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Sample records for addition blood samples

  1. Sampling blood from the lateral tail vein of the rat.

    PubMed

    Lee, Graham; Goosens, Ki A

    2015-01-01

    Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself. PMID:26065632

  2. 7 CFR 27.25 - Additional samples of cotton; drawing.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Additional samples of cotton; drawing. 27.25 Section... CONTAINER REGULATIONS COTTON CLASSIFICATION UNDER COTTON FUTURES LEGISLATION Regulations Inspection and Samples § 27.25 Additional samples of cotton; drawing. In addition to the samples hereinbefore...

  3. 7 CFR 27.25 - Additional samples of cotton; drawing.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Additional samples of cotton; drawing. 27.25 Section... CONTAINER REGULATIONS COTTON CLASSIFICATION UNDER COTTON FUTURES LEGISLATION Regulations Inspection and Samples § 27.25 Additional samples of cotton; drawing. In addition to the samples hereinbefore...

  4. 7 CFR 27.25 - Additional samples of cotton; drawing.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Additional samples of cotton; drawing. 27.25 Section... CONTAINER REGULATIONS COTTON CLASSIFICATION UNDER COTTON FUTURES LEGISLATION Regulations Inspection and Samples § 27.25 Additional samples of cotton; drawing. In addition to the samples hereinbefore...

  5. 7 CFR 27.25 - Additional samples of cotton; drawing.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Additional samples of cotton; drawing. 27.25 Section... CONTAINER REGULATIONS COTTON CLASSIFICATION UNDER COTTON FUTURES LEGISLATION Regulations Inspection and Samples § 27.25 Additional samples of cotton; drawing. In addition to the samples hereinbefore...

  6. 7 CFR 27.25 - Additional samples of cotton; drawing.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Additional samples of cotton; drawing. 27.25 Section... CONTAINER REGULATIONS COTTON CLASSIFICATION UNDER COTTON FUTURES LEGISLATION Regulations Inspection and Samples § 27.25 Additional samples of cotton; drawing. In addition to the samples hereinbefore...

  7. Manual versus automated blood sampling: impact of repeated blood sampling on stress parameters and behavior in male NMRI mice

    PubMed Central

    Kalliokoski, Otto; Sørensen, Dorte B; Hau, Jann; Abelson, Klas S P

    2014-01-01

    Facial vein (cheek blood) and caudal vein (tail blood) phlebotomy are two commonly used techniques for obtaining blood samples from laboratory mice, while automated blood sampling through a permanent catheter is a relatively new technique in mice. The present study compared physiological parameters, glucocorticoid dynamics as well as the behavior of mice sampled repeatedly for 24 h by cheek blood, tail blood or automated blood sampling from the carotid artery. Mice subjected to cheek blood sampling lost significantly more body weight, had elevated levels of plasma corticosterone, excreted more fecal corticosterone metabolites, and expressed more anxious behavior than did the mice of the other groups. Plasma corticosterone levels of mice subjected to tail blood sampling were also elevated, although less significantly. Mice subjected to automated blood sampling were less affected with regard to the parameters measured, and expressed less anxious behavior. We conclude that repeated blood sampling by automated blood sampling and from the tail vein is less stressful than cheek blood sampling. The choice between automated blood sampling and tail blood sampling should be based on the study requirements, the resources of the laboratory and skills of the staff. PMID:24958546

  8. 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.

  9. Automated quantitation of hemoglobin-based blood substitutes in whole blood samples.

    PubMed

    Kunicka, J; Malin, M; Zelmanovic, D; Katzenberg, M; Canfield, W; Shapiro, P; Mohandas, N

    2001-12-01

    It is necessary to develop methods for accurate monitoring of cell-free hemoglobin in circulation. Routine monitoring of circulating cell-free hemoglobin will be useful for evaluating the efficacy of blood substitute administration andfor determining the clearance rates of the blood substitute from circulation. In addition, discriminating between cell-free hemoglobin and cell-associated hemoglobin will enable accurate determination of RBC indices, mean cell hemoglobin and mean corpuscular hemoglobin concentration, in individuals receiving hemoglobin-based blood substitutes. As colorimetric methods used by hematology analyzers to quantitate the hemoglobin value of a blood sample cannot distinguish between cell-associated and cell-free hemoglobin, it is currently not feasible to quantitate the levels of hemoglobin substitutes in circulation. The advent of a technology that measures volume and hemoglobin concentration of individual RBCs provides an alternative strategy for quantitating the cell-associated hemoglobin in a blood sample. We document that the combined use of cell-based and colorimetric hemoglobin measurements provides accurate discrimination between cell-associated and cell-free hemoglobin over a wide range of hemoglobin levels. This strategy should enable rapid and accurate monitoring of the levels of cell-free hemoglobin substitutes in the circulation of recipients of these blood substitutes.

  10. Development of blood extraction system designed by female mosquito's blood sampling mechanism for bio-MEMS

    NASA Astrophysics Data System (ADS)

    Tsuchiya, Kazuyoshi; Nakanishi, Naoyuki; Nakamachi, Eiji

    2005-02-01

    A compact and wearable wristwatch type Bio-MEMS such as a health monitoring system (HMS) to detect blood sugar level for diabetic patient, was newly developed. The HMS consists of (1) a indentation unit with a microneedle to generate the skin penetration force using a shape memory alloy(SMA) actuator, (2) a pumping unit using a bimorph PZT piezoelectric actuator to extract the blood and (3) a gold (Au) electrode as a biosensor immobilized GOx and attached to the gate electrode of MOSFET to detect the amount of Glucose in extracted blood. GOx was immobilized on a self assembled spacer combined with an Au electrode by the cross-link method using BSA as an additional bonding material. The device can extract blood in a few microliter through a painless microneedle with the negative pressure by deflection of the bimorph PZT piezoelectric actuator produced in the blood chamber, by the similar way the female mosquito extracts human blood with muscle motion to flex or relax. The performances of the liquid sampling ability of the pumping unit through a microneedle (3.8mm length, 100μm internal diameter) using the bimorph PZT piezoelectric microactuator were measured. The blood extraction micro device could extract human blood at the speed of 2μl/min, and it is enough volume to measure a glucose level, compared to the amount of commercial based glucose level monitor. The electrode embedded in the blood extraction device chamber could detect electrons generated by the hydrolysis of hydrogen peroxide produced by the reaction between GOx and glucose in a few microliter extracted blood, using the constant electric current measurement system of the MOSFET type hybrid biosensor. The output voltage for the glucose diluted in the chamber was increased lineally with increase of the glucose concentration.

  11. Percutaneous umbilical cord blood sampling - series (image)

    MedlinePlus

    ... the spot where the umbilical cord meets the placenta. He then inserts a needle through your abdomen ... retrieving fetal blood: Placing the needle through the placenta or through the amniotic sac. The placenta's position ...

  12. Whole Genome Amplification from Blood Spot Samples.

    PubMed

    Sørensen, Karina Meden

    2015-01-01

    Whole genome amplification is an invaluable technique when working with DNA extracted from blood spots, as the DNA obtained from this source often is too limited for extensive genetic analysis. Two techniques that amplify the entire genome are common. Here, both are described with focus on the benefits and drawbacks of each system. However, in order to obtain the best possible WGA result the quality of input DNA extracted from the blood spot is essential, but also time consumption, flexibility in format and elution volume and price of the technology are factors influencing system choice. Here, three DNA extraction techniques are described and the above aspects are compared between the systems.

  13. Blood sample stability at room temperature for counting red and white blood cells and platelets.

    PubMed

    Vogelaar, S A; Posthuma, D; Boomsma, D; Kluft, C

    2002-08-01

    Blood handling required for different cellular variables is different. In a practical setting of blood sampling approximately 4 h separated from the first analysis, we compared the analysis of blood cell variables at this 4-h point with analysis of blood stored for approximately 48 h (over the weekend) at room temperature. Blood was collected from 304 apparently healthy individuals aged between 17 and 70 years, with a female/male ratio of 1.8, in K3EDTA. Measurement was performed with a Beckman Coulter Counter Maxm. In addition to the comparison of the data and their correlation on the two time points, we investigated agreement between the data using analysis according to Bland and Altman. Counts of white and red blood cells and platelets were found stable over time and agreement of data was excellent. Platelet mean volume increased as expected between the two time points from 8.8 to 10.3 fl. The white blood cell subpopulations, however, changed over time with a decrease in neutrophils and monocytes and increases in lymphocytes and eosinophils. Apparently, ageing of the sample resulted in the alteration of certain cell characteristics leading to a change in automated cell classification without changing the total number of cells. Among the preanalytical variables recorded, only the time of the year and gender were found to be minor determinants (r < .25) of some of the differences between approximately 4 and approximately 48 h analysis delay. It is concluded that after storage at room temperature over approximately 48 h counts of red, total white cells, platelets and analysis of platelet volume can be combined in one assay session.

  14. 21 CFR 71.4 - Samples; additional information.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... samples of the color additive, articles used as components thereof, or of the food, drug, or cosmetic in... additive, or articles used as components thereof, or of the food, drug, or cosmetic in which the color... respect to the safety of the color additive or the physical or technical effect it produces. The date...

  15. 21 CFR 71.4 - Samples; additional information.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... samples of the color additive, articles used as components thereof, or of the food, drug, or cosmetic in... additive, or articles used as components thereof, or of the food, drug, or cosmetic in which the color... respect to the safety of the color additive or the physical or technical effect it produces. The date...

  16. Payload specialist Reinhard Furrer show evidence of previous blood sampling

    NASA Technical Reports Server (NTRS)

    1985-01-01

    Payload specialist Reinhard Furrer shows evidence of previous blood sampling while Wubbo J. Ockels, Dutch payload specialist (only partially visible), extends his right arm after a sample has been taken. Both men show bruises on their arms.

  17. 21 CFR 868.1100 - Arterial blood sampling kit.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Arterial blood sampling kit. 868.1100 Section 868.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit....

  18. 21 CFR 868.1100 - Arterial blood sampling kit.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Arterial blood sampling kit. 868.1100 Section 868.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit....

  19. 21 CFR 868.1100 - Arterial blood sampling kit.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Arterial blood sampling kit. 868.1100 Section 868.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit....

  20. 21 CFR 868.1100 - Arterial blood sampling kit.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Arterial blood sampling kit. 868.1100 Section 868.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit....

  1. 21 CFR 868.1100 - Arterial blood sampling kit.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Arterial blood sampling kit. 868.1100 Section 868.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit....

  2. Blood oxygen saturation determined by transmission spectrophotometry of hemolyzed blood samples

    NASA Technical Reports Server (NTRS)

    Malik, W. M.

    1967-01-01

    Use of the Lambert-Beer Transmission Law determines blood oxygen saturation of hemolyzed blood samples. This simplified method is based on the difference in optical absorption properties of hemoglobin and oxyhemoglobin.

  3. Analysis of zinc in biological samples by flame atomic absorption spectrometry: use of addition calibration technique.

    PubMed

    Dutra, Rosilene L; Cantos, Geny A; Carasek, Eduardo

    2006-01-01

    The quantification of target analytes in complex matrices requires special calibration approaches to compensate for additional capacity or activity in the matrix samples. The standard addition is one of the most important calibration procedures for quantification of analytes in such matrices. However, this technique requires a great number of reagents and material, and it consumes a considerable amount of time throughout the analysis. In this work, a new calibration procedure to analyze biological samples is proposed. The proposed calibration, called the addition calibration technique, was used for the determination of zinc (Zn) in blood serum and erythrocyte samples. The results obtained were compared with those obtained using conventional calibration techniques (standard addition and standard calibration). The proposed addition calibration was validated by recovery tests using blood samples spiked with Zn. The range of recovery for blood serum and erythrocyte samples were 90-132% and 76-112%, respectively. Statistical studies among results obtained by the addition technique and conventional techniques, using a paired two-tailed Student's t-test and linear regression, demonstrated good agreement among them. PMID:16943611

  4. Are They Bloody Guilty? Blood Doping with Simulated Samples

    ERIC Educational Resources Information Center

    Stuart, Parker E.; Lees, Kelsey D.; Milanick, Mark A.

    2014-01-01

    In this practice-based lab, students are provided with four Olympic athlete profiles and simulated blood and urine samples to test for illegal substances and blood-doping practices. Throughout the course of the lab, students design and conduct a testing procedure and use their results to determine which athletes won their medals fairly. All of the…

  5. Non-terminal blood sampling techniques in guinea pigs.

    PubMed

    Birck, Malene M; Tveden-Nyborg, Pernille; Lindblad, Maiken M; Lykkesfeldt, Jens

    2014-01-01

    Guinea pigs possess several biological similarities to humans and are validated experimental animal models(1-3). However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages(4,5). Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals(6). All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility. PMID:25350490

  6. Non-terminal blood sampling techniques in guinea pigs.

    PubMed

    Birck, Malene M; Tveden-Nyborg, Pernille; Lindblad, Maiken M; Lykkesfeldt, Jens

    2014-01-01

    Guinea pigs possess several biological similarities to humans and are validated experimental animal models(1-3). However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages(4,5). Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals(6). All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility.

  7. Non-Terminal Blood Sampling Techniques in Guinea Pigs

    PubMed Central

    Birck, Malene M.; Tveden-Nyborg, Pernille; Lindblad, Maiken M.; Lykkesfeldt, Jens

    2014-01-01

    Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages4,5. Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals6. All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility. PMID:25350490

  8. Percutaneous umbilical blood sampling and umbilical vein transfusions. Rapid serologic differentiation of fetal blood from maternal blood.

    PubMed

    Steiner, E A; Judd, W J; Oberman, H A; Hayashi, R H; Nugent, C E

    1990-02-01

    Percutaneous umbilical blood samples (PUBS), obtained under ultrasound guidance, are used for prenatal diagnosis and management of hemolytic disease of the newborn (HDN) and other fetal disorders. Rapid testing at the time of sampling is vital to distinguish fetal from maternal blood. Blood typing was performed by slide technique in the treatment room during 38 procedures on 25 patients. Anti-I was used to test 50 presumed PUBS; venous I-positive maternal blood was tested in parallel. Because anti-I cannot detect fetal blood after umbilical vein transfusion (UVT) of I-positive donor blood, ABO and Rh blood typing reagents were used to test 29 samples when maternal and fetal or donor blood groups differed. Monoclonal reagents were used for optimal detection of weak AB antigens in fetal blood. Avid, chemically modified anti-D was used for Rh typing. Blood typing showed 27 (34%) of 79 samples to be maternal blood. Fetal blood was obtained in 8 of 10 cases investigated for fetal disorder and in 16 cases of potential HDN (anti-D, 5; -CD, 5; -cE, 2; -K, 2; -c; -E). The absence of HDN (antigen-negative fetus) was determined in 4 cases. UVT afforded live birth of 9 of 10 infants with HDN and was not indicated in two cases.

  9. Astronaut Joseph Kerwin takes blood sample from Astronaut Charles Conrad

    NASA Technical Reports Server (NTRS)

    1973-01-01

    Scientist-Astronaut Joseph P. Kerwin (right), Skylab 2 science pilot and a doctor of medicine, takes a blood sample from Astronaut Charles Conrad Jr., Sylab 2 commander, as seen in this reproduction taken from a color television transmission made by a TV camera aboard the Skylab 1 and 2 space station cluster in Earth orbit. The blood sampling was part of the Skylab Hematology and Immunology Experiment M110 series.

  10. Factors affecting contamination of blood samples for ethanol determinations.

    PubMed

    Winek, C L; Eastly, T

    1977-01-01

    Contamination of blood samples collected for alcohol analysis from swabbing with an ethanolic antiseptic is minimal (less than 0.6 mg/100 ml or 0.0006 percent ethanol) when routine clinical technique is followed. When technicians were told to be deliberately sloppy, considerable contamination (89 mg/100 ml or 0.09 percent ethanol) occurred. The incidence and extent of contamination from banked blood intended for transfusions are minimal. Two percent of the 1,450 samples analyzed contained alcohol. The average blood alcohol concentration was 26 mg/100 ml or 0.03 percent ethanol. One microliter of rubbing alcohol per milliliter of whole blood, or one-tenth of a drop of rubbing alcohol per milliliter of whole blood, increases the BAC 56.5 mg/100 ml (0.06 percent ethanol) and 67.5 mg/100 ml (0.07 percent ethanol), respectively.

  11. Increased reticulocyte count from cord blood samples using hypotonic lysis.

    PubMed

    Grimberg, Brian T; Scheetz, Emily A; Erickson, John J; Bales, Jacquelyn M; David, Makindi; Daum-Woods, Kathleen; King, Christopher L; Zimmerman, Peter A

    2012-10-01

    Human reticulocytes are one of the fundamental components needed to study the in vitro invasion processes of the human malaria parasite Plasmodium vivax. Additionally examinations of reticulocytes and their binding proteins are difficult in areas of the world that do not have access to advanced equipment or stem cell lines. These issues are particularly relevant to malaria vaccine candidate studies that are directed against surface proteins that the parasites use to gain entry into erythrocytes. Described here is a simple and inexpensive method to increase the reticulocyte count of cord blood samples. Exposure of cord blood to hypotonic saline (0.2%) for 5 min selectively lyses the non-reticulocytes resulting in an average 3.6-fold increase in reticulocyte count. Our studies show that this enrichment process does not damage the hemoglobin of the remaining erythrocytes which are still capable of supporting Plasmodium falciparum invasion and growth. This economical and rapid method of enrichment could facilitate studies of in vitro laboratory culturing of other malaria parasite species which preferentially invade reticulocytes such as P. vivax.

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

    PubMed Central

    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 (R2 > 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

  13. Determination of chlorinated insecticides in blood samples of agricultural workers.

    PubMed

    Rosell, M G; Obiols, J; Berenguer, M J; Guardino, X; López, F; Brosa, J

    1993-11-26

    Lindane, aldrin and p,p'-DDT were determined in blood samples from 71 farmers by means of an analytical method which combines a direct whole-blood extraction with n-hexane and gas chromatography (GC)-electron-capture detection (ECD), using a capillary column, applied to the organic extract. This technique allowed the determination of pesticides at levels varying from 0.1 to 180 micrograms per l of blood, the detection limit for every pesticide being 0.1 microgram/l. GC-mass spectrometry was used to confirm the identity of each pesticide. The advantage of capillary column GC-ECD for pesticide determination is its sensitivity and high resolution, which makes it possible to separate pesticides from a complex n-hexane extract obtained in a very simple pretreatment of the blood sample, which is itself a very complex matrix.

  14. Prospects of Vitamin C as an Additive in Plasma of Stored Blood

    PubMed Central

    Vani, R.; Soumya, R.; Carl, H.; Chandni, V. A.; Neha, K.; Pankhuri, B.; Trishna, S.; Vatsal, D. P.

    2015-01-01

    There is a dire necessity to improve blood storage and prolong shelf-life of blood. Very few studies have focused on oxidative stress (OS) in blood and its influence on plasma with storage. This study attempts to (i) elucidate the continuous changes occurring in plasma during storage through oxidant levels and antioxidant status and (ii) evaluate the influence of vitamin C (VC) as an additive during blood storage. Blood was drawn from male Wistar rats and stored for 25 days at 4°C. Blood samples were divided into control and experimental groups. Plasma was isolated every 5 days and the OS markers, antioxidant enzymes, lipid peroxidation, and protein oxidation products, were studied. Catalase activity increased in all groups with storage. Lipid peroxidation decreased in VC (10) but was maintained in VC (30) and VC (60). Although there were variations in all groups, carbonyls were maintained towards the end of storage. Advanced oxidation protein products (AOPP) increased in VC (30) and were maintained in VC (10) and VC (60). Sulfhydryls were maintained in all groups. Vitamin C could not sufficiently attenuate OS and hence, this opens the possibilities for further studies on vitamin C in combination with other antioxidants, in storage solutions. PMID:26345502

  15. Viscosity measurements on very small capillary blood samples.

    PubMed

    Eugster, M; Häusler, K; Reinhart, W H

    2007-01-01

    Viscosity measurements on very small capillary blood samples could be of considerable clinical interest. We have developed an oscillating viscometer for very small volumes, which consists of a glass capillary containing 7 mul of blood, which is part of an oscillating torsional resonator. The damping of the sinusoidal oscillations depends on the density and viscosity of the fluid, which allows blood viscosity measurements. The instrument was first evaluated in comparison with a standard blood viscometer (Contraves LS 30). Blood from healthy volunteers anticoagulated with EDTA was adjusted to hematocrit levels of 20, 30, 40, 50, and 60%, respectively. A strong correlation was found between hematocrit and oscillating viscosity (y=0.17x-2.05, r=0.969, p<0.0001) and between oscillating and conventional high shear viscosity (y=1.11x-0.62, r=0.971, p<0.0001). Blood viscosity measured in venous or capillary blood of normal subjects was similar (p=0.63). Bedside viscosity measurements on capillary blood drawn from a finger prick during routine blood glucose measurements in patients with diabetes mellitus showed lower blood viscosity than controls (3.62+/-0.87 vs 4.79+/-0.59 mPa.s, p=0.0007), which is in contrast to earlier publications, and may be explained by the lower hematocrit in our diabetic patients (34.7+/-6.0% vs. 43.1+/-1.9%, p<0.0001). Blood viscosity was independent of the actual glucose level (range 3-17 mmol/l). Capillary blood anticoagulated with EDTA was drawn by heel prick from 23 newborns. Blood viscosity was higher (5.66 +/-2.47 mPa.s) than in adult controls (see above), which could be explained by the dependence on the higher hematocrit (46.4 +/-8.6%). We conclude that viscosity measurements can be made on very small samples such as capillary blood from diabetic patients or newborn babies with this new oscillating viscometer. It remains to be determined if such new informations have clinical implications.

  16. Automated blood-sample handling in the clinical laboratory.

    PubMed

    Godolphin, W; Bodtker, K; Uyeno, D; Goh, L O

    1990-09-01

    The only significant advances in blood-taking in 25 years have been the disposable needle and evacuated blood-drawing tube. With the exception of a few isolated barcode experiments, most sample-tracking is performed through handwritten or computer-printed labels. Attempts to reduce the hazards of centrifugation have resulted in air-tight lids or chambers, the use of which is time-consuming and cumbersome. Most commonly used clinical analyzers require serum or plasma, distributed into specialized containers, unique to that analyzer. Aliquots for different tests are prepared by handpouring or pipetting. Moderate to large clinical laboratories perform so many different tests that even multi-analyzers performing multiple analyses on a single sample may account for only a portion of all tests ordered for a patient. Thus several aliquots of each specimen are usually required. We have developed a proprietary serial centrifuge and blood-collection tube suitable for incorporation into an automated or robotic sample-handling system. The system we propose is (a) safe--avoids or prevents biological danger to the many "handlers" of blood; (b) small--minimizes the amount of sample taken and space required to adapt to the needs of satellite and mobile testing, and direct interfacing with analyzers; (c) serial--permits each sample to be treated according to its own "merits," optimizes throughput, and facilitates flexible automation; and (d) smart--ensures quality results through monitoring and intelligent control of patient identification, sample characteristics, and separation process.

  17. Polybrominated diphenyl ethers in maternal and fetal blood samples.

    PubMed Central

    Mazdai, Anita; Dodder, Nathan G; Abernathy, Mary Pell; Hites, Ronald A; Bigsby, Robert M

    2003-01-01

    Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants in consumer goods, such as plastics, electronics, textiles, and construction material. PBDEs have been found in human milk, fat, and blood samples. Rodent studies indicate that PBDEs may be detrimental to neurodevelopment, possibly by lowering thyroid hormone concentrations in blood. In the present study, we determined concentrations of PBDEs and thyroid hormones in human fetal and maternal serum. Patients presenting in labor to Indiana University and Wishard Memorial County hospitals in Indianapolis, who were older than 18 years, were recruited to participate. Twelve paired samples of maternal and cord blood were obtained and analyzed using gas chromatographic mass spectrometry; thyroid hormone concentrations were determined by radioimmunoassay. Six congeners of PBDE were measured in maternal and fetal serum samples. The concentrations of total PBDEs found in maternal sera ranged from 15 to 580 ng/g lipid, and the concentrations found in fetal samples ranged from 14 to 460 ng/g lipid. Individual fetal blood concentrations did not differ from the corresponding maternal concentrations, indicating that measurement of maternal PBDE blood levels is useful in predicting fetal exposure; similarly, other reports have shown a high correlation between PBDE in mother's milk and fetal exposure. In accord with reports on other biologic samples, the tetrabrominated PBDE congener BDE-47 accounted for 53-64% of total PBDEs in the serum. The concentrations of PBDEs found in maternal and fetal serum samples were 20-106-fold higher than the levels reported previously in a similar population of Swedish mothers and infants. In this small sample, there was no apparent correlation between serum PBDEs and thyroid hormone concentrations. Our study shows that human fetuses in the United States may be exposed to relatively high levels of PBDEs. Further investigation is required to determine if these levels are

  18. Diagnosis of Carrion’s Disease by Direct Blood PCR in Thin Blood Smear Negative Samples

    PubMed Central

    Tinco Valdez, Carmen; Pons, Maria J.; del Valle, Luis J.; Oré, Verónica Casabona; Michelena, Denisse Champin; Mayra, Jorge Bazán; Gavidea, Víctor Zavaleta; Vargas, Martha; Ruiz, Joaquim

    2014-01-01

    Bartonella bacilliformis is the etiologic agent of Carrion's disease. This disease has two well established phases, the most relevant being the so called Oroya Fever, in which B. bacilliformis infect the erythrocytes resulting in severe anemia and transient immunosuppression, with a high lethality in the absence of adequate antibiotic treatment. The presence of B. bacilliformis was studied in 113 blood samples suspected of Carrion’s disease based on clinical criteria, despite the absence of a positive thin blood smear, by two different PCR techniques (using Bartonella-specific and universal 16S rRNA gene primers), and by bacterial culture. The specific 16S rRNA gene primers revealed the presence of 21 B. bacilliformis and 1 Bartonella elizabethae, while universal primers showed both the presence of 3 coinfections in which a concomitant pathogen was detected plus Bartonella, in addition to the presence of infections by other microorganisms such as Agrobacterium or Bacillus firmus. These data support the need to implement molecular tools to diagnose Carrion’s disease. PMID:24651298

  19. From clinical sites to biorepositories: effectiveness in blood sample management.

    PubMed

    Lefebvre, Céline; Tremblay, Nancy; Iverson, Bonnie; Wong, David; McWeeny, Kerri; Saghbini, Michael; Martinez, Heather; Hogan, Michael; Gaudet, Daniel; Arsenault, Steve

    2010-12-01

    Today's biobanks must work to take full advantage of collected samples, while maximizing sample quality and minimizing costs to sustain operations for a long period of time. This is a tall order that will require collaboration and compromise for both end-users and collection sites. This article discusses the efforts of the Génome Québec-Centre Hospitalier Affilié Universitaire Régional de Chicoutimi Biobank to fractionate blood samples for the simultaneous preservation of plasma and DNA-containing layers while minimizing resources required for shipping and transport. This article also describes methods for successful reproducible application of the plasma-depleted blood sample to GenPlates (GenVault, Carlsbad, CA).

  20. Improved sample capsule for determination of oxygen in hemolyzed blood

    NASA Technical Reports Server (NTRS)

    Malik, W. M.

    1967-01-01

    Sample capsule for determination of oxygen in hemolyzed blood consists of a measured section of polytetrafluoroethylene tubing equipped at each end with a connector and a stopcock valve. This method eliminates errors from air entrainment or from the use of mercury or syringe lubricant.

  1. Additional sampling directions improve detection range of wireless radiofrequency probes

    PubMed Central

    Mada, Marius; Carpenter, T. Adrian; Sawiak, Stephen J.; Williams, Guy B.

    2015-01-01

    Purpose While MRI is enhancing our knowledge about the structure and function of the human brain, subject motion remains a problem in many clinical applications. Recently, the use of wireless radiofrequency markers with three one‐dimensional (1D) navigators for prospective correction was demonstrated. This method is restricted in the range of motion that can be corrected, however, because of limited information in the 1D readouts. Methods Here, the limitation of techniques for disambiguating marker locations was investigated. It was shown that including more sampling directions extends the tracking range for head rotations. The efficiency of trading readout resolution for speed was explored. Results Tracking of head rotations was demonstrated from −19.2 to 34.4°, −2.7 to 10.0°, and −60.9 to 70.9° in the x‐, y‐, and z‐directions, respectively. In the presence of excessive head motion, the deviation of marker estimates from SPM8 was reduced by 17.1% over existing three‐projection methods. This was achieved by using an additional seven directions, extending the time needed for readouts by a factor of 3.3. Much of this increase may be circumvented by reducing resolution, without compromising accuracy. Conclusion Including additional sampling directions extends the range in which markers can be used, for patients who move a lot. Magn Reson Med 76:913–918, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. PMID:26418189

  2. Dengue-3 outbreak in Paraguay: investigations using capillary blood samples on filter paper.

    PubMed

    Matheus, Severine; Meynard, Jean-Baptiste; Lavergne, Anne; Girod, Romain; Moua, David; Labeau, Bhety; Dussart, Philippe; Lacoste, Vincent; Deparis, Xavier

    2008-11-01

    During a dengue-3 outbreak in Paraguay at the beginning of 2007, capillary blood samples absorbed onto filter papers were collected from 44 suspected cases. These samples were subjected to three molecular and serologic tests, and 31 of the 44 samples gave a positive result by at least one of the techniques used. Molecular analyses detected the dengue-3 serotype in 22 patients and additionally the dengue-2 serotype in two patients. Therefore two different serotypes were co-circulating during this outbreak. Overall, this study validates the use of dried-blood samples for field screening investigations. Indeed, all types of laboratory studies of dengue were possible with samples consisting of a few drops of dried blood from finger pricks.

  3. Improved screening test for abnormal hemoglobins from dried blood samples.

    PubMed

    Altland, K; Kaempfer, M; Granda, H

    1979-01-01

    A method is described wherein blood samples taken from adults or newborns and dried on filter paper can be used for hemoglobin analysis within 2 years after sampling. The samples are eluted in 8 M urea in the presence of 5% 2-mercaptoethanol and 2% of the neutral detergent Nonidet P-40. Then the individual alpha, beta, gamma, and epsilon chains are separated by means of electrofocusing in 8 M urea-PAA gels. Up to 96 samples can be applied to a gel using multiple syringes. Several hundred samples can be analyzed daily by one person. This method may be especially useful for preventive programs against sickle cell anemia as well as for human mutation monitoring systems.

  4. Effects of storage conditions on forensic examinations of blood samples and bloodstains stored for 20 years.

    PubMed

    Hara, M; Nakanishi, H; Yoneyama, K; Saito, K; Takada, A

    2016-01-01

    The effects of various storage conditions on blood identification tests, DNA degradation, and short tandem repeat (STR) typing were evaluated. Bloodstains stored at room temperature, 4 °C, -20 °C, and -80 °C for 20 years; blood samples stored at -20 °C and -80 °C for 20 years; and fresh blood samples were analyzed. Leuco-malachite-green testing, anti-human hemoglobin (Hb) testing (using immunochromatography), and tests for hemoglobin-beta (HBB) mRNA were performed as blood identification tests. DNA degradation was evaluated by quantifying the ratios of 305 and 129 base pair (bp) fragments to 41 bp fragments. STR typing was performed using an AmpFlSTR® Identifiler™ Plus PCR Amplification Kit. All samples were positive in leuco-malachite-green staining and anti-human Hb assays. HBB was not detected in blood samples stored at -20 °C or -80 °C, although this marker was detected in all bloodstains. As indicated by the ratio of 129:41 bp and 305:41 bp DNA fragments, DNA from bloodstains stored at room temperature or 4 °C were significantly degraded compared to DNA from all other samples. STR typing analyses revealed that a portion of the loci was undetected in bloodstains stored at room temperature. Therefore, to prevent DNA degradation during long-term storage, it is recommended that bloodstains and blood be stored at below -20 °C. In addition, because bloodstains are more suitable for detection of blood-specific mRNAs than blood sample, it is desirable that blood is stored as bloodstain for this method.

  5. Effects of storage conditions on forensic examinations of blood samples and bloodstains stored for 20 years.

    PubMed

    Hara, M; Nakanishi, H; Yoneyama, K; Saito, K; Takada, A

    2016-01-01

    The effects of various storage conditions on blood identification tests, DNA degradation, and short tandem repeat (STR) typing were evaluated. Bloodstains stored at room temperature, 4 °C, -20 °C, and -80 °C for 20 years; blood samples stored at -20 °C and -80 °C for 20 years; and fresh blood samples were analyzed. Leuco-malachite-green testing, anti-human hemoglobin (Hb) testing (using immunochromatography), and tests for hemoglobin-beta (HBB) mRNA were performed as blood identification tests. DNA degradation was evaluated by quantifying the ratios of 305 and 129 base pair (bp) fragments to 41 bp fragments. STR typing was performed using an AmpFlSTR® Identifiler™ Plus PCR Amplification Kit. All samples were positive in leuco-malachite-green staining and anti-human Hb assays. HBB was not detected in blood samples stored at -20 °C or -80 °C, although this marker was detected in all bloodstains. As indicated by the ratio of 129:41 bp and 305:41 bp DNA fragments, DNA from bloodstains stored at room temperature or 4 °C were significantly degraded compared to DNA from all other samples. STR typing analyses revealed that a portion of the loci was undetected in bloodstains stored at room temperature. Therefore, to prevent DNA degradation during long-term storage, it is recommended that bloodstains and blood be stored at below -20 °C. In addition, because bloodstains are more suitable for detection of blood-specific mRNAs than blood sample, it is desirable that blood is stored as bloodstain for this method. PMID:26832383

  6. Evaluation of Glucose-6-Phosphate Dehydrogenase stability in stored blood samples

    PubMed Central

    Jalil, Norunaluwar; Azma, Raja Zahratul; Mohamed, Emida; Ithnin, Azlin; Alauddin, Hafiza; Baya, Siti Noor; Othman, Ainoon

    2016-01-01

    Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is the commonest cause of neonatal jaundice in Malaysia. Recently, OSMMR2000-D G6PD Assay Kit has been introduced to quantitate the level of G6PD activity in newborns delivered in Universiti Kebangsaan Malaysia Medical Centre (UKMMC). As duration of sample storage prior to analysis is one of the matters of concern, this study was conducted to identify the stability of G6PD enzyme during storage. A total of 188 cord blood samples from normal term newborns delivered at UKMMC were selected for this study. The cord bloods samples were collected in ethylene-diamine-tetra-acetic acid (EDTA) tubes and refrigerated at 2-8 °C. In addition, 32 out of 188 cord blood samples were spotted on chromatography paper, air-dried and stored at room temperature. G6PD enzyme activities were measured daily for 7 days using the OSMMR2000-D G6PD Assay Kit on both the EDTA blood and dried blood samples. The mean value for G6PD activity was compared between days of analysis using Student Paired T-Test. In this study, 172 out of 188 cord blood samples showed normal enzyme levels while 16 had levels corresponding to severe enzyme deficiency. The daily mean G6PD activity for EDTA blood samples of newborns with normal G6PD activity showed a significant drop on the fourth day of storage (p < 0.005) while for samples with severely deficient G6PD activity, significant drop was seen on third day of storage (p = 0.002). Analysis of dried cord blood showed a significant reduction in enzyme activity as early as the second day of storage (p = 0.001). It was also noted that mean G6PD activity for spotted blood samples were lower compared to those in EDTA tubes for all days (p = 0.001). Thus, EDTA blood samples stored at 2-8 °C appeared to have better stability in terms of their G6PD enzyme level as compared to dried blood samples on filter paper, giving a storage time of up to 3 days. PMID:27103895

  7. Rapid and reliable determination of the halogenating peroxidase activity in blood samples.

    PubMed

    Flemmig, Jörg; Schwarz, Pauline; Bäcker, Ingo; Leichsenring, Anna; Lange, Franziska; Arnhold, Jürgen

    2014-12-15

    By combining easy and fast leukocyte enrichment with aminophenyl-fluorescein (APF) staining we developed a method to quickly and specifically address the halogenating activity of the immunological relevant blood heme peroxidases myeloperoxidase and eosinophil peroxidase, respectively. For leukocyte enrichment a two-fold hypotonic lysis procedure of the blood with Millipore water was chosen which represents a cheap, fast and reliable method to diminish the amount of erythrocytes in the samples. This procedure is shown to be suitable both to human and murine blood micro-samples, making it also applicable to small animal experiments with recurring blood sampling. As all types of leukocytes are kept in the sample during the preparation, they can be analysed separately after discrimination during the flow cytometry analysis. This also holds for all heme peroxidase-containing cells, namely neutrophils, eosinophils and monocytes. Moreover additional parameters (e.g. antibody staining) can be combined with the heme peroxidase activity determination to gain additional information about the different immune cell types. Based on previous results we applied APF for specifically addressing the halogenating activity of leukocyte peroxidases in blood samples. This dye is selectively oxidized by the MPO and EPO halogenation products hypochlorous and hypobromous acid. This approach may provide a suitable tool to gain more insights into the immune-physiological role of the halogenating activity of heme peroxidases.

  8. 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.

  9. 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. PMID:25277089

  10. Bacterial and fungal DNA extraction from blood samples: manual protocols.

    PubMed

    Lorenz, Michael G; Mühl, Helge; Disqué, Claudia

    2015-01-01

    A critical point of molecular diagnosis of systemic infections is the method employed for the extraction of microbial DNA from blood. A DNA isolation method has to be able to fulfill several fundamental requirements for optimal performance of diagnostic assays. First of all, low- and high-molecular-weight substances of the blood inhibitory to downstream analytical reactions like PCR amplification have to be removed. This includes human DNA which is a known source of false-positive results and factor decreasing the analytical sensitivity of PCR assays by unspecific primer binding. At the same time, even extremely low amounts of microbial DNA need to be supplied to molecular diagnostic assays in order to detect low pathogen loads in the blood. Further, considering the variety of microbial etiologies of sepsis, a method should be capable of lysing Gram-positive, Gram-negative, and fungal organisms. Last, extraction buffers, reagents, and consumables have to be free of microbial DNA which leads to false-positive results. Here, we describe manual methods which allow the extraction of microbial DNA from small- and large-volume blood samples for the direct molecular analysis of pathogen.

  11. Effects of experimenters and different blood sampling procedures on blood metabolite values in growing pigs.

    PubMed Central

    Dubreuil, P; Couture, Y; Tremblay, A; Martineau, G P

    1990-01-01

    The aims of the present study were firstly to verify if sera obtained by jugular venipuncture and those obtained from an anterior vena cava cannula were comparable and secondly to observe the effect of different experimenters on the biochemical profile of pigs. In the first experiment, 16 Yorkshire pigs with a mean weight of 48.9 +/- 2.3 kg were venipunctured using either a vacuum tube system or a syringe. The experiment was conducted on two separate days, seven days apart, in a crossover design experiment. At the time of obtaining blood via venipuncture, blood was also withdrawn from the jugular cannula. The tip of the latter was in the anterior vena cava. No effect of the sampling system was observed. However, a significant decrease in serum concentrations of Na, P and CO2 and a significant increase in anion gap, total protein, albumin, globulin, total bilirubin, aspartate amniotransferase, L-gamma glutamyl-transferase and creatine kinase were observed from blood samples obtained by venipuncture. In the second experiment, ten pigs from the first experiment were sampled by an inexperienced manipulator. Variations of the same magnitude as observed in the first experiment were observed in sera obtained by venipuncture and anterior vena cava but 18 of the 22 biochemical variables of sera obtained from the anterior vena cava were significantly different than those from the first experiment. The results show that blood samples obtained by jugular venipuncture in pigs can differ from samples obtained from an anterior vena cava cannula, specially in enzyme concentrations, and the dexterity of the experimenter may have a major effect on blood values. PMID:2379117

  12. Experience of fetal scalp blood sampling during labor.

    PubMed

    Liljeström, Lena; Wikström, Anna-Karin; Skalkidou, Alkistis; Akerud, Helena; Jonsson, Maria

    2014-01-01

    Fetal scalp blood sampling (FBS) is often claimed to be painful for women in labor and difficult for obstetricians to perform. Our aim was to assess women's experience of pain during FBS and obstetricians' experience of difficulty in performing the test. At a tertiary center in Sweden, a questionnaire with answers on a 10-point scale was completed by 51 women and the obstetricians performing the test. Women's experience of pain had a median of 3.5. FBS was well tolerated in women who had epidural analgesia but might be associated with pain in women without. Higher maternal body mass index and less cervical dilation were associated with higher pain ratings. Obstetricians did not generally experience scalp sampling as difficult to perform (median score 3.0). However, the sampling procedure can be more complicated in situations with higher maternal body mass index, less cervical dilation, and a higher station of the fetal head.

  13. 21 CFR 71.4 - Samples; additional information.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... respect to the safety of the color additive or the physical or technical effect it produces. The date used for computing the 90-day limit for the purposes of section 721(d)(1) of the act shall be moved...

  14. 21 CFR 71.4 - Samples; additional information.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... respect to the safety of the color additive or the physical or technical effect it produces. The date used for computing the 90-day limit for the purposes of section 721(d)(1) of the act shall be moved...

  15. Immunoreactive inhibin concentration in blood tested under variable sampling conditions.

    PubMed

    Blaakaer, J; Micic, S; Høgdall, C K

    1996-06-01

    The stability of immunoreactive (i.r.) inhibin in blood samples drawn and handled under different conditions and at different time intervals were studied. Ten serum and plasma samples drawn in 1994 from healthy volunteers were compared to samples collected in 1986 from 10 healthy women admitted for laparoscopic sterilization and analysed 6 years later. All samples were drawn on the twelfth day of the menstrual cycle and handled under identical clinical conditions (22 degrees C). The concentrations in the 1986 samples were similar to the Se-i.r. inhibin levels from 1994. Different clotting temperatures, repetitive freezing and thawing or hemolysis had no effects on the i.r. inhibin values, whereas non-hemolysed samples left at room temperature (22 degrees C) for 3 days were significantly lower, which might be due to a statistical type 2 error. No differences in concentration between serum and plasma i.r. inhibin were demonstrated. In conclusion, i.r. inhibin is a very stable peptide hormone in both serum and plasma if drawn and handled under normal conditions.

  16. 52 additional reference population samples for the 55 AISNP panel.

    PubMed

    Pakstis, Andrew J; Haigh, Eva; Cherni, Lotfi; ElGaaied, Amel Ben Ammar; Barton, Alison; Evsanaa, Baigalmaa; Togtokh, Ariunaa; Brissenden, Jane; Roscoe, Janet; Bulbul, Ozlem; Filoglu, Gonul; Gurkan, Cemal; Meiklejohn, Kelly A; Robertson, James M; Li, Cai-Xia; Wei, Yi-Liang; Li, Hui; Soundararajan, Usha; Rajeevan, Haseena; Kidd, Judith R; Kidd, Kenneth K

    2015-11-01

    Ancestry inference for a person using a panel of SNPs depends on the variation of frequencies of those SNPs around the world and the amount of reference data available for calculation/comparison. The Kidd Lab panel of 55 AISNPs has been incorporated in commercial kits by both Life Technologies and Illumina for massively parallel sequencing. Therefore, a larger set of reference populations will be useful for researchers using those kits. We have added reference population allele frequencies for 52 population samples to the 73 previously entered so that there are now allele frequencies publicly available in ALFRED and FROG-kb for a total of 125 population samples. PMID:26355664

  17. Tritium concentrations of blood samples collected throughout Japan

    SciTech Connect

    Hisamatsu, Shun`ichi; Takizawa, Yukio; Inoue, Yoshikazu

    1995-04-01

    Tritium concentrations were measured for blood samples collected from 20 cities throughout Japan during 1989-1990. The mean {sup 3}H concentration was found to be 1.4 {plus_minus} 0.4 Bq L{sup -1} and 1.0 {plus_minus} 0.4 Bq L{sup -1} (combustion water) for free water {sup 3}H and organically-bound {sup 3}H, respectively, excluding the abnormally high data of one city. The organically-bound {sup 3}H contents clearly depended on the latitudes of sampling locations, although the free water {sup 3}H concentrations showed no correlation with the latitudes. Organically-bound {sup 3}H is considered to be more suitable than free water {sup 3}H as an indicator of long time {sup 3}H exposure to human.

  18. On the improvement of blood sample collection at clinical laboratories

    PubMed Central

    2014-01-01

    Background Blood samples are usually collected daily from different collection points, such hospitals and health centers, and transported to a core laboratory for testing. This paper presents a project to improve the collection routes of two of the largest clinical laboratories in Spain. These routes must be designed in a cost-efficient manner while satisfying two important constraints: (i) two-hour time windows between collection and delivery, and (ii) vehicle capacity. Methods A heuristic method based on a genetic algorithm has been designed to solve the problem of blood sample collection. The user enters the following information for each collection point: postal address, average collecting time, and average demand (in thermal containers). After implementing the algorithm using C programming, this is run and, in few seconds, it obtains optimal (or near-optimal) collection routes that specify the collection sequence for each vehicle. Different scenarios using various types of vehicles have been considered. Unless new collection points are added or problem parameters are changed substantially, routes need to be designed only once. Results The two laboratories in this study previously planned routes manually for 43 and 74 collection points, respectively. These routes were covered by an external carrier company. With the implementation of this algorithm, the number of routes could be reduced from ten to seven in one laboratory and from twelve to nine in the other, which represents significant annual savings in transportation costs. Conclusions The algorithm presented can be easily implemented in other laboratories that face this type of problem, and it is particularly interesting and useful as the number of collection points increases. The method designs blood collection routes with reduced costs that meet the time and capacity constraints of the problem. PMID:24406140

  19. Evaluation of an additive solution for preservation of canine red blood cells.

    PubMed

    Wardrop, K J; Owen, T J; Meyers, K M

    1994-01-01

    The effect of an additive preservative solution on canine red blood cell posttransfusion viability (PTV) and on selected canine red blood cell biochemical parameters was studied. One unit (450 mL) of blood was collected from 6 clinically normal dogs into the anticoagulant citrate phosphate dextrose, centrifuged, and the plasma removed. The red blood cells were then suspended in 100 mL of a saline, adenine, dextrose, and mannitol solution and stored at 4 degrees C. Aliquots were removed for study at 1, 10, 20, 30, 37, and 44 days. The 24-hour PTV of autologous red blood cells was determined using a sodium chromate (51Cr) label. Red blood cell concentrations of 2,3-diphosphoglycerate (2,3-DPG), adenosine-5'-triphosphate (ATP), and pH were also determined. Canine red blood cell PTV, pH, ATP, and 2,3-DPG concentrations decreased during storage (P < .05). The PTV decreased from 94% using day 1 red blood cells to 80% and 75% using day 37 and day 44 red blood cells, respectively (P < .05). Although the mean PTV of the day 44 stored units equaled the Food and Drug Administration (FDA) minimum standard for human red blood cells, the PTV was substandard in 75% of the day 44 units. The FDA standard was exceeded in 83% of the day 37 units. It was concluded that 37-day-old canine red blood cells preserved with a saline, adenine, dextrose, and mannitol solution are of acceptable quality for transfusion.

  20. Analyzing Illumina Gene Expression Microarray Data Obtained From Human Whole Blood Cell and Blood Monocyte Samples.

    PubMed

    Teumer, Alexander; Schurmann, Claudia; Schillert, Arne; Schramm, Katharina; Ziegler, Andreas; Prokisch, Holger

    2016-01-01

    Microarray profiling of gene expression is widely applied to studies in molecular biology and functional genomics. Experimental and technical variations make not only the statistical analysis of single studies but also meta-analyses of different studies very challenging. Here, we describe the analytical steps required to substantially reduce the variations of gene expression data without affecting true effect sizes. A software pipeline has been established using gene expression data from a total of 3358 whole blood cell and blood monocyte samples, all from three German population-based cohorts, measured on the Illumina HumanHT-12 v3 BeadChip array. In summary, adjustment for a few selected technical factors greatly improved reliability of gene expression analyses. Such adjustments are particularly required for meta-analyses of different studies. PMID:26614070

  1. Effects of pre-analytical processes on blood samples used in metabolomics studies.

    PubMed

    Yin, Peiyuan; Lehmann, Rainer; Xu, Guowang

    2015-07-01

    Every day, analytical and bio-analytical chemists make sustained efforts to improve the sensitivity, specificity, robustness, and reproducibility of their methods. Especially in targeted and non-targeted profiling approaches, including metabolomics analysis, these objectives are not easy to achieve; however, robust and reproducible measurements and low coefficients of variation (CV) are crucial for successful metabolomics approaches. Nevertheless, all efforts from the analysts are in vain if the sample quality is poor, i.e. if preanalytical errors are made by the partner during sample collection. Preanalytical risks and errors are more common than expected, even when standard operating procedures (SOP) are used. This risk is particularly high in clinical studies, and poor sample quality may heavily bias the CV of the final analytical results, leading to disappointing outcomes of the study and consequently, although unjustified, to critical questions about the analytical performance of the approach from the partner who provided the samples. This review focuses on the preanalytical phase of liquid chromatography-mass spectrometry-driven metabolomics analysis of body fluids. Several important preanalytical factors that may seriously affect the profile of the investigated metabolome in body fluids, including factors before sample collection, blood drawing, subsequent handling of the whole blood (transportation), processing of plasma and serum, and inadequate conditions for sample storage, will be discussed. In addition, a detailed description of latent effects on the stability of the blood metabolome and a suggestion for a practical procedure to circumvent risks in the preanalytical phase will be given.

  2. Polychlorinated biphenyls in human blood samples of Bombay

    SciTech Connect

    Rao, C.V. ); Banerji, S. )

    1989-11-01

    Polychlorinated biphenyls (PCBs) have received considerable attention in the last two decades, since studies have shown that these are extremely persistent environmental pollutants worldover. Presence of PCBs in human blood has been reported by several investigators. This paper reports the levels of PCBs in the blood of 60 professional and 20 voluntary blood donors.

  3. Raman Spectroscopy: A New Proposal for the Detection of Leukemia Using Blood Samples

    SciTech Connect

    Martinez-Espinosa, J. C.; Gonzalez-Solis, J. L.; Miranda-Beltran, M. L.; Soria-Fregoso, C.; Medina-Valtierra, J.; Sanchez-Gomez, R.

    2008-08-11

    The use of Raman spectroscopy to analyze blood biochemistry and hence distinguish between normal and abnormal blood was investigated. The blood samples were obtained from 6 patients who were clinically diagnosed with leukemia and 6 healthy volunteer. The imprint was put under the microscope and several points were chosen for Raman measurement. All spectra were collected at confocal Raman micro-spectroscopy (Renishaw) with NIR 830 nm laser. It is shown that the serum samples from patients with leukemia and from the control group can be discriminated when the multivariate statistical methods of principal component analysis (PCA) and linear discriminated analysis (LDA) is applied to their Raman spectra. The ratios of some band intensities were analyzed and some band ratios were significant and corresponded to proteins, phospholipids, and polysaccharides. In addition, currently the degree of damage to the bone marrow is estimated through biopsies and therefore it is a very procedure painful. The preliminary results suggest that Raman spectroscopy could be a new technique to study the bone marrow using just blood samples.

  4. Design, construction and six years' experience of an integrated system for automated handling of discrete blood samples.

    PubMed

    Andersson, J L; Schneider, H

    1998-01-01

    The present paper describes the design of an integrated system to aid in the taking and measurement of manual blood samples during nuclear medical examinations requiring blood sampling. In contrast to previously published systems, the present system is not used in the actual sampling of the blood, but aims to aid in all other aspects of handling and measurement. It consists of two main parts. One part is a distributed software system running on the scanner host computer used to register sample times, to display information pertaining to the ongoing examination and to collect data from a number of well crystals. The other main part consists of an industrial robot used to perform the actual weighing, centrifugation, pipetting and measurement of the samples. The system has been operational for 6 years, during which time it has had an "up-time" in excess of 95% and has handled and measured the blood samples from more than 5000 examinations, each comprising an average of 15 blood samples. The throughput of the system is 50 whole blood samples or 21 plasma samples per hour. In addition it has to a large extent removed the "human factor" from the process, thereby increasing the reliability of the data.

