMC confirmed the cytotoxic effects and cell damage caused by AgNC. The present study showed that AgNC produced without coating agents induced significant cytotoxic effects on PBMC owing to their high aspect ratio and active surface area, even at much lower concentrations (<1 µg ml(-1)) than those applied in previous studies, resembling what would occur under real exposure conditions to nanosilver-functionalized consumer products.

  16. Methamidophos induces cytotoxicity and oxidative stress in human peripheral blood mononuclear cells.

    PubMed

    Ramirez-Vargas, Marco Antonio; Huerta-Beristain, Gerardo; Guzman-Guzman, Iris Paola; Alarcon-Romero, Luz Del Carmen; Flores-Alfaro, Eugenia; Rojas-Garcia, Aurora Elizabeth; Moreno-Godinez, Ma Elena

    2017-01-01

    Previous studies have shown that organophosphate pesticide (OP) exposure is associated with oxidative stress. Methamidophos (MET) is an OP widely used in agriculture, which is regarded as a highly toxic pesticide and it is a potent inhibitor of acetylcholinesterase. The aim of this study was to evaluate whether MET can induce oxidative stress at low concentrations in primary cultures of human peripheral blood mononuclear cells (PBMCs). PBMCs from healthy individuals were exposed to MET (0-80 mg/L) for 0-72 h. We performed the MTT and neutral-red assays to assess the cytotoxicity. As indicators of oxidative stress, the levels of reactive oxygen species (ROS) were assessed using flow cytometry, and the malondialdehyde (MDA) and reduced glutathione (GSH) levels were determined. MET decreased the viability of PBMCs in a dose-dependent manner. At concentrations of 3, 10, or 20 mg/L for 24 h, MET increased the ROS production significantly compared with the vehicle control. Similarly, MET increased the levels of MDA at the same concentrations that increased ROS (10 and 20 mg/L); however, no changes in GSH levels were observed. These results suggest that MET increased the generation of oxidative stress in PBMCs. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 147-155, 2017.

  17. Antioxidant enzyme activities of human peripheral blood mononuclear cells exposed to trace elements.

    PubMed

    Kuppusamy, U R; Dharmani, M; Kanthimathi, M S; Indran, M

    2005-07-01

    The trace elements copper, zinc, and selenium are important immune modulators and essential cofactors of the antioxidant enzymes. In the present study, the proliferative effect of human peripheral mononuclear cells (PBMCs) that have been exposed to copper, zinc, and selenium and the corresponding activities of antioxidant enzymes, namely superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase, were determined. Zinc and copper stimulated the PBMC proliferation in a dose-dependent manner within the dose range 25-200 micromol/L. SOD and GPx activities in PBMCs exposed to zinc were inhibited, whereas catalase activity was unaffected. All the three antioxidant enzymes in the cells exposed to copper were inhibited. Selenium exerted more potent inhibition of the cell proliferation while causing stimulation of the antioxidant enzymes at the lowest dose (25 micromol/L) than at the highest dose (200 micromol/L) tested. A significant negative correlation was observed between proliferation and antioxidant enzyme (SOD and GPx) activities in trace-element-exposed PBMC. The present findings substantiate the importance of trace elements as immune modulators and the involvement of enzymatic antioxidant system in the immune cell regulation.

  18. Effect of arsenic, cadmium and lead on the induction of apoptosis of normal human mononuclear cells

    PubMed Central

    DE LA FUENTE, H; PORTALES-PÉREZ, D; BARANDA, L; DÍAZ-BARRIGA, F; SAAVEDRA-ALANÍS, V; LAYSECA, E; GONZÁLEZ-AMARO, R

    2002-01-01

    The aim of this work was to investigate the effect of cadmium, lead and arsenic on the apoptosis of human immune cells. Peripheral blood mononuclear cells (MNC) were incubated with increasing concentrations of these metals and then cellular apoptosis was determined by flow cytometry and by DNA electrophoresis. We found that arsenic induced a significant level of apoptosis at 15 μm after 48h of incubation. Cadmium had a similar effect, but at higher concentrations (65 μm). In addition, cadmium exerted a cytotoxic effect on MNC that seemed to be independent of the induction of apoptosis. In contrast, concentrations of lead as high as 500 μm were nontoxic and did not induce a significant degree of apoptosis. Additional experiments showed that arsenic at concentrations as low as 1·0 μm had a significant pro-apoptotic effect when cells were cultured in the presence of this pollutant for more than 72. Non-T cells were more susceptible than T lymphocytes to the effect of arsenic and cadmium. Interestingly, MNC from children chronically exposed to arsenic showed a high basal rate of apoptosis and a diminished in vitro sensibility to this metalloid. Our results indicate that both arsenic and cadmium are able to induce apoptosis of lymphoid cells, and suggest that this phenomenon may contribute to their immunotoxic effect in vivo. PMID:12100024

  19. Bone resorptive activity of human peripheral blood mononuclear cells after fusion with polyethylene glycol.

    PubMed

    Manrique, Edwin; Castillo, Luz M; Lazala, Oswaldo; Guerrero, Carlos A; Acosta, Orlando

    2017-03-01

    The bone remodeling process occurs through bone formation by osteoblasts and bone resorption by osteoclasts, a process involving the contribution of endocrine and nervous systems. The mechanisms associated to differentiation and proliferation of osteoclasts and osteoblasts are considered a potential therapeutic target for treating some erosive bone diseases. The aim of the present study is to explore the feasibility of generating active osteoclast-like cells from peripheral blood mononuclear cells (PBMCs) following polyethylene glycol (PEG)-induced fusion. PEG-fused PBMCs showed TRAP(+)-multinucleated cells and bone resorption activity, and were also positive for osteoclast markers such as carbonic anhydrase II, calcitonin receptor, vacuolar ATPase, and cathepsin K, when examined by reverse transcription-polymerase chain reaction, immunochemistry and Western blotting. TRAP expression and bone resorptive activity were higher in whole PEG-fused PBMCs than in separated T lymphocytes, B lymphocytes or monocytes. Both TRAP expression and bone resorptive activity were also higher in osteogenesis imperfecta patients compared to PEG-fused PBMCs from healthy individuals. PEG-induced fusion was more efficient in inducing TRAP and bone resorptive activities than macrophage colony-stimulating factor or dexamethasone treatment. Bone resorptive activity of PEG-fused PMBCs was inhibited by bisphosphonates. Evidence is provided that the use of PEG-based cell fusion is a straightforward and amenable method for studying human osteoclast differentiation and testing new therapeutic strategies.

  20. Persistent alterations of gene expression profiling of human peripheral blood mononuclear cells from smokers.

    PubMed

    Weng, Daniel Y; Chen, Jinguo; Taslim, Cenny; Hsu, Ping-Ching; Marian, Catalin; David, Sean P; Loffredo, Christopher A; Shields, Peter G

    2016-10-01

    The number of validated biomarkers of tobacco smoke exposure is limited, and none exist for tobacco-related cancer. Additional biomarkers for smoke, effects on cellular systems in vivo are needed to improve early detection of lung cancer, and to assist the Food and Drug Administration in regulating exposures to tobacco products. We assessed the effects of smoking on the gene expression using human cell cultures and blood from a cross-sectional study. We profiled global transcriptional changes in cultured smokers' peripheral blood mononuclear cells (PBMCs) treated with cigarette smoke condensate (CSC) in vitro (n = 7) and from well-characterized smokers' blood (n = 36). ANOVA with adjustment for covariates and Pearson correlation were used for statistical analysis in this study. CSC in vitro altered the expression of 1 178 genes (177 genes with > 1.5-fold-change) at P < 0.05. In vivo, PBMCs of heavy and light smokers differed for 614 genes (29 with > 1.5-fold-change) at P < 0.05 (309 remaining significant after adjustment for age, race, and gender). Forty-one genes were persistently altered both in vitro and in vivo, 22 having the same expression pattern reported for non-small cell lung cancer. Our data provides evidence that persistent alterations of gene expression in vitro and in vivo may relate to carcinogenic effects of cigarette smoke, and the identified genes may serve as potential biomarkers for cancer. The use of an in vitro model to corroborate results from human studies provides a novel way to understand human exposure and effect. © 2015 Wiley Periodicals, Inc.

  1. Persistent Alterations of Gene Expression Profiling of Human Peripheral Blood Mononuclear Cells From Smokers

    PubMed Central

    Weng, Daniel Y.; Chen, Jinguo; Taslim, Cenny; Hsu, Ping-Ching; Marian, Catalin; David, Sean P.; Loffredo, Christopher A.; Shields, Peter G.

    2016-01-01

    The number of validated biomarkers of tobacco smoke exposure is limited, and none exist for tobacco-related cancer. Additional biomarkers for smoke, effects on cellular systems in vivo are needed to improve early detection of lung cancer, and to assist the Food and Drug Administration in regulating exposures to tobacco products. We assessed the effects of smoking on the gene expression using human cell cultures and blood from a cross-sectional study. We profiled global transcriptional changes in cultured smokers’ peripheral blood mononuclear cells (PBMCs) treated with cigarette smoke condensate (CSC) in vitro (n = 7) and from well-characterized smokers’ blood (n = 36). ANOVA with adjustment for covariates and Pearson correlation were used for statistical analysis in this study. CSC in vitro altered the expression of 1 178 genes (177 genes with > 1.5-fold-change) at P < 0.05. In vivo, PBMCs of heavy and light smokers differed for 614 genes (29 with > 1.5-fold-change) at P < 0.05 (309 remaining significant after adjustment for age, race, and gender). Forty-one genes were persistently altered both in vitro and in vivo, 22 having the same expression pattern reported for non-small cell lung cancer. Our data provides evidence that persistent alterations of gene expression in vitro and in vivo may relate to carcinogenic effects of cigarette smoke, and the identified genes may serve as potential biomarkers for cancer. The use of an in vitro model to corroborate results from human studies provides a novel way to understand human exposure and effect. PMID:26294040

  2. Autologous red blood cells potentiate antibody synthesis by unfractionated human mononuclear cell cultures.

    PubMed

    Rugeles, M T; La Via, M; Goust, J M; Kilpatrick, J M; Hyman, B; Virella, G

    1987-08-01

    We have tried to determine the most favourable conditions for the in vitro induction of specific antibody (Ab) responses to tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH). Human peripheral blood mononuclear cells (PBMNC) were obtained from normal volunteers and stimulated with PWM, TT, KLH, and mixtures of PWM and antigens in the presence or absence of autologous red blood cells (RBC) (1:50 ratio of PBMNC/RBC). The cultures were harvested on day 11; immunoglobulins were determined immunonephelometrically and Ab levels by ELISA with human antibodies used for calibration. While anti-TT responses were easy to induce with PBMNC from recently boosted individuals, the production of anti-TT from PBMNC obtained from non-recently boosted individuals was only possible when PBMNC were stimulated with TT and PWM in the presence of autologous RBC. Similarly, anti-KLH responses were easier to induce with PBMNC from an immune donor; maximal response was observed after stimulation with PWM + KLH in the presence of autologous RBC. Stimulation of primary anti-KLH responses with PBMNC from non-immune donors was only successful when the cells were stimulated with KLH + PWM in the presence of autologous RBC. The potentiation of human B-cell responses with autologous RBC can be abrogated by pretreatment of PBMNC with anti-CD2 antibodies and is associated with increased expression of IL-2 receptors and increased production of gamma interferon (IFN-gamma). However, addition of IFN-gamma in different doses and at different times to PWM-stimulated PBMNC cultures was not as effective as addition of RBC in enhancing the production of immunoglobulin and antibody.

  3. Selected scorpion toxin exposures induce cytokine release in human peripheral blood mononuclear cells.

    PubMed

    Corzo, Gerardo; Espino-Solis, Gerardo Pavel

    2017-03-01

    A cytokine screening on human peripheral blood mononuclear cells (PBMCs) stimulated with selected scorpion toxins (ScTx's) was performed in order to evaluate their effect on human immune cells. The ScTx's chosen for this report were three typical buthid scorpion venom peptides, one with lethal effects on mammals Centruroides suffussus suffusus toxin II (CssII), another, with lethal effects on insects and crustaceans Centruroides noxius toxin 5 (Cn5), and one more without lethal effects Tityus discrepans toxin (Discrepin). A Luminex multiplex analysis was performed in order to determine the amounts chemokines and cytokines IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12-p40, IL-13, interferon alpha (IFN-α), interferon gamma (IFN-γ), tumor necrosis factor alpha TNF-α, and interferon-inducible protein-10 (IP-10) secreted from human PBMCs exposed to these toxins. Although, the ScTx Cn5 is not lethal for mammals, it was able to induce the secretion of cytokines IL-1β, IL-6, and TNF-α, IL-10 and IP-10 in comparison to the lethal CssII, which was able to induce only IP-10 secretion. Discrepin also was able to induce only IP-10. Interestingly, only low amounts of interferons α and β were induced in the presence of the ScTx's assayed. In a synergic experiment, the combination of Discrepin and Cn5 displayed considerable reverse effects on induction of IL-1β, IL-6, IL-10 and TNF-α, but they had a slight synergic effect on IP-10 cytokine production in comparison with the single effect obtained with the Cn5 alone. Thus, the results obtained suggest that the profile of secreted cytokines promoted by ScTx Cn5 is highly related with a cytokine storm event, and also it suggests that the mammalian lethal neurotoxins are not solely responsible of the scorpion envenomation symptomatology.

  4. Combustible and non-combustible tobacco product preparations differentially regulate human peripheral blood mononuclear cell functions.

    PubMed

    Arimilli, Subhashini; Damratoski, Brad E; Prasad, G L

    2013-09-01

    Natural killer (NK) cells and T cells play essential roles in innate and adaptive immune responses in protecting against microbial infections and in tumor surveillance. Although evidence suggests that smoking causes immunosuppression, there is limited information whether the use of smokeless tobacco (ST) products affects immune responses. In this study, we assessed the effects of two preparations of cigarette smoke, ST extract and nicotine on T cell and NK cell responses using Toll-like receptor-ligand stimulated human peripheral blood mononuclear cells (PBMCs). The tobacco product preparations (TPPs) tested included whole smoke conditioned media (WS-CM), total particulate matter (TPM) and a ST product preparation in complete artificial saliva (ST/CAS). The PBMCs were stimulated with polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS). A marked reduction of the expression of intracellular IFN-γ and TNF-α was evident in NK cells and T cells treated with WS-CM and TPM. Consistently, attenuation of ligand-induced secretion of cytokines (IL-1β, IL-10, IL-12 and TNF-α) from PBMCs treated with WS-CM and TPM were observed. While the treatment with TPPs did not alter the expression of the maturation marker CD69, WS-CM and TPM inhibited the cytolytic activity of human PBMCs. Suppression of perforin by WS-CM was also detected. Although interference from the vehicle confounded the interpretation of effects of ST/CAS, some effects were evident only at high concentrations. Nicotine treatment minimally impacted expression of cytokines and cytolytic activity. Data presented herein suggests that the function of NK cells and T cells is influenced by exposure to TPPs (based on equi-nicotine units) in the following order: WS-CM>TPM>ST/CAS. These findings are consistent with the hypothesis put forward by others that chronic smoking leads to immunosuppression, an effect that may contribute to increased microbial infections and cancer incidence among smokers.

  5. Identification of Lactobacillus plantarum genes modulating the cytokine response of human peripheral blood mononuclear cells

    PubMed Central

    2010-01-01

    Background Modulation of the immune system is one of the most plausible mechanisms underlying the beneficial effects of probiotic bacteria on human health. Presently, the specific probiotic cell products responsible for immunomodulation are largely unknown. In this study, the genetic and phenotypic diversity of strains of the Lactobacillus plantarum species were investigated to identify genes of L. plantarum with the potential to influence the amounts of cytokines interleukin 10 (IL-10) and IL-12 and the ratio of IL-10/IL-12 produced by peripheral blood mononuclear cells (PBMCs). Results A total of 42 Lactobacillus plantarum strains isolated from diverse environmental and human sources were evaluated for their capacity to stimulate cytokine production in PBMCs. The L. plantarum strains induced the secretion of the anti-inflammatory cytokine IL-10 over an average 14-fold range and secretion of the pro-inflammatory cytokine IL-12 over an average 16-fold range. Comparisons of the strain-specific cytokine responses of PBMCs to comparative genome hybridization profiles obtained with L. plantarum WCFS1 DNA microarrays (also termed gene-trait matching) resulted in the identification of 6 candidate genetic loci with immunomodulatory capacities. These loci included genes encoding an N-acetyl-glucosamine/galactosamine phosphotransferase system, the LamBDCA quorum sensing system, and components of the plantaricin (bacteriocin) biosynthesis and transport pathway. Deletion of these genes in L. plantarum WCFS1 resulted in growth phase-dependent changes in the PBMC IL-10 and IL-12 cytokine profiles compared with wild-type cells. Conclusions The altered PBMC cytokine profiles obtained with the L. plantarum WCFS1 mutants were in good agreement with the predictions made by gene-trait matching for the 42 L. plantarum strains. This study therefore resulted in the identification of genes present in certain strains of L. plantarum which might be responsible for the stimulation of anti

  6. On-chip Extraction of Intracellular Molecules in White Blood Cells from Whole Blood.

    PubMed

    Choi, Jongchan; Hyun, Ji-chul; Yang, Sung

    2015-10-14

    The extraction of virological markers in white blood cells (WBCs) from whole blood--without reagents, electricity, or instruments--is the most important first step for diagnostic testing of infectious diseases in resource-limited settings. Here we develop an integrated microfluidic chip that continuously separates WBCs from whole blood and mechanically ruptures them to extract intracellular proteins and nucleic acids for diagnostic purposes. The integrated chip is assembled with a device that separates WBCs by using differences in blood cell size and a mechanical cell lysis chip with ultra-sharp nanoblade arrays. We demonstrate the performance of the integrated device by quantitatively analyzing the levels of extracted intracellular proteins and genomic DNAs. Our results show that compared with a conventional method, the device yields 120% higher level of total protein amount and similar levels of gDNA (90.3%). To demonstrate its clinical application to human immunodeficiency virus (HIV) diagnostics, the developed chip was used to process blood samples containing HIV-infected cells. Based on PCR results, we demonstrate that the chip can extract HIV proviral DNAs from infected cells with a population as low as 10(2)/μl. These findings suggest that the developed device has potential application in point-of-care testing for infectious diseases in developing countries.

  7. Detection of heart failure-related biomarker in whole blood with graphene field effect transistor biosensor.

    PubMed

    Lei, Yong-Min; Xiao, Meng-Meng; Li, Yu-Tao; Xu, Li; Zhang, Hong; Zhang, Zhi-Yong; Zhang, Guo-Jun

    2017-05-15

    Since brain natriuretic peptide (BNP) has become internationally recognized biomarkers in the diagnosis and prognosis of heart failure (HF), it is highly desirable to search for a novel sensing tool for detecting the patient's BNP level at the early stage. Here we report a platinum nanoparticles (PtNPs)-decorated reduced graphene oxide (rGO) field effect transistor (FET) biosensor coupled with a microfilter system for label-free and highly sensitive detection of BNP in whole blood. The PtNPs-decorated rGO FET sensor was obtained by drop-casting rGO onto the pre-fabricated FET chip and subsequently assembling PtNPs on the graphene surface. After anti-BNP was bound to the PtNPs surface, BNP was successfully detected by the anti-BNP immobilized FET biosensor. It was found that the developed FET biosensor was able to achieve a low detection limitation of 100fM. Moreover, BNP was successfully detected in human whole blood sample treated by a custom-made microfilter, suggesting the sensor's capability of working in a complex sample matrix. The developed FET biosensor provides a new sensing platform for protein detection, showing its potential applications in clinic sample.

  8. Interferon beta 2/interleukin 6 modulates synthesis of alpha 1-antitrypsin in human mononuclear phagocytes and in human hepatoma cells.

    PubMed Central

    Perlmutter, D H; May, L T; Sehgal, P B

    1989-01-01

    The cytokine IFN beta 2/IL-6 has recently been shown to regulate the expression of genes encoding hepatic acute phase plasma proteins. INF beta 2/IL-6 has also been shown to be identical to MGI-2, a protein that induces differentiation of bone marrow precursor cells toward mature granulocytes and monocytes. Accordingly, we have examined the effect of IFN beta 2/IL-6 on expression of the IL-1- and tumor necrosis factor-unresponsive acute phase protein alpha 1-antitrypsin (alpha 1 AT) in human hepatoma-derived hepatocytes and in human mononuclear phagocytes. Purified human fibroblast and recombinant IFN beta 2/IL-6 each mediate a specific increase in steady-state levels of alpha 1 AT mRNA and a corresponding increase in net synthesis of alpha 1 AT in primary cultures of human peripheral blood monocytes as well as in HepG2 and Hep3B cells. Thus, the effect of IFN beta 2/IL-6 on alpha 1 AT gene expression in these cells is primarily due to an increase in accumulation of alpha 1 AT mRNA and can be distinguished from the direct, predominantly translational effect of bacterial lipopolysaccharide on expression of this gene in monocytes and macrophages. The results indicate that IFN beta 2/IL-6 regulates acute phase gene expression, specifically alpha 1 AT gene expression, in extrahepatic as well as hepatic cell types. Images PMID:2472425

  9. Evidence that leishmania donovani utilizes a mannose receptor on human mononuclear phagocytes to establish intracellular parasitism

    SciTech Connect

    Wilson, M.E.; Pearson, R.D.

    1986-01-01

    The pathogenic protozoan Leishmania donovani must gain entrance into mononuclear phagocytes to successfully parasitize man. The parasite's extracellular promastigote stage is ingested by human peripheral blood monocytes or monocyte-derived macrophages in the absence of serum, in a manner characteristic of receptor-mediated endocytosis. Remarkable similarities have been found between the macrophage receptor(s) for promastigotes and a previously characterized eucaryotic receptor system, the mannose/fucose receptor (MFR), that mediates the binding of zymosan particles and mannose- or fucose-terminal glycoconjugates to macrophages. Ingestion of promastigotes by monocyte-derived macrophages was inhibited by several MFR ligands; that is mannan, mannose-BSA and fucose-BSA. In contrast, promastigote ingestion by monocytes was unaffected by MFR ligands. Furthermore, attachment of promastigotes to macrophages, assessed by using cytochalasin D to prevent phagocytosis, was reduced 49.8% by mannan. Reorientation of the MFR to the ventral surface of the cell was achieved by plating macrophages onto mannan-coated coverslips, reducing MFR activity on the exposed cell surface by 94% as assessed by binding of /sup 125/I-mannose-BSA. Under these conditions, ingestion of promastigotes was inhibited by 71.4%. Internalization of the MFR by exposure of macrophages to zymosan before infection with promastigotes resulted in a 62.3% decrease in parasite ingestion. Additionally, NH/sub 4/Cl decreased macrophage ingestion of promastigotes by 38.2%. Subinhibitory concentration of NH/sub 4/Cl (10 mM) and of mannan (0.25 mg/ml) together inhibited parsite ingestion by 76.4%.

  10. Cytokine-mediated inhibition of fibrillar amyloid-β peptide degradation by human mononuclear phagocytes1

    PubMed Central

    Yamamoto, Masaru; Kiyota, Tomomi; Walsh, Shannon M.; Liu, Jianuo; Kipnis, Jonathan; Ikezu, Tsuneya

    2008-01-01

    Vaccination therapy of AD animal models and patients strongly suggests an active role of brain mononuclear phagocytes in immune-mediated clearance of amyloid-β peptides (Aβ) in brain. Although Aβ uptake by macrophages can be regulated by pro- and anti-inflammatory cytokines, their effects on macrophage-mediated Aβ degradation are poorly understood. To better understand this mechanism of degradation, we examined whether pro- and anti-inflammatory cytokines affect the degradation of Aβ using primary cultured human monocyte-derived macrophages (MDM) and microglia using pulse-chase analysis of fibrillar and oligomer 125I-Aβ40 and Aβ42. Initial uptake of fibrillar Aβ40 and Aβ42 was 40% and its degradation was saturated by 120 hrs in both MDM and microglia, compared to an initial uptake of oligomeric Aβ less than 0.5% and saturation of degradation within 24 hrs. Interferon-γ (IFN-γ) increased the intracellular retention of fibrillar Aβ40 and Aβ42 by inhibiting degradation, whereas interleukin-4 (IL-4), IL-10, and transforming growth factor-β1 (TGF-β1), but not IL-13 and IL-27, enhanced degradation. Fibrillar Aβ degradation in MDM is sensitive to lysosomal and insulin degrading enzyme (IDE) inhibitors but insensitive to proteasomal and neprilysin inhibitors. IFN-γ and TNF-α directly reduced the expression of IDE and chaperone molecules (Hsp70 and Hsc70), which are involved in refolding of aggregated proteins. Co-culture of MDM with activated, but not naïve T cells, suppressed Aβ degradation in MDM, which was partially blocked by a combination of neutralizing antibodies against pro-inflammatory cytokines. These data suggest that pro-inflammatory cytokines suppress Aβ degradation in MDM, whereas select anti-inflammatory and regulatory cytokines antagonize these effects. PMID:18768842

  11. Lipopolysaccharide Stimulates Butyric Acid-Induced Apoptosis in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Kurita-Ochiai, Tomoko; Fukushima, Kazuo; Ochiai, Kuniyasu

    1999-01-01

    We previously reported that butyric acid, an extracellular metabolite from periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat T cells. In this study, we examined the ability of butyric acid to induce apoptosis in peripheral blood mononuclear cells (PBMC) and the effect of bacterial lipopolysaccharide (LPS) on this apoptosis. Butyric acid significantly inhibited the anti-CD3 monoclonal antibody- and concanavalin A-induced proliferative responses in a dose-dependent fashion. This inhibition of PBMC growth by butyric acid depended on apoptosis in vitro. It was characterized by internucleosomal DNA digestion and revealed by gel electrophoresis followed by a colorimetric DNA fragmentation assay to occur in a concentration-dependent fashion. Butyric acid-induced PBMC apoptosis was accompanied by caspase-3 protease activity but not by caspase-1 protease activity. LPS potentiated butyric acid-induced PBMC apoptosis in a dose-dependent manner. Flow-cytometric analysis revealed that LPS increased the proportion of sub-G1 cells and the number of late-stage apoptotic cells induced by butyric acid. Annexin V binding experiments with fractionated subpopulations of PBMC in flow cytometory revealed that LPS accelerated the butyric acid-induced CD3+-T-cell apoptosis followed by similar levels of both CD4+- and CD8+-T-cell apoptosis. The addition of LPS to PBMC cultures did not cause DNA fragmentation, suggesting that LPS was unable to induce PBMC apoptosis directly. These data suggest that LPS, in combination with butyric acid, potentiates CD3+ PBMC T-cell apoptosis and plays a role in the apoptotic depletion of CD4+ and CD8+ cells. PMID:9864191

  12. Olanzapine and aripiprazole differentially affect glucose uptake and energy metabolism in human mononuclear blood cells.

    PubMed

    Stapel, Britta; Kotsiari, Alexandra; Scherr, Michaela; Hilfiker-Kleiner, Denise; Bleich, Stefan; Frieling, Helge; Kahl, Kai G

    2017-05-01

    The use of antipsychotics carries the risk of metabolic side effects, such as weight gain and new onset type-2 diabetes mellitus. The mechanisms of the observed metabolic alterations are not fully understood. We compared the effects of two atypical antipsychotics, one known to favor weight gain (olanzapine), the other not (aripiprazole), on glucose metabolism. Primary human peripheral blood mononuclear cells (PBMC) were isolated and stimulated with olanzapine or aripiprazole for 72 h. Cellular glucose uptake was analyzed in vitro by 18F-FDG uptake. Further measurements comprised mRNA expression of glucose transporter (GLUT) 1 and 3, GLUT1 protein expression, DNA methylation of GLUT1 promoter region, and proteins involved in downstream glucometabolic processes. We observed a 2-fold increase in glucose uptake after stimulation with aripiprazole. In contrast, olanzapine stimulation decreased glucose uptake by 40%, accompanied by downregulation of the cellular energy sensor AMP activated protein kinase (AMPK). GLUT1 protein expression increased, GLUT1 mRNA expression decreased, and GLUT1 promoter was hypermethylated with both antipsychotics. Pyruvat-dehydrogenase (PDH) complex activity decreased with olanzapine only. Our findings suggest that the atypical antipsychotics olanzapine and aripiprazole differentially affect energy metabolism in PBMC. The observed decrease in glucose uptake in olanzapine stimulated PBMC, accompanied by decreased PDH point to a worsening in cellular energy metabolism not compensated by AMKP upregulation. In contrast, aripiprazole stimulation lead to increased glucose uptake, while not affecting PDH complex expression. The observed differences may be involved in the different metabolic profiles observed in aripiprazole and olanzapine treated patients.

  13. How to Motivate Whole Blood Donors to Become Plasma Donors

    PubMed Central

    2014-01-01

    This study tested the efficacy of interventions to recruit new plasma donors among whole blood donors. A sample of 924 donors was randomized to one of three conditions: control; information only by nurse; and information plus self-positive image message by nurse (SPI). Participants in the control condition only received a leaflet describing the plasma donation procedure. In the two experimental conditions the leaflet was explained face-to-face by a nurse. The dependent variables were the proportion of new plasma donors and the number of donations at six months. Overall, 141 (15.3%) new plasma donors were recruited at six months. There were higher proportions of new plasma donors in the two experimental conditions compared to the control condition (P < .001); the two experimental conditions did not differ. Also, compared to the control condition, those in the experimental conditions (all Ps < .001) gave plasma more often (information only by nurse:  d = .26; SPI: d = .32); the SPI intervention significantly outperformed (P < .05) the information only by nurse condition. The results suggest that references to feelings of SPI such as feeling good and being proud and that giving plasma is a rewarding personal experience favor a higher frequency of plasma donation. PMID:25530909

  14. Disease-specific classification using deconvoluted whole blood gene expression

    PubMed Central

    Wang, Li; Oh, William K.; Zhu, Jun

    2016-01-01

    Blood-based biomarker assays have an advantage in being minimally invasive. Diagnostic and prognostic models built on peripheral blood gene expression have been reported for various types of disease. However, most of these studies focused on only one disease type, and failed to address whether the identified gene expression signature is disease-specific or more widely applicable across diseases. We conducted a meta-analysis of 46 whole blood gene expression datasets covering a wide range of diseases and physiological conditions. Our analysis uncovered a striking overlap of signature genes shared by multiple diseases, driven by an underlying common pattern of cell component change, specifically an increase in myeloid cells and decrease in lymphocytes. These observations reveal the necessity of building disease-specific classifiers that can distinguish different disease types as well as normal controls, and highlight the importance of cell component change in deriving blood gene expression based models. We developed a new strategy to develop blood-based disease-specific models by leveraging both cell component changes and cell molecular state changes, and demonstrate its superiority using independent datasets. PMID:27596246

  15. Whole blood glucose analysis based on smartphone camera module

    NASA Astrophysics Data System (ADS)

    Devadhasan, Jasmine Pramila; Oh, Hyunhee; Choi, Cheol Soo; Kim, Sanghyo

    2015-11-01

    Complementary metal oxide semiconductor (CMOS) image sensors have received great attention for their high efficiency in biological applications. The present work describes a CMOS image sensor-based whole blood glucose monitoring system through a point-of-care (POC) approach. A simple poly-ethylene terephthalate (PET) chip was developed to carry out the enzyme kinetic reaction at various concentrations (110-586 mg/dL) of mouse blood glucose. In this technique, assay reagent is immobilized onto amine functionalized silica (AFSiO2) nanoparticles as an electrostatic attraction in order to achieve glucose oxidation on the chip. The assay reagent immobilized AFSiO2 nanoparticles develop a semi-transparent reaction platform, which is technically a suitable chip to analyze by a camera module. The oxidized glucose then produces a green color according to the glucose concentration and is analyzed by the camera module as a photon detection technique; the photon number decreases when the glucose concentration increases. The combination of these components, the CMOS image sensor and enzyme immobilized PET film chip, constitute a compact, accurate, inexpensive, precise, digital, highly sensitive, specific, and optical glucose-sensing approach for POC diagnosis.

  16. How to motivate whole blood donors to become plasma donors.

    PubMed

    Godin, Gaston; Germain, Marc

    2014-01-01

    This study tested the efficacy of interventions to recruit new plasma donors among whole blood donors. A sample of 924 donors was randomized to one of three conditions: control; information only by nurse; and information plus self-positive image message by nurse (SPI). Participants in the control condition only received a leaflet describing the plasma donation procedure. In the two experimental conditions the leaflet was explained face-to-face by a nurse. The dependent variables were the proportion of new plasma donors and the number of donations at six months. Overall, 141 (15.3%) new plasma donors were recruited at six months. There were higher proportions of new plasma donors in the two experimental conditions compared to the control condition (P < .001); the two experimental conditions did not differ. Also, compared to the control condition, those in the experimental conditions (all Ps < .001) gave plasma more often (information only by nurse:  d = .26; SPI: d = .32); the SPI intervention significantly outperformed (P < .05) the information only by nurse condition. The results suggest that references to feelings of SPI such as feeling good and being proud and that giving plasma is a rewarding personal experience favor a higher frequency of plasma donation.

  17. Portable microfluidic cytometer for whole blood cell analysis

    NASA Astrophysics Data System (ADS)

    Grafton, Meggie M.; Zordan, Michael D.; Chuang, Han-Sheng; Rajdev, Pooja; Reece, Lisa M.; Irazoqui, Pedro P.; Wereley, Steven T.; Byrnes, Ron; Todd, Paul; Leary, James F.

    2010-02-01

    Lab-on-a-chip (LOC) systems allow complex laboratory assays to be carried out on a single chip using less time, reagents, and manpower than traditional methods. There are many chips addressing PCR and other DNA assays, but few that address blood cell analysis. Blood analysis, particularly of the cellular component, is highly important in both medical and scientific fields. Traditionally blood samples require a vial of blood, then several processing steps to separate and stain the various components, followed by the preparations for each specific assay to be performed. A LOC system for blood cell analysis and sorting would be ideal. The microfluidic-based system we have developed requires a mere drop of blood to be introduced onto the chip. Once on chip, the blood is mixed with both fluorescent and magnetic labels. The lab-on-a-chip device then uses a syringe drive to push the cells through the chip, while a permanent magnet is positioned to pull the magnetically labeled white blood cells to a separate channel. The white blood cells, labeled with different color fluorescent quantum dots (Qdots) conjugated to antibodies against WBC subpopulations, are analyzed and counted, while a sampling of red blood cells is also counted in a separate channel. This device will be capable of processing whole blood samples on location in a matter of minutes and displaying the cell count and should eventually find use in neonatology, AIDS and remote site applications.

  18. A versatile assay to determine bacterial and host factors contributing to opsonophagocytotic killing in hirudin-anticoagulated whole blood

    PubMed Central

    van der Maten, Erika; de Jonge, Marien I.; de Groot, Ronald; van der Flier, Michiel; Langereis, Jeroen D.

    2017-01-01

    Most bacteria entering the bloodstream will be eliminated through complement activation on the bacterial surface and opsonophagocytosis. However, when these protective innate immune systems do not work optimally, or when bacteria are equipped with immune evasion mechanisms that prevent killing, this can lead to serious infections such as bacteremia and meningitis, which is associated with high morbidity and mortality. In order to study the complement evasion mechanisms of bacteria and the capacity of human blood to opsonize and kill bacteria, we developed a versatile whole blood killing assay wherein both phagocyte function and complement activity can easily be monitored and modulated. In this assay we use a selective thrombin inhibitor hirudin to fully preserve complement activity of whole blood. This assay allows controlled analysis of the requirements for active complement by replacing or heat-inactivating plasma, phagocyte function and bacterial immune evasion mechanisms that contribute to survival in human blood. PMID:28176849

  19. Effects of oral eicosapentaenoic acid versus docosahexaenoic acid on human peripheral blood mononuclear cell gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have beneficial effects on inflammation and cardiovascular disease (CVD). Our aim was to assess the effect of a six-week supplementation with either olive oil, EPA, or DHA on gene expression in peripheral blood mononuclear cells (...

  20. Peculiarities of spectroscopic information of whole blood in atherosclerosis

    NASA Astrophysics Data System (ADS)

    Khairullina, Alphiya Y.; Oleinik, Tatiana V.; Yusupova, Lira B.; Prigoun, Natalia

    1995-01-01

    The coefficient of diffuse reflection and light transmission measurements in an optically thick layer of blood at atherosclerosis conditions under multiple scattering of light in the visual and nearest IR-spectra region (590 -900 nm) were measured for calculation of the absorption coefficients of the material of particles and surrounding medium K((lambda) ) and parameter Q (the latter parameter was defined by the sizes of erythrocytes and aggregates and by refraction coefficient of red cells relative to plasma at atherosclerosis). For the main quantitative spectroscopy of particles the K1((lambda) ) for known value of K((lambda) ) and the parameter Q determinations it is necessary to have the knowledge of relative volume part H occupied by particles. In the case of a high concentration of particles H >= 0.2 as it takes place in the blood the parameters Q and K((lambda) ) are in dependence of H (H - is hematocrit ration for the case of whole blood). It should be noted that spectroscopy of multiple scattering light can give some information out of main absorption bands with the higher accuracy and higher light scattering. The latter value provides the opportunity of determination of faint absorption bands which couldn't be achieved by other methods. The method proposed is characterized by absence of probe preparations, approach to in viva conditions, expressivity, and high informativity of each experiment. A many-fold investigation of the blood of healthy men in the spectral region 650 - 810 nm shows the electron spectrum of absorption of molecular hemoglobin hem is the most optically active blood spectra component K((lambda) ). The broadening of spectral investigations, as in short wave or long wave areas of the spectrum, by the use of multiple scattering methods for calculations of K((lambda) ) and Q((lambda) ) enlarges the number of chromophores studied.

  1. Adaptive Force Sonorheometry for Assessment of Whole Blood Coagulation

    PubMed Central

    Mauldin, F. William; Viola, Francesco; Hamer, Theresa C.; Ahmed, Eman M.; Crawford, Shawna B.; Haverstick, Doris M.; Lawrence, Michael B.; Walker, William F.

    2010-01-01

    Background: Viscoelastic diagnostics that monitor the hemostatic function of whole blood (WB), such as thromboelastography, have been developed with demonstrated clinical utility. By measuring the cumulative effects of all components of hemostasis, viscoelastic diagnostics have circumvented many of the challenges associated with more common tests of blood coagulation. Methods: We describe a new technology, called sonorheometry, that adaptively applies acoustic radiation force to assess coagulation function in WB. The repeatability (precision) of coagulation parameters was assessed using citrated WB samples. A reference range of coagulation parameters, along with corresponding measurements from prothrombin time (PT) and partial thromboplastin time (PTT), were obtained from WB samples of 20 healthy volunteers. In another study, sonorheometry monitored anticoagulation with heparin (0 – 5 IU/ml) and reversal from varied dosages of protamine (0 – 10 IU/ml) in heparinized WB (2 IU/ml). Results: Sonorheometry exhibited low CVs for parameters: clot initiation time (TC1), < 7%; clot stabilization time (TC2), < 6.5%; and clotting angle (θ), < 3.5%. Good correlation was observed between clotting times, TC1 and TC2, and PTT (r = 0.65 and 0.74 respectively; n=18). Linearity to heparin dosage was observed with average linearity r > 0.98 for all coagulation parameters. We observed maximum reversal of heparin anticoagulation at protamine to heparin ratios of 1.4:1 from TC1 (P=0.6) and 1.2:1 from θ (P=0.55). Conclusions: Sonorheometry is a non-contact method for precise assessment of WB coagulation. PMID:20096680

  2. Transplantation of mononuclear cells from human umbilical cord blood promotes functional recovery after traumatic spinal cord injury in Wistar rats

    PubMed Central

    Rodrigues, L.P.; Iglesias, D.; Nicola, F.C.; Steffens, D.; Valentim, L.; Witczak, A.; Zanatta, G.; Achaval, M.; Pranke, P.; Netto, C.A.

    2011-01-01

    Cell transplantation is a promising experimental treatment for spinal cord injury. The aim of the present study was to evaluate the efficacy of mononuclear cells from human umbilical cord blood in promoting functional recovery when transplanted after a contusion spinal cord injury. Female Wistar rats (12 weeks old) were submitted to spinal injury with a MASCIS impactor and divided into 4 groups: control, surgical control, spinal cord injury, and one cell-treated lesion group. Mononuclear cells from umbilical cord blood of human male neonates were transplanted in two experiments: a) 1 h after surgery, into the injury site at a concentration of 5 x 106 cells diluted in 10 µL 0.9% NaCl (N = 8-10 per group); b) into the cisterna magna, 9 days after lesion at a concentration of 5 x 106 cells diluted in 150 µL 0.9% NaCl (N = 12-14 per group). The transplanted animals were immunosuppressed with cyclosporin-A (10 mg/kg per day). The BBB scale was used to evaluate motor behavior and the injury site was analyzed with immunofluorescent markers to label human transplanted cells, oligodendrocytes, neurons, and astrocytes. Spinal cord injury rats had 25% loss of cord tissue and cell treatment did not affect lesion extension. Transplanted cells survived in the injured area for 6 weeks after the procedure and both transplanted groups showed better motor recovery than the untreated ones (P < 0.05). The transplantation of mononuclear cells from human umbilical cord blood promoted functional recovery with no evidence of cell differentiation. PMID:22183246

  3. Whole Blood Gene Expression Profiling in Preclinical and Clinical Cattle Infected with Atypical Bovine Spongiform Encephalopathy.

    PubMed

    Xerxa, Elena; Barbisin, Maura; Chieppa, Maria Novella; Krmac, Helena; Vallino Costassa, Elena; Vatta, Paolo; Simmons, Marion; Caramelli, Maria; Casalone, Cristina; Corona, Cristiano; Legname, Giuseppe

    2016-01-01

    Prion diseases, such as bovine spongiform encephalopathies (BSE), are transmissible neurodegenerative disorders affecting humans and a wide variety of mammals. Variant Creutzfeldt-Jakob disease (vCJD), a prion disease in humans, has been linked to exposure to BSE prions. This classical BSE (cBSE) is now rapidly disappearing as a result of appropriate measures to control animal feeding. Besides cBSE, two atypical forms (named H- and L-type BSE) have recently been described in Europe, Japan, and North America. Here we describe the first wide-spectrum microarray analysis in whole blood of atypical BSE-infected cattle. Transcriptome changes in infected animals were analyzed prior to and after the onset of clinical signs. The microarray analysis revealed gene expression changes in blood prior to the appearance of the clinical signs and during the progression of the disease. A set of 32 differentially expressed genes was found to be in common between clinical and preclinical stages and showed a very similar expression pattern in the two phases. A 22-gene signature showed an oscillating pattern of expression, being differentially expressed in the preclinical stage and then going back to control levels in the symptomatic phase. One gene, SEL1L3, was downregulated during the progression of the disease. Most of the studies performed up to date utilized various tissues, which are not suitable for a rapid analysis of infected animals and patients. Our findings suggest the intriguing possibility to take advantage of whole blood RNA transcriptional profiling for the preclinical identification of prion infection. Further, this study highlighted several pathways, such as immune response and metabolism that may play an important role in peripheral prion pathogenesis. Finally, the gene expression changes identified in the present study may be further investigated as a fingerprint for monitoring the progression of disease and for developing targeted therapeutic interventions.

  4. Investigation of a whole blood fluidized bed Taylor-Couette flow device for enzymatic heparin neutralization.

    PubMed

    Ameer, G A; Harmon, W; Sasisekharan, R; Langer, R

    1999-03-05

    The use of clinical bioreactors will increase as more therapeutic proteins are being cloned, expressed, and produced at a reduced cost. The proposed use of an immobilized heparinase I reactor to make heparin anticoagulation a safer therapy is an example of how the specificity and high activity of an enzyme could be incorporated into a system to ultimately benefit a patient. However, the development of a safe and efficient bioreactor is important for the use of immobilized heparinase I and other therapeutic proteins designed for use in medical extracorporeal procedures. This study examined the possibility of using Taylor-Couette flow and "flow-induced" recirculation of the agarose beads as a way to fluidize agarose-bound heparinase in whole blood. Heparinase I was immobilized onto agarose beads via cyanogen bromide activation. A reactor based on Taylor-Couette flow was designed and modified with a tangential recirculation line. The reactor was tested for efficacy and safety in vitro in human blood. Visualization studies in water and 42% glycerol were used to determine the minimum rotation rate for efficient fluidization. The strategic placement of the recirculation line allowed recirculation of the agarose without the use of an external pump. The device removed 90% of the heparin activity within 2 min from 450 cc of human blood at a blood flow rate of 100 mL/min. Furthermore, the device maintained inlet and outlet clotting times of 269 +/- 10 and 235 +/- 6 s, respectively, demonstrating the potential for regional heparinization. Blood damage was a function of gel volume fraction and rotation rate of the inner cylinder. Hemolysis of the red cells is an important issue when Taylor vortices are combined with macroscopic solid particles such as agarose beads. A modified Taylor-Couette flow device was developed to treat whole blood and operational criteria were established to minimize hemolysis.

  5. Zika Virus Infection and Prolonged Viremia in Whole-Blood Specimens.

    PubMed

    Mansuy, Jean Michel; Mengelle, Catherine; Pasquier, Christophe; Chapuy-Regaud, Sabine; Delobel, Pierre; Martin-Blondel, Guillaume; Izopet, Jacques

    2017-05-15

    We tested whole-blood and plasma samples from immunocompetent patients who had had benign Zika virus infections and found that Zika virus RNA persisted in whole blood substantially longer than in plasma. This finding may have implications for diagnosis of acute symptomatic and asymptomatic infections and for testing of blood donations.

  6. 9 CFR 147.3 - The stained-antigen, rapid, whole-blood test. 3

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false The stained-antigen, rapid, whole-blood test. 3 147.3 Section 147.3 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... Blood Testing Procedures § 147.3 The stained-antigen, rapid, whole-blood test. 3 3 The...

  7. 9 CFR 147.3 - The stained-antigen, rapid, whole-blood test. 3

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false The stained-antigen, rapid, whole-blood test. 3 147.3 Section 147.3 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... Blood Testing Procedures § 147.3 The stained-antigen, rapid, whole-blood test. 3 3 The...

  8. 9 CFR 147.3 - The stained-antigen, rapid, whole-blood test. 3

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false The stained-antigen, rapid, whole-blood test. 3 147.3 Section 147.3 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... Blood Testing Procedures § 147.3 The stained-antigen, rapid, whole-blood test. 3 3 The...

  9. 9 CFR 147.3 - The stained-antigen, rapid, whole-blood test. 3

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false The stained-antigen, rapid, whole-blood test. 3 147.3 Section 147.3 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... Blood Testing Procedures § 147.3 The stained-antigen, rapid, whole-blood test. 3 3 The...

  10. Differentiation of human mononuclear phagocytes increases their innate response to Mycobacterium tuberculosis infection.

    PubMed

    Castaño, Diana; García, Luis F; Rojas, Mauricio

    2014-05-01

    The heterogeneity of mononuclear phagocytes, partially explained by cell differentiation, influences the activation of innate responses. It has been reported that Mycobacterium tuberculosis inhibits monocyte differentiation into either dendritic cells or macrophages. To evaluate whether the activation of effector mechanisms against M. tuberculosis differ between less and more differentiated mononuclear phagocytes, we compared monocytes differentiated in vitro for 24 h (MON24) and 120 h (MDM120) infected with M. tuberculosis H37Rv, H37Ra and the clinical isolate UT127 at different multiplicity of infection. MDM120 phagocytosed more M. tuberculosis, inhibited mycobacterial growth and did not die in response to the infection, compared with MON24. In contrast, MON24 become Annexin V and Propidium iodide positive after 36 h of M. tuberculosis infection. Although, there were striking differences between MON24 and MDM120, there were also some differences in the response to the mycobacterial strains used. Finally, in MDM120 infected with M. tuberculosis H37Rv, a lower percentage of mycobacterial phagosomes accumulated transferrin and a higher percentage co-localized with cathelicidin than in MON24. These results demonstrate that innate responses induced by M. tuberculosis depends upon the stage of differentiation of mononuclear phagocytes and support that terminally differentiated cells are more efficient anti-mycobacterial effectors than the less differentiated ones.

  11. Fluorescence and absorbance analyte sensing in whole blood and plasma based on diffusion separation in silicon-microfabricated flow structures

    NASA Astrophysics Data System (ADS)

    Weigl, Bernhard H.; Hixson, Greg T.; Kenny, Margaret; Zebert, Diane; Dwinnell, Silver; Buj, Todd; Yager, Paul

    1997-05-01

    Based on the recently introduced T-Sensor method, we demonstrate the fluorescence-determination of various analytes directly in whole blood and in serum. The method relies on microfluidic flow in silicon structures, diffusion-based separation, and analyte determination using fluorescent and absorption indicator dyes. Due to extremely small inertial forces in such structures, practically all flow in microstructures is laminar. This allows the movement of different layers of fluid and particles next to each other in a channel without mixing other than by diffusion. A sample solution (e.g., blood), and a receptor solution containing the indicator dye are introduced in a common channel, and flow laminarly next to each other until they exit the structure. Small ions such as H+, and Na+ diffuse rapidly across the channel, whereas larger molecules diffuse more slowly. Larger particles such as blood cells and polymer beads show no significant diffusion within the time the two flow streams are in contact. The fluorescence emission of indicator dyes is a function of the concentration of the analyte molecules and the dye concentration in the interaction zone between the two streams. This device allows continuous monitoring of the concentration of analytes in whole blood without the use of membranes or prior removal of blood cells. This principle is illustrated by the determination of human albumin, total calcium, and pH in whole blood and serum.

  12. Reactive oxygen species formation and apoptosis in human peripheral blood mononuclear cell induced by 900 MHz mobile phone radiation.

    PubMed

    Lu, Yao-Sheng; Huang, Bao-Tian; Huang, Yao-Xiong

    2012-01-01

    We demonstrate that reactive oxygen species (ROS) plays an important role in the process of apoptosis in human peripheral blood mononuclear cell (PBMC) which is induced by the radiation of 900 MHz radiofrequency electromagnetic field (RFEMF) at a specific absorption rate (SAR) of ~0.4 W/kg when the exposure lasts longer than two hours. The apoptosis is induced through the mitochondrial pathway and mediated by activating ROS and caspase-3, and decreasing the mitochondrial potential. The activation of ROS is triggered by the conformation disturbance of lipids, protein, and DNA induced by the exposure of GSM RFEMF. Although human PBMC was found to have a self-protection mechanism of releasing carotenoid in response to oxidative stress to lessen the further increase of ROS, the imbalance between the antioxidant defenses and ROS formation still results in an increase of cell death with the exposure time and can cause about 37% human PBMC death in eight hours.

  13. Metabolic effects of microwave radiation and convection heating on human mononuclear leukocytes. Final report, January 1985-May 1986

    SciTech Connect

    Kiel, J.L.; Wong, L.S.; Erwin, D.N.

    1986-01-01

    Investigated here were the effects of microwave (MW) radiation (2450-MHz, continuous-wave, mean specific absorption rate of 103.5 + or - 4.2 W/kg) and convention heating on the nonphosphorylating oxidative metabolism of human peripheral mononuclear leukocytes (96% lymphocytes, 4% monocytes) at 37 C. Metabolic activity, determined by chemiluminescence (CL) of cells challenged with luminol (5-aminO-2, 3-dihydro-1, 4-phthalazinedione) linked to bovine serum albumin, was detected with a brightness photomer. A significant stimulation after after MW exposure (p < 0.005) over total CL of matched 37 C-incubator controls was observed. A similar degree of stimulation, compared to incubator controls, was also detected after sham treatment. No significant difference existed between changes in total CL or stimulation indices of the MW and sham-exposed groups. Exposure to MW radiation, under normothermic (37 + or - 0.03 C) conditions, appears to have no effect on the oxidative metabolic activity of human peripheral mononuclear leukocytes. However, the significant differences between MW or sham-exposed cells and their respective incubator controls occurred because the temperature of the incubator did not exceed 35.9 C, and 39 minutes were required for the temperature to rise from 22 to 35.9 C. Slow heating of incubator controls must be accounted for in thermal and redio-frequency radiation studies in vitro.

  14. Immunostimulatory acivity of Calophyllum brasiliense, Ipomoea pes-caprae and Matayba elaeagnoides demonstrated by human peripheral blood mononuclear cells proliferation.

    PubMed

    Philippi, Marina Elisa; Duarte, Bruna Momm; Da Silva, Carolina Vieira; De Souza, Michel Thomaz; Niero, Rivaldo; Cechinel Filho, Valdir; Bueno, Edneia Casagranda

    2010-01-01

    This study evaluates the effect of methanol extracts of three Brazilian medicinal plants on in vitro proliferation of human mononuclear cells. Lymphoproliferation assay was carried out by incubating human peripheral blood mononuclear cells from healthy donors (1 x 10(6) cells/mL) with extracts of Calophyllum brasiliense (roots), Ipomoea pes-caprae (whole plant) and Matayba elaeagnoides (bark), both at 10, 50, 100 and 200 microg/mL, alone or with phytohemagglutinin (PHA, 5 microg/mL), in 96-well microplates at 37 degrees C with 5% CO2, for 72 h. The quantification of cell proliferation assay was performed by blue tetrazolium (MTT) reduction with reading at 540 nm. Cells incubated with only the culture medium were used as negative control for cell proliferation, while the positive control consisted of cells and PHA. The results suggest that the extracts of all three studied plants induce T lymphocyte proliferation. I. pes-caprae showed immunostimulatory activity three times higher than the C. brasiliense extract, while that of the M. elaeagnoides extract was 1.5 times higher. The results demonstrate immunostimulatory effects of these three plants, therefore the continuity of these studies is recommended, in order to determine the active principles.

  15. Rapid evaluation of fibrinogen levels using the CG02N whole blood coagulation analyzer.

    PubMed

    Hayakawa, Mineji; Gando, Satoshi; Ono, Yuichi; Mizugaki, Asumi; Katabami, Kenichi; Maekawa, Kunihiko; Miyamoto, Daisuke; Wada, Takeshi; Yanagida, Yuichiro; Sawamura, Atsushi

    2015-04-01

    Rapid evaluation of fibrinogen (Fbg) levels is essential for maintaining homeostasis in patients with massive bleeding during severe trauma and major surgery. This study evaluated the accuracy of fibrinogen levels measured by the CG02N whole blood coagulation analyzer (A&T Corporation, Kanagawa, Japan) using heparinized blood drawn for blood gas analysis (whole blood-Fbg). A total of 100 matched pairs of heparinized blood samples and citrated blood samples were simultaneously collected from patients in the intensive care unit. Whole blood-Fbg results were compared with those of citrated plasma (standard-Fbg). The whole blood coagulation analyzer measured fibrinogen levels within 2 minutes. Strong correlations between standard-Fbg and whole blood-Fbg were observed (ρ = 0.91, p < 0.001). Error grid analysis showed that 88% of the values were clinically acceptable, and 12% were in a range with possible effects on clinical decision-making; none were in a clinically dangerous range without appropriate treatment. Using a fibrinogen cutoff value of 1.5 g/L for standard-Fbg, the area under the receiver operating characteristic curve of whole blood-Fbg was 0.980 (95% confidence interval 0.951-1.000, p < 0.001). The whole blood coagulation analyzer can rapidly measure fibrinogen levels in heparinized blood and could be useful in critical care settings where excessive bleeding is a concern.

  16. Development of a Population Pharmacokinetic Model To Describe Azithromycin Whole-Blood and Plasma Concentrations over Time in Healthy Subjects

    PubMed Central

    Anic-Milic, T.; Oreskovic, K.; Padovan, J.; Brouwer, K. L. R.; Zuo, P.; Schmith, V. D.

    2013-01-01

    Azithromycin (AZI), a broad-spectrum antibiotic, accumulates in polymorphonuclear cells and peripheral blood mononuclear cells. The distribution of AZI in proinflammatory cells may be important to the anti-inflammatory properties. Previous studies have described plasma AZI pharmacokinetics. The objective of this study was to describe the pharmacokinetics of AZI in whole blood (concentration in whole blood [Cb]) and plasma (concentration in plasma [Cp]) of healthy subjects. In this study, 12 subjects received AZI (500 mg once a day for 3 days). AZI Cb and Cp were quantified in serial samples collected up to 3 weeks after the last dose and analyzed using noncompartmental and compartmental methods. After the last dose, Cb was greater than Cp. Importantly, Cb, but not Cp, was quantifiable in all but one subject at 3 weeks. The blood area under the curve during a 24-h dosing interval (AUC24) was ∼2-fold greater than the plasma AUC24, but simulations suggested that Cb was not at steady state by day 3. Upon exploration of numerous models, an empirical 3-compartment model adequately described Cp and Cb, but Cp was somewhat underestimated. Intercompartmental clearance (CL; likely representing cells) was lower than apparent oral CL (18 versus 118 liters/h). Plasma, peripheral, and cell compartmental volumes were 439 liters, 2,980 liters, and 3,084 liters, respectively. Interindividual variability in CL was low (26.2%), while the volume of distribution variability was high (107%). This is the first report to describe AZI Cb in healthy subjects, the distribution parameters between Cp and Cb, and AZI retention in blood for up to 3 weeks following 3 daily doses. The model can be used to predict Cb from Cp for AZI under various dosing regimens. (This study has been registered at ClinicalTrials.gov under registration no. NCT01026064.) PMID:23629714

  17. Effect of 900 MHz Electromagnetic Radiation on the Induction of ROS in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Kazemi, E.; Mortazavi, S. M. J.; Ali-Ghanbari, A.; Sharifzadeh, S.; Ranjbaran, R.; Mostafavi-pour, Z.; Zal, F.; Haghani, M.

    2015-01-01

    Background Despite numerous studies over a decade, it still remains controversial about the biological effects of RF EMF emitted by mobile phone telephony. Objective Here we investigated the effect of 900 MHz GSM on the induction of oxidative stress and the level of intracellular reactive oxygen species (ROS) in human mononuclear cells, monocytes and lymphocytes as defence system cells. Method 6 ml Peripheral Blood samples were obtained from 13 healthy volunteers (21-30 year-old). Each sample was devided into 2 groups: one was exposed RF radiation emitted from a mobile phone simulator for 2 hour and the other used as control group which was not exposed to any fields. After that, mononuclear cells were isolated from peripheral blood by density gradient centrifugation in Ficoll-Paque. The intracellular ROS content in monocytes and lymphocytes was measured by the CM-H2DCFDA fluorescence probe using flowcytometry technique. Results Our results showed significant increase in  ROS production after exposure in population rich in monocytes. This effect was not significant in population rich in lymphocytes in comparison with non exposed cells. Conclusion The results obtained in this study clearly showed the oxidative stress induction capability of RF electromagnetic field in the portion of PBMCs mostly in monocytes, like the case of exposure to micro organisms, although the advantages or disadvantages of this effect should be evaluated. PMID:26396966

  18. The expression and function of human CD300 receptors on blood circulating mononuclear cells are distinct in neonates and adults

    PubMed Central

    Zenarruzabeitia, Olatz; Vitallé, Joana; García-Obregón, Susana; Astigarraga, Itziar; Eguizabal, Cristina; Santos, Silvia; Simhadri, Venkateswara R.; Borrego, Francisco

    2016-01-01

    Neonates are more susceptible to infections than adults. This susceptibility is thought to reflect neonates’ qualitative and quantitative defects in the adaptive and innate immune responses. Differential expression of cell surface receptors may result in altered thresholds of neonatal immune cell activation. We determined whether the expression and function of the lipid-binding CD300 family of receptors are different on neonatal immune cells compared to adult immune cells. A multiparametric flow cytometry analysis was performed to determine the expression of CD300 receptors on adult peripheral blood mononuclear cells and neonatal cord blood mononuclear cells. The expression of the CD300a inhibitory receptor was significantly reduced on cells from the newborn adaptive immune system, and neonatal antigen presenting cells exhibited a different CD300 receptors expression pattern. We also found differential LPS-mediated regulation of CD300 receptors expression on adult monocytes compared to cord blood monocytes, and that CD300c and CD300e-mediated activation was quantitatively different in neonatal monocytes. This is the first complete study examining the expression of CD300 receptors on human neonatal immune cells compared with adult immune cells. Significant differences in the expression and function of CD300 receptors may help to explain the peculiarities and distinctness of the neonatal immune responses. PMID:27595670

  19. Influence of captopril on glucose and fatty acid oxidation in human thrombocytes and mononuclear leucocytes.

    PubMed

    Haeckel, R; Colic, D

    1991-01-01

    Captopril (CAS 62571-86-2) may be beneficial for the treatment of diabetes because of its activating effect on peripheral glucose consumption besides its well known blood pressure degradation. The glucose oxidation has been found to be activated by captopril in thrombocytes and mononuclear leucocytes, cell types which are usually considered to be independent from insulin. Because the oxidation of pyruvate labelled in position C-1 but not of 2-14C-pyruvate and of 1-14C-acetate was enhanced, captopril most probably stimulated the pyruvate decarboxylation reaction. The metabolism of glucose labelled in positions 1 and 6 was equally activated by captopril indicating another step which may be affected by captopril.

  20. PSP activates monocytes in resting human peripheral blood mononuclear cells: immunomodulatory implications for cancer treatment.

    PubMed

    Sekhon, Bhagwant Kaur; Sze, Daniel Man-Yuen; Chan, Wing Keung; Fan, Kei; Li, George Qian; Moore, Douglas Edwin; Roubin, Rebecca Heidi

    2013-06-15

    Polysaccharopeptide (PSP), from Coriolus versicolor, has been used as an adjuvant to chemotherapy, and has demonstrated anti-tumor and immunomodulating effects. However its mechanism remains unknown. To elucidate how PSP affects immune populations, we compared PSP treatments both with and without prior incubation in phytohaemagglutinin (PHA) - a process commonly used in immune population experimentation. We first standardised a capillary electrophoresis fingerprinting technique for PSP identification and characterisation. We then established the proliferative capability of PSP on various immune populations in peripheral blood mononuclear cells, using flow cytometry, without prior PHA treatment. It was found that PSP significantly increased the number of monocytes (CD14(+)/CD16(-)) compared to controls without PHA. This increase in monocytes was confirmed using another antibody panel of CD14 and MHCII. In contrast, proliferations of T-cells, NK, and B-cells were not significantly changed by PSP. Thus, stimulating monocyte/macrophage function with PSP could be an effective therapeutic intervention in targeting tumors.

  1. The state of the science of whole blood: lessons learned at Mayo Clinic

    PubMed Central

    Stubbs, James R.; Zielinski, Martin D.; Jenkins, Donald

    2016-01-01

    AABB Standards specify that ABO group-specific whole blood is the only acceptable choice for whole blood transfusions. Although universal donor group O stored whole blood (SWB) was used extensively by the military during the wars of the mid-twentieth century, its use has fallen out of favor and has never been used to great extent in the civilian trauma population. Interest in the use of whole blood has been renewed, particularly in light of its potential value in far-forward military and other austere environments. Evidence of preserved platelet function in SWB has heightened enthusiasm for a “one stop shop” resuscitation product providing volume, oxygen carrying capacity, and hemostatic effects. Experience with universal donor group O SWB is required to ascertain whether its use will be an advance in trauma care. Described here is the process of establishing a universal donor group O SWB at a civilian trauma center in the United States. PMID:27100754

  2. Effects of clinically relevant alumina ceramic wear particles on TNF-alpha production by human peripheral blood mononuclear phagocytes.

    PubMed

    Hatton, A; Nevelos, J E; Matthews, J B; Fisher, J; Ingham, E

    2003-03-01

    The recent introduction of microseparation of the components of ceramic-on-ceramic hip prostheses during hip simulations has produced clinically relevant wear rates, wear patterns and wear particles. This provided an opportunity to determine the response of primary human peripheral blood mononuclear cells to clinically relevant alumina ceramic wear particles in vitro. Alumina ceramic wear particles were generated in a hip joint simulator under microseparation conditions. The particles showed a bi-modal size distribution with nanometer sized (5-20nm) and larger particles (0.2->10 micrometer). The particles were cultured with human peripheral blood mononuclear cells obtained from six different donors at particle volume to cell number ratios of 1, 10, 100 and 500 micrometer(3). After 24h incubation the viability of the cells and the levels of TNF-alpha were determined. The response to the microseparation wear particles was compared to that of commercially available alumina powder with a uniform morphology and mean size of 0.5 micrometer. All six Donors PBMNC produced significantly elevated levels of TNF-alpha when stimulated with 100 micrometer(3) of the alumina powder per cell. Volumetric concentrations of 10 and 1.0 micrometer(3) per cell failed to stimulate a significant response by the cells from any of the six donors. Three of the six Donors PBMNC secreted significantly elevated levels of TNF-alpha when stimulated with 100 micrometer(3) of the microseparation wear particles, whereas the other three failed to respond to the wear debris at this concentration. All of the Donors PBMNC produced significantly elevated levels of TNF-alpha when stimulated with 500 micrometer(3) of the microseparation wear particles per cell. Thus, a greater volume of the microseparation wear particles was required to activate the PBMNC than the alumina powder. This was probably due to the microseparation wear particles having fewer particles in the critical size range (0.1-1 micrometer

  3. Pathogen Reduction of Fresh Whole Blood for Military and Civilian Use

    DTIC Science & Technology

    2010-04-01

    Pathogen Reduction of Fresh Whole Blood for Military and Civilian Use 24 - 4 RTO-MP-HFM-182 Figure 5: Growth rate of B. cereus in treated and... Bacillus cereus 2/8 Streptococcus pyogenes 4/10 Staphylococcus epidermidis 15/22 3.3 Parasite Reduction Several experiments to test the...RTO-MP-HFM-182 24 - 1 Pathogen Reduction of Fresh Whole Blood for Military and Civilian Use Raymond P. Goodrich, Ph.D., Heather L. Reddy

  4. Whole blood cells loaded with messenger RNA as an anti-tumor vaccine.

    PubMed

    Phua, Kyle K L; Boczkowski, David; Dannull, Jens; Pruitt, Scott; Leong, Kam W; Nair, Smita K

    2014-06-01

    The use of a cell-based vaccine composed of autologous whole blood cells loaded with mRNA is described. Mice immunized with whole blood cells loaded with mRNA encoding antigen develop anti-tumor immunity comparable to DC-RNA immunization. This approach offers a simple and affordable alternative to RNA-based cellular therapy by circumventing complex, laborious and expensive ex vivo manipulations required for DC-based immunizations.

  5. Fiber-optic multiphoton flow cytometry in whole blood and in vivo

    NASA Astrophysics Data System (ADS)

    Chang, Yu-Chung; Ye, Jing Yong; Thomas, Thommey P.; Cao, Zhengyi; Kotlyar, Alina; Tkaczyk, Eric R.; Baker, James R.; Norris, Theodore B.

    2010-07-01

    Circulating tumor cells in the bloodstream are sensitive indicators for metastasis and disease prognosis. Circulating cells have usually been monitored via extraction from blood, and more recently in vivo using free-space optics; however, long-term intravital monitoring of rare circulating cells remains a major challenge. We demonstrate the application of a two-photon-fluorescence optical fiber probe for the detection of cells in whole blood and in vivo. A double-clad fiber was used to enhance the detection sensitivity. Two-channel detection was employed to enable simultaneous measurement of multiple fluorescent markers. Because the fiber probe circumvents scattering and absorption from whole blood, the detected signal strength from fluorescent cells was found to be similar in phosphate-buffered saline (PBS) and in whole blood. The detection efficiency of cells labeled with the membrane-binding dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindoldicarbocyanine, 4-chlorobenzenesulfonate (DiD) was demonstrated to be the same in PBS and in whole blood. A high detection efficiency of green fluorescent protein (GFP)-expressing cells in whole blood was also demonstrated. To characterize in vivo detection, DiD-labeled untransfected and GFP-transfected cells were injected into live mice, and the cell circulation dynamics was monitored in real time. The detection efficiency of GFP-expressing cells in vivo was consistent with that observed ex vivo in whole blood.

  6. Superiority of West Nile Virus RNA Detection in Whole Blood for Diagnosis of Acute Infection.

    PubMed

    Lustig, Yaniv; Mannasse, Batya; Koren, Ravit; Katz-Likvornik, Shiri; Hindiyeh, Musa; Mandelboim, Michal; Dovrat, Sara; Sofer, Danit; Mendelson, Ella

    2016-09-01

    The current diagnosis of West Nile virus (WNV) infection is primarily based on serology, since molecular identification of WNV RNA is unreliable due to the short viremia and absence of detectable virus in cerebrospinal fluid (CSF). Recent studies have shown that WNV RNA can be detected in urine for a longer period and at higher concentrations than in plasma. In this study, we examined the presence of WNV RNA in serum, plasma, whole-blood, CSF, and urine samples obtained from patients diagnosed with acute WNV infection during an outbreak which occurred in Israel in 2015. Our results demonstrate that 33 of 38 WNV patients had detectable WNV RNA in whole blood at the time of diagnosis, a higher rate than in any of the other sample types tested. Overall, whole blood was superior to all other samples, with 86.8% sensitivity, 100% specificity, 100% positive predictive value, and 83.9% negative predictive value. Interestingly, WNV viral load in urine was higher than in whole blood, CSF, serum, and plasma despite the lower sensitivity than that of whole blood. This study establishes the utility of whole blood in the routine diagnosis of acute WNV infection and suggests that it may provide the highest sensitivity for WNV RNA detection in suspected cases.

  7. Superiority of West Nile Virus RNA Detection in Whole Blood for Diagnosis of Acute Infection

    PubMed Central

    Mannasse, Batya; Koren, Ravit; Katz-Likvornik, Shiri; Hindiyeh, Musa; Mandelboim, Michal; Dovrat, Sara; Sofer, Danit; Mendelson, Ella

    2016-01-01

    The current diagnosis of West Nile virus (WNV) infection is primarily based on serology, since molecular identification of WNV RNA is unreliable due to the short viremia and absence of detectable virus in cerebrospinal fluid (CSF). Recent studies have shown that WNV RNA can be detected in urine for a longer period and at higher concentrations than in plasma. In this study, we examined the presence of WNV RNA in serum, plasma, whole-blood, CSF, and urine samples obtained from patients diagnosed with acute WNV infection during an outbreak which occurred in Israel in 2015. Our results demonstrate that 33 of 38 WNV patients had detectable WNV RNA in whole blood at the time of diagnosis, a higher rate than in any of the other sample types tested. Overall, whole blood was superior to all other samples, with 86.8% sensitivity, 100% specificity, 100% positive predictive value, and 83.9% negative predictive value. Interestingly, WNV viral load in urine was higher than in whole blood, CSF, serum, and plasma despite the lower sensitivity than that of whole blood. This study establishes the utility of whole blood in the routine diagnosis of acute WNV infection and suggests that it may provide the highest sensitivity for WNV RNA detection in suspected cases. PMID:27335150

  8. In vitro expansion of Lin{sup +} and Lin{sup −} mononuclear cells from human peripheral blood

    SciTech Connect

    Norhaiza, H. Siti; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul; Rohaya, M. A. W.

    2013-11-27

    Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin{sup −}) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin{sup +}) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin{sup −} cell population. The ability of Lin{sup +} and Lin{sup −} to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin{sup +} and Lin{sup −} were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin{sup +} mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin{sup −} stem cells were not able to survive in proliferation medium however

  9. Interferon-α curbs production of interleukin-22 by human peripheral blood mononuclear cells exposed to live Borrelia burgdorferi.

    PubMed

    Berner, Anika; Bachmann, Malte; Pfeilschifter, Josef; Kraiczy, Peter; Mühl, Heiko

    2015-10-01

    Cytokine networks initiated by means of innate immunity are regarded as a major determinant of host defence in response to acute infection by bacteria including Borrelia burgdorferi. Herein, we demonstrate that interferon (IFN)-α, either endogenously produced after exposure of cells to toll-like receptor-9-activating CpG oligonucleotides or provided as recombinant cytokine, weakens activation of the anti-bacterial interleukin (IL)-1/IL-22 axis in human peripheral blood mononuclear cells exposed to viable B. burgdorferi. As IFN-α has been related to pathological dissemination of the spirochaete, data suggest an immunoregulatory role of type I IFN in this context that is able to significantly modify cytokine profiles thereby possibly determining early course of B. burgdorferi infection.

  10. Beta2-Adrenoceptor Stimulation Suppresses TLR9-Dependent IFNA1 Secretion in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Weisheit, Christina; Knüfermann, Pascal; Baumgarten, Georg; Hoeft, Andreas; Poth, Jens M.

    2013-01-01

    Introduction IFNA1 (interferon alpha) is a key cytokine regulating the activity of numerous immune cells. Plasmacytoid dendritic cells (pDCs) as natural interferon-producing cells play critical roles as sensors of pathogens and link innate to adaptive immunity. CpG motifs within DNA sequences activating toll-like receptor 9 (TLR9) are the main stimuli eliciting IFNA1 secretion from pDCs. Adrenergic substances are capable of differentially modulating the response from various immune cells. Hence, the aim of this study was to examine how adrenoceptor stimulation influences TLR9-induced IFNA1 secretion from human pDCs. Methods PBMCs generated from human whole blood and pDCs enriched from buffy coats were stimulated with LPS and CpG-ODN 2336 in the presence or absence of epinephrine and different adrenoceptor antagonists. Secretion of TNF and IFNA1 was measured by ELISA. Flow cytometry was used to determine efficacy of pDC enrichment and adrenoceptor expression of PBMC subsets. The influence of modified IFNA1 secretion on NK cell activity was evaluated using a colorimetric tumor cell lysis assay. Results TLR9-induced IFNA1 secretion as well as TLR4-induced TNF secretion from PBMCs was dose-dependently attenuated by coincubation with epinephrine. Combination with different specific adrenoceptor antagonists revealed that this effect was mediated by the adrenoceptor β2 (ADRB2). Since flow cytometric analysis could exclude the presence of ADRB2 on pDCs, highly enriched pDCs lacked any visible impact of adrenoceptor stimulation on TLR9-induced IFNA1 release. Combination of pDCs with PBMCs restored the effect, even when they were separated by a permeable membrane. Suppression of TLR9-mediated IFNA1 secretion from PBMCs by adrenoceptor stimulation reduced the lytic activity of NK cells on K562 tumor cells. Conclusion We provide insights into the underlying mechanisms of the interrelation between immune responses and pharmacological agents widely used in clinical practice

  11. Acellular components of Chlamydia pneumoniae stimulate cytokine production in human blood mononuclear cells.

    PubMed

    Netea, M G; Selzman, C H; Kullberg, B J; Galama, J M; Weinberg, A; Stalenhoef, A F; Van der Meer, J W; Dinarello, C A

    2000-02-01

    Accumulating evidence suggest that infection with Chlamydia pneumoniae is associated with atherosclerosis, but the mechanisms involved remain unclear. Inflammation is important in the initial phase of atherogenesis, and cytokines are important in the initiation and progression of inflammation. The aim of this study was to assess the capacity of acellular components of C. pneumoniae to stimulate the production of pro-inflammatory cytokines and chemokines. Peripheral blood mononuclear cells were stimulated in vitro with sonicated C. pneumoniae. Significant amounts of TNF-alpha, IL-1, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) were produced. Inhibition of endotoxin using polymyxin B revealed that chlamydial endotoxin plays a minor role in the cytokine induction. Neutralization of TNF by TNF-binding protein and blockade of IL-1 receptors by IL-1 receptor antagonist revealed that TNF, IL-1 and IL-6 production was independent from each other, whereas IL-8 synthesis was strongly dependent on endogenous TNF and IL-1. In contrast, synthesis of MCP-1 and MIP-1alpha was dependent on endogenous TNF, but not IL-1. In conclusion, acellular components of C. pneumoniae are a potent stimulus for cytokine production, and this mechanism may have an important role in the inflammatory aspects of atherogenesis.

  12. Cardiotrophin-1 induces tumor necrosis factor alpha synthesis in human peripheral blood mononuclear cells.

    PubMed

    Fritzenwanger, Michael; Meusel, Katharina; Jung, Christian; Franz, Marcus; Wang, Zhenhua; Foerster, Martin; Figulla, Hans-R

    2009-01-01

    Chronic heart failure (CHF) is associated with elevated concentrations of tumor necrosis factor (TNF) alpha and cardiotrophin-1 (CT-1) and altered peripheral blood mononuclear cell (PBMC) function. Therefore, we tested whether CT-1 induces TNFalpha in PBMC of healthy volunteers. CT-1 induced in PBMC TNFalpha protein in the supernatant and TNFalpha mRNA in a concentration- and time-dependent manner determined by ELISA and real-time PCR, respectively. Maximal TNFalpha protein was achieved with 100 ng/mL CT-1 after 3-6 hours and maximal TNFalpha mRNA induction after 1 hour. ELISA data were confirmed using immunofluorescent flow cytometry. Inhibitor studies with actinomycin D and brefeldin A showed that both protein synthesis and intracellular transport are essential for CT-1 induced TNFalpha expression. CT-1 caused a dose dependent nuclear factor (NF) kappaB translocation. Parthenolide inhibited both NFkappaB translocation and TNFalpha protein expression indicating that NFkappaB seems to be necessary. We revealed a new mechanism for elevated serum TNFalpha concentrations and PBMC activation in CHF besides the hypothesis of PBMC activation by bacterial translocation from the gut.

  13. Upregulation of human immunodeficiency virus (HIV) replication by CD4 cross-linking in peripheral blood mononuclear cells of HIV-infected adults.

    PubMed Central

    Than, S; Oyaizu, N; Tetali, S; Romano, J; Kaplan, M; Pahwa, S

    1997-01-01

    This study was conducted with peripheral blood mononuclear cells from 67 human immunodeficiency virus (HIV)-infected adults. It supports the hypothesis that cross-linking of CD4 molecules by HIV gp120 can result in HIV upregulation and spread of infection. Underlying mechanisms include activation of latent infection by factors in addition to, or other than, tumor necrosis factor alpha. PMID:9223523

  14. Upregulation of human immunodeficiency virus (HIV) replication by CD4 cross-linking in peripheral blood mononuclear cells of HIV-infected adults.

    PubMed

    Than, S; Oyaizu, N; Tetali, S; Romano, J; Kaplan, M; Pahwa, S

    1997-08-01

    This study was conducted with peripheral blood mononuclear cells from 67 human immunodeficiency virus (HIV)-infected adults. It supports the hypothesis that cross-linking of CD4 molecules by HIV gp120 can result in HIV upregulation and spread of infection. Underlying mechanisms include activation of latent infection by factors in addition to, or other than, tumor necrosis factor alpha.

  15. Impact of Whole-Blood Processing Conditions on Plasma and Serum Concentrations of Cytokines.

    PubMed

    Lee, Jae-Eun; Kim, Jong-Wan; Han, Bok-Ghee; Shin, So-Youn

    2016-02-01

    Pre-analytical variations in plasma and serum samples can occur because of variability in whole-blood processing procedures. The aim of this study was to determine the impact of delayed separation of whole blood on the plasma and serum concentrations of cytokines. The concentrations of 16 cytokines were measured in plasma and serum samples when the centrifugation of whole blood at room temperature was delayed for 4, 6, 24, or 48 h, and the values were compared with those observed after separation within 2 h of whole-blood collection. Receiver operating characteristic (ROC) curve analysis was also performed for cytokines to determine whether cytokine levels in plasma and serum samples can be used to assess delayed separation of whole blood. Plasma concentrations of interleukin (IL)-1β, granulocyte-macrophage colony-stimulating factor (GM-CSF), and soluble CD40 ligand (sCD40L) and serum concentrations of IL-1β, IL-6, IL-8, macrophage inflammatory protein-1α (MIP-1α), and MIP-1β increased significantly (>2-fold) when separation was delayed at room temperature for 24 h. The concentrations of 6 of these cytokines (all except serum IL-1β and IL-6) demonstrated high diagnostic performance (area under the ROC curve >0.8) for delayed separation of whole blood. Furthermore, these cytokine concentrations typically exhibited high sensitivity and specificity at each optimal cutoff point. Conversely, IL-17A was stable in both plasma and serum samples, even when whole-blood centrifugation was delayed at room temperature for 48 h. This study shows that certain cytokines (IL-1β, GM-CSF, sCD40L, IL-8, MIP-1α, and MIP-1β) could be used for assessing the quality of plasma or serum samples.

  16. Investigation of stillbirths, perinatal mortality and weakness in beef calves with low-selenium whole blood concentrations.

    PubMed

    Davis, Anthony J; Myburgh, Jan G

    2016-07-15

    In this on-farm investigation, we report on stillbirths, weakness and perinatal mortality seen in calves on a commercial beef farm in the Roossenekal area, Mpumalanga province, South Africa. Post-mortem examination of these calves and histopathological examination of organ and tissue samples did not indicate an infectious aetiology. Affected calves had marginal to deficient whole blood selenium concentrations. Whole blood samples collected from adult cattle on this farm and five neighbouring farms were deficient in selenium. The potential contributions of other minerals to the symptoms seen are a subject of ongoing investigation, but selenium deficiency was marked in this herd and required urgent correction. Methods to correct the deficiency included the use of injectable products, and an oral selenium supplement chelated to methionine. Selenium availability to plants is primarily determined by the selenium content of the parent bedrock, the presence of other minerals and the pH of the soil. The apparent sudden onset of this problem implicates a soil factor as being responsible for reducing selenium's bioavailability in this area. Selenium deficiency can have a significant impact on human health. HIV and/or AIDS, various forms of cancer and several specific clinical syndromes are associated with selenium deficiency in humans, and the impact on human health in this area also requires further investigation.

  17. Effects of interleukin-10 on human peripheral blood mononuclear cell responses to Cryptococcus neoformans, Candida albicans, and lipopolysaccharide.

    PubMed Central

    Levitz, S M; Tabuni, A; Nong, S H; Golenbock, D T

    1996-01-01

    Deactivation of mononuclear phagocytes is critical to limit the inflammatory response but can be detrimental in the face of progressive infection. We compared the effects of the deactivating cytokine interleukin 10 (IL-10) on human peripheral blood mononuclear cell (PBMC) responses to lipopolysaccharide (LPS), Cryptococcus neoformans, and Candida albicans. IL-10 effected dose-dependent inhibition of tumor necrosis factor alpha (TNF-alpha) release in PBMC stimulated by LPS and C. neoformans, with significant inhibition seen with 0.1 U/ml and greater than 90% inhibition noted with 10 U/ml. In contrast, even at doses as high as 100 U/ml, IL-10 inhibited TNF-alpha release in response to C. albicans by only 50%. IL-10 profoundly inhibited release of IL-1beta from PBMC stimulated by all three stimuli. TNF-alpha mRNA and release was inhibited even if IL-10 was added up to 8 h after cryptococcal stimulation. In contrast, inhibition of IL-1 beta mRNA was of lesser magnitude and occurred only when IL-10 was added within 2 h of cryptococcal stimulation. IL-10 inhibited translocation of NF-kappaB in response to LPS but not the fungal stimuli. All three stimuli induced IL-10 production in PBMC, although over 10-fold less IL-10 was released in response to C. neoformans compared with LPS and C. albicans. Thus, while IL-10 has deactivating effects on PBMC responses to all three stimuli, disparate stimulus- and response-specific patterns of deactivation are seen. Inhibition by IL-10 of proinflammatory cytokine release appears to occur at the level of gene transcription for TNF-alpha and both transcriptionally and posttranscriptionally for IL-1beta. PMID:8641805

  18. A lab-in-a-briefcase for rapid prostate specific antigen (PSA) screening from whole blood.

    PubMed

    Barbosa, Ana I; Castanheira, Ana P; Edwards, Alexander D; Reis, Nuno M

    2014-08-21

    We present a new concept for rapid and fully portable prostate specific antigen (PSA) measurements, termed "lab-in-a-briefcase", which integrates an affordable microfluidic ELISA platform utilising a melt-extruded fluoropolymer microcapillary film (MCF) containing an array of 10 200 μm internal diameter capillaries, a disposable multi-syringe aspirator (MSA), a sample tray pre-loaded with all of the required immunoassay reagents, and a portable film scanner for colorimetric signal digital quantification. Each MSA can perform 10 replicate microfluidic immunoassays on 8 samples, allowing 80 measurements to be made in less than 15 minutes based on semi-automated operation, without the need of additional fluid handling equipment. The assay was optimised for the measurement of a clinically relevant range of PSA of 0.9 to 60.0 ng ml(-1) in 15 minutes with CVs on the order of 5% based on intra-assay variability when read using a consumer flatbed film scanner. The PSA assay performance in the MSA remained robust in undiluted or 1 : 2 diluted human serum or whole blood, and the matrix effect could simply be overcome by extending sample incubation times. The PSA "lab-in-a-briefcase" is particularly suited to a low-resource health setting, where diagnostic labs and automated immunoassay systems are not accessible, by allowing PSA measurement outside the laboratory using affordable equipment.

  19. Lab-on-CD microfluidic platform for rapid separation and mixing of plasma from whole blood.

    PubMed

    Kuo, Ju-Nan; Li, Bo-Shiun

    2014-08-01

    Traditional clinical methods for separating whole blood into blood cells and cell-free plasma are labor intensive and time consuming. Accordingly, the present study proposes a simple compact disk (CD) microfluidic platform for the rapid separation of plasma from whole human blood and the subsequent mixing of the plasma with a suitable reagent. The performance of three CD microfluidic platforms incorporating square-wave mixing channels with different widths is evaluated both numerically and experimentally. The results show that given an appropriate specification of the microchannel geometry and a CD rotation speed of 2000 rpm, a separation efficiency of 95 % can be achieved within 5 ~ 6 s given a diluted blood sample with a hematocrit concentration of 6 %. Moreover, a mixing efficiency of more than 96 % can be obtained within 5 s given a CD rotation speed of 2200 rpm. The practical feasibility of the proposed device is demonstrated by performing a prothrombin time (PT) test. It is shown that while the time required to perform the PT test using a conventional bench top system is around 15 min, the proposed CD microfluidic platform allows the test to be completed within 1 min.

  20. Optimization of direct whole blood PCR amplification with applications on a static thermostat chip.

    PubMed

    Qu, Bai-Yan; Wu, Zhi-Yong; Tian, Xiao-Xi; Chen, Kun; Fang, Fang

    2007-11-01

    In this paper, direct whole blood PCR amplifications on a static chip thermostat without sample purifications are demonstrated; in these amplifications, problems such as cross-interferences and contaminations could be avoided. The amplification conditions, such as the compositions of reagents and thermal programs, were investigated systematically by a GeneAmp PCR system with a native p53 gene segment (about 543 bp) of human genome and an exterior lambda DNA segment (about 500 bp) as targets. Direct amplifications of p53 and K-ras (about 157 bp) gene segments from 0.5 microL blood samples were successfully demonstrated by a static PCR chip with an indium tin oxide glass substrate. The chip thermostat has a typical size of 25 mm x 25 mm, and a polyethylene tube was used as the PCR vial on the glass surface of the chip. Fuzzy proportional integration-differentiation algorithms were adopted in temperature controls of the chip with an aid of a micro-Pt100 sensor. In the direct PCR with the thermostat chip, the whole process only involves automatic thermal programs. This work demonstrated that a chip PCR for field test without desktop facilities is possible either for a point of care test or for forensic analysis.

  1. On-chip Extraction of Intracellular Molecules in White Blood Cells from Whole Blood

    NASA Astrophysics Data System (ADS)

    Choi, Jongchan; Hyun, Ji-Chul; Yang, Sung

    2015-10-01

    The extraction of virological markers in white blood cells (WBCs) from whole blood—without reagents, electricity, or instruments—is the most important first step for diagnostic testing of infectious diseases in resource-limited settings. Here we develop an integrated microfluidic chip that continuously separates WBCs from whole blood and mechanically ruptures them to extract intracellular proteins and nucleic acids for diagnostic purposes. The integrated chip is assembled with a device that separates WBCs by using differences in blood cell size and a mechanical cell lysis chip with ultra-sharp nanoblade arrays. We demonstrate the performance of the integrated device by quantitatively analyzing the levels of extracted intracellular proteins and genomic DNAs. Our results show that compared with a conventional method, the device yields 120% higher level of total protein amount and similar levels of gDNA (90.3%). To demonstrate its clinical application to human immunodeficiency virus (HIV) diagnostics, the developed chip was used to process blood samples containing HIV-infected cells. Based on PCR results, we demonstrate that the chip can extract HIV proviral DNAs from infected cells with a population as low as 102/μl. These findings suggest that the developed device has potential application in point-of-care testing for infectious diseases in developing countries.

  2. Human plasma enhances the infectivity of primary human immunodeficiency virus type 1 isolates in peripheral blood mononuclear cells and monocyte-derived macrophages.