  5. Development of blood vessel searching system using near-infrared light stereo method for clinical blood sampling

    NASA Astrophysics Data System (ADS)

    Cheng, Kai; Morita, Yusuke; Nakamachi, Eiji; Honda, Norihiro; Awazu, Kunio

    2014-10-01

    We developed an accurate three-dimensional blood vessel search (3D BVS) system using NIR light for the clinical blood sampling. In the previous study, the 3D BVS system, which used near-infrared (NIR) light imaging and the stereo method to locate blood vessel accurately in three dimensions has been developed(1). However, as NIR lights could not transmit the human arm, this system could not be used for the subcutaneous blood vessel detection. In this study, we developed a BVS by using the reflecting NIR light for blood sampling assist. The light scattering in human tissue will cause blur of blood vessel edge in image, that makes the diameter of blood vessel became uncertain. In this study, a light propagation simulation and a multilayer phantom were adopted to estimate the measurement error of blood vessel diameter in our BSV system. In the simulation, the optical properties of scattering coefficient, absorption coefficient, and refractive index were set similar with human skin. Next, we fabricated a multilayer phantom, which has the similar structure and optical properties with the human skin to confirm availability of the simulation. Also, the optical properties of our phantom are adjustable in our phantom to imitate the different color of skin. We established the estimation algorithm to detect the blood vessel accurately. Finally, we confirm the availability of our BVS for the blood sampling assist system.

  6. Effect of Sample Storage Temperature and Time Delay on Blood Gases, Bicarbonate and pH in Human Arterial Blood Samples

    PubMed Central

    Mohammadhoseini, Elham; Safavi, Enayat; Seifi, Sepideh; Seifirad, Soroush; Firoozbakhsh, Shahram; Peiman, Soheil

    2015-01-01

    Background: Results of arterial blood gas analysis can be biased by pre-analytical factors, such as time interval before analysis, temperature during storage and syringe type. Objectives: To investigate the effects of samples storage temperature and time delay on blood gases, bicarbonate and PH results in human arterial blood samples. Patients and Methods: 2.5 mL arterial blood samples were drawn from 45 patients via an indwelling Intraarterial catheter. Each sample was divided into five equal samples and stored in multipurpose tuberculin plastic syringes. Blood gas analysis was performed on one of five samples as soon as possible. Four other samples were divided into two groups stored at 22°C and 0°C. Blood gas analyses were repeated at 30 and 60 minutes after sampling. Results: PaO2 of the samples stored at 0°C was increased significantly after 60 minutes (P = 0.007). The PaCO2 of the samples kept for 30 and 60 minutes at 22°C was significantly higher than primary result (P = 0.04, P < 0.001). In samples stored at 22°C, pH decreased significantly after 30 and 60 minutes (P = 0.017, P = 0.001). There were no significant differences in other results of samples stored at 0°C or 22°C after 30 or 60 minutes. Conclusions: In samples stored in plastic syringes, overestimation of PaO2 levels should be noted if samples cooled before analysis. In samples stored in plastic syringes, it is not necessary to store samples in iced water when analysis delayed up to one hour. PMID:26019892

  7. Blood Pressure Associated With Sleep-Disordered Breathing in a Population Sample of Children

    PubMed Central

    Bixler, Edward O.; Vgontzas, Alexandros N.; Lin, Hung-Mo; Liao, Duanping; Calhoun, Susan; Fedok, Fred; Vlasic, Vukmir; Graff, Gavin

    2013-01-01

    The current criteria for sleep-disordered breathing (SDB) in children are not based on a clinically relevant outcome. The purpose of this study was to assess the association of blood pressure with SDB in a random sample of the local elementary school children (kindergarten through grade 5) using a 2-phased strategy. During phase 1, a brief questionnaire was completed for all of the children (N=5740) with a response rate of 78.5%. During phase 2, 700 randomly selected children from phase 1 with a response rate of 70.0% were assessed with a full polysomnograph and a history/physical, including an ECG; ear, nose, and throat; and pulmonary evaluation. We observed a significantly elevated systolic blood pressure associated with the apnea hypopnea index (AHI): AHI ≥1 (2.9 mm Hg); AHI ≥3 (7.1 mm Hg); and AHI ≥5 (12.9 mm Hg). The SDB and blood pressure association remained significant after adjusting for age, sex, race, body mass index percentile or waist circumference, sleep efficiency, percentage of rapid eye movement sleep, and snoring. In addition, older age, body mass index percentile, waist circumference, and snoring were significantly associated with blood pressure, independent of SDB. Based on these findings, our study suggests that SDB is significantly associated with higher levels of systolic blood pressure in children aged 5 to 12 years even after adjusting for the various confounding factors. Clinically, the data support the threshold of AHI ≥5 for the initiation of treatment for SDB. Additional research is indicated to assess the efficacy of SDB treatment on reducing blood pressure. PMID:18838624

  8. Quantification of Rifapentine, a Potent Antituberculosis Drug, from Dried Blood Spot Samples Using Liquid Chromatographic-Tandem Mass Spectrometric Analysis

    PubMed Central

    Parsons, Teresa L.; Marzinke, Mark A.; Hoang, Thuy; Bliven-Sizemore, Erin; Weiner, Marc; Mac Kenzie, William R.; Dorman, Susan E.

    2014-01-01

    The quantification of antituberculosis drug concentrations in multinational trials currently requires the collection of modest blood volumes, centrifugation, aliquoting of plasma, freezing, and keeping samples frozen during shipping. We prospectively enrolled healthy individuals into the Tuberculosis Trials Consortium Study 29B, a phase I dose escalation study of rifapentine, a rifamycin under evaluation in tuberculosis treatment trials. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying rifapentine in whole blood on dried blood spots (DBS) to facilitate pharmacokinetic/pharmacodynamic analyses in clinical trials. Paired plasma and whole-blood samples were collected by venipuncture, and whole blood was spotted on Whatman protein saver 903 cards. The methods were optimized for plasma and then validated for DBS. The analytical measuring range for quantification of rifapentine and its metabolite was 50 to 80,000 ng/ml in whole-blood DBS. The analyte was stable on the cards for 11 weeks with a desiccant at room temperature and protected from light. The method concordance for paired plasma and whole-blood DBS samples was determined after correcting for participant hematocrit or population-based estimates of bias from Bland-Altman plots. The application of either correction factor resulted in acceptable correlation between plasma and whole-blood DBS (Passing-Bablok regression corrected for hematocrit; y = 0.98x + 356). Concentrations of rifapentine may be determined from whole-blood DBS collected via venipuncture after normalization in order to account for the dilutional effects of red blood cells. Additional studies are focused on the application of this methodology to capillary blood collected by finger stick. The simplicity of processing, storage, shipping, and low blood volume makes whole-blood DBS attractive for rifapentine pharmacokinetic evaluations, especially in international and pediatric trials. PMID:25182637

  9. Dried blood spot analysis of creatinine with LC-MS/MS in addition to immunosuppressants analysis.

    PubMed

    Koster, Remco A; Greijdanus, Ben; Alffenaar, Jan-Willem C; Touw, Daan J

    2015-02-01

    In order to monitor creatinine levels or to adjust the dosage of renally excreted or nephrotoxic drugs, the analysis of creatinine in dried blood spots (DBS) could be a useful addition to DBS analysis. We developed a LC-MS/MS method for the analysis of creatinine in the same DBS extract that was used for the analysis of tacrolimus, sirolimus, everolimus, and cyclosporine A in transplant patients with the use of Whatman FTA DMPK-C cards. The method was validated using three different strategies: a seven-point calibration curve using the intercept of the calibration to correct for the natural presence of creatinine in reference samples, a one-point calibration curve at an extremely high concentration in order to diminish the contribution of the natural presence of creatinine, and the use of creatinine-[(2)H3] with an eight-point calibration curve. The validated range for creatinine was 120 to 480 μmol/L (seven-point calibration curve), 116 to 7000 μmol/L (1-point calibration curve), and 1.00 to 400.0 μmol/L for creatinine-[(2)H3] (eight-point calibration curve). The precision and accuracy results for all three validations showed a maximum CV of 14.0% and a maximum bias of -5.9%. Creatinine in DBS was found stable at ambient temperature and 32 °C for 1 week and at -20 °C for 29 weeks. Good correlations were observed between patient DBS samples and routine enzymatic plasma analysis and showed the capability of the DBS method to be used as an alternative for creatinine plasma measurement.

  10. Popliteal Vein Blood Sampling and the Postmortem Redistribution of Diazepam, Methadone, and Morphine.

    PubMed

    Lemaire, Eric; Schmidt, Carl; Denooz, Raphael; Charlier, Corinne; Boxho, Philippe

    2016-07-01

    Postmortem redistribution (PMR) refers to the site- and time-related blood drug concentration variations after death. We compared central blood (cardiac and subclavian) with peripheral blood (femoral and popliteal) concentrations of diazepam, methadone, and morphine. To our knowledge, popliteal blood has never been compared with other sites. Intracardiac blood (ICB), subclavian blood (SB), femoral blood (FB), and popliteal blood (PB) were sampled in 30 cases. To assess PMR, mean concentrations and ratios were compared. Influence of postmortem interval on mean ratios was also assessed. Results show that popliteal mean concentrations were lower than those for other sites for all three drugs, even lower than femoral blood; mean ratios suggested that the popliteal site was less subject to PMR, and estimated postmortem interval did not influence ratios except for diazepam and methadone FB/PB. In conclusion, our study is the first to explore the popliteal site and suggests that popliteal blood is less prone to postmortem redistribution. PMID:27364283

  11. 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. PMID:22273184

  12. Comparison of diagnostic tools for the detection of aspergillosis in blood samples of experimentally infected falcons.

    PubMed

    Fischer, D; Van Waeyenberghe, L; Cray, C; Gross, M; Usleber, E; Pasmans, F; Martel, A; Lierz, M

    2014-12-01

    Antemortem diagnosis of avian aspergillosis is very challenging. Diagnostic assays using blood samples would aid in an early and more definitive diagnosis. In the current study, detection of anti-Aspergillus antibodies, Aspergillus antigen, and Aspergillus toxin (fumigaclavine A), protein electrophoresis and measurement of acute-phase protein concentrations were performed on serum of 18 adult and plasma of 21 juvenile gyr-saker hybrid falcons (Falco rusticolus x Falco cherrug). Adult (n = 15) and juvenile (n = 18) falcons were experimentally inoculated with different dosages of the same strain of Aspergillus fumigatus and an additional three falcons from each age group were used as uninfected control animals. Blood samples were collected prior to inoculation and at 28 days postinoculation. Of the 33 inoculated falcons, 16 demonstrated clinical signs (vomiting, greenish urates, dyspnea, ruffled feathers) commonly associated with aspergillosis and in 14 falcons necropsy revealed aspergillosis granulomas confirmed by mycology and histopathology. Positive galactomannan results were rare, with only 3/15 positive samples from adult falcons and none in the juvenile birds. Most of the inoculated falcons showed an increase of serum amyloid A (66.7%) and haptoglobin (70.4%), but fumigaclavine A was not detected in the blood from any of the experimental animals. Elevated antibody indices were detected in 96.7% of the inoculated birds, but also in 66.7% of the controls. Significant decreases in albumin:globulin ratio were obvious in 81.5% of the inoculated birds, including 100% of the birds with granulomas. Blood from falcons with granulomas demonstrated significantly increased concentration values of alpha 2 and β globulins, decreased percentages of prealbumin and albumin, and increased percentages of alpha 2 and β globulins compared to inoculated falcons without granulomas. In conclusion, acute-phase proteins and the electrophoretic profile of birds challenged with A

  13. Comparison of diagnostic tools for the detection of aspergillosis in blood samples of experimentally infected falcons.

    PubMed

    Fischer, D; Van Waeyenberghe, L; Cray, C; Gross, M; Usleber, E; Pasmans, F; Martel, A; Lierz, M

    2014-12-01

    Antemortem diagnosis of avian aspergillosis is very challenging. Diagnostic assays using blood samples would aid in an early and more definitive diagnosis. In the current study, detection of anti-Aspergillus antibodies, Aspergillus antigen, and Aspergillus toxin (fumigaclavine A), protein electrophoresis and measurement of acute-phase protein concentrations were performed on serum of 18 adult and plasma of 21 juvenile gyr-saker hybrid falcons (Falco rusticolus x Falco cherrug). Adult (n = 15) and juvenile (n = 18) falcons were experimentally inoculated with different dosages of the same strain of Aspergillus fumigatus and an additional three falcons from each age group were used as uninfected control animals. Blood samples were collected prior to inoculation and at 28 days postinoculation. Of the 33 inoculated falcons, 16 demonstrated clinical signs (vomiting, greenish urates, dyspnea, ruffled feathers) commonly associated with aspergillosis and in 14 falcons necropsy revealed aspergillosis granulomas confirmed by mycology and histopathology. Positive galactomannan results were rare, with only 3/15 positive samples from adult falcons and none in the juvenile birds. Most of the inoculated falcons showed an increase of serum amyloid A (66.7%) and haptoglobin (70.4%), but fumigaclavine A was not detected in the blood from any of the experimental animals. Elevated antibody indices were detected in 96.7% of the inoculated birds, but also in 66.7% of the controls. Significant decreases in albumin:globulin ratio were obvious in 81.5% of the inoculated birds, including 100% of the birds with granulomas. Blood from falcons with granulomas demonstrated significantly increased concentration values of alpha 2 and β globulins, decreased percentages of prealbumin and albumin, and increased percentages of alpha 2 and β globulins compared to inoculated falcons without granulomas. In conclusion, acute-phase proteins and the electrophoretic profile of birds challenged with A

  14. Using dried blood spot sampling to improve data quality and reduce animal use in mouse pharmacokinetic studies.

    PubMed

    Wickremsinhe, Enaksha R; Perkins, Everett J

    2015-03-01

    Traditional pharmacokinetic analysis in nonclinical studies is based on the concentration of a test compound in plasma and requires approximately 100 to 200 μL blood collected per time point. However, the total blood volume of mice limits the number of samples that can be collected from an individual animal-often to a single collection per mouse-thus necessitating dosing multiple mice to generate a pharmacokinetic profile in a sparse-sampling design. Compared with traditional methods, dried blood spot (DBS) analysis requires smaller volumes of blood (15 to 20 μL), thus supporting serial blood sampling and the generation of a complete pharmacokinetic profile from a single mouse. Here we compare plasma-derived data with DBS-derived data, explain how to adopt DBS sampling to support discovery mouse studies, and describe how to generate pharmacokinetic and pharmacodynamic data from a single mouse. Executing novel study designs that use DBS enhances the ability to identify and streamline better drug candidates during drug discovery. Implementing DBS sampling can reduce the number of mice needed in a drug discovery program. In addition, the simplicity of DBS sampling and the smaller numbers of mice needed translate to decreased study costs. Overall, DBS sampling is consistent with 3Rs principles by achieving reductions in the number of animals used, decreased restraint-associated stress, improved data quality, direct comparison of interanimal variability, and the generation of multiple endpoints from a single study.

  15. Using dried blood spot sampling to improve data quality and reduce animal use in mouse pharmacokinetic studies.

    PubMed

    Wickremsinhe, Enaksha R; Perkins, Everett J

    2015-03-01

    Traditional pharmacokinetic analysis in nonclinical studies is based on the concentration of a test compound in plasma and requires approximately 100 to 200 μL blood collected per time point. However, the total blood volume of mice limits the number of samples that can be collected from an individual animal-often to a single collection per mouse-thus necessitating dosing multiple mice to generate a pharmacokinetic profile in a sparse-sampling design. Compared with traditional methods, dried blood spot (DBS) analysis requires smaller volumes of blood (15 to 20 μL), thus supporting serial blood sampling and the generation of a complete pharmacokinetic profile from a single mouse. Here we compare plasma-derived data with DBS-derived data, explain how to adopt DBS sampling to support discovery mouse studies, and describe how to generate pharmacokinetic and pharmacodynamic data from a single mouse. Executing novel study designs that use DBS enhances the ability to identify and streamline better drug candidates during drug discovery. Implementing DBS sampling can reduce the number of mice needed in a drug discovery program. In addition, the simplicity of DBS sampling and the smaller numbers of mice needed translate to decreased study costs. Overall, DBS sampling is consistent with 3Rs principles by achieving reductions in the number of animals used, decreased restraint-associated stress, improved data quality, direct comparison of interanimal variability, and the generation of multiple endpoints from a single study. PMID:25836959

  16. Using Dried Blood Spot Sampling to Improve Data Quality and Reduce Animal Use in Mouse Pharmacokinetic Studies

    PubMed Central

    Wickremsinhe, Enaksha R; Perkins, Everett J

    2015-01-01

    Traditional pharmacokinetic analysis in nonclinical studies is based on the concentration of a test compound in plasma and requires approximately 100 to 200 µL blood collected per time point. However, the total blood volume of mice limits the number of samples that can be collected from an individual animal—often to a single collection per mouse—thus necessitating dosing multiple mice to generate a pharmacokinetic profile in a sparse-sampling design. Compared with traditional methods, dried blood spot (DBS) analysis requires smaller volumes of blood (15 to 20 µL), thus supporting serial blood sampling and the generation of a complete pharmacokinetic profile from a single mouse. Here we compare plasma-derived data with DBS-derived data, explain how to adopt DBS sampling to support discovery mouse studies, and describe how to generate pharmacokinetic and pharmacodynamic data from a single mouse. Executing novel study designs that use DBS enhances the ability to identify and streamline better drug candidates during drug discovery. Implementing DBS sampling can reduce the number of mice needed in a drug discovery program. In addition, the simplicity of DBS sampling and the smaller numbers of mice needed translate to decreased study costs. Overall, DBS sampling is consistent with 3Rs principles by achieving reductions in the number of animals used, decreased restraint-associated stress, improved data quality, direct comparison of interanimal variability, and the generation of multiple endpoints from a single study. PMID:25836959

  17. Quantitative photoacoustic blood oxygenation measurement of whole porcine blood samples using a multi-wavelength semiconductor laser system

    NASA Astrophysics Data System (ADS)

    Friedrich, Claus-Stefan; Mienkina, Martin P.; Brenner, Carsten; Gerhardt, Nils C.; Jörger, Manfred; Strauß, Andreas; Beckmann, Martin F.; Schmitz, Georg; Hofmann, Martin R.

    2011-07-01

    We present a photoacoustic measurement system based on semiconductor lasers for blood oxygenation measurements. It permits to use four different optical wavelengths (650nm, 808nm, 850nm, 905nm) to generate photoacoustic signals. As the optical extinction coefficient of oxygenated hemoglobin and deoxygenated hemoglobin is different at specific wavelengths, a blood oxygenation measurement by a multi-wavelength photoacoustic laser system is feasible. Especially at 650nm, the clear difference between the extinction coefficients of the two hemoglobin derivates permits to determine the blood oxygenation in combination with other near infrared wavelengths. A linear model based on tabulated values of extinction coefficients for fully oxygenated and fully deoxygenated hemoglobin is presented. We used heparin stabilized whole porcine blood samples to model the optical behavior of human blood, as the optical absorption behavior of porcine hemoglobin does not differ significantly from human hemoglobin. To determine the real oxygen saturation values of the blood samples, we measured the partial oxygen pressure with an IRMA Trupoint Blood Analysis System. The oxygen saturation values were calculated from a dissociation curve for porcine blood. The results of the photoacoustic measurement are in qualitatively good agreement with the predicted linear model. Further, we analyze the abilities and the limitations of quantitative oxygenation measurements.

  18. 49 CFR 199.111 - Retention of samples and additional testing.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 3 2010-10-01 2010-10-01 false Retention of samples and additional testing. 199... SAFETY DRUG AND ALCOHOL TESTING Drug Testing § 199.111 Retention of samples and additional testing. (a... period, the employee or the employee's representative, the operator, the Administrator, or, if...

  19. Improved age determination of blood and teeth samples using a selected set of DNA methylation markers

    PubMed Central

    Kamalandua, Aubeline

    2015-01-01

    Age estimation from DNA methylation markers has seen an exponential growth of interest, not in the least from forensic scientists. The current published assays, however, can still be improved by lowering the number of markers in the assay and by providing more accurate models to predict chronological age. From the published literature we selected 4 age-associated genes (ASPA, PDE4C, ELOVL2, and EDARADD) and determined CpG methylation levels from 206 blood samples of both deceased and living individuals (age range: 0–91 years). This data was subsequently used to compare prediction accuracy with both linear and non-linear regression models. A quadratic regression model in which the methylation levels of ELOVL2 were squared showed the highest accuracy with a Mean Absolute Deviation (MAD) between chronological age and predicted age of 3.75 years and an adjusted R2 of 0.95. No difference in accuracy was observed for samples obtained either from living and deceased individuals or between the 2 genders. In addition, 29 teeth from different individuals (age range: 19–70 years) were analyzed using the same set of markers resulting in a MAD of 4.86 years and an adjusted R2 of 0.74. Cross validation of the results obtained from blood samples demonstrated the robustness and reproducibility of the assay. In conclusion, the set of 4 CpG DNA methylation markers is capable of producing highly accurate age predictions for blood samples from deceased and living individuals PMID:26280308

  20. Thermal activation of catalytic microjets in blood samples using microfluidic chips.

    PubMed

    Soler, Lluís; Martínez-Cisneros, Cynthia; Swiersy, Anka; Sánchez, Samuel; Schmidt, Oliver G

    2013-11-21

    We demonstrate that catalytic microjet engines can out-swim high complex media composed of red blood cells and serum. Despite the challenge presented by the high viscosity of the solution at room temperature, the catalytic microjets can be activated at physiological temperature and, consequently, self-propel in diluted solutions of blood samples. We prove that these microjets self-propel in 10× diluted blood samples using microfluidic chips.

  1. Thermal activation of catalytic microjets in blood samples using microfluidic chips†

    PubMed Central

    Soler, Lluís; Martínez-Cisneros, Cynthia; Swiersy, Anka; Sánchez, Samuel; Schmidt, Oliver G.

    2014-01-01

    We demonstrate that catalytic microjet engines can out-swim high complex media composed of red blood cells and serum. Despite the challenge presented by the high viscosity of the solution at room temperature, the catalytic microjets can be activated at physiological temperature and, consequently, self-propel in diluted solutions of blood samples. We prove that these microjets self-propel in 10× diluted blood samples using microfluidic chips. PMID:24089195

  2. Utility of the microculture method for Leishmania detection in non-invasive samples obtained from a blood bank.

    PubMed

    Ates, Sezen Canim; Bagirova, Malahat; Allahverdiyev, Adil M; Kocazeybek, Bekir; Kosan, Erdogan

    2013-10-01

    In recent years, the role of donor blood has taken an important place in epidemiology of Leishmaniasis. According to the WHO, the numbers of patients considered as symptomatic are only 5-20% of individuals with asymptomatic leishmaniasis. In this study for detection of Leishmania infection in donor blood samples, 343 samples from the Capa Red Crescent Blood Center were obtained and primarily analyzed by microscopic and serological methods. Subsequently, the traditional culture (NNN), Immuno-chromatographic test (ICT) and Polymerase Chain Reaction (PCR) methods were applied to 21 samples which of them were found positive with at least one method. Buffy coat (BC) samples from 343 blood donors were analyzed: 15 (4.3%) were positive by a microculture method (MCM); and 4 (1.1%) by smear. The sera of these 343 samples included 9 (2.6%) determined positive by ELISA and 7 (2%) positive by IFAT. Thus, 21 of (6.1%) the 343 subjects studied by smear, MCM, IFAT and ELISA techniques were identified as positive for leishmaniasis at least one of the techniques and the sensitivity assessed. According to our data, the sensitivity of the methods are identified as MCM (71%), smear (19%), IFAT (33%), ELISA (42%), NNN (4%), PCR (14%) and ICT (4%). Thus, with this study for the first time, the sensitivity of a MCM was examined in blood donors by comparing MCM with the methods used in the diagnosis of leishmaniasis. As a result, MCM was found the most sensitive method for detection of Leishmania parasites in samples obtained from a blood bank. In addition, the presence of Leishmania parasites was detected in donor bloods in Istanbul, a non-endemic region of Turkey, and these results is a vital importance for the health of blood recipients. PMID:23806567

  3. Utility of the microculture method for Leishmania detection in non-invasive samples obtained from a blood bank.

    PubMed

    Ates, Sezen Canim; Bagirova, Malahat; Allahverdiyev, Adil M; Kocazeybek, Bekir; Kosan, Erdogan

    2013-10-01

    In recent years, the role of donor blood has taken an important place in epidemiology of Leishmaniasis. According to the WHO, the numbers of patients considered as symptomatic are only 5-20% of individuals with asymptomatic leishmaniasis. In this study for detection of Leishmania infection in donor blood samples, 343 samples from the Capa Red Crescent Blood Center were obtained and primarily analyzed by microscopic and serological methods. Subsequently, the traditional culture (NNN), Immuno-chromatographic test (ICT) and Polymerase Chain Reaction (PCR) methods were applied to 21 samples which of them were found positive with at least one method. Buffy coat (BC) samples from 343 blood donors were analyzed: 15 (4.3%) were positive by a microculture method (MCM); and 4 (1.1%) by smear. The sera of these 343 samples included 9 (2.6%) determined positive by ELISA and 7 (2%) positive by IFAT. Thus, 21 of (6.1%) the 343 subjects studied by smear, MCM, IFAT and ELISA techniques were identified as positive for leishmaniasis at least one of the techniques and the sensitivity assessed. According to our data, the sensitivity of the methods are identified as MCM (71%), smear (19%), IFAT (33%), ELISA (42%), NNN (4%), PCR (14%) and ICT (4%). Thus, with this study for the first time, the sensitivity of a MCM was examined in blood donors by comparing MCM with the methods used in the diagnosis of leishmaniasis. As a result, MCM was found the most sensitive method for detection of Leishmania parasites in samples obtained from a blood bank. In addition, the presence of Leishmania parasites was detected in donor bloods in Istanbul, a non-endemic region of Turkey, and these results is a vital importance for the health of blood recipients.

  4. Luminol chemiluminescence biosensor for glycated hemoglobin (HbA1c) in human blood samples.

    PubMed

    Ahn, Kwang-Soo; Lee, JungHoon; Park, Jong-Myeon; Choi, Han Nim; Lee, Won-Yong

    2016-01-15

    Luminol chemiluminescence (CL) biosensor based on boronic acid modified gold substrate has been developed for the determination of glycated hemoglobin (HbA1c) in human blood samples. In order to selectively capture HbA1c in sample, carboxy-EG6-undecanethiol was self-assembled on a gold thin-film substrate, followed by covalent coupling of 3-aminophenyl boronic acid (3-APBA). The captured HbA1c containing four iron heme groups plays as a catalyst for luminol CL reaction in the presence of hydrogen peroxide, and thus the luminol CL response is linearly proportional to the amount of HbA1c captured on the biosensor surface. The present biosensor showed linear dynamic range of HbA1c from 2.5% to 17.0%, which well covers the clinically important concentration range. In addition, the present biosensor exhibited negligible response to interfering species such as hemoglobin, fructose, and sorbitol. The present HbA1c biosensor was applied to the determination of HbA1c in human blood samples and the results were well agreed with that obtained with a conventional method.

  5. MicroRNA Profiling from RSV-Infected Biofluids, Whole Blood, and Tissue Samples.

    PubMed

    Anderson, Lydia; Jorquera, Patricia A; Tripp, Ralph A

    2016-01-01

    Several studies have shown that respiratory syncytial virus (RSV) can modulate the host innate immune response by dysregulation of host microRNAs (miRNAs) related to the antiviral response, a feature that also affects the memory immune response to RSV (Thornburg et al. MBio 3(6), 2012). miRNAs are small, endogenous, noncoding RNAs that function in posttranscriptional gene regulation. Here, we explain a compilation of methods for the purification, quantification, and characterization of miRNA expression profiles in biofluids, whole blood samples, and tissue samples obtained from in vivo studies. In addition, this chapter describes methods for the isolation of exosomal miRNA populations. Understanding alterations in miRNA expression profiles and identifying miRNA targets genes, and their contribution to the pathogenesis of RSV, may help elucidate novel mechanism of host-virus interaction (Rossi et al., Pediatr Pulmonol, 2015). PMID:27464696

  6. Hematologic Assessment in Pet Rats, Mice, Hamsters, and Gerbils: Blood Sample Collection and Blood Cell Identification.

    PubMed

    Lindstrom, Nicole M; Moore, David M; Zimmerman, Kurt; Smith, Stephen A

    2015-09-01

    Hamsters, gerbils, rats, and mice are presented to veterinary clinics and hospitals for prophylactic care and treatment of clinical signs of disease. Physical examination, history, and husbandry practice information can be supplemented greatly by assessment of hematologic parameters. As a resource for veterinarians and their technicians, this article describes the methods for collection of blood, identification of blood cells, and interpretation of the hemogram in mice, rats, gerbils, and hamsters.

  7. What is the right blood hematocrit preparation procedure for standards and quality control samples for dried blood spot analysis?

    PubMed

    Koster, Remco A; Alffenaar, Jan-Willem C; Botma, Rixt; Greijdanus, Ben; Touw, Daan J; Uges, Donald R A; Kosterink, Jos G W

    2015-01-01

    Remco Koster is a research analyst and PhD candidate at the University Medical Center Groningen and University of Groningen. He has been working in the field of bioanalysis for over 13 years, where he has developed numerous analytical methods using LC-MS/MS. His main research focus is the influence of various matrices on the development and performance of analytical methods using LC-MS/MS. The development of high-speed extraction and analysis methods for drugs and drugs of abuse in human matrices like blood, plasma, hair, saliva and dried blood spots often leads to improved procedures for preparation of standards and quality control samples, sample handling and validation. Two hematocrit preparation procedures for standards and quality control samples were evaluated in order to improve the quality of procedures for dried blood spot validation and analysis.

  8. Routine Storage of Red Blood Cell Units in Additive Solution-3: a comprehensive investigation of the RBC metabolome

    PubMed Central

    D'Alessandro, Angelo; Nemkov, Travis; Kelher, Marguerite; West, Bernadette F.; Schwindt, Rani K.; Banerjee, Anirban; Moore, Ernest E; Silliman, Christopher C.; Hansen, Kirk C.

    2014-01-01

    Background In most countries, packed red blood cells (RBCs) can be stored up to 42 days before transfusion. However, observational studies have suggested that storage duration might be associated with increased morbidity and mortality. While clinical trials are underway, impaired metabolism has been documented in RBCs stored in several additive solutions. Here we hypothesize that, despite reported beneficial effects, storage in additive solution-3 (AS-3) results in metabolic impairment weeks before the end of the unit shelf-life. Study design and Methods Five leukocyte-filtered AS-3 RBC units were sampled before, during and after leukoreduction at day0, and then assayed on a weekly basis from storage day1 through day42. RBC extracts and supernatants were assayed using a UHPLC-MS metabolomics workflow. Results Blood bank storage significantly affects metabolic profiles of RBC extracts and supernatants by day14. In addition to energy and redox metabolism impairment, intra- and extracellular accumulation of amino acids was observed proportionally to storage duration, suggesting a role for glutamine and serine metabolism in aging RBCs. Conclusion Metabolomics of stored RBCs could drive the introduction of alternative additive solutions to address some of the storage-dependent metabolic lesions herein reported, thereby increasing the quality of transfused RBCs and minimizing potential links to patient morbidity. PMID:25556331

  9. Acetaminophen and meloxicam inhibit platelet aggregation and coagulation in blood samples from humans.

    PubMed

    Martini, Angela K; Rodriguez, Cassandra M; Cap, Andrew P; Martini, Wenjun Z; Dubick, Michael A

    2014-12-01

    Acetaminophen (Ace) and meloxicam (Mel) are the two types of analgesic and antipyretic medications. This study investigated the dose responses of acetaminophen and meloxicam on platelet aggregation and coagulation function in human blood samples. Blood samples were collected from six healthy humans and processed to make platelet-adjusted (100 × 10 cells/μl) blood samples. Acetaminophen (Tylenol, Q-PAP, 100 mg/ml) was added at the doses of 0 μg/ml (control), 214 μg/ml (the standard dose, 1 ×), 4 ×, 8 ×, 10 ×, 12 ×, 16 ×, and 20 ×. Similarly, meloxicam (Metacam, 5 mg/ml) was added at doses of 0 μg/ml (control), 2.85 μg/ml (the standard dose, 1 ×), 4 ×, 8 ×, 10 ×, 12 ×, 16 ×, and 20 ×. Fifteen minutes after the addition of acetaminophen and/or meloxicam, platelet aggregation was stimulated with collagen (2 μg/ml) or arachidonic acid (0.5 mmol/l) and assessed using a Chrono-Log 700 aggregometer. Coagulation function was assessed by prothrombin time (PT), activated partial thromboplastin time (aPTT), and using Rotem thrombelastogram. A robust inhibition by acetaminophen and/or meloxicam was observed in arachidonic acid-stimulated platelet aggregation starting at 1 × dose. Collagen-stimulated platelet aggregation was inhibited by ACE starting at 1 × (78 ± 10% of control), and by meloxicam starting at 4 × (72 ± 5% of control, both P < 0.05). The inhibitions by acetaminophen and meloxicam combined were similar to those by acetaminophen or meloxicam. aPTT was prolonged by meloxicam starting at 4 ×. No changes were observed in PT or any of Rotem measurements by acetaminophen and/or meloxicam. Acetaminophen and meloxicam compromised platelet aggregation and aPTT. Further effort is warranted to characterize the effects of acetaminophen and meloxicam on bleeding in vivo.

  10. Development of a simple device for processing whole blood samples into measured aliquots of plasma

    SciTech Connect

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

    1986-01-01

    A capillary processor and aliquoter (CPA) has been designed and fabricated that is capable of accepting aliquots of whole blood and automatically processing them into discrete aliquots of plasma. The device consists of two disks, each of which contains 16 individual capillaries and a processing rotor. One of the disks accepts larger capillaries, each of which will hold approx. 100 ..mu..L of whole blood. The second disk, which accepts 2.54-cm-long precision capillaries of varying internal diameter, provides for exact sample volumes ranging from 1 to 10 ..mu..L. The processing rotor consists of 16 individual compartments and chambers to accept both disks. Gravimetric and photometric evaluation of the CPA indicates that it is capable of entraining and delivering microliter volumes of liquids with a degree of precision and accuracy (1 to 2%) approaching that of a state-of-the-art mechanical pipette. In addition, we have demonstrated that aliquots of whole blood can be transferred into the chambers of the processing unit and separated into their cellular and plasma fractions, which can then be analyzed with an acceptable degree of precision (i.e., C.V.s of approx. +-3% for serum enzyme measurements). 15 refs., 6 figs., 4 tbls.

  11. Human milk oligosaccharides and Lewis blood group: individual high-throughput sample profiling to enhance conclusions from functional studies.

    PubMed

    Blank, Dennis; Dotz, Viktoria; Geyer, Rudolf; Kunz, Clemens

    2012-05-01

    Human milk oligosaccharides (HMO) are discussed to play a crucial role in an infant's development. Lewis blood group epitopes, in particular, seem to remarkably contribute to the beneficial effects of HMO. In this regard, large-scale functional human studies could provide evidence of the variety of results from in vitro investigations, although increasing the amount and complexity of sample and data handling. Therefore, reliable screening approaches are needed. To predict the oligosaccharide pattern in milk, the routine serological Lewis blood group typing of blood samples can be applied due to the close relationship between the biosynthesis of HMO and the Lewis antigens on erythrocytes. However, the actual HMO profile of the individual samples does not necessarily correspond to the serological determinations. This review demonstrates the capabilities of merging the traditional serological Lewis blood group typing with the additional information provided by the comprehensive elucidation of individual HMO patterns by means of state-of-the-art analytics. Deduced from the association of the suggested HMO biosynthesis with the Lewis blood group, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiles of oligosaccharides in individual milk samples exemplify the advantages and the limitations of sample assignment to distinct groups.

  12. Prolonged cold storage of red blood cells by oxygen removal and additive usage

    DOEpatents

    Bitensky, M.W.; Yoshida, Tatsuro

    1998-08-04

    Prolonged cold storage of red blood cells by oxygen removal and additive usage. A cost-effective, 4 C storage procedure that preserves red cell quality and prolongs post-transfusion in vivo survival is described. The improved in vivo survival and the preservation of adenosine triphosphate levels, along with reduction in hemolysis and membrane vesicle production of red blood cells stored at 4 C for prolonged periods of time, is achieved by reducing the oxygen level therein at the time of storage; in particular, by flushing the cells with an inert gas, and storing them in an aqueous solution which includes adenine, dextrose, mannitol, citrate ion, and dihydrogen phosphate ion, but no sodium chloride, in an oxygen-permeable container which is located in an oxygen-free environment containing oxygen-scavenging materials. 8 figs.

  13. Prolonged cold storage of red blood cells by oxygen removal and additive usage

    DOEpatents

    Bitensky, Mark W.; Yoshida, Tatsuro

    1998-01-01

    Prolonged cold storage of red blood cells by oxygen removal and additive usage. A cost-effective, 4.degree. C. storage procedure that preserves red cell quality and prolongs post-transfusion in vivo survival is described. The improved in vivo survival and the preservation of adenosine triphosphate levels, along with reduction in hemolysis and membrane vesicle production of red blood cells stored at 4.degree. C. for prolonged periods of time, is achieved by reducing the oxygen level therein at the time of storage; in particular, by flushing the cells with an inert gas, and storing them in an aqueous solution which includes adenine, dextrose, mannitol, citrate ion, and dihydrogen phosphate ion, but no sodium chloride, in an oxygen-permeable container which is located in an oxygen-free environment containing oxygen-scavenging materials.

  14. Use of filter paper blood samples for rabies antibody detection in foxes and raccoon dogs.

    PubMed

    Wasniewski, Marine; Barrat, Jacques; Combes, Benoit; Guiot, Anne Laure; Cliquet, Florence

    2014-08-01

    The effectiveness of oral rabies vaccination in wildlife is usually evaluated by the detection of rabies antibodies. However, the assessment of rabies antibodies has several technical difficulties in the field, such as the collection, storage, transport and titration of blood samples, often of poor quality. The objective of this study was to assess the feasibility of collecting blood on a filter paper (FP) coupled with enzyme-linked immunosorbent assay (ELISA) titration of rabies antibodies in raccoon dogs and red foxes. The FP blood sampling method was found highly specific and repeatable in both species. Overall, results obtained with the FP sampling method were highly concordant with the conventional (venipuncture) sampling methods. Blood eluates from FP samples from foxes and raccoon dogs tested using ELISA showed concordance values of 92% and 95%, respectively, with serum samples tested using the seroneutralisation test and values of 95% and 91%, respectively, when the ELISA was used on both types of sample. The use of FP blood sampling coupled with the titration of rabies antibodies by ELISA provides a reliable alternative to conventional blood sampling and serum testing by seroneutralisation. This simple procedure is particularly attractive and cost-effective for assessing the effectiveness of oral rabies vaccination in field conditions.

  15. Validation of a non-invasive blood-sampling technique for doubly-labelled water experiments.

    PubMed

    Voigt, Christian C; Helversen, Otto Von; Michener, Robert H; Kunz, Thomas H

    2003-04-01

    Two techniques for bleeding small mammals have been used in doubly-labeled water (DLW) studies, including vena puncture and the use of starved nymphal stages of hematophagous reduviid bugs (Reduviidae, Hemiptera). In this study, we tested the validity of using reduviid bugs in doubly-labeled water experiments. We found that the isotope enrichment in initial blood samples collected with bugs was significantly lower compared to isotope enrichment in blood samples obtained using vena puncture. We therefore used the desiccation method for estimating total body water (TBW) in DLW experiments because TBW calculated using the isotope dilution method was overestimated when blood samples were collected using reduviid bugs. In our validation experiment with nectar-feeding bats (Glossophaga soricina), we compared estimates of daily energy expenditure (DEE) using DLW with those derived from the energy balance method. We considered Speakman's equation (controlling for 25% fractionated water loss) as the most appropriate for our study animal and calculated DEE accordingly. On average, DEE estimated with DLW was not significantly different from the mean value obtained with the energy balance method (mean deviation 1.2%). We conclude that although bug hemolymph or intestinal liquids most likely contaminate the samples, estimates of DEE are still valid because the DLW method does not depend on absolute isotope enrichments but on the rate of isotope decrease over time. However, dilution of blood with intestinal liquids or hemolymph from a bug may lead to larger variation in DEE estimates. We also tested how the relative error of DLW estimates changed with varying assumptions about fractionation. We used three additional equations for calculating DEE in DLW experiments. The basic equation for DLW experiments published by Lifson and McClintock (LM-6) assumes no fractionation, resulted in an overestimate of DEE by 10%. Nagy's equation (N-2) controls for changes in body mass but not for

  16. Comparison of blood chemistry values for samples collected from juvenile chinook salmon by three methods

    USGS Publications Warehouse

    Congleton, J.L.; LaVoie, W.J.

    2001-01-01

    Thirteen blood chemistry indices were compared for samples collected by three commonly used methods: caudal transection, heart puncture, and caudal vessel puncture. Apparent biases in blood chemistry values for samples obtained by caudal transection were consistent with dilution with tissue fluids: alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK), triglyceride, and K+ were increased and Na+ and Cl- were decreased relative to values for samples obtained by caudal vessel puncture. Some enzyme activities (ALT, AST, LDH) and K+ concentrations were also greater in samples taken by heart puncture than in samples taken by caudal vessel puncture. Of the methods tested, caudal vessel puncture had the least effect on blood chemistry values and should be preferred for blood chemistry studies on juvenile salmonids.

  17. Validation of a minimally invasive blood-sampling technique for the analysis of hormones in domestic rabbits, Oryctolagus cuniculus (Lagomorpha).

    PubMed

    Voigt, Christian C; Fassbender, Mirja; Dehnhard, Martin; Wibbelt, Gudrun; Jewgenow, Katarina; Hofer, Heribert; Schaub, Günter A

    2004-01-01

    Previous studies in small mammals showed that blood-sucking bugs (Reduviidae, Heteroptera) can be used to obtain blood from veins difficult to access by human experimenters. In the present study, we validated the use of reduviid bugs for endocrinological studies in endotherms using domestic rabbits as a model organism. Two processes could alter the hormone concentrations in the blood ingested by the bug: (1) Mixing of ingested blood with saliva, gut fluid, or hemolymph and (2) digestive processes. We compared concentrations of progesterone, testosterone, and hydrocortisone in blood samples that were acquired from domestic rabbits (Oryctolagus cuniculus) by bugs (Dipetalogaster maxima) with hormone concentrations in blood obtained from the same individual rabbits with a conventional method, i.e., syringe. We found no significant differences in hormone concentrations between the two methods. Thus, the mixing effect is negligible immediately after the blood meal. In addition, we also could not find significant changes in concentrations of progesterone and hydrocortisone for up to 8h after the blood meal. Whereas levels of hydrocortisone remained unchanged for even 24h, progesterone levels significantly increased between eight and 24h. Thus, the bugs' excretory apparatus did not fractionate between water and hormones. Thirdly, we hypothesized that reduviid bugs impose less stress on the rabbits than the conventional method. We showed that deviations in hydrocortisone concentrations between the two blood sampling routines were lower when the bug method was used first and higher when the conventional method was used first. Thus, bugs imposed less stress on the study animals than the conventional method. Overall, we conclude that reduviid bugs present a minimally invasive method for obtaining blood from endotherm animals for endocrinological studies.

  18. Additives

    NASA Technical Reports Server (NTRS)

    Smalheer, C. V.

    1973-01-01

    The chemistry of lubricant additives is discussed to show what the additives are chemically and what functions they perform in the lubrication of various kinds of equipment. Current theories regarding the mode of action of lubricant additives are presented. The additive groups discussed include the following: (1) detergents and dispersants, (2) corrosion inhibitors, (3) antioxidants, (4) viscosity index improvers, (5) pour point depressants, and (6) antifouling agents.

  19. Comparison of three methods of sampling trout blood for measurements of hematocrit

    USGS Publications Warehouse

    Steucke, Erwin W., Jr.; Schoettger, Richard A.

    1967-01-01

    Trout blood is frequently collected for hematocrit measurements by excising the caudal fin (Snieszko, 1960), but this technique is impractical if valuable fish are to be sampled or if repeated observations are desired. Schiffman (1959) and Snieszko (1960) collected blood from the dorsal aorta and the heart, but these methods are relatively slow and require the preparation of needles and syringes. The use of pointed capillary tubes for cardiac punctures increases the speed of sampling, but body fluids may dilute the blood (Perkins, 1957; Larsen and Snieszko, 1961; and Normandau, 1962). There is need for methods of sampling which are rapid and which neither influence hematological determinations nor harm the fish.

  20. 49 CFR 199.111 - Retention of samples and additional testing.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) PIPELINE SAFETY DRUG AND ALCOHOL TESTING Drug Testing § 199.111 Retention of samples and additional testing. (a) Samples that yield positive results on confirmation must be retained by the laboratory in properly...

  1. 40 CFR 80.8 - Sampling methods for gasoline, diesel fuel, fuel additives, and renewable fuels.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... of the Federal Register under 5 U.S.C. 552(a) and 1 CFR part 51. To enforce any edition other than... 40 Protection of Environment 17 2014-07-01 2014-07-01 false Sampling methods for gasoline, diesel... Provisions § 80.8 Sampling methods for gasoline, diesel fuel, fuel additives, and renewable fuels....

  2. Multiplexed flow cytometric sensing of blood electrolytes in physiological samples using fluorescent bulk optode microspheres.

    PubMed

    Xu, Chao; Wygladacz, Katarzyna; Retter, Robert; Bell, Michael; Bakker, Eric

    2007-12-15

    Polymeric bulk optode microsphere ion sensors in combination with suspension array technologies such as analytical flow cytometry may become a power tool for measuring electrolytes in physiological samples. In this work, the methodology for the direct measurement of common blood electrolytes in physiological samples using bulk optode microsphere sensors was explored. The simultaneous determination of Na(+), K(+), and Ca(2+) in diluted sheep blood plasma was demonstrated for the first time, using a random suspension array containing three types of mixed microsphere bulk optodes of similar size, fabricated from the same chromoionophore without additional labeling. Sodium ionophore X, potassium ionophore III, and grafted AU-1 in poly(butyl acrylate) were the ionophores used in the bulk optode microsphere ion sensors for Na(+), K(+), and Ca(2+), respectively, in combination with the cation-exchanger NaTFPB (sodium tetrakis-[3,5-bis(trifluoromethyl)phenyl]borate) and the same concentration of the chromoionophore ETH 5294 (9-(di-ethylamino)-5-octadecanoylimino-5H-benzo[a]phen-oxazine) in plasticized poly(vinyl chloride). Excellent reproducibility was achieved for the sensing of potassium ions. The effect of sample pH was relatively small at near-physiological pH and followed theoretical predictions, yet the sample temperature was found to influence the sensor response to a larger extent. Multiplexed ion sensing was achieved by taking advantage of the chemical tunability of the sensor response, adjusting the sensor compositions so that the three types of ion sensors responded with distinct levels of protonation of the chromoionophore. Consequently, three well-resolved peaks were simultaneously observed in the single-channel histogram during the multiplexed calibration as well as in the subsequent measurement of the three cations in 10-fold-diluted sheep plasma. The assigned peak positions corresponded very well to the physiological range of the measured ions. PMID

  3. 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.

  4. An atypical microfilaria in blood samples from inhabitants of Brazilian Amazon.

    PubMed

    Adami, Y L; Moraes, M A P; Lanfredi, R M; Maia-Herzog, M

    2008-12-01

    An unidentified microfilaria sharing characteristics with Mansonella ozzardi and Onchocerca volvulus was detected in blood samples from seven human volunteers, inhabitants of a community in the border of Amazonas and Acre State. They were detected during epidemiological studies carried out in some communities along Antimary, Acre, and Purus Rivers in the Brazilian Amazon. The most striking difference was presented in the shape of the cephalic space from this microfilaria which was different from those of M. ozzardi and with similarities to O. volvulus in this region, but no remarkable differences were observed at the caudal region. More accurate studies are being carried out in order to provide additional data and supporting evidences before establishment of a new species can be done. PMID:18779979

  5. 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

  6. Selenium levels in related biological samples: human placenta, maternal and umbilical cord blood, hair and nails.

    PubMed

    Lorenzo Alonso, Maria José; Bermejo Barrera, Adela; Cocho de Juan, José Angel; Fraga Bermúdez, José María; Bermejo Barrera, Pilar

    2005-01-01

    A study on selenium levels has been carried out in human placenta, maternal and umbilical cord blood, hair and nails of a group of 50 mothers and in the hair of the newborns. The determinations were perfomed by electrothermal atomic absorption spectrometry. The selenium concentration obtained for each sample type was as follows: For the human placenta the values obtained were between 0.56 and 1.06 microg/g (mean +/- standard deviation: 0.81 +/- 0.02 microg/g). The levels for the umbilical cord blood were 51.1-104.2 microg/l (76.3 +/- 6.5 microg/l). For the maternal blood the values measured were between 57.3 and 117.9 microg/l (90.0 +/- 15.2 microg/l), and for hair and nails were 0.22-1.5 microg/g (0.60 +/- 0.37 microg/g) and 0.46-1.57 microg/g (0.90 +/- 0.27 microg/g), respectively. For the hair of the newborns the values obtained were between 0.40 and 2.53 microg/g (1.04 +/- 0.48 microg/g). The effect of different variables as age, habitat, nutritional index or gestation age of the mothers on the selenium concentration in the samples was studied. The influence of the habitat is significant with a confidence level of 95% for the selenium concentration in maternal blood and umbilical cord blood samples. The influence of the mothers' age is significant with a confidence level of 95% for the selenium concentration in the umbilical cord blood samples. For the placenta samples, the effect of the nutritional index is significant with a confidence level of 95%. There is a positive correlation between samples of umbilical cord blood and the newborns' hair, between placenta and umbilical cord, and between cord blood and maternal blood.

  7. Profiling gene expression in whole blood samples following an in-vitro challenge.

    PubMed

    Spijker, Sabine; van de Leemput, Joyce C H; Hoekstra, Chantal; Boomsma, Dorret I; Smit, August B

    2004-12-01

    Genomics tools (gene- and protein-expression studies) can be used to find possible target genes involved in a quantifiable trait or disease state. However in many instances, cells and tissues directly involved in the trait's expression, for example, brain tissue, are not amenable for gene expression analysis. Whole blood cells share a molecular make-up for cellular communication and gene regulation systems with many other cell types, for example, neuronal cells, and have the advantage of being very accessible for gene profiling. We investigated the feasibility of nationwide blood sample collection for lymphocyte RNA isolation and real-time PCR analysis to quantify genomic responses. We tested several designs for blood collection and storage: blood sampling in PAXgene blood collection tubes and storage at -20 degrees C, blood sampling in heparin tubes and decanting the samples (with or without in-vitro stimulus) into either PAXgene blood collection tubes and storage at -20 degrees C, or polypropylene tubes followed by snap-freezing and storage at -80 degrees C. The latter procedure is the best cost-wise when only small amounts of total RNA are needed for downstream applications. Lymphocyte gene expression studies are most likely hampered by the quality of isolated RNA rather than the sampling method. We show that large-scale nationwide sample collections did not alter RNA quality or gene expression levels when compared to sampling and processing in a more controlled way. To this end, we present an optimized protocol for easy and standardized isolation of high quality RNA using the PAXgene isolation kit. Based on these results, we suggest that whole blood genomic data can be used as a genomic probe in experimental and clinical research.

  8. Determination of lead in paired samples of human blood and synovial fluid

    SciTech Connect

    Villegas-Navarro, A.; Rosales, D.; Bustos, E.; Reyes, R.; Reyes, J.L.; Dieck, T.A.; Heredia, A. )

    1992-09-01

    In spite of the numerous papers published on the toxicity of lead in mammals, little is known about its effects in synovial fluid and bone joints. Our literature search showed a lack of quantitative studies regarding the concentration of lead in human synovial fluid; in addition, normal values regarding the threshold for poisoning by lead in that fluid are unknown. The available literature published corresponds to samples of human wounds by lead bullets localized close to or in a joint. Some of those papers dealing with lead-induced arthritis include symptoms of plumbism. They clearly demonstrate the ability of synovial fluid to dissolve lead and thereby make it available for systemic absorption. The molecular mechanism whereby this process is performed is still unknown, although it would be of interest because of its possible relationship with joint pain, a common problem in patients with lead poisoning that so far has not been fully explained. In a series of experiments with cattle, we found an average ratio of lead between synovial fluid and blood for paired observations of 4.2, although we have not found similar reports, and there is not sufficient information to make a total interpretation of these data. The purpose of this study was to determine the concentration of lead in synovial fluid and blood of corpses and to establish a possible numerical relationship between those two variables. 19 refs., 1 fig., 1 tab.

  9. Electrochemical aptasensor for lung cancer-related protein detection in crude blood plasma samples

    PubMed Central

    Zamay, Galina S.; Zamay, Tatiana N.; Kolovskii, Vasilii A.; Shabanov, Alexandr V.; Glazyrin, Yury E.; Veprintsev, Dmitry V.; Krat, Alexey V.; Zamay, Sergey S.; Kolovskaya, Olga S.; Gargaun, Ana; Sokolov, Alexey E.; Modestov, Andrey A.; Artyukhov, Ivan P.; Chesnokov, Nikolay V.; Petrova, Marina M.; Berezovski, Maxim V.; Zamay, Anna S.

    2016-01-01

    The development of an aptamer-based electrochemical sensor for lung cancer detection is presented in this work. A highly specific DNA-aptamer, LC-18, selected to postoperative lung cancer tissues was immobilized onto a gold microelectrode and electrochemical measurements were performed in a solution containing the redox marker ferrocyanide/ferricyanide. The aptamer protein targets were harvested from blood plasma of lung cancer patients by using streptavidin paramagnetic beads and square wave voltammetry of the samples was performed at various concentrations. In order to enhance the sensitivity of the aptasensor, silica-coated iron oxide magnetic beads grafted with hydrophobic C8 and C4 alkyl groups were used in a sandwich detection approach. Addition of hydrophobic beads increased the detection limit by 100 times. The detection limit of the LC-18 aptasensor was enhanced by the beads to 0.023 ng/mL. The formation of the aptamer – protein – bead sandwich on the electrode surface was visualized by electron microcopy. As a result, the electrochemical aptasensor was able to detect cancer-related targets in crude blood plasma of lung cancer patients. PMID:27694916

  10. Detection of Cervical Cancer Analyzing Blood Samples with Raman Spectroscopy and Multivariate Analysis

    NASA Astrophysics Data System (ADS)

    González-Solís, J. L.; Rodríguez-López, J.; Martínez-Espinosa, J. C.; Frausto-Reyes, C.; Jave-Suárez, L. F.; Aguilar-Lemarroy, A. C.; Vargas-Rodríguez, H.; Martínez-Cano, E.

    2010-05-01

    The use of Raman spectroscopy to analyze blood biochemistry and hence distinguish between normal and abnormal blood was investigated. The blood samples were obtained from 20 patients who were clinically diagnosed with cervical cancer and 10 healthy volunteer. The imprint was put under the Olympus microscope and several points were chosen for Raman measurement. All spectra were collected at a Jobin-Yvon LabRAM HR800 Raman Spectrometer with NIR 830 nm laser. It is shown that the serum samples from patients with cervical cancer and from the control group can be discriminated when the multivariate statistical methods of Principal Component Analysis (PCA) and Linear Discriminated Analysis (LDA) is applied to their Raman spectra. The ratios of some band intensities were analyzed and some band ratios were significant and corresponded to proteins, phospholipids, and polysaccharides. The preliminary results suggest that Raman spectroscopy could be a new technique for the detection using just blood samples.

  11. Perfluoroalkyl Acid Concentrations in Blood Samples Subjected to Transportation and Processing Delay

    PubMed Central

    Bach, Cathrine Carlsen; Henriksen, Tine Brink; Bossi, Rossana; Bech, Bodil Hammer; Fuglsang, Jens; Olsen, Jørn; Nohr, Ellen Aagaard

    2015-01-01

    Background In studies of perfluoroalkyl acids, the validity and comparability of measured concentrations may be affected by differences in the handling of biospecimens. We aimed to investigate whether measured plasma levels of perfluoroalkyl acids differed between blood samples subjected to delay and transportation prior to processing and samples with immediate processing and freezing. Methods Pregnant women recruited at Aarhus University Hospital, Denmark, (n = 88) provided paired blood samples. For each pair of samples, one was immediately processed and plasma was frozen, and the other was delayed and transported as whole blood before processing and freezing of plasma (similar to the Danish National Birth Cohort). We measured 12 perfluoroalkyl acids and present results for compounds with more than 50% of samples above the lower limit of quantification. Results For samples taken in the winter, relative differences between the paired samples ranged between -77 and +38% for individual perfluoroalkyl acids. In most cases concentrations were lower in the delayed and transported samples, e.g. the relative difference was -29% (95% confidence interval -30; -27) for perfluorooctane sulfonate. For perfluorooctanoate there was no difference between the two setups [corresponding estimate 1% (0, 3)]. Differences were negligible in the summer for all compounds. Conclusions Transport of blood samples and processing delay, similar to conditions applied in some large, population-based studies, may affect measured perfluoroalkyl acid concentrations, mainly when outdoor temperatures are low. Attention to processing conditions is needed in studies of perfluoroalkyl acid exposure in humans. PMID:26356420

  12. Paper membrane-based SERS platform for the determination of glucose in blood samples.

    PubMed

    Torul, Hilal; Çiftçi, Hakan; Çetin, Demet; Suludere, Zekiye; Boyacı, Ismail Hakkı; Tamer, Uğur

    2015-11-01

    In this report, we present a paper membrane-based surface-enhanced Raman scattering (SERS) platform for the determination of blood glucose level using a nitrocellulose membrane as substrate paper, and the microfluidic channel was simply constructed by wax-printing method. The rod-shaped gold nanorod particles were modified with 4-mercaptophenylboronic acid (4-MBA) and 1-decanethiol (1-DT) molecules and used as embedded SERS probe for paper-based microfluidics. The SERS measurement area was simply constructed by dropping gold nanoparticles on nitrocellulose membrane, and the blood sample was dropped on the membrane hydrophilic channel. While the blood cells and proteins were held on nitrocellulose membrane, glucose molecules were moved through the channel toward the SERS measurement area. Scanning electron microscopy (SEM) was used to confirm the effective separation of blood matrix, and total analysis is completed in 5 min. In SERS measurements, the intensity of the band at 1070 cm(-1) which is attributed to B-OH vibration decreased depending on the rise in glucose concentration in the blood sample. The glucose concentration was found to be 5.43 ± 0.51 mM in the reference blood sample by using a calibration equation, and the certified value for glucose was 6.17 ± 0.11 mM. The recovery of the glucose in the reference blood sample was about 88 %. According to these results, the developed paper-based microfluidic SERS platform has been found to be suitable for use for the detection of glucose in blood samples without any pretreatment procedure. We believe that paper-based microfluidic systems may provide a wide field of usage for paper-based applications.

  13. Paper membrane-based SERS platform for the determination of glucose in blood samples.

    PubMed

    Torul, Hilal; Çiftçi, Hakan; Çetin, Demet; Suludere, Zekiye; Boyacı, Ismail Hakkı; Tamer, Uğur

    2015-11-01

    In this report, we present a paper membrane-based surface-enhanced Raman scattering (SERS) platform for the determination of blood glucose level using a nitrocellulose membrane as substrate paper, and the microfluidic channel was simply constructed by wax-printing method. The rod-shaped gold nanorod particles were modified with 4-mercaptophenylboronic acid (4-MBA) and 1-decanethiol (1-DT) molecules and used as embedded SERS probe for paper-based microfluidics. The SERS measurement area was simply constructed by dropping gold nanoparticles on nitrocellulose membrane, and the blood sample was dropped on the membrane hydrophilic channel. While the blood cells and proteins were held on nitrocellulose membrane, glucose molecules were moved through the channel toward the SERS measurement area. Scanning electron microscopy (SEM) was used to confirm the effective separation of blood matrix, and total analysis is completed in 5 min. In SERS measurements, the intensity of the band at 1070 cm(-1) which is attributed to B-OH vibration decreased depending on the rise in glucose concentration in the blood sample. The glucose concentration was found to be 5.43 ± 0.51 mM in the reference blood sample by using a calibration equation, and the certified value for glucose was 6.17 ± 0.11 mM. The recovery of the glucose in the reference blood sample was about 88 %. According to these results, the developed paper-based microfluidic SERS platform has been found to be suitable for use for the detection of glucose in blood samples without any pretreatment procedure. We believe that paper-based microfluidic systems may provide a wide field of usage for paper-based applications. PMID:26363778

  14. The ABO blood grouping of a minute hair sample by the immunohistochemical technique.

    PubMed

    Miyasaka, S; Yoshino, M; Sato, H; Miyake, B; Seta, S

    1987-01-01

    The unlabeled antibody (PAP) immunoperoxidase technique was applied to the ABO blood grouping of human scalp hairs. Hair samples were subjected to longitudinal- or cross-sectioning, thus obtaining suitable samples for subsequent immunostaining. The immunostaining was carried out using rabbit anti-A and anti-B sera as the primary antibodies. With this technique, the group-specific staining which is revealed as a dark brown precipitate was clearly observed within the medullae of the hair shaft, and depending on the presence or absence of these precipitates, respective blood groups of unknown hair samples were determined. At the hair root, on the other hand, positive stainings were observed not only in medullary cells but also in some cortical cells of the keratogenous zone. From the present study, it can be safely said that this technique is of practical use for the ABO blood grouping from a minute (less than 3 mm) hair sample.

  15. Alcohol levels in cerebrospinal fluid and blood samples from patients under pathological conditions.

    PubMed

    Agapejev, S; Vassilieff, I; Curi, P R

    1992-11-01

    We measured alcohol levels by the Cordebard method in 148 CSF samples from individuals who had abstained from alcohol for at least 7 days prior to the beginning of the study. Each blood sample was accompanied by a CSF sample from the same patient. CSF samples found to be normal after analysis were used as controls. Mean alcohol concentration in blood did not differ significantly between the control group and the groups with altered CSF. The group with altered CSF had statistically higher alcohol levels in CSF than in blood. CSF lactate, glucose and protein levels were not correlated with alcohol level. The results suggest the presence of endogenous alcohol in the CSF, with levels increasing in the presence of pathological processes involving the nervous system.

  16. Additive quantification on polymer thin films by ToF-SIMS: aging sample effects

    NASA Astrophysics Data System (ADS)

    Poleunis, Claude; Médard, Nicolas; Bertrand, Patrick

    2004-06-01

    Thin films (150 nm) of an amorphous polyester (polyethylene(terephthalate-isophthalate)) containing variable concentrations of an antioxidant (Irgafos™ 168) and a UV-stabilizer (Hostavin™ N30) have been prepared by spin-coating. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) results showed, in the case of a single additive system (antioxidant), that the additive intensity increased on the polymer surface during the first five aging days (exudation phenomenon), followed by an intensity decrease, which was related to the adsorption of hydrocarbon contaminations on the sample surface. This kinetic competition was observed whatever the used additive concentration. In the case of the binary additive system (antioxidant and UV-stabilizer), the antioxidant behavior was similar to the single additive system, whereas, the UV-stabilizer evolution corresponded to an additive depletion, followed by an exudation. These results indicate that it is necessary to be very careful when comparing ToF-SIMS data for additive quantification on polymer surfaces. It is strongly recommended to compare samples having the same aging time, because the surface composition was seen to be strongly dependent of the aging time.

  17. 31. VIEW FROM SOUTHWEST TO CORNER WHERE SAMPLING/CRUSHING ADDITIONS ABUT ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    31. VIEW FROM SOUTHWEST TO CORNER WHERE SAMPLING/CRUSHING ADDITIONS ABUT CRUSHED OXIDIZED ORE BIN. INTACT BARREN SOLUTION TANK VISIBLE IN FRONT OF CRUSHED ORE BIN. - Bald Mountain Gold Mill, Nevada Gulch at head of False Bottom Creek, Lead, Lawrence County, SD

  18. Studies of levels of biogenic amines in meat samples in relation to the content of additives.

    PubMed

    Jastrzębska, Aneta; Kowalska, Sylwia; Szłyk, Edward

    2016-01-01

    The impact of meat additives on the concentration of biogenic amines and the quality of meat was studied. Fresh white and red meat samples were fortified with the following food additives: citric and lactic acids, disodium diphosphate, sodium nitrite, sodium metabisulphite, potassium sorbate, sodium chloride, ascorbic acid, α-tocopherol, propyl 3,4,5-trihydroxybenzoate (propyl gallate) and butylated hydroxyanisole. The content of spermine, spermidine, putrescine, cadaverine, histamine, tyramine, tryptamine and 2-phenylethylamine was determined by capillary isotachophoretic methods in meat samples (fresh and fortified) during four days of storage at 4°C. The results were applied to estimate the impact of the tested additives on the formation of biogenic amines in white and red meat. For all tested meats, sodium nitrite, sodium chloride and disodium diphosphate showed the best inhibition. However, cadaverine and putrescine were characterised by the biggest changes in concentration during the storage time of all the additives. Based on the presented data for the content of biogenic amines in meat samples analysed as a function of storage time and additives, we suggest that cadaverine and putrescine have a significant impact on meat quality. PMID:26515667

  19. Studies of levels of biogenic amines in meat samples in relation to the content of additives.

    PubMed

    Jastrzębska, Aneta; Kowalska, Sylwia; Szłyk, Edward

    2016-01-01

    The impact of meat additives on the concentration of biogenic amines and the quality of meat was studied. Fresh white and red meat samples were fortified with the following food additives: citric and lactic acids, disodium diphosphate, sodium nitrite, sodium metabisulphite, potassium sorbate, sodium chloride, ascorbic acid, α-tocopherol, propyl 3,4,5-trihydroxybenzoate (propyl gallate) and butylated hydroxyanisole. The content of spermine, spermidine, putrescine, cadaverine, histamine, tyramine, tryptamine and 2-phenylethylamine was determined by capillary isotachophoretic methods in meat samples (fresh and fortified) during four days of storage at 4°C. The results were applied to estimate the impact of the tested additives on the formation of biogenic amines in white and red meat. For all tested meats, sodium nitrite, sodium chloride and disodium diphosphate showed the best inhibition. However, cadaverine and putrescine were characterised by the biggest changes in concentration during the storage time of all the additives. Based on the presented data for the content of biogenic amines in meat samples analysed as a function of storage time and additives, we suggest that cadaverine and putrescine have a significant impact on meat quality.

  20. Determination of optimal sampling times for a two blood sample clearance method using (51)Cr-EDTA in cats.

    PubMed

    Vandermeulen, Eva; De Sadeleer, Carlos; Piepsz, Amy; Ham, Hamphrey R; Dobbeleir, André A; Vermeire, Simon T; Van Hoek, Ingrid M; Daminet, Sylvie; Slegers, Guido; Peremans, Kathelijne Y

    2010-08-01

    Estimation of the glomerular filtration rate (GFR) is a useful tool in the evaluation of kidney function in feline medicine. GFR can be determined by measuring the rate of tracer disappearance from the blood, and although these measurements are generally performed by multi-sampling techniques, simplified methods are more convenient in clinical practice. The optimal times for a simplified sampling strategy with two blood samples (2BS) for GFR measurement in cats using plasma (51)chromium ethylene diamine tetra-acetic acid ((51)Cr-EDTA) clearance were investigated. After intravenous administration of (51)Cr-EDTA, seven blood samples were obtained in 46 cats (19 euthyroid and 27 hyperthyroid cats, none with previously diagnosed chronic kidney disease (CKD)). The plasma clearance was then calculated from the seven point blood kinetics (7BS) and used for comparison to define the optimal sampling strategy by correlating different pairs of time points to the reference method. Mean GFR estimation for the reference method was 3.7+/-2.5 ml/min/kg (mean+/-standard deviation (SD)). Several pairs of sampling times were highly correlated with this reference method (r(2) > or = 0.980), with the best results when the first sample was taken 30 min after tracer injection and the second sample between 198 and 222 min after injection; or with the first sample at 36 min and the second at 234 or 240 min (r(2) for both combinations=0.984). Because of the similarity of GFR values obtained with the 2BS method in comparison to the values obtained with the 7BS reference method, the simplified method may offer an alternative for GFR estimation. Although a wide range of GFR values was found in the included group of cats, the applicability should be confirmed in cats suspected of renal disease and with confirmed CKD. Furthermore, although no indications of age-related effect were found in this study, a possible influence of age should be included in future studies. PMID:20452793

  1. l-carnitine as a Potential Additive in Blood Storage Solutions: A Study on Erythrocytes.

    PubMed

    Soumya, R; Carl, H; Vani, R

    2016-09-01

    Erythrocytes undergo various changes during storage (storage lesion) that in turn reduces their functioning and survival. Oxidative stress plays a major role in the storage lesion and antioxidants can be used to combat this stress. This study elucidates the effects of l-carnitine (LC) on erythrocytes of stored blood. Blood was obtained from male Wistar rats and stored (4 °C) for 20 days in CPDA-1 (citrate phosphate dextrose adenine) solution. Samples were divided into-(i) controls (ii) LC 10 (l-carnitine at a concentration of 10 mM) (iii) LC 30 (l-carnitine at a concentration of 30 mM) and (iv) LC 60 (l-carnitine at a concentration of 60 mM). Every fifth day, the biomarkers (haemoglobin, hemolysis, antioxidant enzymes, lipid peroxidation and protein oxidation products) were analysed in erythrocytes. Hemoglobin and protein sulfhydryls were insignificant during storage indicative of the maintenance of hemoglobin and sulfhydryls in all groups. Superoxide dismutase and malondialdehyde levels increased initially and decreased towards the end of storage. The levels of catalase and glutathione peroxidase were lower in experimentals than controls during storage. l-carnitine assisted the enzymes by scavenging the reactive oxygen species produced. Hemolysis increased in all groups with storage, elucidating that l-carnitine could not completely protect lipids and proteins from oxidative stress. Hence, this study opens up new avenues of using l-carnitine as a component of storage solutions with combinations of antioxidants in order to maintain efficacy of erythrocytes.

  2. Using blood samples to estimate persistent organic pollutants and metals in green sea turtles (Chelonia mydas).

    PubMed

    van de Merwe, Jason P; Hodge, Mary; Olszowy, Henry A; Whittier, Joan M; Lee, Shing Y

    2010-04-01

    Persistent organic pollutants (POPs) and heavy metals have been reported in a number of green turtle (Chelonia mydas) populations worldwide. However, due to ethical considerations, these studies have generally been on tissues from deceased and stranded animals. The purpose of this study was to investigate the use of blood samples to estimate the tissue contamination of live C. mydas populations. This study analysed 125 POP compounds and eight heavy metals in the blood, liver, kidney and muscle of 16 C. mydas from the Sea World Sea Turtle Rehabilitation Program, Gold Coast, Australia. Strong correlations were observed between blood and tissue concentrations for a number of POPs and metals. Furthermore, these correlations were observed over large ranges of turtle size, sex and condition. These results indicate that blood samples are a reliable non-lethal method for predicting chemical contamination in C. mydas.

  3. 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. PMID:26186236

  4. Elevated formic acid concentrations in putrefied post-mortem blood and urine samples.

    PubMed

    Viinamäki, Jenni; Rasanen, Ilpo; Vuori, Erkki; Ojanperä, Ilkka

    2011-05-20

    Formic acid (FA) concentration was measured in post-mortem blood and urine samples as methyl formate using a headspace in-tube extraction gas-chromatography-mass-spectrometry method. A total of 113 cases were analyzed, each including a blood and urine sample fortified with 1% sodium fluoride. The cases were divided into three groups: regular (n=59), putrefied (n=30), and methanol-positive (n=22) cases. There was no evidence of ante-mortem methanol consumption in the regular and putrefied cases. In regular cases, the mean (and median) FA concentrations were 0.04 g/l (0.04 g/l) and 0.06 g/l (0.04 g/l) in blood and urine, respectively. In putrefied cases, the mean (and median) FA concentrations were substantially higher, 0.24 g/l (0.22 g/l) and 0.25 g/l (0.15 g/l) in blood and urine, respectively. In three putrefied cases, FA concentration in blood exceeded 0.5 g/l, a level associated with fatal methanol poisoning. Ten putrefied cases were reanalyzed after 3-4 months storage, and no significant changes in FA concentrations were seen. These observations suggest that FA was formed by putrefaction during the post-mortem period, not during sample storage when sodium fluoride was added as a preservative. In methanol-positive cases, the mean (and median) FA concentrations were 0.80 g/l (0.88 g/l) and 3.4 g/l (3.3 g/l) in blood and urine, respectively, and the concentrations ranged from 0.19 to 1.0 g/l in blood and from 1.7 to 5.6 g/l in urine. The mean (and median) methanol concentrations in methanol-positive cases were 3.0 g/l (3.0 g/l) and 4.4 g/l (4.7 g/l) in blood and in urine, respectively. The highest methanol concentrations were 6.0 g/l and 8.7 g/l in blood and urine, respectively. No ethyl alcohol was found in the methanol-positive blood samples. Poor correlation was shown between blood and urine concentrations of FA. Poor correlations were also shown, in both blood and urine, between methanol and FA concentrations. PMID:21112705

  5. Evaluation of pesticide residues in human blood samples from Punjab (India)

    PubMed Central

    Bedi, Jasbir Singh; Gill, J. P. S.; Kaur, P.; Sharma, A.; Aulakh, R. S.

    2015-01-01

    Aim: The present study was undertaken to estimate the current status of residues of organochlorine pesticides (OCPs), organophosphates (OPs) and synthetic pyrethroids (SPs) pesticides in human blood. Materials and Methods: Human blood samples were analyzed by gas chromatography and confirmed by gas chromatography-mass spectrometry in selective ion monitoring mode. Results: The gas chromatographic analysis of human blood samples collected from Punjab revealed the presence of p,p’-dichlorodiphenyl dichloroethylene (DDE), p,p’ dichlorodiphenyl dichloroethane (DDD), o,p’ DDE and β-endosulfan at mean levels of 15.26, 2.71, 5.62 and 4.02 ng/ml, respectively. p,p’ DDE residue was observed in 18.0% blood samples, and it contributes 55% of the total pesticide burden in human blood. The difference of total dichlorordiphenyl trichloroethane (DDT) between different age groups of humans was found to be statistically significant (p<0.05). The difference of DDT and endosulfan between dietary habits, gender and spraying of pesticides was found statistically non-significant, however endosulfan residues were observed only in pesticide sprayer’s population. Conclusion: Occurrence of p,p’ DDE, p,p’ DDD, o,p’ DDE in human blood indicated restricted use of DDT. However, presence of endosulfan residues in occupationally exposed population is a matter of public health concern. PMID:27046999

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

    PubMed

    Bausinger, Julia; Speit, Günter

    2016-09-01

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

  7. Microwaving Blood as a Non-Destructive Technique for Haemoglobin Measurements on Microlitre Samples

    PubMed Central

    Basey-Fisher, Toby H.; Guerra, Nadia; Triulzi, Chiara; Gregory, Andrew; Hanham, Stephen M.; Stevens, Molly M.; Maier, Stefan A.; Klein, Norbert

    2016-01-01

    The non-destructive ex vivo determination of haemoglobin (Hgb) concentration offers the capability to conduct multiple red blood cell haematological measurements on a single sample, an advantage that current optical techniques are unable to offer. Here, a microwave method and device for the accurate and non-destructive determination of Hgb concentration in microlitre blood samples are described. Using broadband microwave spectroscopy, a relationship is established between the dielectric properties of murine blood and Hgb concentration that is utilized to create a technique for the determination of Hgb concentration. Subsequently, a microwave dielectric resonator-microfluidic system is implemented in the analysis of 52 murine samples with microlitre volumes and Hgb concentrations ranging from 0 to 17 g dL−1. Using the characterized relationship, independent and minimally invasive Hgb measurements are made on nine healthy mice as well as seven with mutations in the Adenomatous polyposis coli (APC) gene that leads to colorectal cancer and consequently anaemia. PMID:24002989

  8. A sample-to-result system for blood coagulation tests on a microfluidic disk analyzer.

    PubMed

    Lin, Chia-Hui; Liu, Cheng-Yuan; Shih, Chih-Hsin; Lu, Chien-Hsing

    2014-09-01

    In this report, we describe in detail a microfluidic analyzer, which is able to conduct blood coagulation tests using whole blood samples. Sample preparation steps, such as whole blood aliquoting and metering, plasma separation, decanting, and mixing with reagents were performed in sequence through microfluidic functions integrated on a disk. Both prothrombin time (PT) and activated partial thromboplastin time (aPTT) were carried out on the same platform and the test results can be reported in 5 min. Fifty clinical samples were tested for both PT and aPTT utilizing the microfluidic disk analyzer and the instrument used in hospitals. The test results showed good correlation and agreement between the two instruments.

  9. A sample-to-result system for blood coagulation tests on a microfluidic disk analyzer

    PubMed Central

    Lin, Chia-Hui; Liu, Cheng-Yuan; Shih, Chih-Hsin; Lu, Chien-Hsing

    2014-01-01

    In this report, we describe in detail a microfluidic analyzer, which is able to conduct blood coagulation tests using whole blood samples. Sample preparation steps, such as whole blood aliquoting and metering, plasma separation, decanting, and mixing with reagents were performed in sequence through microfluidic functions integrated on a disk. Both prothrombin time (PT) and activated partial thromboplastin time (aPTT) were carried out on the same platform and the test results can be reported in 5 min. Fifty clinical samples were tested for both PT and aPTT utilizing the microfluidic disk analyzer and the instrument used in hospitals. The test results showed good correlation and agreement between the two instruments. PMID:25332733

  10. No effect of blood sampling or phytohaemagglutinin injection on postfledging survival in a wild songbird.

    PubMed

    Bowers, Emerson Keith; Sakaluk, Scott K; Thompson, Charles F

    2016-05-01

    The injection of phytohaemagglutinin (PHA) and sampling of blood are widely used in studies of wild vertebrates to assess components of immune and endocrine function and health state and to obtain genetic material. Despite the pervasive use of these techniques in the life sciences, their potential effects on survival are rarely considered. For example, whether injection of the immunogen PHA into body parts critical for locomotion (e.g., the prepatagium, or wing web, in birds) affects survival has not been tested. Here, we test whether injection of PHA into the wing web and blood sampling from nestling house wrens affects their subsequent recruitment and survival as breeding adults. Capture-mark-recapture analysis on a large sample of young (N = 20,152 fledglings from 3959 broods) treated over 10 years revealed that neither PHA injection nor blood sampling affected individual survival and detection probability. Recruitment as a breeder varied among years, but this variation was not attributable to sampling effort, or the percent of all adults identified at the nest during a given year. Variation in the percent of adults identified was primarily attributable to the effect of nest depredation on our ability to capture nesting pairs. Our results indicating lack of an effect of blood sampling and immune stimulation on survival are encouraging, but we recommend further work to assess the potential negative effects of all commonly used techniques on the survival of study subjects in the wild, including the potential costs associated with mounting various immunological responses.

  11. Effects of atmospheric precipitation additions on phytoplankton photosynthesis in Lake Michigan water samples

    SciTech Connect

    Parker, J.I.; Tisue, G.T.; Kennedy, C.W.; Seils, C.A.

    1981-01-01

    The effects of incremental additions (0.1 to 50% v/v) of atmospheric precipitation on phytoplankton photosynthesis (/sup 14/C uptake) were tested in Lake Michigan water samples. Wet deposition was used in experiments I, III, and IV, and a melted snow core was used in experiment II. Additions of precipitation significantly reduced photosynthesis in the first three experiments, starting at about the 5 to 15% treatment level. No significant difference occurred in experiment IV, but photosynthesis was greater than in the control samples and this precipitation sample appeared to stimulate primary productivity. Soluble reactive phosphate, nitrate, and ammonia levels in the precipitation samples exceeded the lake water averages by factors of 10, 2, and 50, respectively. Silicon levels in precipitation reduced pH very little and no consistent relationship was observed with reduced photosynthesis. Alkalinity was greatly reduced in the treated samples and special precautions were required in ce, Ti, Be, Co, Cu, Mo, Ni, P,f the Pd crystals of about 30 A. Possible mechanisms are discussed for isotope exchange in CO molecules in these catalysts and for the promoting effect of Pd on the activity of CuO.

  12. Microfluidic, marker-free isolation of circulating tumor cells from blood samples.

    PubMed

    Karabacak, Nezihi Murat; Spuhler, Philipp S; Fachin, Fabio; Lim, Eugene J; Pai, Vincent; Ozkumur, Emre; Martel, Joseph M; Kojic, Nikola; Smith, Kyle; Chen, Pin-i; Yang, Jennifer; Hwang, Henry; Morgan, Bailey; Trautwein, Julie; Barber, Thomas A; Stott, Shannon L; Maheswaran, Shyamala; Kapur, Ravi; Haber, Daniel A; Toner, Mehmet

    2014-03-01

    The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen-independent microfluidic CTC-iChip technology. The CTC-iChip uses deterministic lateral displacement, inertial focusing and magnetophoresis to sort up to 10⁷ cells/s. By using two-stage magnetophoresis and depletion antibodies against leukocytes, we achieve 3.8-log depletion of white blood cells and a 97% yield of rare cells with a sample processing rate of 8 ml of whole blood/h. The CTC-iChip is compatible with standard cytopathological and RNA-based characterization methods. This protocol describes device production, assembly, blood sample preparation, system setup and the CTC isolation process. Sorting 8 ml of blood sample requires 2 h including setup time, and chip production requires 2-5 d.

  13. Microfluidic, marker-free isolation of circulating tumor cells from blood samples

    PubMed Central

    Karabacak, Nezihi Murat; Spuhler, Philipp S; Fachin, Fabio; Lim, Eugene J; Pai, Vincent; Ozkumur, Emre; Martel, Joseph M; Kojic, Nikola; Smith, Kyle; Chen, Pin-i; Yang, Jennifer; Hwang, Henry; Morgan, Bailey; Trautwein, Julie; Barber, Thomas A; Stott, Shannon L; Maheswaran, Shyamala; Kapur, Ravi; Haber, Daniel A; Toner, Mehmet

    2014-01-01

    The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen–independent microfluidic CTC-iChip technology. The CTC-iChip uses deterministic lateral displacement, inertial focusing and magnetophoresis to sort up to 107 cells/s. By using two-stage magnetophoresis and depletion antibodies against leukocytes, we achieve 3.8-log depletion of white blood cells and a 97% yield of rare cells with a sample processing rate of 8 ml of whole blood/h. The CTC-iChip is compatible with standard cytopathological and RNA-based characterization methods. This protocol describes device production, assembly, blood sample preparation, system setup and the CTC isolation process. Sorting 8 ml of blood sample requires 2 h including setup time, and chip production requires 2–5 d. PMID:24577360

  14. Rapid Microbial Sample Preparation from Blood Using a Novel Concentration Device

    PubMed Central

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

    2015-01-01

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

  15. Additive non-uniform random sampling in superimposed fiber Bragg grating strain gauge

    NASA Astrophysics Data System (ADS)

    Ma, Y. C.; Liu, H. Y.; Yan, S. B.; Yang, Y. H.; Yang, M. W.; Li, J. M.; Tang, J.

    2013-05-01

    This paper demonstrates an additive non-uniform random sampling and interrogation method for dynamic and/or static strain gauge using a reflection spectrum from two superimposed fiber Bragg gratings (FBGs). The superimposed FBGs are designed to generate non-equidistant space of a sensing pulse train in the time domain during dynamic strain gauge. By combining centroid finding with smooth filtering methods, both the interrogation speed and accuracy are improved. A 1.9 kHz dynamic strain is measured by generating an additive non-uniform randomly distributed 2 kHz optical sensing pulse train from a mean 500 Hz triangular periodically changing scanning frequency.

  16. Method and apparatus for automated processing and aliquoting of whole blood samples for analysis in a centrifugal fast analyzer

    DOEpatents

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

    1985-08-05

    A rotor and disc assembly for use in a centrifugal fast analyzer. The assembly is designed to process multiple samples of whole blood followed by aliquoting of the resultant serum into precisely measured samples for subsequent chemical analysis. The assembly requires minimal operator involvement with no mechanical pipetting. The system comprises: (1) a whole blood sample disc; (2) a serum sample disc; (3) a sample preparation rotor; and (4) an analytical rotor. The blood sample disc and serum sample disc are designed with a plurality of precision bore capillary tubes arranged in a spoked array. Samples of blood are loaded into the blood sample disc by capillary action and centrifugally discharged into cavities of the sample preparation rotor where separation of serum and solids is accomplished. The serum is loaded into the capillaries of the serum sample disc by capillary action and subsequently centrifugally expelled into cuvettes of the analyticaly rotor for conventional methods. 5 figs.

  17. Method and apparatus for automated processing and aliquoting of whole blood samples for analysis in a centrifugal fast analyzer

    DOEpatents

    Burtis, Carl A.; Johnson, Wayne F.; Walker, William A.

    1988-01-01

    A rotor and disc assembly for use in a centrifugal fast analyzer. The assembly is designed to process multiple samples of whole blood followed by aliquoting of the resultant serum into precisely measured samples for subsequent chemical analysis. The assembly requires minimal operator involvement with no mechanical pipetting. The system comprises (1) a whole blood sample disc, (2) a serum sample disc, (3) a sample preparation rotor, and (4) an analytical rotor. The blood sample disc and serum sample disc are designed with a plurality of precision bore capillary tubes arranged in a spoked array. Samples of blood are loaded into the blood sample disc in capillary tubes filled by capillary action and centrifugally discharged into cavities of the sample preparation rotor where separation of serum and solids is accomplished. The serum is loaded into the capillaries of the serum sample disc by capillary action and subsequently centrifugally expelled into cuvettes of the analytical rotor for analysis by conventional methods.

  18. The future of doping control in athletes. Issues related to blood sampling.

    PubMed

    Birkeland, K I; Hemmersbach, P

    1999-07-01

    When current antidoping programmes were developed, the most frequently used doping agents were xenobiotics, such as stimulants and anabolic steroids, that are readily detectable in urine with the use of gas chromatography and mass spectrometry. As control of traditional doping agents became effective, some athletes turned to other means to improve performance, including blood doping and the application of recombinant peptide hormones such as erythropoietin and growth hormone. Doping with these agents is not easily detected in urine samples, and therefore new strategies must be developed as a supplement to those already in use. Such strategies will probably include analysing blood samples, as several of the most promising methods that are able to detect modern doping agents use blood as the analytical matrix. Non-autologous blood doping results in an admixture of self and foreign red blood cells that can be detected in a blood sample with the methods available. Methods to indicate doping with erythropoietin include the indirect finding of an elevated level of soluble transferrin receptor in serum, or a direct demonstration of a shift from the normal to an abnormal spectrum of erythropoietin isoforms. To indicate doping with growth hormone, a set of serum parameters including insulin growth factors and their binding proteins are under investigation as indirect evidence. A direct method using isotopic differences between endogenous and recombinant growth hormones is being investigated. A similar method has been established to detect the administration of testosterone esters. Several legal and ethical questions must be solved before blood sampling can become a part of routine doping control, but the major ethical question is whether sport can continue as today without proper methods to detect many modern doping agents.

  19. Rapid Diagnosis of Staphylococcal Catheter-Related Bacteraemia in Direct Blood Samples by Real-Time PCR.

    PubMed

    Zboromyrska, Yuliya; De la Calle, Cristina; Soto, Marcelo; Sampietro-Colom, Laura; Soriano, Alex; Alvarez-Martínez, Míriam José; Almela, Manel; Marco, Francesc; Arjona, Ruth; Cobos-Trigueros, Nazaret; Morata, Laura; Mensa, José; Martínez, José Antonio; Mira, Aurea; Vila, Jordi

    2016-01-01

    Catheter-related bacteremia (CRB) is an important cause of morbidity and mortality among hospitalized patients, being staphylococci the main etiologic agents. The objective of this study was to assess the use of a PCR-based assay for detection of staphylococci directly from blood obtained through the catheter to diagnose CRB caused by these microorganisms and to perform a cost-effectiveness analysis. A total of 92 patients with suspected CRB were included in the study. Samples were obtained through the catheter. Paired blood cultures were processed by standard culture methods and 4 ml blood samples were processed by GeneXpert-MRSA assay for the detection of methicillin-susceptible (MSSA) or methicillin-resistant (MRSA) Staphylococcus aureus, and methicillin-resistant coagulase-negative staphylococci (MR-CoNS). Sixteen CRB caused by staphylococci were diagnosed among 92 suspected patients. GeneXpert detected 14 out of 16 cases (87.5%), including 4 MSSA and 10 MR-CoNS in approximately 1 hour after specimen receipt. The sensitivity and specificity of GeneXpert were 87.5% (CI 95%: 60.4-97.8) and 92.1% (CI 95%: 83-96.7), respectively, compared with standard culture methods. The sensitivity of GeneXpert for S. aureus was 100%. Regarding a cost-effectiveness analysis, the incremental cost of using GeneXpert was of 31.1€ per patient while the incremental cost-effectiveness ratio of GeneXpert compared with blood culture alones was about 180€ per life year gained. In conclusion, GeneXpert can be used directly with blood samples obtained through infected catheters to detect S. aureus and MR-CoNS in approximately 1h after sampling. In addition, it is cost-effective especially in areas with high prevalence of staphylococcal CRB. PMID:27571200

  20. Rapid Diagnosis of Staphylococcal Catheter-Related Bacteraemia in Direct Blood Samples by Real-Time PCR

    PubMed Central

    Soto, Marcelo; Sampietro-Colom, Laura; Soriano, Alex; Alvarez-Martínez, Míriam José; Almela, Manel; Marco, Francesc; Arjona, Ruth; Cobos-Trigueros, Nazaret; Morata, Laura; Mensa, José; Martínez, José Antonio; Mira, Aurea

    2016-01-01

    Catheter-related bacteremia (CRB) is an important cause of morbidity and mortality among hospitalized patients, being staphylococci the main etiologic agents. The objective of this study was to assess the use of a PCR-based assay for detection of staphylococci directly from blood obtained through the catheter to diagnose CRB caused by these microorganisms and to perform a cost-effectiveness analysis. A total of 92 patients with suspected CRB were included in the study. Samples were obtained through the catheter. Paired blood cultures were processed by standard culture methods and 4 ml blood samples were processed by GeneXpert-MRSA assay for the detection of methicillin-susceptible (MSSA) or methicillin-resistant (MRSA) Staphylococcus aureus, and methicillin-resistant coagulase-negative staphylococci (MR-CoNS). Sixteen CRB caused by staphylococci were diagnosed among 92 suspected patients. GeneXpert detected 14 out of 16 cases (87.5%), including 4 MSSA and 10 MR-CoNS in approximately 1 hour after specimen receipt. The sensitivity and specificity of GeneXpert were 87.5% (CI 95%: 60.4–97.8) and 92.1% (CI 95%: 83–96.7), respectively, compared with standard culture methods. The sensitivity of GeneXpert for S. aureus was 100%. Regarding a cost-effectiveness analysis, the incremental cost of using GeneXpert was of 31.1€ per patient while the incremental cost-effectiveness ratio of GeneXpert compared with blood culture alones was about 180€ per life year gained. In conclusion, GeneXpert can be used directly with blood samples obtained through infected catheters to detect S. aureus and MR-CoNS in approximately 1h after sampling. In addition, it is cost-effective especially in areas with high prevalence of staphylococcal CRB. PMID:27571200

  1. A sample extraction method for faster, more sensitive PCR-based detection of pathogens in blood culture.

    PubMed

    Regan, John F; Furtado, Manohar R; Brevnov, Maxim G; Jordan, Jeanne A

    2012-01-01

    Three mechanistically different sample extraction methodologies, namely, silica spin columns, phenol-chloroform, and an automated magnetic capture of polymer-complexed DNA (via an Automate Express instrument), were compared for their abilities to purify nucleic acids from blood culture fluids for use in TaqMan assays for detection of Staphylococcus aureus. The extracts from silica columns required 100- to 1000-fold dilutions to sufficiently reduce the powerful PCR inhibitory effects of the anticoagulant sodium polyanetholsulfonate, a common additive in blood culture media. In contrast, samples extracted by either phenol-chloroform or the Automate Express instrument required little or no dilution, respectively, allowing for an approximate 100-fold improvement in assay sensitivity. Analysis of 60 blood culture bottles indicated that these latter two methodologies could be used to detect lower numbers of pathogens and that a growing S. aureus culture could be detected 2 hours earlier than when using silica columns. Of the three tested methodologies, the Automate Express instrument had the shortest time to result, requiring only approximately 80 minutes to process 12 samples. These findings highlight the importance of considering the mechanism when selecting a DNA extraction methodology, given that certain PCR inhibitors act in a similar fashion to DNA in certain chemical environments, resulting in copurification, whereas other methodologies use different chemistries that have advantages during the DNA purification of certain types of samples.

  2. 1-Hydroxypyrene Levels in Blood Samples of Rats After Exposure to Generator Fumes

    PubMed Central

    Ifegwu, Clinton; Igwo-Ezikpe, Miriam N.; Anyakora, Chimezie; Osuntoki, Akinniyi; Oseni, Kafayat A.; Alao, Eragbae O.

    2013-01-01

    Polynuclear Aromatic Hydrocarbons (PAHs) are a major component of fuel generator fumes. Carcinogenicity of these compounds has long been established. In this study, 37 Swiss albino rats were exposed to generator fumes at varied distances for 8 hours per day for a period of 42 days and the level of 1-hydroxypyrene in their blood was evaluated. This study also tried to correlate the level of blood 1-hyroxypyrene with the distance from the source of pollution. Plasma was collected by centrifuging the whole blood sample followed by complete hydrolysis of the conjugated 1-hydroxypyrene glucuronide to yield the analyte of interest, 1-hydroxypyrene, which was achieved using beta glucuronidase. High performance liquid chromatography (HPLC) with UV detector was used to determine the 1-hydroxypyrene concentrations in the blood samples. The mobile phase was water:methanol (12:88 v/v) isocratic run at the flow rate of 1.2 mL/min with CI8 stationary phase at 250 nm. After 42 days of exposure, blood concentration level of 1-hydroxypyrene ranged from 34 μg/mL to 26.29 μg/mL depending on the distance from source of exposure. The control group had no 1-hydroxypyrene in their blood. After the period of exposure, percentage of death correlated with the distance from the source of exposure. Percentage of death ranged from 56% to zero depending on the proximity to source of pollution. PMID:24179393

  3. [Comparative validation of manual and automated methods for mixing and volume control of total blood samples].

    PubMed

    Folléa, G; Bigey, F; Jacob, D; Cazenave, J P

    1997-07-01

    During blood collection, agitation and volume limitations are critical to ensure thorough mixing of the blood with the anticoagulant and obtention of the predetermined volume. These 2 factors are essential to prevent blood activation and to obtain well standardized blood products. The objective of this study was to compare the quality of the blood collected using 2 types of collection method: tripping of a scale at a predetermined volume limit of 450 mL in the presence of manual agitation, and the 3 blood collection monitors currently available in France. A minimum of 100 collection procedures was performed for each of the 4 methods tested. Results were found to be equivalent using either the manual or the automated procedures with regard to both the accuracy and reproducibility of the blood volumes obtained and the collection times and flow rates. The characteristics of the red blood cell concentrates, platelet concentrates and plasma units prepared from the first 30 collections of each group were assessed and compared to regulatory requirements. The quality of all these products was found to be comparable to that currently observed at quality control and no product was rejected at the release control for reasons of poor collection. An assessment of the practicability of the different methods showed that the automated devices are subject to practical difficulties involving transport and battery loading. In addition, the cost of this equipment is approximately 5 times higher than that of the scales. In conclusion, the results of this study show that in our hands, no significant advantage could be expected from the use of automated blood collection monitors as compared to simple scales with manual mixing. These results further raise the question of the applicability to labile blood products of the comparative validations currently accepted in the pharmaceutical industry, in order to allow the use of correctly validated alternative methods.

  4. Glycosylated hemoglobin determination from capillary blood samples. Utility in an epidemiologic survey of diabetes.

    PubMed

    Ferrell, R E; Hanis, C L; Aguilar, L; Tulloch, B; Garcia, C; Schull, W J

    1984-02-01

    Total glycosylated hemoglobin was measured from capillary blood specimens obtained from a sample of 1880 individuals of Mexican-American ancestry residing in Starr County, Texas, between January 1981 and February 1982, as part of an epidemiologic survey to assess the prevalence of noninsulin-dependent diabetes mellitus (Type II). No significant difference was found between males and females. Diabetics were found to have significantly higher levels of glycosylated hemoglobin than nondiabetics. However, among diabetics, there was no significant difference between newly diagnosed and known diabetics, and known diabetics taking medication did not differ significantly from those not taking medication. An analysis of the specificity and sensitivity of glycosylated hemoglobin, fasting blood glucose, and casual blood glucose determinations as screening devices in a survey of diabetes prevalence reveals that glycosylated hemoglobin is superior to casual blood glucose determination. The conditions under which various screening devices might be more effective are discussed. PMID:6695895

  5. Small and cheap: accurate differential blood count with minimal sample volume by laser scanning cytometry (LSC)

    NASA Astrophysics Data System (ADS)

    Mittag, Anja; Lenz, Dominik; Smith, Paul J.; Pach, Susanne; Tarnok, Attila

    2005-04-01

    Aim: In patients, e.g. with congenital heart diseases, a differential blood count is needed for diagnosis. To this end by standard automatic analyzers 500 μl of blood is required from the patients. In case of newborns and infants this is a substantial volume, especially after operations associated with blood loss. Therefore, aim of this study was to develop a method to determine a differential blood picture with a substantially reduced specimen volume. Methods: To generate a differential blood picture 10 μl EDTA blood were mixed with 10 μl of a DRAQ5 solution (500μM, Biostatus) and 10 μl of an antibody mixture (CD45-FITC, CD14-PE, diluted with PBS). 20 μl of this cell suspension was filled into a Neubauer counting chamber. Due to the defined volume of the chamber it is possible to determine the cell count per volume. The trigger for leukocyte counting was set on DRAQ5 signal in order to be able to distinguish nucleated white blood cells from erythrocytes. Different leukocyte subsets could be distinguished due to the used fluorescence labeled antibodies. For erythrocyte counting cell suspension was diluted another 150 times. 20 μl of this dilution was analyzed in a microchamber by LSC with trigger set on forward scatter signal. Results: This method allows a substantial decrease of blood sample volume for generation of a differential blood picture (10 μl instead of 500μl). There was a high correlation between our method and the results of routine laboratory (r2=0.96, p<0.0001 n=40). For all parameters intra-assay variance was less than 7 %. Conclusions: In patients with low blood volume such as neonates and in critically ill infants every effort has to be taken to reduce the blood volume needed for diagnostics. With this method only 2% of standard sample volume is needed to generate a differential blood picture. Costs are below that of routine laboratory. We suggest this method to be established in paediatric cardiology for routine diagnostics and for

  6. Detection of the BLV provirus from nasal secretion and saliva samples using BLV-CoCoMo-qPCR-2: Comparison with blood samples from the same cattle.

    PubMed

    Yuan, Yuan; Kitamura-Muramatsu, Yuri; Saito, Susumu; Ishizaki, Hiroshi; Nakano, Miwa; Haga, Satoshi; Matoba, Kazuhiro; Ohno, Ayumu; Murakami, Hironobu; Takeshima, Shin-Nosuke; Aida, Yoko

    2015-12-01

    Bovine leukemia virus (BLV) induces enzootic bovine leukosis, which is the most common neoplastic disease in cattle. Sero-epidemiological studies show that BLV infection occurs worldwide. Direct contact between infected and uninfected cattle is thought to be one of the risk factors for BLV transmission. Contact transmission occurs via a mixture of natural sources, blood, and exudates. To confirm that BLV provirus is detectable in these samples, matched blood, nasal secretion, and saliva samples were collected from 50 cattle, and genomic DNA was extracted. BLV-CoCoMo-qPCR-2, an assay developed for the highly sensitive detection of BLV, was then used to measure the proviral load in blood (n=50), nasal secretions (n=48), and saliva (n=47) samples. The results showed that 35 blood samples, 14 nasal secretion samples, and 6 saliva samples were positive for the BLV provirus. Matched blood samples from cattle that were positive for the BLV provirus (either in nasal secretion or saliva samples) were also positive in their blood. The proviral load in the positive blood samples was >14,000 (copies/1×10(5) cells). Thus, even though the proviral load in the nasal secretion and saliva samples was much lower (<380 copies/1×10(5) cells) than that in the peripheral blood, prolonged direct contact between infected and healthy cattle may be considered as a risk factor for BLV transmission. PMID:26298004

  7. Detection of the BLV provirus from nasal secretion and saliva samples using BLV-CoCoMo-qPCR-2: Comparison with blood samples from the same cattle.

    PubMed

    Yuan, Yuan; Kitamura-Muramatsu, Yuri; Saito, Susumu; Ishizaki, Hiroshi; Nakano, Miwa; Haga, Satoshi; Matoba, Kazuhiro; Ohno, Ayumu; Murakami, Hironobu; Takeshima, Shin-Nosuke; Aida, Yoko

    2015-12-01

    Bovine leukemia virus (BLV) induces enzootic bovine leukosis, which is the most common neoplastic disease in cattle. Sero-epidemiological studies show that BLV infection occurs worldwide. Direct contact between infected and uninfected cattle is thought to be one of the risk factors for BLV transmission. Contact transmission occurs via a mixture of natural sources, blood, and exudates. To confirm that BLV provirus is detectable in these samples, matched blood, nasal secretion, and saliva samples were collected from 50 cattle, and genomic DNA was extracted. BLV-CoCoMo-qPCR-2, an assay developed for the highly sensitive detection of BLV, was then used to measure the proviral load in blood (n=50), nasal secretions (n=48), and saliva (n=47) samples. The results showed that 35 blood samples, 14 nasal secretion samples, and 6 saliva samples were positive for the BLV provirus. Matched blood samples from cattle that were positive for the BLV provirus (either in nasal secretion or saliva samples) were also positive in their blood. The proviral load in the positive blood samples was >14,000 (copies/1×10(5) cells). Thus, even though the proviral load in the nasal secretion and saliva samples was much lower (<380 copies/1×10(5) cells) than that in the peripheral blood, prolonged direct contact between infected and healthy cattle may be considered as a risk factor for BLV transmission.

  8. Blood

    MedlinePlus

    ... solid part of your blood contains red blood cells, white blood cells, and platelets. Red blood cells (RBC) deliver oxygen from your lungs to your tissues and organs. White blood cells (WBC) fight infection and are part of your ...

  9. Organochlorine pesticide residues in blood samples of agriculture and sheep wool workers in Bangalore (rural), India.

    PubMed

    Dhananjayan, V; Ravichandran, B; Rajmohan, H R

    2012-04-01

    To describe exposure level of organochlorine pesticides (OCP) among workers occupationally engaged in agriculture and sheep wool associated jobs, the present study was carried out in rural neighborhood of Bangalore city, India. Thirty participants were interviewed and obtained informed consent before blood sample collection. The maximum concentrations of OCP were detected in blood samples of agriculture workers than sheep wool workers. Among the metabolites of HCH and DDT, lindane (γ-HCH) and p,p'-DDE were the most contributed to the total OCP. There were no differences in pesticide residues found between sex and work groups. It was observed that about 30% of samples exceeded the tolerance limits of 10 μg/L prescribed for HCH under the prevention of food adulteration act. Therefore, the present study recommends continuous monitoring with larger sample size.

  10. Insight into normal thymic activity by assessment of peripheral blood samples.

    PubMed

    Machnes-Maayan, Diti; Lev, Atar; Katz, Uriel; Mishali, David; Vardi, Amir; Simon, Amos J; Somech, Raz

    2015-03-01

    The thymus is a highly specialized organ for T cell receptor (TCR) rearrangement and selection mechanisms that ensure the formation of functional and self-tolerant cells. Little is known about how peripheral blood assessment of thymic function reflects thymus activity during infancy. We compared thymic function-related markers in the thymus with those in peripheral blood in order to check their correlations. We concomitantly blood samples from immunocompetent infants who underwent cardiac surgery that involved thymectomy. The studied thymic markers included TCR excision circles (TRECs), four different TCRD (TCR delta chain) gene rearrangements, the TCR repertoire, regulatory T cells (Tregs, defined as the CD4+CD25+FOXP3+ cell population) and real-time quantitative polymerase chain reaction (RQ-PCR) mRNA expression of forkhead box P3 (FOXP3). Twenty patients were enrolled in this study. Their mean age at the time of the surgery was 3 months/5 days ± 3 months/18 days. There was a significant correlation between thymic and peripheral blood levels of TREC, all four TCRD gene rearrangements and the amount of Tregs. The levels of these parameters were significantly higher in the thymus than those detected in the peripheral blood. The TCR repertoire distribution in both samples was similar. FOXP3 mRNA levels in the thymus and peripheral blood correlated well. Our findings demonstrated a strong and significant correlation between peripheral blood and intra-thymic activity parameters during infancy. Assessment of these parameters in peripheral blood can be used to accurately estimate different intra-thymic capacities for assessing T cell function in health and disease.