    PubMed Central

    Wu, S C; Spouge, J L; Conley, S R; Tsai, W P; Merges, M J; Nara, P L

    1995-01-01

    Physiological microenvironments such as blood, seminal plasma, mucosal secretions, or lymphatic fluids may influence the biology of the virus-host cell and immune interactions for human immunodeficiency virus type 1 (HIV-1). Relative to media, physiological levels of human plasma were found to enhance the infectivity of HIV-1 primary isolates in both phytohemagglutinin-stimulated peripheral blood mononuclear cells and monocyte-derived macrophages. Enhancement was observed only when plasma was present during the virus-cell incubation and resulted in a 3- to 30-fold increase in virus titers in all of the four primary isolates tested. Both infectivity and virion binding experiments demonstrated a slow, time-dependent process generally requiring between 1 and 10 h. Human plasma collected in anticoagulants CPDA-1 and heparin, but not EDTA, exhibited this effect at concentrations from 90 to 40%. Furthermore, heat-inactivated plasma resulted in a loss of enhancement in peripheral blood mononuclear cells but not in monocyte-derived macrophages. Physiological concentrations of human plasma appear to recruit additional infectivity, thus increasing the infectious potential of the virus inoculum. PMID:7666510

  3. Increased basal production of interleukin-10 by peripheral blood mononuclear cells in human alveolar echinococcosis.

    PubMed

    Godot, V; Harraga, S; Deschaseaux, M; Bresson-Hadni, S; Gottstein, B; Emilie, D; Vuitton, D A

    1997-12-01

    The secretion of IL-10 by peripheral blood mononuclear cells (PBMC) and the expression of IL-10 mRNA in fractionated CD4+ and CD8+ lymphocyte subsets and non-B-non-T cells, with and without stimulation by the mitogen phytohemagglutinin-C (PHA-C) and specific Echinococcus multilocularis (E. multilocularis) antigens, were assessed in 7 patients with alveolar echinococcosis (AE) and 6 healthy subjects. Results of studies on IL-10 were compared to those on IFN-gamma, IL-4 and IL-5 in the same patients and control subjects. IL-10 production was significantly higher in patient PBMC-culture supernatants than in the control group supernatants, both at the basal level and after mitogen or specific E. multilocularis antigen stimulation. Both CD4+ and CD8+ lymphocyte populations and non-B-non-T cells of AE patients and controls expressed IL-10 mRNA. Semi-quantification of IL-10 mRNA revealed a significantly higher transcript level in unstimulated-CD8+ T cells from AE patients in comparison with CD8+ T cells of healthy donors. PBMC from patients produced very low levels of IL-4 but the production of IFN-gamma was not significantly depressed compared to the controls. PBMC, isolated from 4 AE patients and 4 control subjects stimulated with specific E. multilocularis antigens, secreted IL-5; IL-5 mRNA was only detected in the CD4+ lymphocyte subset. The secretion of IL-5 and the expression of IL-5 mRNA in healthy subjects could be due to the presence of non-specific mitogenic parasitic factors. This non-specific mitogenic activity of the parasite, besides inducing a high secretion of IL-10 in patients with evolutive AE, may contribute to the lack of host control of parasite growth and to the persistence of granulomatous lesions, due to the inhibition of an efficient Th1 immune response.

  4. Vis-NIR spectroscopy with moving-window PLS method applied to rapid analysis of whole blood viscosity.

    PubMed

    Chen, Jiemei; Yin, Zhiwei; Tang, Yi; Pan, Tao

    2017-02-21

    A rapid analytical method of human whole blood viscosity with low, medium, and high shear rates [WBV(L), WBV(M), and WBV(H), respectively] was developed using visible and near-infrared (Vis-NIR) spectroscopy combined with a moving-window partial least squares (MW-PLS) method. Two groups of peripheral blood samples were collected for modeling and validation. Separate analytical models were established for male and female groups to avoid interference in different gender groups and improve the homogeneity and prediction accuracy. Modeling was performed for multiple divisions of calibration and prediction sets to avoid over-fitting and achieve parameter stability. The joint analysis models for three indicators were selected through comprehensive evaluation of MW-PLS. The selected joint analysis models were 812-1278 nm for males and 670-1146 nm for females. The root-mean-square errors (SEP) and the correlation coefficients of prediction (RP) for all validation samples were 0.54 mPa•s and 0.91 for WBV(L), 0.25 mPa•s and 0.92 for WBV(M), and 0.22 mPa•s and 0.90 for WBV(H). Results indicated high prediction accuracy, with prediction values similar to the clinically measured values. Overall, the findings confirmed the feasibility of whole blood viscosity quantification based on Vis-NIR spectroscopy with MW-PLS. The proposed rapid and simple technique is a promising tool for surveillance, control, and treatment of cardio-cerebrovascular diseases in large populations. Graphical Abstract The caption/legend of the online abstract figure: The selected wavebands and the prediction effects for the three indicators of whole blood viscosity.

  5. HSP27 modulates survival signaling in endosulfan-exposed human peripheral blood mononuclear cells treated with curcumin.

    PubMed

    Ahmed, T; Banerjee, B D

    2016-07-01

    Endosulfan, a well-known organochlorine pesticide, induces apoptosis and depletion of reduced glutathione (GSH) in human peripheral blood mononuclear cells (PBMC). Thus, for the amelioration of its effect, antioxidant and antiapoptotic potential of curcumin was evaluated. For ascertaining the attenuating effect of curcumin, various biochemical indices of cell damage such as cytotoxicity, oxidative stress, apoptosis (phosphatidylserine externalization, DNA fragmentation, and cytochrome c) in human PBMC was evaluated following endosulfan exposure (0-100 µM). To assess the role of HSP27 on endosulfan-induced apoptosis, the expression of HSP27 was examined. Curcumin (25 µM) increased cell viability significantly. As evident from the restoration of GSH, antiapoptotic potential was directly proportional to their antioxidant nature of curcumin. The present study indicates that the beneficial effect of curcumin on endosulfan-induced cytotoxicity is related to the induced synthesis of HSP27, emphasizing its antioxidant and therapeutic potential as well as underscoring the mechanism of pesticide-induced toxicity at cellular level. Taken together, these findings suggest that curcumin protects against endosulfan-induced immunotoxicity in human PBMC by attenuating apoptosis.

  6. Gene expression profiles in human peripheral blood mononuclear cells as biomarkers for nutritional in vitro and in vivo investigations

    PubMed Central

    Klenow, Stefanie; Borowicki, Anke; Gill, Chris I. R.; Pool-Zobel, Beatrice L.; Glei, Michael

    2010-01-01

    Identification of chemopreventive substances may be achieved by measuring biological endpoints in human cells in vitro. Since generally only tumour cells are available for such investigations, our aim was to test the applicability of peripheral blood mononuclear cells (PBMC) as an in vitro primary cell model since they mimic the human in vivo situation and are relatively easily available. Cell culture conditions were refined, and the basal variation of gene expression related to drug metabolism and stress response was determined. Results were compared with profiles of an established human colon cell line (HT29) as standard. For biomarker development of nutritional effects, PBMC and HT29 cells were treated with potentially chemopreventive substances (chrysin and butyrate), and gene expression was determined. Key results were that relevant stress response genes, such as glutathione S-transferase T2 (GSTT2) and GSTM2, were modulated by butyrate in PBMC as in HT29 cells, but the blood cells were less sensitive and responded with high individual differences. We conclude that these cells may serve as a surrogate tissue in dietary investigations and the identified differentially expressed genes have the potential to become marker genes for population studies on biological effects. PMID:21189867

  7. Kinetics of taurine depletion and repletion in plasma, serum, whole blood and skeletal muscle in cats.

    PubMed

    Pacioretty, L; Hickman, M A; Morris, J G; Rogers, Q R

    2001-12-01

    The relationship between taurine concentrations of plasma, whole blood, serum and skeletal muscle during taurine depletion and repletion was investigated in cats, to identify the most useful indicators of taurine status. Sixteen cats were fed a purified diet containing either 0 or 0.15 g/kg taurine for 5 months. Treatments were then reversed and the taurine concentration was measured during repletion and depletion phases. Plasma taurine exhibited the fastest rate (slow component) of depletion (t 1/2 = 4.8 wk), followed by serum (5.3 wk), whole blood (6.2 wk), and skeletal muscle (11.2 wk). Whole blood taurine was the first to replete at a rate of 0.74 wk to 1/2 maximal repletion, followed by serum (2.1 wk), skeletal muscle (3.5 wk), and plasma (3.5 wk). Whole blood more closely reflected skeletal muscle taurine concentrations than plasma during depletion, while plasma taurine concentrations appear to be the most valuable predictor of skeletal muscle taurine concentrations during repletion. This study suggests that the best clinical method to evaluate the taurine status of the cat is the determination and interpretation of both plasma and whole blood taurine concentrations.

  8. Evidence for direct transfer of tissue factor from monocytes to platelets in whole blood.

    PubMed

    Sovershaev, Mikhail A; Egorina, Elena M; Osterud, Bjarne; Hansen, John-Bjarne

    2012-06-01

    Varying specificity of anti-tissue factor (anti-TF) antibodies gives rise to erroneous conclusions on TF positivity of platelets. Although monocytes are a well established source of TF in whole blood, there is no consensus whether platelets express or acquire TF from external sources. To test whether platelets can acquire TF expressed in monocytes, we studied a transfer of TF-yellow fluorescent protein (TF-YFP) from monocytes nucleofected with TF-YFP to platelets in a whole blood model. Platelets isolated from whole blood were found positive for TF when immunostained with anti-TF antibody from one supplier, whereas no platelet TF antigen was found in whole blood immunostained with anti-TF antibody from another supplier. Both antibodies recognized TF in monocytes. Platelets isolated from whole blood reconstituted with monocytes expressing TF-YFP fusion protein were found positive for TF-YFP only after stimulation with lipopolysaccharide (LPS). Taken together, TF protein could be transferred from monocytes upon stimulation with LPS.

  9. An indirect spectrophotometric method for the determination of silicon in serum, whole blood and erythrocytes.

    PubMed

    Tamada, Tomoko

    2003-09-01

    An indirect method for the determination of silicon in blood samples has been developed. The proposed method overcame interference from a large amount of salts and phosphate in blood samples, and enabled us to determine the silicon contents in serum and whole blood by the same operation. After blood samples were digested by microwave heating, silicon, present as silicate in the sample solution, was reacted with molybdate to form a silicomolybdate complex. The complex was then separated from unreacted molybdate by a cation-exchange resin column. The molybdate liberated from the complex was spectrophotometrically determined in place of silicon. Since the method is not affected the composition of matrices between serum and whole blood, it could achieve good precision and accuracy, and could also estimate the silicon contents in erythrocytes from those in serum and whole blood. The sensitivity of the method was almost equal to that of the conventional silicomolybdenum blue method, and the calibration curve was linear up to 50 micromol l(-1) of silicon with a detection limit of 1.1 micromol l(-1) in whole blood. The mean concentrations of silicon in five healthy subjects were 11 micromol l(-1) for serum, 28 micromol l(-1) for whole blood and 50 micromol l(-1) for erythrocytes. Thus, the obtained distribution ratio between serum and erythrocytes was in the range of 0.15-0.39, and was found to be included in a narrow range.

  10. The changes in serum and whole blood rheological properties of rabbits during the progression of atherosclerosis.

    PubMed

    K Abdelhalim, Mohamed Anwar; Al-Ayed, Mohammed Suliman; Abdelmottaleb Moussa, Sherif A; Al-Mohy, Yanallah Hussain

    2016-05-01

    This study aimed to evaluate the role of zinc (Zn)-supplemented with high cholesterol diet (HCD) on the serum and whole blood rheological properties of rabbits fed a HCD. Twenty-four New Zealand white rabbits were divided into three groups. The HCD group was fed a diet with 1.0% cholesterol and 1.0% olive oil. The HCD + Zn group was fed a diet with 1.0% cholesterol, 1.0% olive oil, and Zn. Blood viscosity, shear stress, and torque (%) were measured at shear rates ranging from 225 to 1875 s-1 for serum and 75-900 s-1 for whole blood. Serum viscosity and shear stress in HCD rabbits were significantly higher at all shear rates compared to controls; while whole blood viscosity and shear stress in HCD rabbits were significantly lower at all shear rates compared to controls. Viscosity and shear stress in both serum and whole blood from rabbits in the HCD + Zn group returned to normal values at all shear rates. The Zn supplemented to HCD rabbits, delays the progression of atherosclerosis. Changes in blood serum viscosity could reflect changes in non-clotting proteins, glucose, nutrients and trace elements; while changes in whole blood viscosity could result from changes in hematocrit, hemoglobin, and erythrocyte count. One of the factors responsible for increasing the serum viscosity values of HCD rabbits might be attributed to increase in Fe and decrease in Zn levels in the blood serum.

  11. Persistent organochlorine pesticides in blood serum and whole blood

    SciTech Connect

    Waliszewski, S.M. ); Szymczynski, G.A. )

    1991-06-01

    Since organochlorine pesticides were introduced for plant protection and sanitation, they have been of great benefit in the control of pest populations and in combating the spread of infectious diseases. Unfortunately, they accumulate in the environment and this has resulted in a ban on their use. Nevertheless, they are still widely used in tropical countries as the insecticides of choice. An analytical procedure was elaborated to find out the extent of contamination of the human body by persistent residues of organochlorine pesticides and to determine the gradient between adipose tissue and biological fluids, which correlates with bioaccumulation and dissipation processes. The method has two important advantages: it is a simple, low-cost semi-micro, and it makes it possible to determine free and bound pesticides.

  12. Effect of human polymorphonuclear and mononuclear leukocytes on chromosomal and plasmid DNA of Escherichia coli. Role of acid DNase

    SciTech Connect

    Rozenberg-Arska, M.; van Strijp, J.A.; Hoekstra, W.P.; Verhoef, J.

    1984-05-01

    Phagocytosis and killing by polymorphonuclear and mononuclear leukocytes are important host resistance factors against invading microorganisms. Evidence showing that killing is rapidly followed by degradation of bacterial components is limited. Therefore, we studied the fate of Escherichia coli DNA following phagocytosis of E. coli by polymorphonuclear and mononuclear leukocytes. (/sup 3/H)Thymidine-labeled, unencapsulated E. coli PC2166 and E. coli 048K1 were incubated in serum, washed, and added to leukocytes. Uptake and killing of the bacteria and degradation of DNA were measured. Although phagocytosis and killing by mononuclear leukocytes was less efficient than that by polymorphonuclear leukocytes, only mononuclear leukocytes were able to degrade E. coli PC2166 DNA. Within 2 h, 60% of the radioactivity added to mononuclear leukocytes was released into the supernate, of which 40% was acid soluble. DNA of E. coli 048K1 was not degraded. To further analyze the capacity of mononuclear leukocytes to degrade E. coli DNA, chromosomal and plasmid DNA was isolated from ingested bacteria and subjected to agarose gel-electrophoresis. Only chromosomal DNA was degraded after phagocytosis. Plasmid DNA of E. coli carrying a gene coding for ampicillin resistance remained intact for a 2-h period after ingestion, and was still able to transform recipient E. coli cells after this period. Although we observed no DNA degradation during phagocytosis by polymorphonuclear leukocytes, lysates of both polymorphonuclear and mononuclear leukocytes contained acid-DNase activity with a pH optimum of 4.9. However, the DNase activity of mononuclear leukocytes was 20 times higher than that of polymorphonuclear leukocytes. No difference was observed between DNase activity from polymorphonuclear and mononuclear leukocytes from a chronic granulomatous disease patient with DNase activity from control polymorphonuclear and mononuclear leukocytes.

  13. Fluorescence detection of the anticancer drug topotecan and related camptothecins in plasma and whole blood by two-photon excitation

    NASA Astrophysics Data System (ADS)

    Burke, Thomas G.; Malak, Henryk M.; Gryczynski, Ignacy; Lakowicz, Joseph R.

    1997-05-01

    Recent FDA-approval of topotecan (9-dimethylaminomethyl-10- hydroxycamptothecin) and camptosar (CPT-11) along with the accelerated clinical development of related camptothecin drugs provides new hope for the successful treatment of human cancer, including neoplasms for which no effective treatments currently exist. Current clinical efforts worldwide are aimed at optimizing the therapeutic efficacies of the camptothecins, with the major focus on the determination of the most effective dosing schedules. To this end, technological advances which provide a direct and rapid means of measuring plasma drug levels (i.e. such that correlations between plasma drug levels and clinical responses can be sought) would be of great utility. Here we report on the direct fluorescence detection of topotecan and SN-38 in human plasma and topotecan in whole blood at micro molar levels using two-photon excitation at 730 or 820 nm. Topotecan was detected at concentrations as low as 0.05 and 1 (mu) M in plasma and whole blood, respectively. Since skin, blood and other tissues are translucent at long wavelengths, our results suggest the attractive possibility of homogeneous or noninvasive clinical sensing of camptothecins in situ using two-photon excitation.

  14. A hybrid poly(dimethylsiloxane) microsystem for on-chip whole blood filtration optimized for steroid screening.

    PubMed

    Thorslund, Sara; Klett, Oliver; Nikolajeff, Fredrik; Markides, Karin; Bergquist, Jonas

    2006-03-01

    Miniaturized biochemical devices in glass, silicon and polymer materials are starting to find their way from the academic laboratories to real-life applications. However, most attention has been given to miniaturize the downstream functions of various microfluidic systems, leaving the sample introduction and preparation steps to more conventional, bulkier solutions. For point-of-care diagnostics in particular, it becomes crucial to be able to handle complex human samples in a miniaturized format.In this work, we report on a microsystem for on-chip sample preparation that is able to remove blood cells from whole blood. The hybrid system consists of a commercially available membrane filter incorporated into a poly(dimethylsiloxane) (PDMS) casted device. Membrane materials were evaluated on the bases of low nonspecific adsorption of free and protein-bound testosterone as analyte substance. The hybrid system including a hydrophilic polypropylene filter successfully removed blood cells from diluted human whole blood. Surface oxidation was sufficient to make the plasma filtrate flow through the membrane filter and the channel system by capillary force alone and thus no external pumping source was needed.

  15. Microfluidic whole blood testing of platelet response to pharmacological agents.

    PubMed

    Li, Ruizhi; Grosser, Tilo; Diamond, Scott L

    2017-01-19

    Platelets present a number of intracellular and transmembrane targets subject to pharmacological modulation, either for cardiovascular disease reduction or as an unintended drug response. Microfluidic devices allow human blood to clot on a defined surface under controlled hemodynamic and pharmacological conditions. The potencies of a number of antiplatelet and anticancer drugs have been tested with respect to platelet deposition on collagen under flow. Inhibitors of cyclooxygenase-1 (COX-1) reduce platelet deposition, either when added ex vivo to blood or ingested orally by patients prior to testing. Some individuals display a functional "aspirin-insensitivity" in microfluidic assay. When certain nonsteroidal anti-inflammatory drugs (NSAIDs) are taken orally, they block COX-1 acetylation by aspirin with concomitant reduction of aspirin efficacy against platelets in microfluidic assay. Both P2Y1 and P2Y12 inhibitors reduce platelet deposition under flow, as do NO donors and iloprost that target the guanylate cyclase and the prostacyclin receptor, respectively. In a microfluidic assay of 37 kinase inhibitors, dasatinib had potent antiplatelet activity, while bosutinib was less potent. Dasatinib and bosutinib have known profiles against numerous kinases, revealing overlapping and nonoverlapping activities relevant to their unique actions against platelets. Also, dasatinib caused a marked and specific inhibition of GPVI signaling induced by convulxin, consistent with a dasatinib-associated bleeding risk. Microfluidic devices facilitate drug library screening, dose-response testing, and drug-drug interaction studies. Kinase inhibitors developed as anticancer agents may present antiplatelet activities that are detectable by microfluidic assay and potentially linked to bleeding risks.

  16. Ambient overnight hold of whole blood prior to the manufacture of blood components.

    PubMed

    Thomas, Stephen

    2010-12-01

    Blood services routinely separate whole blood into components that are then stored under different conditions. The storage conditions used for whole blood prior to separation must therefore be a compromise between the needs of the red cells (which benefit from refrigeration) and plasma and platelets (which are better preserved at ambient temperature). For many years, the approach has been to manufacture plasma and platelet components on the day of blood collection, and to refrigerate any unprocessed blood for manufacture into red cell components on the following day. However, this can make it challenging to maintain adequate stocks of all components. The European practice of 'ambient hold' of whole blood for up to 24 hours prior to processing allows greater flexibility in blood component manufacture, and the data reviewed suggest there is relatively little impact on the quality of red cell or plasma components, and an improvement in the quality of platelet components.

  17. [Whole-blood transfusion for hemorrhagic shock resuscitation: two cases in Djibouti].

    PubMed

    Cordier, P Y; Eve, O; Dehan, C; Topin, F; Menguy, P; Bertani, A; Massoure, P L; Kaiser, E

    2012-01-01

    Hemorrhagic shock requires early aggressive treatment, including transfusion of packed red blood cells and hemostatic resuscitation. In austere environments, when component therapy is not available, warm fresh whole-blood transfusion is a convenient treatment. It provides red blood cells, clotting factors, and functional platelets. Therefore it is commonly used in military practice to treat hemorrhagic shock in combat casualties. At Bouffard Hospital Center in Djibouti, the supply of packed red blood cells is limited, and apheresis platelets are unavailable. We used whole blood transfusion in two civilian patients with life-threatening non-traumatic hemorrhages. One had massive bleeding caused by disseminated intravascular coagulation due to septic shock; the second was a 39 year-old pregnant woman with uterine rupture. In both cases, whole blood transfusion (twelve and ten 500 mL bags respectively), combined with etiological treatment, enabled coagulopathy correction, hemorrhage control, and satisfactory recovery.

  18. Blood leukocyte and spleen lymphocyte immune response of spleen lymphocytes and whole blood leukocytes of hamsters

    SciTech Connect

    Peters, B.A.; Sothmann, M.; Wehrenberg, W.B. )

    1989-01-01

    This study was designed to evaluate the effects of chronic physical activity on the immune response of spleen lymphocytes and whole blood leukocytes of hamsters. Animals were kept sedentary or allowed to exercise spontaneously on running wheels for eight weeks. Physically active animals averaged 12 kilometers per day. The immune response of spleen lymphocytes whole blood leukocytes was evaluated by {sup 3}H-thymidine incorporation in response to Concanavalin A or lipopolysaccharide. There was no treatment effect between physically active and sedentary hamster in response of spleen lymphocytes. The immune response of whole blood leukocytes to these mitogens was significantly greater in physically active vs. sedentary hamsters. These results demonstrate that chronic physical activity has the capacity to modulate immunoresponses.

  19. Plastic containers and the whole-blood clotting test: glass remains the best option.

    PubMed

    Stone, Richard; Seymour, Jamie; Marshall, Oliver

    2006-12-01

    This is the first study to identify normal whole-blood clotting times in various plastic containers and to identify the effect of the addition of various concentrations of Pseudechis australis (Mulga snake) venom on the clotting time in glass and plastic. Polycarbonate was identified as a potential alternative to glass as a testing container owing to a whole-blood clotting time within acceptable limits for a bedside test (mean 29.5 min) and equivalent performance to glass in the presence of P. australis venom. Other plastic containers (such as polypropylene and polyethylene) were found to be unsuitable owing to very prolonged clotting times (>60 min) or impaired performance in the presence of venom. Overall, owing to the variation between the performance of different plastics and the difficulty in differentiating between them, plastic containers cannot be recommended as an alternative to glass when performing the whole-blood clotting test for envenomed patients.

  20. Human Peripheral Blood Mononuclear Cells Cultured in Normal and Hyperglycemic Media in Simulated Microgravity Using NASA Bioreactors

    NASA Technical Reports Server (NTRS)

    Lawless, DeSales

    2003-01-01

    We sought answers to several questions this summer at NASA Johnson Space Center. Initial studies involved the in vitro culture of human peripheral blood mononuclear in cells in different conditioned culture media. Several human cancer clones were similarly studied to determine responses to aberrant glycosylation by the argon laser. The cells were grown at unit gravity in flasks and in simulated microgravity using NASA bioreactors. The cells in each instance were analyzed by flow cytometry. Cell cycle analysis was acquired by staining nuclear DNA with propidium iodide. Responses to the laser stimulation was measured by observing autofluorescence emitted in the green and red spectra after stimulation. Extent of glycosylation correlated with the intensity of the laser stimulated auto-fluorescence. Our particular study was to detect and monitor aberrant glycosylation and its role in etiopathogenesis. Comparisons were made between cells known to be neoplastic and normal cell controls using the same Laser Induced Autofluorescence technique. Studies were begun after extensive literature searches on using the antigen presenting potential of dendritic cells to induce proliferation of antigen specific cytotoxic T-cells. The Sendai virus served as the antigen. Our goal is to generate sufficient numbers of such cells in the simulated microgravity environment for use in autologous transplants of virally infected individuals including those positive for hepatitis and HIV.

  1. The effects of calpain inhibition upon IL-2 and CD25 expression in human peripheral blood mononuclear cells.

    PubMed

    Schaecher, K E; Goust, J M; Banik, N L

    2001-10-01

    Calcium is an important contributor to T cell activation; it is also the major factor in the activation of the calcium-activated neutral proteinase, calpain. For this reason, we wanted to investigate if calpain has a role in T cell activation and what aspects of this activation calpain affects. As measured by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), calpain inhibition decreased interleukin-2 (IL-2) and CD25 mRNA expression in a dose-dependent manner, at early time points following the initial activation, and over extended periods of time in activated human peripheral blood mononuclear cells (PBMCs). Using an enzyme-linked immuno-sorbent assay (ELISA) specific for human IL-2, we found that calpain inhibition decreased IL-2 secretion in a dose-dependent manner, shortly after activation, and continuously over time. Inhibiting calpain caused a dose-dependent inhibition of CD25 cell surface expression and also inhibited expression shortly after activation and for at least 48 h. This study showed that calpain has an integral role in the synthesis of the two important T cell activation factors, IL-2 and CD25.

  2. Attachment, ingestion and intracellular killing of Helicobacter pylori by human peripheral blood mononuclear leukocytes and mouse peritoneal inflammatory macrophages.

    PubMed

    Chmiela, M; Paziak-Domanska, B; Wadström, T

    1995-02-01

    The different steps of phagocytosis, attachment, ingestion and intracellular killing of cells of Helicobacter pylori strain 17874 (expressing sialic acid-specific haemagglutinin) and cells of H. pylori strain 17875 (expressing non-sialic acid-specific haemagglutinin) have been studied. More cells of sialopositive H. pylori strain 17874 have been found attached to human peripheral blood mononuclear leukocytes (PBM) and mouse peritoneal inflammatory macrophages (PIM) than cells of sialonegative H. pylori strain 17875. Binding of cells of H. pylori strain 17874 has been significantly inhibited by treatment of phagocytes with neuraminidase. Inhibition of adhesion of these bacteria preincubated with foetuin to normal phagocytic cells has also been found. Well adhering cells of H. pylori strain 17874 were more resistant to killing mechanisms of human PBM and mouse PIM than cells of strain 17875. Good, probably sialic acid-specific haemagglutinin dependent, adhesion of H. pylori bacteria to phagocytes can be considered as an important virulence factor which facilitates the pathogen to avoid the defence mechanisms.

  3. Development of Multiscale Materials in Microfluidic Devices: Case Study for Viral Separation from Whole Blood

    NASA Astrophysics Data System (ADS)

    Surawathanawises, Krissada

    prior to anodization is evaluated to control the growth of AAO. Together with controlled anodizing voltages and electrolytic concentrations, AAO pore and cell sizes are shown to be tunable and controllable with narrow size distributions within submicron range. A high degree of order of AAO pore arrangement is also demonstrated. In addition, overall anodization becomes more time-efficient and stable at high anodizing voltages. Secondly, a three-dimensional (3D) assembly of microbeads is used as a template to fabricate a spherical pore network with small interconnected openings. After depositing and drying a suspension containing both micro- and nanobeads, the microbeads assemble into a 3D close-packed structure while the nanobeads fill the interstitial space. When the nanobeads are melted and microbeads are removed, a spherical pore matrix then form with small interconnected openings. Such the opening size is in submicron range can be adjusted depending on the size of microbead. The advantages of the two macroporous materials are not only controllable and tunable pore size, but also high surface-to-volume ratio due to the nanoscale features. With a ratio on the order of ~1 microm-1, the porous materials provide a significantly large binding surface. Computational and experimental results reveal that porous materials with a pore size matching the nanoparticle size are suitable for their capture. Separation of human immunodeficiency virus (HIV) is used as a model and capture yields of ~99 % and ~80 % are achieved in the nanopost structure and spherical pore network, respectively, after treated with a functional chemistry. Hence, the properties of these two macroporous materials are suitable as a size-exclusion and affinity separation for viral particles. To further explore multiscale separation, i.e. capturing viruses from whole blood, micropatterned arrays of macroporous materials have been designed. In this design, a microscale gap allows the passage of microparticles

  4. Enrichment of cancer cells from whole blood using a microfabricated porous filter.

    PubMed

    Kim, Eun Hye; Lee, Jong Kil; Kim, Byung Chul; Rhim, Sung Han; Kim, Jhin Wook; Kim, Kyung Hee; Jung, Sung Mok; Park, Pyeong Soo; Park, Hee Chul; Lee, Jason; Jeon, Byung Hee

    2013-09-01

    Enrichment of circulating tumor cells (CTCs) from whole blood is very challenging due to its rarity. We have developed a new CTC enrichment method using a microfabricated filter. The filter was designed to fractionate tumor cells by cell size and optimized to have high porosity and proper pore distribution. When cancer cells were spiked in whole blood, the average recovery rate was 82.0 to 86.7% and the limit of detection by filtration process was approximately 2 cancer cells in a testing volume of blood. The results indicate that the microfabricated filter-based enrichment would be useful to retrieve and analyze CTCs in practice.

  5. Development of a Whole Blood Staining Device for use During Space Shuttle Flights

    NASA Technical Reports Server (NTRS)

    Sams, Clarence F.; Crucian, Brian E.; Clift, Vaughan L.; Meinelt, Ellen M.

    1999-01-01

    Exposure to microgravity during space flight results in profound physiologic changes. Numerous studies have shown changes in circulating populations of peripheral blood immune cells immediately after space flight. It is currently unknown if these changes result from exposure to microgravity or are caused by the stress of reentry and readaptation to gravity. We have developed the whole blood staining device as a system for the staining of whole blood collected during space flight for subsequent flow cytometric analysis, This device contains all liquids to address safety issues concerned with space flight and also moves the cells through the staining, lyse/fixation and dilution steps.

  6. Enrichment of reticulocytes from whole blood using aqueous multiphase systems of polymers.

    PubMed

    Kumar, Ashok A; Lim, Caeul; Moreno, Yovany; Mace, Charles R; Syed, Abeer; Van Tyne, Daria; Wirth, Dyann F; Duraisingh, Manoj T; Whitesides, George M

    2015-01-01

    This paper demonstrates the enrichment of reticulocytes by centrifuging whole blood through aqueous multiphase systems (AMPSs)-immiscible phases of solutions of polymers that form step-gradients in density. The interfaces of an AMPS concentrate cells; this concentration facilitates the extraction of blood enriched for reticulocytes. AMPS enrich reticulocytes from blood from both healthy and hemochromatosis donors. Varying the osmolality and density of the phases of AMPS provides different levels of enrichment and yield of reticulocytes. A maximum enrichment of reticulocytemia of 64 ± 3% was obtained from donors with hemochromatosis. When used on peripheral blood from normal donors, AMPS can provide a higher yield of enriched reticulocytes and a higher proportion of reticulocytes expressing CD71 than differential centrifugation followed by centrifugation over Percoll. Blood enriched for reticulocytes by AMPS could be useful for research on malaria. Several species of malaria parasites show a preference to invade young erythrocytes and reticulocytes; this preference complicates in vitro cultivation of these species in human blood. Plasmodium knowlesi malaria parasites invade normal human blood enriched for reticulocytes by AMPSs at a rate 2.2 times greater (P < 0.01) than they invade unenriched blood. Parasite invasion in normal blood enriched by AMPS was 1.8 times greater (P < 0.05) than in blood enriched to a similar reticulocytemia by differential centrifugation followed by centrifugation over Percoll. The enrichment of reticulocytes that are invaded by malaria parasites demonstrates that AMPSs can provide a label-free method to enrich cells for biological research.

  7. Rapid determination of three anticoagulant rodenticides in whole blood by liquid chromatography coupled with electrospray ionization mass spectrometry.

    PubMed

    Jin, Mi-Cong; Chen, Xiao-Hong

    2006-01-01

    A rapid, sensitive and selective method for the simultaneous determination of bromadiolone, flocoumafen and brodifacoum in whole blood using warfarin as internal standard (IS) by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESI-MS) has been developed and validated. The target compounds were extracted from the whole blood with ethyl acetate and separated on an XDB C18 column (150 mm x 2.1 mm i.d. x 5 microm) by using a mobile phase consisting of 0.2% acetic acid/methanol (12/88, v/v) at a constant flow rate of 0.50 mL/min. The analytes were detected using negative ESI-MS in the selected ion monitoring (SIM) mode. The molecular ions [M-H]- of m/z 527, 541,523 and 307 were selected for the quantification for bromadiolone, flocoumafen, brodifacoum and the IS, respectively. The calibration curves were linear (r2 > 0.995) in the concentration range of 0.50-100.00 ng/mL. The method showed a satisfactory sensitivity (0.05-0.5 ng/mL using 200 microL blood), precision (RSD < 11.9%), accuracy (recovery: 82.0-96.1%) and selectivity. This method was successfully applied to the determination of the analytes for the diagnoses of poisoned human beings and animals.

  8. Red wine consumption and prevention of atherosclerosis: an in vitro model using human peripheral blood mononuclear cells.

    PubMed

    Magrone, T; Tafaro, A; Jirillo, F; Panaro, M A; Cuzzuol, P; Cuzzuol, A C; Pugliese, V; Amati, L; Jirillo, E; Covelli, V

    2007-01-01

    Evidence has been provided that red wine possesses antiatherogenic activities in virtue of its content in polyphenols (flavonoids and non-flavonoids substances). Here, some red wines (Negroamaro, Primitivo and Lambrusco) were tested for their ability to trigger nitric oxide (NO) production from human healthy peripheral blood mononuclear cells (PBMC). Negroamaro was the strongest inducer of NO from PBMC and deprivation of polyphenols did not influence its NO generation capacity. This fact supports the involvement of polyphenols in the NO production even in the absence of alcohol, which also per se does not exert any significant activity. These results are also corroborated by the evidence that PBMC inducible-nitric oxide synthase expression occurred by the effect of samples containing polyphenols but this expression was very weak when polyphenols were removed from the whole Negroamaro. In synthesis, flavonoids and resveratrol, major constituents of red wine, once absorbed at intestinal level, enter circulation and trigger monocytes for NO production. To the best of our knowledge, this is the first demonstration of a direct effect of red wine on monocytes for NO release to occur. On the other hand, also the macrophage contingent from gut-associated lymphoid tissue can contribute to NO generation, besides the aliquot produced by endothelial cells, as previously demonstrated by various authors. Taken together, these results support the concept that moderate intake of red wine can prevent atherosclerosis via production of NO, a potent vasodilator of terminal vessels.

  9. Antigenotoxic potential of aqueous extracts from the chanterelle mushroom, Cantharellus cibarius (higher Basidiomycetes), on human mononuclear cell cultures.

    PubMed

    Mendez-Espinoza, Claudia; Garcia-Nieto, Edelmira; Esquivel, Adriana Montoya; Gonzalez, Monica Montiel; Bautista, Efrain Velasco; Ezquerro, Carmen Calderon; Santacruz, Libertad Juarez

    2013-01-01

    Cantharellus cibarius is one of the most important wild, edible, and ectomycorrhizal mushrooms growing at La Malinche National Park, Tlaxcala, Mexico; therefore, the assessment of its biological properties is of great interest to know its potential as an alternative treatment to chemopreventive strategies when it is consumed as part of a diet. Comet assay was used to evaluate the antigenotoxic properties of several concentrations of aqueous extracts (0.0125, 0.025, 0.05, 0.1, and 0.2% w/v) prepared at room temperature (22 ± 2°C). As a test system we used human mononuclear cells exposed to methyl methanesulphonate (MMS) in vitro according to 3 different protocols: previous, simultaneous, and posterior. Previous (0.0125%) and simultaneous (0.1%) treatments resulted in the highest inhibitory efficiency. In the former, the cells assessed showed a tail length of 94.9 ± 64 µm; in the latter, the tails measured 106.2 ± 40 µm. Resulting percentages of reduction in damage were 236% and 196.1%, respectively. We did not obtain a dose-dependent response. The mean tail length for each protocol (previous, 133.1 ± 80 µm; simultaneous, 127.8 ± 57 µm; posterior, 146.3 ± 74 µm) was statistically significant with regard to the positive control (MMS).

  10. Accumulation of defective viral genomes in peripheral blood mononuclear cells of human immunodeficiency virus type 1-infected individuals.

    PubMed Central

    Sanchez, G; Xu, X; Chermann, J C; Hirsch, I

    1997-01-01

    Human immunodeficiency virus type 1 (HIV-1) genomes present in peripheral blood mononuclear cells (PBMCs) of infected persons or in lymphocytes infected in vitro were studied by long-distance PCR (LD-PCR) using primers localized in the HIV-1 long terminal repeats. The full-length 9-kb DNA was the only LD-PCR product obtained in peripheral and cord blood lymphocytes from seronegative donors infected in vitro. However, a high proportion (27% to 66%) of distinct populations of extensively deleted HIV-1 genomes of variable size was detected in PBMCs of 15 of 16 HIV-1-infected persons. Physical mapping of defective genomes showed that the frequency of deletions is proportional to their proximity to the central part of HIV-1 genome, which is consistent with a deletion mechanism involving a single polymerase jump during reverse transcription. Sequencing of deletion junctions revealed the presence of short direct repeats of three or four nucleotides. The number of defective HIV-1 genomes decreased after in vitro activation of PBMCs. Persistence of full-length and deleted genomes in in vitro activated PBMCs correlated with isolation of an infectious virus. Our results represent the first quantitative assessment of intragenomic rearrangements in HIV-1 genomes in PBMCs of infected persons and demonstrate that, in contrast to in vitro infection, defective genomes accumulate in PBMCs of infected persons. PMID:9032358

  11. Identification of Immunopotentiating Lactic Acid Bacteria that Induce Antibody Production by in vitro Stimulated Human Peripheral Blood Mononuclear Cells.

    PubMed

    Yamashita, Makiko; Hitaka, Akira; Fujino, Himiko; Matsumoto, Takashi; Hasegawa, Takanori; Morimatsu, Fumiki; Fujiki, Tsukasa; Katakura, Yoshinori

    2012-01-01

    L-leucyl-L-leucine methyl ester (LLME) is known to remove lysosome-rich cells from human peripheral blood mononuclear cells (PBMCs). To evaluate the immunopotentiating ability of lactic acid bacteria (LAB), we adopted the in vitro stimulation protocol of LLME-treated PBMCs as a model assay system and monitored the level of antibody produced by stimulated PBMCs. The results indicated that several LAB strains have immunopotentiating ability against PBMCs, as evidenced by the enhanced antibody production and increased number of antigen-specific B cells. Next, we identified T cells as the direct target cells of the immunopotentiating LAB strain L32, suggesting that L32 induced antibody production by PBMCs through T-cell activation. Finally, we tested the immunopotentiating ability of ligands for Toll-like receptor 2 (TLR2), which is known to mediate the LAB signal, and observed that both L32 and one of the TLR2 ligands, LTA-BS, induced antigen-specific antibody production by in vitro stimulated PBMC. This suggests that L32 and LTA-BS can be used as an adjuvant for stimulating immune reaction in PBMCs.

  12. In vitro effects of 'designer' amphetamines on human peripheral blood mononuclear leukocytes proliferation and on natural killer cell activity.

    PubMed

    Gagnon, L; Lacroix, F; Chan, J; Buttar, H S

    1992-12-01

    Human peripheral blood mononuclear leukocytes (PBML) proliferation was measured in the presence or absence of amphetamines. Proliferation in response to T-cell mitogen PHA was suppressed from 22 to 34% by d- and dl-amphetamine, respectively, contrarily to 1-form which did not affect proliferation of PHA-stimulated PBML. The 'designer' amphetamines appeared to be more potent inhibitors of PBML proliferation induced by both PHA and PWM stimulation than those of the racemic and isomeric forms of amphetamine. A wide variation was seen in the suppressive actions of the 'designer' amphetamines, and the mean percentages of suppression varied from 12 to 45% compared with the control values. 4-Propoxy-amphetamine (4-PA) was found to be the most active among the 'designer' drugs. In vitro effects of d-, 1- and dl-amphetamine were also studied on natural killer (NK) cell activity. A marked increase in the NK cell activity was observed only in the presence of very low concentrations (10(-12) to 10(-10) M) of dl-amphetamine, however, the activity of the NK cell remained within the control limits in the presence of d- or 1-forms. The findings suggest that the abuse of amphetamines, especially the 'designer' drugs, may adversely affect the activity of immunoregulatory cells and might lead to a compromised immune system in amphetamine abusers.

  13. Effect of BjcuL, a lectin isolated from Bothrops jararacussu, on human peripheral blood mononuclear cells.

    PubMed

    Pires, Weverson Luciano; de Castro, Onassis Boeri; Kayano, Anderson Makoto; da Silva Setúbal, Sulamita; Pontes, Adriana Silva; Nery, Neriane Monteiro; Paloschi, Mauro Valentino; Dos Santos Pereira, Soraya; Stábeli, Rodrigo Guerino; Fernandes, Carla Freire Celedônio; Soares, Andreimar Martins; Zuliani, Juliana Pavan

    2017-02-08

    BjcuL is a C-type lectin with specificity for the binding of β-d-galactose units isolated from Bothrops jararacussu venom. It triggers cellular infiltration in post capillary venules, increases edema and vascular permeability in murine models, contributes to in vitro neutrophil activation and modulates macrophage functional activation towards an M1 state. The purpose of this study was to investigate the effect of BjcuL on human peripheral blood mononuclear cells (PBMCs) activation with a focus on PBMCs proliferation and inflammatory mediators release. Results showed that BjcuL is not toxic to PBMCs, that BjcuL inhibits PBMCs proliferation and that it stimulates PBMCs to produce superoxide anion and hydrogen peroxide, primarily via lymphocyte stimulation, but does not stimulate the production of nitric oxide and PGE2. These results demonstrate that BjcuL has an immunomodulatory effect on PBMCs. Further studies are needed to confirm the immunomodulatory effect of BjcuL, to elucidate the molecular mechanisms of action responsible for its effects and to determine its potential application as an immunopharmacological and biotechnological tool.

  14. The Phenotypic Characterization of the Human Renal Mononuclear Phagocytes Reveal a Co-Ordinated Response to Injury

    PubMed Central

    Leone, Dario A.; Kozakowski, Nicolas; Kornauth, Christoph; Waidacher, Theresa; Neudert, Barbara; Loeffler, Agnes G.; Haitel, Andrea; Rees, Andrew J.; Kain, Renate

    2016-01-01

    Mammalian tissues contain networks of mononuclear phagocytes (MPh) that sense injury and orchestrate the response to it. In mice, this is affected by distinct populations of dendritic cells (DC), monocytes and macrophages and recent studies suggest the same is true for human skin and intestine but little is known about the kidney. Here we describe the analysis of MPh populations in five human kidneys and show they are highly heterogeneous and contain discrete populations of DC, monocytes and macrophages. These include: plasmacytoid DC (CD303+) and both types of conventional DC—cDC1 (CD141+ cells) and CD2 (CD1c+ cells); classical, non-classical and intermediate monocytes; and macrophages including a novel population of CD141+ macrophages clearly distinguishable from cDC1 cells. The relative size of the MPh populations differed between kidneys: the pDC population was bi-modally distributed being less than 2% of DC in two kidneys without severe injury and over 35% in the remaining three with low grade injury in the absence of morphological evidence of inflammation. There were profound differences in the other MPh populations in kidneys with high and low numbers of pDC. Thus, cDC1 cells were abundant (55 and 52.3%) when pDC were sparse and sparse (12.8–12.5%) when pDC were abundant, whereas the proportions of cDC2 cells and classical monocytes increased slightly in pDC high kidneys. We conclude that MPh are highly heterogeneous in human kidneys and that pDC infiltration indicative of low-grade injury does not occur in isolation but is part of a co-ordinated response affecting all renal DC, monocyte and macrophage populations. PMID:26999595

  15. CHARACTERIZATION OF CYTOKINE-INDUCED MYELOID-DERIVED SUPPRESSOR CELLS FROM NORMAL HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS

    PubMed Central

    Lechner, Melissa G.; Liebertz, Daniel J.; Epstein, Alan L.

    2010-01-01

    Introduction Tumor immune tolerance can derive from the recruitment of suppressor cell populations, including myeloid-derived suppressor cells (MDSC). In cancer patients, increased MDSC correlate with more aggressive disease and a poor prognosis. Experimental Design Expression of 15 immune factors (TGFβ, IL-1β, IL-4, IL-6, IL-10, GM-CSF, M-CSF, IDO, FLT3L, c-kit L, iNOS, ARG-1, TNFα, COX2, VEGF) by MDSC-inducing human solid tumor cell lines was evaluated by RT-PCR. Based upon these data, cytokine mixtures were then tested for their ability to generate suppressive CD33+ cells from healthy donor PBMC in vitro by measuring their ability to inhibit the proliferation of, and IFNγ production by, fresh autologous human T cells after CD3/CD28 stimulation. Induced MDSC were characterized with respect to their morphology, surface phenotype, and gene expression profile. Results MDSC-inducing cancer cell lines demonstrated multiple pathways for MDSC generation, including over-expression of IL-6, IL-1β, COX2, M-CSF, and IDO. CD33+ cells with potent suppressive capacity were best generated in vitro by GM-CSF and IL-6, and secondarily by GM-CSF + IL-1β, PGE2, TNFα, or VEGF. Characterization studies of cytokine-induced suppressive cells revealed CD33+CD11b+CD66b+HLA-DRlowIL-13Rα2int large mononuclear cells with abundant basophilic cytoplasm. Expression of iNOS, TGFβ, NOX2, VEGF, and/or ARG-1 was also up-regulated and transwell studies showed suppression of autologous T cells to be contact dependent. Conclusion Suppressive CD33+ cells generated from PBMC by GM-CSF and IL-6 were consistent with human MDSC. This study suggests that these cytokines are potential therapeutic targets for the inhibition of MDSC induction in cancer patients. PMID:20644162

  16. Effects of strenuous exercise on Th1/Th2 gene expression from human peripheral blood mononuclear cells of marathon participants.