  11. [Automated serial diagnosis of donor blood samples. Ergonomic and economic organization structure].

    PubMed

    Stoll, T; Fischer-Fröhlich, C L; Mayer, G; Hanfland, P

    1990-01-01

    A comprehensive computer-aided administration-system for blood-donors is presented. Ciphered informations of barcode-labels allow the automatic and nevertheless selective pipetting of samples by pipetting-robots. Self-acting analysis-results are transferred to a host-computer in order to actualize a donor data-base.

  12. Alkylphenol polyethoxylate derivatives in groundwater and blood samples collected from pig herds in Taiwan.

    PubMed

    Chiu, Tai-Shun; Hsieh, Chi-Ying; Miaw, Chang-Ling; Lin, Chao-Nan; Chang, Tsung-Chou; Yen, Chia-Hung; Chiou, Ming-Tang

    2014-07-01

    Alkylphenol polyethoxylate (APEO) derivatives, such as nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), nonylphenol (NP) and octylphenol (OP), have been detected in the surface water, sediment, food and groundwater of numerous countries. Because groundwater is the main source of water for pig herds, the aim of this study was to measure the concentrations of APEO derivatives in groundwater and blood samples that were collected from pig herds raised near the Wuluo River in Southern Taiwan. The mean concentrations of NP, OP, NP1EO and NP2EO in the groundwater supply for 10 pig herds were 0.04 µg/l, 0.26 ± 0.23 µg/l, 0.74 ± 0.69 µg/l and 0.17 ± 0.22 µg/l, respectively. NP was detected in all blood samples collected from 5 of the 10 pig herds. The highest concentrations detected in the blood samples collected from six-week-old piglets and sows were 12.00 µg/l and 56.94 µg/l, respectively. Blood samples from 4 of the 5 herds showed OP contamination. The highest OP concentrations detected in 6-week-old piglets and sows were 275.58 µg/l and 566.32 µg/l, respectively. These results indicate that APEO derivatives accumulated in the groundwater supply and the bloodstreams of the pigs.

  13. Effect of human blood addition on dendritic growth of cupric chloride crystals in aqueous solutions

    NASA Astrophysics Data System (ADS)

    Shibata, Takashi; Shirasaka, Ryukoh; Ogawa, Tomoya; Takakuwa, Yuichi; Furiya, Kei; Tanaka, Akemi; Kogure, Mitsuko; Obata, Hiroshi

    1994-09-01

    An extremely small amount of 0.2% or less (volume ratio) of human blood influences the dendritic growth of cupric chloride crystals in an aqueous solution, with some researchers claiming that the growth depends upon any disease the blood donor might be carrying. This is a very surprising phenomenon. Dendrites grown in a blood-added CuCl 2 ⋯ 2H 2O solution were classified into groups of blue and green by color; all the dendrites grown in an aqueous solution without blood were a single color: blue. A very clear difference between the blue and green dendrites was obtained by thermo-gravimetry/differential thermal analysis, because of positional and numerical difference of water molecules in the cupric chloride crystals. Many tiny granules were observed on facets of the dendrites grown in the blood-added aqueous solutions. Surfaces of the dendrites were surveyed by an electron probe X-ray micro-analyzer and by an X-ray photo-electron spectroscope, and chemical shifts of copper, chlorine, nitrogen, carbon and oxygen signals were found on those dendrites grown in blood-added CuCl 2 ⋯ 2H 2O solutions. This evidence suggests that components of blood including amino acid, peptide and/or protein or some composition of them were chemisorbed on the dendrite surfaces.

  14. Direct RNA-based detection of CTX-M β-lactamases in human blood samples.

    PubMed

    Stein, Claudia; Makarewicz, Oliwia; Pfeifer, Yvonne; Brandt, Christian; Pletz, Mathias W

    2015-05-01

    Bloodstream infections with ESBL-producers are associated with increased mortality, which is due to delayed appropriate treatment resulting in clinical failure. Current routine diagnostics for detection of bloodstream infections consists of blood culture followed by species identification and susceptibility testing. In attempts to improve and accelerate diagnostic procedures, PCR-based methods have been developed. These methods focus on species identification covering only a limited number of ESBL coding genes. Therefore, they fail to cover the steadily further evolving genetic diversity of clinically relevant β-lactamases. We have recently designed a fast and novel RNA targeting method to detect and specify CTX-M alleles from bacterial cultures, based on an amplification-pyrosequencing approach. We further developed this assay towards a diagnostic tool for clinical use and evaluated its sensitivity and specificity when applied directly to human blood samples. An optimized protocol for mRNA isolation allows detection of specific CTX-M groups from as little as 100 CFU/mL blood via reverse transcription, amplification, and pyrosequencing directly from human EDTA blood samples as well as from pre-incubated human blood cultures with a turnaround time for test results of <7 h.

  15. On the asymptotic improvement of supervised learning by utilizing additional unlabeled samples - Normal mixture density case

    NASA Technical Reports Server (NTRS)

    Shahshahani, Behzad M.; Landgrebe, David A.

    1992-01-01

    The effect of additional unlabeled samples in improving the supervised learning process is studied in this paper. Three learning processes. supervised, unsupervised, and combined supervised-unsupervised, are compared by studying the asymptotic behavior of the estimates obtained under each process. Upper and lower bounds on the asymptotic covariance matrices are derived. It is shown that under a normal mixture density assumption for the probability density function of the feature space, the combined supervised-unsupervised learning is always superior to the supervised learning in achieving better estimates. Experimental results are provided to verify the theoretical concepts.

  16. Multiplex short tandem repeat amplification of low template DNA samples with the addition of proofreading enzymes.

    PubMed

    Davis, Carey P; Chelland, Lynzee A; Pavlova, Victoria R; Illescas, María J; Brown, Kelly L; Cruz, Tracey Dawson

    2011-05-01

    With <100 pg of template DNA, routine short tandem repeat (STR) analysis often fails, resulting in no or partial profiles and increased stochastic effects. To overcome this, some have investigated preamplification methods that include the addition of proofreading enzymes to the PCR cocktail. This project sought to determine whether adding proofreading polymerases directly in the STR amplification mixture would improve the reaction when little template DNA is available. Platinum Taq High Fidelity and GeneAmp High Fidelity were tested in Profiler Plus™ STR reactions alone and in combination with AmpliTaq(®) Gold. All reactions included the additional step of a post-PCR purification step. With both pristine low template DNA and casework samples, the addition of these polymerases resulted in comparable or no improvement in the STR amplification signal. Further, stochastic effects and artifacts were observed equally across all enzyme conditions. Based on these studies, the addition of these proofreading enzymes to a multiplex STR amplification is not recommended for low template DNA work.

  17. Detection of Leukemia with Blood Samples Using Raman Spectroscopy and Multivariate Analysis

    NASA Astrophysics Data System (ADS)

    Martínez-Espinosa, J. C.; González-Solís, J. L.; Frausto-Reyes, C.; Miranda-Beltrán, M. L.; Soria-Fregoso, C.; Medina-Valtierra, J.

    2009-06-01

    The use of Raman spectroscopy to analyze blood biochemistry and hence distinguish between normal and abnormal blood was investigated. Blood samples were obtained from 6 patients who were clinically diagnosed with leukemia and 6 healthy volunteers. The imprint was put under the microscope and several points were chosen for Raman measurement. All the spectra were collected by a confocal Raman micro-spectroscopy (Renishaw) with a NIR 830 nm laser. It is shown that the serum samples from patients with leukemia and from the control group can be discriminated when the multivariate statistical methods of principal component analysis (PCA) and linear discriminated analysis (LDA) are applied to their Raman spectra. The ratios of some band intensities were analyzed and some band ratios were significant and corresponded to proteins, phospholipids, and polysaccharides. The preliminary results suggest that Raman Spectroscopy could be a new technique to study the degree of damage to the bone marrow using just blood samples instead of biopsies, treatment very painful for patients.

  18. Evaluation of PCR Approaches for Detection of Bartonella bacilliformis in Blood Samples

    PubMed Central

    Gomes, Cláudia; Martinez-Puchol, Sandra; Pons, Maria J.; Bazán, Jorge; Tinco, Carmen; del Valle, Juana; Ruiz, Joaquim

    2016-01-01

    Background The lack of an effective diagnostic tool for Carrion’s disease leads to misdiagnosis, wrong treatments and perpetuation of asymptomatic carriers living in endemic areas. Conventional PCR approaches have been reported as a diagnostic technique. However, the detection limit of these techniques is not clear as well as if its usefulness in low bacteriemia cases. The aim of this study was to evaluate the detection limit of 3 PCR approaches. Methodology/Principal Findings We determined the detection limit of 3 different PCR approaches: Bartonella-specific 16S rRNA, fla and its genes. We also evaluated the viability of dry blood spots to be used as a sample transport system. Our results show that 16S rRNA PCR is the approach with a lowest detection limit, 5 CFU/μL, and thus, the best diagnostic PCR tool studied. Dry blood spots diminish the sensitivity of the assay. Conclusions/Significance From the tested PCRs, the 16S rRNA PCR-approach is the best to be used in the direct blood detection of acute cases of Carrion’s disease. However its use in samples from dry blood spots results in easier management of transport samples in rural areas, a slight decrease in the sensitivity was observed. The usefulness to detect by PCR the presence of low-bacteriemic or asymptomatic carriers is doubtful, showing the need to search for new more sensible techniques. PMID:26959642

  19. Genetic characterization of atypical Mansonella (Mansonella) ozzardi microfilariae in human blood samples from northeastern Peru.

    PubMed

    Marcos, Luis A; Arrospide, Nancy; Recuenco, Sergio; Cabezas, Cesar; Weil, Gary J; Fischer, Peter U

    2012-09-01

    DNA sequence comparisons are useful for characterizing proposed new parasite species or strains. Microfilariae with an atypical arrangement of nuclei behind the cephalic space have been recently described in human blood samples from the Amazon region of Peru. Three blood specimens containing atypical microfilariae were genetically characterized using three DNA markers (5S ribosomal DNA, 12S ribosomal DNA, and cytochrome oxidase I). All atypical microfilariae were clustered into the Mansonella group and indistinguishable from M. ozzardi based on these DNA markers. PMID:22826497

  20. Analysis of carboxyhemoglobin and lead in blood samples from HANES III examinations (Hispanic HANES). Final report

    SciTech Connect

    Radford, E.P.

    1983-01-01

    Blood samples from a population of Hispanic background were measured for carboxyhemoglobin (COHb), thiocyanate (SCN) and lead (Pb). Preliminary analysis of 2121 results indicates that combination of measurements of COHb with SCN helps distinguish smokers from non-smokers, and sharpens the definition of persons likely to have been exposed to environmental sources of carbon monoxide. Higher values of blood lead were more common among those exposed to environmental sources of carbon monoxide than in those with no evidence of such exposure. 4 references, 1 figure, 3 tables. (ACR)

  1. Interlaboratory comparison of PCR-based identification of Candida and Aspergillus DNA in spiked blood samples.

    PubMed

    Reichard, Utz; Buchheidt, Dieter; Lass-Flörl, Cornelia; Loeffler, Juergen; Lugert, Raimond; Ruhnke, Markus; Tintelnot, Kathrin; Weig, Michael; Groß, Uwe

    2012-09-01

    Despite PCR per se being a powerful and sensitive technique, regarding the detection of fungi in patients' blood, no consensus for a standardised PCR protocol yet exists. To complement other ongoing or accomplished studies which tackle this problem, the German Reference Center for Systemic Mycoses conducted an interlaboratory comparison starting with blood samples spiked with fungal cell elements. Altogether, six laboratories using in-house PCR-protocols from Germany and Austria participated in the trial. Blood samples were spiked with vital cells of Candida albicans or Aspergillus fumigatus. Candida was used in the yeast form, whereas Aspergillus cells were either spiked as conidia or as very young germlings, also known as smoo cells. Spiked blood samples contained between 10 and 10 000 cells ml(-1). Depending on the techniques used for fungal cell disruption and DNA-amplification, detection quality was variable between laboratories, but also differed within single laboratories in different trials particularly for samples spiked with less than 100 cells ml(-1). Altogether, at least regarding the detection of A. fumigatus, two of six laboratories showed constant reliable test results also with low fungal cell number spiked samples. Protocols used by these labs do not differ substantially from others. However, as particularities, one protocol included a conventional phenol chloroform extraction during the DNA preparation process and the other included a real time PCR-protocol based on FRET probes. Other laboratory comparisons on the basis of clinical samples should follow to further evaluate the procedures. The difficulties and problems of such trials in general are discussed.

  2. Interlaboratory comparison of PCR-based identification of Candida and Aspergillus DNA in spiked blood samples.

    PubMed

    Reichard, Utz; Buchheidt, Dieter; Lass-Flörl, Cornelia; Loeffler, Juergen; Lugert, Raimond; Ruhnke, Markus; Tintelnot, Kathrin; Weig, Michael; Groß, Uwe

    2012-09-01

    Despite PCR per se being a powerful and sensitive technique, regarding the detection of fungi in patients' blood, no consensus for a standardised PCR protocol yet exists. To complement other ongoing or accomplished studies which tackle this problem, the German Reference Center for Systemic Mycoses conducted an interlaboratory comparison starting with blood samples spiked with fungal cell elements. Altogether, six laboratories using in-house PCR-protocols from Germany and Austria participated in the trial. Blood samples were spiked with vital cells of Candida albicans or Aspergillus fumigatus. Candida was used in the yeast form, whereas Aspergillus cells were either spiked as conidia or as very young germlings, also known as smoo cells. Spiked blood samples contained between 10 and 10 000 cells ml(-1). Depending on the techniques used for fungal cell disruption and DNA-amplification, detection quality was variable between laboratories, but also differed within single laboratories in different trials particularly for samples spiked with less than 100 cells ml(-1). Altogether, at least regarding the detection of A. fumigatus, two of six laboratories showed constant reliable test results also with low fungal cell number spiked samples. Protocols used by these labs do not differ substantially from others. However, as particularities, one protocol included a conventional phenol chloroform extraction during the DNA preparation process and the other included a real time PCR-protocol based on FRET probes. Other laboratory comparisons on the basis of clinical samples should follow to further evaluate the procedures. The difficulties and problems of such trials in general are discussed. PMID:22248125

  3. An investigation of dust lead sampling locations and children's blood lead levels.

    PubMed

    Wilson, Jonathan; Dixon, Sherry; Galke, Warren; McLaine, Patricia

    2007-01-01

    The objective of this study is to provide guidance on where to collect dust lead wipe samples in homes to best characterize the risk of a resident child having a blood lead level at or above the CDC level of concern (10 microg/dl). In 1998, the Milwaukee Health Department enrolled 72 children living in pre-1950 buildings: 34 had elevated (i.e., > or = 10 microg/dl) blood lead levels (EBL); and 38 had non-elevated blood lead levels (non-EBL). This study explored dust lead sampling locations by examining loading differences between homes where children with EBL and non-EBL lived. Floor, windowsill, and window trough samples were collected in the living room, kitchen, bathroom, and child's bedroom and play area. Floor samples were collected at four locations: room entry; center of the room; under a window; and against the wall opposite the window (perimeter). Geometric mean floor dust lead levels were generally two to three times higher in homes of EBL children than homes of non-EBL children. Sampling the floor at the room entry or center is preferable to sampling under the window or from the perimeter of the room. When the central floor average was used, the room combinations that had the greatest differences between homes of EBL children and non-EBL children all included a sample from the child's bedroom and excluded the bathroom. When the entry floor average was used, the greatest differences also excluded bathrooms, but otherwise included a mix of all of the other rooms. Window samples did not distinguish where children with EBLs versus non-EBLs resided. This paper is based on Milwaukee alone, so generalizing results to other locations should be done with caution. PMID:16823397

  4. Sample stability for complete blood cell count using the Sysmex XN haematological analyser

    PubMed Central

    Daves, Massimo; Zagler, Elmar M.; Cemin, Roberto; Gnech, Flora; Joos, Alexandra; Platzgummer, Stefan; Lippi, Giuseppe

    2015-01-01

    Background Sample stability is a crucial aspect for the quality of results of a haematology laboratory. This study was conducted to investigate the reliability of haematological testing using Sysmex XN in samples stored for up to 24 h at different temperatures. Materials and methods Haematological tests were performed on whole blood samples collected from 16 ostensibly healthy outpatients immediately after collection and 3 h, 6 h or 24 h afterwards, with triple aliquots kept at room temperature, 4 °C or 37 °C. Results No meaningful bias was observed after 3 h under different storage conditions, except for red blood cell distribution width (RDW) and platelet count (impedance technique, PLT-I) at 37 °C. After 6 h, meaningful bias was observed for mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) at room temperature, red blood cell (RBC) count, mean corpuscular haemoglobin concentration (MCHC), MCH, MCV and PLT-I at 4 °C, and RBC, RDW, MCHC, MCH and PLT-I at 37 °C. After 24 h, a meaningful bias was observed for MCHC, MCV, platelet count (fluorescent technique, PLT-F) and mean platelet volume (MPV) at room temperature, MCHC, MCV, PLT-I and MPV at 4 °C, and all parameters except RBC count and MPV at 37 °C. Discussion Great caution should be observed when analysing results of haematological tests conducted more than 3 h after sample collection. PMID:26057491

  5. Quantitative determination of valproic acid in postmortem blood samples--evidence of strong matrix dependency and instability.

    PubMed

    Kiencke, Verena; Andresen-Streichert, Hilke; Müller, Alexander; Iwersen-Bergmann, Stefanie

    2013-11-01

    Most of the daily work of forensic toxicologists deals with fatal cases resulting from overdoses of licit and illicit drugs. However, another reason for fatalities in patients suffering from epilepsy can be undetectable or subtherapeutic levels of antiepileptic drugs. Some studies have shown a correlation between "sudden unexpected death in epilepsy" (SUDEP) and the ineffective treatment of epilepsy. Low levels of antiepileptic drugs may be a risk factor for SUDEP. The death of a psychiatric patient also suffering from epilepsy inspired the investigation. Subsequent to the death of the patient, the doctor was accused of providing inadequate therapy for epilepsy. The patient was to be treated with valproic acid. We developed and validated a simple method of determining valproic acid levels by gas chromatography-mass spectrometry for serum, but a transfer of the method from serum to postmortem whole blood failed. The method had to be modified and revalidated for postmortem whole blood specimens. A stability study of valproic acid in postmortem blood was conducted, showing a decline of valproic acid levels by 85 % after storage at room temperature for 28 days. During the storage time, the blood samples showed changes in consistency. Depending on the stage of decomposition, it is necessary to perform a determination by standard addition with an equilibration time of 4 h before extraction to achieve reliable results. For a proper interpretation of quantitative results, it is necessary to keep the postmortem decline of valproic acid concentrations in mind.

  6. Sample to answer visualization pipeline for low-cost point-of-care blood cell counting

    NASA Astrophysics Data System (ADS)

    Smith, Suzanne; Naidoo, Thegaran; Davies, Emlyn; Fourie, Louis; Nxumalo, Zandile; Swart, Hein; Marais, Philip; Land, Kevin; Roux, Pieter

    2015-03-01

    We present a visualization pipeline from sample to answer for point-of-care blood cell counting applications. Effective and low-cost point-of-care medical diagnostic tests provide developing countries and rural communities with accessible healthcare solutions [1], and can be particularly beneficial for blood cell count tests, which are often the starting point in the process of diagnosing a patient [2]. The initial focus of this work is on total white and red blood cell counts, using a microfluidic cartridge [3] for sample processing. Analysis of the processed samples has been implemented by means of two main optical visualization systems developed in-house: 1) a fluidic operation analysis system using high speed video data to determine volumes, mixing efficiency and flow rates, and 2) a microscopy analysis system to investigate homogeneity and concentration of blood cells. Fluidic parameters were derived from the optical flow [4] as well as color-based segmentation of the different fluids using a hue-saturation-value (HSV) color space. Cell count estimates were obtained using automated microscopy analysis and were compared to a widely accepted manual method for cell counting using a hemocytometer [5]. The results using the first iteration microfluidic device [3] showed that the most simple - and thus low-cost - approach for microfluidic component implementation was not adequate as compared to techniques based on manual cell counting principles. An improved microfluidic design has been developed to incorporate enhanced mixing and metering components, which together with this work provides the foundation on which to successfully implement automated, rapid and low-cost blood cell counting tests.

  7. Determination of cathinones and related ephedrines in forensic whole-blood samples by liquid-chromatography-electrospray tandem mass spectrometry.

    PubMed

    Sørensen, Lambert K

    2011-04-01

    A liquid-chromatography-tandem-mass-spectrometry method using pneumatically assisted electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of cathinone, methcathinone, ethcathinone, amfepramone, mephedrone, flephedrone, methedrone, methylone, butylone, cathine, norephedrine, ephedrine, pseudoephedrine, methylephedrine and methylpseudoephedrine in human live and post-mortem whole blood. The blood proteins were precipitated by the addition of methanol, and the extract was purified by ultrafiltration. The separation of diastereomeric ephedrines was achieved on an ethyl-linked phenyl column. Matrix-matched calibrants combined with the isotope dilution of selected substances were used for quantitative analysis. The relative intra-laboratory reproducibility standard deviations were generally better than 7% at concentrations of 20 μg/L, and the mean true recoveries were 87-106% in the concentration range of 10-250 μg/L. The detection limits were in the range of 0.5-3 μg/L. The cathinones were unstable in whole blood and sample extracts under neutral conditions, but the stability could be improved by the acidification of the sample matrix. PMID:21376674

  8. Relationship between gonadal steroids and corticosterone during blood sampling in saker falcons.

    PubMed

    al-Ankar, A R

    1998-07-01

    Blood sampling in manually restrained or ketamine (15 mg/kg given intramuscularly) treated saker falcons (Falco cherrug) induced an increased concentration in plasma corticosterone. Elevated plasma progesterone, oestradiol 17 beta, and testosterone concentrations also were observed in some of these birds. An inverse relationship was demonstrated between levels of corticosterone and progesterone, but not with the levels of other hormones. It is suggested that progesterone measurement should be taken into consideration when studying the influence of stressors in falcons.

  9. Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples.

    PubMed

    Flemmig, Jörg; Schwarz, Pauline; Bäcker, Ingo; Leichsenring, Anna; Lange, Franziska; Arnhold, Jürgen

    2016-01-01

    In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 µl makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments. Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes. Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases. PMID:27501318

  10. Active tracking of rejected dried blood samples in a large program in Nigeria

    PubMed Central

    Inalegwu, Auchi; Phillips, Sunny; Datir, Rawlings; Chime, Christopher; Ozumba, Petronilla; Peters, Samuel; Ogbanufe, Obinna; Mensah, Charles; Abimiku, Alash’Le; Dakum, Patrick; Ndembi, Nicaise

    2016-01-01

    AIM: To study the impact of rejection at different levels of health care by retrospectively reviewing records of dried blood spot samples received at the molecular laboratory for human immunodeficiency virus (HIV) early infant diagnosis (EID) between January 2008 and December 2012. METHODS: The specimen rejection rate, reasons for rejection and the impact of rejection at different levels of health care was examined. The extracted data were cleaned and checked for consistency and then de-duplicated using the unique patient and clinic identifiers. The cleaned data were ciphered and exported to SPSS version 19 (SPSS 2010 IBM Corp, New York, United States) for statistical analyses. RESULTS: Sample rejection rate of 2.4% (n = 786/32552) and repeat rate of 8.8% (n = 69/786) were established. The mean age of infants presenting for first HIV molecular test among accepted valid samples was 17.83 wk (95%CI: 17.65-18.01) vs 20.30 wk (95%CI: 16.53-24.06) for repeated samples. HIV infection rate was 9.8% vs 15.9% for accepted and repeated samples. Compared to tertiary healthcare clinics, secondary and primary clinics had two-fold and three-fold higher likelihood of sample rejection, respectively (P < 0.05). We observed a significant increase in sample rejection rate with increasing number of EID clinics (r = 0.893, P = 0.041). The major reasons for rejection were improper sample collection (26.3%), improper labeling (16.4%) and insufficient blood (14.8%). CONCLUSION: Programs should monitor pre-analytical variables and incorporate continuous quality improvement interventions to reduce errors associated with sample rejection and improve patient retention. PMID:27175352

  11. Optical detection of Trypanosoma cruzi in blood samples for diagnosis purpose

    NASA Astrophysics Data System (ADS)

    Alanis, Elvio; Romero, Graciela; Alvarez, Liliana; Martinez, Carlos C.; Basombrio, Miguel A.

    2004-10-01

    An optical method for detection of Trypanosoma Cruzi (T. cruzi) parasites in blood samples of mice infected with Chagas disease is presented. The method is intended for use in human blood, for diagnosis purposes. A thin layer of blood infected by T. cruzi parasites, in small concentrations, is examined in an interferometric microscope in which the images of the vision field are taken by a CCD camera and temporarily stored in the memory of a host computer. The whole sample is scanned displacing the microscope plate by means of step motors driven by the computer. Several consecutive images of the same field are taken and digitally processed by means of image temporal differentiation in order to detect if a parasite is eventually present in the field. Each field of view is processed in the same fashion, until the full area of the sample is covered or until a parasite is detected, in which case an acoustical warning is activated and the corresponding image is displayed permitting the technician to corroborate the result visually. A discussion of the reliability of the method as well as a comparison with other well established techniques are presented.

  12. Shelf life of cooked goat blood sausage prepared with the addition of heart and kidney.

    PubMed

    Silva, F A P; Amaral, D S; Guerra, I C D; Arcanjo, N M O; Bezerra, T K A; Ferreira, V C S; Araújo, I B S; Dalmás, P S; Madruga, M S

    2014-08-01

    This study focused on the effect of two packaging formats (vacuum packaging and over-wrap packaging) on the shelf life of cooked sausage prepared with blood, heart, kidney and goat meat fragments under storage at 4±1°C for a period of 90 days. The storage time and type of packaging significantly affected the chemical (pH, moisture, protein and TBARS number), physical (shear force) and microbial (mould and yeast) parameters of cooked sausage. Vacuum packaging maintained the microbiological and chemical qualities of cooked goat blood sausage for a longer period of time (63 days) compared to over-wrap packaging (41 days) and could be a viable alternative to refrigerated storage of the product for quality maintenance. PMID:24769873

  13. Shelf life of cooked goat blood sausage prepared with the addition of heart and kidney.

    PubMed

    Silva, F A P; Amaral, D S; Guerra, I C D; Arcanjo, N M O; Bezerra, T K A; Ferreira, V C S; Araújo, I B S; Dalmás, P S; Madruga, M S

    2014-08-01

    This study focused on the effect of two packaging formats (vacuum packaging and over-wrap packaging) on the shelf life of cooked sausage prepared with blood, heart, kidney and goat meat fragments under storage at 4±1°C for a period of 90 days. The storage time and type of packaging significantly affected the chemical (pH, moisture, protein and TBARS number), physical (shear force) and microbial (mould and yeast) parameters of cooked sausage. Vacuum packaging maintained the microbiological and chemical qualities of cooked goat blood sausage for a longer period of time (63 days) compared to over-wrap packaging (41 days) and could be a viable alternative to refrigerated storage of the product for quality maintenance.

  14. Array CGH Analysis of Paired Blood and Tumor Samples from Patients with Sporadic Wilms Tumor

    PubMed Central

    del Carmen Crespo, María; Vallespín, Elena; Palomares-Bralo, María; Martin-Arenas, Rubén; Rueda-Arenas, Inmaculada; Silvestre de Faria, Paulo Antonio; García-Miguel, Purificación; Lapunzina, Pablo; Regla Vargas, Fernando; Seuanez, Hector N.; Martínez-Glez, Víctor

    2015-01-01

    Wilms tumor (WT), the most common cancer of the kidney in infants and children, has a complex etiology that is still poorly understood. Identification of genomic copy number variants (CNV) in tumor genomes provides a better understanding of cancer development which may be useful for diagnosis and therapeutic targets. In paired blood and tumor DNA samples from 14 patients with sporadic WT, analyzed by aCGH, 22% of chromosome abnormalities were novel. All constitutional alterations identified in blood were segmental (in 28.6% of patients) and were also present in the paired tumor samples. Two segmental gains (2p21 and 20q13.3) and one loss (19q13.31) present in blood had not been previously described in WT. We also describe, for the first time, a small, constitutive partial gain of 3p22.1 comprising 2 exons of CTNNB1, a gene associated to WT. Among somatic alterations, novel structural chromosomal abnormalities were found, like gain of 19p13.3 and 20p12.3, and losses of 2p16.1-p15, 4q32.5-q35.1, 4q35.2-q28.1 and 19p13.3. Candidate genes included in these regions might be constitutively (SIX3, SALL4) or somatically (NEK1, PIAS4, BMP2) operational in the development and progression of WT. To our knowledge this is the first report of CNV in paired blood and tumor samples in sporadic WT. PMID:26317783

  15. EFFECT OF STORAGE TIME AND STORAGE CONDITIONS ON ANTIBODY DETECTION IN BLOOD SAMPLES COLLECTED ON FILTER PAPER.

    PubMed

    Bevins, Sarah; Pappert, Ryan; Young, John; Schmit, Brandon; Kohler, Dennis; Baeten, Laurie

    2016-07-01

    Using filter paper to collect blood from wildlife for antibody analysis can be a powerful technique to simplify the collection, transport, and storage of blood samples. Despite these advantages, there are limited data that detail how long these samples can be stored and how storage conditions affect antibody longevity. We used blood samples collected on filter paper from coyotes experimentally infected with Yersinia pestis to determine optimum sample storage conditions over time. Blood samples collected on filter paper were stored for 454 d or more in four groups: 1) at ambient temperature and at ambient relative humidity, 2) at ambient temperature with desiccant, 3) at 4 C with desiccant, and 4) at -20 C with desiccant. Samples stored at 4 C or -20 C with desiccant had detectable antibody for a longer period of time than the samples stored at room temperature. PMID:27187032

  16. Glycosyltransferases as marker genes for the quantitative polymerase chain reaction-based detection of circulating tumour cells from blood samples of patients with breast cancer undergoing adjuvant therapy.

    PubMed

    Kölbl, Alexandra C; Hiller, Roman A; Ilmer, Mathias; Liesche, Friederike; Heublein, Sabine; Schröder, Lennard; Hutter, Stefan; Friese, Klaus; Jeschke, Udo; Andergassen, Ulrich

    2015-08-01

    Altered glycosylation is a predominant feature of tumour cells; it serves for cell adhesion and detachment, respectively, and facilitates the immune escape of these cells. Therefore changes in the expression of glycosyltransferase genes could help to identify circulating tumour cells (CTCs) in the blood samples of cancer patients using a quantitative polymerase chain reaction (PCR) approach. Blood samples of healthy donors were inoculated with certain numbers of established breast cancer cell line cells, thus creating a model system. These samples were analysed by quantitative PCR for the expression of six different glycosyltransferase genes. The three genes with the best results in the model system were consecutively applied to samples from adjuvant breast cancer patients and of healthy donors. FUT3 and GALNT6 showed the highest increase in relative expression, while GALNT6 and ST3GAL3 were the first to reach statistically significant different ∆CT-values comparing the sample with and without addition of tumour cells. These three genes were applied to patient samples, but did not show any significant results that may suggest the presence of CTCs in the blood. Although the relative expression of some of the glycosyltransferase genes exhibited reasonable results in the model system, their application to breast cancer patient samples will have to be further improved, e.g. by co-analysis of patient blood samples by gold-standard methods.

  17. An improved, PCR-based strategy for the detection of Trypanosoma cruzi in human blood samples.

    PubMed

    Ribeiro-dos-Santos, G; Nishiya, A S; Sabino, E C; Chamone, D F; Saez-Alquézar, A

    1999-10-01

    Attempts were made to improve the PCR-based detection of Trypanosoma cruzi in blood samples, primarily for screening blood donors. Samples were obtained from candidate donors who were reactive in one or two of three serological tests for Chagas disease (and therefore considered 'indeterminate') or in all three tests (3+). Each sample was then examined using three different, PCR-based techniques: 'PCR-I' (in which the target DNA is a nuclear repetitive sequence); 'PCR-II' [amplifying a conserved region of the T. cruzi kinetoplast DNA (kDNA)]; and 'PCR-III' (a new strategy in which the target kDNA is amplified by 'nested' PCR). Among the samples from 3+ individuals, PCR-I, PCR-II and PCR-III amplified two (3.8%) out of 52, four (4.5%) out of 88, and 27 (25.7%) out of 105 samples tested, respectively. Seven, 69 and 70 samples from 'indeterminate' subjects were tested by PCR-I, PCR-II and PCR-III, respectively; there was not a single positive result by PCR-I or PCR-II, but three (4.3%) of the samples tested by PCR-III were positive. In a reconstruction experiment, in conditions in which PCR-I and PCR-II could not detect 10,000 parasites/ml, PCR-III was able to detect one parasite/ml. Although all three PCR-based strategies examined had rather poor sensitivities, PCR-III was far more sensitive than PCR-I or PCR-II. PMID:10715696

  18. Behavior of optical properties of coagulated blood sample at 633 nm wavelength

    NASA Astrophysics Data System (ADS)

    Morales Cruzado, Beatriz; Vázquez y Montiel, Sergio; Delgado Atencio, José Alberto

    2011-03-01

    Determination of tissue optical parameters is fundamental for application of light in either diagnostics or therapeutical procedures. However, in samples of biological tissue in vitro, the optical properties are modified by cellular death or cellular agglomeration that can not be avoided. This phenomena change the propagation of light within the biological sample. Optical properties of human blood tissue were investigated in vitro at 633 nm using an optical setup that includes a double integrating sphere system. We measure the diffuse transmittance and diffuse reflectance of the blood sample and compare these physical properties with those obtained by Monte Carlo Multi-Layered (MCML). The extraction of the optical parameters: absorption coefficient μa, scattering coefficient μs and anisotropic factor g from the measurements were carried out using a Genetic Algorithm, in which the search procedure is based in the evolution of a population due to selection of the best individual, evaluated by a function that compares the diffuse transmittance and diffuse reflectance of those individuals with the experimental ones. The algorithm converges rapidly to the best individual, extracting the optical parameters of the sample. We compare our results with those obtained by using other retrieve procedures. We found that the scattering coefficient and the anisotropic factor change dramatically due to the formation of clusters.

  19. Determination of DDT and related compounds in blood samples from agricultural workers.

    PubMed

    Guardino, X; Serra, C; Obiols, J; Rosell, M G; Berenguer, M J; López, F; Brosa, J

    1996-01-01

    An analytical method combining a solid-phase (C18) clean-up and GC-electron-capture detection using a capillary column, was implemented to determine p,p'-DDT and its metabolites (p,p'-DDD and p,p'-DDE), as well as other organochlorine pesticides in whole blood samples from 30 farmers and 24 non-occupationally exposed workers. The average concentrations for the quantified pesticides, p,p'-DDT, p,p'-DDD and p,p'-DDE, were 0.9, 1.5 and 8.0 micrograms/l whole blood for exposed workers and 0.3, 0.5 and 3.3 micrograms/l for unexposed workers, respectively. GC-MS was used to confirm the identity of the pesticides found. Solid-phase extraction and the protocol used give a cleaner analytical matrix, not only improving sensitivity and resolution, but also allowing analyses with smaller blood samples as compared to other methods.

  20. Dried blood spot sampling for detection of monoclonal immunoglobulin gene rearrangement.

    PubMed

    Petrara, Maria Raffaella; Elefanti, Lisa; Quaggio, Monica; Zanchetta, Marisa; Scaini, Maria Chiara; Masalu, Nestory; De Rossi, Anita; Menin, Chiara

    2013-10-01

    Molecular methods are important tools for diagnosis and monitoring of many lymphoproliferative disorders. The reliability of lymphoma diagnoses is strikingly different between developed and developing countries, partly due to lack of access to these advanced molecular analyses. To overcome these problems, we propose a new application of dried blood spots (DBS) for detecting clonal B-cell populations in peripheral blood (PB). We ensured that the DBS contained sufficient lymphocytes to perform a PCR-based clonality assay without producing false positives. Using the Namalwa B-cell line, we established that the assay is sensitive enough to detect 200 clonal cells in the analyzed sample. Very similar clonal results were obtained between DNA from DBS and fresh whole blood from patients with B-cell chronic lymphocytic leukemia. B-cell clonality can also be detected in DBS from African children with EBV-associated diseases. This is the first study demonstrating that clonality testing can be performed on DBS samples, thus improving the diagnostic and monitoring options for lymphoproliferative diseases in resource-limited settings. PMID:23965169

  1. Less invasive blood sampling in the animal laboratory: clinical chemistry and haematology of blood obtained by the Triatominae bug Dipetalogaster maximus.

    PubMed

    Markvardsen, S N; Kjelgaard-Hansen, M; Ritz, C; Sørensen, D B

    2012-04-01

    Dipetalogaster maximus (Dipmax), a blood-sucking bug belonging to the family Reduviidae, has been used to obtain blood samples, for example for clinical chemistry and haematology, in a variety of zoo animals and wildlife. Using this bug allows stress-free blood sampling as the bug is able to draw blood without the mammal noticing the bug. In laboratory animal science, the need for blood samples from unstressed animals may arise, especially in animal behaviour research. The use of Dipmax bugs may prove a valuable tool for this purpose. To validate the method, we compared an array of standard blood parameters sampled from New Zealand White rabbits, sampled either by the use of bugs or by the conventional method; puncture of vena auricularis caudalis. The overall hypothesis was that there was no significant difference in clinical chemistry and haematological parameters between the bug method and the conventional method. A total of 17 clinical parameters as well as 12 haematological parameters were measured and compared in New Zealand White rabbits. The results showed that for 13 of these 29 analysed parameters, the bug method and the conventional method did not give significantly different results, and the obtained results were thus directly comparable. For the remaining parameters the obtained results were significantly different. However, all parameters were measurable in the bug samples. The influences of the bug metabolism on these parameters are discussed.

  2. Isolation of Aeromonas salmonicida from Human Blood Sample: A Case Report.

    PubMed

    Tewari, Rachna; Dudeja, Mridu; Nandy, Shyamasree; Das, Ayan Kumar

    2014-02-01

    Aeromonas salmonicida belonging to the genus Aeromonas, is a common pathogen that causes furunculosis and septicaemia in variety of fishes. It infects cold blooded vertebrates living at low temperatures mainly salmonid fish hence named salmonicida. Untill recently Aeromanas salmonicida is considered to be a fish pathogen. A. salmonicida is considered to be non-pathogenic for humans as it cannot grow at 37ºC. "However, In our laboratory culture plates and broths were incubated twice at 37ºC and each time same type of colonies were isolated which were identified as A. samonicida by Vitek 2 compact system bioMerieux, Inc. (Durham, N.C.)". By far no report has been received regarding its isolation from humans biological sample. Here we present the first report of A. salmonicida isolated from the human blood. PMID:24701507

  3. Direct detection of Theileria annulata in bovine blood samples using standard and isothermal DNA amplification approaches.

    PubMed

    Gomes, Jacinto; Inácio, João

    2015-01-01

    Tropical theileriosis is a tick-borne disease responsible for important health problems in cattle, caused by the hemoprotozoan Theileria annulata. Traditionally, detection of Theileria pathogens in infected animals requires the microscopic examination of stained-blood smears and serological methods. Molecular diagnostic assays have been developed for the detection of Theileria parasites, including PCR-based and reverse line blotting approaches, but these methods usually demand qualified personnel, complex instrumentation, and expensive materials. Loop-mediated isothermal amplification (LAMP) can facilitate the design of molecular assays independent of the use of sophisticated equipment. In this chapter we describe the application of two molecular assays for the direct detection of T. annulata in bovine blood samples, based in real-time PCR and LAMP, both targeting the Tams1-encoding gene of this parasite.

  4. Blood pressure interacts with APOE ε4 to predict memory performance in a midlife sample

    PubMed Central

    Oberlin, Lauren E.; Manuck, Stephen B.; Gianaros, Peter J.; Ferrell, Robert E.; Muldoon, Matthew F.; Jennings, J. Richard; Flory, Janine D.; Erickson, Kirk I.

    2015-01-01

    Objective Elevated blood pressure and the Apolipoprotein ε4 allele (APOE ε4) are independent risk factors for Alzheimer’s disease. We sought to determine whether the combined presence of the APOE ε4 allele and elevated blood pressure is associated with lower cognitive performance in cognitively healthy middle-aged adults. Methods A total of 975 participants aged 30–54 (mean age = 44.47) were genotyped for APOE. Cardiometabolic risk factors including blood pressure, lipids, and glucose were assessed and cognitive function was measured using the Trail Making Test and the Visual Reproduction and Logical Memory subtests from the Wechsler Memory Scale. Results Multivariable regression analysis showed that the association between APOE ε4 and episodic memory performance varied as a function of systolic blood pressure (SBP), such that elevated SBP was predictive of poorer episodic memory performance only in APOE ε4 carriers (β = −.092; t = −2.614; p = .009). Notably, this association was apparent at prehypertensive levels (≥ 130 mm Hg), even after adjusting for physical activity, depression, smoking, and other cardiometabolic risk factors. Conclusions The joint presence of APOE ε4 and elevated SBP, even at prehypertensive levels, is associated with lower cognitive performance in healthy, middle-aged adults. Results of this study suggest that the combination of APOE ε4 and elevated SBP may synergistically compromise memory function well before the appearance of clinically significant impairments. Interventions targeting blood pressure control in APOE ε4 carriers during midlife should be studied as a possible means to reduce the risk of cognitive decline in genetically susceptible samples. PMID:25730733

  5. [Study of molecular mechanism for a blood sample with A3 phenotype].

    PubMed

    Liang, Wei; Yang, Liang; Mei, Chuanliang; Xu, Deyi; Deng, Gang; He, Yunlei; Liu, Yiyu; Zhang, Zhe

    2015-10-01

    OBJECTIVE To explore the molecular mechanism for a blood sample with mixed-field hemagglutination upon determination of ABO blood group. METHODS Serological techniques were employed to identify the erythrocyte phenotype. The A and B antigens were detected by flow cytometry. The preliminary genotype of ABO gene was assayed with sequence-specific primer-polymerase chain reaction (PCR-SSP). Exons 6 and 7 of the ABO gene were amplified with PCR and analyzed by direct sequencing. Haplotypes of the ABO gene were analyzed by cloning sequencing as well. RESULTS The serological reaction pattern has supported an O phenotype when all the tubes were centrifuged for the first time. However, a mixed-field hemagglutination of red blood cells (RBCs) with anti-A antibodies was present after the tube was centrifuged five times later. A antigens were detected on the surface of partial red blood cells of the sample by flow cytometry. PCR- SSP results have shown that the preliminary ABO genotype was A/O. Analysis of the fragments of exons 6 and 7 of the ABO gene has indicated that heterozygosis lied as follows: 261G/A, 425T/T, 467C/T, 646A/T, 681A/G, 745C/T, 771C/T, 829A/G, conjecturing the genotype to be A307/O02, which was confirmed by haplotype sequence analysis. Compared with A101 allele, A307 allele has two missense mutations, 467C> T and 745C> T, which have resulted in substitutions Pro156Leu and Arg249Trp in the A glycosyltransferase polypeptide chain. CONCLUSION A variant allele (A307) has been identified for the first time in mainland China, which is responsible for the formation of A3 phenotype. PMID:26418996

  6. Capillary blood sampling: national recommendations on behalf of the Croatian Society of Medical Biochemistry and Laboratory Medicine.

    PubMed

    Krleza, Jasna Lenicek; Dorotic, Adrijana; Grzunov, Ana; Maradin, Miljenka

    2015-01-01

    Capillary blood sampling is a medical procedure aimed at assisting in patient diagnosis, management and treatment, and is increasingly used worldwide, in part because of the increasing availability of point-of-care testing. It is also frequently used to obtain small blood volumes for laboratory testing because it minimizes pain. The capillary blood sampling procedure can influence the quality of the sample as well as the accuracy of test results, highlighting the need for immediate, widespread standardization. A recent nationwide survey of policies and practices related to capillary blood sampling in medical laboratories in Croatia has shown that capillary sampling procedures are not standardized and that only a small proportion of Croatian laboratories comply with guidelines from the Clinical Laboratory Standards Institute (CLSI) or the World Health Organization (WHO). The aim of this document is to provide recommendations for capillary blood sampling. This document has been produced by the Working Group for Capillary Blood Sampling within the Croatian Society of Medical Biochemistry and Laboratory Medicine. Our recommendations are based on existing available standards and recommendations (WHO Best Practices in Phlebotomy, CLSI GP42-A6 and CLSI C46-A2), which have been modified based on local logistical, cultural, legal and regulatory requirements. We hope that these recommendations will be a useful contribution to the standardization of capillary blood sampling in Croatia.

  7. Capillary blood sampling: national recommendations on behalf of the Croatian Society of Medical Biochemistry and Laboratory Medicine

    PubMed Central

    Krleza, Jasna Lenicek; Dorotic, Adrijana; Grzunov, Ana; Maradin, Miljenka

    2015-01-01

    Capillary blood sampling is a medical procedure aimed at assisting in patient diagnosis, management and treatment, and is increasingly used worldwide, in part because of the increasing availability of point-of-care testing. It is also frequently used to obtain small blood volumes for laboratory testing because it minimizes pain. The capillary blood sampling procedure can influence the quality of the sample as well as the accuracy of test results, highlighting the need for immediate, widespread standardization. A recent nationwide survey of policies and practices related to capillary blood sampling in medical laboratories in Croatia has shown that capillary sampling procedures are not standardized and that only a small proportion of Croatian laboratories comply with guidelines from the Clinical Laboratory Standards Institute (CLSI) or the World Health Organization (WHO). The aim of this document is to provide recommendations for capillary blood sampling. This document has been produced by the Working Group for Capillary Blood Sampling within the Croatian Society of Medical Biochemistry and Laboratory Medicine. Our recommendations are based on existing available standards and recommendations (WHO Best Practices in Phlebotomy, CLSI GP42-A6 and CLSI C46-A2), which have been modified based on local logistical, cultural, legal and regulatory requirements. We hope that these recommendations will be a useful contribution to the standardization of capillary blood sampling in Croatia. PMID:26524965

  8. Identification of a suitable internal control for fluorescence analysis on canine peripheral blood samples.

    PubMed

    Riondato, F; Martini, V; Poggi, A; Rota, A; Comazzi, S; Sulce, M; Bruno, B; Borrelli, A; Miniscalco, B

    2016-04-01

    Reliable detection of fluorescence intensity (FI) by flow cytometry (FC) is fundamental. FI depends on instrument settings and sample processing procedures: thus, measurements should be done using internal controls with known FI. Commercially available beads-based standards are expensive, thus reducing their usability in the veterinary practice. Cell subsets with stable mean FI (MFI) within the population have been proposed as acceptable surrogates in human medicine. In veterinary medicine, no data exist about stability of antigen expression among different subjects or upon sample storage. The aim of the present study was to evaluate MFI variability of main lymphocytes antigens among the lymphoid cells within each subject, among different subjects, and upon 24-h storage, in order to identify the antigen most suitable as stable internal control in MFI analyses. Peripheral blood samples from 18 healthy dogs were analysed by FC within 3h from sampling to assess the expression of CD3, CD5, CD4, CD8, CD21 and cyCD79b using conjugated monoclonal antibodies. Analyses were restricted to the lymphoid population. Fluorescent microbeads were added to each tube, and antigen MFI was calculated as Relative Fluorescence Intensity RFI (CD/beads). Fluorescence histogram CV (fhCV) for each CD was regarded as an index of the variability of expression among lymphocytes within each subject (cell-to-cell variability); whereas the CV of RFI was regarded as an index of inter-subjects variability (dog-to-dog variability). In 11 cases, FC analyses were repeated after 24h storage at 4°C and RFI and CVs of fresh and stored samples were compared to assess variability linked to storage. CD4 was identified as the best antigen to be used as an internal control for MFI analyses in canine peripheral blood samples because of low cell-to-cell and dog-to-dog variability, and optimal stability upon 24-h storage. Blood samples from a second group of 21 healthy dogs were labelled only with CD4, in order

  9. Comparison of blood plasma sample preparation methods for combined LC-MS lipidomics and metabolomics.

    PubMed

    Patterson, Rainey E; Ducrocq, Antoine J; McDougall, Danielle J; Garrett, Timothy J; Yost, Richard A

    2015-10-01

    The goal of this research was to find the most comprehensive lipid extraction of blood plasma, while also providing adequate aqueous preparation for metabolite analysis. Comparisons have been made previously of the Folch, Bligh-Dyer, and Matyash lipid extractions; furthermore, this paper provides an additional comparison of a phospholipid removal plate for analysis. This plate was used for lipid extraction rather than its intended use in lipid removal for polar analysis, and it proves to be robust for targeted lipid analysis. Folch and Matyash provided reproducible recovery over a range of lipid classes, however the Matyash aqueous layer compared well to a typical methanol preparation for polar metabolite analysis. Thus, the Matyash method is the best choice for an untargeted biphasic extraction for metabolomics and lipidomics in blood plasma. PMID:26343017

  10. Does volumetric absorptive microsampling eliminate the hematocrit bias for caffeine and paraxanthine in dried blood samples? A comparative study.

    PubMed

    De Kesel, Pieter M M; Lambert, Willy E; Stove, Christophe P

    2015-06-30

    Volumetric absorptive microsampling (VAMS) is a novel sampling technique that allows the straightforward collection of an accurate volume of blood (approximately 10μL) from a drop or pool of blood by dipping an absorbent polymeric tip into it. The resulting blood microsample is dried and analyzed as a whole. The aim of this study was to evaluate the potential of VAMS to overcome the hematocrit bias, an important issue in the analysis of dried blood microsamples. An LC-MS/MS method for analysis of the model compounds caffeine and paraxanthine in VAMS samples was fully validated and fulfilled all pre-established criteria. In conjunction with previously validated procedures for dried blood spots (DBS) and blood, this allowed us to set up a meticulous comparative study in which both compounds were determined in over 80 corresponding VAMS, DBS and liquid whole blood samples. These originated from authentic human patient samples, covering a wide hematocrit range (0.21-0.50). By calculating the differences with reference whole blood concentrations, we found that analyte concentrations in VAMS samples were not affected by a bias that changed over the evaluated hematocrit range, in contrast to DBS results. However, VAMS concentrations tend to overestimate whole blood concentrations, as a consistent positive bias was observed. A different behavior of VAMS samples prepared from incurred and spiked blood, combined with a somewhat reduced recovery of caffeine and paraxanthine from VAMS tips at high hematocrit values, an effect that was not observed for DBS using a very similar extraction procedure, was found to be at the basis of the observed VAMS-whole blood deviations. Based on this study, being the first in which the validity and robustness of VAMS is evaluated by analyzing incurred human samples, it can be concluded that VAMS effectively assists in eliminating the effect of hematocrit. PMID:26041521

  11. Does volumetric absorptive microsampling eliminate the hematocrit bias for caffeine and paraxanthine in dried blood samples? A comparative study.