    PubMed

    Xiang, Lianbin; Rehm, Kristina E; Marshall, Gailen D

    2014-08-01

    Physical stressors, such as strenuous exercise, can have numerous effects on the human body including the immune system. The aim of this study was to evaluate the gene expression profile of Th1/Th2 cytokines and related transcription factor genes in order to investigate possible immune imbalances before and after a marathon. Blood samples were collected from 16 normal volunteers 24-48 h before and one week after completing a marathon race. Gene expression of Th1 and Th2 related cytokines from human peripheral blood mononuclear cells (PBMC) was analyzed using Human Th1-Th2-Th3 RT(2) Profiler PCR Array and qRT-PCR that measured the transcript levels of 84 genes related to T cell activation. We found that PBMC express a characteristic Th2-like gene profile one week post-marathon compared to pre-marathon. The majority of genes up-regulated one week post-marathon such as IL-4, GATA3, and CCR4 were Th2 associated. For Th1-related genes, CXCR3 and IRF1 were up-regulated one week post-marathon. There was a trend of down-regulation of two Th1 related genes, T-bet and STAT1. Th3-related gene expression patterns did not change in the study. The ratios of both IFN-γ/IL-4 and T-bet/GATA3 gene expressions were significantly lower one week after marathon. These findings suggest that a Th1/Th2 immune imbalance persisted at least 1 week after completion of a marathon which offers a mechanistic rationale for the increased risk of upper respiratory tract infections often reported after strenuous exercise.

  17. Kit for the selective labeling of red blood cells in whole blood with .sup.9 TC

    DOEpatents

    Srivastava, Suresh C.; Babich, John W.; Straub, Rita; Richards, Powell

    1992-01-01

    Disclosed herein are a method and kit for the preparation of .sup.99m Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available therein for reduction of technetium.

  18. New method for the selective labeling of erythrocytes in whole blood with Tc-99m

    DOEpatents

    Srivastava, S.C.; Babich, J.W.; Straub, R.; Richards, P.

    1984-01-27

    Method and kit are described for the preparation of /sup 99m/Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available therein for the reduction of technetium.

  19. The effect of storage on whole blood chemiluminescence measurement of equine neutrophils.

    PubMed

    Krumrych, Wiesław; Skórzewski, Radosław; Malinowski, Edward

    2013-01-01

    The aim of this study was to determine the effect of duration and temperature of sample storage on whole blood chemiluminescence measurement results. Venous blood from 18 clinically healthy Polish half-bred horses aged 4 to 11 years were used in the study. Luminol dependent chemiluminescence (CL) was used to measure neutrophil oxygen metabolism in whole blood. Blood samples were examined for spontaneous CL and stimulated by a surface receptor stimulus as well as extra-receptor stimulus. The assay was performed in two parallel experimental sets with samples stored at 4 and 22 °C, respectively. Whole blood CL was estimated at 2, 6, 24, 48, 72, 96 and 120 h after collection. The study demonstrated that temperature and duration of sample storage are factors that determine the quality of CL measurements of whole blood in horses. The study concluded that samples should be stored at 4 °C and the assay should be performed as early as possible. It was also shown that the viability period of horse blood for CL assays is relatively long. Material stored at room temperature for 24 h and even up to 48 h at 4 °C did not show any significant decrease in spontaneous or stimulated chemiluminescence.

  20. Impact of bone lead and bone resorption on plasma and whole blood lead levels during pregnancy.

    PubMed

    Téllez-Rojo, Martha María; Hernández-Avila, Mauricio; Lamadrid-Figueroa, Héctor; Smith, Donald; Hernández-Cadena, Leticia; Mercado, Adriana; Aro, Antonio; Schwartz, Joel; Hu, Howard

    2004-10-01

    The authors tested the hypotheses that maternal bone lead burden is associated with increasing maternal whole blood and plasma lead levels over the course of pregnancy and that this association is modified by rates of maternal bone resorption. A total of 193 Mexican women were evaluated (1997-1999) in the first, second, and third trimesters of pregnancy. Whole blood lead and plasma lead levels were measured in each trimester. Urine was analyzed for cross-linked N-telopeptides (NTx) of type I collagen, a biomarker of bone resorption. Patella and tibia lead levels were measured at 4 weeks postpartum. The relation between whole blood, plasma, and bone lead and NTx was assessed using mixed models. Plasma lead concentrations followed a U-shape, while NTx levels increased significantly during pregnancy. In a multivariate model, the authors observed a significant and positive interaction between NTx and bone lead when plasma lead was used as the outcome variable. Dietary calcium intake was inversely associated with plasma lead. Results for whole blood lead were similar but less pronounced. These results confirm previous evidence that bone resorption increases during pregnancy, with a consequential significant release of lead from bone, constituting an endogenous source of prenatal exposure. They also provide a rationale for testing strategies (e.g., nutritional supplementation with calcium) aimed at decreasing prenatal lead exposure.

  1. Prolonged Detection of Zika Virus in Vaginal Secretions and Whole Blood

    PubMed Central

    Gorchakov, Rodion; Carlson, Anna R.; Berry, Rebecca; Lai, Lilin; Natrajan, Muktha; Garcia, Melissa N.; Correa, Armando; Patel, Shital M.; Aagaard, Kjersti; Mulligan, Mark J.

    2017-01-01

    Infection with Zika virus is an emerging public health crisis. We observed prolonged detection of virus RNA in vaginal mucosal swab specimens and whole blood for a US traveler with acute Zika virus infection who had visited Honduras. These findings advance understanding of Zika virus infection and provide data for additional testing strategies. PMID:27748649

  2. Neutrophil function is preserved in a pooled granulocyte component prepared from whole blood donations.

    PubMed

    Bashir, Saber; Stanworth, Simon; Massey, Edwin; Goddard, Fred; Cardigan, Rebecca

    2008-03-01

    Whole blood-derived granulocytes (buffy coats) are issued as an alternative to apheresis donations, but are heavily contaminated with red cells and platelets and there is minimal in vitro data describing their functionality. We developed a purer pooled granulocyte component (PGC) from whole blood donations by pooling 10 ABO-matched buffy coats with 400 ml of platelet additive solution (SSP+) and re-centrifuging. The PGC was irradiated (25-50 Gy) and neutrophil viability, chemotaxis, phagocytosis and respiratory burst activity were determined by flow cytometry. Results from 13 PGC at 16-20 h following donation were compared with those obtained from 20 standard individual buffy coats and with fresh whole blood. The PGC contained similar numbers of neutrophils (approximately 0.9 x 10(10)) with a reduced volume and haemoglobin content when compared with 10 individual buffy coats. Neutrophils in the PGC maintained >90% viability, oxidative burst and phagocytic activity and their ability to migrate towards a chemoattractant 16-20 h following donation, which is similar to results obtained with either fresh whole blood or standard buffy coats. Therefore, neutrophil function in the PGC was preserved 16-20 h following donation, but this product had significantly lower red cell contamination compared with 10 buffy coats, which are currently transfused.

  3. 9 CFR 147.3 - The stained-antigen, rapid, whole-blood test. 3

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...-blood test. 3 147.3 Section 147.3 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... Blood Testing Procedures § 147.3 The stained-antigen, rapid, whole-blood test. 3 3 The procedure... Secretary of Agriculture. (b) A loop for measuring the correct quantity of blood can usually be...

  4. Brief Report: Whole Blood Serotonin Levels and Gastrointestinal Symptoms in Autism Spectrum Disorder

    ERIC Educational Resources Information Center

    Marler, Sarah; Ferguson, Bradley J.; Lee, Evon Batey; Peters, Brittany; Williams, Kent C.; McDonnell, Erin; Macklin, Eric A.; Levitt, Pat; Gillespie, Catherine Hagan; Anderson, George M.; Margolis, Kara Gross; Beversdorf, David Q.; Veenstra-VanderWeele, Jeremy

    2016-01-01

    Elevated whole blood serotonin levels are observed in more than 25% of children with autism spectrum disorder (ASD). Co-occurring gastrointestinal (GI) symptoms are also common in ASD but have not previously been examined in relationship with hyperserotonemia, despite the synthesis of serotonin in the gut. In 82 children and adolescents with ASD,…

  5. THz spectroscopy of whole blood, plasma and cells in mice of SHR line with various pathology

    NASA Astrophysics Data System (ADS)

    Panchenko, A.; Tyndyk, M.; Smolyanskaya, O.; Sulatskiy, M.; Kravtsenyuk, O.; Balbekin, N.; Khodzitsky, M.

    2016-08-01

    This paper is devoted to studying of optical properties of whole blood and blood plasma in SHR mice grafted Ehrlich's carcinoma and mice with chronic inflammation at the terahertz frequency range. Additionally physiological saline solution suspension of ascites Ehrlich's carcinoma cells was explored.

  6. Kit for the selective labeling of red blood cells in whole blood with [sup 99]Tc

    DOEpatents

    Srivastava, S.C.; Babich, J.W.; Straub, R.; Richards, P.

    1992-05-26

    Disclosed herein are a method and kit for the preparation of [sup 99m]Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available therein for reduction of technetium. No Drawings

  7. Isolation of Salmonella typhi from Standard Whole Blood Culture versus Blood-Clot Cultures

    DTIC Science & Technology

    1988-12-01

    The use of 10% oxgall and bile broth medium, both supplemented with freshly prepared 100 u/ml streptokinase, for isolating Salmonella typhi by clot...significantly better rate of isolation than the clot culture methods. Keywords: Cultures biology; Clot cultures; Salmonella typhi ; Isolation of S. typhi; Whole blood culture; Blood-clot culture; Reprints.

  8. Results of an international round robin for serum and whole-blood folate.

    PubMed

    Gunter, E W; Bowman, B A; Caudill, S P; Twite, D B; Adams, M J; Sampson, E J

    1996-10-01

    Because of the increasing significance of folate nutriture to public health, a "round robin" interlaboratory comparison study was conducted to assess differences among methods. Twenty research laboratories participated in a 3-day analysis of six serum and six whole-blood pools. Overall means, SDs, and CVs derived from these results were compared within and across method types. Results reported for serum and whole-blood folate demonstrated overall CVs of 27.6% and 35.7%, respectively, across pools and two- to ninefold differences in concentrations between methods, with the greatest variation occurring at critical low folate concentrations. Although results for serum pools were less variable than those for whole-blood pools, substantial intermethod variation still occurred. The overall results underscore the urgent need for developing and validating reference methods for serum and whole-blood folate and for properly characterized reference materials. For evaluating study or clinical data, method-specific reference ranges (established with clinical confirmation of values for truly folate-deficient individuals) must be used.

  9. Assay for Listeria monocytogenes cells in whole blood using isotachophoresis and recombinase polymerase amplification.

    PubMed

    Eid, Charbel; Santiago, Juan G

    2016-12-19

    We present a new approach which enables lysis, extraction, and detection of inactivated Listeria monocytogenes cells from blood using isotachophoresis (ITP) and recombinase polymerase amplification (RPA). We use an ITP-compatible alkaline and proteinase K approach for rapid and effective lysis. We then perform ITP purification to separate bacterial DNA from whole blood contaminants using a microfluidic device that processes 25 μL sample volume. Lysis, mixing, dispensing, and on-chip ITP purification are completed in a total of less than 50 min. We transfer extracted DNA directly into RPA master mix for isothermal incubation and detection, an additional 25 min. We first validate our assay in the detection of purified genomic DNA spiked into whole blood, and demonstrate a limit of detection of 16.7 fg μL(-1) genomic DNA, the equivalent of 5 × 10(3) cells per mL. We then show detection of chemically-inactivated L. monocytogenes cells spiked into whole blood, and demonstrate a limit of detection of 2 × 10(4) cells per mL. Lastly, we show preliminary experimental data demonstrating the feasibility of the integration of ITP purification with RPA detection on a microfluidic chip. Our results suggest that ITP purification is compatible with RPA detection, and has potential to extend the applicability of RPA to whole blood.

  10. A new on-chip whole blood/plasma separator driven by asymmetric capillary forces.

    PubMed

    Lee, Kang Kug; Ahn, Chong H

    2013-08-21

    A new on-chip whole blood/plasma separator driven by asymmetric capillary forces, which are produced through a microchannel with sprayed nanobead multilayers, has been designed, fabricated and fully characterized. The silica nanobead multilayers revealing as superhydrophilic surfaces have been fabricated using a spray layer-by-layer (LbL) nano-assembly method. This new on-chip blood plasma separator has been targeted for a sample-to-answer (S-to-A) microfluidic lab-on-a-chip (LOC) toward point-of-care clinical testing (POCT). Effective plasma separation from undiluted whole blood was achieved through the microchannel which was composed of asymmetric superhydrophilic surfaces with a 10 mm hydrophobic patch. Blood cells were continuously accumulated over the hydrophobic patch while the blood plasma was able to flow over the patch. Therefore, the blood plasma was successfully separated from the whole blood throughout the accumulated blood cells which worked as a so-called 'self-built-in blood cell microfilter'. The separated plasma was approximately 102 nL from a single drop of 3 μL whole blood within 10 min, which is very suitable for single-use disposable POCT devices.

  11. Determination of whole blood and plasma viscosity by means of flow curve analysis.

    PubMed

    Ruef, Peter; Gehm, Jutta; Gehm, Lothar; Felbinger, Claudia; Pöschl, Johannes; Kuss, Navina

    2014-01-01

    The LS300 viscometer permits automated measurements of viscosity at several shear rates of non-Newtonian fluids. We determined whole blood and plasma viscosity, aggregation, red blood cell deformability, and hematocrit of 66 healthy adults. The effects of the anticoagulants EDTA, heparin and citrate, and of centrifugation on blood viscosity (n=12) and red blood cell geometry (n=5) were investigated. With regard to the whole blood viscosity of adults, the best agreement was obtained by Casson's calculation compared to the methods of Ostwald, Bingham and Newton. The approximated flow curve of plasma showed only marginal differences between the method of Newton and Ostwald, whereas the latter gave the best quality of approximation. Centrifugation and the anticoagulants had a significant impact on whole blood viscosity and yield shear stress, whereas erythrocyte geometry remained unaffected. By linear regression of hematocrit with viscosity and yield shear stress, its impact on blood viscosity could be calculated in a hematocrit range of 0.32-0.50. Determination of whole blood viscosity should be performed in a standardized manner at several shear rates and without centrifugation of the blood samples.

  12. Influence of 2(3-methyl-cinnamyl-hydrazono)-propionate on glucose and palmitate oxidation in human mononuclear leukocytes. Hydrazonopropionic acids, a new class of hypoglycaemic substances, VII.

    PubMed

    Haeckel, R; Fink, P C; Oellerich, M

    1987-09-01

    2-(3-Methyl-cinnamyl-hydrozono)-propionate stimulated glucose oxidation in human mononuclear leukocytes and the stimulation was similar to that by concanavalin A. Both substances must affect glucose metabolism at two sites, the first site being before the pyruvate dehydrogenase step because of the increase of lactate plus pyruvate concentration. The second site is related to pyruvate oxidation. The hydrazone inhibited the conversion of plamitate to CO2. This effect could have caused an activation of the pyruvate dehydrogenase complex, resulting from a decrease acetyl-CoA/CoA ratio. Concanavalin A did not influence fatty acid oxidation. Both substances did not affect the CO2 formation from acetate. Mononuclear leukocytes appear to be a suitable model for the investigation of the influence of hypoglycaemic substances on glucose and fatty acid metabolism in living human cells.

  13. Whole Blood Activation Results in Altered T Cell and Monocyte Cytokine Production Profiles by Flow Cytometry

    NASA Technical Reports Server (NTRS)

    Crucian, Brian E.; Sams, Clarence F.

    2001-01-01

    An excellent monitor of the immune balance of peripheral circulating cells is to determine their cytokine production patterns in response to stimuli. Using flow cytometry, a positive identification of cytokine producing cells in a mixed culture may be achieved. Recently, the ability to assess cytokine production following a whole-blood activation culture has been described. In this study, whole blood activation was compared to traditional PBMC activation and the individual cytokine secretion patterns for both T cells, T cell subsets and monocytes was determined by flow cytometry. RESULTS: For T cell cytokine assessment (IFNg/IL-10 and IL-21/L-4) following PMA +ionomycin activation: (1) a Significantly greater percentages of T cells producing IFNgamma and IL-2 were observed following whole-blood culture and (2) altered T cell cytokine production kinetics were observed by varying whole blood culture times. Four-color analysiS was used to allow assessment of cytokine production by specific T cell subsets. It was found that IFNgamma production was significantly elevated in the CD3+/CD8+ T cell population as compared to the CD3+/CD8- population following five hours of whole blood activation. Conversely, IL-2 and IL-10 production were Significantly elevated in the CD3+/CD8- T cell population as compared to the CD3+/CD8+ population. Monocyte cytokine production was assessed in both culture systems following LPS activation for 24 hours. A three-color flow cytometric was used to assess two cytokines (IL-1a/IL-12 and TNFa/IL-10) in conjunction with CD14. Nearly all monocytes were stimulated to produce IL-1a, IL-12 and TNFa. equally well in both culture systems, however monocyte production of IL-10 was significantly elevated in whole blood culture as compared to PBMC culture. IL-12 producing monocytes appeared to be a distinct subpopulation of the IL-1a producing set, whereas IL-10 and TNFa producing monocytes were largely mutually exclusive. IL-10 and TNFa producing

  14. Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro

    DTIC Science & Technology

    2013-03-11

    variability. Male volunteers, who had been screened to be HIV and Hepatitis B negative ranged from 19-61 years of age. Human monocytes and lymphocytes...generation anthrax vaccines [62]. Addressing this concern , our study identified a host of potential targets associated with biofunctions, such as... b . ABSTRACT c. THIS PAGE 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON 19b. TELEPHONE NUMBER (Include area

  15. Associations Between Whole Blood and Dietary Omega-3 Polyunsaturated Fatty Acid Levels in Collegiate Athletes.

    PubMed

    Wilson, Patrick B; Madrigal, Leilani A

    2016-12-01

    Omega-3 polyunsaturated fatty acids (PUFAs) have important physiological functions and may offer select benefits for athletic performance and recovery. The purpose of this investigation was to assess dietary and whole blood omega-3 PUFAs among collegiate athletes. In addition, a brief questionnaire was evaluated as a valid tool for quantifying omega-3 PUFA intake. Fifty-eight athletes (9 males, 49 females) completed a 21-item questionnaire developed to assess omega-3 PUFA intake and provided dried whole blood samples to quantify α-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and the HS-Omega-3 Index. Geometric means (95% confidence intervals) for the HS-Omega-3 Index were 4.79% (4.37-5.25%) and 4.75% (4.50-5.01%) for males and females, respectively. Median dietary intakes of ALA, EPA, and DHA were all below 100 mg. Among females, several dietary omega-3 PUFA variables were positively associated with whole blood EPA, with total EPA (rho = 0.67, p < .001) and total DHA (rho = 0.69, p < .001) intakes showing the strongest correlations. Whole blood DHA among females showed positive associations with dietary intakes, with total EPA (rho = 0.62, p < .001) and total DHA (rho = 0.64, p < .001) intakes demonstrating the strongest correlations. The HS-Omega-3 Index in females was positively correlated with all dietary variables except ALA. Among males, the only significant correlation was between food and whole blood EPA (rho = 0.83, p < .01). Collegiate athletes had relatively low intakes of omega-3 PUFAs. A 21-item questionnaire may be useful for screening female athletes for poor omega-3 PUFA status.

  16. Whole blood NAD and NADP concentrations are not depressed in subjects with clinical pellagra.

    PubMed

    Creeke, Paul I; Dibari, Filippo; Cheung, Edith; van den Briel, Tina; Kyroussis, Eustace; Seal, Andrew J

    2007-09-01

    Population surveys for niacin deficiency are normally based on clinical signs or on biochemical measurements of urinary niacin metabolites. Status may also be determined by measurement of whole blood NAD and NADP concentrations. To compare these methods, whole blood samples and spot urine samples were collected from healthy subjects (n = 2) consuming a western diet, from patients (n = 34) diagnosed with pellagra and attending a pellagra clinic in Kuito (central Angola, where niacin deficiency is endemic), and from female community control subjects (n = 107) who had no clinical signs of pellagra. Whole blood NAD and NADP concentrations were measured by microtiter plate-based enzymatic assays and the niacin urinary metabolites 1-methyl-2-pyridone-5-carboxamide (2-PYR) and 1-methylnicotinamide (1-MN) by HPLC. In healthy volunteers, inter- and intra-day variations for NAD and NADP concentrations were much lower than for the urinary metabolites, suggesting a more stable measure of status. However, whole blood concentrations of NAD and NADP or the NAD:NADP ratio were not significantly depressed in clinical pellagra. In contrast, the concentrations of 2-PYR and 1-MN, expressed relative to either creatinine or osmolality, were lower in pellagra patients and markedly higher following treatment. The use of the combined cut-offs (2-PYR <3.0 micromol/mmol creatinine and 1-MN <1.3 micromol/mmol creatinine) gave a sensitivity of 91% and specificity of 72%. In conclusion, whole blood NAD and NADP concentrations gave an erroneously low estimate of niacin deficiency. In contrast, spot urine sample 2-PYR and 1-MN concentrations, relative to creatinine, were a sensitive and specific measure of deficiency.

  17. Automated processing of whole blood units: operational value and in vitro quality of final blood components

    PubMed Central

    Jurado, Marisa; Algora, Manuel; Garcia-Sanchez, Félix; Vico, Santiago; Rodriguez, Eva; Perez, Sonia; Barbolla, Luz

    2012-01-01

    Background The Community Transfusion Centre in Madrid currently processes whole blood using a conventional procedure (Compomat, Fresenius) followed by automated processing of buffy coats with the OrbiSac system (CaridianBCT). The Atreus 3C system (CaridianBCT) automates the production of red blood cells, plasma and an interim platelet unit from a whole blood unit. Interim platelet unit are pooled to produce a transfusable platelet unit. In this study the Atreus 3C system was evaluated and compared to the routine method with regards to product quality and operational value. Materials and methods Over a 5-week period 810 whole blood units were processed using the Atreus 3C system. The attributes of the automated process were compared to those of the routine method by assessing productivity, space, equipment and staffing requirements. The data obtained were evaluated in order to estimate the impact of implementing the Atreus 3C system in the routine setting of the blood centre. Yield and in vitro quality of the final blood components processed with the two systems were evaluated and compared. Results The Atreus 3C system enabled higher throughput while requiring less space and employee time by decreasing the amount of equipment and processing time per unit of whole blood processed. Whole blood units processed on the Atreus 3C system gave a higher platelet yield, a similar amount of red blood cells and a smaller volume of plasma. Discussion These results support the conclusion that the Atreus 3C system produces blood components meeting quality requirements while providing a high operational efficiency. Implementation of the Atreus 3C system could result in a large organisational improvement. PMID:22044958

  18. 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

    2017-02-10

    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).

  19. Effective use of frozen donor peripheral blood mononuclear cells for human immunodeficiency virus type 1 isolation from vertically infected pediatric patients.

    PubMed Central

    Paul, M O; Tetali, S; Pahwa, S

    1994-01-01

    In this study, we examined variables related to human immunodeficiency virus (HIV) isolation utilizing samples from 51 HIV-infected (153 plasma and 122 peripheral blood mononuclear cell [PBMC] samples) and 57 uninfected (182 plasma and 163 PBMC samples) infants. Our chief observation was that cryopreservation of donor PBMCs does not significantly alter their sensitivity or specificity for isolation of HIV from patient PBMCs or plasma. PMID:8051275

  20. Effective use of frozen donor peripheral blood mononuclear cells for human immunodeficiency virus type 1 isolation from vertically infected pediatric patients.

    PubMed

    Paul, M O; Tetali, S; Pahwa, S

    1994-05-01

    In this study, we examined variables related to human immunodeficiency virus (HIV) isolation utilizing samples from 51 HIV-infected (153 plasma and 122 peripheral blood mononuclear cell [PBMC] samples) and 57 uninfected (182 plasma and 163 PBMC samples) infants. Our chief observation was that cryopreservation of donor PBMCs does not significantly alter their sensitivity or specificity for isolation of HIV from patient PBMCs or plasma.

  1. Long-term three-dimensional perfusion culture of human adult bone marrow mononuclear cells in bioreactors.

    PubMed

    Schmelzer, Eva; Finoli, Anthony; Nettleship, Ian; Gerlach, Jörg C

    2015-04-01

    The construction and long-term maintenance of three-dimensional in vitro bone marrow models is of great interest but still quite challenging. Here we describe the use of a multi-compartment hollow-fiber membrane based three-dimensional perfusion bioreactor for long-term culture of whole human bone marrow mononuclear cells. We also investigated bioreactors with incorporated open-porous foamed hydroxyapatite scaffolds, mimicking the in vivo bone matrix. Cells in bioreactors with and without scaffolds were cultured to 6 weeks and compared to Petri dish controls. Cells were analyzed for gene expression, surface markers by flow cytometry, metabolic activity, hematopoietic potential, viability, and attachment by immunocytochemistry. Cells in bioreactors were metabolic active during long-term culture. The percentages of hematopoietic stem cell and mature endothelial cell fractions were maintained in bioreactors. The expression of most of the analyzed genes stabilized and increased after long-term culture of 6 weeks. Compared to Petri dish culture controls, bioreactor perfusion culture improved in both the short and long-term, the colony formation unit capacity of hematopoietic progenitors. Cells attached to the ample surface area provided by hydroxyapatite scaffolds. The implementation of a hydroxyapatite scaffold did not influence colony formation capacity, percentages of cell type specific fractions, gene expression, cell viability or metabolic turnover when compared to control cells cultured in bioreactors without scaffolds. In conclusion, three-dimensional perfusion bioreactor culture enables long-term maintenance of primary human bone marrow cells, with hydroxyapatite scaffolds providing an in vivo-like scaffold for three-dimensional culture.

  2. Effects of red grape juice polyphenols in NADPH oxidase subunit expression in human neutrophils and mononuclear blood cells.

    PubMed

    Dávalos, Alberto; de la Peña, Gema; Sánchez-Martín, Carolina C; Teresa Guerra, M; Bartolomé, Begoña; Lasunción, Miguel A

    2009-10-01

    The NADPH oxidase enzyme system is the main source of superoxide anions in phagocytic and vascular cells. NADPH oxidase-dependent superoxide generation has been found to be abnormally enhanced in several chronic diseases. Evidence is accumulating that polyphenols may have the potential to improve cardiovascular health, although the mechanism is not fully established. Consumption of concentrated red grape juice, rich in polyphenols, has been recently shown to reduce NADPH oxidase activity in circulating neutrophils from human subjects. In the present work we studied whether red grape juice polyphenols affected NADPH oxidase subunit expression at the transcription level. For this, we used human neutrophils and mononuclear cells from peripheral blood, HL-60-derived neutrophils and the endothelial cell line EA.hy926.Superoxide production was measured with 2'7'-dichlorofluorescein diacetate or lucigenin, mRNA expression by real-time RT-PCR and protein expression by Western blot. Each experiment was performed at least three times. In all cell types tested, red grape juice, dealcoholised red wine and pure polyphenols decreased superoxide anion production. Red grape juice and dealcoholised red wine selectively reduced p47phox, p22phox and gp91phox expression at both mRNA and protein levels, without affecting the expression of p67phox. Pure polyphenols, particularly quercetin, also reduced NADPH oxidase subunit expression, especially p47phox, in all cell types tested. The present results showing that red grape juice polyphenols reduce superoxide anion production provide an alternative mechanism by which consumption of grape derivatives may account for a reduction of oxidative stress associated with cardiovascular and/or inflammatory diseases related to NADPH oxidase superoxide overproduction.

  3. Thrombin potently stimulates cytokine production in human vascular smooth muscle cells but not in mononuclear phagocytes.

    PubMed

    Kranzhöfer, R; Clinton, S K; Ishii, K; Coughlin, S R; Fenton, J W; Libby, P

    1996-08-01

    Thrombosis frequently occurs during atherogenesis and in response to vascular injury. Accumulating evidence supports a role for inflammation in the same situation. The present study therefore sought links between thrombosis and inflammation by determining whether thrombin, which is present in active form at sites of thrombosis, can elicit inflammatory functions of human monocytes and vascular smooth muscle cells (SMCs), two major constituents of advanced atheroma. Human alpha-thrombin (EC50, approximately equal to 500 pmol/L) potently induced interleukin (IL)-6 release from SMCs. The tethered-ligand thrombin receptor appeared to mediate this effect. Furthermore, alpha-thrombin also rapidly increased levels of mRNA encoding IL-6 and monocyte chemotactic protein-1 (MCP-1) in SMCs. In contrast, only alpha-thrombin concentrations of > or = 100 nmol/L could stimulate release of IL-6 or tumor necrosis factor-alpha (TNF alpha) in peripheral blood monocytes or monocyte-derived macrophages. Lipid loading of macrophages did not augment thrombin responsiveness. Likewise, only alpha-thrombin concentrations of > or = 100 nmol/L increased levels of IL-6, IL-1 beta, MCP-1, or TNF alpha mRNA in monocytes. Differential responses of SMCs and monocytes to thrombin extended to early agonist-mediated increases in [Ca2+]i. SMCs and endothelial cells, but not monocytes, contained abundant mRNA encoding the thrombin receptor and displayed cell surface thrombin receptor expression detected with a novel monoclonal antibody. Thus, the level of thrombin receptors appeared to account for the differential thrombin susceptibility of SMCs and monocytes. These data suggest that SMCs may be more sensitive than monocytes/macrophages to thrombin activation in human atheroma. Cytokines produced by thrombin-activated SMCs may contribute to ongoing inflammation in atheroma complicated by thrombosis or subjected to angioplasty.

  4. Phase I study of the adoptive immunotherapy of human cancer with lectin activated autologous mononuclear cells.

    PubMed

    Mazumder, A; Eberlein, T J; Grimm, E A; Wilson, D J; Keenan, A M; Aamodt, R; Rosenberg, S A

    1984-02-15

    In previous in vitro studies, the authors showed that phytohemagglutinin (PHA) stimulated peripheral blood lymphocytes (PBL) from cancer patients to generate cells that were lytic for fresh autologous tumor but not for lymphocytes or lymphoblasts. Thus, after IRB approval, a phase I clinical protocol was instituted in cancer patients who had failed all other therapy to determine the toxicity and effects, in vivo, of the infusion of large numbers of such PHA activated autologous PBL. Ten patients were treated on the protocol, six with sarcoma, one with melanoma, and three with colorectal cancer. Up to a total of 1.7 X 10(11) PBL were obtained from 7 to 15 successive leukaphereses, the cells from each leukapheresis being incubated in vitro in medium containing PHA and human AB serum for 2 days and then reinfused following the next leukapheresis 2 days later. Toxicity encountered included fever and chills in 10/10 patients, headaches in 5/10, nausea and vomiting in 3/10, and requirement for erythrocyte transfusion in 8/10. No evidence for autoimmune disease, abnormal serum chemical or coagulation studies, or pulmonary emboli was found. 111Indium trafficing studies showed distribution of infused cells mainly to the spleen and liver, with some accumulation in the lungs and tumor especially after repeated infusions. In 9/10 patients, activated PBL were detected in the peripheral circulation by the sixth leukapheresis. Evidence for this was found by assaying the incorporation of tritiated thymidine (3H-Tdr) into, and lysis of fresh tumor cells by, unstimulated PBL from successive leukaphereses. No tumor regression was seen in these patients with bulk disease. These studies demonstrated that large numbers of PHA-activated PBL can be safely obtained and infused into humans, achieving an increase in the number of circulating activated cells with evidence of migration of cells to tumor, lungs, liver and spleen. Further studies of the use of activated lymphocyte infusion in

  5. Intra- and Intersubject Whole Blood/Plasma Cannabinoid Ratios Determined by 2-Dimensional, Electron Impact GC-MS with Cryofocusing

    PubMed Central

    Schwilke, Eugene W.; Karschner, Erin L.; Lowe, Ross H.; Gordon, Ann M.; Cadet, Jean Lud; Herning, Ronald I.; Huestis, Marilyn A.

    2011-01-01

    BACKGROUND Whole-blood concentrations of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC(THCCOOH)are approximately half of those in plasma due to high plasma protein binding and poor cannabinoid distribution into erythrocytes. Whole blood is frequently the only specimen available in forensic investigations; controlled cannabinoid administration studies provide scientific data for interpretation of cannabinoid tests but usually report plasma concentrations. Whole-blood/plasma cannabinoid ratios from simultaneously collected authentic specimens are rarely reported. METHODS We collected whole blood for 7 days from 32 individuals residing on a closed research unit. Part of the whole blood was processed to obtain plasma, and the whole blood and plasma were stored at −20°C until analysis by validated 2-dimensional GC-MS methods. RESULTS We measured whole-blood/plasma cannabinoid ratios in 187 specimen pairs. Median (interquartile range) whole-blood/plasma ratios were 0.39 (0.28–0.48) for THC (n = 75), 0.56 (0.43–0.73) for 11-OH-THC (n = 17), and 0.37 (0.24–0.56) for THCCOOH (n = 187). Intrasubject variability was determined for the first time: 18.1%–56.6% CV (THC) and 10.8%–38.2% CV (THCCOOH). The mean whole-blood/plasma THC ratio was significantly lower than the THCCOOH ratio (P = 0.0001; 4 participants’ mean THCCOOH ratios were >0.8). CONCLUSION Intra- and intersubject whole-blood/plasma THC and THCCOOH ratios will aid interpretation of whole-blood cannabinoid data. PMID:19264857

  6. Quercetin protects human peripheral blood mononuclear cells from OTA-induced oxidative stress, genotoxicity, and inflammation.

    PubMed

    Periasamy, Ramyaa; Kalal, Iravathy Goud; Krishnaswamy, Rajashree; Viswanadha, VijayaPadma

    2016-07-01

    Ochratoxin A (OTA) is one of the most abundant food-contaminating mycotoxins world wide, and is detrimental to human and animal health. This study evaluated the protective effect of quercetin against OTA-induced cytotoxicity, genotoxicity, and inflammatory response in lymphocytes. Cytotoxicity determined by MTT assay revealed IC20 value of OTA to be 20 µM, which was restored to near control values by pretreatment with quercetin. Oxidative stress parameters such as antioxidant enzymes, LPO and PCC levels indicated that quercetin exerted a protective effect on OTA-induced oxidative stress. Quercetin exerted an antigenotoxic effect on OTA-induced genotoxicity, by significantly reducing the number of structural aberrations in chromosomes and comet parameters like, % olive tail moment from 2.76 ± 0.02 to 0.56 ± 0.02 and % tail DNA from 56.23 ± 2.56 to 12.36 ± 0.56 as determined by comet assay. OTA-induced NO, TNF-α, IL-6, and IL-8 were significantly reduced in the quercetin pretreated samples indicating its anti-inflammatory role. Our results demonstrate for the first time that quercetin exerts a cytoprotective effect against OTA-induced oxidative stress, genotoxicity, and inflammation in lymphocytes. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 855-865, 2016.

  7. Human Peripheral Blood Mononuclear Cell Function and Dendritic Cell Differentiation Are Affected by Bisphenol-A Exposure

    PubMed Central

    Ariemma, Fabiana; Cimmino, Ilaria; Bruzzese, Dario; Scerbo, Roberta; Picascia, Stefania; D’Esposito, Vittoria; Beguinot, Francesco; Formisano, Pietro

    2016-01-01

    Environmental pollutants, including endocrine disruptor chemicals (EDCs), interfere on human health, leading to hormonal, immune and metabolic perturbations. Bisphenol-A (BPA), a main component of polycarbonate plastics, has been receiving increased attention due to its worldwide distribution with a large exposure. In humans, BPA, for its estrogenic activity, may have a role in autoimmunity, inflammatory and allergic diseases. To this aim, we assessed the effect of low BPA doses on functionality of human peripheral blood mononuclear cells (PBMCs), and on in vitro differentiation of dendritic cells from monocytes (mDCs). Fresh peripheral blood samples were obtained from 12 healthy adult volunteers. PBMCs were left unstimulated or were activated with the mitogen phytohemagglutinin (PHA) or the anti-CD3 and anti-CD28 antibodies and incubated in presence or absence of BPA at 0.1 and 1nM concentrations. The immune-modulatory effect of BPA was assessed by evaluating the cell proliferation and the levels of interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-10 (IL-10) and interleukin-13 (IL-13) secreted by PBMCs. mDCs were differentiated with IL-4 and GC-CSF with or without BPA and the expression of differentiation/maturation markers (CD11c, CD1a, CD86, HLA-DR) was evaluated by flow cytometry; furthermore, a panel of 27 different cytokines, growth factors and chemokines were assayed in the mDC culture supernatants. PBMCs proliferation significantly increased upon BPA exposure compared to BPA untreated cells. In addition, a significant decrease in IL-10 secretion was observed in PBMCs incubated with BPA, either in unstimulated or mitogen-stimulated cells, and at both 0.1 and 1nM BPA concentrations. Similarly, IL-13 was reduced, mainly in cells activated by antiCD3/CD28. By contrast, no significant changes in IFN-γ and IL-4 production were found in any condition assayed. Finally, BPA at 1nM increased the density of dendritic cells expressing CD1a and concomitantly

  8. [Determination of Al, Be, Cd, Co, Cr, Mn, Ni, Pb, Se and Tl in whole blood by atomic absorption spectrometry without preliminary sample digestion].

    PubMed

    Ivanenko, N B; Ivanenko, A A; Solov'ev, N D; Navolotskiĭ, D V; Pavlova, O V; Ganeev, A A

    2014-01-01

    Methods of whole blood trace element determination by Graphite furnace atomic absorption spectrometry (in the variant of Zeeman's modulation polarization spectrometry) have been proposed. They do not require preliminary sample digestion. Furnace programs, modifiers and blood dilution factors were optimized. Seronorm™ human whole blood reference materials were used for validation. Dynamic ranges (for undiluted blood samples) were: Al 8 ¸ 210 мg/L; Be 0.3 ¸ 50 мg/L; Cd 0.2 ¸ 75 мg/L; Сo 5 ¸ 350 мg/L; Cr 10 ¸ 100 мg/L; Mn 6 ¸ 250 мg/L; Ni 10 ¸ 350 мg/L; Pb 3 ¸ 240 мg/L; Se 10 ¸ 500 мg/L; Tl 2 ¸ 600 мg/L. Precision (RSD) for the middle of dynamic range ranged from 5% for Mn to 11 for Se.

  9. The expression of pluripotency genes and neuronal markers after neurodifferentiation in fibroblasts co-cultured with human umbilical cord blood mononuclear cells.

    PubMed

    Marinowic, D R; Domingues, M F; Machado, D C; DaCosta, J C

    2015-01-01

    Human umbilical cord blood is an attractive source of stem cells; however, it has a heterogeneous cell population with few mesenchymal stem cells. Cell reprogramming induced by different methodologies can confer pluripotency to differentiated adult cells. The objective of this study was to evaluate the reprogramming of fibroblasts and their subsequent neural differentiation after co-culture with umbilical cord blood mononuclear cells. Cells were obtained from four human umbilical cords. The mononuclear cells were cultured for 7 d and subsequently co-cultured with mouse fibroblast NIH-3T3 cells for 6 d. The pluripotency of the cells was evaluated by RT-PCR using primers specific for pluripotency marker genes. The pluripotency was also confirmed by adipogenic and osteogenic differentiation. Neural differentiation of the reprogrammed cells was evaluated by immunofluorescence. All co-cultured cells showed adipogenic and osteogenic differentiation capacity. After co-cultivation, cells expressed the pluripotency gene KLF4. Statistically significant differences in cell area, diameter, optical density, and fractal dimension were observed by confocal microscopy in the neurally differentiated cells. Contact in the form of co-cultivation of fibroblasts with umbilical cord blood mononuclear fraction for 6 d promoted the reprogramming of these cells, allowing the later induction of neural differentiation.

  10. Human Umbilical Cord Blood Mononuclear Cells in a Double-Hit Model of Bronchopulmonary Dysplasia in Neonatal Mice

    PubMed Central

    Mildau, Céline; Shen, Jie; Kasoha, Mariz; Laschke, Matthias W.; Roolfs, Torge; Schmiedl, Andreas; Tschernig, Thomas; Bieback, Karen; Gortner, Ludwig

    2013-01-01

    Background Bronchopulmonary dysplasia (BPD) presents a major threat of very preterm birth and treatment options are still limited. Stem cells from different sources have been used successfully in experimental BPD, induced by postnatal hyperoxia. Objectives We investigated the effect of umbilical cord blood mononuclear cells (MNCs) in a new double-hit mouse model of BPD. Methods For the double-hit, date mated mice were subjected to hypoxia and thereafter the offspring was exposed to hyperoxia. Human umbilical cord blood MNCs were given intraperitoneally by day P7. As outcome variables were defined: physical development (auxology), lung structure (histomorphometry), expression of markers for lung maturation and inflammation on mRNA and protein level. Pre- and postnatal normoxic pups and sham treated double-hit pups served as control groups. Results Compared to normoxic controls, sham treated double-hit animals showed impaired physical and lung development with reduced alveolarization and increased thickness of septa. Electron microscopy revealed reduced volume density of lamellar bodies. Pulmonary expression of mRNA for surfactant proteins B and C, Mtor and Crabp1 was reduced. Expression of Igf1 was increased. Treatment with umbilical cord blood MNCs normalized thickness of septa and mRNA expression of Mtor to levels of normoxic controls. Tgfb3 mRNA expression and pro-inflammatory IL-1β protein concentration were decreased. Conclusion The results of our study demonstrate the therapeutic potential of umbilical cord blood MNCs in a new double-hit model of BPD in newborn mice. We found improved lung structure and effects on molecular level. Further studies are needed to address the role of systemic administration of MNCs in experimental BPD. PMID:24069341

  11. A Potential Role for Mononuclear Phagocytes in Cutaneous Ulcer Development in Human Immunodeficiency Virus–Leishmania braziliensis Coinfection

    PubMed Central

    Guimarães, Luiz H.; Saldanha, Maíra; Menezes, Taís; Moreno, Lis; Torres, Alex; Costa, Rúbia; Passos, Sara; Badaró, Roberto; Arruda, Sérgio; Carvalho, Lucas P.

    2015-01-01

    Skin ulcer development in cutaneous leishmaniasis due to Leishmania braziliensis infection is associated with a mononuclear cell infiltrate and high levels of tumor necrosis factor (TNF). Herein, we show that despite the absence of Leishmania-driven TNF, a cutaneous leishmaniasis patient with acquired immunodeficiency syndrome developed a skin ulcer. The presence of mononuclear phagocytes and high levels of TNF, chemokine (C-C motif) ligand 2 (CCL2), and metalloproteinase-9 in tissue are identified as potential contributors to immunopathology observed in L. braziliensis-infected patients. PMID:26483124

  12. Abnormal MicroRNA Expression in Ts65Dn Hippocampus and Whole Blood: Contributions to Down Syndrome Phenotypes

    PubMed Central

    Keck-Wherley, Jennifer; Grover, Deepak; Bhattacharyya, Sharmistha; Xu, Xiufen; Holman, Derek; Lombardini, Eric D.; Verma, Ranjana; Biswas, Roopa; Galdzicki, Zygmunt

    2011-01-01

    Down syndrome (DS; trisomy 21) is one of the most common genetic causes of intellectual disability, which is attributed to triplication of genes located on chromosome 21. Elevated levels of several microRNAs (miRNAs) located on chromosome 21 have been reported in human DS heart and brain tissues. The Ts65Dn mouse model is the most investigated DS model with a triplicated segment of mouse chromosome 16 harboring genes orthologous to those on human chromosome 21. Using ABI TaqMan miRNA arrays, we found a set of miRNAs that were significantly up- or downregulated in the Ts65Dn hippocampus compared to euploid controls. Furthermore, miR-155 and miR-802 showed significant overexpression in the Ts65Dn hippocampus, thereby confirming results of previous studies. Interestingly, miR-155 and miR-802 were also overexpressed in the Ts65Dn whole blood but not in lung tissue. We also found overexpression of the miR-155 precursors, pri- and pre-miR-155 derived from the miR-155 host gene, known as B cell integration cluster, suggesting enhanced biogenesis of miR-155. Bioinformatic analysis revealed that neurodevelopment, differentiation of neuroglia, apoptosis, cell cycle, and signaling pathways including ERK/MAPK, protein kinase C, phosphatidylinositol 3-kinase, m-TOR and calcium signaling are likely targets of these miRNAs. We selected some of these potential gene targets and found downregulation of mRNA encoding Ship1, Mecp2 and Ezh2 in Ts65Dn hippocampus. Interestingly, the miR-155 target gene Ship1 (inositol phosphatase) was also downregulated in Ts65Dn whole blood but not in lung tissue. Our findings provide insights into miRNA-mediated gene regulation in Ts65Dn mice and their potential contribution to impaired hippocampal synaptic plasticity and neurogenesis, as well as hemopoietic abnormalities observed in DS. PMID:22042248

  13. A smart pipette for equipment-free separation and delivery of plasma for on-site whole blood analysis.

    PubMed

    Im, Sung B; Kim, Sang C; Shim, Joon S

    2016-02-01

    A novel device of smart pipette has been suggested to extract and deliver plasma from whole blood in a disposable format. By operating an on-chip disposable micropump, approximately 30 μL of plasma was obtained from 100 μL of whole blood within 5 min without any external equipment for point-of-care blood analysis.