    PubMed

    De Kesel, Pieter M M; Lambert, Willy E; Stove, Christophe P

    2015-06-30

    Volumetric absorptive microsampling (VAMS) is a novel sampling technique that allows the straightforward collection of an accurate volume of blood (approximately 10μL) from a drop or pool of blood by dipping an absorbent polymeric tip into it. The resulting blood microsample is dried and analyzed as a whole. The aim of this study was to evaluate the potential of VAMS to overcome the hematocrit bias, an important issue in the analysis of dried blood microsamples. An LC-MS/MS method for analysis of the model compounds caffeine and paraxanthine in VAMS samples was fully validated and fulfilled all pre-established criteria. In conjunction with previously validated procedures for dried blood spots (DBS) and blood, this allowed us to set up a meticulous comparative study in which both compounds were determined in over 80 corresponding VAMS, DBS and liquid whole blood samples. These originated from authentic human patient samples, covering a wide hematocrit range (0.21-0.50). By calculating the differences with reference whole blood concentrations, we found that analyte concentrations in VAMS samples were not affected by a bias that changed over the evaluated hematocrit range, in contrast to DBS results. However, VAMS concentrations tend to overestimate whole blood concentrations, as a consistent positive bias was observed. A different behavior of VAMS samples prepared from incurred and spiked blood, combined with a somewhat reduced recovery of caffeine and paraxanthine from VAMS tips at high hematocrit values, an effect that was not observed for DBS using a very similar extraction procedure, was found to be at the basis of the observed VAMS-whole blood deviations. Based on this study, being the first in which the validity and robustness of VAMS is evaluated by analyzing incurred human samples, it can be concluded that VAMS effectively assists in eliminating the effect of hematocrit.

  12. Assessing skin blood flow dynamics in older adults using a modified sample entropy approach.

    PubMed

    Liao, Fuyuan; Jan, Yih-Kuen

    2014-01-01

    The aging process may result in attenuated microvascular reactivity in response to environmental stimuli, which can be evaluated by analyzing skin blood flow (SBF) signals. Among various methods for analyzing physiological signals, sample entropy (SE) is commonly used to quantify the degree of regularity of time series. However, we found that for temporally correlated data, SE value depends on the sampling rate. When data are oversampled, SE may give misleading results. To address this problem, we propose to modify the definition of SE by using time-lagged vectors in the calculation of the conditional probability that any two vectors of successive data points are within a tolerance r for m points remain within the tolerance at the next point. The lag could be chosen as the first minimum of the auto mutual information function. We tested the performance of modified SE using simulated signals and SBF data. The results showed that modified SE is able to quantify the degree of regularity of the signals regardless of sampling rate. Using this approach, we observed a more regular behavior of blood flow oscillations (BFO) during local heating-induced maximal vasodilation period compared to the baseline in young and older adults and a more regular behavior of BFO in older adults compared to young adults. These results suggest that modified SE may be useful in the study of SBF dynamics.

  13. Physiological and Pathological Impact of Blood Sampling by Retro-Bulbar Sinus Puncture and Facial Vein Phlebotomy in Laboratory Mice

    PubMed Central

    Holst, Birgitte; Hau, Jann; Rozell, Björn; Abelson, Klas Stig Peter

    2014-01-01

    Retro-bulbar sinus puncture and facial vein phlebotomy are two widely used methods for blood sampling in laboratory mice. However, the animal welfare implications associated with these techniques are currently debated, and the possible physiological and pathological implications of blood sampling using these methods have been sparsely investigated. Therefore, this study was conducted to assess and compare the impacts of blood sampling by retro-bulbar sinus puncture and facial vein phlebotomy. Blood was obtained from either the retro-bulbar sinus or the facial vein from male C57BL/6J mice at two time points, and the samples were analyzed for plasma corticosterone. Body weights were measured at the day of blood sampling and the day after blood sampling, and the food consumption was recorded automatically during the 24 hours post-procedure. At the end of study, cheeks and orbital regions were collected for histopathological analysis to assess the degree of tissue trauma. Mice subjected to facial vein phlebotomy had significantly elevated plasma corticosterone levels at both time points in contrast to mice subjected to retro-bulbar sinus puncture, which did not. Both groups of sampled mice lost weight following blood sampling, but the body weight loss was higher in mice subjected to facial vein phlebotomy. The food consumption was not significantly different between the two groups. At gross necropsy, subcutaneous hematomas were found in both groups and the histopathological analyses revealed extensive tissue trauma after both facial vein phlebotomy and retro-bulbar sinus puncture. This study demonstrates that both blood sampling methods have a considerable impact on the animals' physiological condition, which should be considered whenever blood samples are obtained. PMID:25426941

  14. Description of a new non-injectable connector to reduce the complications of arterial blood sampling.

    PubMed

    Mariyaselvam, M Z; Heij, R E; Laba, D; Richardson, J A; Hodges, E J; Maduakor, C A; Carter, J J; Young, P J

    2015-01-01

    Arterial cannulation is associated with complications including bacterial contamination, accidental intra-arterial injection and blood spillage. We performed a series of audits and experiments to gauge the potential for these, as well as assess the possible contribution of a new device, the Needle-Free Arterial Non-Injectable Connector (NIC), in reducing these risks. The NIC comprises a needle-free connector that prevents blood spillage and a one-way valve allowing aspiration only; once screwed onto the side port of a three-way tap, the device can only be removed with difficulty. We performed a clinical audit of arterial monitoring systems in our intensive care unit, which showed an incidence of bacterial colonisation of five in 86 (6%) three-way tap ports. We constructed a manikin simulation experiment of the management of acute bradycardia, in which trainee doctors were required to inject atropine intravenously. Ten of 15 (66%) doctors injected the drug into the three-way tap of the arterial monitoring system rather than into the intravenous cannula or the central venous catheter. In a laboratory study, we replicated the arterial blood sampling and flushing sequence from a three-way tap, with the syringes attached either directly to the three-way tap port or to a NIC attached to the port. The first (discard) syringe attached to the three-way tap was contaminated with bacteria. Bacterial growth was found in 17 of 20 (85%) downstream flushed samples (corresponding to the patient's circulation) when the three-way tap was accessed directly, compared to none of 20 accessed via the NIC (p < 0.0001). Growth was found on all of 20 (100%) ports accessed directly compared to none of 20 accessed via the NIC (p < 0.0001). The NIC effectively prevents bacteria from contaminating sampling lines. As its design also prevents accidental intra-arterial injection, we suggest that it can reduce complications of arterial monitoring.

  15. Comparison of 2 electronic cowside tests to detect subclinical ketosis in dairy cows and the influence of the temperature and type of blood sample on the test results.

    PubMed

    Iwersen, M; Klein-Jöbstl, D; Pichler, M; Roland, L; Fidlschuster, B; Schwendenwein, I; Drillich, M

    2013-01-01

    The objective of this study was to determine the suitability of 2 electronic hand-held devices [FreeStyle Precision (FSP), Abbott GmbH & Co. KG, Wiesbaden, Germany and GlucoMen LX Plus (GLX), A. Menarini GmbH, Vienna, Austria] for measuring β-hydroxybutyrate (BHBA) in dairy cows. Three experiments were conducted to evaluate (1) the diagnostic performance of the devices, (2) the effect of the type of blood sample, and (3) the influence of the ambient temperature on the determined results. A total of 415 blood samples from lactating Holstein and Simmental cows were collected and analyzed with both devices (whole blood) and in a laboratory (serum). Correlation coefficients between whole-blood and serum BHBA concentrations were highly significant, with 94% for the FSP and 80% for the GLX device. Based on thresholds for subclinical ketosis of 1.2 and 1.4 mmol of BHBA/L, results obtained with the hand-held devices were evaluated by receiver operating characteristics analyses. This resulted in adjusted thresholds of 1.2 and 1.4 mmol/L for the FSP and 1.1 and 1.3 mmol/L for the GLX device. Applying these thresholds, sensitivities were 98 and 100% for the FSP and 80 and 86% for the GLX device, respectively. Corresponding specificities were 90 and 97% for the FSP and 87 and 96% for the GLX device, respectively. Additionally, concentrations of BHBA were tested with both devices in whole blood, EDTA-added whole blood, and in their resulting serum and plasma, collected from 65 animals. Determined BHBA concentrations were similar within each device for whole and EDTA-added blood, and in serum and plasma, but differed between whole blood and serum and between EDTA-added blood and plasma. Blood samples with low (0.4 mmol/L), medium (1.1 mmol/L), and high (1.6 mmol/L) BHBA concentrations were stored between +5 to +32°C and analyzed repeatedly at temperature levels differing by 4°C. Additionally, devices and test strips were stored at equal conditions and used for measurement

  16. Does Pneumatic Tube System Transport Contribute to Hemolysis in ED Blood Samples?

    PubMed Central

    Phelan, Michael P.; Reineks, Edmunds Z.; Hustey, Fredric M.; Berriochoa, Jacob P.; Podolsky, Seth R.; Meldon, Stephen; Schold, Jesse D.; Chamberlin, Janelle; Procop, Gary W.

    2016-01-01

    Introduction Our goal was to determine if the hemolysis among blood samples obtained in an emergency department and then sent to the laboratory in a pneumatic tube system was different from those in samples that were hand-carried. Methods The hemolysis index is measured on all samples submitted for potassium analysis. We queried our hospital laboratory database system (SunQuest®) for potassium results for specimens obtained between January 2014 and July 2014. From facility maintenance records, we identified periods of system downtime, during which specimens were hand-carried to the laboratory. Results During the study period, 15,851 blood specimens were transported via our pneumatic tube system and 92 samples were hand delivered. The proportions of hemolyzed specimens in the two groups were not significantly different (13.6% vs. 13.1% [p=0.90]). Results were consistent when the criterion was limited to gross (3.3% vs 3.3% [p=0.99]) or mild (10.3% vs 9.8% [p=0.88]) hemolysis. The hemolysis rate showed minimal variation during the study period (12.6%–14.6%). Conclusion We found no statistical difference in the percentages of hemolyzed specimens transported by a pneumatic tube system or hand delivered to the laboratory. Certain features of pneumatic tube systems might contribute to hemolysis (e.g., speed, distance, packing material). Since each system is unique in design, we encourage medical facilities to consider whether their method of transport might contribute to hemolysis in samples obtained in the emergency department. PMID:27625719

  17. Does Pneumatic Tube System Transport Contribute to Hemolysis in ED Blood Samples?

    PubMed Central

    Phelan, Michael P.; Reineks, Edmunds Z.; Hustey, Fredric M.; Berriochoa, Jacob P.; Podolsky, Seth R.; Meldon, Stephen; Schold, Jesse D.; Chamberlin, Janelle; Procop, Gary W.

    2016-01-01

    Introduction Our goal was to determine if the hemolysis among blood samples obtained in an emergency department and then sent to the laboratory in a pneumatic tube system was different from those in samples that were hand-carried. Methods The hemolysis index is measured on all samples submitted for potassium analysis. We queried our hospital laboratory database system (SunQuest®) for potassium results for specimens obtained between January 2014 and July 2014. From facility maintenance records, we identified periods of system downtime, during which specimens were hand-carried to the laboratory. Results During the study period, 15,851 blood specimens were transported via our pneumatic tube system and 92 samples were hand delivered. The proportions of hemolyzed specimens in the two groups were not significantly different (13.6% vs. 13.1% [p=0.90]). Results were consistent when the criterion was limited to gross (3.3% vs 3.3% [p=0.99]) or mild (10.3% vs 9.8% [p=0.88]) hemolysis. The hemolysis rate showed minimal variation during the study period (12.6%–14.6%). Conclusion We found no statistical difference in the percentages of hemolyzed specimens transported by a pneumatic tube system or hand delivered to the laboratory. Certain features of pneumatic tube systems might contribute to hemolysis (e.g., speed, distance, packing material). Since each system is unique in design, we encourage medical facilities to consider whether their method of transport might contribute to hemolysis in samples obtained in the emergency department.

  18. Effects on animal wellbeing and sample quality of 2 techniques for collecting blood from the facial vein of mice.

    PubMed

    Francisco, Cassie C; Howarth, Gordon S; Whittaker, Alexandra L

    2015-01-01

    When sampling blood from mice, several different techniques can be used, with retroorbital sinus sampling traditionally being the most common. Given the severe tissue trauma caused by retroorbital sampling, alternative methods such as the facial vein route have been developed. The aim of this study was to evaluate 2 techniques for facial vein bleeding in conscious mice to ascertain whether differences in clinical outcomes, practicability of sample collection, and hematologic parameters were apparent. Blood samples were obtained from the facial vein of 40 BALB/c mice by using either a 21-gauge needle or a lancet. Subsequently, the protocol was repeated with isoflurane-anesthetized mice sampled by using the lancet method (n = 20). Behavior immediately after sampling was observed, and sample quantity, sampling time, and time until bleeding ceased were measured. Clinical pathology data and hematoma diameter at necropsy were analyzed also. The mean sample quantity collected (approximately 0.2 mL) was comparable among methods, but sampling was much more rapid when mice were anesthetized by using isoflurane. The only other noteworthy finding was a significantly reduced number of platelets in samples from anesthetized mice. Adverse, ongoing clinical signs were rare regardless of the method used. The results revealed no significant differences in welfare implications or blood sample quality among the methods or between conscious and anesthetized mice. Therefore, any of the methods we evaluated for obtaining blood samples from the facial vein are appropriate for use in research studies.

  19. Effects on Animal Wellbeing and Sample Quality of 2 Techniques for Collecting Blood from the Facial Vein of Mice

    PubMed Central

    Francisco, Cassie C; Howarth, Gordon S; Whittaker, Alexandra L

    2015-01-01

    When sampling blood from mice, several different techniques can be used, with retroorbital sinus sampling traditionally being the most common. Given the severe tissue trauma caused by retroorbital sampling, alternative methods such as the facial vein route have been developed. The aim of this study was to evaluate 2 techniques for facial vein bleeding in conscious mice to ascertain whether differences in clinical outcomes, practicability of sample collection, and hematologic parameters were apparent. Blood samples were obtained from the facial vein of 40 BALB/c mice by using either a 21-gauge needle or a lancet. Subsequently, the protocol was repeated with isoflurane-anesthetized mice sampled by using the lancet method (n = 20). Behavior immediately after sampling was observed, and sample quantity, sampling time, and time until bleeding ceased were measured. Clinical pathology data and hematoma diameter at necropsy were analyzed also. The mean sample quantity collected (approximately 0.2 mL) was comparable among methods, but sampling was much more rapid when mice were anesthetized by using isoflurane. The only other noteworthy finding was a significantly reduced number of platelets in samples from anesthetized mice. Adverse, ongoing clinical signs were rare regardless of the method used. The results revealed no significant differences in welfare implications or blood sample quality among the methods or between conscious and anesthetized mice. Therefore, any of the methods we evaluated for obtaining blood samples from the facial vein are appropriate for use in research studies. PMID:25651095

  20. Concentrations of persistent organic pollutants (POPs) in human blood samples from Mexico City, Mexico.

    PubMed

    Orta-García, Sandra; Pérez-Vázquez, Francisco; González-Vega, Carolina; Varela-Silva, José Antonio; Hernández-González, Lidia; Pérez-Maldonado, Iván

    2014-02-15

    Studies in Mexico have demonstrated exposure to persistent organic pollutants (POPs) in people living in different sites through the country. However, studies evaluating exposure to POPs in people living in Mexico City (one of most contaminated places in the world) are scarce. Therefore, the aim of this study was to assess the levels of polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT) and its metabolite dichlorodiphenyldichloroethylene (DDE) in the blood as exposure biomarkers in people living in Mexico City. A total of 123 participants (blood donors aged 20-60 years) were recruited during 2010 in Mexico City. Quantitative analyses of blood samples were performed using gas chromatography coupled with mass spectrometry. Levels of the assessed compounds ranged from non-detectable (

  1. Concentrations of persistent organic pollutants (POPs) in human blood samples from Mexico City, Mexico.

    PubMed

    Orta-García, Sandra; Pérez-Vázquez, Francisco; González-Vega, Carolina; Varela-Silva, José Antonio; Hernández-González, Lidia; Pérez-Maldonado, Iván

    2014-02-15

    Studies in Mexico have demonstrated exposure to persistent organic pollutants (POPs) in people living in different sites through the country. However, studies evaluating exposure to POPs in people living in Mexico City (one of most contaminated places in the world) are scarce. Therefore, the aim of this study was to assess the levels of polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT) and its metabolite dichlorodiphenyldichloroethylene (DDE) in the blood as exposure biomarkers in people living in Mexico City. A total of 123 participants (blood donors aged 20-60 years) were recruited during 2010 in Mexico City. Quantitative analyses of blood samples were performed using gas chromatography coupled with mass spectrometry. Levels of the assessed compounds ranged from non-detectable (

  2. A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples.

    PubMed

    Batty, Elizabeth M; Wong, T H Nicholas; Trebes, Amy; Argoud, Karène; Attar, Moustafa; Buck, David; Ip, Camilla L C; Golubchik, Tanya; Cule, Madeleine; Bowden, Rory; Manganis, Charis; Klenerman, Paul; Barnes, Eleanor; Walker, A Sarah; Wyllie, David H; Wilson, Daniel J; Dingle, Kate E; Peto, Tim E A; Crook, Derrick W; Piazza, Paolo

    2013-01-01

    To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.

  3. Additional diversity within the B70 serotype: identification of B*1580 in a Caucasoid blood donor.

    PubMed

    Kervaire, B; Tiercy, J-M

    2004-03-01

    Anew B70 variant, B*1580, has been identified in a Swiss Caucasoid blood donor. Sequencing of exons 2 and 3 revealed that the HLA-B*1580 differs from its closest matching allele B*1518 by two substitutions in exon 3, leading to two amino acid changes, threonine to isoleucine and leucine to isoleucine at codons 94 and 95, respectively. The complete human leukocyte antigen type of the donor is: A*2402, A*2601; B*5101, B*1580; Cw*0704, Cw*1402/05; DRB1*0801, DQB1*0402. The B*1580 is a new member of the B70 cluster, characterized by the SEE motif at positions 24, 45, and 46 in the alpha1-domain. Substitutions at codons 94 and 95, also found in some B62 and B75 alleles, do not appear to interfere with the B70 serological reactivity. Based on sequence similarity and linkage with Cw*0704, the rare alleles B*1509, B*1529, B*1564, and B*1580 are possibly derived from the B*1518 haplotype.

  4. Evidence That Certain Waste Tank Headspace Vapor Samples Were Contaminated by Semivolatile Polymer Additives

    SciTech Connect

    Huckaby, James L.

    2006-02-09

    Vapor samples collected from the headspaces of the Hanford Site high-level radioactive waste tanks in 1994 and 1995 using the Vapor Sampling System (VSS) were reported to contain trace levels of phthalates, antioxidants, and certain other industrial chemicals that did not have a logical origin in the waste. This report examines the evidence these chemicals were sampling artifacts (contamination) and identifies the chemicals reported as headspace constituents that may instead have been contaminants. Specific recommendations are given regarding the marking of certain chemicals as suspect on the basis they were sampling manifold contaminants.

  5. Environmental contaminants in Texas, USA, wetland reptiles: Evaluation using blood samples

    USGS Publications Warehouse

    Clark, D.R.; Bickham, J.W.; Baker, D.L.; Cowman, D.F.

    2000-01-01

    Four species of reptiles (diamondback water snake [Nerodia rhombifer], blotched water snake [N. erythrogaster], cottonmouth [Agkistrodon piscivorus], and red-eared slider [Trachemys scripta]) were collected at two contaminated and three reference sites in Texas, USA. Old River Slough has received intensive applications of agricultural chemicals since the 1950s. Municipal Lake received industrial arsenic wastes continuously from 1940 to 1993. Blood samples were analyzed for organochlorines, potentially toxic elements, genetic damage, and plasma cholinesterase (ChE). Dichlorodiphenyldichloroethylene (DDE) concentrations reached as high as 3.0 ppm (wet weight) in whole blood of a diamondback water snake at Old River Slough, a level probably roughly equivalent to the maximum concentration found in plasma of peregrine falcons (Falco peregrinus) in 1978 to 1979 when DDE peaked in this sensitive species. Possible impacts on diamondback water snakes are unknown, but at least one diamondback water snake was gravid when captured, indicating active reproduction. Arsenic was not found in red-eared sliders (only species sampled) from Municipal Lake. Red-eared sliders of both sexes at Old River Slough showed declining levels of ChE with increasing mass, suggesting a life-long decrease of ChE levels. Possible negative population consequences are unknown, but no evidence was found in body condition (mass relative to carapace length) that red-eared sliders at either contaminated site were harmed.

  6. Pattern recognition of neuron specific enolase and carcinoembryonic antigen in whole blood samples.

    PubMed

    Stefan-van Staden, Raluca-Ioana; Comnea-Stancu, Ionela Raluca; Surdu-Bob, Carmen Cristina; Stanciu-Gavan, Camelia

    2015-02-01

    New tools and methods for pattern recognition of neuron specific enolase (NSE) and carcinoembryonic antigen (CEA) were proposed for the screening of whole blood samples. The new tools were based on stochastic sensors designed using nanoporous gold microspheres, graphite, graphene, diamond paste as well as α-CDs, and 5,10,15,20-tetraphenyl-21H,23H-porphyrin. The best sensor for the assay of CEA was the one based on P/graphite (the limit of determination was 16 fg/ml and sensitivity was 2.32 × 10(7)  s mg(-1)  ml), while for the assay of NSE the, best sensor was the one based on P/graphene (the limit of determination was 7.45 pg/ml and sensitivity was 2.49 × 10(8)  s mg(-1)  ml). The sensor of choice for simultaneous detection of NSE and CEA is the one based on P/graphene because we need high sensitivity and low limit of determination for NSE. To our knowledge, this is the only one screening test for early detection of lung cancer, by identification of NSE and CEA in whole blood samples. PMID:25604868

  7. Variation of Peripheral Blood Mononuclear Cell RNA Quality in Archived Samples

    PubMed Central

    Kozlakidis, Zisis; Mant, Christine; Abdinur, Fartun; Cope, Andrew; Steiner, Szabi; Peakman, Mark; Hayday, Adrian

    2011-01-01

    The Infectious Diseases BioBank (IDB) has consistently archived peripheral blood mononuclear cell (PBMNC) RNA for transcriptome analyses. RNA is particularly labile, and hence, these samples provide a sensitive indicator for assessing the IDB's quality-assurance measures. Independent analyses of 104 PBMNC RNA specimens from 26 volunteers revealed that the mean RNA integrity number (RIN) was high (9.02), although RIN ranged between scores of 7 and 10. This variation of RIN values was not associated with ischemic time, PBMNC quality, number of samples processed per day, self-medication after immunization, freezer location, donor characteristics, differential white blood cell counts, or daily variation in RNA extractions (all P>0.05). RIN values were related to the date of collection, with those processed during mid-summer having highest RIN scores (P=0.0001). Amongst specimens with the lowest RIN scores, no common feature could be identified. Thus, no technical explanation for the variation in RNA quality could be ascertained and these may represent normal physiological variations. These data provide strong evidence that current IDB protocols for the isolation and preservation PBMNC RNA are robust. PMID:21977241

  8. Prevalence of synthetic cannabinoids in blood samples from Norwegian drivers suspected of impaired driving during a seven weeks period.

    PubMed

    Tuv, Silja Skogstad; Krabseth, Hege; Karinen, Ritva; Olsen, Kirsten M; Øiestad, Elisabeth L; Vindenes, Vigdis

    2014-01-01

    From early year 2000 different herbal products containing synthetic cannabinoids (SC) have appeared on the drug market all over the world, and new substances are frequently introduced. The prevalence of SC use in different populations is however still mainly unknown, also in Norway. This information is difficult to obtain, but studies of drivers suspected of driving under the influence of drugs (DUID), might provide important information. The aim of this study was to assess the prevalence of SC in drivers suspected of being under the influence of drugs in Norway, and investigate if SCs impair driving performance. For two periods of three and four weeks all blood samples from drivers suspected of DUID in Norway were analyzed for the presence of 12 and 18 different SCs, respectively. A new ultra performance liquid chromatography tandem mass spectrometry method was developed. A total of 726 cases were analyzed during our study period, and SCs were detected in 16 cases (2.2%) in total. The mean age of these drivers was 29.6 years. High concentrations of other psychoactive drugs were detected in all the blood samples where a SC was found. AM-2201 and JWH-018 were the most frequently detected SCs, each found in five cases. In addition RSC-4, JWH-122, JWH-081 and JWH-250 were detected. None of the drivers had reported using SCs prior to driving. Despite the limited number of SCs investigated in this 7 week study period, a considerable percent of the cases were positive. Other psychoactive drugs of abuse were always found concomitant with the SCs, and the age of these drivers indicates that experienced drug users also ingest SCs. Since other drugs were found in all the samples, the psychomotor impairment caused by the SCs is difficult to estimate. Our study shows the importance of screening analyses of biological samples from different populations to assess the prevalence of drug use, since self-reporting might be encumbered with significant under-reporting.

  9. 19 CFR 151.11 - Request for samples or additional examination packages after release of merchandise.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY (CONTINUED) EXAMINATION, SAMPLING... of this chapter. For purposes of determining admissibility, representatives of the Food and Drug Administration may obtain samples of any food, drug, device, or cosmetic, the importation of which is governed...

  10. 19 CFR 151.11 - Request for samples or additional examination packages after release of merchandise.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY (CONTINUED) EXAMINATION, SAMPLING... of this chapter. For purposes of determining admissibility, representatives of the Food and Drug Administration may obtain samples of any food, drug, device, or cosmetic, the importation of which is governed...

  11. 19 CFR 151.11 - Request for samples or additional examination packages after release of merchandise.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY (CONTINUED) EXAMINATION, SAMPLING... of this chapter. For purposes of determining admissibility, representatives of the Food and Drug Administration may obtain samples of any food, drug, device, or cosmetic, the importation of which is governed...

  12. 19 CFR 151.11 - Request for samples or additional examination packages after release of merchandise.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY (CONTINUED) EXAMINATION, SAMPLING... of this chapter. For purposes of determining admissibility, representatives of the Food and Drug Administration may obtain samples of any food, drug, device, or cosmetic, the importation of which is governed...

  13. Comparison of Sample Fixation and the use of LDS-751 or anti-CD45 for Leukocyte Identification in Mouse Whole Blood for Flow Cytometry.

    PubMed Central

    Maes, Melissa L.; Davidson, Lisa; McDonagh, Paul F.; Ritter, Leslie S.

    2007-01-01

    Flow cytometry methods used to measure leukocyte function often entail sample preparation procedures that cause artifactual cell activation. To avoid leukocyte activation by isolation techniques, some preparation methods use fluorescent markers to discriminate leukocytes from erythrocytes in whole blood. One of these markers, laser dye styryl–751(LDS-751), has been used to distinguish leukocytes by staining nucleic acid, but has been found to stain other blood cells and dead cells indiscriminately. Thus, LDS-751 may not be an appropriate reagent for leukocyte identification in whole blood. Fixing samples with formaldehydes increases cell permeability and causes surface protein cross-linking that may alter staining of both intra- and extracellular markers. The degree of this sample alteration by formaldehyde fixation, however, remains in question. In addition, little is known about flow cytometry and sample preparation methods in mouse whole blood. The purpose of this study was to determine if labeling leukocytes with a monoclonal antibody specific to leukocyte common antigen (CD45) was superior to labeling with LDS-751 and to determine the effect of sample fixation on a mouse whole blood preparation for flow cytometry. Samples were incubated with CD16/CD32 Fc receptor blocker, and either 10 μg/ml LDS-751 or phosphate buffered saline (PBS). The samples were then fixed with paraformaldehyde or diluted with PBS followed by incubation with 5ug/ml PerCP-conjugated anti-CD45, 5ug/ml FITC-conjugated anti-CD11b, or 80 μM dichlorofluorescein diacetate. We found that samples labeled with LDS-751 demonstrated decreased fluorescence intensity for granulocyte CD11b expression and ROS production compared to samples labeled with anti-CD45. In addition, sample fixation decreased mean fluorescence intensity in samples labeled with either LDS-751 or anti-CD45. We conclude that labeling leukocytes with monoclonal antibody CD45 in a mouse whole blood preparation is preferable, as

  14. Hematological parameters in Polish mixed breed rabbits with addition of meat breed blood in the annual cycle.

    PubMed

    Tokarz-Deptuła, B; Niedźwiedzka-Rystwej, P; Adamiak, M; Hukowska-Szematowicz, B; Trzeciak-Ryczek, A; Deptuła, W

    2015-01-01

    In the paper we studied haematologic values, such as haemoglobin concentration, haematocrit value, thrombocytes, leucocytes: lymphocytes, neutrophils, basophils, eosinophils and monocytes in the pheral blood in Polish mixed-breeds with addition of meat breed blood in order to obtain the reference values which are until now not available for this animals. In studying this indices we took into consideration the impact of the season (spring, summer, autumn, winter), and sex of the animals. The studies have shown a high impact of the season of the year on those rabbits, but only in spring and summer. Moreover we observed that the sex has mean impact on the studied values of haematological parameters in those rabbits. According to our knowledge, this is the first paper on haematologic values in this widely used group of rabbits, so they may serve as reference values. PMID:26812808

  15. Bridging the gap between sample collection and laboratory analysis: using dried blood spots to identify human exposure to chemical agents

    NASA Astrophysics Data System (ADS)

    Hamelin, Elizabeth I.; Blake, Thomas A.; Perez, Jonas W.; Crow, Brian S.; Shaner, Rebecca L.; Coleman, Rebecca M.; Johnson, Rudolph C.

    2016-05-01

    Public health response to large scale chemical emergencies presents logistical challenges for sample collection, transport, and analysis. Diagnostic methods used to identify and determine exposure to chemical warfare agents, toxins, and poisons traditionally involve blood collection by phlebotomists, cold transport of biomedical samples, and costly sample preparation techniques. Use of dried blood spots, which consist of dried blood on an FDA-approved substrate, can increase analyte stability, decrease infection hazard for those handling samples, greatly reduce the cost of shipping/storing samples by removing the need for refrigeration and cold chain transportation, and be self-prepared by potentially exposed individuals using a simple finger prick and blood spot compatible paper. Our laboratory has developed clinical assays to detect human exposures to nerve agents through the analysis of specific protein adducts and metabolites, for which a simple extraction from a dried blood spot is sufficient for removing matrix interferents and attaining sensitivities on par with traditional sampling methods. The use of dried blood spots can bridge the gap between the laboratory and the field allowing for large scale sample collection with minimal impact on hospital resources while maintaining sensitivity, specificity, traceability, and quality requirements for both clinical and forensic applications.

  16. Improvement of precision for pipetting blood serum samples into a graphite furnace

    NASA Astrophysics Data System (ADS)

    Bohrer, D.; do Nascimento, P. C.; Binotto, R.; Borges da Costa, J. A. T.; Szlachta, T.

    2002-12-01

    Graphite furnace atomic absorption spectrometry is a well-established technique for trace metal determination in blood and serum samples. For this kind of samples, a R.S.D. of up to 10% is considered acceptable, especially for those elements, the concentrations of which do not allow high dilution. Among the reasons that contribute to an increase of the S.D. is the retention of proteins (and analyte) in the sample dispenser capillary, because proteins might be adsorbed on polymeric surfaces. In the present work, the interaction protein/polymer was studied, considering the amount of protein that could be retained by the capillary, the influence of sample dilution, time of contact and the possible co-adsorption of metals. Serum proteins were retained in the capillary, depending on the material (Tygon>silicone rubber>poly(tetrafluorethylene)); dilution did not prevent the adsorption, and only 30 s of contact were enough for the adsorption to occur. Using a column filled with PTFE powder (60 mesh), it was possible to observe that metals were co-adsorbed to a large extent. Water, diluted nitric acid or aqueous solutions of Triton X-100 were not able to promote the complete desorption of the proteins retained by the polymeric materials. Total elution was achieved with methanol, and its use as rinse solution decreased the R.S.D. ( n=10) for aluminium, manganese and chromium determination in serum to <10%.

  17. 21 CFR 640.53 - Testing the blood.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Testing the blood. 640.53 Section 640.53 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Cryoprecipitate § 640.53 Testing the blood. (a) Blood... sample of blood collected at the time of collecting the source blood, and such sample container shall...

  18. 21 CFR 640.53 - Testing the blood.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Testing the blood. 640.53 Section 640.53 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Cryoprecipitate § 640.53 Testing the blood. (a) Blood... sample of blood collected at the time of collecting the source blood, and such sample container shall...

  19. 21 CFR 640.53 - Testing the blood.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Testing the blood. 640.53 Section 640.53 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Cryoprecipitate § 640.53 Testing the blood. (a) Blood... sample of blood collected at the time of collecting the source blood, and such sample container shall...

  20. 21 CFR 640.53 - Testing the blood.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Testing the blood. 640.53 Section 640.53 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Cryoprecipitate § 640.53 Testing the blood. (a) Blood... sample of blood collected at the time of collecting the source blood, and such sample container shall...

  1. 21 CFR 640.53 - Testing the blood.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Testing the blood. 640.53 Section 640.53 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Cryoprecipitate § 640.53 Testing the blood. (a) Blood... sample of blood collected at the time of collecting the source blood, and such sample container shall...

  2. DNA Damage Focus Analysis in Blood Samples of Minipigs Reveals Acute Partial Body Irradiation

    PubMed Central

    Lamkowski, Andreas; Forcheron, Fabien; Agay, Diane; Ahmed, Emad A.; Drouet, Michel; Meineke, Viktor; Scherthan, Harry

    2014-01-01

    Radiation accidents frequently involve acute high dose partial body irradiation leading to victims with radiation sickness and cutaneous radiation syndrome that implements radiation-induced cell death. Cells that are not lethally hit seek to repair ionizing radiation (IR) induced damage, albeit at the expense of an increased risk of mutation and tumor formation due to misrepair of IR-induced DNA double strand breaks (DSBs). The response to DNA damage includes phosphorylation of histone H2AX in the vicinity of DSBs, creating foci in the nucleus whose enumeration can serve as a radiation biodosimeter. Here, we investigated γH2AX and DNA repair foci in peripheral blood lymphocytes of Göttingen minipigs that experienced acute partial body irradiation (PBI) with 49 Gy (±6%) Co-60 γ-rays of the upper lumbar region. Blood samples taken 4, 24 and 168 hours post PBI were subjected to γ-H2AX, 53BP1 and MRE11 focus enumeration. Peripheral blood lymphocytes (PBL) of 49 Gy partial body irradiated minipigs were found to display 1–8 DNA damage foci/cell. These PBL values significantly deceed the high foci numbers observed in keratinocyte nuclei of the directly γ-irradiated minipig skin regions, indicating a limited resident time of PBL in the exposed tissue volume. Nonetheless, PBL samples obtained 4 h post IR in average contained 2.2% of cells displaying a pan-γH2AX signal, suggesting that these received a higher IR dose. Moreover, dispersion analysis indicated partial body irradiation for all 13 minipigs at 4 h post IR. While dose reconstruction using γH2AX DNA repair foci in lymphocytes after in vivo PBI represents a challenge, the DNA damage focus assay may serve as a rapid, first line indicator of radiation exposure. The occurrence of PBLs with pan-γH2AX staining and of cells with relatively high foci numbers that skew a Poisson distribution may be taken as indicator of acute high dose partial body irradiation, particularly when samples are available early after

  3. Nested PCR detection of malaria directly using blood filter paper samples from epidemiological surveys

    PubMed Central

    2014-01-01

    Background Nested PCR is considered a sensitive and specific method for detecting malaria parasites and is especially useful in epidemiological surveys. However, the preparation of DNA templates for PCR is often time-consuming and costly. Methods A simplified PCR method was developed to directly use a small blood filter paper square (2 × 2 mm) as the DNA template after treatment with saponin. This filter paper-based nested PCR method (FP-PCR) was compared to microscopy and standard nested PCR with DNA extracted by using a Qiagen DNA mini kit from filter paper blood spots of 204 febrile cases. The FP-PCR technique was further applied to evaluate malaria infections in 1,708 participants from cross-sectional epidemiological surveys conducted in Myanmar and Thailand. Results The FP-PCR method had a detection limit of ~0.2 parasites/μL blood, estimated using cultured Plasmodium falciparum parasites. With 204 field samples, the sensitivity of the FP-PCR method was comparable to that of the standard nested PCR method, which was significantly higher than that of microscopy. Application of the FP-PCR method in large cross-sectional studies conducted in Myanmar and Thailand detected 1.9% (12/638) and 6.2% (66/1,070) asymptomatic Plasmodium infections, respectively, as compared to the detection rates of 1.3% (8/638) and 0.04% (4/1,070) by microscopy. Conclusion This FP-PCR method was much more sensitive than microscopy in detecting Plasmodium infections. It drastically increased the detection sensitivity of asymptomatic infections in cross-sectional surveys conducted in Thailand and Myanmar, suggesting that this FP-PCR method has a potential for future applications in malaria epidemiology studies. PMID:24884761

  4. Continuous quality control of the blood sampling procedure using a structured observation scheme

    PubMed Central

    Seemann, Tine Lindberg; Nybo, Mads

    2016-01-01

    Introduction An observational study was conducted using a structured observation scheme to assess compliance with the local phlebotomy guideline, to identify necessary focus items, and to investigate whether adherence to the phlebotomy guideline improved. Materials and methods The questionnaire from the EFLM Working Group for the Preanalytical Phase was adapted to local procedures. A pilot study of three months duration was conducted. Based on this, corrective actions were implemented and a follow-up study was conducted. All phlebotomists at the Department of Clinical Biochemistry and Pharmacology were observed. Three blood collections by each phlebotomist were observed at each session conducted at the phlebotomy ward and the hospital wards, respectively. Error frequencies were calculated for the phlebotomy ward and the hospital wards and for the two study phases. Results A total of 126 blood drawings by 39 phlebotomists were observed in the pilot study, while 84 blood drawings by 34 phlebotomists were observed in the follow-up study. In the pilot study, the three major error items were hand hygiene (42% error), mixing of samples (22%), and order of draw (21%). Minor significant differences were found between the two settings. After focus on the major aspects, the follow-up study showed significant improvement for all three items at both settings (P < 0.01, P < 0.01, and P = 0.01, respectively). Conclusion Continuous quality control of the phlebotomy procedure revealed a number of items not conducted in compliance with the local phlebotomy guideline. It supported significant improvements in the adherence to the recommended phlebotomy procedures and facilitated documentation of the phlebotomy quality. PMID:27812302

  5. Antidepressants detection and quantification in whole blood samples by GC-MS/MS, for forensic purposes.

    PubMed

    Truta, Liliana; Castro, André L; Tarelho, Sónia; Costa, Pedro; Sales, M Goreti F; Teixeira, Helena M

    2016-09-01

    Depression is among the most prevalent psychiatric disorders of our society, leading to an increase in antidepressant drug consumption that needs to be accurately determined in whole blood samples in Forensic Toxicology Laboratories. For this purpose, this work presents a new gas chromatography tandem mass spectrometry (GC-MS/MS) method targeting the simultaneous and rapid determination of 14 common Antidepressants in whole blood: 13 Antidepressants (amitriptyline, citalopram, clomipramine, dothiepin, fluoxetine, imipramine, mianserin, mirtazapine, nortryptiline, paroxetine, sertraline, trimipramine and venlafaxine) and 1 Metabolite (N-desmethylclomipramine). Solid-phase extraction was used prior to chromatographic separation. Chromatographic and MS/MS parameters were selected to improve sensitivity, peak resolution and unequivocal identification of the eluted analyte. The detection was performed on a triple quadrupole tandem MS in selected ion monitoring (SIM) mode in tandem, using electronic impact ionization. Clomipramine-D3 and trimipramine-D3 were used as deutered internal standards. The validation parameters included linearity, limits of detection, lower limit of quantification, selectivity/specificity, extraction efficiency, carry-over, precision and robustness, and followed internationally accepted guidelines. Limits of quantification and detection were lower than therapeutic and sub-therapeutic concentration ranges. Overall, the method offered good selectivity, robustness and quick response (<16min) for typical concentration ranges, both for therapeutic and lethal levels. PMID:27376459

  6. Putative Epimutagens in Maternal Peripheral and Cord Blood Samples Identified Using Human Induced Pluripotent Stem Cells

    PubMed Central

    Arai, Yoshikazu; Hayakawa, Koji; Arai, Daisuke; Ito, Rie; Iwasaki, Yusuke; Saito, Koichi; Akutsu, Kazuhiko; Takatori, Satoshi; Ishii, Rie; Hayashi, Rumiko; Izumi, Shun-Ichiro; Sugino, Norihiro; Kondo, Fumio; Horie, Masakazu; Nakazawa, Hiroyuki; Makino, Tsunehisa; Hirosawa, Mitsuko; Shiota, Kunio; Ohgane, Jun

    2015-01-01

    The regulation of transcription and genome stability by epigenetic systems are crucial for the proper development of mammalian embryos. Chemicals that disturb epigenetic systems are termed epimutagens. We previously performed chemical screening that focused on heterochromatin formation and DNA methylation status in mouse embryonic stem cells and identified five epimutagens: diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se), and octachlorodipropyl ether (S-421). Here, we used human induced pluripotent stem cells (hiPSCs) to confirm the effects of 20 chemicals, including the five epimutagens, detected at low concentrations in maternal peripheral and cord blood samples. Of note, these individual chemicals did not exhibit epimutagenic activity in hiPSCs. However, because the fetal environment contains various chemicals, we evaluated the effects of combined exposure to chemicals (DEP, Hg, cotinine, Se, and S-421) on hiPSCs. The combined exposure caused a decrease in the number of heterochromatin signals and aberrant DNA methylation status at multiple gene loci in hiPSCs. The combined exposure also affected embryoid body formation and neural differentiation from hiPSCs. Therefore, DEP, Hg, cotinine, Se, and S-421 were defined as an “epimutagen combination” that is effective at low concentrations as detected in maternal peripheral and cord blood. PMID:26339649

  7. Identification of malaria infected red blood samples by digital holographic quantitative phase microscope

    NASA Astrophysics Data System (ADS)

    Patel, Nimit R.; Chhaniwal, Vani K.; Javidi, Bahram; Anand, Arun

    2015-07-01

    Development of devices for automatic identification of diseases is desired especially in developing countries. In the case of malaria, even today the gold standard is the inspection of chemically treated blood smears through a microscope. This requires a trained technician/microscopist to identify the cells in the field of view, with which the labeling chemicals gets attached. Bright field microscopes provide only low contrast 2D images of red blood cells and cell thickness distribution cannot be obtained. Quantitative phase contrast microscopes can provide both intensity and phase profiles of the cells under study. The phase information can be used to determine thickness profile of the cell. Since cell morphology is available, many parameters pertaining to the 3D shape of the cell can be computed. These parameters in turn could be used to decide about the state of health of the cell leading to disease diagnosis. Here the investigations done on digital holographic microscope, which provides quantitative phase images, for comparison of parameters obtained from the 3D shape profile of objects leading to identification of diseased samples is described.

  8. PERT: a method for expression deconvolution of human blood samples from varied microenvironmental and developmental conditions.

    PubMed

    Qiao, Wenlian; Quon, Gerald; Csaszar, Elizabeth; Yu, Mei; Morris, Quaid; Zandstra, Peter W

    2012-01-01

    The cellular composition of heterogeneous samples can be predicted using an expression deconvolution algorithm to decompose their gene expression profiles based on pre-defined, reference gene expression profiles of the constituent populations in these samples. However, the expression profiles of the actual constituent populations are often perturbed from those of the reference profiles due to gene expression changes in cells associated with microenvironmental or developmental effects. Existing deconvolution algorithms do not account for these changes and give incorrect results when benchmarked against those measured by well-established flow cytometry, even after batch correction was applied. We introduce PERT, a new probabilistic expression deconvolution method that detects and accounts for a shared, multiplicative perturbation in the reference profiles when performing expression deconvolution. We applied PERT and three other state-of-the-art expression deconvolution methods to predict cell frequencies within heterogeneous human blood samples that were collected under several conditions (uncultured mono-nucleated and lineage-depleted cells, and culture-derived lineage-depleted cells). Only PERT's predicted proportions of the constituent populations matched those assigned by flow cytometry. Genes associated with cell cycle processes were highly enriched among those with the largest predicted expression changes between the cultured and uncultured conditions. We anticipate that PERT will be widely applicable to expression deconvolution strategies that use profiles from reference populations that vary from the corresponding constituent populations in cellular state but not cellular phenotypic identity.

  9. Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples

    PubMed Central

    Zhou, Liqing; Jones, Claire; Gibani, Malick M.; Dobinson, Hazel; Thomaides-Brears, Helena; Shrestha, Sonu; Blohmke, Christoph J.; Darton, Thomas C.; Pollard, Andrew J.

    2016-01-01

    Background Enteric fever remains an important cause of morbidity in many low-income countries and Salmonella Paratyphi A has emerged as the aetiological agent in an increasing proportion of cases. Lack of adequate diagnostics hinders early diagnosis and prompt treatment of both typhoid and paratyphoid but development of assays to identify paratyphoid has been particularly neglected. Here we describe the development of a rapid and sensitive blood culture PCR method for detection of Salmonella Paratyphi A from blood, potentially allowing for appropriate diagnosis and antimicrobial treatment to be initiated on the same day. Methods Venous blood samples from volunteers experimentally challenged orally with Salmonella Paratyphi A, who subsequently developed paratyphoid, were taken on the day of diagnosis; 10 ml for quantitative blood culture and automated blood culture, and 5 ml for blood culture PCR. In the latter assay, bacteria were grown in tryptone soy broth containing 2.4% ox bile and micrococcal nuclease for 5 hours (37°C) before bacterial DNA was isolated for PCR detection targeting the fliC-a gene of Salmonella Paratyphi A. Results An optimized broth containing 2.4% ox bile and micrococcal nuclease, as well as a PCR test was developed for a blood culture PCR assay of Salmonella Paratyphi A. The volunteers diagnosed with paratyphoid had a median bacterial burden of 1 (range 0.1–6.9) CFU/ml blood. All the blood culture PCR positive cases where a positive bacterial growth was shown by quantitative blood culture had a bacterial burden of ≥ 0.3 CFU/ ml blood. The blood culture PCR assay identified an equal number of positive cases as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood), but utilized only half the volume of specimens. Conclusions The blood culture PCR method for detection of Salmonella Paratyphi A can be completed within 9 hours and offers the potential for same-day diagnosis of enteric fever. Using 5 ml blood, it exhibited a

  10. Perfluoroalkyl substances in the blood of wild rats and mice from 47 prefectures in Japan: use of samples from nationwide specimen bank.

    PubMed

    Taniyasu, Sachi; Senthilkumar, Kurunthachalam; Yamazaki, Eriko; Yeung, Leo W Y; Guruge, Keerthi S; Kannan, Kurunthachalam; Yamashita, Nobuyoshi

    2013-07-01

    Numerous studies have reported on the global distribution, persistence, fate, and toxicity of perfluoroalkyl and polyfluoroalkyl substances (PFASs). However, studies on PFASs in terrestrial mammals are scarce. Rats can be good sentinels of human exposure to toxicants because of their habitat, which is in close proximity to humans. Furthermore, exposure data measured for rats can be directly applied for risk assessment because many toxicological studies use rodent models. In this study, a nationwide survey of PFASs in the blood of wild rats as well as surface water samples collected from rats' habitats from 47 prefectures in Japan was conducted. In addition to known PFASs, combustion ion chromatography technique was used for analysis of total fluorine concentrations in the blood of rats. In total, 216 blood samples representing three species of wild rats (house rat, Norway rats, and field mice) were analyzed for 23 PFASs. Perfluorooctanesulfonate (PFOS; concentration range <0.05-148 ng/mL), perfluorooctane sulfonamide (PFOSA; <0.1-157), perfluorododecanoate (<0.05-5.8), perfluoroundecanoate (PFUnDA; <0.05-51), perfluorodecanoate (PFDA; <0.05-9.7), perfluorononanoate (PFNA; <0.05-249), and perfluorooctanoate (PFOA) (<0.05-60) were detected >80 % of the blood samples. Concentrations of several PFASs in rat blood were similar to those reported for humans. PFSAs (mainly PFOS) accounted for 45 % of total PFASs, whereas perfluoroalkyl carboxylates (PFCAs), especially PFUnDA and PFNA, accounted for 20 and 10 % of total PFASs, respectively. In water samples, PFCAs were the predominant compounds with PFOA and PFNA found in >90 % of the samples. There were strong correlations (p < 0.001 to p < 0.05) between human population density and levels of PFOS, PFNA, PFOA, and PFOSA in wild rat blood. PMID:23494483

  11. Sulfatide Analysis by Mass Spectrometry for Screening of Metachromatic Leukodystrophy in Dried Blood and Urine Samples

    PubMed Central

    Spacil, Zdenek; Kumar, Arun Babu; Liao, Hsuan-Chieh; Auray-Blais, Christiane; Stark, Samantha; Suhr, Teryn R.; Scott, C. Ronald; Turecek, Frantisek; Gelb, Michael H.

    2016-01-01

    BACKGROUND Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder caused by deficiency in arylsulfatase A activity, leading to accumulation of sulfatide substrates. Diagnostic and monitoring procedures include demonstration of reduced arylsulfatase A activity in peripheral blood leukocytes or detection of sulfatides in urine. However, the development of a screening test is challenging because of instability of the enzyme in dried blood spots (DBS), the widespread occurrence of pseudodeficiency alleles, and the lack of available urine samples from newborn screening programs. METHODS We measured individual sulfatide profiles in DBS and dried urine spots (DUS) from MLD patients with LC-MS/MS to identify markers with the discriminatory power to differentiate affected individuals from controls. We also developed a method for converting all sulfatide molecular species into a single species, allowing quantification in positive-ion mode upon derivatization. RESULTS In DBS from MLD patients, we found up to 23.2-fold and 5.1-fold differences in total sulfatide concentrations for early- and late-onset MLD, respectively, compared with controls and pseudodeficiencies. Corresponding DUS revealed up to 164-fold and 78-fold differences for early- and late-onset MLD patient samples compared with controls. The use of sulfatides converted to a single species simplified the analysis and increased detection sensitivity in positive-ion mode, providing a second option for sulfatide analysis. CONCLUSIONS This study of sulfatides in DBS and DUS suggests the feasibility of the mass spectrometry method for newborn screening of MLD and sets the stage for a larger-scale newborn screening pilot study. PMID:26585924

  12. Croatian Society of Medical Biochemistry and Laboratory Medicine: national recommendations for venous blood sampling

    PubMed Central

    Nikolac, Nora; Šupak-Smolčić, Vesna; Šimundić, Ana-Maria; Ćelap, Ivana

    2013-01-01

    Phlebotomy is one of the most complex medical procedures in the diagnosis, management and treatment of patients in healthcare. Since laboratory test results are the basis for a large proportion (60–80%) of medical decisions, any error in the phlebotomy process could have serious consequences. In order to minimize the possibility of errors, phlebotomy procedures should be standardised, well-documented and written instructions should be available at every workstation. Croatia is one of the few European countries that have national guidelines for phlebotomy, besides the universally used CLSI (Clinical Laboratory Standards Institute) H3-A6 Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; approved Standard-Sixth Edition (CLSI, 2007) and WHO (World Health Organization) guidelines on drawing blood: best practices in phlebotomy (WHO, 2010). However, the growing body of evidence in importance of preanalytical phase management resulted in a need for evidence based revision and expansion of existing recommendations. The Croatian Society for Medical Biochemistry and Laboratory Medicine, Working Group for the Preanalytical Phase issued this recommendation. This document is based on the CLSI guideline H3-A6, with significant differences and additional information. PMID:24266294

  13. Preterm Cord Blood Contains a Higher Proportion of Immature Hematopoietic Progenitors Compared to Term Samples

    PubMed Central

    Podestà, Marina; Bruschettini, Matteo; Cossu, Claudia; Sabatini, Federica; Dagnino, Monica; Romantsik, Olga; Spaggiari, Grazia Maria; Ramenghi, Luca Antonio; Frassoni, Francesco

    2015-01-01

    Background Cord blood contains high number of hematopoietic cells that after birth disappear. In this paper we have studied the functional properties of the umbilical cord blood progenitor cells collected from term and preterm neonates to establish whether quantitative and/or qualitative differences exist between the two groups. Methods and Results Our results indicate that the percentage of total CD34+ cells was significantly higher in preterm infants compared to full term: 0.61% (range 0.15–4.8) vs 0.3% (0.032–2.23) p = 0.0001 and in neonates <32 weeks of gestational age (GA) compared to those ≥32 wks GA: 0.95% (range 0.18–4.8) and 0.36% (0.15–3.2) respectively p = 0.0025. The majority of CD34+ cells co-expressed CD71 antigen (p<0.05 preterm vs term) and grew in vitro large BFU-E, mostly in the second generation. The subpopulations CD34+CD38- and CD34+CD45- resulted more represented in preterm samples compared to term, conversely, Side Population (SP) did not show any difference between the two group. The absolute number of preterm colonies (CFCs/10microL) resulted higher compared to term (p = 0.004) and these progenitors were able to grow until the third generation maintaining an higher proportion of CD34+ cells (p = 0.0017). The number of colony also inversely correlated with the gestational age (Pearson r = -0.3001 p<0.0168). Conclusions We found no differences in the isolation and expansion capacity of Endothelial Colony Forming Cells (ECFCs) from cord blood of term and preterm neonates: both groups grew in vitro large number of endothelial cells until the third generation and showed a transitional phenotype between mesenchymal stem cells and endothelial progenitors (CD73, CD31, CD34 and CD144)The presence, in the cord blood of preterm babies, of high number of immature hematopoietic progenitors and endothelial/mesenchymal stem cells with high proliferative potential makes this tissue an important source of cells for developing new cells therapies

  14. Comparative value of blood and skin samples for diagnosis of spotted fever group rickettsial infection in model animals.