  14. Members of the Candida parapsilosis Complex and Candida albicans are Differentially Recognized by Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Estrada-Mata, Eine; Navarro-Arias, María J.; Pérez-García, Luis A.; Mellado-Mojica, Erika; López, Mercedes G.; Csonka, Katalin; Gacser, Attila; Mora-Montes, Héctor M.

    2016-01-01

    The systemic infections caused by members of the Candida parapsilosis complex are currently associated to high morbility and mortality rates, and are considered as relevant as those caused by Candida albicans. Since the fungal cell wall is the first point of contact with the host cells, here we performed a comparison of this organelle in members of the C. parapsilosis complex, and its relevance during interaction with human peripheral blood mononuclear cells (PBMCs). We found that the wall of the C. parapsilosis complex members is similar in composition, but differs to that from C. albicans, with less mannan content and more β-glucan and porosity levels. Furthermore, lectin-based analysis showed increased chitin and β1,3-glucan exposure at the surface of C. parapsilosis sensu lato when compared to C. albicans. Yeast cells of members of the C. parapsilosis complex stimulated more cytokine production by human PBMCs than C. albicans cells; and this significantly changed upon removal of O-linked mannans, indicating this wall component plays a significant role in cytokine stimulation by C. parapsilosis sensu lato. When inner wall components were exposed on the wall surface, C. parapsilosis sensu stricto and C. metapsilosis, but not C. orthopsilosis, stimulated higher cytokine production. Moreover, we found a strong dependency on β1,3-glucan recognition for the members of the C. parapsilosis complex, but not for live C. albicans cells; whereas TLR4 was required for TNFα production by the three members of the complex, and stimulation of IL-6 by C. orthopsilosis. Mannose receptor had a significant role during TNFα and IL-1β stimulation by members of the complex. Finally, we demonstrated that purified N- and O-mannans from either C. parapsilosis sensu lato or C. albicans are capable to block the recognition of these pathogens by human PBMCs. Together; our results suggest that the innate immune recognition of the members of the C. parapsilosis complex is differential

  15. Comparison of manual and automated nucleic acid extraction from whole-blood samples.

    PubMed

    Riemann, Kathrin; Adamzik, Michael; Frauenrath, Stefan; Egensperger, Rupert; Schmid, Kurt W; Brockmeyer, Norbert H; Siffert, Winfried

    2007-01-01

    Nucleic acid extraction and purification from whole blood is a routine application in many laboratories. Automation of this procedure promises standardized sample treatment, a low error rate, and avoidance of contamination. The performance of the BioRobot M48 (Qiagen) and the manual QIAmp DNA Blood Mini Kit (Qiagen) was compared for the extraction of DNA from whole blood. The concentration and purity of the extracted DNAs were determined by spectrophotometry. Analytical sensitivity was assessed by common PCR and genotyping techniques. The quantity and quality of the generated DNAs were slightly higher using the manual extraction method. The results of downstream applications were comparable to each other. Amplification of high-molecular-weight PCR fragments, genotyping by restriction digest, and pyrosequencing were successful for all samples. No cross-contamination could be detected. While automated DNA extraction requires significantly less hands-on time, it is slightly more expensive than the manual extraction method.

  16. Neurocysticercosis: validity of ELISA after storage of whole blood and cerebrospinal fluid on paper.

    PubMed

    Fleury, A; Bouteille, B; Garcia, E; Marquez, C; Preux, P M; Escobedo, F; Sotelo, J; Dumas, M

    2001-09-01

    Cysticercosis is an infestation of Cysticercus cellulosae. When it occurs in the brain, chronic neurological complications can ensue, most commonly seizures. Neurocysticercosis is usually diagnosed by neuroimaging, a technique not available in most endemic countries. Hence immunological tests are valuable for diagnosis and epidemiological surveys. We evaluated the suitability of paper for storing blood and cerebrospinal fluid (CSF) until subsequent testing by enzyme-linked immunosorbent assay (ELISA), by testing whole blood samples on filter paper from 305 patients and CSF samples from 117 patients stored on ordinary white typing paper and on filter paper. Optimal preservation of biological samples is achieved when whole blood is stored on filter paper, CSF on white paper, and when samples are frozen within 1 week after collection. Our results could improve diagnostic capabilities and facilitate epidemiological surveys in endemic countries where immunodiagnostic tests cannot be rapidly performed because of inadequate laboratory infrastructure.

  17. Device and method for automated separation of a sample of whole blood into aliquots

    DOEpatents

    Burtis, Carl A.; Johnson, Wayne F.

    1989-01-01

    A device and a method for automated processing and separation of an unmeasured sample of whole blood into multiple aliquots of plasma. Capillaries are radially oriented on a rotor, with the rotor defining a sample chamber, transfer channels, overflow chamber, overflow channel, vent channel, cell chambers, and processing chambers. A sample of whole blood is placed in the sample chamber, and when the rotor is rotated, the blood moves outward through the transfer channels to the processing chambers where the blood is centrifugally separated into a solid cellular component and a liquid plasma component. When the rotor speed is decreased, the plasma component backfills the capillaries resulting in uniform aliquots of plasma which may be used for subsequent analytical procedures.

  18. Brief Report: Whole Blood Serotonin Levels and Gastrointestinal Symptoms in Autism Spectrum Disorder.

    PubMed

    Marler, Sarah; Ferguson, Bradley J; Lee, Evon Batey; Peters, Brittany; Williams, Kent C; McDonnell, Erin; Macklin, Eric A; Levitt, Pat; Gillespie, Catherine Hagan; Anderson, George M; Margolis, Kara Gross; Beversdorf, David Q; Veenstra-VanderWeele, Jeremy

    2016-03-01

    Elevated whole blood serotonin levels are observed in more than 25% of children with autism spectrum disorder (ASD). Co-occurring gastrointestinal (GI) symptoms are also common in ASD but have not previously been examined in relationship with hyperserotonemia, despite the synthesis of serotonin in the gut. In 82 children and adolescents with ASD, we observed a correlation between a quantitative measure of lower GI symptoms and whole blood serotonin levels. No significant association was seen between functional constipation diagnosis and serotonin levels in the hyperserotonemia range, suggesting that this correlation is not driven by a single subgroup. More specific assessment of gut function, including the microbiome, will be necessary to evaluate the contribution of gut physiology to serotonin levels in ASD.

  19. Brief Report: Whole Blood Serotonin Levels and Gastrointestinal Symptoms in Autism Spectrum Disorder

    PubMed Central

    Marler, Sarah; Ferguson, Bradley J.; Lee, Evon Batey; Peters, Brittany; Williams, Kent C.; McDonnell, Erin; Macklin, Eric A.; Levitt, Pat; Gillespie, Catherine Hagan; Anderson, George M.; Margolis, Kara Gross; Beversdorf, David Q.; Veenstra-VanderWeele, Jeremy

    2016-01-01

    Elevated whole blood serotonin levels are observed in more than 25 % of children with autism spectrum disorder (ASD). Co-occurring gastrointestinal (GI) symptoms are also common in ASD but have not previously been examined in relationship with hyperserotonemia, despite the synthesis of serotonin in the gut. In 82 children and adolescents with ASD, we observed a correlation between a quantitative measure of lower GI symptoms and whole blood serotonin levels. No significant association was seen between functional constipation diagnosis and serotonin levels in the hyperserotonemia range, suggesting that this correlation is not driven by a single subgroup. More specific assessment of gut function, including the microbiome, will be necessary to evaluate the contribution of gut physiology to serotonin levels in ASD. PMID:26527110

  20. Integrated separation of blood plasma from whole blood for microfluidic paper-based analytical devices.

    PubMed

    Yang, Xiaoxi; Forouzan, Omid; Brown, Theodore P; Shevkoplyas, Sergey S

    2012-01-21

    Many diagnostic tests in a conventional clinical laboratory are performed on blood plasma because changes in its composition often reflect the current status of pathological processes throughout the body. Recently, a significant research effort has been invested into the development of microfluidic paper-based analytical devices (μPADs) implementing these conventional laboratory tests for point-of-care diagnostics in resource-limited settings. This paper describes the use of red blood cell (RBC) agglutination for separating plasma from finger-prick volumes of whole blood directly in paper, and demonstrates the utility of this approach by integrating plasma separation and a colorimetric assay in a single μPAD. The μPAD was fabricated by printing its pattern onto chromatography paper with a solid ink (wax) printer and melting the ink to create hydrophobic barriers spanning through the entire thickness of the paper substrate. The μPAD was functionalized by spotting agglutinating antibodies onto the plasma separation zone in the center and the reagents of the colorimetric assay onto the test readout zones on the periphery of the device. To operate the μPAD, a drop of whole blood was placed directly onto the plasma separation zone of the device. RBCs in the whole blood sample agglutinated and remained in the central zone, while separated plasma wicked through the paper substrate into the test readout zones where analyte in plasma reacted with the reagents of the colorimetric assay to produce a visible color change. The color change was digitized with a portable scanner and converted to concentration values using a calibration curve. The purity and yield of separated plasma was sufficient for successful operation of the μPAD. This approach to plasma separation based on RBC agglutination will be particularly useful for designing fully integrated μPADs operating directly on small samples of whole blood.

  1. Selected epidemiological aspects of fresh whole blood application in the Polish Field Hospital in Afghanistan.

    PubMed

    Olszewski, Adam; Korzeniewski, Krzysztof; Lass, Anna

    2014-01-01

    Minimisation of blood transmitted diseases is a basic element of all blood transfusion strategies. Civilian health service standards used in peacetime may be difficult to implement in a battlefield. The risk of blood-borne diseases depends on the applied donor qualification procedures and the epidemiological situation in the areas of military operations. The authors discuss various epidemiological aspects considered when selecting potential donors of fresh whole blood for a Walking Blood Bank at the Polish Field Hospital in Afghanistan.

  2. Performance characteristics of the HemoCue B-Glucose analyzer using whole-blood samples.

    PubMed

    Voss, E M; Cembrowski, G S

    1993-07-01

    We evaluated the HemoCue B-Glucose (HemoCue Inc, Mission Viejo, Calif) analyzer for accuracy, precision, linearity, and recovery. One hundred eighteen capillary whole-blood samples were analyzed in duplicate on the HemoCue B-Glucose and the YSI 2300 STAT Glucose/L-Lactate (Yellow Springs [Ohio] Instruments) analyzers; corresponding plasma glucose levels were measured in duplicate on the Roche Cobas MIRA (Roche Diagnostic Systems, Nutley, NJ) analyzer. Plasma glucose levels were converted to whole-blood equivalent glucose levels by using a factor of 1.11. The following regression equations were obtained: HemoCue = 1.02 (YSI) + 0.19, Sy/x = 0.52, r2 = .984; and HemoCue = 0.98 (whole-blood equivalent glucose levels) + 0.26, Sy/x = 0.55, r2 = .982. Within-run coefficients of variation were 4.0%, 3.5%, 2.2%, and 1.0% at glucose concentrations of 3.9, 5.4, 8.7, and 17.1 mmol/L (71, 97, 156, and 308 mg/dL), respectively. Between-run imprecision and total imprecision using lyopholized materials with three lot numbers of cuvettes were 4.2% and 5.6% at 2.1 mmol/L (37 mg/dL) and 2.4% and 2.7% at 5.2 mmol/L (95 mg/dL), respectively. The HemoCue B-Glucose analyzer displayed linearity between 0 and 22.2 mmol/L (0 and 400 mg/dL), and the percent recovery averaged 98.7% +/- 4.5% (mean +/- SD). The HemoCue B-Glucose analyzer is a rapid, simple, and reliable method for determinations of whole-blood glucose levels.

  3. Acoustofluidics and whole-blood manipulation in surface acoustic wave counterflow devices.

    PubMed

    Travagliati, Marco; Shilton, Richie J; Pagliazzi, Marco; Tonazzini, Ilaria; Beltram, Fabio; Cecchini, Marco

    2014-11-04

    On-chip functional blocks for sample preprocessing are necessary elements for the implementation of fully portable micrototal analysis systems (μTAS). We demonstrate and characterize the microparticle and whole-blood manipulation capabilities of surface acoustic wave (SAW) driven counterflow micropumps. The motion of suspended cells in this system is governed by the two dominant acoustic forces associated with the scattered SAW (of wavelength λf): acoustic-radiation force and acoustic-streaming Stokesian drag force. We show that by reducing the microchannel height (h) beyond a threshold value the balance of these forces is shifted toward the acoustic-radiation force and that this yields control of two different regimes of microparticle dynamics. In the regime dominated by the acoustic radiation force (h ≲ λf), microparticles are collected in the seminodes of the partial standing sound-wave arising from reflections off microchannel walls. This enables the complete separation of plasma and corpuscular components of whole blood in periodical predetermined positions without any prior sample dilution. Conversely, in the regime dominated by acoustic streaming (h ≫ λf), the microbeads follow vortical streamlines in a pattern characterized by three different phases during microchannel filling. This makes it possible to generate a cell-concentration gradient within whole-blood samples, a behavior not previously reported in any acoustic-streaming device. By careful device design, a new class of SAW pumping devices is presented that allows the manipulation and pretreatment of whole-blood samples for portable and integrable biological chips and is compatible with hand-held battery-operated devices.

  4. Whole blood chloroquine concentrations with Plasmodium vivax infection in Irian Jaya, Indonesia.

    PubMed

    Baird, J K; Leksana, B; Masbar, S; Suradi; Sutanihardja, M A; Fryauff, D J; Subianto, B

    1997-06-01

    Whole blood concentrations of self-administered chloroquine (CQ) and its metabolite desethylchloroquine (DCQ) were measured in 168 patients with microscopically confirmed infection by Plasmodium vivax in northeastern Irian Jaya, Indonesia. The study consisted of both survey and passive case detection in four separate villages between 1992 and 1994. The subjects were Javanese people 4-51 years old who had lived in the Arso region for up to two years. The sum of CQ and DCQ ranged from 0 to 8,342 ng/ml of whole blood, and 122 subjects (73%) had > or = 100 ng/ml of CQ plus DCQ, the estimated minimally effective concentration (MEC) in whole blood against chloroquine-sensitive P. vivax. Among 56 subjects reporting to a clinic with symptoms of malaria, 53 (95%) had ordinarily effective levels of chloroquine in blood. Among 109 largely asymptomatic malaria patients found by survey case detection, 69 (63%) had chloroquine blood levels greater than the MEC. Virtually all clinical and most subclinical vivax malaria in this region occurs despite ordinarily effective levels of chloroquine in blood.

  5. Adrenergic Effect on Cytokine Release After Ex Vivo Healthy Volunteers' Whole Blood LPS Stimulation.

    PubMed

    Papandreou, Vasiliki; Kavrochorianou, Nadia; Katsoulas, Theodoros; Myrianthefs, Pavlos; Venetsanou, Kyriaki; Baltopoulos, George

    2016-06-01

    Catecholamines are molecules with immunomodulatory properties in health and disease. Several studies showed the effect of catecholamines when administered to restore hemodynamic stability in septic patients. This study investigates the effect of norepinephrine and dobutamine on whole blood cytokine release after ex vivo lipopolysaccharide (LPS) stimulation. Whole blood collected from healthy individuals was stimulated with LPS, in the presence of norepinephrine or dobutamine at different concentrations, with or without metoprolol, a β1 receptor antagonist. Cytokine measurement was performed in isolated cell culture supernatants with ELISA. Results are expressed as mean ± SEM and compared with Mann-Whitney rank-sum test. Both norepinephrine and dobutamine significantly reduced TNF-α and IL-6 production after ex vivo LPS stimulation of whole blood in a dose-dependent manner, and this effect was partially reversed by the presence of metoprolol. Norepinephrine and dobutamine reduce the LPS-induced production of pro-inflammatory cytokines, thus possibly contributing to altered balance between the inflammatory and anti-inflammatory responses, which are vital for a successful host response to severe disease, shock, and sepsis.

  6. An electrochemical Lab-on-a-CD system for parallel whole blood analysis.

    PubMed

    Li, Tingjie; Fan, Yaxi; Cheng, Yang; Yang, Jun

    2013-07-07

    Lab-on-a-CD, as a main branch of Lab-on-a-chip technology, has led to several very successful commercial products. Most of these existing Lab-on-a-CD systems present complex system designs and thus are relatively expensive. In this work, we have developed a simple but robust Lab-on-a-CD system for parallel whole blood analyses. This Lab-on-a-CD system incorporates electrochemical bioanalysis and a simple blood sample separation mechanism into the centrifugal platform, and thus reduces the system's complexity. To demonstrate the applicability, the system was applied to perform basic metabolic panel tests, for example, the concentrations of glucose, lactate and uric acids of whole blood samples. Using only 16 μL of whole blood, within a few minutes, the Lab-on-a-CD system could produce results that agreed in general with the data by a conventional system. Therefore, this proof-of-concept Lab-on-a-CD system has demonstrated the potential to become a robust and simple-to-use device for parallel blood analyses.

  7. Whole Blood Transcriptome Sequencing Reveals Gene Expression Differences between Dapulian and Landrace Piglets.

    PubMed

    Hu, Jiaqing; Yang, Dandan; Chen, Wei; Li, Chuanhao; Wang, Yandong; Zeng, Yongqing; Wang, Hui

    2016-01-01

    There is little genomic information regarding gene expression differences at the whole blood transcriptome level of different pig breeds at the neonatal stage. To solve this, we characterized differentially expressed genes (DEGs) in the whole blood of Dapulian (DPL) and Landrace piglets using RNA-seq (RNA-sequencing) technology. In this study, 83 DEGs were identified between the two breeds. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified immune response and metabolism as the most commonly enriched terms and pathways in the DEGs. Genes related to immunity and lipid metabolism were more highly expressed in the DPL piglets, while genes related to body growth were more highly expressed in the Landrace piglets. Additionally, the DPL piglets had twofold more single nucleotide polymorphisms (SNPs) and alternative splicing (AS) than the Landrace piglets. These results expand our knowledge of the genes transcribed in the piglet whole blood of two breeds and provide a basis for future research of the molecular mechanisms underlying the piglet differences.

  8. Measurement of platelet aggregation functions using whole blood migration ratio in a microfluidic chip.

    PubMed

    Seo, Hong Seog; Choi, Sung Hyuk; Han, Miran; Kim, Kyeong Ah; Cho, Chi Hyun; An, Seong Soo A; Lim, Chae Seung; Shin, Sehyun

    2016-01-01

    Platelets play a major role in maintaining endothelial integrity and hemostasis. Of the various soluble agonists, ADP is an important in vivo stimulus for inducing platelet aggregation. In this study, a simple, rapid, and affordable method was designed for testing bleeding time (BT) and platelet aggregation with a two-channel microfluidic chip. Whole blood migration ratio (MR) from a microchip system was evaluated in comparison to the closure time (CT) from PFA-100 assays (Siemens, Germany) and CD62P expression on platelets. To induce platelet aggregation, a combination of collagen (1.84 mg/ml) and ADP (37.5 mg/ml) were used as agonists. After adding the agonists to samples, whole blood MR from the microchip system was measured. The outcome of the assessment depended on reaction time and agonist concentration. MR of whole blood from the microchip system was significantly correlated with CT from PFA-100 (r = 0.61, p <  0.05, n = 60). In addition, MR was negatively correlated with CD62P expression (r =-0.95, p <  0.05, n = 60). These results suggest that the measurement of MR using agonists is an easy, simple and efficient method for monitoring platelet aggregation in normal and ADP-receptors defective samples, along with the BT test. Thus, usage of the current microfluidic method could expand to diverse applications, including efficacy assessments in platelet therapy.

  9. Comprehensive characterization of chondrocyte cultures in plasma and whole blood biomatrices for cartilage tissue engineering.

    PubMed

    Schulz, Ronny M; Haberhauer, Marcus; Zernia, Göran; Pösel, Claudia; Thümmler, Christian; Somerson, Jeremy S; Huster, Daniel

    2014-07-01

    Many synthetic polymers and biomaterials have been used as matrices for 3D chondrocyte seeding and transplantation in the field of cartilage tissue engineering. To develop a fully autologous carrier for chondrocyte cultivation, we examined the feasibility of allogeneic plasma and whole blood-based matrices and compared them to agarose constructs. Primary articular chondrocytes isolated from 12-month-old pigs were embedded into agarose, plasma and whole blood matrices and cultivated under static-free swelling conditions for up to four weeks. To evaluate the quality of the synthesized extracellular matrix (ECM), constructs were subjected to weekly examinations using histological staining, spectrophotometry, immunohistochemistry and biochemical analysis. In addition, gene expression of cartilage-specific markers such as aggrecan, Sox9 and collagen types I, II and X was determined by RT-PCR. Chondrocyte morphology was assessed via scanning electron microscopy and viability staining, including proliferation and apoptosis assays. Finally, (13)  C NMR spectroscopy provided further evidence of synthesis of ECM components. It was shown that chondrocyte cultivation in allogeneic plasma and whole-blood matrices promoted sufficient chondrocyte viability and differentiation behaviour, resulting in neo-formation of a hyaline-like cartilage matrix.

  10. [Determination of strontium content in whole blood and urine by icp-ms].

    PubMed

    Ulanova, T S; Gileva, O V; Stenno, E V; Veikhman, G A; Nedochitova, A V

    2015-01-01

    Parameters of strontium determination in the whole blood and urine of children living near ore deposits containing up to 20% strontium sulfate have been determined. The average strontium content in the whole blood of two children groups of 109.52 ± 11.07 mg/L and 131.62 ± 12.95 mg/L, significantly exceeded the level in the comparison group 44.2 ± 4.24 mg/L. The average strontium contents of two groups of children in urine were 1252.3 ± 332.2 mg/L and 1341.5 ± 241.8 mg/L, these values were 4.2 and 4.5 times higher than in the comparison group 296.4 ± 61.5 mg/L. The conditions for blood and urine sample preparation were optimized to reduce measure errors and to determine strontium at the reference concentration level. The accuracy of the results has been confirmed by analysis of the standard samples Seronorm™ Whole Blood L1, L2, L3 and Seronorm™ Urine.

  11. Whole Blood Transcriptome Sequencing Reveals Gene Expression Differences between Dapulian and Landrace Piglets

    PubMed Central

    Yang, Dandan; Li, Chuanhao; Wang, Yandong

    2016-01-01

    There is little genomic information regarding gene expression differences at the whole blood transcriptome level of different pig breeds at the neonatal stage. To solve this, we characterized differentially expressed genes (DEGs) in the whole blood of Dapulian (DPL) and Landrace piglets using RNA-seq (RNA-sequencing) technology. In this study, 83 DEGs were identified between the two breeds. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified immune response and metabolism as the most commonly enriched terms and pathways in the DEGs. Genes related to immunity and lipid metabolism were more highly expressed in the DPL piglets, while genes related to body growth were more highly expressed in the Landrace piglets. Additionally, the DPL piglets had twofold more single nucleotide polymorphisms (SNPs) and alternative splicing (AS) than the Landrace piglets. These results expand our knowledge of the genes transcribed in the piglet whole blood of two breeds and provide a basis for future research of the molecular mechanisms underlying the piglet differences. PMID:28105431

  12. The effect of ex vivo anticoagulants on whole blood platelet aggregation.

    PubMed

    Kalb, Madeleine L; Potura, Lukasz; Scharbert, Gisela; Kozek-Langenecker, Sibylle A

    2009-02-01

    Pre- and intraoperative platelet function monitoring is increasingly recommended in order to detect risk factors for bleeding and to target coagulation management. The ideal anticoagulant for accurate platelet aggregometry remains controversial. The aim of this experimental trial was to compare platelet aggregability in whole blood stored in citrate, heparin and direct thrombin inhibitors. Whole blood was drawn from 11 healthy adult volunteers who had not taken any medication in the previous 14 days. Blood was stored in trisodium citrate, unfractionated heparin, melagatran, lepirudin and argatroban. Platelet aggregation was performed using the impedance aggregometer Multiplate (Dynabyte, Munich, Germany) with adenosine diphosphate (ADP), thrombin receptor activating peptide (TRAP), collagen, arachidonic acid and ristocetin as agonists. Samples were analysed immediately after blood sampling (baseline), as well as 30 and 120 min afterwards. At baseline there were no significant differences in aggregability between samples containing direct thrombin inhibitors and heparin. In contrast, aggregation in response to all agonists except for ristocetin was significantly impaired in citrated blood. During storage the response to arachidonic acid and collagen was maintained by direct thrombin inhibitors and heparin, whereas ADP-, TRAP- and ristocetin-induced aggregation varied considerably over time in all ex vivo anticoagulants tested. Pre-analytical procedures should be standardized because storage duration and anticoagulants significantly affect platelet aggregability in whole blood. For point-of-care monitoring with immediate analysis after blood withdrawal all tested direct thrombin inhibitors as well as unfractionated heparin can be used as anticoagulants whereas citrate is not recommended.

  13. Association between DNA Methylation in Whole Blood and Measures of Glucose Metabolism: KORA F4 Study

    PubMed Central

    Wahl, Simone; Kunze, Sonja; Molnos, Sophie; Volkova, Nadezda; Schramm, Katharina; Carstensen-Kirberg, Maren; Waldenberger, Melanie; Gieger, Christian; Peters, Annette; Illig, Thomas; Prokisch, Holger; Roden, Michael; Grallert, Harald

    2016-01-01

    Epigenetic regulation has been postulated to affect glucose metabolism, insulin sensitivity and the risk of type 2 diabetes. Therefore, we performed an epigenome-wide association study for measures of glucose metabolism in whole blood samples of the population-based Cooperative Health Research in the Region of Augsburg F4 study using the Illumina HumanMethylation 450 BeadChip. We identified a total of 31 CpG sites where methylation level was associated with measures of glucose metabolism after adjustment for age, sex, smoking, and estimated white blood cell proportions and correction for multiple testing using the Benjamini-Hochberg (B-H) method (four for fasting glucose, seven for fasting insulin, 25 for homeostasis model assessment-insulin resistance [HOMA-IR]; B-H-adjusted p-values between 9.2x10-5 and 0.047). In addition, DNA methylation at cg06500161 (annotated to ABCG1) was associated with all the aforementioned phenotypes and 2-hour glucose (B-H-adjusted p-values between 9.2x10-5 and 3.0x10-3). Methylation status of additional three CpG sites showed an association with fasting insulin only after additional adjustment for body mass index (BMI) (B-H-adjusted p-values = 0.047). Overall, effect strengths were reduced by around 30% after additional adjustment for BMI, suggesting that this variable has an influence on the investigated phenotypes. Furthermore, we found significant associations between methylation status of 21 of the aforementioned CpG sites and 2-hour insulin in a subset of samples with seven significant associations persisting after additional adjustment for BMI. In a subset of 533 participants, methylation of the CpG site cg06500161 (ABCG1) was inversely associated with ABCG1 gene expression (B-H-adjusted p-value = 1.5x10-9). Additionally, we observed an enrichment of the top 1,000 CpG sites for diabetes-related canonical pathways using Ingenuity Pathway Analysis. In conclusion, our study indicates that DNA methylation and diabetes-related traits

  14. Estimation of Cell-Type Composition Including T and B Cell Subtypes for Whole Blood Methylation Microarray Data.

    PubMed

    Waite, Lindsay L; Weaver, Benjamin; Day, Kenneth; Li, Xinrui; Roberts, Kevin; Gibson, Andrew W; Edberg, Jeffrey C; Kimberly, Robert P; Absher, Devin M; Tiwari, Hemant K

    2016-01-01

    DNA methylation levels vary markedly by cell-type makeup of a sample. Understanding these differences and estimating the cell-type makeup of a sample is an important aspect of studying DNA methylation. DNA from leukocytes in whole blood is simple to obtain and pervasive in research. However, leukocytes contain many distinct cell types and subtypes. We propose a two-stage model that estimates the proportions of six main cell types in whole blood (CD4+ T cells, CD8+ T cells, monocytes, B cells, granulocytes, and natural killer cells) as well as subtypes of T and B cells. Unlike previous methods that only estimate overall proportions of CD4+ T cell, CD8+ T cells, and B cells, our model is able to estimate proportions of naïve, memory, and regulatory CD4+ T cells as well as naïve and memory CD8+ T cells and naïve and memory B cells. Using real and simulated data, we are able to demonstrate that our model is able to reliably estimate proportions of these cell types and subtypes. In studies with DNA methylation data from Illumina's HumanMethylation450k arrays, our estimates will be useful both for testing for associations of cell type and subtype composition with phenotypes of interest as well as for adjustment purposes to prevent confounding in epigenetic association studies. Additionally, our method can be easily adapted for use with whole genome bisulfite sequencing (WGBS) data or any other genome-wide methylation data platform.

  15. A microarray gene analysis of peripheral whole blood in normal adult male rats after long-term GH gene therapy.

    PubMed

    Qin, Ying; Tian, Ya-Ping

    2010-06-01

    The main aims of this study were to determine the effects of GH gene abuse/misuse in normal animals and to discover genes that could be used as candidate biomarkers for the detection of GH gene therapy abuse/misuse in humans. We determined the global gene expression profile of peripheral whole blood from normal adult male rats after long-term GH gene therapy using CapitalBio 27 K Rat Genome Oligo Arrays. Sixty one genes were found to be differentially expressed in GH gene-treated rats 24 weeks after receiving GH gene therapy, at a two-fold higher or lower level compared to the empty vector group (p < 0.05). These genes were mainly associated with angiogenesis, oncogenesis, apoptosis, immune networks, signaling pathways, general metabolism, type I diabetes mellitus, carbon fixation, cell adhesion molecules, and cytokine-cytokine receptor interaction. The results imply that exogenous GH gene expression in normal subjects is likely to induce cellular changes in the metabolism, signal pathways and immunity. A real-time qRT-PCR analysis of a selection of the genes confirmed the microarray data. Eight differently expressed genes were selected as candidate biomarkers from among these 61 genes. These 8 showed five-fold higher or lower expression levels after the GH gene transduction (p < 0.05). They were then validated in real-time PCR experiments using 15 single-treated blood samples and 10 control blood samples. In summary, we detected the gene expression profiles of rat peripheral whole blood after long-term GH gene therapy and screened eight genes as candidate biomarkers based on the microarray data. This will contribute to an increased mechanistic understanding of the effects of chronic GH gene therapy abuse/misuse in normal subjects.

  16. Induction of RET Dependent and Independent Pro-Inflammatory Programs in Human Peripheral Blood Mononuclear Cells from Hirschsprung Patients

    PubMed Central

    Rusmini, Marta; Griseri, Paola; Lantieri, Francesca; Matera, Ivana; Hudspeth, Kelly L.; Roberto, Alessandra; Mikulak, Joanna; Avanzini, Stefano; Rossi, Valentina; Mattioli, Girolamo; Jasonni, Vincenzo; Ravazzolo, Roberto; Pavan, William J.; Pini-Prato, Alessio

    2013-01-01

    Hirschsprung disease (HSCR) is a rare congenital anomaly characterized by the absence of enteric ganglia in the distal intestinal tract. While classified as a multigenic disorder, the altered function of the RET tyrosine kinase receptor is responsible for the majority of the pathogenesis of HSCR. Recent evidence demonstrate a strong association between RET and the homeostasis of immune system. Here, we utilize a unique cohort of fifty HSCR patients to fully characterize the expression of RET receptor on both innate (monocytes and Natural Killer lymphocytes) and adaptive (B and T lymphocytes) human peripheral blood mononuclear cells (PBMCs) and to explore the role of RET signaling in the immune system. We show that the increased expression of RET receptor on immune cell subsets from HSCR individuals correlates with the presence of loss-of-function RET mutations. Moreover, we demonstrate that the engagement of RET on PBMCs induces the modulation of several inflammatory genes. In particular, RET stimulation with glial-cell line derived neurotrophic factor family (GDNF) and glycosyl-phosphatidylinositol membrane anchored co-receptor α1 (GFRα1) trigger the up-modulation of genes encoding either for chemokines (CCL20, CCL2, CCL3, CCL4, CCL7, CXCL1) and cytokines (IL-1β, IL-6 and IL-8) and the down-regulation of chemokine/cytokine receptors (CCR2 and IL8-Rα). Although at different levels, the modulation of these “RET-dependent genes” occurs in both healthy donors and HSCR patients. We also describe another set of genes that, independently from RET stimulation, are differently regulated in healthy donors versus HSCR patients. Among these “RET-independent genes”, there are CSF-1R, IL1-R1, IL1-R2 and TGFβ-1, whose levels of transcripts were lower in HSCR patients compared to healthy donors, thus suggesting aberrancies of inflammatory responses at mucosal level. Overall our results demonstrate that immune system actively participates in the physiopathology of

  17. NOD-scid IL2R γnull mice engrafted with human peripheral blood mononuclear cells as a model to test therapeutics targeting human signaling pathways

    PubMed Central

    2013-01-01

    Background Animal models of human inflammatory diseases have limited predictive quality for human clinical trials for various reasons including species specific activation mechanisms and the immunological background of the animals which markedly differs from the genetically heterogeneous and often aged patient population. Objective Development of an animal model allowing for testing therapeutics targeting pathways involved in the development of Atopic Dermatitis (AD) with better translatability to the patient. Methods NOD-scid IL2R γnull mice engrafted with human peripheral blood mononuclear cells (hPBMC) derived from patients suffering from AD and healthy volunteers were treated with IL-4 and the antagonistic IL-4 variant R121/Y124D (Pitrakinra). Levels of human (h)IgE, amount of B-, T- and plasma- cells and ratio of CD4 : CD8 positive cells served as read out for induction and inhibition of cell proliferation and hIgE secretion. Results were compared to in vitro analysis. Results hIgE secretion was induced by IL-4 and inhibited by the IL-4 antagonist Pitrakinra in vivo when formulated with methylcellulose. B-cells proliferated in response to IL-4 in vivo; the effect was abrogated by Pitrakinra. IL-4 shifted CD4 : CD8 ratios in vitro and in vivo when hPBMC derived from healthy volunteers were used. Pitrakinra reversed the effect. Human PBMC derived from patients with AD remained inert and engrafted mice reflected the individual responses observed in vitro. Conclusion NOD-scid IL2R γnull mice engrafted with human PBMC reflect the immunological history of the donors and provide a complementary tool to in vitro studies. Thus, studies in this model might provide data with better translatability from bench to bedside. PMID:23294516

  18. Quantification of 33 antidepressants by LC-MS/MS--comparative validation in whole blood, plasma, and serum.

    PubMed

    Montenarh, Deborah; Wernet, Mathias P; Hopf, Markus; Maurer, Hans H; Schmidt, Peter H; Ewald, Andreas H

    2014-09-01

    In the present study, a liquid chromatography-mass spectrometry (LC-MS/MS) multi-analyte approach based on a simple liquid-liquid extraction was developed for fast target screening and quantification of 33 antidepressants in whole blood, plasma, and serum. The method was validated with respect to selectivity, matrix effects, recovery, process efficiency, accuracy and precision, stabilities, and limits. In addition, cross-calibration between the three biosamples was done to assess the impact of the different matrices on the calibration. Whole blood, plasma, and serum (500 μL each) were extracted twice at pH 7.4 and at pH 10 with ether-ethyl acetate (1:1). Separation, detection, and quantification were performed using LC-MS/MS with electrospray ionization in positive mode. For accuracy and precision, full calibration was performed with ranges from subtherapeutic to toxic concentrations. The approach was sensitive and selective for 33 analytes in whole blood and 31 analytes in plasma and serum and accurate and precise for 30 of the 33 tested drugs in whole blood, 31 in plasma, and 28 in serum. Cross-calibration was successful only for 13 analytes in whole blood and 16 analytes in serum calculated over a calibration curve made in plasma, 12 analytes in whole blood and 15 analytes in plasma calculated over a calibration curve made in serum, and 10 analytes in plasma and 15 analytes in serum calculated over a calibration curve made in whole blood.

  19. The Impact of Glyphosate, Its Metabolites and Impurities on Viability, ATP Level and Morphological changes in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Kwiatkowska, Marta; Jarosiewicz, Paweł; Michałowicz, Jaromir; Koter-Michalak, Maria; Huras, Bogumiła; Bukowska, Bożena

    2016-01-01

    The toxicity of herbicides to animals and human is an issue of worldwide concern. The present study has been undertaken to assess toxic effect of widely used pesticide—glyphosate, its metabolites: aminomethylphosphonic acid (AMPA) and methylphosphonic acid and its impurities: N-(phosphonomethyl)iminodiacetic acid (PMIDA), N-methylglyphosate, hydroxymethylphosphonic acid and bis-(phosphonomethyl)amine on human peripheral blood mononuclear cells (PBMCs). We have evaluated the effect of those compounds on viability, ATP level, size (FSC-A parameter) and granulation (SSC-A parameter) of the cells studied. Human peripheral blood mononuclear cells were exposed to different concentrations of glyphosate, its metabolites and impurities (0.01–10 mM) for 4 and 24 h. It was found that investigated compounds caused statistically significant decrease in viability and ATP level of PBMCs. The strongest changes in cell viability and ATP level were observed after 24 h incubation of PBMCs with bis-(phosphonomethyl)amine, and particularly PMIDA. Moreover, all studied compounds changed cell granularity, while PMIDA and bis-(phosphonomethyl)amine altered PBMCs size. It may be concluded that bis-(phosphonomethyl)amine, and PMIDA caused a slightly stronger damage to PBMCs than did glyphosate. Changes in the parameters studied in PBMCs were observed only at high concentrations of the compounds examined, which clearly shows that they may occur in this cell type only as a result of acute poisoning of human organism with these substances. PMID:27280764

  20. Ellagic Acid, a Dietary Polyphenol, Inhibits Tautomerase Activity of Human Macrophage Migration Inhibitory Factor and Its Pro-inflammatory Responses in Human Peripheral Blood Mononuclear Cells.

    PubMed

    Sarkar, Souvik; Siddiqui, Asim A; Mazumder, Somnath; De, Rudranil; Saha, Shubhra J; Banerjee, Chinmoy; Iqbal, Mohd S; Adhikari, Susanta; Alam, Athar; Roy, Siddhartha; Bandyopadhyay, Uday

    2015-05-27

    Ellagic acid (EA), a phenolic lactone, inhibited tautomerase activity of human macrophage migration inhibitory factor (MIF) noncompetitively (Ki = 1.97 ± 0.7 μM). The binding of EA to MIF was determined by following the quenching of tryptophan fluorescence. We synthesized several EA derivatives, and their structure-activity relationship studies indicated that the planar conjugated lactone moiety of EA was essential for MIF inhibition. MIF induces nuclear translocation of NF-κB and chemotaxis of peripheral blood mononuclear cells (PBMCs) to promote inflammation. We were interested in evaluating the effect of EA on nuclear translocation of NF-κB and chemotactic activity in human PBMCs in the presence of MIF. The results showed that EA inhibited MIF-induced NF-κB nuclear translocation in PBMCs, as evident from confocal immunofluorescence microscopic data. EA also inhibited MIF-mediated chemotaxis of PBMCs. Thus, we report MIF-inhibitory activity of EA and inhibition of MIF-mediated proinflammatory responses in PBMCs by EA.

  1. Thresholds of whole-blood β-hydroxybutyrate and glucose concentrations measured with an electronic hand-held device to identify ovine hyperketonemia.

    PubMed

    Pichler, M; Damberger, A; Schwendenwein, I; Gasteiner, J; Drillich, M; Iwersen, M

    2014-03-01

    Metabolic disorders, especially hyperketonemia, are very common in dairy sheep. The whole-blood concentrations of β-hydroxybutyrate (BHBA) and glucose can be determined by commercially available electronic hand-held devices, which are used in human medicine and for the detection of ketosis in dairy cows. The aim of this study was to evaluate the suitability of the hand-held device Precision Xceed (PX; Abbott Diabetes Care Inc., Abbott Park, IL) to detect hyperketonemia in ewes. An additional objective of this study was to evaluate the agreement between samples obtained by minimal invasive venipuncture of an ear vein and measurements of whole-blood samples from the jugular vein (vena jugularis, v. jug.). Blood samples taken from the v. jug. were collected from 358 ewes on 4 different farms. These samples and a blood drop obtained from an ear vein were analyzed simultaneously on farm with the PX. For method comparison, the samples obtained from the v. jug. were also analyzed by standard methods, which served as the gold standard at the Central Laboratory of the University of Veterinary Medicine Vienna, Austria. The correlation coefficients between the serum BHBA concentration and the concentrations measured with the hand-held meter in the whole blood from an ear vein and the v. jug. were 0.94 and 0.96, respectively. The correlation coefficients of plasma and whole-blood glucose concentration were 0.68 for the v. jug. and 0.47 for the ear vein. The mean glucose concentration was significantly lower in animals classified as hyperketonemic (BHBA ≥ 1.6 mmol/L) compared with healthy ewes. Whole-blood concentrations of BHBA and glucose measured with the PX from v. jug. showed a constant negative bias of 0.15 mmol/L and 8.4 mg/dL, respectively. Hence, a receiver operating characteristic analysis was performed to determine thresholds for the PX to detect hyperketonemia in ewes. This resulted in thresholds for moderate ketosis of BHBA concentrations of 0.7 mmol/L in blood

  2. Cytogenetic comparison of the responses of mouse and human peripheral blood lymphocytes to /sup 60/Co gamma radiation

    SciTech Connect

    Kligerman, A.D.; Halperin, E.C.; Erexson, G.L.; Honore, G.; Westbrook-Collins, B.; Allen, J.W.

    1988-08-01

    Experiments were conducted to compare the chromosome damaging effects of /sup 60/Co gamma radiation on mouse and human peripheral blood lymphocytes (PBLs). Either whole blood or isolated and pelleted mononuclear leucocytes (MNLs) were irradiated with a /sup 60/Co unit to yield exposures of 1, 2, 3, or 4 Gy. In addition, mice were whole-body irradiated in vivo with the same doses so that an in vitro-in vivo comparison could be made. The results indicate that mouse PBLs irradiated in whole blood, whether in vivo or in vitro, respond similarly to /sup 60/Co gamma rays as measured by dicentric chromosome formation. In addition, mouse and human PBLs showed a similar radiosensitivity, but because the mouse PBL data were best fitted to an exponential function and the human PBL data to a quadratic function, direct comparisons were difficult to make. Pelleted MNLs from mice were much less sensitive to the clastogenic effects of gamma radiation than whole blood. This is believed to be due to hypoxic conditions that developed during irradiation and transport. Human PBLs did not show a marked difference whether irradiated in whole blood or as pelleted MNLs in tissue culture medium.

  3. Whole Blood Donation Affects the Interpretation of Hemoglobin A1c

    PubMed Central

    Lenters-Westra, Erna; de Kort, Wim; Bokhorst, Arlinke G.; Bilo, Henk J. G.; Slingerland, Robbert J.; Vos, Michel J.

    2017-01-01

    Introduction Several factors, including changed dynamics of erythrocyte formation and degradation, can influence the degree of hemoglobin A1c (HbA1c) formation thereby affecting its use in monitoring diabetes. This study determines the influence of whole blood donation on HbA1c in both non-diabetic blood donors and blood donors with type 2 diabetes. Methods In this observational study, 23 non-diabetic blood donors and 21 blood donors with type 2 diabetes donated 475 mL whole blood and were followed prospectively for nine weeks. Each week blood samples were collected and analyzed for changes in HbA1c using three secondary reference measurement procedures. Results Twelve non-diabetic blood donors (52.2%) and 10 (58.8%) blood donors with type 2 diabetes had a significant reduction in HbA1c following blood donation (reduction >-4.28%, P < 0.05). All non-diabetic blood donors with a normal ferritin concentration predonation had a significant reduction in HbA1c. In the non-diabetic group the maximum reduction was -11.9%, in the type 2 diabetes group -12.0%. When eligible to donate again, 52.2% of the non-diabetic blood donors and 41.2% of the blood donors with type 2 diabetes had HbA1c concentrations significantly lower compared to their predonation concentration (reduction >-4.28%, P < 0.05). Conclusion Patients with type 2 diabetes contributing to whole blood donation programs can be at risk of falsely lowered HbA1c. This could lead to a wrong interpretation of their glycemic control by their general practitioner or internist. PMID:28118412

  4. Automated high-throughput purification of genomic DNA from whole blood using Promega's MagneSilTM paramagnetic particles with either the Max Yield or MagneSilTM ONE normalized purification methods

    NASA Astrophysics Data System (ADS)

    Bitner, Rex M.; Koller, Susan C.; Sankbeil, Jacqui

    2003-07-01

    Two different methods of automated high throughput purification of genomic DNA from human whole blood in 96 well plates are described. One method uses MagneSilTM paramagnetic particles to purify a maximal amount of the DNA present in the sample. Another method, the MagnesilTM ONE system, allows for the purification of a predetermined amount of DNA from human whole blood. Protocols for the purification of 100 ng or, alternatively 1 ug, of human genomic DNA from whole blood using MagneSilTM paramagnetic particles and a Beckman BioMekTM FX robot are described. With the maximal yield purification system, typical DNA yields fall in the range of 4-9 ug of DNA from 200ul of human whole blood, depending upon the white cell content of donor sample. For situations where DNA achiving is desired, or when the number of downstream sample applications is not clearly defined (e.g. multiple SNP analyses) the maximal yield method is usually preferred. However, in situations with a defined downstream application (e.g. criminal databasing or use of a defined set of amplifications) where purifying DNA in a narrow concentrate range streamlines the high throughput purification and analysis process, the automated MagneSilTM ONE purification system is the method of choice. DNA from either method is suitable for applications such as PCR, STR, READITTM SNP analysis, and multiplexed PCR systems such as Promega's Y-chromosome deletion detection system.

  5. Specific binding of magnetic nanoparticle probes to platelets in whole blood detected by magnetorelaxometry

    NASA Astrophysics Data System (ADS)

    Eberbeck, Dietmar; Wiekhorst, Frank; Steinhoff, Uwe; Schwarz, Kay Oliver; Kummrow, Andreas; Kammel, Martin; Neukammer, Jörg; Trahms, Lutz

    2009-05-01

    The binding of monoclonal antibodies labelled with magnetic nanoparticles to CD61 surface proteins expressed by platelets in whole blood samples was measured by magnetorelaxometry. This technique is sensitive to immobilization of the magnetic labels upon binding. Control experiments with previous saturation of the epitopes on the platelet surfaces demonstrated the specificity of the binding. The kinetics of the antibody antigen reaction is accessible with a temporal resolution of 12 s. The minimal detectable platelet concentration is about 2000 μL -1 (sample volume 150 μL). The proportionality of the magnetic relaxation amplitude to the number of bound labels allows a quantification of the antibody binding capacity.

  6. Parallel artificial liquid membrane extraction of new psychoactive substances in plasma and whole blood.

    PubMed

    Vårdal, Linda; Askildsen, Hilde-Merete; Gjelstad, Astrid; Øiestad, Elisabeth Leere; Edvardsen, Hilde Marie Erøy; Pedersen-Bjergaard, Stig

    2017-03-24

    Parallel artificial liquid membrane extraction (PALME) was combined with ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and the potential for screening of new psychoactive substances (NPS) was investigated for the first time. PALME was performed in 96-well format comprising a donor plate, a supported liquid membrane (SLM), and an acceptor plate. Uncharged NPS were extracted from plasma or whole blood, across an organic SLM, and into an aqueous acceptor solution, facilitated by a pH gradient. MDAI (5,6-methylenedioxy-2-aminoindane), methylone, PFA (para-fluoroamphetamine), mCPP (meta-chlorophenylpiperazine), pentedrone, methoxetamine, MDPV (methylenedioxypyrovalerone), ethylphenidate, 2C-E (2,5-dimethoxy-4-ethylphenethylamine), bromo-dragonfly, and AH-7921 (3,4-dichloro-N-{[1-(dimethylamino)cyclohexyl]methyl}benzamide) were selected as representative NPS. Optimization of operational parameters was necessary as the NPS were novel to PALME, and because PALME was performed from whole blood for the very first time. In the PALME method developed for plasma, NPS were extracted from a 250μL alkalized donor solution consisting of 125μL plasma sample, 115μL 40mM NaOH, and 10μL internal standard. In the PALME method from whole blood, the 250μL alkalized donor solution consisted of 100μL whole blood, 50μL deionized water, 75μL 80mM NaOH, and 25μL internal standard. In both methods, extraction was accomplished across an SLM of 5μL dodecyl acetate with 1% trioctylamine (w/w), and further into an acidic acceptor solution of 50μL 20mM formic acid. The extraction was promoted by agitation at 900rpm and was carried out for 120min. Method validation was performed and the following parameters were considered: linearity, limits of quantification (LOQ), intra- and inter-day precision, accuracy, extraction recoveries, carry-over, and matrix effects. The validation results were in accordance with FDA guidelines.

  7. Whole blood and apheresis donors in Quebec, Canada: Demographic differences and motivations to donate.

    PubMed

    Charbonneau, Johanne; Cloutier, Marie-Soleil; Carrier, Élianne

    2015-12-01

    This study sought to compare demographics and donation motivations among plasma/platelet donors (PPDs) and whole blood donors (WBDs), in a voluntary and non-remunerated context. Motives to donate blood and demographic characteristics were collected through questionnaires completed by 795 WBDs and 473 PPDs. Comparison of WBDs and PPDs under chi-square tests showed that 17 out of 23 motivators were statistically different according to various demographic variables. These results demonstrate the existence of specific donor profiles both for WBDs and PPDs. Agencies should develop new recruitment strategies tailored to these donors, especially if they wish to convince WBDs to convert to apheresis donation.

  8. Homologous whole blood transfusion during treatment of severe anemia in a chimpanzee (Pan troglodytes).

    PubMed

    Debenham, John James; Atencia, Rebeca

    2014-09-01

    A 12-yr-old female chimpanzee (Pan troglodytes) was presented as severely emaciated and with generalized muscle weakness. Hematology and biochemistry revealed severe anemia and hypokalemia. The chimpanzee was treated supportively and symptomatically; although initially stable, the animal deteriorated rapidly on day 5, becoming depressed and jaundiced with further deterioration of anemia. To address the decline, a prompt transfusion of compatible and cross-matched fresh whole blood from a healthy adult male chimpanzee was administered over 120 min. During transfusion, an immediate reduction in the recipient's tachycardia was noted and substantial clinical improvement continued over 24 hr posttransfusion; no adverse transfusion reactions were observed.

  9. Oxidative stress induces DNA damage and inhibits the repair of DNA lesions induced by N-acetoxy-2-acetylaminofluorene in human peripheral mononuclear leukocytes.

    PubMed

    Pero, R W; Anderson, M W; Doyle, G A; Anna, C H; Romagna, F; Markowitz, M; Bryngelsson, C

    1990-08-01

    Human mononuclear leukocytes were exposed to prooxidants such as H2O2, phorbol-12-myristate-13-acetate, and 4-nitroquinoline-N-oxide, and the effects on induction of DNA damage and repair were evaluated. ADP ribosylation was activated by prooxidant exposure and the response was bimodal with peaks of activation occurring at about 30 min and 4-5 h. Other evidence for prooxidant-induced DNA damage was provided by nucleoid sedimentation assays. Unscheduled DNA synthesis (UDS) was only slightly induced by prooxidant exposure which suggested that either the DNA lesions were repaired by a short patch mechanism involving little UDS, or the repair process was inhibited by prooxidant exposures, or some combination of both. This point was clarified by the fact that the repair of DNA lesions induced by N-acetoxy-2-acetylaminofluorene, an inducer of large patch DNA repair, was inhibited in a dose-dependent manner by exposure to H2O2 and the inhibition was dependent on ADP ribosylation. In contrast, the repair of DNA strand breaks induced by prooxidant exposures as identified above were complete within about 8 h and the repair was independent of ADP ribosylation. Both ADP ribosylation and N-acetoxy-2-acetylaminofluorene-induced UDS were shown to be up- and down-regulated by the redox state of human mononuclear leukocytes indicating a unique mechanism of cellular control over DNA repair.

  10. Microfiltration platform for continuous blood plasma protein extraction from whole blood during cardiac surgery

    PubMed Central

    Aran, Kiana; Fok, Alex; Sasso, Lawrence A.; Kamdar, Neal; Guan, Yulong; Sun, Qi; Ündar, Akif

    2015-01-01

    This report describes the design, fabrication, and testing of a cross-flow filtration microdevice, for the continuous extraction of blood plasma from a circulating whole blood sample in a clinically relevant environment to assist in continuous monitoring of a patient’s inflammatory response during cardiac surgeries involving cardiopulmonary bypass (CPB) procedures (about 400 000 adult and 20 000 pediatric patients in the United States per year). The microfiltration system consists of a two-compartment mass exchanger with two aligned sets of PDMS microchannels, separated by a porous polycarbonate (PCTE) membrane. Using this microdevice, blood plasma has been continuously separated from blood cells in a real-time manner with no evidence of bio-fouling or cell lysis. The technology is designed to continuously extract plasma containing diagnostic plasma proteins such as complements and cytokines using a significantly smaller blood volume as compared to traditional blood collection techniques. The microfiltration device has been tested using a simulated CPB circulation loop primed with donor human blood, in a manner identical to a clinical surgical setup, to collect plasma fractions in order to study the effects of CPB system components and circulation on immune activation during extracorporeal circulatory support. The microdevice, with 200 nm membrane pore size, was connected to a simulated CPB circuit, and was able to continuously extract ~15% pure plasma volume (100% cell-free) with high sampling frequencies which could be analyzed directly following collection with no need to further centrifuge or modify the fraction. Less than 2.5 ml total plasma volume was collected over a 4 h sampling period (less than one Vacutainer blood collection tube volume). The results tracked cytokine concentrations collected from both the reservoir and filtrate samples which were comparable to those from direct blood draws, indicating very high protein recovery of the microdevice

  11. Microfiltration platform for continuous blood plasma protein extraction from whole blood during cardiac surgery.

    PubMed

    Aran, Kiana; Fok, Alex; Sasso, Lawrence A; Kamdar, Neal; Guan, Yulong; Sun, Qi; Ündar, Akif; Zahn, Jeffrey D

    2011-09-07

    This report describes the design, fabrication, and testing of a cross-flow filtration microdevice, for the continuous extraction of blood plasma from a circulating whole blood sample in a clinically relevant environment to assist in continuous monitoring of a patient's inflammatory response during cardiac surgeries involving cardiopulmonary bypass (CPB) procedures (about 400,000 adult and 20,000 pediatric patients in the United States per year). The microfiltration system consists of a two-compartment mass exchanger with two aligned sets of PDMS microchannels, separated by a porous polycarbonate (PCTE) membrane. Using this microdevice, blood plasma has been continuously separated from blood cells in a real-time manner with no evidence of bio-fouling or cell lysis. The technology is designed to continuously extract plasma containing diagnostic plasma proteins such as complements and cytokines using a significantly smaller blood volume as compared to traditional blood collection techniques. The microfiltration device has been tested using a simulated CPB circulation loop primed with donor human blood, in a manner identical to a clinical surgical setup, to collect plasma fractions in order to study the effects of CPB system components and circulation on immune activation during extracorporeal circulatory support. The microdevice, with 200 nm membrane pore size, was connected to a simulated CPB circuit, and was able to continuously extract ~15% pure plasma volume (100% cell-free) with high sampling frequencies which could be analyzed directly following collection with no need to further centrifuge or modify the fraction. Less than 2.5 ml total plasma volume was collected over a 4 h sampling period (less than one Vacutainer blood collection tube volume). The results tracked cytokine concentrations collected from both the reservoir and filtrate samples which were comparable to those from direct blood draws, indicating very high protein recovery of the microdevice

  12. Suppression of immunoglobulin production in human peripheral blood mononuclear cells by monocytes via secretion of heavy-chain ferritin.

    PubMed

    Yamashita, Makiko; Harada, Gakuro; Matsumoto, Shin-ei; Aiba, Yoshihiro; Ichikawa, Akira; Fujiki, Tsukasa; Udono, Miyako; Kabayama, Shigeru; Yoshida, Tadashi; Zhang, Pingbo; Fujii, Hiroshi; Shirahata, Sanetaka; Katakura, Yoshinori

    2014-02-01

    In vitro antigen stimulation of peripheral blood mononuclear cells (PBMCs) does not induce immunoglobulin (Ig) production. However, pretreatment of PBMCs with l-leucyl-l-leucine methyl ester (LLME) prior to in vitro stimulation removes the suppression of Ig production. In the present study, we attempted to identify the target cells of LLME and determine the mechanisms by which Ig production in PBMCs is suppressed. We found that CD14(+) monocytes are involved in the suppression of Ig production in PBMCs. Furthermore, we confirmed that heavy-chain ferritin derived from CD14(+) monocytes suppresses Ig production in PBMCs, possibly through iron sequestration.

  13. [Whole blood viscosity measurement in acute myocardial infarction: feasibility and significance].

    PubMed

    Philippe, F; Lacombe, C; Bucherer, C; Drobinski, G; Montalescot, G; Thomas, D

    2001-10-01

    Thrombolytic agents and new antiplatelet drugs used in acute myocardial infarction (AMI) could change whole blood viscosity. The aim of this pilot trial is to compare blood viscosity at four shear rate levels among three groups of patients: AMI receiving thrombolysis with alteplase (n: 10), AMI eligible for primary angioplasty with abciximab (n: 10), healthy volunteers (n: 10). Viscosity measurement was obtained in 30 minutes with a Couette hemoviscosimeter. At baseline, blood viscosity level was higher in patients with acute coronary syndromes than in healthy volunteers (72 +/- 32 mPa.s versus 51 +/- 13 mPa.s, p<0.05). After thrombolysis, viscosity was higher at 90 minutes than at third day, paradoxically with fibrinogen elevation (72 +/- 32 mPa.s versus 58 +/- 27 mPa.s, p=0.01). In primary angioplasty with abciximab, viscosity decreased significantly (56 +/- 28 mPa.s versus 43 +/- 13 mPa.s, p=0.01). The effects of ionic contrast agent and abciximab are discussed. In healthy volunteers group, 100 mg aspirin once a day during 7 days reduces blood viscosity at high shear stress. The small size of the study population restricts correlation analysis with major clinical adverse events. A larger trial is necessary to evaluate the predictive value of whole blood viscosity in reocclusive and/or hemorrhagic events in those reperfusion strategies but also in case of thrombolytic agent and abciximab combination.

  14. Portable simultaneous multiple analyte whole-blood analyzer for point-of-care testing.

    PubMed

    Schembri, C T; Ostoich, V; Lingane, P J; Burd, T L; Buhl, S N

    1992-09-01

    We describe a portable clinical chemistry analyzer for point-of-care measurements of multiple analytes in less than 10 min from approximately 40 microL of whole blood (fingerstick or venous). Whole blood is applied directly to a 7.9-cm-diameter, single-use plastic rotor containing liquid diluent and greater than or equal to 4-12 tests in the form of 1- to 2-mm-diameter dry reagent beads. The reagent/rotor is immediately placed in a portable instrument along with a ticket/label results card. As the instrument spins the rotor, capillary and rotational forces process the blood into diluted plasma, distribute the patient's diluted sample to cuvettes containing the reagent beads, and mix the diluted sample with the reagents. The instrument monitors the chemical reactions optically at nine wavelengths; sample volume and temperature are also measured optically. The calibration data for each reagent are read from a bar code on the periphery of each rotor. The instrument processes all the measurements to calculate, store, print, and communicate the results. Each reagent/rotor contains an enzymatic control that must be within a defined range before the results from that analysis are reported.

  15. A time-resolved immunofluorometric assay for porcine C-reactive protein quantification in whole blood.

    PubMed

    Martinez-Subiela, S; Eckersall, P D; Campbell, F M; Parra, M D; Fuentes, P; Ceron, J J

    2007-01-01

    A time-resolved immunofluorometric assay (TR-IFMA) for C-reactive protein (CRP) determination in whole blood of pigs was developed and validated. CRP was isolated from porcine acute-phase serum by affinity chromatography on agarose, coupled with phosphorylethanolamine and polyclonal antibodies to porcine CRP were purified from antiserum raised in sheep immunized with porcine CRP. Intra- and inter-assay coefficients of variation (CVs) were in the range 3.13-7.19% and 7.06-15.66%, respectively, showing good precision. The assay measured the CRP values in a proportional and linear manner (r=0.99); additionally, CRP concentrations measured in whole blood by the present TR-IFMA and in serum by an established immunoturbidimetric assay were highly correlated (R(2)=0.97). The limit of detection of the method was 0.0028 mg/L. Significantly lower CRP concentrations were observed after 7 days of sample storage at 4 degrees C. The injection of turpentine oil caused a significant increase in CRP concentrations and significantly higher CRP concentrations were observed in pigs with pathological processes compared to healthy animals.

  16. Major and trace elements in whole blood of phlebotomized patients with porphyria cutanea tarda.

    PubMed

    Dinya, Mariann; Székely, E; Szentmihályi, K; Tasnádi, Gy; Blázovics, A

    2005-01-01

    Porphyria cutanea tarda (PCT) is a disorder of hem biosynthesis resulting from a decreased activity of the uroporphyrinogen decarboxylase enzyme. Hem precursors are accumulated in the blood, liver and skin. Inherited and acquired factors also contribute to the pathogenesis of PCT. Hem precursors and porphyrins are excreted with urine and faeces. Whole blood of 8 PCT patients and 6 volunteers of Caucasian origin were analysed. In addition to routine laboratory measurements, 19 elements (Al, B, Ba, Ca, Cd, Co, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, P, S, V, Zn) were determined by means of inductively coupled plasma optical emission spectrometry (ICP-OES). Mg, P and S concentrations in whole blood were decreased significantly (p<0.05), whereas Ba was increased in PCT patients compared to controls. Metabolic alterations are reflected in the correlation of parameters. Positive correlations were found between the element pairs of Zn-Al, Zn-Mg, Zn-Mn, B-S, Fe-Mg, K-P, Mg-Mn for PCT patients, whereas in the control group Al-Mn, Ca-Cu, Ca-Na, Cu-Mg, Fe-K, Mg-Na, Zn-P showed positive correlations.

  17. Keishibukuryogan, a Traditional Japanese Medicine, Inhibits Platelet Aggregation in Guinea Pig Whole Blood

    PubMed Central

    Terawaki, Kiyoshi; Noguchi, Masamichi; Yuzurihara, Mitsutoshi; Omiya, Yuji; Ikarashi, Yasushi; Kase, Yoshio

    2015-01-01

    Effects of keishibukuryogan (KBG) on platelet aggregation were investigated. To ensure the specificity of KBG, tokishakuyakusan (TSS) and kamisyoyosan (KSS), which are known to have platelet aggregation-inhibiting effects, and rikkunshito (RKT) and shakuyakukanzoto (SKT), which are considered to be devoid of such effects, were used for comparison. The platelet aggregation of each test drug was measured by the screen filtration pressure method using whole blood of guinea pigs and expressed as a collagen-induced pressure rate (%) or a collagen concentration required for 50% increase in the pressure rate (PATI value). KBG suppressed the collagen-induced whole blood pressure rate increase and increased the PATI value, like TSS and KSS. Neither RKT nor SKT showed these effects. The Moutan cortex and Cinnamomi cortex, the constituent crude drugs of KBG, showed KBG-like pressure rate suppression and PATI-increasing effects. Furthermore, paeonol, a representative component of Moutan cortex, and aspirin which is known to have platelet aggregation-inhibiting activity (COX-1 inhibitor) also showed similar effects. These results suggest that the platelet aggregation-inhibiting activity of the constituent crude drugs Moutan cortex and Cinnamomi cortex is involved in the improving effects of KBG on impaired microcirculation and that paeonol plays a role in these effects. PMID:26379740

  18. Determination of buprenorphine, fentanyl and LSD in whole blood by UPLC-MS-MS.

    PubMed

    Berg, Thomas; Jørgenrud, Benedicte; Strand, Dag Helge

    2013-04-01

    A sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method has been developed and validated for the quantification of buprenorphine, fentanyl and lysergic acid diethylamide (LSD) in whole blood. Sample preparation was performed by liquid-liquid extraction (LLE) with methyl tert-butyl ether. UPLC-MS-MS analysis was performed with a mobile phase consisting of ammonium formate (pH 10.2) and methanol. Positive electrospray ionization MS-MS detection was performed with two multiple reaction monitoring transitions for each of the analytes and the deuterium labeled internal standards. Limit of detection values of buprenorphine, fentanyl and LSD were 0.28, 0.044 and 0.0097 ng/mL and limit of quantification values were 0.94, 0.14 and 0.036 ng/mL, respectively. Most phospholipids were removed during LLE. No or only minor matrix effects were observed. The method has been routinely used at the Norwegian Institute of Public Health since September 2011 for qualitative and quantitative detections of buprenorphine, fentanyl and/or LSD in more than 400 whole blood samples with two replicates per sample.

  19. [Comparison of rheologic properties between Ca-alginate hydrogel microspheres suspension and whole blood].

    PubMed

    Xu, Pei; Wang, Xiang; Li, Yaojin; Wang, Feifei; Duan, Ming; Yang, Li

    2013-02-01

    Starting from the form of red blood cells and the hematocrit (Hct, about 45 vol% of whole blood), we tried to prepare a kind of microspheres suspension to imitate non-Newtonian fluid property of whole blood, exploring its potentiality to be applied in blood viscosity quality control substance. In our study, we produced Ca-alginate hydrogel microspheres using emulsion polymerization, then we suspended the microspheres in 0.9 wt% NaCl solution to obtain a kind of liquid sample with the microspheres taking 45% volume. Then we used two types of viscometers to measure and analyse the changes of sample viscosity at different shear rate. We observed the forms of Ca-alginate hydrogel microspheres with microscope, and found them to be relatively complete, and their diameters to be normally distributed. Diameters of about 90% of the microspheres were distributed in a range from 6 to 22 micron. The samples were examined with viscometer FASCO-3010 and LG-R-80c respectively, both of which have shown a shear-thinning effect. After 5-week stability test, the CV of viscosity results corresponding to the two instruments were 7.3% to 13.8% and 8.9% to 14.2%, respectively. Although some differences existed among the results under the same shear rate, the general variation trends of the corresponding results were consistent, so the sample had the potentiality to be widely used in calibrating a different type of blood viscometer.

  20. 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.

  1. Comparison of Proteins in Whole Blood and Dried Blood Spot Samples by LC/MS/MS

    NASA Astrophysics Data System (ADS)

    Chambers, Andrew G.; Percy, Andrew J.; Hardie, Darryl B.; Borchers, Christoph H.

    2013-09-01

    Dried blood spot (DBS) sampling methods are desirable for population-wide biomarker screening programs because of their ease of collection, transportation, and storage. Immunoassays are traditionally used to quantify endogenous proteins in these samples but require a separate assay for each protein. Recently, targeted mass spectrometry (MS) has been proposed for generating highly-multiplexed assays for biomarker proteins in DBS samples. In this work, we report the first comparison of proteins in whole blood and DBS samples using an untargeted MS approach. The average number of proteins identified in undepleted whole blood and DBS samples by liquid chromatography (LC)/MS/MS was 223 and 253, respectively. Protein identification repeatability was between 77 %-92 % within replicates and the majority of these repeated proteins (70 %) were observed in both sample formats. Proteins exclusively identified in the liquid or dried fluid spot format were unbiased based on their molecular weight, isoelectric point, aliphatic index, and grand average hydrophobicity. In addition, we extended this comparison to include proteins in matching plasma and serum samples with their dried fluid spot equivalents, dried plasma spot (DPS), and dried serum spot (DSS). This work begins to define the accessibility of endogenous proteins in dried fluid spot samples for analysis by MS and is useful in evaluating the scope of this new approach.

  2. [Platelet concentrates from whole-blood donations (buffy-coat) or apheresis: which one to use?].

    PubMed

    Lozano, María Luisa; Rivera, José; Vicente, Vicente

    2012-05-05

    Platelet concentrates (PCs) prepared either from whole-blood donations by the buffy-coat method (BC), or by plateletpheresis are indicated to prevent or treat acute hemorrhage secondary to thrombocytopenia, and there is an ongoing debate about which platelet product should be used. Usage of each of these two products is highly heterogeneous among countries and individual institutions, ranging from 10 to 90%, with a 50:50 ratio in Europe. In comparison of pooled platelets prepared by the BC method and apheresis PCs, data suggest similar efficacy of the products. Regarding recipients' adverse reactions, there is no advantage for apheresis concentrates. From the donor's point of view, evidence favours using the abundance of platelets available from whole-blood donation. As residual viral transmission risk continues to fall, the advantage of apheresis products related to the decrease to donor exposure lessens. While the cost-effectiveness of apheresis products is comparable to that of other accepted blood safety interventions, in case of emerging pathogens, probably pathogen inactivation of pooled BC PCs would be a more desirable strategy.

  3. Isolation of plasma from whole blood using planar microfilters for lab-on-a-chip applications.

    PubMed

    Crowley, Timothy A; Pizziconi, Vincent

    2005-09-01

    Researchers are actively developing devices for the microanalysis of complex fluids, such as blood. These devices have the potential to revolutionize biological analysis in a manner parallel to the computer chip by providing very high throughput screening of complex samples and massively parallel bioanalytical capabilities. A necessary step performed in clinical chemistry is the isolation of plasma from whole blood, and effective sample preparation techniques are needed for the development of miniaturized clinical diagnostic devices. This study demonstrates the use of passive, operating entirely on capillary action, transverse-flow microfilter devices for the microfluidic isolation of plasma from whole blood. Using these planar microfilters, blood can be controllably fractionated with minimal cell lysis. A characterization of the device performance reveals that plasma filter flux is dependent upon the wall shear rate of blood in the filtration channel, and this result is consistent with macroscale blood filtration using microporous membranes. Also, an innovative microfluidic layout is demonstrated that extends device operation time via capillary action from seconds to minutes. Efficiency of these microfilters is approximately three times higher than the separation efficiencies predicted for microporous membranes under similar conditions. As such, the application of the microscale blood filtration designs used in this study may have broad implications in the design of lab-on-a-chip devices, as well as the field of separation science.

  4. Determination of cotinine in pericardial fluid and whole blood by liquid chromatography-tandem mass spectrometry.

    PubMed

    Hegstad, S; Stray-Pedersen, A; Olsen, L; Vege, A; Rognum, T O; Mørland, J; Christophersen, A S

    2009-05-01

    Cotinine is the main metabolite of nicotine and is used as an indicator of exposure to tobacco smoke. A method has been developed for quantification of cotinine in pericardial fluid and whole blood collected from autopsy casework involving cases of infant death. Sample clean-up was achieved by solid-phase extraction with a mixed-mode column. Cotinine was quantified by liquid chromatography-tandem mass spectrometry. Positive ionization was performed in the multiple reaction monitoring mode. Two transitions were monitored for the analyte and one for the internal standard, cotinine-d(3). The calibration range was 0.9-176 ng/mL for cotinine in both matrixes. The recovery of the analyte ranged from 86 to 92%, and the between-assay precisions ranged from 4 to 6% relative standard deviation. Whole blood and pericardial fluid samples from 95 infant deaths obtained during autopsy were analyzed. A strong correlation (R(2) = 0.97) was found between the cotinine concentrations in pericardial fluid and blood. The correlation was not affected by the postmortem time interval. This study demonstrates that pericardial fluid may be an alternative specimen to blood for quantification of cotinine in forensic autopsies.

  5. Whole Blood Gene Expression Profiles to Assess Pathogenesis and Disease Severity in Infants with Respiratory Syncytial Virus Infection

    PubMed Central

    Mejias, Asuncion; Dimo, Blerta; Suarez, Nicolas M.; Garcia, Carla; Suarez-Arrabal, M. Carmen; Jartti, Tuomas; Blankenship, Derek; Jordan-Villegas, Alejandro; Ardura, Monica I.; Xu, Zhaohui; Banchereau, Jacques; Chaussabel, Damien; Ramilo, Octavio

    2013-01-01

    Background Respiratory syncytial virus (RSV) is the leading cause of viral lower respiratory tract infection (LRTI) and hospitalization in infants. Mostly because of the incomplete understanding of the disease pathogenesis, there is no licensed vaccine, and treatment remains symptomatic. We analyzed whole blood transcriptional profiles to characterize the global host immune response to acute RSV LRTI in infants, to characterize its specificity compared with influenza and human rhinovirus (HRV) LRTI, and to identify biomarkers that can objectively assess RSV disease severity. Methods and Findings This was a prospective observational study over six respiratory seasons including a cohort of infants hospitalized with RSV (n = 135), HRV (n = 30), and influenza (n = 16) LRTI, and healthy age- and sex-matched controls (n = 39). A specific RSV transcriptional profile was identified in whole blood (training cohort, n = 45 infants; Dallas, Texas, US) and validated in three different cohorts (test cohort, n = 46, Dallas, Texas, US; validation cohort A, n = 16, Turku, Finland; validation cohort B, n = 28, Columbus, Ohio, US) with high sensitivity (94% [95% CI 87%–98%]) and specificity (98% [95% CI 88%–99%]). It classified infants with RSV LRTI versus HRV or influenza LRTI with 95% accuracy. The immune dysregulation induced by RSV (overexpression of neutrophil, inflammation, and interferon genes, and suppression of T and B cell genes) persisted beyond the acute disease, and immune dysregulation was greatly impaired in younger infants (<6 mo). We identified a genomic score that significantly correlated with outcomes of care including a clinical disease severity score and, more importantly, length of hospitalization and duration of supplemental O2. Conclusions Blood RNA profiles of infants with RSV LRTI allow specific diagnosis, better understanding of disease pathogenesis, and assessment of disease severity. This study opens new avenues for

  6. Use of a whole blood competitive immunoassay for the assessment of worker exposures to propylene oxide at three manufacturing facilities.

    PubMed

    Jones, Alan L; Van der Woord, Miriam; Bourrillon, François

    2005-04-01

    The level of N-(2-hydroxypropyl)valine adducts in haemoglobin has been shown to correlate well with workplace exposure to propylene oxide (PO). However, the analytical method, using the modified Edman degradation procedure, is prohibitively time-consuming and expensive for use as a routine workplace exposure measurement tool. As an alternative, AB Biomonitoring Ltd of Cardiff, Wales, developed a competitive immunoassay for the determination of N-(2-hydroxypropyl)valine adducts in human haemoglobin. Studies showed that whole blood samples analysed using an enzyme linked immunosorbent assay (ELISA) and the modified Edman degradation procedure over the concentration range 3.7-992 nmol N-(2-hydroxypropyl)valine g(-1) haemoglobin are in good agreement (correlation coefficient 0.998, n = 10). The intervariance and intravariance data indicate the repeatability of the ELISA method over the assay conditions employed and show that it is robust over its working range [2-200 pmol N-(2-hydroxypropyl)valine g(-1) haemoglobin]. The assay employs a whole blood matrix and has a working range of 2-6000 pmol g(-1) Hb (equivalent to up to 5 ppm PO exposure, 8 h per day, 5 days per week, over 4 months). The practicality of the assay was tested by assessing exposures to PO at three world-scale manufacturing sites in France and The Netherlands. Over 800 samples were taken over a 2 year period from operators, maintenance fitters and office staff. The data, typically <50 pmol g(-1) globin, indicate that exposures were significantly <0.1 ppm at all times (The Dutch occupational exposure limit is 2.5 ppm over 8 h). Samples were taken after a major turnaround and also before and after the start-up of a newly commissioned plant. All data indicated that high levels of control were effective in minimizing exposure. This study has shown that the immunoassay is a powerful tool for the exposure component of future epidemiology studies, as well as a definitive demonstration of the effectiveness of

  7. Ochratoxin A inhibits the production of tissue factor and plasminogen activator inhibitor-2 by human blood mononuclear cells: Another potential mechanism of immune-suppression

    SciTech Connect

    Rossiello, Maria R.; Rotunno, Crescenzia; Coluccia, Addolorata; Carratu, Maria R.; Di Santo, Angelomaria; Evangelista, Virgilio; Semeraro, Nicola; Colucci, Mario

    2008-06-01

    The mycotoxin ochratoxin A (OTA), an ubiquitous contaminant of food products endowed with a wide spectrum of toxicity, affects several functions of mononuclear leukocytes. Monocytes/macrophages play a major role in fibrin accumulation associated with immune-inflammatory processes through the production of tissue factor (TF) and plasminogen activator inhibitor 2 (PAI-2). We studied the effect of OTA on TF and PAI-2 production by human blood mononuclear cells (MNC). The cells were incubated for 3 or 18 h at 37 deg. C with non toxic OTA concentrations in the absence and in the presence of lipopolysaccharide (LPS) or other inflammatory agents. TF activity was measured by a one-stage clotting test. Antigen assays were performed by specific ELISAs in cell extracts or conditioned media and specific mRNAs were assessed by RT-PCR. OTA had no direct effect on TF and PAI-2 production by MNC. However, OTA caused a dose-dependent reduction in LPS-induced TF (activity, antigen and mRNA) and PAI-2 (antigen and mRNA) production with > 85% inhibition at 1 {mu}g/ml. Similar results were obtained when monocyte-enriched preparations were used instead of MNC. TF production was also impaired by OTA (1 {mu}g/ml) when MNC were stimulated with phorbol myristate acetate (98% inhibition), IL-1{beta} (83%) or TNF-{alpha} (62%). The inhibition of TF and PAI-2 induction might represent a hitherto unrecognized mechanism whereby OTA exerts immunosuppressant activity.

  8. Distinct Epigenetic Effects of Tobacco Smoking in Whole Blood and among Leukocyte Subtypes

    PubMed Central

    Porter, Devin K.; Pittman, Gary S.; Bennett, Brian D.; Wan, Ma; Crowl, Christopher L.; Adamski, Kelly N.; Huang, Zhiqing; Murphy, Susan K.; Bell, Douglas A.

    2016-01-01

    Tobacco smoke exposure dramatically alters DNA methylation in blood cells and may mediate smoking-associated complex diseases through effects on immune cell function. However, knowledge of smoking effects in specific leukocyte subtypes is limited. To better characterize smoking–associated methylation changes in whole blood and leukocyte subtypes, we used Illumina 450K arrays and Reduced Representation Bisulfite Sequencing (RRBS) to assess genome-wide DNA methylation. Differential methylation analysis in whole blood DNA from 172 smokers and 81 nonsmokers revealed 738 CpGs, including 616 previously unreported CpGs, genome-wide significantly associated with current smoking (p <1.2x10-7, Bonferroni correction). Several CpGs (MTSS1, NKX6-2, BTG2) were associated with smoking duration among heavy smokers (>22 cigarettes/day, n = 86) which might relate to long-term heavy-smoking pathology. In purified leukocyte subtypes from an independent group of 20 smokers and 14 nonsmokers we further examined methylation and gene expression for selected genes among CD14+ monocytes, CD15+ granulocytes, CD19+ B cells, and CD2+ T cells. In 10 smokers and 10 nonsmokers we used RRBS to fine map differential methylation in CD4+ T cells, CD8+ T cells, CD14+, CD15+, CD19+, and CD56+ natural killer cells. Distinct cell-type differences in smoking-associated methylation and gene expression were identified. AHRR (cg05575921), ALPPL2 (cg21566642), GFI1 (cg09935388), IER3 (cg06126421) and F2RL3 (cg03636183) showed a distinct pattern of significant smoking-associated methylation differences across cell types: granulocytes> monocytes>> B cells. In contrast GPR15 (cg19859270) was highly significant in T and B cells and ITGAL (cg09099830) significant only in T cells. Numerous other CpGs displayed distinctive cell-type responses to tobacco smoke exposure that were not apparent in whole blood DNA. Assessing the overlap between these CpG sites and differential methylated regions (DMRs) with RRBS in 6

  9. The effectiveness of cooling conditions on temperature of canine EDTA whole blood samples

    PubMed Central

    Sun, Xiaocun; Flatland, Bente

    2016-01-01

    Background Preanalytic factors such as time and temperature can have significant effects on laboratory test results. For example, ammonium concentration will increase 31% in blood samples stored at room temperature for 30 min before centrifugation. To reduce preanalytic error, blood samples may be placed in precooled tubes and chilled on ice or in ice water baths; however, the effectiveness of these modalities in cooling blood samples has not been formally evaluated. The purpose of this study was to evaluate the effectiveness of various cooling modalities on reducing temperature of EDTA whole blood samples. Methods Pooled samples of canine EDTA whole blood were divided into two aliquots. Saline was added to one aliquot to produce a packed cell volume (PCV) of 40% and to the second aliquot to produce a PCV of 20% (simulated anemia). Thirty samples from each aliquot were warmed to 37.7 °C and cooled in 2 ml allotments under one of three conditions: in ice, in ice after transfer to a precooled tube, or in an ice water bath. Temperature of each sample was recorded at one minute intervals for 15 min. Results Within treatment conditions, sample PCV had no significant effect on cooling. Cooling in ice water was significantly faster than cooling in ice only or transferring the sample to a precooled tube and cooling it on ice. Mean temperature of samples cooled in ice water was significantly lower at 15 min than mean temperatures of those cooled in ice, whether or not the tube was precooled. By 4 min, samples cooled in an ice water bath had reached mean temperatures less than 4 °C (refrigeration temperature), while samples cooled in other conditions remained above 4.0 °C for at least 11 min. For samples with a PCV of 40%, precooling the tube had no significant effect on rate of cooling on ice. For samples with a PCV of 20%, transfer to a precooled tube resulted in a significantly faster rate of cooling than direct placement of the warmed tube onto ice. Discussion Canine

  10. Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights

    SciTech Connect

    Katika, Madhumohan R.; Hendriksen, Peter J.M.; Shao, Jia; Loveren, Henk van; Peijnenburg, Ad

    2012-10-01

    Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examined gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5 μM DON for 3, 6 and 24 h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4 μM DON for 6 and 24 h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs. -- Highlights: ► The human T cell line Jurkat and human

  11. Immunomodulatory properties of human serum immunoglobulin A: anti-inflammatory and pro-inflammatory activities in human monocytes and peripheral blood mononuclear cells

    PubMed Central

    Olas, K; Butterweck, H; Teschner, W; Schwarz, H P; Reipert, B M

    2005-01-01

    Our study investigated the immunomodulatory activities of human plasma-derived serum immunoglobulin (Ig)A. Previous findings seem contradictory indicating either pro- or anti-inflammatory activities. We used serum IgA purified from large plasma pools and studied the modulation of the release of cytokines and chemokines from resting and lipopolysaccharide (LPS, endotoxin)-stimulated human adherent monocytes and human peripheral blood mononuclear cells (PBMC). Our results indicate that IgA down-modulates the release of the pro-inflammatory chemokines monocyte chemoattractant protein (MCP) 1, macrophage inflammatory protein (MIP) 1α and MIP1β from LPS-stimulated PBMC and the release of MCP1, MIP1α and MIP1β from LPS-stimulated monocytes. Furthermore, we confirmed previous reports that plasma-derived serum IgA down-modulates the release of the pro-inflammatory cytokines, interleukin (IL)-6 and tumour necrosis factor (TNF)-α, from LPS-stimulated monocytes and PBMC, and up-regulates the release of IL-1 receptor antagonist (IL-1RA) from resting and LPS-stimulated monocytes and resting PBMC. This IgA-mediated up-regulation of IL-1RA is independent of the simultaneous up-regulation of IL-1β release, as shown by blocking the biological activity of IL-1β with a neutralizing antibody. On the other hand, we also found an IgA-induced pro-inflammatory activity, namely IgA-mediated up-regutation of the release of pro-inflammatory IL-1β as well as down-regulation of the anti-inflammatory cytokines IL-10 and IL-12p40 from LPS-stimulated monocytes and PBMC and a down-regulation of transforming growth factor (TGF)-β from resting and LPS-stimulated PBMC. We conclude that human serum IgA has both an anti-inflammatory and a pro-inflammatory capacity and this dual capacity might contribute to the feedback mechanisms maintaining a balance between pro-inflammatory and anti-inflammatory activities. PMID:15932509

  12. Effects of active bufadienolide compounds on human cancer cells and CD4+CD25+Foxp3+ regulatory T cells in mitogen-activated human peripheral blood mononuclear cells.

    PubMed

    Yuan, Bo; He, Jing; Kisoh, Keishi; Hayashi, Hideki; Tanaka, Sachiko; Si, Nan; Zhao, Hai-Yu; Hirano, Toshihiko; Bian, Baolin; Takagi, Norio

    2016-09-01

    The growth inhibitory effects of bufadienolide compounds were investigated in two intractable cancer cells, a human glioblastoma cell line U-87 and a pancreatic cancer cell line SW1990. Among four bufadienolide compounds, a dose-dependent cytotoxicity was observed in these cancer cells after treatment with gamabufotalin and arenobufagin. The IC50 values of the two compounds were 3-5 times higher in normal peripheral blood mononuclear cells (PBMCs) than these values for both cancer cell lines. However, similar phenomena were not observed for two other bufadienolide compounds, telocinobufagin and bufalin. These results thus suggest that gamabufotalin and arenobufagin possess selective cytotoxic activity against tumor cells rather than normal cells. Moreover, a clear dose-dependent lactate dehydrogenase (LDH) release, a well-known hallmark of necrosis, was observed in both cancer cells treated with gamabufotalin, suggesting that gamabufotalin-mediated cell death is predominantly associated with a necrosis-like phenotype. Of most importance, treatment with as little as 8 ng/ml of gamabufotalin, even an almost non-toxic concentration to PBMCs, efficiently downregulated the percentages of CD4+CD25+Foxp3+ regulator T (Treg) cells in mitogen-activated PBMCs. Given that Treg cells play a critical role in tumor immunotolerance by suppressing antitumor immunity, these results suggest that gamabufotalin may serve as a promising candidate, as an adjuvant therapeutic agent by manipulating Treg cells to enhance the efficacy of conventional anticancer drugs and lessen their side-effects. These findings provide insights into the clinical application of gamabufotalin for cancer patients with glioblastoma/pancreatic cancer based on its cytocidal effect against tumor cells as well as its depletion of Treg cells.

  13. Methylome-wide association study of whole blood DNA in the Norfolk Island isolate identifies robust loci associated with age.

    PubMed

    Benton, Miles C; Sutherland, Heidi G; Macartney-Coxson, Donia; Haupt, Larisa M; Lea, Rodney A; Griffiths, Lyn R

    2017-02-28

    Epigenetic regulation of various genomic functions, including gene expression, provide mechanisms whereby an organism can dynamically respond to changes in its environment and modify gene expression accordingly. One epigenetic mechanism implicated in human aging and age-related disorders is DNA methylation. Isolated populations such as Norfolk Island (NI) should be advantageous for the identification of epigenetic factors related to aging due to reduced genetic and environmental variation. Here we conducted a methylome-wide association study of age using whole blood DNA in 24 healthy female individuals from the NI genetic isolate (aged 24-47 years). We analysed 450K methylation array data using a machine learning approach (GLMnet) to identify age-associated CpGs. We identified 497 CpG sites, mapping to 422 genes, associated with age, with 11 sites previously associated with age. The strongest associations identified were for a single CpG site in MYOF and an extended region within the promoter of DDO. These hits were validated in curated public data from 2316 blood samples (MARMAL-AID). This study is the first to report robust age associations for MYOF and DDO, both of which have plausible functional roles in aging. This study also illustrates the value of genetic isolates to reveal new associations with epigenome-level data.

  14. Quantitation of tacrolimus in whole blood using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS).

    PubMed

    Donaldson, Keri J; Shaw, Leslie M

    2010-01-01

    We describe a multiple reaction monitoring positive ion HPLC/tandem mass spectrometric method for quantification of tacrolimus in human whole blood with online extraction and cleanup. Included in this procedure: API 2000 triple quadrupole mass spectrometer with turbo-ion spray source (Applied Biosystems, Foster City, CA); 10-port diverter/switching valve (Valco, Houston, TX); HPLC system (Agilent Technologies series 1100, Wilmington, DE); 10 mm (C(18)) guard cartridge (Perkin Elmer, Norwalk, CT) used as an extraction column; a Nova-Pak C18 analytical column (2.1 x 150 mm I.D., 4 microm, Waters Corp, Milford, MA); washing solution, methanol: 30 mM ammonium acetate pH 5.1 (80:20); eluting solution, methanol:30 mM ammonium acetate pH 5.1 (97:3); flow rate 0.8 mL/min; and a run-time of 2.8 min. The first and third quadrupoles were set to detect the ammonium adduct ion and a high mass fragment of tacrolimus (m/z 821.5-->768.3), and of an internal standard (ascomycin) (m/z 901.8-->834.4). The lower limit of quantification of this method is 3.75 mg/L. The concentration of drug is determined by comparing peak-area ratios for tacrolimus and internal standard to a standard curve constructed using non-weighted linear through zero regression.

  15. The in vitro effects of artificial and natural sweeteners on the immune system using whole blood culture assays.

    PubMed

    Rahiman, F; Pool, E J

    2014-01-01

    This article investigates the effects of commercially available artificial (aspartame, saccharin, sucralose) and natural sweeteners (brown sugar, white sugar, molasses) on the immune system. Human whole blood cultures were incubated with various sweeteners and stimulated in vitro with either phytohemagglutinin or endotoxin. Harvested supernatants were screened for cytotoxicity and cytokine release. Results showed that none of the artificial or natural sweeteners proved to be cytotoxic, indicating that no cell death was induced in vitro. The natural sweetener, sugar cane molasses (10 ug/mL), enhanced levels of the inflammatory biomarker IL-6 while all artificial sweeteners (10 ug/mL) revealed a suppressive effect on IL-6 secretion (P < 0.001). Exposure of blood cells to sucralose-containing sweeteners under stimulatory conditions reduced levels of the biomarker of humoral immunity, Interleukin-10 (P < 0.001). The cumulative suppression of Interleukin-6 and Interleukin-10 levels induced by sucralose may contribute to the inability in mounting an effective humoral response when posed with an exogenous threat.