    PubMed

    Levin, Michael L; Snellgrove, Alyssa N; Zemtsova, Galina E

    2016-07-01

    The definitive diagnosis of spotted fever group (SFG) rickettsioses in humans is challenging due to the retrospective nature and cross reactivity of the serological methods and the absence of reliable and consistent samples for molecular diagnostics. Existing data indicate the transient character of bacteremia in experimentally infected animals. The ability of arthropod vectors to acquire rickettsial infection from the laboratory animals in the absence of systemic infection and known tropism of rickettsial agents to endothelial cells of peripheral blood vessels underline the importance of local infection and consequently the diagnostic potential of skin samples. In order to evaluate the diagnostic sensitivity of rickettsial DNA detection in blood and skin samples, we compared results of PCR testing in parallel samples collected from model laboratory animals infected with Rickettsia rickettsii, Rickettsia parkeri and Rickettsia slovaca-like agent at different time points after infection. Skin samples were collected from ears - away from the site of tick placement and without eschars. Overall, testing of skin samples resulted in a higher proportion of positive results than testing of blood samples. Presented data from model animals demonstrates that testing of skin samples from sites of rickettsial proliferation can provide definitive molecular diagnosis of up to 60-70% of tick-borne SFG rickettsial infections during the acute stage of illness. Detection of pathogen DNA in cutaneous samples is a valuable alternative to blood-PCR at least in model animals. PMID:27282078

  15. Serum levels of perfluoroalkyl compounds in human maternal and umbilical cord blood samples

    SciTech Connect

    Monroy, Rocio; Morrison, Katherine; Teo, Koon; Atkinson, Stephanie; Kubwabo, Cariton; Stewart, Brian; Foster, Warren G.

    2008-09-15

    Perfluoroalkyl compounds (PFCs) are end-stage metabolic products from industrial flourochemicals used in the manufacture of plastics, textiles, and electronics that are widely distributed in the environment. The objective of the present study was to quantify exposure to perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), perfluorodecanoic acid (PFDeA), perfluorohexane sulfonate (PFHxS), perfluoroheptanoic acid (PFHpA), and perfluorononanoic acid (PFNA) in serum samples collected from pregnant women and the umbilical cord at delivery. Pregnant women (n=101) presenting for second trimester ultrasound were recruited and PFC residue levels were quantified in maternal serum at 24-28 weeks of pregnancy, at delivery, and in umbilical cord blood (UCB; n=105) by liquid chromatography-mass spectrometry. Paired t-test and multiple regression analysis were performed to determine the relationship between the concentrations of each analyte at different sample collection time points. PFOA and PFOS were detectable in all serum samples analyzed including the UCB. PFOS serum levels (mean{+-}S.D.) were significantly higher (p<0.001) in second trimester maternal serum (18.1{+-}10.9 ng/mL) than maternal serum levels at delivery (16.2{+-}10.4 ng/mL), which were higher than the levels found in UCB (7.3{+-}5.8 ng/mL; p<0.001). PFHxS was quantifiable in 46/101 (45.5%) maternal and 21/105 (20%) UCB samples with a mean concentration of 4.05{+-}12.3 and 5.05{+-}12.9 ng/mL, respectively. There was no association between serum PFCs at any time point studied and birth weight. Taken together our data demonstrate that although there is widespread exposure to PFCs during development, these exposures do not affect birth weight.

  16. A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections

    DOE PAGES

    Moen, Scott T.; Hatcher, Christopher L.; Singh, Anup K.

    2016-04-07

    We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 μL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. Ultimately, this platform has great potential in both medical diagnostics and researchmore » applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing.« less

  17. A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections

    PubMed Central

    Moen, Scott T.; Hatcher, Christopher L.; Singh, Anup K.

    2016-01-01

    We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 μL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. This platform has great potential in both medical diagnostics and research applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing. PMID:27054764

  18. A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections.

    PubMed

    Moen, Scott T; Hatcher, Christopher L; Singh, Anup K

    2016-01-01

    We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 μL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. This platform has great potential in both medical diagnostics and research applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing. PMID:27054764

  19. Analysis of Tank 43H Samples at the Conclusion of Uranyl Carbonate Addition

    SciTech Connect

    Oji, L.N.

    2002-10-15

    Tank 43H serves as the feed Tank to the 2H evaporator. In the months of July and August 2001, about 21,000 gallons of a depleted uranyl carbonate solution were added to Tank 43H and agitated with two Flygt mixers. The depleted uranium addition served to decrease the U-235 enrichment in the Tank 43H supernate so that the supernate could be evaporated with no risk of accumulating enriched uranium.

  20. A New Blood Collection Device Minimizes Cellular DNA Release During Sample Storage and Shipping When Compared to a Standard Device

    PubMed Central

    Norton, Sheila E; Luna, Kristin K; Lechner, Joel M; Qin, Jianbing; Fernando, M Rohan

    2013-01-01

    Background Cell-free DNA (cfDNA) circulating in blood is currently used for noninvasive diagnostic and prognostic tests. Minimizing background DNA is vital for detection of low abundance cfDNA. We investigated whether a new blood collection device could reduce background levels of genomic DNA (gDNA) in plasma compared to K3EDTA tubes, when subjected to conditions that may occur during sample storage and shipping. Methods Blood samples were drawn from healthy donors into K3EDTA and Cell-Free DNA™ BCT (BCT). To simulate shipping, samples were shaken or left unshaken. In a shipping study, samples were shipped or not shipped. To assess temperature variations, samples were incubated at 6°C, 22°C, and 37°C. In all cases, plasma was harvested by centrifugation and total plasma DNA (pDNA) assayed by quantitative real-time polymerase chain reaction (qPCR). Results Shaking and shipping blood in K3EDTA tubes showed significant increases in pDNA, whereas no change was seen in BCTs. Blood in K3EDTA tubes incubated at 6°C, 22°C, and 37°C showed increases in pDNA while pDNA from BCTs remained stable. Conclusions BCTs prevent increases in gDNA levels that can occur during sample storage and shipping. This new device permits low abundance DNA target detection and allows accurate cfDNA concentrations. PMID:23852790

  1. Rapid Method for Screening Dried Blood Samples on Filter Paper for Human Immunodeficiency Virus Type 1 DNA

    PubMed Central

    Panteleeff, Dana DeVange; John, Grace; Nduati, Ruth; Mbori-Ngacha, Dorothy; Richardson, Barbra; Kreiss, Joan; Overbaugh, Julie

    1999-01-01

    PCR is a highly sensitive method for the detection of human immunodeficiency virus type 1 (HIV-1) nucleic acids in blood mononuclear cells and plasma. However, blood separation techniques require extensive laboratory support systems and are difficult when a limited volume of blood is available, which is often the case for infants. The use of blood samples stored on filter paper has many advantages for the detection of perinatal HIV-1 infection, but current methods require extraction and purification of target DNA prior to PCR amplification. We report a highly sensitive and rapid method for the extraction and detection of HIV-1 DNA in infant blood samples stored on filter papers. Because this rapid protocol does not involve steps for the removal of potential inhibitors of the PCR, the highest sensitivity is achieved by testing the filter paper lysate in quadruplicate. Assays for HIV-1 DNA were done by using nested PCR techniques that amplify HIV-1 gag DNA from blood spot samples on filter paper and from corresponding viably frozen mononuclear cells separated from venous blood samples obtained from 111 infants born to HIV-1-seropositive mothers. PCR results with blood from filter papers showed 100% specificity (95% confidence internal [CI] 93.1 to 100%) and 96% (95% CI, 88.65 to 98.9%) and 88% (95% CI, 79.2 to 94.5%) sensitivity (for quadruplicate and duplicate tests, respectively) compared to PCR results with blood mononuclear cells. Moreover, this method could detect HIV-1 sequences of multiple subtypes. PMID:9889216

  2. Standard addition flow method for potentiometric measurements at low concentration levels: application to the determination of fluoride in food samples.

    PubMed

    Galvis-Sánchez, Andrea C; Santos, João Rodrigo; Rangel, António O S S

    2015-02-01

    A standard addition method was implemented by using a flow manifold able to perform automatically multiple standard additions and in-line sample treatment. This analytical strategy was based on the in-line mixing of sample and standard addition solutions, using a merging zone approach. The flow system aimed to exploit the standard addition method to quantify the target analyte particularly in cases where the analyte concentration in the matrix is below the lower limit of linear response of the detector. The feasibility of the proposed flow configuration was assessed through the potentiometric determination of fluoride in sea salts of different origins and different types of coffee infusions. The limit of quantification of the proposed manifold was 5×10(-6) mol L(-1), 10-fold lower than the lower limit of linear response of the potentiometric detector used. A determination rate of 8 samples h(-1) was achieved considering an experimental procedure based on three standard additions per sample. The main advantage of the proposed strategy is the simple approach to perform multiple standard additions, which can be implemented with other ion selective electrodes, especially in cases when the primary ion is below the lower limit of linear response of the detector.

  3. SYBR green-based detection of Leishmania infantum DNA using peripheral blood samples.

    PubMed

    Ghasemian, Mehrdad; Gharavi, Mohammad Javad; Akhlaghi, Lame; Mohebali, Mehdi; Meamar, Ahmad Reza; Aryan, Ehsan; Oormazdi, Hormozd; Ghayour, Zahra

    2016-03-01

    Parasitological methods for the diagnosis of visceral leishmaniasis (VL) require invasive sampling procedures. The aim of this study was to detect Leishmania infantum (L. infantum) DNA by real time-PCR method in peripheral blood of symptomatic VL patient and compared its performance with nested PCR, an established molecular method with very high diagnostic indices. 47 parasitologically confirmed VL patients diagnosed by direct agglutination test (DAT > 3200), bone marrow aspiration and presented characteristic clinical features (fever, hepatosplenomegaly, and anemia) and 40 controls (non-endemic healthy control-30, Malaria-2, Toxoplasma gondii-2, Mycobacterium tuberculosis-2, HBV-1, HCV-1, HSV-1 and CMV-1) were enrolled in this study. SYBR-green based real time-PCR and nested PCR was performed to amplify the Kinetoplast DNA minicircle gene using the DNA extracted from Buffy coat. From among 47 patients, 45 (95.7 %) were positive by both nested-PCR and real time-PCR. These results indicate that real time-PCR was not only as sensitive as a nested-PCR assay for detection of Leishmania kDNA in clinical sample, but also more rapid. The advantage of real time-PCR based methods over nested-PCR is simple to perform, more faster in which nested-PCR requires post-PCR processing and reducing contamination risk.

  4. On-chip acoustophoretic isolation of microflora including S. typhimurium from raw chicken, beef and blood samples.

    PubMed

    Ngamsom, Bongkot; Lopez-Martinez, Maria J; Raymond, Jean-Claude; Broyer, Patrick; Patel, Pradip; Pamme, Nicole

    2016-04-01

    Pathogen analysis in food samples routinely involves lengthy growth-based pre-enrichment and selective enrichment of food matrices to increase the ratio of pathogen to background flora. Similarly, for blood culture analysis, pathogens must be isolated and enriched from a large excess of blood cells to allow further analysis. Conventional techniques of centrifugation and filtration are cumbersome, suffer from low sample throughput, are not readily amenable to automation and carry a risk of damaging biological samples. We report on-chip acoustophoresis as a pre-analytical technique for the resolution of total microbial flora from food and blood samples. The resulting 'clarified' sample is expected to increase the performance of downstream systems for the specific detection of the pathogens. A microfluidic chip with three inlets, a central separation channel and three outlets was utilized. Samples were introduced through the side inlets, and buffer solution through the central inlet. Upon ultrasound actuation, large debris particles (10-100 μm) from meat samples were continuously partitioned into the central buffer channel, leaving the 'clarified' outer sample streams containing both, the pathogenic cells and the background flora (ca. 1 μm) to be collected over a 30 min operation cycle before further analysis. The system was successfully tested with Salmonella typhimurium-spiked (ca. 10(3)CFU mL(-1)) samples of chicken and minced beef, demonstrating a high level of the pathogen recovery (60-90%). When applied to S. typhimurium contaminated blood samples (10(7)CFU mL(-1)), acoustophoresis resulted in a high depletion (99.8%) of the red blood cells (RBC) which partitioned in the buffer stream, whilst sufficient numbers of the viable S. typhimurium remained in the outer channels for further analysis. These results indicate that the technology may provide a generic approach for pre-analytical sample preparation prior to integrated and automated downstream detection of

  5. On-chip acoustophoretic isolation of microflora including S. typhimurium from raw chicken, beef and blood samples.

    PubMed

    Ngamsom, Bongkot; Lopez-Martinez, Maria J; Raymond, Jean-Claude; Broyer, Patrick; Patel, Pradip; Pamme, Nicole

    2016-04-01

    Pathogen analysis in food samples routinely involves lengthy growth-based pre-enrichment and selective enrichment of food matrices to increase the ratio of pathogen to background flora. Similarly, for blood culture analysis, pathogens must be isolated and enriched from a large excess of blood cells to allow further analysis. Conventional techniques of centrifugation and filtration are cumbersome, suffer from low sample throughput, are not readily amenable to automation and carry a risk of damaging biological samples. We report on-chip acoustophoresis as a pre-analytical technique for the resolution of total microbial flora from food and blood samples. The resulting 'clarified' sample is expected to increase the performance of downstream systems for the specific detection of the pathogens. A microfluidic chip with three inlets, a central separation channel and three outlets was utilized. Samples were introduced through the side inlets, and buffer solution through the central inlet. Upon ultrasound actuation, large debris particles (10-100 μm) from meat samples were continuously partitioned into the central buffer channel, leaving the 'clarified' outer sample streams containing both, the pathogenic cells and the background flora (ca. 1 μm) to be collected over a 30 min operation cycle before further analysis. The system was successfully tested with Salmonella typhimurium-spiked (ca. 10(3)CFU mL(-1)) samples of chicken and minced beef, demonstrating a high level of the pathogen recovery (60-90%). When applied to S. typhimurium contaminated blood samples (10(7)CFU mL(-1)), acoustophoresis resulted in a high depletion (99.8%) of the red blood cells (RBC) which partitioned in the buffer stream, whilst sufficient numbers of the viable S. typhimurium remained in the outer channels for further analysis. These results indicate that the technology may provide a generic approach for pre-analytical sample preparation prior to integrated and automated downstream detection of

  6. Inactivation of Geobacillus stearothermophilus in canned food and coconut milk samples by addition of enterocin AS-48.

    PubMed

    Viedma, Pilar Martínez; Abriouel, Hikmate; Ben Omar, Nabil; López, Rosario Lucas; Valdivia, Eva; Gálvez, Antonio

    2009-05-01

    The cyclic bacteriocin enterocin AS-48 was tested on a cocktail of two Geobacillus stearothermophilus strains in canned food samples (corn and peas), and in coconut milk. AS-48 (7 microg/g) reduced viable cell counts below detection levels in samples from canned corn and peas stored at 45 degrees C for 30 days. In coconut milk, bacterial inactivation by AS-48 (1.75 microg/ml) was even faster. In all canned food and drink samples inoculated with intact G. stearothermophilus endospores, bacteriocin addition (1.75 microg per g or ml of food sample) rapidly reduced viable cell counts below detection levels and avoided regrowth during storage. After a short-time bacteriocin treatment of endospores, trypsin addition markedly increased G. stearothermophilus survival, supporting the effect of residual bacteriocin on the observed loss of viability for endospores. Results from this study support the potential of enterocin AS-48 as a biopreservative against G. stearothermophilus. PMID:19269571

  7. Rapid Detection of Candida albicans by Polymerase Spiral Reaction Assay in Clinical Blood Samples.

    PubMed

    Jiang, Xiaoqun; Dong, Derong; Bian, Lihong; Zou, Dayang; He, Xiaoming; Ao, Da; Yang, Zhan; Huang, Simo; Liu, Ningwei; Liu, Wei; Huang, Liuyu

    2016-01-01

    Candida albicans is the most common human yeast pathogen which causes mucosal infections and invasive fungal diseases. Early detection of this pathogen is needed to guide preventative and therapeutic treatment. The aim of this study was to establish a polymerase spiral reaction (PSR) assay that rapidly and accurately detects C. albicans and to assess the clinical applicability of PSR-based diagnostic testing. Internal transcribed spacer 2 (ITS2), a region between 5.8S and 28S fungal ribosomal DNA, was used as the target sequence. Four primers were designed for amplification of ITS2 with the PSR method, which was evaluated using real time turbidity monitoring and visual detection using a pH indicator. Fourteen non-C. albicans yeast strains were negative for detection, which indicated the specificity of PSR assay was 100%. A 10-fold serial dilution of C. albicans genomic DNA was subjected to PSR and conventional polimerase chain reaction (PCR) to compare their sensitivities. The detection limit of PSR was 6.9 pg/μl within 1 h, 10-fold higher than that of PCR (69.0 pg/μl). Blood samples (n = 122) were collected from intensive care unit and hematological patients with proven or suspected C. albicans infection at two hospitals in Beijing, China. Both PSR assay and the culture method were used to analyze the samples. Of the 122 clinical samples, 34 were identified as positive by PSR. The result was consistent with those obtained by the culture method. In conclusion, a novel and effective C. albicans detection assay was developed that has a great potential for clinical screening and point-of-care testing. PMID:27379048

  8. Rapid Detection of Candida albicans by Polymerase Spiral Reaction Assay in Clinical Blood Samples

    PubMed Central

    Jiang, Xiaoqun; Dong, Derong; Bian, Lihong; Zou, Dayang; He, Xiaoming; Ao, Da; Yang, Zhan; Huang, Simo; Liu, Ningwei; Liu, Wei; Huang, Liuyu

    2016-01-01

    Candida albicans is the most common human yeast pathogen which causes mucosal infections and invasive fungal diseases. Early detection of this pathogen is needed to guide preventative and therapeutic treatment. The aim of this study was to establish a polymerase spiral reaction (PSR) assay that rapidly and accurately detects C. albicans and to assess the clinical applicability of PSR-based diagnostic testing. Internal transcribed spacer 2 (ITS2), a region between 5.8S and 28S fungal ribosomal DNA, was used as the target sequence. Four primers were designed for amplification of ITS2 with the PSR method, which was evaluated using real time turbidity monitoring and visual detection using a pH indicator. Fourteen non-C. albicans yeast strains were negative for detection, which indicated the specificity of PSR assay was 100%. A 10-fold serial dilution of C. albicans genomic DNA was subjected to PSR and conventional polimerase chain reaction (PCR) to compare their sensitivities. The detection limit of PSR was 6.9 pg/μl within 1 h, 10-fold higher than that of PCR (69.0 pg/μl). Blood samples (n = 122) were collected from intensive care unit and hematological patients with proven or suspected C. albicans infection at two hospitals in Beijing, China. Both PSR assay and the culture method were used to analyze the samples. Of the 122 clinical samples, 34 were identified as positive by PSR. The result was consistent with those obtained by the culture method. In conclusion, a novel and effective C. albicans detection assay was developed that has a great potential for clinical screening and point-of-care testing. PMID:27379048

  9. The post-occipital spinal venous sinus of the Nile crocodile Crocodylus niloticus: its anatomy and use for blood sample collection and intravenous infusions.

    PubMed

    Myburgh, Jan G; Kirberger, Robert M; Steyl, Johan C A; Soley, John T; Booyse, Dirk G; Huchzermeyer, Fritz W; Lowers, Russel H; Guillette, Louis J

    2014-05-05

    The post-occipital sinus of the spinal vein is often used for the collection of blood samples from crocodilians. Although this sampling method has been reported for several crocodilian species, the technique and associated anatomy has not been described in detail in any crocodilian, including the Nile crocodile (Crocodylus niloticus). The anatomy of the cranial neck region was investigated macroscopically, microscopically, radiographically and by means of computed tomography. Latex was injected into the spinal vein and spinal venous sinus of crocodiles to visualise the regional vasculature. The spinal vein ran within the vertebral canal, dorsal to and closely associated with the spinal cord and changed into a venous sinus cranially in the post-occipital region. For blood collection, the spinal venous sinus was accessed through the interarcuate space between the atlas and axis (C1 and C2) by inserting a needle angled just off the perpendicular in the midline through the craniodorsal cervical skin, just cranial to the cranial borders of the first cervical osteoderms. The most convenient method of blood collection was with a syringe and hypodermic needle. In addition, the suitability of the spinal venous sinus for intravenous injections and infusions in live crocodiles was evaluated. The internal diameter of the commercial human epidural catheters used during these investigations was relatively small, resulting in very slow infusion rates. Care should be taken not to puncture the spinal cord or to lacerate the blood vessel wall using this route for blood collection or intravenous infusions.

  10. The relation between perceived unfair treatment and blood pressure in a racially/ethnically diverse sample of women.

    PubMed

    Brown, Charlotte; Matthews, Karen A; Bromberger, Joyce T; Chang, Yuefang

    2006-08-01

    Elevated blood pressure is an important public health problem in midlife women, especially among minority groups. Few studies have examined the impact of perceived unfair treatment due to different factors such as racism, sexism, or ageism on blood pressure. By use of a racially/ethnically diverse community sample of nearly 3,300 midlife women enrolled in the longitudinal, multisite Study of Women's Health across the Nation between 1995 and 1997, this study examined whether perceived unfair treatment varied by race/ethnicity and whether it was associated with blood pressure levels. Overall, unfair treatment was reported by 65% of African-American women, 60% of Chinese women, 36% of Japanese women, 47% of White women, and 27% of Hispanic women. Although racial/ethnic differences in blood pressure were evident, high levels of perceived unfair treatment were not a correlate of elevated blood pressure.

  11. Absolute quantification of transferrin in blood samples of harbour seals using HPLC-ICP-MS.

    PubMed

    Grebe, Mechthild; Pröfrock, Daniel; Kakuschke, Antje; Broekaert, Jose A C; Prange, Andreas

    2011-02-01

    Harbour seals (Phoca vitulina) are bio-indicators for the assessment of their habitat and environmental changes. Besides population parameters and trends (survival, age structure, sex ratio), the individual health status represents a further important parameter for this assessment. The health status of seals is a complex and vague term, determined by a wide range of diagnostic parameters. Quantities of important blood proteins such as transferrin (Tf), as well as altered distribution patterns of its glycoforms, are frequently used as biomarkers in clinical diagnosis. Within this context Tf quantities and a varying pattern of its glycoforms are used as indicator for e.g. certain liver diseases, which also represents one of the most frequently observed pathological indication in harbour seals of the North Sea. Currently, most assay based quantification methods for Tf are limited since they often provide only information regarding the total Tf concentration rather than information of its different glycoforms. Due to a lack of suitable seal Tf antibodies also the application of more specific antibody based approaches is not possible. Within this background a new approach for the absolute quantification of the iron-transport protein Tf in the blood of harbour seals using its characteristic iron content and HPLC-ICP-MS detection is described. Method validation was performed using a certified human serum reference material (ERM-DA470K/IFCC). A Tf concentration of 2.33 ± 0.03 g L(-1) (sum of all quantified glycoforms) has been calculated, which is in good agreement with the certified total Tf concentration of 2.35 ± 0.08 g L(-1), confirming the accuracy of the proposed analytical method. Finally, different seal samples were analysed to demonstrate the suitability of the procedure for the quantification of Tf in real samples as well as to observe modified glycoform patterns. Compared to our previous studies for the first time it was possible to quantify the serum Tf

  12. The CERN antiproton source: Controls aspects of the additional collector ring and fast sampling devices

    NASA Astrophysics Data System (ADS)

    Chohan, V.

    1990-08-01

    The upgrade of the CERN antiproton source, meant to gain an order of magnitude in antiproton flux, required the construction of an additional ring to complement the existing antiproton accumulator (AA) and an entire rebuild of the target zone. The AA also needed major modifications to handle the increased flux and perform purely as an accumulator, preceded by collection in the collector ring (AC). The upgrade, known as the ACOL (antiproton collector) project, was approved under strict time and budgetary constraints and the existing AA control system, based on the Proton Synchrotron (PS) Divisional norms of CAMAC and Norsk-Data computers, had to be extended in the light of this. The limited (9 months) installation period for the whole upgrade meant that substantial preparatory and planning activities had to be carried out during the normal running of the AA. Advantage was taken of the upgrade to improve and consolidate the AA. Some aspects of the control system related to this upgrade are discussed together with the integration of new applications and instrumentation. The overall machine installation and running-in was carried out within the defined milestones and the project has now achieved the physics design goals.

  13. Determining the Volume of Additive Solution and Residual Plasma in Whole Blood Filtered and Buffy Coat Processed Red Cell Concentrates

    PubMed Central

    Jordan, Andrew; Acker, Jason P.

    2016-01-01

    Summary Background Residual plasma in transfused red cell concentrates (RCCs) has been associated with adverse transfusion outcomes. Despite this, there is no consensus on the standard procedure for measuring residual plasma volume. Methods The volumes of residual plasma and additive solution were measured in RCCs processed using two separation methods: whole blood filtration (WBF) and buffy coat (BC)/RCC filtration. The concentration of mannitol and albumin in RCC components was measured using colorimetric assays. Mannitol concentration was used to calculate additive solution volume. Residual plasma volume was calculated using two methods. Results Calculated RCC supernatant volumes were much lower in BC-processed components compared to WBF-processed components (BC = 97 ± 6 ml, WBF = 109 ± 4 ml; p < 0.05). Calculated additive solution volumes were greater in WBF- than in BC-processed components (BC = 81 ± 4 ml, WBF = 105 ± 2 ml; p < 0.05). Absolute residual plasma volume varied significantly based on the calculation method used. Conclusion Disparity between plasma volume calculation methods was observed. Efforts should be made to standardize residual plasma volume measurement methods in order to accurately assess the impact of residual plasma on transfusion outcomes. PMID:27330533

  14. Single blood-Hg samples can result in exposure misclassification: temporal monitoring within the Japanese community (United States)

    PubMed Central

    2012-01-01

    Background The most prominent non-occupational source of exposure to methylmercury is the consumption of fish. In this study we examine a fish consuming population to determine the extent of temporal exposure and investigate the extent to which single time estimates of methylmercury exposure based on blood-Hg concentration can provide reliable estimates of longer-term average exposure. Methods Blood-mercury levels were obtained from a portion of the Arsenic Mercury Intake Biometric Study (AMIBS) cohort. Specifically, 56 Japanese women residing in the Puget Sound area of Washington State, US were sampled on three occasions across a one-year period. Results An average of 135 days separated samples, with mean blood-mercury levels for the visits being 5.1, 6.6 and 5.0 μg/l and geometric means being 2.7, 4.5 and 3.1 μg/l. The blood-mercury levels in this group exceed national averages with geometric means for two of the visits being between the 90th and 95th percentiles of nationally observed levels and the lowest geometric mean being between the 75th and 90th percentile. Group means were not significantly different across sampling periods suggesting that exposure of combined subjects remained relatively constant. Comparing intra-individual results over time did not reveal a strong correlation among visits (r = 0.19, 0.50, 0.63 between 1st and 2nd, 2nd and 3rd, and 1st and 3rd sample results, respectively). In comparing blood-mercury levels across two sampling interval combinations (1st and 2nd, 2nd and 3rd, and 1st and 3rd visits, respectively), 58% (n = 34), 53% (n = 31) and 29% (n = 17) of the individuals had at least a 100% difference in blood-Hg levels. Conclusions Point estimates of blood-mercury, when compared with three sample averages, may not reflect temporal variability and individual exposures estimated on the basis of single blood samples should be treated with caution as indicators of long-term exposure. Reliance on single blood

  15. Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples.

    PubMed

    Zubakov, Dmitry; Hanekamp, Eline; Kokshoorn, Mieke; van Ijcken, Wilfred; Kayser, Manfred

    2008-03-01

    Human body fluids such as blood and saliva represent the most common source of biological material found at a crime scene. Reliable tissue identification in forensic science can reveal significant insights into crime scene reconstruction and can thus contribute toward solving crimes. Limitations of existing presumptive tests for body fluid identification in forensics, which are usually based on chemoluminescence or protein analysis, are expected to be overcome by RNA-based methods, provided that stable RNA markers with tissue-specific expression patterns are available. To generate sets of stable RNA markers for reliable identification of blood and saliva stains we (1) performed whole-genome gene expression analyses on a series of time-wise degraded blood and saliva stain samples using the Affymetrix U133 plus2 GeneChip, (2) consulted expression databases to obtain additional information on tissue specificity, and (3) confirmed expression patterns of the most promising candidate genes by quantitative real-time polymerase chain reaction including additional forensically relevant tissues such as semen and vaginal secretion. Overall, we identified nine stable mRNA markers for blood and five stable mRNA markers for saliva detection showing tissue-specific expression signals in stains aged up to 180 days of age, expectedly older. Although, all of the markers were able to differentiate blood/saliva from semen samples, none of them could differentiate vaginal secretion because of the complex nature of vaginal secretion and the biological similarity of buccal and vaginal mucosa. We propose the use of these 14 stable mRNA markers for identification of blood and saliva stains in future forensic practice. PMID:17579879

  16. Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples.

    PubMed

    Zubakov, Dmitry; Hanekamp, Eline; Kokshoorn, Mieke; van Ijcken, Wilfred; Kayser, Manfred

    2008-03-01

    Human body fluids such as blood and saliva represent the most common source of biological material found at a crime scene. Reliable tissue identification in forensic science can reveal significant insights into crime scene reconstruction and can thus contribute toward solving crimes. Limitations of existing presumptive tests for body fluid identification in forensics, which are usually based on chemoluminescence or protein analysis, are expected to be overcome by RNA-based methods, provided that stable RNA markers with tissue-specific expression patterns are available. To generate sets of stable RNA markers for reliable identification of blood and saliva stains we (1) performed whole-genome gene expression analyses on a series of time-wise degraded blood and saliva stain samples using the Affymetrix U133 plus2 GeneChip, (2) consulted expression databases to obtain additional information on tissue specificity, and (3) confirmed expression patterns of the most promising candidate genes by quantitative real-time polymerase chain reaction including additional forensically relevant tissues such as semen and vaginal secretion. Overall, we identified nine stable mRNA markers for blood and five stable mRNA markers for saliva detection showing tissue-specific expression signals in stains aged up to 180 days of age, expectedly older. Although, all of the markers were able to differentiate blood/saliva from semen samples, none of them could differentiate vaginal secretion because of the complex nature of vaginal secretion and the biological similarity of buccal and vaginal mucosa. We propose the use of these 14 stable mRNA markers for identification of blood and saliva stains in future forensic practice.

  17. Comparative determination of methyl mercury in whole blood samples using GC-ICP-MS and GC-MS techniques.

    PubMed

    Hippler, J; Hoppe, H W; Mosel, F; Rettenmeier, A W; Hirner, A V

    2009-08-15

    Two methods for the determination of methyl mercury (MeHg) in whole blood samples based on different mass spectrometric detection techniques are compared. The methods were employed in two studies in which the internal exposure of a group of mercury-exposed workers to total mercury and MeHg was investigated. Blood samples of these workers were analysed for MeHg independently from each other in two laboratories using similar extraction procedures but different detection techniques, viz. coupled GC-EI-MS/ICP-MS and GC-MS using D(3)-MeHg as internal standard. MeHg was detected in all blood samples in concentrations ranging from 0.3 to 9.0 microg/L. Though different detection techniques were employed, the results obtained by the two laboratories were in relatively good agreement.

  18. The identification of menstrual blood in forensic samples by logistic regression modeling of miRNA expression.

    PubMed

    Hanson, Erin K; Mirza, Mohid; Rekab, Kamel; Ballantyne, Jack

    2014-11-01

    We report the identification of sensitive and specific miRNA biomarkers for menstrual blood, a tissue that might provide probative information in certain specialized instances. We incorporated these biomarkers into qPCR assays and developed a quantitative statistical model using logistic regression that permits the prediction of menstrual blood in a forensic sample with a high, and measurable, degree of accuracy. Using the developed model, we achieved 100% accuracy in determining the body fluid of interest for a set of test samples (i.e. samples not used in model development). The development, and details, of the logistic regression model are described. Testing and evaluation of the finalized logistic regression modeled assay using a small number of samples was carried out to preliminarily estimate the limit of detection (LOD), specificity in admixed samples and expression of the menstrual blood miRNA biomarkers throughout the menstrual cycle (25-28 days). The LOD was <1 ng of total RNA, the assay performed as expected with admixed samples and menstrual blood was identified only during the menses phase of the female reproductive cycle in two donors.

  19. Trace samples of human blood in mosquitoes as a forensic investigation tool.

    PubMed

    Rabêlo, K C N; Albuquerque, C M R; Tavares, V B; Santos, S M; Souza, C A; Oliveira, T C; Oliveira, N C L; Crovella, S

    2015-01-01

    Investigations of any type of crime invariably starts at the crime scene by collecting evidence. Thus, the purpose of this research was to collect and analyze an entomological trace from an environment that is similar to those of indoor crime scenes. Hematophagous mosquitoes were collected from two residential units; saliva of volunteers that were residents in the units was also collected for genetic analysis as reference samples. We examined the allele frequencies of 15 short tandem repeat loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA) and amelogenin. A total of 26 female hematophagous mosquitoes were identified as Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus; we were able to obtain 11 forensically valid genetic profiles, with a minimum of 0.028203 ng/μL of human DNA. Thus, the results of this study showed that it was possible to correlate human genetic information from mosquitoes with the volunteer reference samples, which validates the use of this information as forensic evidence. Furthermore, we observed mixed genetic profiles from one mosquito. Therefore, it is clearly important to collect these insects indoors where crimes were committed, because it may be possible to find intact genetic profiles of suspects in the blood found in the digestive tract of hematophagous mosquitoes for later comparison to identify an offender and/or exclude suspects. PMID:26600546

  20. Trace samples of human blood in mosquitoes as a forensic investigation tool.

    PubMed

    Rabêlo, K C N; Albuquerque, C M R; Tavares, V B; Santos, S M; Souza, C A; Oliveira, T C; Oliveira, N C L; Crovella, S

    2015-11-23

    Investigations of any type of crime invariably starts at the crime scene by collecting evidence. Thus, the purpose of this research was to collect and analyze an entomological trace from an environment that is similar to those of indoor crime scenes. Hematophagous mosquitoes were collected from two residential units; saliva of volunteers that were residents in the units was also collected for genetic analysis as reference samples. We examined the allele frequencies of 15 short tandem repeat loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA) and amelogenin. A total of 26 female hematophagous mosquitoes were identified as Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus; we were able to obtain 11 forensically valid genetic profiles, with a minimum of 0.028203 ng/μL of human DNA. Thus, the results of this study showed that it was possible to correlate human genetic information from mosquitoes with the volunteer reference samples, which validates the use of this information as forensic evidence. Furthermore, we observed mixed genetic profiles from one mosquito. Therefore, it is clearly important to collect these insects indoors where crimes were committed, because it may be possible to find intact genetic profiles of suspects in the blood found in the digestive tract of hematophagous mosquitoes for later comparison to identify an offender and/or exclude suspects.

  1. Identification and Characterization of Persistent Human Erythrovirus Infection in Blood Donor Samples

    PubMed Central

    Candotti, Daniel; Etiz, Nermin; Parsyan, Armen; Allain, Jean-Pierre

    2004-01-01

    The presence of human erythrovirus DNA in 2,440 blood donations from the United Kingdom and sub-Saharan Africa (Ghana, Malawi, and South Africa) was screened. Sensitive qualitative and real-time quantitative PCR assays revealed a higher prevalence of persistent infection with the simultaneous presence of immunoglobulin G (IgG) and viral DNA (0.55 to 1.3%) than previously reported. This condition was characterized by a low viral load (median, 558 IU/ml; range, 42 to 135,000 IU/ml), antibody-complexed virus, free specific IgG, and potentially infectious free virus. Human erythrovirus genotype 1 (formerly parvovirus B19) was prevalent in the United Kingdom, Malawi, and South Africa. In contrast, only human erythrovirus genotype 3 (erythrovirus variant V9) was prevalent in Ghana. Genotype 3 had considerable genetic diversity, clustering in two probable subtypes. Genotype 1-based antibody assays failed to detect 38.5% of Ghanaian samples containing antibodies to genotype 3 virus but did not fail to detect cases of persistent infection. This study indicates a potential African origin of genotype 3 human erythrovirus and considerable shortcomings in the tools currently used to diagnose erythrovirus infection. PMID:15507603

  2. Concentrations of drugs determined in blood samples collected from suspected drugged drivers in England and Wales.

    PubMed

    Burch, Hilary J; Clarke, Elizabeth J; Hubbard, Alison M; Scott-Ham, Michael

    2013-05-01

    This communication reports the blood concentrations of alcohol and drugs from 376 cases of alleged driving under the influence of drugs analysed at the Forensic Science Service Chorley and London laboratories between February 2010 and March 2011. The samples were analysed for alcohol, amphetamine, benzodiazepines, cocaine, MDMA, opiates, γ-hydroxybutyrate (GHB), ketamine, methadone and methylmethcathinone (the 4-isomer of which is known as mephedrone). The results were interpreted with respect to the number and type of drugs of abuse detected and the concentrations measured. Alcohol was quantified in 113 cases (30%), and of these a level in excess of the prescribed UK limit for driving of 80 mg% was present in 90 cases. In 80 cases, only the concentration of alcohol was measured, the concentrations of both drugs and alcohol were measured in 33 cases. In the remaining 263 cases, only the concentrations of relevant drugs of abuse were measured. The most common drug of abuse quantified was cocaine which was detected in 92 cases, either as the active drug or as its major metabolite benzoylecgonine, followed by diazepam which was quantified in 76 cases. Concentrations of some new drugs, and drugs rarely reported in driving under the influence cases are also presented.

  3. Apicomplexa primers amplify Proteromonas (Stramenopiles, Slopalinida, Proteromonadidae) in tissue and blood samples from lizards.

    PubMed

    Maia, João P M C; Gómez-Díaz, Elena; Harris, D James

    2012-12-01

    Microscopy has traditionally been the most common method in parasitological studies, but in recent years molecular screening has become increasingly frequent to detect protozoan parasites in a wide range of vertebrate hosts and vectors. During routine molecular screening of apicomplexan parasites in reptiles using the 18S rRNA gene, we have amplified and sequenced Proteromonas parasites from three lizard hosts (less than 1% prevalence). We conducted phylogenetic analysis to confirm the taxonomic position and infer their relationships with other stramenopiles. Although our phylogeny is limited due to scarcity of molecular data on these protists, our results confirm they are closely related to Proteromonas lacertae. Our findings show that unexpected parasites can be amplified from host samples (blood and tissue) using general procedures to detect hemoparasites, and stress that positive PCR amplifications alone should not be considered as definitive proof of infection by particular parasites. Further validation by sequence confirmation and thorough phylogenetic assessment will not only avoid false positives and biased prevalence estimates but also provide valuable information on the biodiversity and phylogenetic relationships of other parasitic organisms. More generally, our results illustrate the perils of general diagnosis protocols in parasitological studies and the need of cross-validation procedures.

  4. Apparatus for direct addition of reagents into a nuclear magnetic resonance (NMR) sample in the NMR probe

    NASA Astrophysics Data System (ADS)

    Perrin, Charles L.; Rivero, Ignacio A.

    1999-04-01

    Nuclear magnetic resonance (NMR) is a widely used tool in chemistry and biochemistry. It is occasionally necessary to add small aliquots of solvents or reagents repeatedly into the NMR tube. Ordinarily this is accomplished only by ejecting the sample and carrying out the addition outside the probe. It would be preferable to add the aliquot directly into the sample. We have designed and implemented a delivery system to accomplish this. This apparatus is particularly applicable to a recent NMR titration method for measuring relative pK's and to experiments where temperature must also be varied. This apparatus provides a safe, simple, and inexpensive method for repeated aliquot addition directly into the sample in the NMR probe.

  5. Chemical concentration measurement in blood serum and urine samples using liquid-core optical fiber Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Qi, Dahu; Berger, Andrew J.

    2007-04-01

    We report measurements of chemical concentrations in clinical blood serum and urine samples using liquid-core optical fiber (LCOF) Raman spectroscopy to increase the collected signal strength. Both Raman and absorption spectra were acquired in the near-infrared region using the LCOF geometry. Spectra of 71 blood serum and 61 urine samples were regressed via partial least squares against reference analyzer values. Significant correlation was found between predicted and reference concentrations for 13 chemicals. Using absorption data to normalize the LCOF enhancement made the results more accurate. The experimental geometry is well suited for high-volume and automated chemical analysis of clear biofluids.

  6. Procalcitonin Predicts Real-Time PCR Results in Blood Samples from Patients with Suspected Sepsis

    PubMed Central

    Mencacci, Antonella; Leli, Christian; Cardaccia, Angela; Meucci, Marta; Moretti, Amedeo; D'Alò, Francesco; Farinelli, Senia; Pagliochini, Rita; Barcaccia, Mariella; Bistoni, Francesco

    2012-01-01

    Background Early diagnosis and rapid bacterial identification are of primary importance for outcome of septic patients. SeptiFast® (SF) real-time PCR assay is of potential utility in the etiological diagnosis of sepsis, but it cannot replace blood culture (BC) for routine use in clinical laboratory. Procalcitonin (PCT) is a marker of sepsis and can predict bacteremia in septic patients. The aim of the present study was to investigate whether PCT serum levels could predict SF results, and could help screening febrile patients in which a SF assay can improve the etiological diagnosis of sepsis. Methods From 1009 febrile patients with suspected sepsis, 1009 samples for BC, SF real-time PCR, and PCT determination were obtained simultaneously, and results were compared and statistically analysed. Receiver operating characteristic (ROC) curves were generated to determine the area under the curve and to identify which cut-off of PCT value produced the best sensitivity to detect SF results. Results Mean PCT values of sera drawn simultaneously with samples SF positive (35.42±61.03 ng/ml) or BC positive (23.14±51.56 ng/ml) for a pathogen were statistically higher than those drawn simultaneously with SF negative (0.84±1.67 ng/ml) or BC negative (2.79±16.64 ng/ml) samples (p<0.0001). For SF, ROC analysis showed an area under the curve of 0.927 (95% confidence interval: 0.899–0.955, p<0.0001). The PCT cut-off value of 0.37 ng/ml showed a negative predictive value of 99%, reducing the number of SF assays of 53.9%, still identifying the 96.4% of the pathogens. Conclusion PCT can be used in febrile patients with suspected sepsis to predict SF positive or negative results. A cut-off value of 0.37 ng/ml can be considered for optimal sensitivity, so that, in the routine laboratory activity, SF assay should not be used for diagnosis of sepsis in an unselected patient population with a PCT value <0.37 ng/ml. PMID:23300907

  7. Sampling blood from big brown bats (Eptesicus fuscus) in the field with and without anesthesia: Impacts on survival

    USGS Publications Warehouse

    Ellison, L.E.; O'Shea, T.J.; Wimsatt, J.; Pearce, R.D.; Neubaum, D.J.; Neubaum, M.A.; Bowen, R.A.

    2006-01-01

    Blood was collected from wild big brown bats (Eptesicus fuscus) with and without anesthesia in Fort Collins, Colorado in 2004 to assess the impacts of these procedures on short-term survival and 1-yr return rates. Short-term survival and 1-yr return rates after release were passively monitored using PIT tag detection hoops placed at selected buildings. Comparison of 14-day maximum likelihood survival estimates from bats not bled (142 adult females, 62 volant juveniles), and bats sampled for blood with anesthesia (96 adult females, 23 volant juveniles) and without anesthesia (112 adult females, 22 volant juveniles) indicated no adverse effects of either treatment (juveniles: X2=53.38, df=41, P=0.09; adults: X2=39.09, df=44, P=0.68). Return rates of bats one year after sampling were similar among adult female controls (75.4%, n=142, 95% CI=67.4-82.2%), females sampled for blood with anesthesia (83.0%, n=112, 95% CI=74.8-89.5%), and females sampled without anesthesia (87.5%, n=96, 95% CI=79.2-93.4%). Lack of an effect was also noted in 1-yr return rates of juvenile females. These data suggest that the use of anesthesia during sampling of blood has no advantages in terms of enhancement of survival in big brown bats. ?? Wildlife Disease Association 2006.

  8. Dried blood spot proteomics: surface extraction of endogenous proteins coupled with automated sample preparation and mass spectrometry analysis.

    PubMed

    Martin, Nicholas J; Bunch, Josephine; Cooper, Helen J

    2013-08-01

    Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.

  9. Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

    NASA Astrophysics Data System (ADS)

    Martin, Nicholas J.; Bunch, Josephine; Cooper, Helen J.

    2013-08-01

    Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.

  10. Use of liquid heparin for blood gas sampling in pediatric intensive care unit: A comparative study of effects of varying volumes of heparin on blood gas parameters

    PubMed Central

    Chhapola, Viswas; Kumar, Sandeep; Goyal, Pallavi; Sharma, Rajni

    2013-01-01

    Background and Aims: Pre-analytical errors in sample collection affect the reliability of blood gas (BG) analysis. Amount of liquid heparin as anticoagulant in samples for BG can affect results by its dilutional direct binding and compositional effects. The aim of this study was to examine the effect of varying amounts of heparin in blood samples on results. Materials and Methods: The prospective study was conducted on 15 children at a pediatric intensive care unit (PICU). Three different heparinized syringes were used containing minimal, 60 IU and 120 IU of heparin. A total volume of 1 ml blood in each syringe was taken and was analyzed by blood gas analyzer. Statistical analysis used related samples Friedman's test and Wilcoxon signed ranks test for paired comparisons. The observed bias was also compared with the desirable bias according to specifications by Ricos et al. Results: There was a significant difference (P < 0.05) in values of pH, pCO2, HCO3−, Hb and Na+ in the three syringes. The pCO2, HCO3− and Na+ levels decreased with the increasing amount of heparin. The observed percentage bias was more than desirable percentage bias specifications for pCO2, HCO3−, Hb, Na+, K+ and Cl− levels. Conclusions: Syringes with minimal liquid heparin are most appropriate for studying BG parameters as they have the least effect on these parameters. There is a need to standardize the procedure of syringe preparation for BG analysis. Further studies are needed to compare minimal amounts of heparin with commercially available dry balanced heparin syringes. PMID:24501486

  11. PCBs, PCDD/Fs, and PBDEs in blood samples of a rural population in South Germany.

    PubMed

    Fromme, Hermann; Albrecht, Michael; Appel, Markus; Hilger, Bettina; Völkel, Wolfgang; Liebl, Bernhard; Roscher, Eike

    2015-01-01

    The body burden of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), dioxin-like (dl-PCBs) and non-dioxin-like (ndl-PCBs) polychlorinated biphenyls, and polybrominated diphenyl ethers (PBDEs) was determined in blood samples from 70 subjects between 4 and 76 years old. The participants of the study were recruited in the neighborhood of a reclamation plant located in a rural area in Southern Germany. The median concentrations (95th percentiles in parentheses), expressed as WHO2005-TEQ (toxic equivalents), for PCDD/Fs and dl-PCBs were 4.5 (17.9)pgg(-1) l.w. and 2.6 (13.2)pgg(-1) l.w., respectively. The dl-PCBs contributed 40% of the total TEQ (median values), and the most abundant congener was PCB 156. Combined, the sum of the 6 non-dioxin-like PCBs had a median of 0.773μgL(-1) and a 95th percentile of 4.895μgL(-1). For the six tetra to hepta PBDE congeners, the median was 1.8ngg(-1) l.w. (95th percentile: 16.2ngg(-1) l.w.). None of our study subjects had a body burden that exceeded the biomonitoring equivalents for dioxins or PBDE congener 99 or the human biomonitoring values for ndl-PCBs. Likewise the study group did not exceed German reference values or values obtained in similar investigations. Overall, our study did not exhibit elevated internal exposures. The results also hint further decreasing tendencies for PCDD/Fs, PCBs, and PBDEs in Germany and demonstrates that people in the vicinity of a reclamation plant with no indication of an environmental contamination did not exhibit elevated internal exposures.

  12. Sample data processing in an additive and reproducible taxonomic workflow by using character data persistently linked to preserved individual specimens

    PubMed Central

    Kilian, Norbert; Henning, Tilo; Plitzner, Patrick; Müller, Andreas; Güntsch, Anton; Stöver, Ben C.; Müller, Kai F.; Berendsohn, Walter G.; Borsch, Thomas

    2015-01-01

    We present the model and implementation of a workflow that blazes a trail in systematic biology for the re-usability of character data (data on any kind of characters of pheno- and genotypes of organisms) and their additivity from specimen to taxon level. We take into account that any taxon characterization is based on a limited set of sampled individuals and characters, and that consequently any new individual and any new character may affect the recognition of biological entities and/or the subsequent delimitation and characterization of a taxon. Taxon concepts thus frequently change during the knowledge generation process in systematic biology. Structured character data are therefore not only needed for the knowledge generation process but also for easily adapting characterizations of taxa. We aim to facilitate the construction and reproducibility of taxon characterizations from structured character data of changing sample sets by establishing a stable and unambiguous association between each sampled individual and the data processed from it. Our workflow implementation uses the European Distributed Institute of Taxonomy Platform, a comprehensive taxonomic data management and publication environment to: (i) establish a reproducible connection between sampled individuals and all samples derived from them; (ii) stably link sample-based character data with the metadata of the respective samples; (iii) record and store structured specimen-based character data in formats allowing data exchange; (iv) reversibly assign sample metadata and character datasets to taxa in an editable classification and display them and (v) organize data exchange via standard exchange formats and enable the link between the character datasets and samples in research collections, ensuring high visibility and instant re-usability of the data. The workflow implemented will contribute to organizing the interface between phylogenetic analysis and revisionary taxonomic or monographic work

  13. Addition of Autologous Mesenchymal Stem Cells to Whole Blood for Bio-Enhanced ACL Repair has No Benefit in the Porcine Model

    PubMed Central

    Proffen, Benedikt L.; Vavken, Patrick; Haslauer, Carla M.; Fleming, Braden C.; Harris, Chad E.; Machan, Jason T.; Murray, Martha M.

    2015-01-01

    Background Co-culture of mesenchymal stem cells (MSCs) from the retropatellar fat pad and peripheral blood has been shown to stimulate anterior cruciate ligament (ACL) fibroblast proliferation and collagen production in vitro. Current techniques of bio-enhanced ACL repair in animal studies involve adding a biologic scaffold, in this case an extracellular matrix based scaffold saturated with autologous whole blood, to a simple suture repair of the ligament. Whether the enrichment of whole blood with MSCs would further improve the in vivo results of bio-enhanced ACL repair was investigated. Hypothesis/Purpose The hypothesis was that the addition of MSCs derived from adipose tissue or peripheral blood to the blood-extracellular matrix composite, which is used in bio-enhanced ACL repair to stimulate healing, would improve the biomechanical properties of a bio-enhanced ACL repair after 15 weeks of healing. Study Design Controlled laboratory study. Methods Twenty-four adolescent Yucatan mini-pigs underwent ACL transection followed by: 1) bio-enhanced ACL repair, 2) bio-enhanced ACL repair with the addition of autologous adipose-derived MSCs and 3) bio-enhanced ACL repair with the addition of autologous peripheral blood derived MSCs. After fifteen weeks of healing, structural properties of the ACL (yield & failure load, linear stiffness) were measured. Cell and vascular density were measured in the repaired ACL via histology, and its tissue structure was qualitatively evaluated using the Advanced Ligament Maturity Index. Results After fifteen weeks of healing, there were no significant improvements in the biomechanical or histological properties with the addition of adipose-derived MSCs. The only significant change with the addition of peripheral blood MSCs was an increase in knee anteroposterior (AP) laxity when measured at 30 degrees of flexion. Conclusions These findings suggest that the addition of adipose or peripheral blood MSCs to whole blood prior to saturation of

  14. Blood oxygen content in microliter samples using an easy-to-build galvanic oxygen cell.

    PubMed

    Grubb, B R; Mills, C D

    1981-02-01

    We have designed a simple, inexpensive, easy-to-build and operate apparatus for measuring blood oxygen content. The galvanic oxygen cell (fuel cell) requires as little as 1 microliter of blood and has a measuring time of 1-3 min. It is well suited for measuring oxygen content in fluids low in oxygen inasmuch as the sensitivity of the instrument is variable. Either air or water (at a known temperature and oxygen tension) can be used for calibration. No significant differences in blood oxygen content measured with our cell or the Van Slyke manometric method were found.