  16. Analysis of Valproic Acid, Salicylic Acid and Ibuprofen in Whole Blood by GC-MS.

    PubMed

    Stephenson, Jon B; Flater, Melanie L; Bain, Lisa T

    2016-10-01

    The Georgia Bureau of Investigation utilized a silylation method of analysis for low molecular weight carboxylic acids in the past. Due to the negative impact such derivatizations can have on gas chromatography-mass spectrometry (GC-MS) systems an alternative means of analysis was investigated. The described method is a whole blood solid phase extraction of valproic acid, salicylic acid and ibuprofen utilizing butylation for sensitivity and improved chromatography by GC-MS. The method produced a limit of detection and limit of quantitation at 1 mg/L for valproic acid, 2 mg/L for salicylic acid and 0.25 mg/L for ibuprofen. The variability based upon the middle of the calibration curve estimated to be 7% for valproic acid, 8% for salicylic acid and 11% for ibuprofen established upon a 95% confidence interval, with the highest percent coefficient of variation being 5.3% for ibuprofen.

  17. Evaluation of a simple Theileria annulata culture protocol from experimentally infected bovine whole blood.

    PubMed

    Gharbi, M; Latrach, R; Sassi, L; Darghouth, M A

    2012-08-01

    We have evaluated a new simple technique using whole blood from experimentally infected cattle for the isolation and cultivation of Theileria annulata. The study was carried out on 20 Holstein-Frisian bovines that had been experimentally infected with a virulent lethal dose of Theileria annulata. This technique has been compared to the classical peripheral blood monocyte isolation with Ficoll carried out on 22 experimentally infected Holstein-Friesian calves. The effectiveness of the reference technique was estimated to 86.4%, whilst the effectiveness of the new technique was 100%. Moreover, this new technique leads to time and money saving estimated to € 3.06 per sample. It decreases the contamination risks by reducing the steps of sample manipulation.

  18. Trace element levels in whole blood samples from residents of the city Badajoz, Spain.

    PubMed

    Moreno, M A; Marin, C; Vinagre, F; Ostapczuk, P

    1999-05-19

    Copper, lead, cadmium, and zinc were determined by anodic stripping voltammetry after sample digestion and potentiometric stripping analysis was used for Pb and Cd determination in original samples. Selenium was determined by cathodic stripping voltammetry or hydride generation AAS. Element levels found in the whole blood sample in a group of 82 people are for Cd: 0.98 +/- 0.94 ng/ml; for Pb: 46.7 +/- 28.6 ng/ml; for Cu: 1.07 +/- 0.12 micrograms/ml; for Zn: 6.95 +/- 1.08 micrograms/ml, and for Se: 116 +/- 25 ng/ml. Analytical data have been correlated to age, sex, smokers or non-smokers, drinking and food habits.

  19. Psychoneuroimmunology and natural killer cells: the chromium release whole blood assay.

    PubMed

    Fletcher, Mary Ann; Barnes, Zachary; Broderick, Gordon; Klimas, Nancy G

    2012-01-01

    Natural killer (NK) cells are an essential component of innate immunity. These lymphocytes are also sensitive barometers of the effects of endogenous and exogenous stressors on the immune system. This chapter will describe a chromium ((51)Cr) release bioassay designed to measure the target cell killing capacity of NK cells (NKCC). Key features of the cytotoxicity assay are that it is done with whole blood and that numbers of effector cells are determined for each sample by flow cytometry and lymphocyte count. Effector cells are defined as CD3-CD56+ lymphocytes. Target cells are the K562 eyrthroleukemia cell line. Killing capacity is defined as number of target cells killed per effector cell, at an effector cell/target cell ratio of 1:1 during a 4 h in vitro assay.

  20. Standardized Whole-Blood Transcriptional Profiling Enables the Deconvolution of Complex Induced Immune Responses.

    PubMed

    Urrutia, Alejandra; Duffy, Darragh; Rouilly, Vincent; Posseme, Céline; Djebali, Raouf; Illanes, Gabriel; Libri, Valentina; Albaud, Benoit; Gentien, David; Piasecka, Barbara; Hasan, Milena; Fontes, Magnus; Quintana-Murci, Lluis; Albert, Matthew L

    2016-09-06

    Systems approaches for the study of immune signaling pathways have been traditionally based on purified cells or cultured lines. However, in vivo responses involve the coordinated action of multiple cell types, which interact to establish an inflammatory microenvironment. We employed standardized whole-blood stimulation systems to test the hypothesis that responses to Toll-like receptor ligands or whole microbes can be defined by the transcriptional signatures of key cytokines. We found 44 genes, identified using Support Vector Machine learning, that captured the diversity of complex innate immune responses with improved segregation between distinct stimuli. Furthermore, we used donor variability to identify shared inter-cellular pathways and trace cytokine loops involved in gene expression. This provides strategies for dimension reduction of large datasets and deconvolution of innate immune responses applicable for characterizing immunomodulatory molecules. Moreover, we provide an interactive R-Shiny application with healthy donor reference values for induced inflammatory genes.

  1. Dry film preparation from whole blood, plasma and serum for quantitative infrared diffuse reflectance spectroscopy

    NASA Astrophysics Data System (ADS)

    Bittner, A.; Heise, H. M.

    1998-06-01

    The potential of infrared spectroscopy in the analysis of biotic fluids for the determination of important clinical parameters such as glucose and other blood substrates has been investigated. For this purpose dried films from whole blood, blood plasma and serum were prepared on diffusely reflecting gold-coated substrates from sandpaper of different grades. This enabled measurements in the mid and near infrared spectral ranges by using special diffuse reflectance accessories. The removal of water leads to a considerable enrichment of the fluid constituents. Due to the reduced sample complexity a considerable gain in spectral information is obtained. This is especially valid for measurements in the near infrared where the problems associated with variability in the spectra of aqueous samples due to several parameters, i.e., temperature, electrolyte content etc., are well known. Additionally, mid infrared studies were carried out into the stability of dried samples.

  2. Pretreatment of whole blood using hydrogen peroxide and UV irradiation. Design of the advanced oxidation process.

    PubMed

    Bragg, Stefanie A; Armstrong, Kristie C; Xue, Zi-Ling

    2012-08-15

    A new process to pretreat blood samples has been developed. This process combines the Advanced Oxidation Process (AOP) treatment (using H(2)O(2) and UV irradiation) with acid deactivation of the enzyme catalase in blood. A four-cell reactor has been designed and built in house. The effect of pH on the AOP process has been investigated. The kinetics of the pretreatment process shows that at high C(H(2)O(2),t=0), the reaction is zeroth order with respect to C(H(2)O(2)) and first order with respect to C(blood). The rate limiting process is photon flux from the UV lamp. Degradation of whole blood has been compared with that of pure hemoglobin samples. The AOP pretreatment of the blood samples has led to the subsequent determination of chromium and zinc concentrations in the samples using electrochemical methods.

  3. Micro-elastometry on whole blood clots using actuated surface-attached posts (ASAPs)

    PubMed Central

    Judith, Robert M.; Fisher, Jay K.; Spero, Richard Chasen; Fiser, Briana L.; Turner, Adam; Oberhardt, Bruce; Taylor, R.M.; Falvo, Michael R.; Superfine, Richard

    2015-01-01

    We present a novel technology for microfluidic elastometry and demonstrate its ability to measure stiffness of blood clots as they form. A disposable micro-capillary strip draws small volumes (20 μL) of whole blood into a chamber containing a surface-mounted micropost array. The posts are magnetically actuated, thereby applying a shear stress to the blood clot. The posts’ response to magnetic field changes as the blood clot forms; this response is measured by optical transmission. We show that a quasi-static model correctly predicts the torque applied to the microposts. We experimentally validate the ability of the system to measure clot stiffness by correlating our system with a commercial thromboelastograph. We conclude that actuated surface-attached post (ASAP) technology addresses a clinical need for point-of-care and small-volume elastic haemostatic assays. PMID:25592158

  4. Rapid Detection of Thrombin and Other Protease Activity Directly in Whole Blood

    NASA Astrophysics Data System (ADS)

    Yu, Johnson Chung Sing

    Thrombin is a serine protease that plays a key role in the clotting cascade to promote hemostasis following injury to the endothelium. From a clinical diagnostic perspective, in-vivo thrombin activity is linked to various blood clotting disorders, as well as cardiovascular disease (DVT, arteriosclerosis, etc). Thus, the ability to rapidly measure protease activity directly in whole blood will provide important new diagnostics, and clinical researchers with a powerful tool to further elucidate the relationship between circulating protease levels and disease. The ultimate goal is to design novel point of care (POC) diagnostic devices that are capable of monitoring protease activities directly in whole blood and biological sample. A charge-changing substrate specific to the thrombin enzyme was engineered and its functionality was confirmed by a series of experiments. This led to the preliminary design, construction, and testing of two device platforms deemed fully functional for the electrophoretic separation and focusing of charged peptide fragments. The concept of using the existing charge-changing substrate platform for bacterial protease detection was also investigated. Certain strains of E coli are associated with severe symptoms such as abdominal cramps, bloody diarrhea, and vomiting. The OmpT protease is expressed on the outer membrane of E coli and plays a role in the cleavage of antimicrobial peptides, the degradation of recombinant heterologous proteins, and the activation of plasminogen in the host. Thus, a synthetic peptide substrate specific to the OmpT protease was designed and modeled for the purpose of detecting E coli in biological sample.

  5. Whole blood gene expression profiles distinguish clinical phenotypes of venous thromboembolism☆

    PubMed Central

    Lewis, Deborah A.; Suchindran, Sunil; Beckman, Michele G.; Hooper, W. Craig; Grant, Althea M.; Heit, John A.; Manco-Johnson, Marilyn; Moll, Stephan; Philipp, Claire S.; Kenney, Kristy; De Staercke, Christine; Pyle, Meredith E.; Chi, Jen-Tsan; Ortel, Thomas L.

    2015-01-01

    Introduction Recurrent venous thromboembolism (VTE) occurs infrequently following a provoked event but occurs in up to 30% of individuals following an initial unprovoked event. There is limited understanding of the biological mechanisms that predispose patients to recurrent VTE. Objectives To identify whole blood gene expression profiles that distinguished patients with clinically distinct patterns of VTE. Patients/Methods We studied 107 patients with VTE separated into 3 groups: (1) ‘low-risk’ patients had one or more provoked VTE; (2) ‘moderate-risk’ patients had a single unprovoked VTE; (3) ‘high-risk’ patients had ≥2 unprovoked VTE. Each patient group was also compared to twenty-five individuals with no personal history of VTE. Total RNA from whole blood was isolated and hybridized to Illumina HT-12 V4 Beadchips to assay whole genome expression. Results Using class prediction analysis, we distinguished high-risk patients from low-risk patients and healthy controls with good receiver operating curve characteristics (AUC = 0.81 and 0.84, respectively). We also distinguished moderate-risk individuals and low-risk individuals from healthy controls with AUC’s of 0.69 and 0.80, respectively. Using differential expression analysis, we identified several genes previously implicated in thrombotic disorders by genetic analyses, including SELP, KLKB1, ANXA5, and CD46. Protein levels for several of the identified genes were not significantly different between the different groups. Conclusion Gene expression profiles are capable of distinguishing patients with different clinical presentations of VTE, and genes relevant to VTE risk are frequently differentially expressed in these comparisons. PMID:25684211

  6. Quantitation of opioids in whole blood by electron impact-gas chromatography-mass spectrometry.

    PubMed

    Tiscione, Nicholas B; Shan, Xiaoqin; Alford, Ilene; Yeatman, Dustin Tate

    2011-03-01

    Opioids are frequently encountered in Forensic Toxicology casework. A PubMed literature search was conducted to find a method using electron impact-gas chromatography-mass spectrometry to examine whole blood specimens. A previously published method was identified, and an updated version was provided by the State of North Carolina Office of the Chief Medical Examiner. This procedure was used as a starting point for development and validation of a refined procedure to be used in the Palm Beach County Sheriff's Office Forensic Toxicology laboratory for routine analysis of antemortem forensic toxicology case samples. Materials and instrumentation common to most forensic toxicology laboratories were utilized while obtaining detection limits from 1 to 10 ng/mL and quantitation limits of 2.5 to 10 ng/mL using 1 mL of whole blood. Target compounds were chosen based on applicability to the method as well as availability and common use in the United States and include dihydrocodeine, codeine, morphine, hydrocodone, 6-monoacetylmorphine, hydromorphone, oxycodone, and oxymorphone. Each analyte demonstrated two zero-order linear ranges (r(2) > 0.990) over the concentrations evaluated (from 2.5 to 500 ng/mL). The coefficient of variation of replicate analyses was less than 12%. Quantitative accuracy was within ± 27% at 2.5 ng/mL, ± 11% at 10 ng/mL, and ± 8% at 50 ng/mL. The validated method provides a more sensitive procedure for the quantitation of common opioids in blood using standard laboratory equipment and a small amount of sample.

  7. Long-Term Blood Alcohol Stability in Forensic Antemortem Whole Blood Samples.

    PubMed

    Tiscione, Nicholas B; Vacha, Ruth E; Alford, Ilene; Yeatman, Dustin Tate; Shan, Xiaoqin

    2015-01-01

    The effect of long-term room temperature storage on the stability of ethanol in whole blood specimens was investigated. One hundred and seventeen preserved whole blood case samples (110 of 117 with two tubes of blood in each case) were used for this study. One tube from each case was initially tested for blood alcohol concentration (BAC) for criminal driving under the influence proceedings. Cases positive for ethanol ranged in BAC from 0.023 to 0.281 g/dL. The second tube, if present, remained sealed. All blood samples were then stored at room temperature. After 5.4-10.3 years, the opened tubes were reanalyzed for BAC by the same laboratory that performed the initial testing using the same method and same instrumentation. After the same storage period, the unopened tubes were sent to a different laboratory, using a different method and different instrumentation, and reanalyzed for BAC after a total of 5.6-10.5 years of room temperature storage. Seven samples initially negative for alcohol remained negative. All samples initially positive for ethanol demonstrated a decrease in BAC over time with a statistically significant difference in loss observed based on blood sample volume and whether or not the tube had been previously opened. The decrease in BAC ranged from 0.005 to 0.234 g/dL. Tubes that were not previously opened and were more than half full demonstrated better BAC stability with 89% of these tubes demonstrating a loss of BAC between 0.01 and 0.05 g/dL.

  8. DNA-AP sites generation by Etoposide in whole blood cells

    PubMed Central

    2009-01-01

    Background Etoposide is currently one of the most commonly used antitumor drugs. The mechanisms of action proposed for its antitumor activity are based mainly on its interaction with topoisomerase II. Etoposide effects in transformed cells have been described previously. The aim of the present study was to evaluate the genotoxic effects of this drug in non-transformed whole blood cells, such as occurs as collateral damage induced by some chemotherapies. Methods To determine etoposide genotoxicity, we employed Comet assay in two alkaline versions. To evaluate single strand breaks and delay repair sites we use pH 12.3 conditions and pH >13 to evidence alkali labile sites. With the purpose to quantified apurinic or apyrimidine (AP) sites we employed a specific restriction enzyme. Etoposide effects were determined on whole blood cells cultured in absence or presence of phytohemagglutinin (PHA) treated during 2 and 24 hours of cultured. Results Alkaline (pH > 13) single cell gel electrophoresis (SCGE) assay experiments revealed etoposide-induced increases in DNA damage in phytohemaglutinine (PHA)-stimulated blood and non-stimulated blood cells. When the assay was performed at a less alkaline pH, 12.3, we observed DNA damage in PHA-stimulated blood cells consistent with the existence of alkali labile sites (ALSs). In an effort to elucidate the molecular events underlying this result, we applied exonuclease III (Exo III) in conjunction with a SCGE assay, enabling detection of DNA-AP sites along the genome. More DNA AP-sites were revealed by Exo III and ALSs were recognized by the SCGE assay only in the non-stimulated blood cells treated with etoposide. Conclusion Our results indicate that etoposide induces DNA damage specifically at DNA-AP sites in quiescent blood cells. This effect could be involved in the development of secondary malignancies associated with etoposide chemotherapy. PMID:19917085

  9. Whole blood gene expression profile associated with spontaneous preterm birth in women with threatened preterm labor.

    PubMed

    Heng, Yujing Jan; Pennell, Craig Edward; Chua, Hon Nian; Perkins, Jonathan Edward; Lye, Stephen James

    2014-01-01

    Threatened preterm labor (TPTL) is defined as persistent premature uterine contractions between 20 and 37 weeks of gestation and is the most common condition that requires hospitalization during pregnancy. Most of these TPTL women continue their pregnancies to term while only an estimated 5% will deliver a premature baby within ten days. The aim of this work was to study differential whole blood gene expression associated with spontaneous preterm birth (sPTB) within 48 hours of hospital admission. Peripheral blood was collected at point of hospital admission from 154 women with TPTL before any medical treatment. Microarrays were utilized to investigate differential whole blood gene expression between TPTL women who did (n = 48) or did not have a sPTB (n = 106) within 48 hours of admission. Total leukocyte and neutrophil counts were significantly higher (35% and 41% respectively) in women who had sPTB than women who did not deliver within 48 hours (p<0.001). Fetal fibronectin (fFN) test was performed on 62 women. There was no difference in the urine, vaginal and placental microbiology and histopathology reports between the two groups of women. There were 469 significant differentially expressed genes (FDR<0.05); 28 differentially expressed genes were chosen for microarray validation using qRT-PCR and 20 out of 28 genes were successfully validated (p<0.05). An optimal random forest classifier model to predict sPTB was achieved using the top nine differentially expressed genes coupled with peripheral clinical blood data (sensitivity 70.8%, specificity 75.5%). These differentially expressed genes may further elucidate the underlying mechanisms of sPTB and pave the way for future systems biology studies to predict sPTB.

  10. Tetrahydrocannabinol and two of its metabolites in whole blood using liquid chromatography-tandem mass spectrometry.

    PubMed

    Coulter, Cynthia; Miller, Elizabeth; Crompton, Katherine; Moore, Christine

    2008-10-01

    An analytical procedure for the determination of Delta9-tetrahydrocannabinol (THC), 11-nor-9-carboxy-Delta9-tetrahydrocannabinol (THCA), and 11-hydroxy-Delta9-tetrahydrocannabinol (11-OH-THC) in whole blood has been developed and validated using liquid chromatography with tandem mass spectral detection (MS). Cannabinoids present in the blood samples were quantified using solid-phase extraction followed by MS detection in positive electrospray ionization mode. For confirmation, two transitions were monitored and one ratio determined. Samples being reported as positive were required to have both transitions present, the ratio of quantifying transition to qualifying transition being within 20% of that determined from known calibration standards. The monitoring of the qualifying transition and requirement for its presence within a specific ratio to the primary ion has the potential of limiting the sensitivity of the assay, however, the additional confidence in the final result as well as forensic defensibility were considered to be of greater importance. The limit of quantitation was 0.5 ng/mL for THC, 5 ng/mL for THCA, and 2 ng/mL for 11-OH-THC. The limit of detection was 0.5 ng/mL for THC, 4 ng/mL for THCA, and 1 ng/mL for 11-OH-THC. The percentage recovery of the cannabinoids from whole blood at a concentration of 5 ng/mL was 71.5% for THC, 64.5% for 11-OH-THC, and 61.2% for THCA (n = 3).

  11. Determination of cannabinoids in whole blood by UPLC-MS-MS.

    PubMed

    Jamey, Carole; Szwarc, Esther; Tracqui, Antoine; Ludes, Bertrand

    2008-06-01

    An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method was developed and validated for the simultaneous determination of Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in whole blood. Samples were prepared by protein precipitation followed by solid-phase extraction. Data were acquired using positive electrospray ionization and multiple reaction monitoring. Two transitions were selected for THC (m/z 315.0 > 193.0 and m/z 315.0 > 122.7) and THC-COOH (m/z 345.0 > 299.0 and m/z 345.0 > 327.0), and one transition was chosen for 11-OH-THC (m/z 331.0 > 313.0). Deuterated analogues of each analyte were used as internal standards for quantification. Run time was 10 min. Limits of quantification (LOQ) were 0.05 ng/mL for THC, 0.1 ng/mL for 11-OH-THC, and 0.2 ng/mL for THC-COOH. Linearity was established from LOQ to 50 ng/mL for each substance (r(2) always > 0.999). Accuracy ranged from 96 to 106%, and imprecision was less than 10% for all analytes. The UPLC-MS-MS method was found to be sensitive, specific, and rapid because it requires no derivatization step. It can be an alternative to gas chromatography-mass spectrometry for the determination of cannabinoids in whole blood.

  12. Aprotinin prolongs activated and nonactivated whole blood clotting time and potentiates the effect of heparin in vitro.

    PubMed

    Despotis, G J; Filos, K S; Levine, V; Alsoufiev, A; Spitznagel, E

    1996-06-01

    This study was designed to evaluate the effect of aprotinin on activated versus nonactivated whole blood clotting time using two different on-site methods and to quantify these anticoagulant properties when compared to heparin in a controlled, in vitro environment. Blood specimens were obtained prior to heparin administration from 56 patients undergoing cardiac surgery. Specimens obtained from the first consecutive 20 patients were mixed with either normal saline (NS) or aprotinin (400 kallikrein inhibiting units (KIU)/mL), inserted into Hemochron tubes containing either NS or heparin (0.3 or 0.6 U/mL) and then used to measure celite-activated (celite ACT) and nonactivated whole blood clotting time (WBCT1) using four Hemochron instruments. Accordingly, specimens obtained from the second consecutive 20 patients were mixed with either NS or aprotinin, inserted into Automated Clot Timer cartridges containing either NS or heparin (0.06, 0.13, or 0.25 U/mL) and then used to measure kaolin-activated (kaolin ACT) or nonactivated whole blood clotting times (WBCT2) using four Automated Clot Timer instruments. Specimens obtained from the last 16 patients were mixed with either incrementally larger doses of aprotinin (0, 100, 200, 300, or 400 KIU/mL) or heparin (0, 0.12, 0.24, 0.36, 0.48, or 0.72 U/mL) and were then used for measurement of whole blood clotting time (WBCT2) using six Automated Clot Timer instruments. Aprotinin significantly prolonged activated or nonactivated whole blood clotting time and potentiated the prolongation of whole blood clotting time by heparin. The linear relationship between whole blood clotting time and either heparin concentration (WBCT2 = H x 357 + 280, mean adjusted r2 = 0.88) or aprotinin concentration (WBCT2 = A x 0.97 + 300, mean adjusted r2 = 0.94) was variable among patients. On average, 200 KIU/mL of aprotinin prolonged WBCT2 to the same extent as 0.69 +/- 0.28 U/mL of heparin using linear regression models within each patient

  13. Microwave-accelerated surface plasmon-coupled directional luminescence 2: a platform technology for ultra fast and sensitive target DNA detection in whole blood.

    PubMed

    Aslan, Kadir; Previte, Michael J R; Zhang, Yongxia; Geddes, Chris D

    2008-02-29

    The application of Microwave-Accelerated Surface Plasmon-Coupled Luminescence (MA-SPCL) to fast and sensitive DNA hybridization assays in buffer and whole blood is presented. In this regard, a model DNA hybridization assay whereby a fluorophore-labeled target ssDNA specific to human immunodeficiency, Hepatitis C (Hep C), is probed by an anchor probe immobilized on thin gold films, is driven to completion within 1 min with microwave heating, as compared to an identical assay completed in approximately 4 h at room temperature. Finite-Difference Time-Domain calculations show that gold disks are preferentially heated around the edges creating a temperature gradient along the disks, which in turn results in the larger influx of complementary DNA towards anchor probe-modified surface. Thermal images of the assay platform during microwave heating also provide additional information on the microwave heating pattern in the microwave cavity. Finally, the effects of low power microwave heating on the ability of DNA to re-hybridize with the complimentary target on the surface gold films, which allows the multiple re-use of the gold films, is demonstrated. The MA-SPCL technique offers an alternative approach to current DNA based detection technologies, especially when speed and sensitivity are required, such as in the identification of DNA or even RNA-based diseases using whole blood samples that affect human health.

  14. Comparative study of the effect of chloro-, dichloro-, bromo-, and dibromoacetic acid on necrotic, apoptotic and morphological changes in human peripheral blood mononuclear cells (in vitro study).

    PubMed

    Michałowicz, Jaromir; Wróblewski, Wojciech; Mokra, Katarzyna; Maćczak, Aneta; Kwiatkowska, Marta

    2015-10-01

    In this study, the effect of monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), monobromoacetic acid (MBAA) and dibromoacetic acid (DBAA) on human peripheral blood mononuclear cells (PBMCs) was assessed. HAAs studied induced at millimolar concentrations necrotic alterations in PBMCs with the strongest effect noted for MBAA and DBAA. Chloro- and bromoacetic acids also provoked changes in PBMCs morphology because they caused a strong decrease in cell size (particularly DCAA and DBAA) and increase in cell granulation (mainly MBAA and DBAA). All HAAs studied, and DCAA and DBAA in particular (at lower concentrations than those, which caused necrosis) induced apoptotic changes, which was confirmed by analysis of alterations in cell membrane permeability and caspase 8, 9 and 3 activation. Moreover, HAAs examined (mainly dihalogenated acids) strongly increased transmembrane mitochondrial potential and enhanced ROS (mainly hydroxyl radical) formation, which was possibly associated with apoptotic changes provoked by those substances. The results showed that DBAA exhibited the strongest effects on PBMCs.

  15. Microfluidic Chip-Based Detection and Intraspecies Strain Discrimination of Salmonella Serovars Derived from Whole Blood of Septic Mice

    PubMed Central

    Patterson, Adriana S.; Heithoff, Douglas M.; Ferguson, Brian S.; Soh, H. Tom; Mahan, Michael J.

    2013-01-01

    Salmonella is a zoonotic pathogen that poses a considerable public health and economic burden in the United States and worldwide. Resultant human diseases range from enterocolitis to bacteremia to sepsis and are acutely dependent on the particular serovar of Salmonella enterica subsp. enterica, which comprises over 99% of human-pathogenic S. enterica isolates. Point-of-care methods for detection and strain discrimination of Salmonella serovars would thus have considerable benefit to medical, veterinary, and field applications that safeguard public health and reduce industry-associated losses. Here we describe a single, disposable microfluidic chip that supports isothermal amplification and sequence-specific detection and discrimination of Salmonella serovars derived from whole blood of septic mice. The integrated microfluidic electrochemical DNA (IMED) chip consists of an amplification chamber that supports loop-mediated isothermal amplification (LAMP), a rapid, single-temperature amplification method as an alternative to PCR that offers advantages in terms of sensitivity, reaction speed, and amplicon yield. The amplification chamber is connected via a microchannel to a detection chamber containing a reagentless, multiplexed (here biplex) sensing array for sequence-specific electrochemical DNA (E-DNA) detection of the LAMP products. Validation of the IMED device was assessed by the detection and discrimination of S. enterica subsp. enterica serovars Typhimurium and Choleraesuis, the causative agents of enterocolitis and sepsis in humans, respectively. IMED chips conferred rapid (under 2 h) detection and discrimination of these strains at clinically relevant levels (<1,000 CFU/ml) from whole, unprocessed blood collected from septic animals. The IMED-based chip assay shows considerable promise as a rapid, inexpensive, and portable point-of-care diagnostic platform for the detection and strain-specific discrimination of microbial pathogens. PMID:23354710

  16. The Interchangeability of Plasma and Whole Blood Metal Ion Measurement in the Monitoring of Metal on Metal Hips

    PubMed Central

    Malek, Ibrahim A.; Rogers, Joanne; King, Amanda Christina; Clutton, Juliet; Winson, Daniel; John, Alun

    2015-01-01

    One hundred and twenty six paired samples of plasma and whole blood were measured with inductively coupled plasma mass spectrometry technique for metal ions analysis to determine a relationship between them. There was a significant difference between the mean plasma and whole blood concentrations of both cobalt (Co) and chromium (Cr) (p < 0.0001 for both Co and Cr). The mean ratio between plasma and whole blood Cr and Co was 1.56 (range: 0.39–3.85) and 1.54 (range: 0.64–18.26), respectively, but Bland and Altman analysis illustrated that this relationship was not universal throughout the range of concentrations. There was higher variability at high concentrations for both ions. We conclude that both these concentrations should not be used interchangeably and conversion factors are unreliable due to concentration dependent variability. PMID:26798516

  17. Rates of vaso-vagal reactions among first time teenaged whole blood, double red cell, and plateletpheresis donors.

    PubMed

    Reiss, Robert F; Harkin, Ruth; Lessig, Marvin; Mascari, Julie

    2009-01-01

    Given the paucity of published data regarding reaction rates in younger teenaged donors, we evaluated the reaction rates in all of our first time teenaged donors after New York Blood Center lowered the minimum permissible age for blood donations from 17 to 16 yr in 2005. The overall rates of vaso-vagal reactions in donors aged 16 to 19, and those resulting in syncope, occurring in 72,769 consecutive first time whole blood, 3,822 double red cell, and 777 platelet apheresis donations were calculated. They were correlated with age and compared to those found in donors aged 20-29. Separate rates were calculated by gender, age in yr, and donation type, and then compared to each other. The overall reaction rate among first time teenaged whole blood donors was 8.2% and was significantly greater than among plateletpheresis donors (4.0%; p <0.0002). The rate in female whole blood donors (10.0%) was significantly higher than in males (6.4%; p <0.0002). In male double red cell donors the overall reaction rate of 3.5% was significantly lower than that found in male whole blood donors (p <0.002). Among both male and female whole blood donors a significant correlation with decreasing donor age between 19 and 16 yr was found (r(2) = 0.981; p = 0.01) and (r(2) = 0.988; p = 0.006), respectively. We conclude that teenaged donors have increased reaction rates when compared to adults and the reaction rates increase with decreasing age. In addition, females have higher reaction rates than males. Finally, reaction rates associated with apheresis donations are significantly lower than those associated with whole blood donations.

  18. Comparison of whole blood, serum, and plasma for early detection of candidemia by multiplex-tandem PCR.

    PubMed

    Lau, Anna; Halliday, Catriona; Chen, Sharon C-A; Playford, E Geoffrey; Stanley, Keith; Sorrell, Tania C

    2010-03-01

    We applied multiplex-tandem PCR (MT-PCR) to 255 EDTA whole-blood specimens, 29 serum specimens, and 24 plasma specimens from 109 patients with Candida bloodstream infection (candidemia) to determine whether a diagnosis could be expedited in comparison with the time to diagnosis by the use of standard blood culture. Overall, the MT-PCR performed better than blood culture with DNA extracted from whole blood from 52/74 (70%) patients, accelerating the time to detection (blood culture flagging) and determination of the pathogenic species (by use of the API 32C system [bioMérieux, Marcy l'Etoile, France]) by up to 4 days (mean, 2.2 days; range, 0.5 to 8 days). Candida DNA was detected more often in serum (71%) and plasma (75%) than in whole blood (54%), although relatively small numbers of serum and plasma specimens were tested. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay with whole blood were 75%, 97%, 95%, and 85%, respectively. Fungal DNA was not detected by MT-PCR in 6/24 (25%) whole-blood samples drawn simultaneously with the positive blood culture sample. MT-PCR performed better with whole-blood specimens stored at -20 degrees C (75%) and when DNA was extracted within 1 week of sampling (66%). The molecular and culture identification results correlated for 61 of 66 patients (92%); one discrepant result was due to misidentification by culture. All but one sample from 53 patients who were at high risk of candidemia but did not have proven disease were negative by MT-PCR. The results demonstrate the good potential of MT-PCR to detect candidemia, to provide Candida species identification prior to blood culture positivity, and to provide improved sensitivity when applied to with serum and plasma specimens.

  19. Human mononuclear cell function after 4 degrees C storage during 1-G and microgravity conditions of spaceflight

    NASA Technical Reports Server (NTRS)

    Meehan, R.; Taylor, G.; Lionetti, F.; Neale, L.; Curren, T.

    1989-01-01

    Future space missions of long duration may require that autologous leukocytes be stored in flight for infusion to restore normal immune competence in crewmembers. Peripheral blood mononuclear cells (PBMNCs), as leukocyte concentrates in autologous plasma and 2% dextrose, were stored in the microgravity conditions provided by the U.S. Space Shuttle Columbia (Mission 61-C). Activity of PBMNC after space flight was compared with that from a series of preflight ground control experiments, which demonstrated in culture a progressive daily loss in mitogen-stimulated protein synthesis at 24 h and thymidine uptake at 72 h after storage for 7 d at 4 degrees C. Post-storage viabilities were at least 90% as determined by trypan dye exclusion. A progressive reduction in the percentage of PBMNC expressing cell-surface phenotype markers, which was similar for monocytes, B cells, and T-cell subsets, also occurred after storage. The ability of PBMNC, stored for 8 d in Columbia's middeck, to become activated and proliferate in vitro was similar to that of cells that remained in identical flight lockers on the ground as 1-G controls, thus indicating that PBMNCs were not adversely affected by storage under microgravity conditions.

  20. Effects of alcohol-induced human peripheral blood mononuclear cell (PBMC) pretreated whey protein concentrate (WPC) on oxidative damage.

    PubMed

    Tseng, Yang-Ming; Chen, Sheng-Yi; Chen, Chien-Hung; Jin, Yi-Ru; Tsai, Shih-Meng; Chen, Ing-Jun; Lee, Jang-Hwa; Chiu, Chzng-Cheng; Tsai, Li-Yu

    2008-09-10

    Excessive alcohol consumption can induce apoptosis in a variety of tissues and influence the antioxidant status in peripheral blood mononuclear cells (PBMC). This paper investigates the effects of whey protein concentrate (WPC) pretreated in PBMC on the apoptosis and antioxidant status after the treatment of alcohol. The results show that the percentages of apoptotic cells in the alcohol-treated group were higher than those in the group without alcohol treatment. Additionally, there was higher glutathione (GSH) peroxidase (GPx) activity when the PBMC were treated with 300 mg/dL of alcohol. With regard to the activity of GSH reductase (GRx), there was higher activity in the group pretreated with WPC than in the group with the treatment of alcohol only. On the contrary, the levels of GSH were reduced after the treatment of alcohol, but there was a higher level of GSH in the group pretreated with WPC. In this study, it was found that the increased level of GSH in PBMC might not be attributed to the effect of GRx because there was still a higher level of GSH in the group with the treatment of WPC and BCNU (a GRx inhibitor) in this study. The results indicated that PBMC pretreated with WPC might ameliorate alcohol-induced effects such as imbalance of the antioxidant status.

  1. Human mononuclear cell function after 4 degrees C storage during 1-G and microgravity conditions of spaceflight.

    PubMed

    Meehan, R; Taylor, G; Lionetti, F; Neale, L; Curren, T

    1989-07-01

    Future space missions of long duration may require that autologous leukocytes be stored in flight for infusion to restore normal immune competence in crewmembers. Peripheral blood mononuclear cells (PBMNCs), as leukocyte concentrates in autologous plasma and 2% dextrose, were stored in the microgravity conditions provided by the U.S. Space Shuttle Columbia (Mission 61-C). Activity of PBMNC after space flight was compared with that from a series of preflight ground control experiments, which demonstrated in culture a progressive daily loss in mitogen-stimulated protein synthesis at 24 h and thymidine uptake at 72 h after storage for 7 d at 4 degrees C. Post-storage viabilities were at least 90% as determined by trypan dye exclusion. A progressive reduction in the percentage of PBMNC expressing cell-surface phenotype markers, which was similar for monocytes, B cells, and T-cell subsets, also occurred after storage. The ability of PBMNC, stored for 8 d in Columbia's middeck, to become activated and proliferate in vitro was similar to that of cells that remained in identical flight lockers on the ground as 1-G controls, thus indicating that PBMNCs were not adversely affected by storage under microgravity conditions.

  2. Flow cytometry analysis of hormone receptors on human peripheral blood mononuclear cells to identify stress-induced neuroendocrine effects

    NASA Technical Reports Server (NTRS)

    Meehan, R. T.

    1986-01-01

    Understanding the role of circulating peptide hormones in the pathogenesis of space-flight induced disorders would be greatly facilitated by a method which monitors chronic levels of hormones and their effects upon in vivo cell physiology. Single and simultaneous multiparameter flow cytometry analysis was employed to identify subpopulations of mononuclear cells bearing receptors for ACTH, Endorphin, and Somatomedin-C using monoclonal antibodies and monospecific antisera with indirect immunofluorescence. Blood samples were obtained from normal donors and subjects participating in decompression chamber studies (acute stress), medical student academic examination (chronic stress), and a drug study (Dexamethasone). Preliminary results indicate most ACTH and Endorphin receptor positive cells are monocytes and B-cells, exhibit little diurnal variation but the relative percentages of receptor positive cells are influenced by exposure to various stressors and ACTH inhibition. This study demonstrates the capability of flow cytometry analysis to study cell surface hormone receptor regulation which should allow insight into neuroendocrine modulation of the immune and other cellular systems during exposure to stress or microgravity.

  3. In vivo tracing of indium-111 oxine-labeled human peripheral blood mononuclear cells in patients with lymphatic malignancies

    SciTech Connect

    Mueller, C.Z.; Zielinski, C.C.; Linkesch, W.; Ludwig, H.; Sinzinger, H.

    1989-06-01

    The in vivo migration of (/sup 111/In)oxine-labeled peripheral mononuclear cells (PMNC) was studied in 20 patients with various lymphatic malignancies and palpable enlarged lymph nodes. The maximal labeling dose of 10 microCi (0.37 MBq) (/sup 111/In)oxine/10(8) PMNC was found not to adversely influence either cell viability or lymphocyte proliferation in vitro. For in vivo studies, 1.5 X 10(9) PMNC were gained by lymphapheresis and reinjected intravenously after radioactive labeling, 150 microCi (5.55 MBq). The labeling of enlarged palpable lymph nodes was achieved in three out of three patients with Hodgkin's disease and in five out of five with high-malignant lymphoma, whereas three out of seven patients with low malignant lymphoma and no patient with chronic lymphatic leukemia had positive lymph node imaging. We thus conclude that PMNC retain their ability to migrate after (/sup 111/In)oxine labeling and that these cells traffic to involved lymph nodes of some, but not all hematologic malignancies.

  4. Human Intervention Study to Assess the Effects of Supplementation with Olive Leaf Extract on Peripheral Blood Mononuclear Cell Gene Expression

    PubMed Central

    Boss, Anna; Kao, Chi Hsiu-Juei; Murray, Pamela M.; Marlow, Gareth; Barnett, Matthew P. G.; Ferguson, Lynnette R.

    2016-01-01

    Olive leaf extract (OLE) has been used for many years for its putative health benefits, but, to date, scientific evidence for the basis of these effects has been weak. Although recent literature has described a link between ailments such as cardiovascular disease, diabetes and cancer and a protective effect of polyphenols in the OLE, the mode of action is still unclear. Here, we describe a double-blinded placebo (PBO)-controlled trial, in which gene expression profiles of peripheral blood mononuclear cells from healthy male volunteers (n = 29) were analysed to identify genes that responded to OLE, following an eight-week intervention with 20 mL daily consumption of either OLE or PBO. Differences between groups were determined using an adjusted linear model. Subsequent analyses indicated downregulation of genes important in inflammatory pathways, lipid metabolism and cancer as a result of OLE consumption. Gene expression was verified by real-time PCR for three genes (EGR1, COX-2 and ID3). The results presented here suggest that OLE consumption may result in health benefits through influencing the expression of genes in inflammatory and metabolic pathways. Future studies with a larger study group, including male and female participants, looking into direct effects of OLE on lipid metabolism and inflammation are warranted. PMID:27918443

  5. Expression levels of seven candidate genes in human peripheral blood mononuclear cells and their association with preeclampsia

    PubMed Central

    Garza-Veloz, I.; Carrillo-Sanchez, K.; Martinez-Gaytan, V.; Cortes-Flores, R.; Ochoa-Torres, M. A.; Guerrero, G. G.; Rodriguez-Sanchez, I. P.; Cancela-Murrieta, C. O.; Zamudio-Osuna, M.; Badillo-Almaraz, J. I.; Castruita-De la Rosa, C.

    2014-01-01

    Objective To evaluate the peripheral blood mononuclear cell (PBMC) expression levels of hemeoxygenase 1 (HMOX-1), superoxide dismutase 1 (SOD-1), vascular endothelial growth factor A (VEGF-A), transforming growth factor beta 1 (TGF-β1), interleukin (IL)-6, IL-15 and AdipoQ genes to study their association with preeclampsia (PE). Methods A total of 177 pregnant women were recruited: 108 cases and 69 controls. Quantification of gene expression was measured by quantitative real-time polymerase chain reaction (PCR) using TaqMan probes. Results Underexpression of VEGF-A and TGF-β1 was a constant in most of the cases (80.91% and 76.36%, respectively) and their expression was associated with onset and/or severity of disease (p values < 0.05). IL-6, IL-15 and AdipoQ, showed low or no expression in PBMC samples evaluated. Conclusion PBMC underexpression of VEGF-A and TGF-β1 is a hallmark of PE in the study population. PMID:24295154

  6. Adhesion of mononuclear cells from multiple sclerosis patients to cerebral vessels in cryostat sections of normal human brain.

    PubMed

    Zaffaroni, M; Martinazzi, S; Crivelli, F; Ghezzi, A; Zampieri, A; Martinazzi, M; Zibetti, A; Canal, N

    1999-09-01

    Leukocyte extravasation across the blood-brain barrier is a critical event in the pathogenesis of multiple sclerosis (MS). This complex multistep process includes the adhesion of leukocytes to the endothelial cells of the central nervous system microvasculature. To investigate this phenomenon in MS, we developed a modified version of the frozen-section assay. Peripheral blood mononuclear cells (PBM) from 26 MS patients, 26 healthy controls and 10 patients with other inflammatory non- neurological diseases (OIND) were co-incubated with cryostat sections of normal brain white matter, immunohistochemically labelled with anti-CD45 antibody and counterstained with Giemsa stain. CD45-positive PBM adherent to transected microvasculature were counted with an automated image analyzer. MS patients showed an increased number of vessel-bound PBM (48.8 +/- 36.4) with respect to healthy controls (27.4 +/- 20.7, P = 0.01) and OIND patients (22.6 +/- 7.8, P = 0.01). Significant differences were also obtained counting the number of vessel-bound PBM as a percent of total vascular cells between MS patients (12.7 +/- 7.2%) and healthy controls (6.9 +/- 5.4%, P = 0.002) or OIND patients (7.4 +/- 4.4%, P = 0.03). We confirm that PBM from MS patients show an increased potential of binding to cerebral vessels. The frozen-section assay provides a unique tool to study in situ the molecular interactions of leukocytes with brain vascular structures.

  7. Whole Blood Activation Results in Enhanced Detection of T Cell and Monocyte Cytokine Production by Flow Cytometry

    NASA Technical Reports Server (NTRS)

    Sams, Clarence F.; Crucian, Brian E.

    2001-01-01

    An excellent monitor of the immune balance of peripheral circulating cells is to determine their cytokine production patterns in response to stimuli. Using flow cytometry a positive identification of cytokine producing cells in a mixed culture may be achieved. Recently, the ability to assess cytokine production following a wholeblood activation culture has been described. We compared whole blood culture to standard PBMC culture and determined the individual cytokine secretion patterns for both T cells and monocytes via flow cytometry. For T cells cytokine assessment following PMA +ionomycin activation: (1) a significantly greater percentages of T cells producing IFNgamma and IL-2 were observed following whole-blood culture; (2) altered T cell cytokine production kinetics were observed by varying whole blood culture times. In addition, a four-color cytometric analysis was used to allow accurate phenotyping and quantitation of cytokine producing lymphocyte populations. Using this technique we found IFNgamma production to be significantly elevated in the CD3+/CD8+ T cell population as compared to the CD3+/CD8- population following five hours of whole blood activation. Conversely, IL-2 and IL-10 production were significantly elevated in the CD3+/CD8- T cell population as compared to the CD3+/CD8+ population. Monocyte cytokine production was assessed in both culture systems following LPS activation for 24 hours. A three-color flow cytometric was used to assess two cytokines in conjunction with CD 14. The cytokine pairs used for analysis were IL-1a/IL-12, and IL-10ITNFa. Nearly all monocytes were stimulated to produce IL-1a, IL-12 and TNFalpha equally well in both culture systems. Monocyte production of IL-10 was significantly elevated following whole blood culture as compared to PBMC culture. IL-12 producing monocytes appeared to be a distinct subpopulation of the IL-1a producing set, whereas IL-10 and TNFa producing monocytes were largely mutually exclusive. IL-10 and

  8. Genetic analysis of DNA methylation and gene expression levels in whole blood of healthy human subjects

    PubMed Central

    2012-01-01

    Background The predominant model for regulation of gene expression through DNA methylation is an inverse association in which increased methylation results in decreased gene expression levels. However, recent studies suggest that the relationship between genetic variation, DNA methylation and expression is more complex. Results Systems genetic approaches for examining relationships between gene expression and methylation array data were used to find both negative and positive associations between these levels. A weighted correlation network analysis revealed that i) both transcriptome and methylome are organized in modules, ii) co-expression modules are generally not preserved in the methylation data and vice-versa, and iii) highly significant correlations exist between co-expression and co-methylation modules, suggesting the existence of factors that affect expression and methylation of different modules (i.e., trans effects at the level of modules). We observed that methylation probes associated with expression in cis were more likely to be located outside CpG islands, whereas specificity for CpG island shores was present when methylation, associated with expression, was under local genetic control. A structural equation model based analysis found strong support in particular for a traditional causal model in which gene expression is regulated by genetic variation via DNA methylation instead of gene expression affecting DNA methylation levels. Conclusions Our results provide new insights into the complex mechanisms between genetic markers, epigenetic mechanisms and gene expression. We find strong support for the classical model of genetic variants regulating methylation, which in turn regulates gene expression. Moreover we show that, although the methylation and expression modules differ, they are highly correlated. PMID:23157493

  9. A simple and rapid DNA extraction method from whole blood for highly sensitive detection and quantitation of HIV-1 proviral DNA by real-time PCR.