  15. Development and validation of an on-line multidimensional SPE-LC-MS/MS method for the quantitation of Tetrandrine in blood samples.

    PubMed

    Caglar, Sena; Morello, Rosa; Boos, Karl-Siegfried

    2015-04-15

    On-line solid-phase extraction (SPE) is becoming an increasingly widespread technique in the clean-up of complex matrices such as body fluids, prior to chromatographic analysis. The use of small SPE columns instead of disposable SPE cartridges allows multiple injections and complete automation. In addition, it decreases the cost of consumables and improves the quality of the overall analysis. Coupling of SPE with HPLC combines sample preparation and separation in one system. In this paper a validated on-line multidimensional (MD) SPE-LC-MS/MS method is described for the determination of Tetrandrine (model drug) in human blood samples. The developed method showed the applicability of direct injection of plasma samples to an on-line MD-SPE-LC-MS/MS system to determine small molecules i.e. drugs. The experimental design is unique. Quantification was through tandem mass spectrometry with positive electrospray ionization (ESI) and multiple reactions monitoring (MRM). The limit of detection was calculated as 31.98 ng/mL. The linear range of the method was between 40.0 and 800.0 ng/mL. Pharmacokinetic parameters are usually determined by analysis of drug concentrations in plasma rather than whole blood. Parameters determined using plasma data may be misleading if concentrations of drug differ between plasma and red blood cells. We successfully applied the developed method for the determination of the distribution coefficient of the model drug Tetrandrine between human red blood cells and blood plasma proteins. The determination of distribution coefficient study results demonstrated that the developed method can provide direct and accurate measurement of RBC partitioning in a model drug and could be applied for screening of other compounds for potential high RBC partition, predicting potential drug toxicity and investigating mechanisms associated with RBC partitions. PMID:25746132

  16. Reaction of homopiperazine with endogenous formaldehyde: a carbon hydrogen addition metabolite/product identified in rat urine and blood.

    PubMed

    Martin, Scott; Lenz, Eva M; Temesi, Dave; Wild, Martin; Clench, Malcolm R

    2012-08-01

    Drug reactivity and bioactivation are of major concern to the development of potential drug candidates in the pharmaceutical industry (Chem Res Toxicol 17:3-16, 2004; Chem Res Toxicol 19:889-893, 2006). Identifying potentially problematic compounds as soon as possible in the discovery process is of great importance, so often early in vitro screening is used to speed up attrition. Identification of reactive moieties is relatively straightforward with appropriate in vitro trapping experiments; however, on occasion unexpected reactive intermediates can be found later during more detailed in vivo studies. Here, we present one such example involving a series of compounds from an early drug discovery campaign. These compounds were found to react with endogenous formaldehyde from a rat in vivo study, resulting in the formation of novel +13-Da bridged homopiperazine products (equivalent to the addition of one carbon and one hydrogen atom), which were detected in urine and blood. The identification of these +13-Da products and their origin and mechanism of formation are described in detail through analyses of a representative homopiperazine compound [N-(3-(3-fluorophenyl)-1,2,4-thiadiazol-5-yl)-4-(4-isopropyl-1,4-diaze-pane-2-carbonyl)piperazine-1-carboxamide (AZX)] by liquid chromatography-UV-mass spectrometry, (1)H NMR, and chemical tests. PMID:22550270

  17. Effects of music therapy on pain responses induced by blood sampling in premature infants: A randomized cross-over trial

    PubMed Central

    Shabani, Fidan; Nayeri, Nahid Dehghan; Karimi, Roghiyeh; Zarei, Khadijeh; Chehrazi, Mohammad

    2016-01-01

    Background: Premature infants are subjected to many painful procedures during care and treatment. The aim of this study was to assess the effect of music therapy on physiological and behavioral pain responses of premature infants during and after blood sampling. Materials and Methods: This study was a cross-over clinical trial conducted on 20 infants in a hospital affiliated to Tehran University of Medical Sciences for a 5-month period in 2011. In the experimental group, Transitions music was played from 5 min before until 10 min after blood sampling. The infants’ facial expressions and physiological measures were recorded from 10 min before until 10 min after sampling. All steps and measurements, except music therapy, were the same for the control group. Data were analyzed using SAS and SPSS software through analysis of variance (ANOVA) and Chi-square tests. Results: There were significant differences between the experimental and control groups (P = 0.022) in terms of heart rate during needle extraction and at the first 5 min after sampling (P = 0.005). Considering the infant's sleep–wake state in the second 5 min before sampling, the statistical difference was significant (P = 0.044). Difference was significant (P = 0.045) during injection of the needle, in the first 5 min after sampling (P = 0.002), and in the second 5 min after sampling (P = 0.005). There were significant difference in infants’ facial expressions of pain in the first 5 min after sampling (P = 0.001). Conclusions: Music therapy reduces the physiological and behavioral responses of pain during and after blood sampling. PMID:27563323

  18. Enhanced Stability of Blood Matrices Using a Dried Sample Spot Assay to Measure Human Butyrylcholinesterase Activity and Nerve Agent Adducts

    PubMed Central

    Perez, Jonas W.; Pantazides, Brooke G.; Watson, Caroline M.; Thomas, Jerry D.; Blake, Thomas A.; Johnson, Rudolph C.

    2015-01-01

    Dried matrix spots are safer to handle and easier to store than wet blood products, but factors such as intra-spot variability and unknown sample volumes have limited their appeal as a sampling format for quantitative analyses. In this work, we introduce a dried spot activity assay for quantifying butyrylcholinesterase (BChE) specific activity which is BChE activity normalized to the total protein content in a sample spot. The method was demonstrated with blood, serum, and plasma spotted on specimen collection devices (cards) which were extracted to measure total protein and BChE activity using a modified Ellman assay. Activity recovered from dried spots was ∼80% of the initial spotted activity for blood and >90% for plasma and serum. Measuring total protein in the sample and calculating specific activity substantially improved quantification and reduced intra-spot variability. Analyte stability of nerve agent adducts was also evaluated, and the results obtained via BChE-specific activity measurements were confirmed by quantification of BChE adducts using a previously established LC-MS/MS method. The spotted samples were up to 10-times more resistant to degradation compared to unspotted control samples when measuring BChE inhibition by the nerve agents sarin and VX. Using this method, both BChE activity and adducts can be accurately measured from a dried sample spot. This use of a dried sample spot with normalization to total protein is robust, demonstrates decreased intra-spot variability without the need to control for initial sample volume, and enhances analyte stability. PMID:25955132

  19. Detection of JCPyV microRNA in blood and urine samples of multiple sclerosis patients under natalizumab therapy.

    PubMed

    Giovannelli, Irene; Martelli, Francesco; Repice, Anna; Massacesi, Luca; Azzi, Alberta; Giannecchini, Simone

    2015-12-01

    Polyomavirus JC (JCPyV) reactivation and development of progressive multifocal leukoencephalopathy is a health concern in multiple sclerosis patients under natalizumab therapy. Here, the JCPyV microRNA-J1-3p and microRNA-J1-5p expressions and genomic variability were investigated in blood and urine samples of multiple sclerosis patients before and under natalizumab therapy and in healthy controls. The two JCPyV microRNAs were detected in the JCPyV-DNA-positive peripheral blood mononuclear cell samples and in the exosomes derived from plasma and urine obtained from JCPyV-DNA-positive and JCPyV-DNA-negative patients. In particular, the increased JCPyV microRNA expression in samples of multiple sclerosis patients under natalizumab therapy was consistent with the high JCPyV-DNA positivity observed in these samples. Moreover, JCPyV microRNA genomic region showed few nucleotide differences in samples obtained from blood and urine of multiple sclerosis patients and healthy controls. Overall, these data suggest a potential role of the JCPyV microRNA expression in counteracting the viral reactivation to maintain JCPyV asymptomatic persistence in the host.

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

    PubMed Central

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

    2011-01-01

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

  1. Persistent organochlorine pesticides detected in blood and tissue samples of vultures from different localities in South Africa.

    PubMed

    van Wyk, E; Bouwman, H; van der Bank, H; Verdoorn, G H; Hofmann, D

    2001-07-01

    Gas chromatography was used to establish the presence of quantifiable residues of 14 persistent chlorinated hydrocarbon pollutants in whole blood, clotted blood, heart, kidney, liver and muscle samples obtained from individual African whitebacked (Pseudogyps africanus), Cape griffon (Gyps coprotheres) and Lappetfaced (Torgos tracheliotos) vultures from different localities in South Africa. The levels of pesticides measured in whole blood samples of live specimens were compared between nestlings from two natural breeding colonies, adults from a wildlife area and birds held in captivity. Statistically significant (P<0.05) differences between populations were detected in geometric means calculated for gamma-BHC (lindane), alpha(cis)-chlordane and alpha-endosulfan. Five of the organochlorine contaminants displayed significant variations between concentrations detected in the clotted blood, organs and muscles excised from vulture carcasses. This includes residues of gamma-BHC, alpha-chlordane, dieldrin, beta-endosulfan and heptachlor epoxide. Values of the respective biocides measured in vulture samples were generally low in comparison to results documented for a number of avian species. Although no threat is posed by any of the organochloride pesticides, continual monitoring of especially breeding colonies is recommended. Furthermore, the suitability of African whitebacked vulture nestlings as basic bioindicators is highly advocated. PMID:11461840

  2. [Use of C-arm CT for improving the hit rate for selective blood sampling from adrenal veins].

    PubMed

    Georgiades, C; Kharlip, J; Valdeig, S; Wacker, F K; Hong, K

    2009-09-01

    Primary hyperaldosteronism is the most common curable cause of hypertension with a prevalence of up to 12% among patients with hypertension. Selective blood sampling from adrenal veins is considered the diagnostic gold standard. However, it is underutilized due to the high technical failure rate. The use of C-arm CT during the sampling procedure can reduce or even eliminate this failure rate. If adrenal vein sampling is augmented by native C-arm CT to check for the correct catheter position, the technical success rate increases substantially. General use of this technique will result in correct diagnosis and treatment for patients with primary hyperaldosteronism.

  3. Particle size distribution and chemical composition of total mixed rations for dairy cattle: water addition and feed sampling effects.

    PubMed

    Arzola-Alvarez, C; Bocanegra-Viezca, J A; Murphy, M R; Salinas-Chavira, J; Corral-Luna, A; Romanos, A; Ruíz-Barrera, O; Rodríguez-Muela, C

    2010-09-01

    Four dairy farms were used to determine the effects of water addition to diets and sample collection location on the particle size distribution and chemical composition of total mixed rations (TMR). Samples were collected weekly from the mixing wagon and from 3 locations in the feed bunk (top, middle, and bottom) for 5 mo (April, May, July, August, and October). Samples were partially dried to determine the effect of moisture on particle size distribution. Particle size distribution was measured using the Penn State Particle Size Separator. Crude protein, neutral detergent fiber, and acid detergent fiber contents were also analyzed. Particle fractions 19 to 8, 8 to 1.18, and <1.18 mm were judged adequate in all TMR for rumen function and milk yield; however, the percentage of material>19 mm was greater than recommended for TMR, according to the guidelines of Cooperative Extension of Pennsylvania State University. The particle size distribution in April differed from that in October, but intermediate months (May, July, and August) had similar particle size distributions. Samples from the bottom of the feed bunk had the highest percentage of particles retained on the 19-mm sieve. Samples from the top and middle of the feed bunk were similar to that from the mixing wagon. Higher percentages of particles were retained on >19, 19 to 8, and 8 to 1.18 mm sieves for wet than dried samples. The reverse was found for particles passing the 1.18-mm sieve. Mean particle size was higher for wet than dried samples. The crude protein, neutral detergent fiber, and acid detergent fiber contents of TMR varied with month of sampling (18-21, 40-57, and 21-34%, respectively) but were within recommended ranges for high-yielding dairy cows. Analyses of TMR particle size distributions are useful for proper feed bunk management and formulation of diets that maintain rumen function and maximize milk production and quality. Water addition may help reduce dust associated with feeding TMR. PMID

  4. Differential expression of minimal residual disease markers in peripheral blood and bone marrow samples from high-risk neuroblastoma patients

    PubMed Central

    YAMAMOTO, NOBUYUKI; KOZAKI, AIKO; HARTOMO, TRI BUDI; YANAI, TOMOKO; HASEGAWA, DAIICHIRO; KAWASAKI, KEIICHIRO; KOSAKA, YOSHIYUKI; MATSUO, MASAFUMI; HIRASE, SATOSHI; MORI, TAKESHI; HAYAKAWA, AKIRA; IIJIMA, KAZUMOTO; NISHIO, HISAHIDE; NISHIMURA, NORIYUKI

    2015-01-01

    Neuroblastoma is an aggressive solid tumor that leads to tumor relapse in more than half of high-risk patients. Minimal residual disease (MRD) is primarily responsible for tumor relapses and may be detected in peripheral blood (PB) and bone marrow (BM) samples. To evaluate the disease status and treatment response, a number of MRD detection protocols based on either common or distinct markers for PB and BM samples have been reported. However, the correlation between the expression of MRD markers in PB and BM samples remains elusive in the clinical samples. In the present study, the expression of 11 previously validated MRD markers (CHRNA3, CRMP1, DBH, DCX, DDC, GABRB3, GAP43, ISL1, KIF1A, PHOX2B and TH) was determined in 23 pairs of PB and BM samples collected from seven high-risk neuroblastoma patients at the same time point, and the sample was scored as MRD-positive if one of the MRD markers exceeded the normal range. Although the number of MRD-positive samples was not significantly different between PB and BM samples, the two most sensitive markers for PB samples (CRMP1 and KIF1A) were different from those for BM samples (PHOX2B and DBH). There was no statistically significant correlation between the expression of MRD markers in the PB and BM samples. These results suggest that MRD markers were differentially expressed in PB and BM samples from high-risk neuroblastoma patients. PMID:26722317

  5. Rapid Treponema pallidum clearance from blood and ulcer samples following single dose benzathine penicillin treatment of early syphilis.

    PubMed

    Tipple, Craig; Jones, Rachael; McClure, Myra; Taylor, Graham

    2015-02-01

    Currently, the efficacy of syphilis treatment is measured with anti-lipid antibody tests. These can take months to indicate cure and, as a result, syphilis treatment trials require long periods of follow-up. The causative organism, Treponema pallidum (T. pallidum), is detectable in the infectious lesions of early syphilis using DNA amplification. Bacteraemia can likewise be identified, typically in more active disease. We hypothesise that bacterial clearance from blood and ulcers will predict early the standard serology-measured treatment response and have developed a qPCR assay that could monitor this clearance directly in patients with infectious syphilis. Patients with early syphilis were given an intramuscular dose of benzathine penicillin. To investigate the appropriate sampling timeframe samples of blood and ulcer exudate were collected intensively for T. pallidum DNA (tpp047 gene) and RNA (16S rRNA) quantification. Sampling ended when two consecutive PCRs were negative. Four males were recruited. The mean peak level of T. pallidum DNA was 1626 copies/ml whole blood and the mean clearance half-life was 5.7 hours (std. dev. 0.53). The mean peak of 16S rRNA was 8879 copies/ml whole blood with a clearance half-life of 3.9 hours (std. dev. 0.84). From an ulcer, pre-treatment, 67,400 T. pallidum DNA copies and 7.08 x 107 16S rRNA copies were detected per absorbance strip and the clearance half-lives were 3.2 and 4.1 hours, respectively. Overall, T. pallidum nucleic acids were not detected in any sample collected more than 56 hours (range 20-56) after treatment. All patients achieved serologic cure. In patients with active early syphilis, measuring T. pallidum levels in blood and ulcer exudate may be a useful measure of treatment success in therapeutic trials. These laboratory findings need confirmation on a larger scale and in patients receiving different therapies.

  6. Validation and Clinical Evaluation of a Novel Method To Measure Miltefosine in Leishmaniasis Patients Using Dried Blood Spot Sample Collection

    PubMed Central

    Rosing, H.; Hillebrand, M. J. X.; Blesson, S.; Mengesha, B.; Diro, E.; Hailu, A.; Schellens, J. H. M.; Beijnen, J. H.

    2016-01-01

    To facilitate future pharmacokinetic studies of combination treatments against leishmaniasis in remote regions in which the disease is endemic, a simple cheap sampling method is required for miltefosine quantification. The aims of this study were to validate a liquid chromatography-tandem mass spectrometry method to quantify miltefosine in dried blood spot (DBS) samples and to validate its use with Ethiopian patients with visceral leishmaniasis (VL). Since hematocrit (Ht) levels are typically severely decreased in VL patients, returning to normal during treatment, the method was evaluated over a range of clinically relevant Ht values. Miltefosine was extracted from DBS samples using a simple method of pretreatment with methanol, resulting in >97% recovery. The method was validated over a calibration range of 10 to 2,000 ng/ml, and accuracy and precision were within ±11.2% and ≤7.0% (≤19.1% at the lower limit of quantification), respectively. The method was accurate and precise for blood spot volumes between 10 and 30 μl and for Ht levels of 20 to 35%, although a linear effect of Ht levels on miltefosine quantification was observed in the bioanalytical validation. DBS samples were stable for at least 162 days at 37°C. Clinical validation of the method using paired DBS and plasma samples from 16 VL patients showed a median observed DBS/plasma miltefosine concentration ratio of 0.99, with good correlation (Pearson's r = 0.946). Correcting for patient-specific Ht levels did not further improve the concordance between the sampling methods. This successfully validated method to quantify miltefosine in DBS samples was demonstrated to be a valid and practical alternative to venous blood sampling that can be applied in future miltefosine pharmacokinetic studies with leishmaniasis patients, without Ht correction. PMID:26787691

  7. Post mortem concentrations of endogenous gamma hydroxybutyric acid (GHB) and in vitro formation in stored blood and urine samples.

    PubMed

    Busardò, Francesco Paolo; Bertol, Elisabetta; Vaiano, Fabio; Baglio, Giovanni; Montana, Angelo; Barbera, Nunziata; Zaami, Simona; Romano, Guido

    2014-10-01

    Gamma-hydroxybutyrate (GHB) is a central nervous system depressant, primarily used as a recreational drug of abuse with numerous names. It has also been involved in various instances of drug-facilitated sexual assault due to its potential incapacitating effects. The first aim of this paper is to measure the post-mortem concentration of endogenous GHB in whole blood and urine samples of 30 GHB free-users, who have been divided according to the post-mortem interval (PMI) in three groups (first group: 24-36h; second group: 37-72h; third group: 73-192h), trying to evaluate the role of PMI in affecting post mortem levels. Second, the Authors have evaluated the new formation of GHB in vitro in blood and urine samples of the three groups, which have been stored at -20°C, 4°C and 20°C over a period of one month. The concentrations were measured by GC-MS after liquid-liquid extraction according to the method validated and published by Elliot (For. Sci. Int., 2003). For urine samples, GHB concentrations were creatinine-normalized. In the first group the GHB mean concentration measured after autopsy was: 2.14mg/L (range 0.54-3.21mg/L) in blood and 3.90mg/g (range 0.60-4.81mg/g) in urine; in the second group it was: 5.13mg/L (range 1.11-9.60mg/L) in blood and 3.93mg/g (range 0.91-7.25mg/g) in urine; in the third group it was: 11.8mg/L (range 3.95-24.12mg/L) in blood and 9.83mg/g (range 3.67-21.90mg/g) in urine. The results obtained in blood and urine samples showed a statistically significant difference among groups (p<0.001) in the first analysis performed immediately after autopsy. Throughout the period of investigation up to 4 weeks, the comparison of storage temperatures within each group showed in blood and urine samples a mean difference at 20°C compared to -20°C not statistically significant at the 10% level. These findings allow us to affirm that the PMI strongly affects the post mortem production of GHB in blood and urine samples. Regarding the new formation of

  8. Top-Down Proteomics and Direct Surface Sampling of Neonatal Dried Blood Spots: Diagnosis of Unknown Hemoglobin Variants

    NASA Astrophysics Data System (ADS)

    Edwards, Rebecca L.; Griffiths, Paul; Bunch, Josephine; Cooper, Helen J.

    2012-11-01

    We have previously shown that liquid microjunction surface sampling of dried blood spots coupled with high resolution top-down mass spectrometry may be used for screening of common hemoglobin variants HbS, HbC, and HbD. In order to test the robustness of the approach, we have applied the approach to unknown hemoglobin variants. Six neonatal dried blood spot samples that had been identified as variants, but which could not be diagnosed by current screening methods, were analyzed by direct surface sampling top-down mass spectrometry. Both collision-induced dissociation and electron transfer dissociation mass spectrometry were employed. Four of the samples were identified as β-chain variants: two were heterozygous Hb D-Iran, one was heterozygous Hb Headington, and one was heterozygous Hb J-Baltimore. The fifth sample was identified as the α-chain variant heterozygous Hb Phnom Penh. Analysis of the sixth sample suggested that it did not in fact contain a variant. Adoption of the approach in the clinic would require speed in both data collection and interpretation. To address that issue, we have compared manual data analysis with freely available data analysis software (ProsightPTM). The results demonstrate the power of top-down proteomics for hemoglobin variant analysis in newborn samples.

  9. Generation of dimethylnitrosamine in water purification systems. Detection in human blood samples during hemodialysis.

    PubMed

    Simenhoff, M L; Dunn, S R; Fiddler, W; Pensabene, J W; Smiley, J

    1983-10-21

    Dimethylnitrosamine (DMNA), a carcinogen, was detected at levels up to 32 micrograms/L in dialysate from five of 16 dialysis units surveyed. Blood drawn from patients at one of these units in which DMNA was raised in the dialysate showed a significant increase in the amount of DMNA in the patient's blood when predialysis levels were compared with 15-minute intradialysis levels. The presence of a mixed-bed deionizer without an antecedent carbon filter appeared to be necessary for DMNA production. These data suggest that DMNA is generated in certain water purification systems and may then diffuse into the patient's blood. Guidelines for deionizer-treated water should be revised to include an activated carbon filter.

  10. Smart oxygen cuvette for optical monitoring of dissolved oxygen in biological blood samples

    NASA Astrophysics Data System (ADS)

    Dabhi, Harish; Alla, Suresh Kumar; Shahriari, Mahmoud R.

    2010-02-01

    A smart Oxygen Cuvette is developed by coating the inner surface of a cuvette with oxygen sensitive thin film material. The coating is glass like sol-gel based sensor that has an embedded ruthenium compound in the glass film. The fluorescence of the ruthenium is quenched depending on the oxygen level. Ocean Optics phase fluorometer, NeoFox is used to measure this rate of fluorescence quenching and computes it for the amount of oxygen present. Multimode optical fibers are used for transportation of light from an LED source to cuvette and from cuvette to phase fluorometer. This new oxygen sensing system yields an inexpensive solution for monitoring the dissolved oxygen in samples for biological and medical applications. In addition to desktop fluorometers, smart oxygen cuvettes can be used with the Ocean Optics handheld Fluorometers, NeoFox Sport. The Smart Oxygen Cuvettes provide a resolution of 4PPB units, an accuracy of less than 5% of the reading, and 90% response in less than 10 seconds.

  11. Yeast-containing feed additive alters gene expression profiles associated with innate immunity in whole blood of a rodent model.

    PubMed

    Branson, Jennifer A; McLean, Derek J; Forsberg, Neil E; Bobe, Gerd

    2016-05-01

    Feeding a yeast-containing additive (YCA; OmniGen-AF) improves immune responses in ruminant livestock and reduces subsequent production losses. The objective was to identify molecular pathways by which dietary YCA may modify immune responses using a rodent model. Thirty-seven healthy, unchallenged CD rats received a diet containing 0 (control; n = 5, only 28 d), 0.5% (n = 15) or 1% (n = 17) YCA for 7 (n = 4/group), 14 (n = 3 or 4/group), 21 (n = 3 or 4/group) or 28 (n = 5/group) d. At the end of the feeding periods, whole blood was collected and the isolated RNA was analyzed for the expression of 84 genes involved in innate and cell-mediated adaptive immune responses. Three bacterial pattern recognition receptors TLR1 (0.5%: + 2.01; 1%: + 2.38), TLR6 (0.5%: + 2.11; 1%: + 2.34) and NOD2 (0.5%: + 2.32; 1%: + 2.23), two APC surface receptors CD1D1 (0.5%: + 1.75; 1%: + 2.33) and CD80 (0.5%: +2.45; 1%: +3.00), and the cell signaling molecule MAPK8 (0.5%: +1.87; 1%: +2.35) were significantly up-regulated by YCA at both inclusion rates. In conclusion, feeding YCA may potentially increase recognition and responses to bacterial pathogens and T-cell activation and differentiation and thereby maintain health and prevent production losses. PMID:27033362

  12. Role of therapeutic drug monitoring in pulmonary infections: use and potential for expanded use of dried blood spot samples.

    PubMed

    Hofman, Susan; Bolhuis, Mathieu S; Koster, Remco A; Akkerman, Onno W; van Assen, Sander; Stove, Christophe; Alffenaar, Jan-Willem C

    2015-01-01

    Respiratory tract infections are among the most common infections in men. We reviewed literature to document their pharmacological treatments, and the extent to which therapeutic drug monitoring (TDM) is needed during treatment. We subsequently examined potential use of dried blood spots as sample procedure for TDM. TDM was found to be an important component of clinical care for many (but not all) pulmonary infections. For gentamicin, linezolid, voriconazole and posaconazole dried blood spot methods and their use in TDM were already evident in literature. For glycopeptides, β-lactam antibiotics and fluoroquinolones it was determined that development of a dried blood spot (DBS) method could be useful. This review identifies specific antibiotics for which development of DBS methods could support the optimization of treatment of pulmonary infections.

  13. Extended acclimatization is required to eliminate stress effects of periodic blood-sampling procedures on vasoactive hormones and blood volume in beagle dogs.

    PubMed

    Slaughter, M R; Birmingham, J M; Patel, B; Whelan, G A; Krebs-Brown, A J; Hockings, P D; Osborne, J A

    2002-10-01

    Important in all experimental animal studies is the need to control stress stimuli associated with environmental change and experimental procedures. As the stress response involves alterations in levels of vasoactive hormones, ensuing changes in cardiovascular parameters may confound experimental outcomes. Accordingly, we evaluated the duration required for dogs (n = 4) to acclimatized to frequent blood sampling that involved different procedures. On each sampling occasion during a 6-week period, dogs were removed from their pen to a laboratory area and blood was collected either by venepuncture (days 2, 15, 34, 41) for plasma renin activity (PRA), epinephrine (EPI), norepinephrine, aldosterone, insulin, and atrial natriuretic peptide, or by cannulation (dogs restrained in slings; days 1, 8, 14, 22, 30, 33, 37, 40) for determination of haematocrit (HCT) alone (days 1 to 22) or HCT with plasma volume (PV; days 30 to 40). PRA was higher on days 2 and 15 compared with days 34 and 41 and had decreased by up to 48% by the end of the study (day 41 vs day 15; mean/SEM: 1.18/0.27 vs 2.88/0.79 ng ANG I/ml/h, respectively). EPI showed a time-related decrease from days 2 to 34, during which mean values had decreased by 51% (mean/SEM: 279/29 vs 134/20.9 pg/ml for days 2 and 34, respectively), but appeared stable from then on. None of the other hormones showed any significant variability throughout the course of the study. HCT was relatively variable between days 1 to 22 but stabilized from day 30, after which all mean values were approximately 6% lower than those between days 1 and 8. We conclude that an acclimatization period of at least 4 weeks is required to eliminate stress-related effects in dogs associated with periodic blood sampling. PMID:12396283

  14. How well do blood folate concentrations predict dietary folate intakes in a sample of Canadian lactating women exposed to high levels of folate? An observational study

    PubMed Central

    Houghton, Lisa A; Sherwood, Kelly L; O'Connor, Deborah L

    2007-01-01

    Background In 1998, mandatory folic acid fortification of white flour and select cereal grain products was implemented in Canada with the intention to increase dietary folate intakes of reproducing women. Folic acid fortification has produced a dramatic increase in blood folate concentrations among reproductive age women, and a reduction in neural tube defect (NTD)-affected pregnancies. In response to improved blood folate concentrations, many health care professionals are asking whether a folic acid supplement is necessary for NTD prevention among women with high blood folate values, and how reliably high RBC folate concentrations predict folate intakes shown in randomized controlled trials to be protective against NTDs. The objective of this study was to determine how predictive blood folate concentrations and folate intakes are of each other in a sample of well-educated lactating Canadian women exposed to high levels of synthetic folate. Methods The relationship between blood folate concentrations and dietary folate intakes, determined by weighed food records, were assessed in a sample of predominantly university-educated lactating women (32 ± 4 yr) at 4-(n = 53) and 16-wk postpartum (n = 55). Results Median blood folate concentrations of all participants were well above plasma and RBC folate cut-off levels indicative of deficiency (6.7 and 317 nmol/L, respectively) and all, except for 2 subjects, were above the cut-off for NTD-risk reduction (>906 nmol/L). Only modest associations existed between total folate intakes and plasma (r = 0.46, P < 0.001) and RBC (r = 0.36, P < 0.01) folate concentrations at 16-wk postpartum. Plasma and RBC folate values at 16-wk postpartum correctly identified the quartile of folate intake of only 26 of 55 (47%) and 18 of 55 (33%) of subjects, respectively. The mean RBC folate concentration of women consuming 151–410 μg/d of synthetic folate (2nd quartile of intake) did not differ from that of women consuming >410 μg/d (3rd and

  15. Analysis of hemoglobin adducts from acrylamide, glycidamide, and ethylene oxide in paired mother/cord blood samples from Denmark.

    PubMed

    von Stedingk, Hans; Vikström, Anna C; Rydberg, Per; Pedersen, Marie; Nielsen, Jeanette K S; Segerbäck, Dan; Knudsen, Lisbeth E; Törnqvist, Margareta

    2011-11-21

    The knowledge about fetal exposure to acrylamide/glycidamide from the maternal exposure through food is limited. Acrylamide, glycidamide, and ethylene oxide are electrophiles and form adducts with hemoglobin (Hb), which could be used for in vivo dose measurement. In this study, a method for analysis of Hb adducts by liquid chromatography-mass spectrometry, the adduct FIRE procedure, was applied to measurements of adducts from these compounds in maternal blood samples (n = 87) and umbilical cord blood samples (n = 219). The adduct levels from the three compounds, acrylamide, glycidamide, and ethylene oxide, were increased in tobacco smokers. Highly significant correlations were found between cord and maternal blood with regard to measured adduct levels of the three compounds. The mean cord/maternal hemoglobin adduct level ratios were 0.48 (range 0.27-0.86) for acrylamide, 0.38 (range 0.20-0.73) for glycidamide, and 0.43 (range 0.17-1.34) for ethylene oxide. In vitro studies with acrylamide and glycidamide showed a lower (0.38-0.48) rate of adduct formation with Hb in cord blood than with Hb in maternal blood, which is compatible with the structural differences in fetal and adult Hb. Together, these results indicate a similar life span of fetal and maternal erythrocytes. The results showed that the in vivo dose in fetal and maternal blood is about the same and that the placenta gives negligible protection of the fetus to exposure from the investigated compounds. A trend of higher levels of the measured adducts in cord blood with gestational age was observed, which may reflect the gestational age-related change of the cord blood Hb composition toward a higher content of adult Hb. The results suggest that the Hb adduct levels measured in cord blood reflect the exposure to the fetus during the third trimester. The evaluation of the new analytical method showed that it is suitable for monitoring of background exposures of the investigated electrophilic compounds in large

  16. A nested PCR assay exhibits enhanced sensitivity for detection of Theileria parva infections in bovine blood samples from carrier animals.

    PubMed

    Odongo, David O; Sunter, Jack D; Kiara, Henry K; Skilton, Robert A; Bishop, Richard P

    2010-01-01

    Theileria parva causes East Coast fever, an economically important disease of cattle in sub-Saharan Africa. We describe a nested polymerase chain reaction (nPCR) assay for the detection of T. parva DNA in cattle blood spotted onto filter paper using primers derived from the T. parva-specific 104-kDa antigen (p104) gene. The sensitivity of this assay was compared to a previously described p104-based PCR and also the reverse line blot (RLB) technique, using serial dilutions of blood from a calf with known T. parva piroplasm parasitaemia. The relative sensitivities of the three assays were 0.4, 1.4 and 4 parasites/microl corresponding to blood parasitaemias of 9.2 x 10(-6)%, 2.8 x 10(-5)% and 8.3 x 10(-5)%, respectively. The three assays were applied to samples from two calves infected with the T. parva Muguga stock. Parasite DNA was consistently detectable by the two p104 PCR assays until 48 and 82 days post-infection, respectively, and thereafter sporadically. RLB detected parasite DNA in the two infected calves until days 43 and 45. Field samples from 151 Kenyan cattle exhibited 37.7% positivity for T. parva by regular p104 PCR and 42.3% positivity using p104 nPCR. Among 169 cattle blood samples from Southern Sudan, 36% were positive for T. parva using nPCR. The nPCR assay represents a highly sensitive tool for detection and monitoring of asymptomatic carrier state infections of T. parva in the blood of cattle. PMID:19902251

  17. Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

    PubMed Central

    Ramírez, Juan Carlos; Cura, Carolina Inés; Moreira, Otacilio da Cruz; Lages-Silva, Eliane; Juiz, Natalia; Velázquez, Elsa; Ramírez, Juan David; Alberti, Anahí; Pavia, Paula; Flores-Chávez, María Delmans; Muñoz-Calderón, Arturo; Pérez-Morales, Deyanira; Santalla, José; Guedes, Paulo Marcos da Matta; Peneau, Julie; Marcet, Paula; Padilla, Carlos; Cruz-Robles, David; Valencia, Edward; Crisante, Gladys Elena; Greif, Gonzalo; Zulantay, Inés; Costales, Jaime Alfredo; Alvarez-Martínez, Miriam; Martínez, Norma Edith; Villarroel, Rodrigo; Villarroel, Sandro; Sánchez, Zunilda; Bisio, Margarita; Parrado, Rudy; Galvão, Lúcia Maria da Cunha; da Câmara, Antonia Cláudia Jácome; Espinoza, Bertha; de Noya, Belkisyole Alarcón; Puerta, Concepción; Riarte, Adelina; Diosque, Patricio; Sosa-Estani, Sergio; Guhl, Felipe; Ribeiro, Isabela; Aznar, Christine; Britto, Constança; Yadón, Zaida Estela; Schijman, Alejandro G.

    2015-01-01

    An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease. PMID:26320872

  18. Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients.

    PubMed

    Ramírez, Juan Carlos; Cura, Carolina Inés; da Cruz Moreira, Otacilio; Lages-Silva, Eliane; Juiz, Natalia; Velázquez, Elsa; Ramírez, Juan David; Alberti, Anahí; Pavia, Paula; Flores-Chávez, María Delmans; Muñoz-Calderón, Arturo; Pérez-Morales, Deyanira; Santalla, José; Marcos da Matta Guedes, Paulo; Peneau, Julie; Marcet, Paula; Padilla, Carlos; Cruz-Robles, David; Valencia, Edward; Crisante, Gladys Elena; Greif, Gonzalo; Zulantay, Inés; Costales, Jaime Alfredo; Alvarez-Martínez, Miriam; Martínez, Norma Edith; Villarroel, Rodrigo; Villarroel, Sandro; Sánchez, Zunilda; Bisio, Margarita; Parrado, Rudy; Maria da Cunha Galvão, Lúcia; Jácome da Câmara, Antonia Cláudia; Espinoza, Bertha; Alarcón de Noya, Belkisyole; Puerta, Concepción; Riarte, Adelina; Diosque, Patricio; Sosa-Estani, Sergio; Guhl, Felipe; Ribeiro, Isabela; Aznar, Christine; Britto, Constança; Yadón, Zaida Estela; Schijman, Alejandro G

    2015-09-01

    An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease. PMID:26320872

  19. Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients.

    PubMed

    Ramírez, Juan Carlos; Cura, Carolina Inés; da Cruz Moreira, Otacilio; Lages-Silva, Eliane; Juiz, Natalia; Velázquez, Elsa; Ramírez, Juan David; Alberti, Anahí; Pavia, Paula; Flores-Chávez, María Delmans; Muñoz-Calderón, Arturo; Pérez-Morales, Deyanira; Santalla, José; Marcos da Matta Guedes, Paulo; Peneau, Julie; Marcet, Paula; Padilla, Carlos; Cruz-Robles, David; Valencia, Edward; Crisante, Gladys Elena; Greif, Gonzalo; Zulantay, Inés; Costales, Jaime Alfredo; Alvarez-Martínez, Miriam; Martínez, Norma Edith; Villarroel, Rodrigo; Villarroel, Sandro; Sánchez, Zunilda; Bisio, Margarita; Parrado, Rudy; Maria da Cunha Galvão, Lúcia; Jácome da Câmara, Antonia Cláudia; Espinoza, Bertha; Alarcón de Noya, Belkisyole; Puerta, Concepción; Riarte, Adelina; Diosque, Patricio; Sosa-Estani, Sergio; Guhl, Felipe; Ribeiro, Isabela; Aznar, Christine; Britto, Constança; Yadón, Zaida Estela; Schijman, Alejandro G

    2015-09-01

    An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.

  20. Concentrations of cadmium, lead, and zinc in fish from mining-influenced waters of northeastern Oklahoma: Sampling of blood, carcass, and liver for aquatic biomonitoring

    USGS Publications Warehouse

    Brumbaugh, W.G.; Schmitt, C.J.; May, T.W.

    2005-01-01

    The Tri-States Mining District (TSMD) of Missouri (MO), Kansas (KS), and Oklahoma (OK), USA, was mined for lead (Pb) and zinc (Zn) for more than a century. Mining ceased more than 30 years ago, but wastes remain widely distributed in the region, and there is evidence of surface- and groundwater contamination in the Spring River-Neosho River (SR-NR) system of northeastern OK. In October 2001, we collected a total of 74 fish from six locations in the SR-NR system that included common carp (Cyprinus carpio), channel- and flathead catfish (Ictalurus punctatus and Pylodictis olivaris), largemouth- and spotted bass (Micropterus salmoides and Micropterus punctulatus), and white crappie (Pomoxis annularis). We obtained additional fish from locations in MO that included three reference sites and one site that served as a "positive control" (heavily contaminated by Pb). Blood, carcass (headed, eviscerated, and scaled) and liver (carp only) samples were analyzed for cadmium (Cd), Pb, and Zn. Our objectives were to assess the degree to which fish from the OK portion of the SR-NR system are contaminated by these elements and to evaluate fish blood sampling for biomonitoring. Concentrations of Cd and Pb in carp and catfish from OK sites were elevated and Pb concentrations of some approached those of the highly contaminated site in MO, but concentrations in bass and crappie were relatively low. For Zn, correlations were weak among concentrations in the three tissues and none of the samples appeared to reflect site contamination. Variability was high for Cd in all three tissues of carp; differences between sites were statistically significant (p < 0.05) only for blood even though mean liver concentrations were at least 100-fold greater than those in blood. Blood concentrations of Cd and Pb were positively correlated (r 2 = 0.49 to 0.84) with the concentration of the same element in carp and catfish carcasses or in carp livers, and the corresponding multiple regression models were

  1. Concentrations of cadmium, lead, and zinc in fish from mining-influenced waters of northeastern Oklahoma: sampling of blood, carcass, and liver for aquatic biomonitoring.

    PubMed

    Brumbaugh, William G; Schmitt, Christopher J; May, Thomas W

    2005-07-01

    The Tri-States Mining District (TSMD) of Missouri (MO), Kansas (KS), and Oklahoma (OK), USA, was mined for lead (Pb) and zinc (Zn) for more than a century. Mining ceased more than 30 years ago, but wastes remain widely distributed in the region, and there is evidence of surface- and groundwater contamination in the Spring River-Neosho River (SR-NR) system of northeastern OK. In October 2001, we collected a total of 74 fish from six locations in the SR-NR system that included common carp (Cyprinus carpio), channel- and flathead catfish (Ictalurus punctatus and Pylodictis olivaris), largemouth- and spotted bass (Micropterus salmoides and Micropterus punctulatus), and white crappie (Pomoxis annularis). We obtained additional fish from locations in MO that included three reference sites and one site that served as a "positive control" (heavily contaminated by Pb). Blood, carcass (headed, eviscerated, and scaled) and liver (carp only) samples were analyzed for cadmium (Cd), Pb, and Zn. Our objectives were to assess the degree to which fish from the OK portion of the SR-NR system are contaminated by these elements and to evaluate fish blood sampling for biomonitoring. Concentrations of Cd and Pb in carp and catfish from OK sites were elevated and Pb concentrations of some approached those of the highly contaminated site in MO, but concentrations in bass and crappie were relatively low. For Zn, correlations were weak among concentrations in the three tissues and none of the samples appeared to reflect site contamination. Variability was high for Cd in all three tissues of carp; differences between sites were statistically significant (p < 0.05) only for blood even though mean liver concentrations were at least 100-fold greater than those in blood. Blood concentrations of Cd and Pb were positively correlated (r2 = 0.49 to 0.84) with the concentration of the same element in carp and catfish carcasses or in carp livers, and the corresponding multiple regression models were

  2. How You Can Help Medical Research: Donating Your Blood, Tissue, and Other Samples

    MedlinePlus

    ... sample to make sure the proposed research is ethical, useful, and based on good science. After the ... discuss further? • Am I comfortable with experts making decisions about how my samples will be used in ...

  3. A one-step extraction procedure for the screening of cocaine, amphetamines and cannabinoids in postmortem blood samples.

    PubMed

    Pelição, Fabrício Souza; Peres, Mariana Dadalto; Pissinate, Jauber Fornaciari; De Martinis, Bruno Spinosa

    2014-01-01

    A gas chromatography-mass spectrometric (GC-MS) method was developed and validated for the simultaneous detection and quantification in postmortem whole blood samples of cocaine (COC), amphetamines (AMPs) and cannabis; the main drugs involved in cases of impaired driving in Brazil. The analytes were extracted by solid-phase extraction by means of Bond-Elute Certify cartridges, derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide at 80°C for 30 min and analyzed by GC-MS. Linearity ranged from 10 to 500 ng/mL, except for ecgonine methyl ester, for which linearity ranged from 10 to 100 ng/mL. Inter- and intra-day imprecision ranged from 2.8 to 18.4% and from 1.5 to 14.9%, respectively. Accuracy values lay between 86.9 and 104.4%. The limit of quantitation for all drugs was 10 ng/mL and recoveries were >74% for all analytes, except for cannabinoids, which showed poor recovery (∼30%). The developed method was applied to real samples collected from deceased victims due to traffic accidents. These samples were selected according to the results obtained in immunoassay screening on collected urine samples. Five samples were positive for the presence of COC and metabolites, four samples were positive for cannabinoids, six samples were positive for AMPs and two samples were drug negative. Some samples were positive for more than one class of drug. Results obtained from whole blood samples showed good agreement with urine screening. The developed method proved capable of quantifying all three classes of drugs of abuse proposed in this study, through a one-step extraction procedure.

  4. Successful Generation of Human Induced Pluripotent Stem Cell Lines from Blood Samples Held at Room Temperature for up to 48 hr.

    PubMed

    Agu, Chukwuma A; Soares, Filipa A C; Alderton, Alex; Patel, Minal; Ansari, Rizwan; Patel, Sharad; Forrest, Sally; Yang, Fengtang; Lineham, Jonathan; Vallier, Ludovic; Kirton, Christopher M

    2015-10-13

    The collection sites of human primary tissue samples and the receiving laboratories, where the human induced pluripotent stem cells (hIPSCs) are derived, are often not on the same site. Thus, the stability of samples prior to derivation constrains the distance between the collection site and the receiving laboratory. To investigate sample stability, we collected blood and held it at room temperature for 5, 24, or 48 hr before isolating peripheral blood mononuclear cells (PBMCs) and reprogramming into IPSCs. Additionally, PBMC samples at 5- and 48-hr time points were frozen in liquid nitrogen for 4 months and reprogrammed into IPSCs. hIPSC lines derived from all time points were pluripotent, displayed no marked difference in chromosomal aberration rates, and differentiated into three germ layers. Reprogramming efficiency at 24- and 48-hr time points was 3- and 10-fold lower, respectively, than at 5 hr; the freeze-thaw process of PBMCs resulted in no obvious change in reprogramming efficiency. PMID:26388286

  5. Pattern recognition of monocyte chemoattractant protein-1 (MCP-1) in whole blood samples using new platforms based on nanostructured materials.

    PubMed

    Stefan-van Staden, Raluca-Ioana; Gugoasa, Livia Alexandra; Biris, Alexandru Radu

    2015-09-28

    Four stochastic microsensors based on nanostructured materials (graphene, maltodextrin (MD), and diamond) integrated in miniaturized platforms were proposed. Monocyte chemoattractant protein-1 (MCP-1) is a pro-inflammatory cytokine whose main function is to regulate cell trafficking. It is correlated with the incidence of cardiovascular diseases and obesity, and was used as the model analyte in this study. The screening of whole blood samples for MCP-1 can be done for concentrations ranging from 10(-12) to 10(-8) g mL(-1). The method was used for both qualitative and quantitative assessments of MCP-1 in whole blood samples. The lowest quantification limits for the assay of MCP-1 (1 pg mL(-1)) were reached when the microsensors based on protoporphyrin IX/Graphene-Au-3 and on MD/Graphene were employed in the platform design. PMID:26183340

  6. Pattern recognition of monocyte chemoattractant protein-1 (MCP-1) in whole blood samples using new platforms based on nanostructured materials

    NASA Astrophysics Data System (ADS)

    Stefan-van Staden, Raluca-Ioana; Gugoasa, Livia Alexandra; Biris, Alexandru Radu

    2015-09-01

    Four stochastic microsensors based on nanostructured materials (graphene, maltodextrin (MD), and diamond) integrated in miniaturized platforms were proposed. Monocyte chemoattractant protein-1 (MCP-1) is a pro-inflammatory cytokine whose main function is to regulate cell trafficking. It is correlated with the incidence of cardiovascular diseases and obesity, and was used as the model analyte in this study. The screening of whole blood samples for MCP-1 can be done for concentrations ranging from 10-12 to 10-8 g mL-1. The method was used for both qualitative and quantitative assessments of MCP-1 in whole blood samples. The lowest quantification limits for the assay of MCP-1 (1 pg mL-1) were reached when the microsensors based on protoporphyrin IX/Graphene-Au-3 and on MD/Graphene were employed in the platform design.

  7. Measuring the level of agreement in hematologic and biochemical values between blood sampling sites in leatherback sea turtles (Dermochelys coriacea).

    PubMed

    Stewart, Kimberly; Mitchell, Mark A; Norton, Terry; Krecek, Rosina C

    2012-12-01

    Conservation programs to protect endangered sea turtles are being instituted worldwide. A common practice in these programs is to collect blood to evaluate the health of the turtles. Several different venipuncture sites are used to collect blood from sea turtles for hematologic and biochemistry tests, depending on the species. To date, it is unknown what affect venipuncture site may have on sample results. The purpose of this study was to measure the level of agreement between hematologic and biochemistry values collected from the dorsal cervical sinus and the interdigital vein of leatherback (Dermochelys coriacea) sea turtles. Paired heparinized blood samples were obtained from the dorsal cervical sinus and the interdigital vein of 12 adult female nesting leatherback sea turtles on Keys Beach, St. Kitts, West Indies. Even though the sample population was small, the data for each chemistry were normally distributed, except for creatine kinase (CK). There was no significant difference when comparing biochemistry or hematologic values by venipuncture site, except for CK (P = 0.02). The level of agreement between sampling sites was considered good for albumin, calcium, globulin, glucose, packed cell volume, phosphorus, potassium, sodium, total protein, total solids, uric acid, white blood cell count, and all of the individual white cell types, while the level of agreement for aspartate aminotransferase and CK were considered poor. This information, coupled with the fact that the interdigital vein affords a less-invasive procedure, demonstrates that the interdigital vein is an appropriate location to use when establishing a hematologic and biochemical profile for leatherback sea turtles.

  8. Measuring the level of agreement in hematologic and biochemical values between blood sampling sites in leatherback sea turtles (Dermochelys coriacea).

    PubMed

    Stewart, Kimberly; Mitchell, Mark A; Norton, Terry; Krecek, Rosina C

    2012-12-01

    Conservation programs to protect endangered sea turtles are being instituted worldwide. A common practice in these programs is to collect blood to evaluate the health of the turtles. Several different venipuncture sites are used to collect blood from sea turtles for hematologic and biochemistry tests, depending on the species. To date, it is unknown what affect venipuncture site may have on sample results. The purpose of this study was to measure the level of agreement between hematologic and biochemistry values collected from the dorsal cervical sinus and the interdigital vein of leatherback (Dermochelys coriacea) sea turtles. Paired heparinized blood samples were obtained from the dorsal cervical sinus and the interdigital vein of 12 adult female nesting leatherback sea turtles on Keys Beach, St. Kitts, West Indies. Even though the sample population was small, the data for each chemistry were normally distributed, except for creatine kinase (CK). There was no significant difference when comparing biochemistry or hematologic values by venipuncture site, except for CK (P = 0.02). The level of agreement between sampling sites was considered good for albumin, calcium, globulin, glucose, packed cell volume, phosphorus, potassium, sodium, total protein, total solids, uric acid, white blood cell count, and all of the individual white cell types, while the level of agreement for aspartate aminotransferase and CK were considered poor. This information, coupled with the fact that the interdigital vein affords a less-invasive procedure, demonstrates that the interdigital vein is an appropriate location to use when establishing a hematologic and biochemical profile for leatherback sea turtles. PMID:23272336

  9. Large-Scale Prospective T Cell Function Assays in Shipped, Unfrozen Blood Samples: Experiences from the Multicenter TRIGR Trial

    PubMed Central

    Cheung, Roy K.; Becker, Dorothy J.; Girgis, Rose; Palmer, Jerry P.; Cuthbertson, David; Krischer, Jeffrey P.

    2014-01-01

    Broad consensus assigns T lymphocytes fundamental roles in inflammatory, infectious, and autoimmune diseases. However, clinical investigations have lacked fully characterized and validated procedures, equivalent to those of widely practiced biochemical tests with established clinical roles, for measuring core T cell functions. The Trial to Reduce Insulin-dependent diabetes mellitus in the Genetically at Risk (TRIGR) type 1 diabetes prevention trial used consecutive measurements of T cell proliferative responses in prospectively collected fresh heparinized blood samples shipped by courier within North America. In this article, we report on the quality control implications of this simple and pragmatic shipping practice and the interpretation of positive- and negative-control analytes in our assay. We used polyclonal and postvaccination responses in 4,919 samples to analyze the development of T cell immunocompetence. We have found that the vast majority of the samples were viable up to 3 days from the blood draw, yet meaningful responses were found in a proportion of those with longer travel times. Furthermore, the shipping time of uncooled samples significantly decreased both the viabilities of the samples and the unstimulated cell counts in the viable samples. Also, subject age was significantly associated with the number of unstimulated cells and T cell proliferation to positive activators. Finally, we observed a pattern of statistically significant increases in T cell responses to tetanus toxin around the timing of infant vaccinations. This assay platform and shipping protocol satisfy the criteria for robust and reproducible long-term measurements of human T cell function, comparable to those of established blood biochemical tests. We present a stable technology for prospective disease-relevant T cell analysis in immunological diseases, vaccination medicine, and measurement of herd immunity. PMID:24334687

  10. Serum biochemical characteristics of Beluga, Huso huso (L.), in response to blood sampling after clove powder solution exposure.