    PubMed

    McFall, Sally M; Wagner, Robin L; Jangam, Sujit R; Yamada, Douglas H; Hardie, Diana; Kelso, David M

    2015-03-01

    Early diagnosis and access to treatment for infants with human immunodeficiency virus-1 (HIV-1) is critical to reduce infant mortality. The lack of simple point-of-care tests impedes the timely initiation of antiretroviral therapy. The development of FINA, filtration isolation of nucleic acids, a novel DNA extraction method that can be performed by clinic personnel in less than 2 min has been reported previously. In this report, significant improvements in the DNA extraction and amplification methods are detailed that allow sensitive quantitation of as little as 10 copies of HIV-1 proviral DNA and detection of 3 copies extracted from 100 μl of whole blood. An internal control to detect PCR inhibition was also incorporated. In a preliminary field evaluation of 61 South African infants, the FINA test demonstrated 100% sensitivity and specificity. The proviral copy number of the infant specimens was quantified, and it was established that 100 microliters of whole blood is required for sensitive diagnosis of infants.

  10. Determination of Nitrite in Whole Blood by High-Performance Liquid Chromatography with Electrochemical Detection and a Case of Nitrite Poisoning.

    PubMed

    Yan, Hui; Zhuo, Xiangyi; Shen, Baohua; Xiang, Ping; Shen, Min

    2016-01-01

    Although nitrite is widely used in meat processing, it is a major toxicity hazard to children and is responsible for the blue-baby syndrome. A simple and effective method to determine nitrite in whole blood has been devised using ion chromatography with suppressed conductivity detection. The blood sample was deproteinized by adding acetonitrile and purified with mini-cartridges to remove hydrophobic compounds, chloride ions, and metal ions. An aliquot of the filtrate was injected onto the ion chromatography. The retention time for nitrite was 13.8 min and the detection limit of nitrite in whole blood was 0.4 μmol/L. The calibration curve was linear (r(2) = 0.9999) over the concentration working range. The blood nitrite concentration of a victim who attempted suicide by ingesting sodium nitrite powder was determined using the present method. The basal levels for nitrite in human blood was determined with 7.1 ± 0.9 μmol/L (n = 12).

  11. Ionized calcium in normal serum, ultrafiltrates, and whole blood determined by ion-exchange electrodes

    PubMed Central

    Moore, Edward W.

    1970-01-01

    Ion-exchange calcium electrodes represent the first practical method for the direct measurement of ionized calcium [Ca++] in biologic fluids. Using both “static” and “flow-through” electrodes, serum [Ca++] was within a rather narrow range: 0.94-1.33 mmoles/liter (mean, 1.14 mmoles/liter). Within a given individual, [Ca++] varied only about 6% over a several month period. Consistent pH effects on [Ca++] were observed in serum and whole blood, [Ca++] varying inversely with pH. Less consistent pH effects were also noted in ultrafiltrates, believed to largely represent precipitation of certain calcium complexes from a supersaturated solution. Heparinized whole blood [Ca++] was significantly less than in corresponding serum at normal blood pH, related to the formation of a calcium-heparin complex. [Ca++] in ultrafiltrates represented a variable fraction (66.7-90.2%) of total diffusible calcium. There was no apparent correlation between serum ionized and total calcium concentrations. Thus, neither serum total calcium nor total ultrafiltrable calcium provided a reliable index of serum [Ca++]. Change in serum total calcium was almost totally accounted for by corresponding change in protein-bound calcium [CaProt]. About 81% of [CaProt] was estimated to be bound to albumin and about 19% to globulins. From observed pH, serum protein, and [CaProt] data, a nomogram was developed for estimating [CaProt] without ultrafiltration. Data presented elsewhere indicate that calcium binding by serum proteins obeys the mass-law equation for a monoligand association. This was indicated in the present studies by a close correspondence of observed serum [Ca++] values with those predicted by the McLean-Hastings nomogram. While these electrodes allow study of numerous problems not possible previously, they have not been perfected to the same degree of reliability obtainable with current pH electrodes. The commercial (Orion flow-through) electrode is: (a) expensive. (b) requires

  12. Is platelet function as measured by Thrombelastograph monitoring in whole blood affected by platelet inhibitors?

    PubMed

    Bailey, Lori A; Sistino, Joseph J; Uber, Walter E

    2005-03-01

    Platelet inhibitors, especially the glycoprotein (GP) IIb/IIIa receptor antagonists, have demonstrated their effectiveness in reducing the acute ischemic complications of percutaneous coronary intervention (PCI) and in improving clinical outcomes in patients with acute coronary crisis. Three common platelet inhibitors observed in emergent cardiopulmonary bypass (CPB) for failed PCI are abciximab, eptifibatide, and tirofiban. An in vitro model was constructed in two parts to determine whether platelet aggregation inhibition induced by platelet inhibitors would be demonstrated by the Thrombelastograph (TEG) monitor when compared with baseline samples with no platelet inhibitor. In part A, 20 mL of fresh whole blood was divided into four groups: group I = baseline, group A = abcix-imab microg/mL, group E = eptifibatide ng/mL, and group T = tirofiban ng/mL. Platelet inhibitor concentrations in whole blood were derived starting with reported serum concentrations with escalation to achieve 80% platelet inhibition using the Medtronic hemoSTATUS and/or Lumi-aggregometer. A concentration range determined by our in vitro tests were chosen for each drug using concentrations achieving less than, equal to, or greater than 80% platelet inhibition. In part B, TEG analysis was then performed using baseline and concentrations for each drug derived in part A. Parameters measured were clot formation reaction time (R), coagulation time (K), maximum amplitude (MA) and alpha angle (A). Groups E1000 and E2000 extended R over control by 37% and 23%, respectively (p = 0.01 and 0.03). Groups E1000 and E2000 increased K times by 45% and 58% (p = .02 and .04). T160 samples prolonged K by 20% (p = 0.01). The angle or clot strength (A) was decreased in groups T160 and E1000 by 23% (+ 7.06 SD) and 18% (+ 11.23 SD), respectively (p = 0.001 and 0.01). The MA decrease was statistically significant in the T160, E1000 and E2000 by 9%, 6% and 13% respectively (p = 0.01). Samples treated with abciximab

  13. Measurement of pH in whole blood by near-infrared spectroscopy

    SciTech Connect

    Alam, M. Kathleen; Maynard, John D.; Robinson, M. Ries Niemczyk, Thomas M. Rohrscheib, Mark R.

    1999-03-01

    Whole blood pH has been determined {ital in vitro} by using near-infrared spectroscopy over the wavelength range of 1500 to 1785 nm with multivariate calibration modeling of the spectral data obtained from two different sample sets. In the first sample set, the pH of whole blood was varied without controlling cell size and oxygen saturation (O{sub 2} Sat) variation. The result was that the red blood cell (RBC) size and O{sub 2} Sat correlated with pH. Although the partial least-squares (PLS) multivariate calibration of these data produced a good pH prediction cross-validation standard error of prediction (CVSEP)=0.046, R{sup 2}=0.982, the spectral data were dominated by scattering changes due to changing RBC size that correlated with the pH changes. A second experiment was carried out where the RBC size and O{sub 2} Sat were varied orthogonally to the pH variation. A PLS calibration of the spectral data obtained from these samples produced a pH prediction with an R{sup 2} of 0.954 and a cross-validated standard error of prediction of 0.064 pH units. The robustness of the PLS calibration models was tested by predicting the data obtained from the other sets. The predicted pH values obtained from both data sets yielded R{sup 2} values greater than 0.9 once the data were corrected for differences in hemoglobin concentration. For example, with the use of the calibration produced from the second sample set, the pH values from the first sample set were predicted with an R{sup 2} of 0.92 after the predictions were corrected for bias and slope. It is shown that spectral information specific to pH-induced chemical changes in the hemoglobin molecule is contained within the PLS loading vectors developed for both the first and second data sets. It is this pH specific information that allows the spectra dominated by pH-correlated scattering changes to provide robust pH predictive ability in the uncorrelated data, and visa versa. {copyright} {ital 1999} {ital Society for Applied

  14. Interactions of allogeneic human mononuclear cells in the two-way mixed leucocyte culture (MLC): influence of cell numbers, subpopulations and cyclosporin

    PubMed Central

    Sato, T; Deiwick, A; Raddatz, G; Koyama, K; Schlitt, H J

    1999-01-01

    With organ allografts considerable numbers of donor-type mononuclear cells are transferred to the recipient, leading to bilateral immunological interactions between donor and recipient lymphocytes. To study such bilateral immune reactions in detail, human two-way MLC were performed. In this model proliferation kinetics, patterns of activation, and survival of the two populations were analysed, and the relevance of initial cell subset composition, relative cell numbers, and the effect of immunosuppression on this co-culture were evaluated. It could be demonstrated that with an initial 50:50 ratio of two populations of allogeneic cells one population dominated after 21 days of co-culture in 78 out of 80 combinations (97%) tested; the other population decreased markedly after an initially stable phase of 6–7 days. With unequal starting conditions the larger population dominated when resting cells were used, but small populations of preactivated cells or separated CD8+ cells could also dominate. Depletion of CD16+ natural killer (NK) cells and of CD2− cells (B cell and monocytes) had no effect on domination. Addition of cyclosporin delayed or blocked the domination process while addition of IL-2 accelerated it. Disappearance of one population was associated with detection of apoptotic cells. The findings indicate that co-cultures of allogeneic mononuclear cells are generally not stable for more than 1 week, but lead to active elimination of one population. CD8+ cells and particularly preactivated cells seem to play the most important role in that process, while NK cells are of less importance. Cyclosporin can prolong survival of allogeneic cells in co-culture. These observations suggest that under the conditions of clinical organ transplantation even small amounts of immunocompetent donor cells transferred by the graft may persist for some time and may, thereby, have the chance to exert immunomodulatory functions. PMID:9933457

  15. Can Melatonin Act as an Antioxidant in Hydrogen Peroxide-Induced Oxidative Stress Model in Human Peripheral Blood Mononuclear Cells?

    PubMed Central

    Emamgholipour, Solaleh; Hossein-Nezhad, Arash; Ansari, Mohammad

    2016-01-01

    Purpose. We aimed to investigate the possible effects of melatonin on gene expressions and activities of MnSOD and catalase under conditions of oxidative stress induced by hydrogen peroxide (H2O2) in peripheral blood mononuclear cells (PBMCs). Materials and Methods. PBMCs were isolated from healthy subjects and treated as follows: (1) control (only with 0.1% DMSO for 12 h); (2) melatonin (1 mM) for 12 h; (3) H2O2 (250 μM) for 2 h; (4) H2O2 (250 μM) for 2 h following 10 h pretreatment with melatonin (1 mM). The gene expression was evaluated by real-time PCR. MnSOD and catalase activities in PBMCs were determined by colorimetric assays. Results. Pretreatment of PBMCs with melatonin significantly augmented expression and activity of MnSOD which were diminished by H2O2. Melatonin treatment of PBMCs caused a significant upregulation of catalase by almost 2-fold in comparison with untreated cells. However, activity and expression of catalase increased by 1.5-fold in PBMCs under H2O2-induced oxidative stress compared with untreated cell. Moreover, pretreatment of PBMCs with melatonin resulted in a significant 1.8-fold increase in catalase expression compared to PBMCs treated only with H2O2. Conclusion. It seems that melatonin could prevent from undesirable impacts of H2O2-induced oxidative stress on MnSOD downregulation. Moreover, melatonin could promote inductive effect of H2O2 on catalase mRNA expression. PMID:26881079

  16. TLR7 Agonist GS-9620 Is a Potent Inhibitor of Acute HIV-1 Infection in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Bam, Rujuta A.; Hansen, Derek; Irrinki, Alivelu; Mulato, Andrew; Jones, Gregg S.; Hesselgesser, Joseph; Frey, Christian R.; Cihlar, Tomas

    2016-01-01

    ABSTRACT GS-9620 is a potent and selective oral Toll-like receptor 7 (TLR7) agonist that directly activates plasmacytoid dendritic cells (pDCs). GS-9620 suppressed hepatitis B virus (HBV) in animal models of chronic infection and transiently activated HIV expression ex vivo in latently infected peripheral blood mononuclear cells (PBMCs) from virally suppressed patients. Currently, GS-9620 is under clinical evaluation for treating chronic HBV infection and for reducing latent reservoirs in virally suppressed HIV-infected patients. Here, we investigated the in vitro anti-HIV-1 activity of GS-9620. GS-9620 potently inhibited viral replication in PBMCs, particularly when it was added 24 to 48 h prior to HIV infection (50% effective concentration = 27 nM). Depletion of pDCs but not other immune cell subsets from PBMC cultures suppressed GS-9620 antiviral activity. Although GS-9620 was inactive against HIV in purified CD4+ T cells and macrophages, HIV replication was potently inhibited by conditioned medium derived from GS-9620-treated pDC cultures when added to CD4+ T cells prior to infection. This suggests that GS-9620-mediated stimulation of PBMCs induced the production of a soluble factor(s) inhibiting HIV replication in trans. GS-9620-treated PBMCs primarily showed increased production of interferon alpha (IFN-α), and cotreatment with IFN-α-blocking antibodies reversed the HIV-1-inhibitory effect of GS-9620. Additional studies demonstrated that GS-9620 inhibited a postentry event in HIV replication at a step coincident with or prior to reverse transcription. The simultaneous activation of HIV-1 expression and inhibition of HIV-1 replication are important considerations for the clinical evaluation of GS-9620 since these antiviral effects may help restrict potential local HIV spread upon in vivo latency reversal. PMID:27799218

  17. Selective suppression of cytokine secretion in whole blood cell cultures of patients with colorectal cancer.

    PubMed Central

    Lahm, H.; Schindel, M.; Frikart, L.; Cerottini, J. P.; Yilmaz, A.; Givel, J. C.; Fischer, J. R.

    1998-01-01

    We have investigated the secretion of interferon alpha (IFN-alpha), IFN-gamma, interleukin-1alpha (IL-1alpha), IL-1beta, IL-2 and tumour necrosis factor alpha (TNF-alpha) in whole blood cell cultures (WBCCs) of colorectal cancer patients upon mitogen stimulation. Whereas the values for IL-1beta and TNF-alpha remained virtually unchanged in comparison with healthy control subjects, WBCCs of colorectal cancer patients secreted significantly lower amounts of IFN-alpha (P < 0.005), IFN-gamma (P < 0.0001), IL-1alpha (P < 0.0001) and IL-2 (P < 0.05). This reduction correlated with the progression of the disease. The total leucocyte and monocyte population were almost identical in both groups. In contrast, a dramatic depletion of lymphocytes was observed in colorectal cancer patients, which affected both lymphocyte counts (P < 0.0005) and their distribution (P < 0.0001). Our results suggest a selective suppression of cytokines in colorectal cancer patients that is related to tumour burden. Several mechanisms might account for this phenomenon, one of which might be lymphocyte depletion. PMID:9792144

  18. Smart point-of-care systems for molecular diagnostics based on nanotechnology: whole blood glucose analysis

    NASA Astrophysics Data System (ADS)

    Devadhasan, Jasmine P.; Kim, Sanghyo

    2015-07-01

    Complementary metal oxide semiconductor (CMOS) image sensors are received great attention for their high efficiency in biological applications. The present work describes a CMOS image sensor-based whole blood glucose monitoring system through a point-of-care (POC) approach. A simple poly-ethylene terephthalate (PET) film chip was developed to carry out the enzyme kinetic reaction at various concentrations of blood glucose. In this technique, assay reagent was adsorbed onto amine functionalized silica (AFSiO2) nanoparticles in order to achieve glucose oxidation on the PET film chip. The AFSiO2 nanoparticles can immobilize the assay reagent with an electrostatic attraction and eased to develop the opaque platform which was technically suitable chip to analyze by the camera module. The oxidized glucose then produces a green color according to the glucose concentration and is analyzed by the camera module as a photon detection technique. The photon number decreases with increasing glucose concentration. The simple sensing approach, utilizing enzyme immobilized AFSiO2 nanoparticle chip and assay detection method was developed for quantitative glucose measurement.

  19. A comparative evaluation of four DNA extraction protocols from whole blood sample.

    PubMed

    Ghaheri, M; Kahrizi, D; Yari, K; Babaie, A; Suthar, R S; Kazemi, E

    2016-03-31

    All organisms have Deoxyribonucleic acid (DNA) within their cells. DNA is a complex molecule that contains all of the information necessary to build and maintain an organism. DNA extraction is one of the most basic and essential techniques in the study of DNA that allow huge advances in molecular biology, biotechnology and bioinformatics laboratories. Whole blood samples are one of the main sources used to obtain DNA and there are many different protocols available in this issue. In current research, compared four DNA extraction protocols from blood samples; include modified phenol-chloroform protocol, two salting-out and enzyme free method and from commercial kit. The extracted DNAs by these protocols were analyzed according to their time demands, quality and quantity, toxicity and functionality in PCR method. Also the quality and quantity of the extracted DNA were surveyed by gel electrophoresis and Nanodrop spectrophotometry methods. It was observed that there are not significantly differences between these methods about DNA Purity (A260/A280), but the DNA yield (ng DNA/μl) of phenol/chloroform method was higher than other methods. In addition, phenol/chloroform was the most toxic method and it takes more time than other methods. Roche diagnostics GmbH kit was the most expensive among the four methods but the least extraction time was required and it was the safest method.

  20. The effect of smoking on MAOA promoter methylation in DNA prepared from lymphoblasts and whole blood.

    PubMed

    Philibert, Robert A; Beach, Steven R H; Gunter, Tracy D; Brody, Gene H; Madan, Anup; Gerrard, Meg

    2010-03-05

    Prior work using lymphoblast DNA prepared from 192 subjects from the Iowa Adoption Studies (IAS) demonstrated that decreased MAOA promoter methylation was associated with lifetime symptom count for nicotine dependence (ND) and provided suggestive evidence that the amount of methylation is genotype dependent. In the current investigation, we replicate and extend these prior findings in three ways using another 289 IAS subjects and the same methodologies. First, we show that methylation is dependent on current smoking status. Second, we introduce a factor analytic approach to DNA methylation, highlighting three distinct regions of the promoter that may function in somewhat different ways for males and females. Third, we directly compare the methylation signatures in DNA prepared from whole blood and lymphoblasts from a subset of these subjects and provide suggestive evidence favoring the use of lymphoblast DNA. We conclude that smoking reliably decreases MAOA methylation, but exact characterization of effects on level of methylation depend on genotype, smoking history, current smoking status, gender, and region of the promoter-associated CpG Island examined.

  1. Comparison of plasma with whole blood prothrombin time and fibrinogen on the same instrument.

    PubMed

    Amukele, Timothy K; Ferrell, Chris; Chandler, Wayne L

    2010-04-01

    We compared plasma with whole blood (WB) international normalized ratio (INR) and fibrinogen using the same instrument and reagents. WBINRs were 50% higher than plasma INRs. After increasing the WB sample volume 40% and adjusting the International Sensitivity Index, WBINRs were similar to plasma INRs [adjusted WBINR = 0.99(plasma INR) - 0.02; r(2) = 0.98; n = 155], but the average difference in WB vs plasma INR was 4-fold higher than duplicate plasma INRs. Variation in hematocrit was a major determinant of the accuracy of the WBINR, with increased error at high INRs. The WB fibrinogen assay was highly dependent on the sample hematocrit (r(2) = 0.83), even after the sample volume was adjusted. Accurate WB fibrinogen measurements required a mathematical hematocrit correction. We conclude that WBINR and fibrinogen assays can be performed on point-of-care or automated analyzers, but sample volume must be adjusted to account for hematocrit. Accuracy is limited by variations in hematocrit with worsening accuracy for samples with high INRs or low fibrinogen levels.

  2. Rapid detection of cancer related DNA nanoparticulate biomarkers and nanoparticles in whole blood

    NASA Astrophysics Data System (ADS)

    Heller, Michael J.; Krishnan, Raj; Sonnenberg, Avery

    2010-08-01

    The ability to rapidly detect cell free circulating (cfc) DNA, cfc-RNA, exosomes and other nanoparticulate disease biomarkers as well as drug delivery nanoparticles directly in blood is a major challenge for nanomedicine. We now show that microarray and new high voltage dielectrophoretic (DEP) devices can be used to rapidly isolate and detect cfc-DNA nanoparticulates and nanoparticles directly from whole blood and other high conductance samples (plasma, serum, urine, etc.). At DEP frequencies of 5kHz-10kHz both fluorescent-stained high molecular weight (hmw) DNA, cfc-DNA and fluorescent nanoparticles separate from the blood and become highly concentrated at specific DEP highfield regions over the microelectrodes, while blood cells move to the DEP low field-regions. The blood cells can then be removed by a simple fluidic wash while the DNA and nanoparticles remain highly concentrated. The hmw-DNA could be detected at a level of <260ng/ml and the nanoparticles at <9.5 x 109 particles/ml, detection levels that are well within the range for viable clinical diagnostics and drug nanoparticle monitoring. Disease specific cfc-DNA materials could also be detected directly in blood from patients with Chronic Lymphocytic Leukemia (CLL) and confirmed by PCR genotyping analysis.

  3. Immediate adverse reactions to platelet transfusions: whole blood derived versus apheresis platelets.

    PubMed

    Salam, A; Hosain, G M; Hosain, M M; Narvios, A; Sazama, K; Lichtiger, B

    2013-01-01

    The transfusion of whole blood derived platelets (WBDPs) or apheresis platelets (APs) is standard support for cancer patients. However, disputes remain about which type of platelets are ideal in terms of efficacy, cost, and the risk of adverse reactions. This cross sectional study included 141 cancer patients who underwent chemotherapy or hematopoietic progenitor cell transplantation and received platelet transfusions at The University of Texas M.D. Anderson Cancer Center between 2002 and 2003 were retrospectively evaluated. A total of 141 patients who did not differ significantly in terms of age or sex had a reaction to transfusions (WBDPs, n=123; APs, n=18), for a frequency of 0.66% in patients who received WBDPs and 0.45% in patients who received APs, but this difference was not statistically significant (p=0.13). More WBDP-related reactions occurred in patients transfused with older platelets (>2 days old) than in patients transfused with fresh platelets, but the difference compared with AP-associated reactions was not statistically significant. However, the rate of reactions to WBDP may increase if WBDPs are stored for a prolonged time (>2 days). Until evidence becomes available that clearly refutes this; the more fresh platelets as possible may be used.

  4. Assessment of erythrocyte aggregation in whole blood samples by light backscattering: clinical applications

    NASA Astrophysics Data System (ADS)

    Priezzhev, Alexander V.; Firsov, Nikolai N.; Vyshlova, Marina G.; Lademann, Juergen; Richter, Heike; Kiesewetter, Holger; Mueller, Gerhard J.

    1999-05-01

    We report on the results of a collaborative effort made in the field of optical diagnostics of whole blood samples to study the ability of red blood cells to aggregate in a Couette chamber. We studied a possibility to quantitatively measure this ability as a function of the physiological state of blood donors. The aggregometer designed by the Russian coauthors of this paper and described in their earlier publications (see e.g. Proc SPIE 1884, 2100, 2678, 2982) was extensively used in the experiments performed in the Rheumatology Institute in Moscow and in the Charite Clinic in Berlin. The following parameters were measured: two characteristic times of RBC aggregation and the average spontaneous aggregation rate in the state of stasis, the average hydrodynamic strength of all aggregates and that of the largest aggregates. Different algorithms of the remission signal processing for the quantitative evaluation of the above parameters were compared. Reproducible alterations of the parameters from their normal values were obtained for blood samples from individuals suffering auto-immune disease and diabetes. Statistical data is reported proving high efficiency of the technique for the diagnostics of rheological disorders. Basing on these data the quantitative criteria of the heaviness of hemorheological state of the patients are proposed that are important for choosing specific therapies for which the patient is minimally resistant.

  5. Investigation of electrolyte measurement in diluted whole blood using spectroscopic and chemometric methods

    NASA Technical Reports Server (NTRS)

    Soller, Babs R.; Favreau, Janice; Idwasi, Patrick O.

    2003-01-01

    The feasibility of using near-infrared (NIR) spectroscopy in combination with partial least-squares (PLS) regression was explored to measure electrolyte concentration in whole blood samples. Spectra were collected from diluted blood samples containing randomized, clinically relevant concentrations of Na+, K+, and Ca2+. Sodium was also studied in lysed blood. Reference measurements were made from the same samples using a standard clinical chemistry instrument. Partial least squares (PLS) was used to develop calibration models for each ion with acceptable results (Na+, R2 = 0.86, CVSEP = 9.5 mmol/L; K+, R2 = 0.54, CVSEP = 1.4 mmol/L; Ca2+, R2 = 0.56, CVSEP = 0.18 mmol/L). Slightly improved results were obtained using a narrower wavelength region (470-925 nm) where hemoglobin, but not water, absorbed indicating that ionic interaction with hemoglobin is as effective as water in causing measurable spectral variation. Good models were also achieved for sodium in lysed blood, illustrating that cell swelling, which is correlated with sodium concentration, is not required for calibration model development.

  6. Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

    NASA Astrophysics Data System (ADS)

    Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

    2016-09-01

    Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis.

  7. Temperature and the respiratory properties of whole blood in two reptiles, Pogona barbata and Emydura signata.

    PubMed

    Stawski, Clare Y; Grigg, Gordon C; Booth, David T; Beard, Lyn A

    2006-02-01

    We investigated the capacity of two reptiles, an agamid lizard Pogona barbata and a chelid turtle Emydura signata, to compensate for the effects of temperature by making changes in their whole blood respiratory properties. This was accomplished by measuring the P50 (at 10, 20 and 30 degrees C), hematocrit (Hct), haemoglobin concentration ([Hb]) and mean cell haemoglobin concentration (MCHC) in field acclimatised and laboratory acclimated individuals. The acute effect of temperature on P50 in P. barbata, expressed as heat of oxygenation (deltaH), ranged from -16.8+/-1.84 to -28.5+/-2.73 kJ/mole. P50 of field acclimatised P. barbata increased significantly from early spring to summer at the test temperatures of 20 degrees C (43.1+/-1.2 to 48.8+/-2.1 mmHg) and 30 degrees C (54.7+/-1.2 to 65.2+/-2.3 mmHg), but showed no acclimation under laboratory conditions. For E. signata, deltaH ranged from -31.1+/-6.32 to -48.2+/-3.59 kJ/mole. Field acclimatisation and laboratory acclimation of P50 did not occur. However, in E. signata, there was a significant increase in [Hb] and MCHC from early spring to summer in turtles collected from the wild (1.0+/-0.1 to 1.7+/-0.2 mmol/L and 4.0+/-0.3 to 6.7+/-0.7 mmol/L, respectively).

  8. A fast LC-APCI/MS method for analyzing benzodiazepines in whole blood using monolithic support.

    PubMed

    Bugey, Aurélie; Rudaz, Serge; Staub, Christian

    2006-03-07

    A simple and fast procedure was developed for the simultaneous determination of eight benzodiazepines (BZDs) in whole blood using liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS). Sample pretreatment was carried out using a simple liquid-liquid extraction (LLE) with n-butylchloride, and chromatographic separation was performed using a monolithic silica column to speed up the analytical process. APCI and electrospray ionization (ESI) were compared. Whereas both ionization techniques appeared suitable for BZDs, APCI was found to be slightly more sensitive, especially for the determination of frequently low-dosed compounds. The method was validated according to the guidelines of the "Société Française des Sciences et Techniques Pharmaceutiques" (SFSTP) in the concentration range of 2.5-500 microg/L. The limit of quantification (LOQ) was 2.5 microg/L for all the compounds. Validation data including linearity, precision, and trueness were obtained, allowing subtherapeutic quantification of frequently low-dosed BZDs. The high selectivity of the mass spectrometer, along with the properties of the monolithic support, allowed unequivocal analysis of the eight compounds in less than 5 min. To demonstrate the potential of the method, it was used for the analysis of benzodiazepines in postmortem blood samples.

  9. Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

    PubMed Central

    Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

    2016-01-01

    Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis. PMID:27596736

  10. Pulsed photoacoustic flow imaging of whole blood with low frequency detection

    NASA Astrophysics Data System (ADS)

    van den Berg, Pim J.; Daoudi, Khalid; Steenbergen, Wiendelt

    2016-03-01

    Ultrasound flow imaging is widely used for quantification of blood flow in vivo, but estimating low flow velocities remains challenging. Pulsed photoacoustic flowmetry could be an alternative - but has not been shown capable of deep in vivo imaging so far. A new photoacoustic system is proposed, that has more potential for deep in-vivo applications. In this work the system is tested in vitro. In contrast to earlier research, 1064 nm NIR laser irradiation is used, that would allow deeper in-vivo light penetration. For detection, a 15 MHz transducer with lower in-vivo tissue ultrasound attenuation is used. Both changes are not trivial, as they reduce the overall visibility of the photoacoustic signals from red blood cells. This work shows the flow estimation performance of this system in vitro, and aims to serve as a point of reference when moving to an in-vivo application. Using this 15 MHz transducer, 1D flow profiles of flowing blood were extracted. The jitter (standard error) in the velocity estimation over the profiles was 10% when estimating flow of particles and 20% with whole blood; the bias in flow estimation was roughly 30%.

  11. A Novel Ultrasound-Based Method to Evaluate Hemostatic Function of Whole Blood

    PubMed Central

    Viola, Francesco; Mauldin, F. William; Lin-Schmidt, Xiefan; Haverstick, Doris M.; Lawrence, Michael B.; Walker, William F.

    2009-01-01

    Background: Unregulated hemostasis represents a leading cause of mortality and morbidity in the developed world. Being able to recognize and quantify defects of the hemostatic process is critical to reduce mortality and implement appropriate treatment. Methods: We describe a novel ultrasound-based technology, named sonorheometry, which can assess hemostasis function from a small sample of blood. Sonorheometry uses the phenomenon of acoustic radiation force to measure the dynamic changes in blood viscoelasticity during clot formation and clot dissolution. We performed in vitro experiments using whole blood samples of 1ml to demonstrate that sonorheometry is indicative of hemostatic functions that depend on plasma coagulation factors, platelets, and plasma fibrinolytic factors. Results: Sonorheometry measurements show titration effects to compounds known to alter the coagulation factors (GPRP peptide, 0 to 8 mmol/l), platelets (abciximab, 0 to 12 ug/ml), and fibrinolytic factors (urokinase, 0 to 200U). Repeated measurements of blood samples from the same subjects yielded reproducibility errors on the order of 5%. Conclusions: These data indicate that sonorheometry accurately quantifies the functional role of the components of hemostasis in vitro. PMID:19861121

  12. Differential DNA methylation patterns of polycystic ovarian syndrome in whole blood of Chinese women.

    PubMed

    Li, Shuxia; Zhu, Dongyi; Duan, Hongmei; Ren, Anran; Glintborg, Dorte; Andersen, Marianne; Skov, Vibe; Thomassen, Mads; Kruse, Torben; Tan, Qihua

    2016-05-12

    As a universally common endocrinopathy in women of reproductive age, the polycystic ovarian syndrome is characterized by composite clinical phenotypes reflecting the contributions of reproductive impact of ovarian dysfunction and metabolic abnormalities with widely varying symptoms resulting from interference of the genome with the environment through integrative biological mechanisms including epigenetics. We have performed a genome-wide DNA methylation analysis on polycystic ovarian syndrome and identified a substantial number of genomic sites differentially methylated in the whole blood of PCOS patients and healthy controls (52 sites, false discovery rate < 0.05 and corresponding p value < 5.68e-06), highly consistently replicating biological pathways extensively implicated in immunity and immunity-related inflammatory disorders (false discovery rate < 0.05) that were reportedly regulated in the DNA methylome from ovarian tissue under PCOS condition. Most importantly, our genome-wide profiling focusing on PCOS patients revealed a large number of DNA methylation sites and their enriched functional pathways significantly associated with diverse clinical features (levels of prolactin, estradiol, progesterone and menstrual cycle) that could serve as novel molecular basis of the clinical heterogeneity observed in PCOS women.

  13. Reactive Oxygen Species Formation and Apoptosis in Human Peripheral Blood Mononuclear Cell Induced by 900 MHz Mobile Phone Radiation

    PubMed Central

    Lu, Yao-Sheng; Huang, Bao-Tian; Huang, Yao-Xiong

    2012-01-01

    We demonstrate that reactive oxygen species (ROS) plays an important role in the process of apoptosis in human peripheral blood mononuclear cell (PBMC) which is induced by the radiation of 900 MHz radiofrequency electromagnetic field (RFEMF) at a specific absorption rate (SAR) of ~0.4 W/kg when the exposure lasts longer than two hours. The apoptosis is induced through the mitochondrial pathway and mediated by activating ROS and caspase-3, and decreasing the mitochondrial potential. The activation of ROS is triggered by the conformation disturbance of lipids, protein, and DNA induced by the exposure of GSM RFEMF. Although human PBMC was found to have a self-protection mechanism of releasing carotenoid in response to oxidative stress to lessen the further increase of ROS, the imbalance between the antioxidant defenses and ROS formation still results in an increase of cell death with the exposure time and can cause about 37% human PBMC death in eight hours. PMID:22778799

  14. Engraftment of tonsillar mononuclear cells in human skin/SCID mouse chimera--validation of a novel xenogeneic transplantation model for autoimmune diseases.

    PubMed

    Yamanaka, N; Yamamoto, Y; Kuki, K

    2001-01-01

    Pustulosis palmaris et plantaris (PPP) has been considered as one of the typical tonsillar focal infections, based on the marked clinical improvement of the skin lesions after tonsillectomy. Despite the accumulation of data showing the clinical efficacy of tonsillectomy for this skin lesion, fundamental etiological and pathophysiological issues have yet to be addressed. One primary obstacle hindering investigators has been the lack of an appropriate animal model for this human skin disorder. In the early stage of PPP, it has been reported that lymphocytes, predominantly CD4+ T lymphocytes, infiltrate the palmar and plantar skins. However, the origin and mechanism of infiltration by these lymphocytes is not clear and there are very few reports on whether tonsillar mononuclear cells react directly with the skin. We have been intrigued by the ability to engraft human cells onto severe combined immunodeficiency (SCID) mice, together with the opportunity for long-term graft survival and ability to adoptively transfer various human immunocompetent cells. In this review, we addressed the existing deficiencies in our understanding of the relationship between tonsils and PPP by using emerging transplantation technology involving SCID mice.

  15. An improved LC-MS/MS method for quantitation of indapamide in whole blood: application for a bioequivalence study.

    PubMed

    Pinto, Guilherme Araújo; Pastre, Kátia Isabel Fercondini; Bellorio, Karini Bruno; de Souza Teixeira, Leonardo; de Souza, Weidson Carlo; de Abreu, Fernanda Crunivel; de Santana E Silva Cardoso, Fabiana Fernandes; Pianetti, Gerson Antônio; César, Isabela Costa

    2014-09-01

    An improved LC-MS/MS method for the quantitation of indapamide in human whole blood was developed and validated. Indapamide-d3 was used as internal standard (IS) and liquid-liquid extraction was employed for sample preparation. LC separation was performed on Synergi Polar RP-column (50 × 4.6 mm i.d.; 4 µm) and mobile phase composed of methanol and 5 mm aqueous ammonium acetate containing 1 mm formic acid (60:40), at flow rate of 1 mL/min. The run time was 3.0 min and the injection volume was 20 μL. Mass spectrometric detection was performed using electrospray ion source in negative ionization mode, using the transitions m/z 364.0 → m/z 188.9 and m/z 367.0 → m/z 188.9 for indapamide and IS, respectively. Calibration curve was constructed over the range 0.25-50 ng/mL. The method was precise and accurate, and provided recovery rates >80% for indapamide and IS. The method was applied to determine blood concentrations of indapamide in a bioequivalence study with two sustained release tablet formulations. The 90% confidence interval for the geometric mean ratios for maximum concentration was 95.78% and for the area under the concentration-time curve it was 97.91%. The tested indapamide tablets (Eurofarma Laboratórios S.A.) were bioequivalent to Natrilix®, according to the rate and extent of absorption.

  16. Characterization of a new ion selective electrode for ionized magnesium in whole blood, plasma, serum, and aqueous samples.

    PubMed

    Altura, B T; Shirey, T L; Young, C C; Dell'Orfano, K; Hiti, J; Welsh, R; Yeh, Q; Barbour, R L; Altura, B M

    1994-01-01

    Results from a novel ion selective electrode (ISE) for ionized magnesium (Mg2+) correlate well with atomic absorption spectroscopy on aqueous solutions containing from 0.1-3.0 mmol MgCl2/L. Day to day precision (coefficient of variation) of the electrode on protein-based controls is < 4%; aqueous-based controls < 6%. The new ISE is selective for Mg2+ with a selectivity constant for Ca2+ (KMgCa) of 8 x 10(-2). Adding pathophysiologic concentrations of Cd2+, Ca2+, Cu2+, Fe3+, K+, Na+, or Zn2+ to serum and aqueous solutions gave negligible to minimal changes in measured Mg2+. Ligand binding studies in aqueous solution indicate that pathophysiologic concentrations of different anions (e.g. heparin, lactate, bicarbonate, phosphate, acetate and sulfate) bind to Mg2+, effectively reducing its concentration in solution. Likewise, silicon (as either found in Vacuutainer tubes or as chlorosilane) failed to exert any significant effect on measured Mg2+. Addition of Intralipid (up to 500 mg/dL) gave negligible to minimal changes in Mg2+. Mg2+ measurements on whole blood, plasma, and serum for a given human subject's samples are virtually identical, at least within the reference range for Mg2+. Typically, Mg2+ is 71% of TMg, but varies from subject to subject; i.e. Mg2+ cannot be predicted from TMg. Clinical studies revealed that the Mg2+/TMg ratio could be remarkably consistent in sequential samples (e.g., throughout the course of coronary bypass surgery) taken from one patient, but that this ratio could differ dramatically from the ratio in sequential samples taken from another. Mg2+ is held within a narrow range (0.53-0.67 mmol/L) in normal, healthy subjects when compared to TMg (0.70-0.96 mmol/L).

  17. Biomarker-based classification of bacterial and fungal whole-blood infections in a genome-wide expression study

    PubMed Central

    Dix, Andreas; Hünniger, Kerstin; Weber, Michael; Guthke, Reinhard; Kurzai, Oliver; Linde, Jörg

    2015-01-01

    Sepsis is a clinical syndrome that can be caused by bacteria or fungi. Early knowledge on the nature of the causative agent is a prerequisite for targeted anti-microbial therapy. Besides currently used detection methods like blood culture and PCR-based assays, the analysis of the transcriptional response of the host to infecting organisms holds great promise. In this study, we aim to examine the transcriptional footprint of infections caused by the bacterial pathogens Staphylococcus aureus and Escherichia coli and the fungal pathogens Candida albicans and Aspergillus fumigatus in a human whole-blood model. Moreover, we use the expression information to build a random forest classifier to classify if a sample contains a bacterial, fungal, or mock-infection. After normalizing the transcription intensities using stably expressed reference genes, we filtered the gene set for biomarkers of bacterial or fungal blood infections. This selection is based on differential expression and an additional gene relevance measure. In this way, we identified 38 biomarker genes, including IL6, SOCS3, and IRG1 which were already associated to sepsis by other studies. Using these genes, we trained the classifier and assessed its performance. It yielded a 96% accuracy (sensitivities >93%, specificities >97%) for a 10-fold stratified cross-validation and a 92% accuracy (sensitivities and specificities >83%) for an additional test dataset comprising Cryptococcus neoformans infections. Furthermore, the classifier is robust to Gaussian noise, indicating correct class predictions on datasets of new species. In conclusion, this genome-wide approach demonstrates an effective feature selection process in combination with the construction of a well-performing classification model. Further analyses of genes with pathogen-dependent expression patterns can provide insights into the systemic host responses, which may lead to new anti-microbial therapeutic advances. PMID:25814982

  18. Biomarker-based classification of bacterial and fungal whole-blood infections in a genome-wide expression study.

    PubMed

    Dix, Andreas; Hünniger, Kerstin; Weber, Michael; Guthke, Reinhard; Kurzai, Oliver; Linde, Jörg

    2015-01-01

    Sepsis is a clinical syndrome that can be caused by bacteria or fungi. Early knowledge on the nature of the causative agent is a prerequisite for targeted anti-microbial therapy. Besides currently used detection methods like blood culture and PCR-based assays, the analysis of the transcriptional response of the host to infecting organisms holds great promise. In this study, we aim to examine the transcriptional footprint of infections caused by the bacterial pathogens Staphylococcus aureus and Escherichia coli and the fungal pathogens Candida albicans and Aspergillus fumigatus in a human whole-blood model. Moreover, we use the expression information to build a random forest classifier to classify if a sample contains a bacterial, fungal, or mock-infection. After normalizing the transcription intensities using stably expressed reference genes, we filtered the gene set for biomarkers of bacterial or fungal blood infections. This selection is based on differential expression and an additional gene relevance measure. In this way, we identified 38 biomarker genes, including IL6, SOCS3, and IRG1 which were already associated to sepsis by other studies. Using these genes, we trained the classifier and assessed its performance. It yielded a 96% accuracy (sensitivities >93%, specificities >97%) for a 10-fold stratified cross-validation and a 92% accuracy (sensitivities and specificities >83%) for an additional test dataset comprising Cryptococcus neoformans infections. Furthermore, the classifier is robust to Gaussian noise, indicating correct class predictions on datasets of new species. In conclusion, this genome-wide approach demonstrates an effective feature selection process in combination with the construction of a well-performing classification model. Further analyses of genes with pathogen-dependent expression patterns can provide insights into the systemic host responses, which may lead to new anti-microbial therapeutic advances.

  19. Effects of oral N-acetylcysteine on plasma homocysteine and whole blood glutathione levels in healthy, non-pregnant women.

    PubMed

    Roes, Eva Maria; Raijmakers, Maarten T M; Peters, Wilbert H M; Steegers, Eric A P

    2002-05-01

    Oral N-acetylcysteine supplementation in nine young healthy females induced a quick and highly significant decrease in plasma homocysteine levels and an increase in whole blood concentration of the antioxidant glutathione. N-acetylcysteine impresses as an efficient drug in lowering homocysteine concentration and might be beneficial for individuals with hyperhomocysteinemia who are at increased risk of cardiovascular disease.

  20. Method and kit for the selective labeling of red blood cells in whole blood with TC-99M

    DOEpatents

    Srivastava, Suresh C.; Babich, John W.; Straub, Rita; Richards, Powell

    1988-01-01

    Disclosed herein are a method and kit for the preparation of .sup.99m Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available therein for the reduction of technetium.

  1. Optimization of a Whole-Blood Gamma Interferon Assay for Detection of Mycobacterium bovis-Infected Cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antigens of Mycobacterium bovis elicit a cell-mediated immune response upon intradermalinjection in cattle. In vitro, such antigens stimulate the production of interferon (IFN)-gamma by bovine T-cells in whole blood culture (IFN-gamma assay). We have analyzed various parameters of the in vitro IFN-g...

  2. Rapid on-chip recalcification and drug dosing of citrated whole blood using microfluidic buffer sheath flow.

    PubMed

    Muthard, Ryan W; Diamond, Scott L

    2014-01-01

    Millions of clotting tests each year require recalcification of blood treated with sodium citrate, a calcium chelator that prevents prothrombinase assembly. We validated a converging trifurcated microfluidic device to measure platelet and fibrin accumulation following on-chip recalcification of citrated whole blood. Recalcification was accomplished by sheathing the blood with Ca2+ buffer. Fluorescein rapidly diffused across the buffer-blood interface (achieving 62.5% of maximum centerline concentration within ~4 cm of flow), while albumin remained relatively unchanged in blood due to its lower diffusivity (<20% decrease). Since Ca2+ diffuses faster than fluorescein, full recalcification of whole blood was achieved within ~1 cm of flow prior to encountering a collagen/tissue surface. Pla