    PubMed

    Hoseini, Seyyed Morteza; Hosseini, Seyed Abbas; Nodeh, Ali Jafar

    2011-09-01

    In order to investigate the effect of anesthesia on serum parameters, Beluga, Huso huso (L.) were blood-sampled immediately without anesthesia (control) or subjected to following anesthesia procedure: 40, 120, and 240 s exposure to 3,000, 700, and 500 mg l⁻¹ clove solution, respectively. Blood samples were collected after these periods, when fish were immobile and reached stage 4 anesthesia. Results showed that cortisol and glucose levels were significantly high in 700 and 500 but not 3,000 mg l⁻¹ group compared to control. Serum lactate levels were significantly high in 500 mg l⁻¹ group compared to control group. Lactate levels were not significantly differed between control, 3,000, and 700 mg l⁻¹ groups. There were no significant differences in serum levels of cholesterol, total protein, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, Na⁺, Cl⁻, K⁺, and Ca²⁺. Results suggest that rapid anesthesia with higher dose is better than slow anesthesia with lower dose for blood sampling in Beluga.

  11. Lack of leptin activity in blood samples of Adélie penguin and bar-tailed godwit.

    PubMed

    Yosefi, Sara; Hen, Gideon; Rosenblum, Charles I; Cerasale, David J; Beaulieu, Michaël; Criscuolo, Francois; Friedman-Einat, Miriam

    2010-10-01

    Unsuccessful attempts to identify the leptin gene in birds are well documented, despite the characterization of its receptor (LEPR). Since leptin and LEPR have poor sequence conservation among vertebrates, we speculated that a functional assay should represent the best way to detect leptin in birds. Using a leptin bioassay that is based on activation of the chicken LEPR in cultured cells, blood samples from wild birds with extreme seasonal variation in voluntary food intake and fat deposition (Adélie penguins and bar-tailed godwits) were tested for leptin activity. In these experiments, blood samples collected during the pre-incubation and the chick-rearing periods of Adélie penguins, and during the migratory flight and refueling stages of bar-tailed godwits, were found to contain no detectable leptin activity, while the sensitivity of the assay to activation by human blood samples from donor subjects representing a variety of body mass indices and fat contents was clearly demonstrated. These results suggest that in birds, an alternative control mechanism to that of mammals operates in the communication between the body fat tissues and the central control on energy homeostasis.

  12. Evaluation of dried blood spots as sample matrix for gas chromatography/mass spectrometry based metabolomic profiling.

    PubMed

    Kong, Sing Teang; Lin, Hai-Shu; Ching, Jianhong; Ho, Paul C

    2011-06-01

    We propose using dried blood spots (DBS) as sample matrix for gas chromatography/mass spectrometry (GC/MS) based metabolomic profiling for the benefits of higher sample stability, more convenient sample acquisition with DBS, higher analyte separation power, and more readily biomarker identification with GC/MS. To establish this proposition, the metabolomic profiles generated from DBS were compared with that obtained from the conventional whole blood and plasma matrixes and also with dried plasma spots (DPS) as another covariate control. Our findings indicated that whole blood produced the most number of detectable markers (866), whereas DPS yielded the least number (614). DBS and plasma matrix, on the other hand, produced the most similar numbers of detectable (695 vs 749) and identifiable markers (137 vs 147, matching with Fiehn library). From the analysis of the DBS and plasma metabolomic profiles, it was concluded that when l-lysine 2, iminodiacetic acid 2, dl-threo-beta-hydroxyaspartic acid, citric acid, or adenosine-5-monophosphate 2 are not involved as markers, DBS could be a suitable substitute for plasma for metabolomic profiling.

  13. Preconcentration and determination of lead and cadmium levels in blood samples of adolescent workers consuming smokeless tobacco products in Pakistan.

    PubMed

    Arain, Sadaf Sadia; Kazi, Tasneem Gul; Afridi, Hassan Imran; Brahman, Kapil Dev; Naeemullah; Khan, Sumaira; Panhwar, Abdul Haleem; Kamboh, Muhammad Afzal; Memon, Jamil R

    2015-05-01

    The present study was aimed to evaluate the cadmium (Cd) and lead (Pb) levels in the blood samples of adolescent boys, chewing different smokeless tobacco (SLT) products in Pakistan. For comparative purpose, boys of the same age group (12-15 years), not consumed any SLT products were selected as referents. To determine trace levels of Cd and Pb in blood samples, a preconcentration method, vortex-assisted liquid-liquid microextraction (VLLME) has been developed, prior to analysis by flame atomic absorption spectrometry. The hydrophobic chelates of Cd and Pb with ammonium pyrrolidinedithiocarbamate were extracted into the fine droplets of ionic liquid (IL) 1-butyl-3-methylimidazolium hexafluorophosphate, while nonionic surfactant, Triton X-114 was used as a dispersing medium. The main factors affecting the recoveries of Cd and Pb, such as concentration of APDC, centrifugation time, volume of IL and TX-114, were investigated in detail. It was also observed that adolescent boys who consumed different SLT products have 2- to 3-fold higher levels of Cd and Pb in their blood samples as compared to referent boys (p < 0.001). PMID:25930204

  14. Estimation of a partially linear additive model for data from an outcome-dependent sampling design with a continuous outcome.

    PubMed

    Tan, Ziwen; Qin, Guoyou; Zhou, Haibo

    2016-10-01

    Outcome-dependent sampling (ODS) designs have been well recognized as a cost-effective way to enhance study efficiency in both statistical literature and biomedical and epidemiologic studies. A partially linear additive model (PLAM) is widely applied in real problems because it allows for a flexible specification of the dependence of the response on some covariates in a linear fashion and other covariates in a nonlinear non-parametric fashion. Motivated by an epidemiological study investigating the effect of prenatal polychlorinated biphenyls exposure on children's intelligence quotient (IQ) at age 7 years, we propose a PLAM in this article to investigate a more flexible non-parametric inference on the relationships among the response and covariates under the ODS scheme. We propose the estimation method and establish the asymptotic properties of the proposed estimator. Simulation studies are conducted to show the improved efficiency of the proposed ODS estimator for PLAM compared with that from a traditional simple random sampling design with the same sample size. The data of the above-mentioned study is analyzed to illustrate the proposed method.

  15. Estimation of a partially linear additive model for data from an outcome-dependent sampling design with a continuous outcome.

    PubMed

    Tan, Ziwen; Qin, Guoyou; Zhou, Haibo

    2016-10-01

    Outcome-dependent sampling (ODS) designs have been well recognized as a cost-effective way to enhance study efficiency in both statistical literature and biomedical and epidemiologic studies. A partially linear additive model (PLAM) is widely applied in real problems because it allows for a flexible specification of the dependence of the response on some covariates in a linear fashion and other covariates in a nonlinear non-parametric fashion. Motivated by an epidemiological study investigating the effect of prenatal polychlorinated biphenyls exposure on children's intelligence quotient (IQ) at age 7 years, we propose a PLAM in this article to investigate a more flexible non-parametric inference on the relationships among the response and covariates under the ODS scheme. We propose the estimation method and establish the asymptotic properties of the proposed estimator. Simulation studies are conducted to show the improved efficiency of the proposed ODS estimator for PLAM compared with that from a traditional simple random sampling design with the same sample size. The data of the above-mentioned study is analyzed to illustrate the proposed method. PMID:27006375

  16. Hepatitis B Virus DNA in Blood Samples Positive for Antibodies to Core Antigen and Negative for Surface Antigen

    PubMed Central

    Gutiérrez, C.; León, G.; Loureiro, C. L.; Uzcátegui, N.; Liprandi, F.; Pujol, F. H.

    1999-01-01

    Anti-hepatitis B core antigen (HBcAg)-positive hepatitis B surface antigen (HBsAg)-negative plasma samples from blood donors were tested by nested PCR. DNA positivity was more significantly associated with high levels of anti-HBcAg than with low levels of anti-HBsAg antibodies. Analysis of a dilution of anti-HBcAg antibodies might result in a more rational exclusion of anti-HBcAg-positive HBsAg-negative samples, reducing the number of donations discarded and enabling more countries to incorporate anti-HBcAg testing. PMID:10473534

  17. [Viral infections and pregnancy: contribution of amniotic fluid and blood samples].

    PubMed

    Grangeot-Keros, L; Cointe, D

    2001-12-01

    The main viral infections prenatally detected in fetuses are: cytomegalovirus, parvovirus B19, rubella virus and varicellazoster virus infections. Prenatal diagnosis is based on the direct detection of the virus by culture (CMV), of its antigens or of its genome, essentially by PCR. This direct detection can be done either on fetal blood or on amniotic fluid. Prenatal diagnosis can also be performed by detection of specific IgM in fetal blood (rubella). Non specific markers of viral infection can also help in diagnosis. At the present time, prenatal diagnosis is essentially based on the detection of the viral genome in amniotic fluid. In order to better appreciate the severity of fetal infections, some groups have tried to identify prognostic markers of these infections. The viral load as well as the level of specific IgM could play a role in certain infections (CMV). PMID:11802552

  18. Optimal blood sampling time windows for parameter estimation using a population approach: design of a phase II clinical trial.

    PubMed

    Chenel, Marylore; Ogungbenro, Kayode; Duval, Vincent; Laveille, Christian; Jochemsen, Roeline; Aarons, Leon

    2005-12-01

    The objective of this paper is to determine optimal blood sampling time windows for the estimation of pharmacokinetic (PK) parameters by a population approach within the clinical constraints. A population PK model was developed to describe a reference phase II PK dataset. Using this model and the parameter estimates, D-optimal sampling times were determined by optimising the determinant of the population Fisher information matrix (PFIM) using PFIM_ _M 1.2 and the modified Fedorov exchange algorithm. Optimal sampling time windows were then determined by allowing the D-optimal windows design to result in a specified level of efficiency when compared to the fixed-times D-optimal design. The best results were obtained when K(a) and IIV on K(a) were fixed. Windows were determined using this approach assuming 90% level of efficiency and uniform sample distribution. Four optimal sampling time windows were determined as follow: at trough between 22 h and new drug administration; between 2 and 4 h after dose for all patients; and for 1/3 of the patients only 2 sampling time windows between 4 and 10 h after dose, equal to [4 h-5 h 05] and [9 h 10-10 h]. This work permitted the determination of an optimal design, with suitable sampling time windows which was then evaluated by simulations. The sampling time windows will be used to define the sampling schedule in a prospective phase II study.

  19. Analysis of tumor template from multiple compartments in a blood sample provides complementary access to peripheral tumor biomarkers

    PubMed Central

    Strauss, William M.; Carter, Chris; Simmons, Jill; Klem, Erich; Goodman, Nathan; Vahidi, Behrad; Romero, Juan; Masterman-Smith, Michael; O'Regan, Ruth; Gogineni, Keerthi; Schwartzberg, Lee; Austin, Laura K.; Dempsey, Paul W.; Cristofanilli, Massimo

    2016-01-01

    Targeted cancer therapeutics are promised to have a major impact on cancer treatment and survival. Successful application of these novel treatments requires a molecular definition of a patient's disease typically achieved through the use of tissue biopsies. Alternatively, allowing longitudinal monitoring, biomarkers derived from blood, isolated either from circulating tumor cell derived DNA (ctcDNA) or circulating cell-free tumor DNA (ccfDNA) may be evaluated. In order to use blood derived templates for mutational profiling in clinical decisions, it is essential to understand the different template qualities and how they compare to biopsy derived template DNA as both blood-based templates are rare and distinct from the gold-standard. Using a next generation re-sequencing strategy, concordance of the mutational spectrum was evaluated in 32 patient-matched ctcDNA and ccfDNA templates with comparison to tissue biopsy derived DNA template. Different CTC antibody capture systems for DNA isolation from patient blood samples were also compared. Significant overlap was observed between ctcDNA, ccfDNA and tissue derived templates. Interestingly, if the results of ctcDNA and ccfDNA template sequencing were combined, productive samples showed similar detection frequency (56% vs 58%), were temporally flexible, and were complementary both to each other and the gold standard. These observations justify the use of a multiple template approach to the liquid biopsy, where germline, ctcDNA, and ccfDNA templates are employed for clinical diagnostic purposes and open a path to comprehensive blood derived biomarker access. PMID:27049831

  20. Blood lead levels in a representative sample of the Spanish adult population: the BIOAMBIENT.ES project.

    PubMed

    Cañas, Ana I; Cervantes-Amat, Marta; Esteban, Marta; Ruiz-Moraga, Montserrat; Pérez-Gómez, Beatriz; Mayor, Juan; Castaño, Argelia

    2014-01-01

    This paper provides the first baseline information on a national scale regarding lead exposure in the Spanish adult population. Blood lead levels were measured in a representative sample of the Spanish working population (1880 subjects aged 18-65 years) in order to help establish reference levels, follow temporal trends, identify high-exposure groups and to enable comparisons with other countries. All participants completed an epidemiological questionnaire including gender, age, occupational sector, geographic area, and dietary and lifestyle information. We found that the geometric mean of blood lead levels in the study population was 24.0μg/L (95% CI: 23.0-25.1μg/L), with women having significantly lower levels than men, 19.5μg/L (18.5-20.5μg/L) compared to 28.3μg/L (26.7-30.0μg/L), respectively. Mean blood lead levels were higher in elder groups in both genders. Women of a childbearing age had blood levels of 18.0μg/L (GM). Reference values (95%) for lead in blood in the studied population was 56.80μg/L, with -64.00μg/L, 44.80μg/L and 36.00μg/L for man, women and women of childbearing age, respectively. Workers from the service sector had lower blood lead levels than those from the construction, agricultural and industry sectors. Small, although significant, geographical differences had been found. In an European comparison, the Spanish population studied herein had lead levels similar to populations in countries such as France and Belgium, and slightly lower levels than Italian, Czech, German or UK populations.

  1. Isolation of endothelial colony-forming cells from blood samples collected from the jugular and cephalic veins of healthy adult horses.

    PubMed

    Sharpe, Ashley N; Seeto, Wen J; Winter, Randolph L; Zhong, Qiao; Lipke, Elizabeth A; Wooldridge, Anne A

    2016-10-01

    OBJECTIVE To evaluate optimal isolation of endothelial colony-forming cells (ECFCs) from peripheral blood of horses. SAMPLE Jugular and cephalic venous blood samples from 17 adult horses. PROCEDURES Each blood sample was divided; isolation was performed with whole blood adherence (WBA) and density gradient centrifugation (DGC). Isolated cells were characterized by uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-Ac-LDL), vascular tubule formation, and expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor-2, and von Willebrand factor) and hematopoietic (CD14) cell markers by use of indirect immunofluorescence assay (IFA) and flow cytometry. RESULTS Colonies with cobblestone morphology were isolated from 15 of 17 horses. Blood collected from the cephalic vein yielded colonies significantly more often (14/17 horses) than did blood collected from the jugular vein (8/17 horses). Of 14 cephalic blood samples with colonies, 13 were obtained with DGC and 8 with WBA. Of 8 jugular blood samples with colonies, 8 were obtained with DGC and 4 with WBA. Colony frequency (colonies per milliliter of blood) was significantly higher for cephalic blood samples and samples isolated with DGC. Cells formed vascular tubules, had uptake of DiI-Ac-LDL, and expressed endothelial markers by use of IFA and flow cytometry, which confirmed their identity as ECFCs. CONCLUSIONS AND CLINICAL RELEVANCE Maximum yield of ECFCs was obtained for blood samples collected from both the jugular and cephalic veins and use of DGC to isolate cells. Consistent yield of ECFCs from peripheral blood of horses will enable studies to evaluate diagnostic and therapeutic uses.

  2. Isolation of endothelial colony-forming cells from blood samples collected from the jugular and cephalic veins of healthy adult horses.

    PubMed

    Sharpe, Ashley N; Seeto, Wen J; Winter, Randolph L; Zhong, Qiao; Lipke, Elizabeth A; Wooldridge, Anne A

    2016-10-01

    OBJECTIVE To evaluate optimal isolation of endothelial colony-forming cells (ECFCs) from peripheral blood of horses. SAMPLE Jugular and cephalic venous blood samples from 17 adult horses. PROCEDURES Each blood sample was divided; isolation was performed with whole blood adherence (WBA) and density gradient centrifugation (DGC). Isolated cells were characterized by uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-Ac-LDL), vascular tubule formation, and expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor-2, and von Willebrand factor) and hematopoietic (CD14) cell markers by use of indirect immunofluorescence assay (IFA) and flow cytometry. RESULTS Colonies with cobblestone morphology were isolated from 15 of 17 horses. Blood collected from the cephalic vein yielded colonies significantly more often (14/17 horses) than did blood collected from the jugular vein (8/17 horses). Of 14 cephalic blood samples with colonies, 13 were obtained with DGC and 8 with WBA. Of 8 jugular blood samples with colonies, 8 were obtained with DGC and 4 with WBA. Colony frequency (colonies per milliliter of blood) was significantly higher for cephalic blood samples and samples isolated with DGC. Cells formed vascular tubules, had uptake of DiI-Ac-LDL, and expressed endothelial markers by use of IFA and flow cytometry, which confirmed their identity as ECFCs. CONCLUSIONS AND CLINICAL RELEVANCE Maximum yield of ECFCs was obtained for blood samples collected from both the jugular and cephalic veins and use of DGC to isolate cells. Consistent yield of ECFCs from peripheral blood of horses will enable studies to evaluate diagnostic and therapeutic uses. PMID:27668588

  3. Persistent organic pollutants in blood samples of Southern Giant Petrels (Macronectes giganteus) from the South Shetland Islands, Antarctica.

    PubMed

    Colabuono, Fernanda I; Vander Pol, Stacy S; Huncik, Kevin M; Taniguchi, Satie; Petry, Maria V; Kucklick, John R; Montone, Rosalinda C

    2016-09-01

    Seabirds play an important role as top consumers in the food web and can be used as biomonitors of exposure to pollutants. Contamination studies involving non-destructive sampling methods are of considerable importance, allowing better evaluation of the levels of pollutants and their toxic effects. In the present study, organohalogen contaminants were analyzed in 113 blood samples from Southern Giant Petrel (Macronectes giganteus) adults and chicks collected in the austral summer of 2011/2012 and 2012/2013 from colonies on Elephant and Livingston Islands, South Shetland, Antarctica. Polychlorinated biphenyls (PCBs), hexachlorobenzene (HCB), pentachlorobenzene (PeCB), mirex, dichlorodiphenyltrichloroetane and derivatives (DDTs) and chlordanes were detected in all birds, whereas polybrominated diphenyl ethers (PBDEs) were not detected in any blood samples. No significant differences were found in organochlorine levels between sampling events. Adults exhibited significantly higher levels than chicks, except for PeCB. PCBs, HCB, mirex and DDTs were statistically similar in males and females from Elephant Island. Females on Livingston Island exhibited higher HCB values than males, but no sex differences were found regarding other organochlorines. The similarity in organochlorine levels between sexes in birds with very marked sexual segregation in feeding habits during the breeding season may indicate that significant amounts of contaminants are acquired during migration to lower latitudes, when the diets of males and females are similar. Birds sampled on Livingston Island exhibited significantly lower levels of PCBs, HCB, DDTs, mirex and chlordanes in comparison to those on Elephant Island, which could be the result of distinct foraging patterns between the two colonies. Organochlorine levels were similar between years in birds captured in two consecutive breeding seasons. Blood samples from Southern Giant Petrels adults and chicks proved to be useful for the comparison

  4. Frequency of enterovirus detection in blood samples of neonates admitted to hospital with sepsis-like illness in Kuwait.

    PubMed

    Ahmad, Suhail; Dalwai, Ajmal; Al-Nakib, Widad

    2013-07-01

    This study investigated the role of enteroviruses in sepsis-like illness among neonates in Kuwait. Serum samples from 139 consecutive neonates presenting with sepsis-like illness during a three and a half-year-period whose blood cultures were negative for bacterial pathogens were tested. Enterovirus RNA was detected by single-step reverse-transcription PCR (RT-PCR). Specific genotypes were identified by direct DNA sequencing of enteroviral genome. Serotype-specific antibodies in serum samples from some selected patients were detected by virus neutralization test using coxsackievirus B types (CBVs). All 139 neonates presented with sepsis-like illness and blood samples were uniformly negative for aerobic/anaerobic bacterial cultures. Fifty-six (40%) neonates had further complications of sepsis including carditis (n = 34) and multi-organ involvement (n = 22). Enterovirus RNA was detected by RT-PCR in 34 of 139 (24%) serum samples which is among the highest frequency reported so far in non-epidemic settings. Genotyping identified CBVs as most common enteroviruses, causing 19 of 34 (56%) enteroviral sepsis episodes in neonates. Of 34 carditis cases, 18 were positive for CBVs by serotyping including all 10 enterovirus RNA-positive samples. Only one fatality was observed due to liver failure in a neonate with hepatitis. Our data showed that enteroviruses are responsible for 24% of neonatal sepsis cases due to non-bacterial causes in Kuwait. The data indicate that enteroviruses should be considered in the differential diagnosis of sepsis-like illness among neonates, particularly those with negative blood cultures for bacterial pathogens.

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

    PubMed Central

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

    2003-01-01

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

  6. Immunoelectrophoresis - blood

    MedlinePlus

    IEP - serum; Immunoglobulin electrophoresis - blood; Gamma globulin electrophoresis; Serum immunoglobulin electrophoresis ... A blood sample is needed. For information on how this is done, see: Venipuncture

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

    PubMed

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

    2011-04-01

    We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO liquid handler mounted with the Te-MagS magnetic separation device (Tecan, Männedorf, Switzerland). The protocols were validated for accredited forensic genetic work according to ISO 17025 using the Qiagen MagAttract DNA Mini M48 kit (Qiagen GmbH, Hilden, Germany) from fresh whole blood and blood from deceased individuals. The workflow was simplified by returning the DNA extracts to the original tubes minimizing the risk of misplacing samples. The tubes that originally contained the samples were washed with MilliQ water before the return of the DNA extracts. The PCR was setup in 96-well microtiter plates. The methods were validated for the kits: AmpFℓSTR Identifiler, SGM Plus and Yfiler (Applied Biosystems, Foster City, CA), GenePrint FFFL and PowerPlex Y (Promega, Madison, WI). The automated protocols allowed for extraction and addition of PCR master mix of 96 samples within 3.5h. In conclusion, we demonstrated that (1) DNA extraction with magnetic beads and (2) PCR setup for accredited, forensic genetic short tandem repeat typing can be implemented on a simple automated liquid handler leading to the reduction of manual work, and increased quality and throughput. PMID:21609694

  8. Concentrations of polybrominated diphenyl ethers, polychlorinated dibenzo-p-dioxins and dibenzofurans, and polychlorinated biphenyls in human blood samples from Korea.

    PubMed

    Kim, Byung-Hoon; Ikonomou, Michael G; Lee, Se-Jin; Kim, Hee-Sun; Chang, Yoon-Seok

    2005-01-01

    Analysis of blood samples is an effective way of evaluating contamination by persistent pollutants such as polybrominated diphenyl ethers (PBDEs), polychlorinated dibenzo-p-dioxin/furans (PCDD/Fs), and polychlorinated biphenyls (PCBs) in human population. Concentrations of PBDEs, PCDD/Fs and PCBs were measured in the blood of laborers (n = 13) working full time in two different municipal waste incinerator (MWI) plants and residents from the general population (n = 22) living in areas near MWIs in Korea. The concentrations of PBDEs were found to be slightly higher in the blood of incineration workers (8.61-46.05 ng/g lipid; mean, 19.33 ng/g lipid; median, 15.94 ng/g lipid) in comparison to that of residents from the general population (7.24-28.89 ng/g lipid; mean, 15.06 ng/g lipid; median, 14.34 ng/g lipid). The total average PCDD/Fs and PCB TEQ concentration was 20.11 pg/g lipid, averaged over incineration workers (17.73 pg/g lipid) and the general population (21.52 pg/g lipid). In addition, the average total crude concentration of PCDD/Fs was 7.40 ng/g lipids, which was 4.1 times greater than for PBDEs. Congener specific analysis confirmed that BDE 47 was a predictive indicator for total PBDE concentration (correlation coefficient r = 0.912), and that PCB 153 was a predictive indicator for total PCB concentration (r = 0.967). The PBDE levels in human blood in Korea are much higher than those reported in other countries. The presence of the BDE 183 congener was characteristic in the blood of workers from an electronic dismantling facility in MWIs.

  9. Towards a phylogenetic generic classification of Thelypteridaceae: Additional sampling suggests alterations of neotropical taxa and further study of paleotropical genera.

    PubMed

    Almeida, Thaís Elias; Hennequin, Sabine; Schneider, Harald; Smith, Alan R; Batista, João Aguiar Nogueira; Ramalho, Aline Joseph; Proite, Karina; Salino, Alexandre

    2016-01-01

    Thelypteridaceae is one of the largest fern families, having about 950 species and a cosmopolitan distribution but with most species occurring in tropical and subtropical regions. Its generic classification remains controversial, with different authors recognizing from one up to 32 genera. Phylogenetic relationships within the family have not been exhaustively studied, but previous studies have confirmed the monophyly of the lineage. Thus far, sampling has been inadequate for establishing a robust hypothesis of infrafamilial relationships within the family. In order to understand phylogenetic relationships within Thelypteridaceae and thus to improve generic reclassification, we expand the molecular sampling, including new samples of Old World taxa and, especially, many additional neotropical representatives. We also explore the monophyly of exclusively or mostly neotropical genera Amauropelta, Goniopteris, Meniscium, and Steiropteris. Our sampling includes 68 taxa and 134 newly generated sequences from two plastid genomic regions (rps4-trnS and trnL-trnF), plus 73 rps4 and 72 trnL-trnF sequences from GenBank. These data resulted in a concatenated matrix of 1980 molecular characters for 149 taxa. The combined data set was analyzed using maximum parsimony and bayesian inference of phylogeny. Our results are consistent with the general topological structure found in previous studies, including two main lineages within the family: phegopteroid and thelypteroid. The thelypteroid lineage comprises two clades; one of these included the segregates Metathelypteris, Coryphopteris, and Amauropelta (including part of Parathelypteris), whereas the other comprises all segregates of Cyclosorus s.l., such as Goniopteris, Meniscium, and Steiropteris (including Thelypteris polypodioides, previously incertae sedis). The three mainly neotropical segregates were found to be monophyletic but nested in a broadly defined Cyclosorus. The fourth mainly neotropical segregate, Amauropelta

  10. Towards a phylogenetic generic classification of Thelypteridaceae: Additional sampling suggests alterations of neotropical taxa and further study of paleotropical genera.

    PubMed

    Almeida, Thaís Elias; Hennequin, Sabine; Schneider, Harald; Smith, Alan R; Batista, João Aguiar Nogueira; Ramalho, Aline Joseph; Proite, Karina; Salino, Alexandre

    2016-01-01

    Thelypteridaceae is one of the largest fern families, having about 950 species and a cosmopolitan distribution but with most species occurring in tropical and subtropical regions. Its generic classification remains controversial, with different authors recognizing from one up to 32 genera. Phylogenetic relationships within the family have not been exhaustively studied, but previous studies have confirmed the monophyly of the lineage. Thus far, sampling has been inadequate for establishing a robust hypothesis of infrafamilial relationships within the family. In order to understand phylogenetic relationships within Thelypteridaceae and thus to improve generic reclassification, we expand the molecular sampling, including new samples of Old World taxa and, especially, many additional neotropical representatives. We also explore the monophyly of exclusively or mostly neotropical genera Amauropelta, Goniopteris, Meniscium, and Steiropteris. Our sampling includes 68 taxa and 134 newly generated sequences from two plastid genomic regions (rps4-trnS and trnL-trnF), plus 73 rps4 and 72 trnL-trnF sequences from GenBank. These data resulted in a concatenated matrix of 1980 molecular characters for 149 taxa. The combined data set was analyzed using maximum parsimony and bayesian inference of phylogeny. Our results are consistent with the general topological structure found in previous studies, including two main lineages within the family: phegopteroid and thelypteroid. The thelypteroid lineage comprises two clades; one of these included the segregates Metathelypteris, Coryphopteris, and Amauropelta (including part of Parathelypteris), whereas the other comprises all segregates of Cyclosorus s.l., such as Goniopteris, Meniscium, and Steiropteris (including Thelypteris polypodioides, previously incertae sedis). The three mainly neotropical segregates were found to be monophyletic but nested in a broadly defined Cyclosorus. The fourth mainly neotropical segregate, Amauropelta

  11. Corrections of arterial input function for dynamic H215O PET to assess perfusion of pelvic tumours: arterial blood sampling versus image extraction

    NASA Astrophysics Data System (ADS)

    Lüdemann, L.; Sreenivasa, G.; Michel, R.; Rosner, C.; Plotkin, M.; Felix, R.; Wust, P.; Amthauer, H.

    2006-06-01

    Assessment of perfusion with 15O-labelled water (H215O) requires measurement of the arterial input function (AIF). The arterial time activity curve (TAC) measured using the peripheral sampling scheme requires corrections for delay and dispersion. In this study, parametrizations with and without arterial spillover correction for fitting of the tissue curve are evaluated. Additionally, a completely noninvasive method for generation of the AIF from a dynamic positron emission tomography (PET) acquisition is applied to assess perfusion of pelvic tumours. This method uses a volume of interest (VOI) to extract the TAC from the femoral artery. The VOI TAC is corrected for spillover using a separate tissue TAC and for recovery by determining the recovery coefficient on a coregistered CT data set. The techniques were applied in five patients with pelvic tumours who underwent a total of 11 examinations. Delay and dispersion correction of the blood TAC without arterial spillover correction yielded in seven examinations solutions inconsistent with physiology. Correction of arterial spillover increased the fitting accuracy and yielded consistent results in all patients. Generation of an AIF from PET image data was investigated as an alternative to arterial blood sampling and was shown to have an intrinsic potential to determine the AIF noninvasively and reproducibly. The AIF extracted from a VOI in a dynamic PET scan was similar in shape to the blood AIF but yielded significantly higher tissue perfusion values (mean of 104.0 ± 52.0%) and lower partition coefficients (-31.6 ± 24.2%). The perfusion values and partition coefficients determined with the VOI technique have to be corrected in order to compare the results with those of studies using a blood AIF.

  12. A sup 125 I-radioimmunoassay for measuring androstenedione in serum and in blood-spot samples from neonates

    SciTech Connect

    Thomson, S.; Wallace, A.M.; Cook, B. )

    1989-08-01

    We developed a radioimmunoassay with a gamma-emitting radioligand to measure androstenedione in human serum and in dried blood-spot samples from newborns. Antisera were raised in rabbits against androstenedione linked to bovine serum albumin at positions 3, 6, or 11 on the steroid nucleus. Radioligands were prepared by linking ({sup 125}I)iodohistamine at positions 3, 6, or 11. Linkages were through either carboxymethyloxime or hemisuccinate bridges. All label and antibody combinations were examined, and the most sensitive and specific combination (antiserum raised against androstenedione-3-carboxymethyloxime-bovine serum albumin with an androstenedione-carboxymethyloxime-({sup 125}I)iodohistamine label) was selected for full evaluation. We report the performance of these selected reagents in an immunoassay for androstenedione in both serum and dried blood-spot samples from neonates. We measured concentrations of androstenedione in serum under normal and pathological conditions such as congenital adrenal hyperplasia and polycystic ovarian disease. Diurnal variation in normal men was observed. Androstenedione was measured in blood spots from neonates born at term or prematurely, with respiratory distress syndrome, or with congenital adrenal hyperplasia.

  13. Growth and parameters of microflora in intestinal and faecal samples of piglets due to application of a phytogenic feed additive.

    PubMed

    Muhl, A; Liebert, F

    2007-10-01

    A commercial phytogenic feed additive (PFA), containing the fructopolysaccharide inulin, an essential oil mix (carvacrol, thymol), chestnut meal (tannins) and cellulose powder as carrier substance, was examined for effects on growth and faecal and intestinal microflora of piglets. Two experiments (35 days) were conducted, each with 40 male castrated weaned piglets. In experiment 1, graded levels of the PFA were supplied (A1: control; B1: 0.05% PFA; C1: 0.1% PFA; D1: 0.15% PFA) in diets based on wheat, barley, soybean meal and fish meal with lysine as the limiting amino acid. In experiment 2, a similar diet with 0.1% of the PFA (A2: control; B2: 0.1% PFA; C2: +0.35% lysine; D2: 0.1% PFA + 0.35% lysine) and lysine supplementation was utilized. During experiment 1, no significant effect of the PFA on growth, feed intake and feed conversion rate was observed (p > 0.05). Lysine supplementation in experiment 2 improved growth performance significantly, but no significant effect of the PFA was detected. Microbial counts in faeces (aerobes, Gram negatives, anaerobes and lactobacilli) during the first and fifth week did not indicate any significant PFA effect (p > 0.05). In addition, microflora in intestinal samples was not significantly modified by supplementing the PFA (p > 0.05). Lysine supplementation indicated lysine as limiting amino acid in the basal diet, but did not influence the microbial counts in faeces and small intestine respectively.

  14. Capillary sample

    MedlinePlus

    ... using capillary blood sampling. Disadvantages to capillary blood sampling include: Only a limited amount of blood can be drawn using this method. The procedure has some risks (see below). Capillary ...

  15. A rapid paper-based test for quantifying sickle hemoglobin in blood samples from patients with sickle cell disease.

    PubMed

    Piety, Nathaniel Z; Yang, Xiaoxi; Lezzar, Dalia; George, Alex; Shevkoplyas, Sergey S

    2015-06-01

    Quantification of sickle hemoglobin (HbS) in patients with sickle cell disease (SCD) undergoing hydroxyurea or chronic transfusion therapy is essential to monitoring the effectiveness of these therapies. The clinical monitoring of %HbS using conventional laboratory methods is limited by high per-test costs and long turnaround times usually associated with these methods. Here we demonstrate a simple, rapid, inexpensive paper-based assay capable of quantifying %HbS in blood samples from patients with SCD. A 20 μL droplet of whole blood and hemoglobin solubility buffer was deposited on chromatography paper. The relative color intensities of regions of the resulting blood stain, determined by automated image analysis, are used to estimate %HbS. We compared the paper-based assay with hemoglobin electrophoresis (comparison method) using blood samples from 88 subjects. The test shows high correlation (R(2)  = 0.86) and strong agreement (standard deviation of difference = 7%HbS) with conventional Hb electrophoresis measurement of %HbS, and closely approximates clinically predicted change in %HbS with transfusion therapy (mean difference 2.6%HbS, n = 5). The paper-based assay can be completed in less than 35 min and has a per-test cost less than $0.25. The assay is accurate across a wide range of HbS levels (10-97%) and hemoglobin concentrations (5.6-12.9 g/dL) and is unaffected by high levels of HbF (up to 80.6%). This study demonstrates the feasibility of the paper-based %HbS assay. The paper-based test could improve clinical care for SCD, particularly in resource-limited settings, by enabling more rapid and less expensive %HbS monitoring.

  16. Dairy cows with metritis: Coxiella burnetii test results in uterine, blood and bulk milk samples.

    PubMed

    Muskens, J; van Maanen, C; Mars, M H

    2011-01-10

    In cattle, Coxiella burnetii infections are generally asymptomatic but can also be associated with reproductive disorders. Metritis is considered as one of the symptoms of C. burnetii infections, but reliable information is lacking. Therefore, information on the presence of C. burnetii in the uterine content of cows with metritis is important to increase our knowledge on this pathogen. In this study, the uterine content of 45 dairy cows with metritis belonging to 12 herds was tested for C. burnetii with a real-time PCR assay. Only one uterine sample tested PCR (highly) positive, all other samples were PCR negative. The PCR positive cow tested also positive for antibodies. Three other cows from other herds tested antibody positive. The bulk milk (BM) samples of these 12 herds were tested by real-time PCR assay and antibody-ELISA. Six BM samples (50%) were positive in PCR and 10 (83%) were positive in ELISA. Culturing the uterus samples by bacteriology, the most frequently cultured bacteria were arcanobacterium (n=24), E. coli (n=16), other streptococci (n=10), Streptococcus uberis (n=8) and Streptococcus dysgalactiae (n=5). This study indicates that C. burnetii is not an important cause for metritis in dairy herds, although apparently C. burnetii was or had been present in most of these herds.

  17. Assessment of the levels of polybrominated diphenyl ethers in blood samples from Guadalajara, Jalisco, Mexico.

    PubMed

    Orta-Garcia, Sandra Teresa; León-Moreno, Lilia Carolina; González-Vega, Carolina; Dominguez-Cortinas, Gabriela; Espinosa-Reyes, Guillermo; Pérez-Maldonado, Iván N

    2012-10-01

    The purpose of this study was to measure levels of polybrominated diphenyl ethers (PBDEs) in the blood of children (50 individuals) living in Guadalajara, Jalisco, Mexico. We analyzed six PBDE congeners by gas chromatography-mass spectrometry. Total PBDE levels ranged from not detectable (nd) to 15.2 μg/L on a whole-weight basis and from nd to 6,435 ng/g lipid on a lipid-weight basis. The dominant congener in our study was BDE-153, followed by BDE-154, BDE-99, BDE-100, and BDE-47. Levels of BDE-209 were below the detection limit. Our data indicate that children living in the areas studied in this work are exposed to high levels of PBDEs.

  18. [Immunobiological blood parameters in rabbits after addition to the diet suspensions of chlorella, sodium sulfate, citrate and chromium chloride].

    PubMed

    Lesyk, Ia V; Fedoruk, R S; Dolaĭchuk, O P

    2013-01-01

    We studied the content of glycoproteins and their individual carbohydrate components, the phagocyte activity of neutrophils, phagocyte index, phagocyte number lizotsym and bactericidal activity of the serum concentration of circulating immune complexes and middle mass molecules in the blood of rabbits following administration into the diet chlorella suspension, sodium sulfate, chromium citrate and chromium chloride. The studies were conducted on rabbits weighing 3.7-3.9 kg with altered diet from the first day of life to 118 days old. Rabbits were divided into five groups: the control one and four experimental groups. We found that in the blood of rabbits of experimental groups recieved sodium sulphate, chromium chloride and chromium citrate, the content of glycoprotein's and their carbohydrate components was significantly higher during the 118 days of the study compared with the control group. Feeding rabbits with mineral supplements likely reflected the differences compared with the control parameters of nonspecific resistance in the blood for the study period, which was more pronounced in the first two months of life.

  19. Kinetic quantitation of cerebral PET-FDG studies without concurrent blood sampling: statistical recovery of the arterial input function.

    PubMed

    O'Sullivan, F; Kirrane, J; Muzi, M; O'Sullivan, J N; Spence, A M; Mankoff, D A; Krohn, K A

    2010-03-01

    Kinetic quantitation of dynamic positron emission tomography (PET) studies via compartmental modeling usually requires the time-course of the radio-tracer concentration in the arterial blood as an arterial input function (AIF). For human and animal imaging applications, significant practical difficulties are associated with direct arterial sampling and as a result there is substantial interest in alternative methods that require no blood sampling at the time of the study. A fixed population template input function derived from prior experience with directly sampled arterial curves is one possibility. Image-based extraction, including requisite adjustment for spillover and recovery, is another approach. The present work considers a hybrid statistical approach based on a penalty formulation in which the information derived from a priori studies is combined in a Bayesian manner with information contained in the sampled image data in order to obtain an input function estimate. The absolute scaling of the input is achieved by an empirical calibration equation involving the injected dose together with the subject's weight, height and gender. The technique is illustrated in the context of (18)F -Fluorodeoxyglucose (FDG) PET studies in humans. A collection of 79 arterially sampled FDG blood curves are used as a basis for a priori characterization of input function variability, including scaling characteristics. Data from a series of 12 dynamic cerebral FDG PET studies in normal subjects are used to evaluate the performance of the penalty-based AIF estimation technique. The focus of evaluations is on quantitation of FDG kinetics over a set of 10 regional brain structures. As well as the new method, a fixed population template AIF and a direct AIF estimate based on segmentation are also considered. Kinetics analyses resulting from these three AIFs are compared with those resulting from radially sampled AIFs. The proposed penalty-based AIF extraction method is found to

  20. Age-specific discrimination of blood plasma samples of healthy and ovarian cancer prone mice using laser-induced breakdown spectroscopy

    NASA Astrophysics Data System (ADS)

    Melikechi, Noureddine; Markushin, Yuri; Connolly, Denise C.; Lasue, Jeremie; Ewusi-Annan, Ebo; Makrogiannis, Sokratis

    2016-09-01

    Epithelial ovarian cancer (EOC) mortality rates are strongly correlated with the stage at which it is diagnosed. Detection of EOC prior to its dissemination from the site of origin is known to significantly improve the patient outcome. However, there are currently no effective methods for early detection of the most common and lethal subtype of EOC. We sought to determine whether laser-induced breakdown spectroscopy (LIBS) and classification techniques such as linear discriminant analysis (LDA) and random forest (RF) could classify and differentiate blood plasma specimens from transgenic mice with ovarian carcinoma and wild type control mice. Herein we report results using this approach to distinguish blood plasma samples obtained from serially bled (at 8, 12, and 16 weeks) tumor-bearing TgMISIIR-TAg transgenic and wild type cancer-free littermate control mice. We have calculated the age-specific accuracy of classification using 18,000 laser-induced breakdown spectra of the blood plasma samples from tumor-bearing mice and wild type controls. When the analysis is performed in the spectral range 250 nm to 680 nm using LDA, these are 76.7 (± 2.6)%, 71.2 (± 1.3)%, and 73.1 (± 1.4)%, for the 8, 12 and 16 weeks. When the RF classifier is used, we obtain values of 78.5 (± 2.3)%, 76.9 (± 2.1)% and 75.4 (± 2.0)% in the spectral range of 250 nm to 680 nm, and 81.0 (± 1.8)%, 80.4 (± 2.1)% and 79.6 (± 3.5)% in 220 nm to 850 nm. In addition, we report, the positive and negative predictive values of the classification of the two classes of blood plasma samples. The approach used in this study is rapid, requires only 5 μL of blood plasma, and is based on the use of unsupervised and widely accepted multivariate analysis algorithms. These findings suggest that LIBS and multivariate analysis may be a novel approach for detecting EOC.

  1. Isotope ratio analysis of lead in blood and environmental samples by multi-collector inductively coupled plasma mass spectrometry.

    PubMed

    Takagi, Mai; Yoshinaga, Jun; Tanaka, Atsushi; Seyama, Haruhiko

    2011-01-01

    It is widely recognized that lead (Pb) affects children's cognitive function, even at relatively low blood lead levels (<10 µg dL(-1)). The determination of the source of Pb in children is essential for effective risk management. The use of multi-collector ICPMS (MC-ICPMS) for isotope ratio measurements of Pb in environmental and biological samples was examined for this purpose. MC-ICPMS with an instrumental mass fractionation correction by Tl allowed accurate isotope ratio measurements of the Pb isotopic reference material NIST SRM 981. However, the presence of matrix elements (Al, Ca, Fe and Na) at more than 10 mg kg(-1) in the sample solution significantly deteriorated the accuracy. The separation of Pb from the matrix is necessary for accurate measurements of the isotope ratio of Pb in environmental and biological samples. Bromide-complexation, followed by anion exchange was found to be satisfactory in terms of the recovery of Pb (90 to 104%) and the efficiency of matrix separation. The procedure was applied to a preliminary source analysis of Pb in the blood of Japanese children, and a significant contribution of indoor dust was demonstrated.

  2. Determination of gamma-hydroxybutyrate (GHB) and its precursors in blood and urine samples: a salting-out approach.

    PubMed

    Kankaanpää, Aino; Liukkonen, Raija; Ariniemi, Kari

    2007-08-01

    Gamma-hydroxybutyrate (GHB) is an increasingly popular drug of abuse that causes stimulation, euphoria, anxiolysis or hypnosis, depending on the dose used. Low doses of the drug are used recreationally, and also implicated in drug-facilitated sexual assaults. Because of the unusually steep dose-response curves, accidental GHB overdosing, leading to coma, seizures or death can occur. Being a controlled substance, GHB is often substituted with its non-scheduled precursors gamma-butyrolactone (GBL) and 1,4-butanediol (BD), which are rapidly metabolized into GHB in the body. Here we describe an assay for GHB, GBL and BD in blood and/or urine samples. GHB and BD were extracted from diluted 200 microL aliquots of samples with t-butylmethylether (plus internal standard benzyl alcohol) in test tubes preloaded with NaCl. After acidification and centrifugation the solvent phase was transferred to a test tube preloaded with Na(2)SO(4), incubated for 30 min, centrifuged again, and evaporated in vacuum. The residue was mixed with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) in acetonitrile, and injected into a GC-MS. When analyzing GBL, the salting-out step was omitted, and analysis was performed with a GC-FID apparatus. As revealed by the validation data this procedure is suitable for quantitative determination of GHB and its precursors in blood and/or urine samples. PMID:17658710

  3. Determination of gamma-hydroxybutyrate (GHB) and its precursors in blood and urine samples: a salting-out approach.

    PubMed

    Kankaanpää, Aino; Liukkonen, Raija; Ariniemi, Kari

    2007-08-01

    Gamma-hydroxybutyrate (GHB) is an increasingly popular drug of abuse that causes stimulation, euphoria, anxiolysis or hypnosis, depending on the dose used. Low doses of the drug are used recreationally, and also implicated in drug-facilitated sexual assaults. Because of the unusually steep dose-response curves, accidental GHB overdosing, leading to coma, seizures or death can occur. Being a controlled substance, GHB is often substituted with its non-scheduled precursors gamma-butyrolactone (GBL) and 1,4-butanediol (BD), which are rapidly metabolized into GHB in the body. Here we describe an assay for GHB, GBL and BD in blood and/or urine samples. GHB and BD were extracted from diluted 200 microL aliquots of samples with t-butylmethylether (plus internal standard benzyl alcohol) in test tubes preloaded with NaCl. After acidification and centrifugation the solvent phase was transferred to a test tube preloaded with Na(2)SO(4), incubated for 30 min, centrifuged again, and evaporated in vacuum. The residue was mixed with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) in acetonitrile, and injected into a GC-MS. When analyzing GBL, the salting-out step was omitted, and analysis was performed with a GC-FID apparatus. As revealed by the validation data this procedure is suitable for quantitative determination of GHB and its precursors in blood and/or urine samples.

  4. Sequencing CYP2D6 for the detection of poor-metabolizers in post-mortem blood samples with tramadol.

    PubMed

    Fonseca, Suzana; Amorim, António; Costa, Heloísa Afonso; Franco, João; Porto, Maria João; Santos, Jorge Costa; Dias, Mário

    2016-08-01

    Tramadol concentrations and analgesic effect are dependent on the CYP2D6 enzymatic activity. It is well known that some genetic polymorphisms are responsible for the variability in the expression of this enzyme and in the individual drug response. The detection of allelic variants described as non-functional can be useful to explain some circumstances of death in the study of post-mortem cases with tramadol. A Sanger sequencing methodology was developed for the detection of genetic variants that cause absent or reduced CYP2D6 activity, such as *3, *4, *6, *8, *10 and *12 alleles. This methodology, as well as the GC/MS method for the detection and quantification of tramadol and its main metabolites in blood samples was fully validated in accordance with international guidelines. Both methodologies were successfully applied to 100 post-mortem blood samples and the relation between toxicological and genetic results evaluated. Tramadol metabolism, expressed as its metabolites concentration ratio (N-desmethyltramadol/O-desmethyltramadol), has been shown to be correlated with the poor-metabolizer phenotype based on genetic characterization. It was also demonstrated the importance of enzyme inhibitors identification in toxicological analysis. According to our knowledge, this is the first study where a CYP2D6 sequencing methodology is validated and applied to post-mortem samples, in Portugal. The developed methodology allows the data collection of post-mortem cases, which is of primordial importance to enhance the application of these genetic tools to forensic toxicology and pathology.

  5. Estimation of the Time Interval between the Administration of Heroin and the Sampling of Blood in Chronic Inhalers.

    PubMed

    Dubois, Nathalie; Hallet, Claude; Seidel, Laurence; Demaret, Isabelle; Luppens, David; Ansseau, Marc; Rozet, Eric; Albert, Adelin; Hubert, Philippe; Charlier, Corinne

    2015-05-01

    To develop a model for estimating the time delay between last heroin consumption and blood sampling in chronic drug users. Eleven patients, all heroin inhalers undergoing detoxification, were included in the study. Several plasma samples were collected during the detoxification procedure and analyzed for the heroin metabolites 6-acetylmorphine (6AM), morphine (MOR), morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G), according to a UHPLC/MSMS method. The general linear mixed model was applied to time-related concentrations and a pragmatic four-step delay estimation approach was proposed based on the simultaneous presence of metabolites in plasma. Validation of the model was carried out using the jackknife technique on the 11 patients, and on a group of 7 test patients. Quadratic equations were derived for all metabolites except 6AM. The interval delay estimation was 2-4 days when only M3G present in plasma, 1-2 days when M6G and M3G were both present, 0-1 day when MOR, M6G and M3G were present and <2 h for all metabolites present. The 'jackknife' correlation between declared and actual estimated delays was 0.90. The overall precision of the delay estimates was 8-9 h. The delay between last heroin consumption and blood sampling in chronic drug users can be satisfactorily predicted from plasma heroin metabolites.

  6. Development of an Automated and Sensitive Microfluidic Device for Capturing and Characterizing Circulating Tumor Cells (CTCs) from Clinical Blood Samples.

    PubMed

    Gogoi, Priya; Sepehri, Saedeh; Zhou, Yi; Gorin, Michael A; Paolillo, Carmela; Capoluongo, Ettore; Gleason, Kyle; Payne, Austin; Boniface, Brian; Cristofanilli, Massimo; Morgan, Todd M; Fortina, Paolo; Pienta, Kenneth J; Handique, Kalyan; Wang, Yixin

    2016-01-01

    Current analysis of circulating tumor cells (CTCs) is hindered by sub-optimal sensitivity and specificity of devices or assays as well as lack of capability of characterization of CTCs with clinical biomarkers. Here, we validate a novel technology to enrich and characterize CTCs from blood samples of patients with metastatic breast, prostate and colorectal cancers using a microfluidic chip which is processed by using an automated staining and scanning system from sample preparation to image processing. The Celsee system allowed for the detection of CTCs with apparent high sensitivity and specificity (94% sensitivity and 100% specificity). Moreover, the system facilitated rapid capture of CTCs from blood samples and also allowed for downstream characterization of the captured cells by immunohistochemistry, DNA and mRNA fluorescence in-situ hybridization (FISH). In a subset of patients with prostate cancer we compared the technology with a FDA-approved CTC device, CellSearch and found a higher degree of sensitivity with the Celsee instrument. In conclusion, the integrated Celsee system represents a promising CTC technology for enumeration and molecular characterization.

  7. Development of an Automated and Sensitive Microfluidic Device for Capturing and Characterizing Circulating Tumor Cells (CTCs) from Clinical Blood Samples

    PubMed Central

    Gogoi, Priya; Sepehri, Saedeh; Zhou, Yi; Gorin, Michael A.; Paolillo, Carmela; Capoluongo, Ettore; Gleason, Kyle; Payne, Austin; Boniface, Brian; Cristofanilli, Massimo; Morgan, Todd M.; Fortina, Paolo; Pienta, Kenneth J.; Handique, Kalyan; Wang, Yixin

    2016-01-01

    Current analysis of circulating tumor cells (CTCs) is hindered by sub-optimal sensitivity and specificity of devices or assays as well as lack of capability of characterization of CTCs with clinical biomarkers. Here, we validate a novel technology to enrich and characterize CTCs from blood samples of patients with metastatic breast, prostate and colorectal cancers using a microfluidic chip which is processed by using an automated staining and scanning system from sample preparation to image processing. The Celsee system allowed for the detection of CTCs with apparent high sensitivity and specificity (94% sensitivity and 100% specificity). Moreover, the system facilitated rapid capture of CTCs from blood samples and also allowed for downstream characterization of the captured cells by immunohistochemistry, DNA and mRNA fluorescence in-situ hybridization (FISH). In a subset of patients with prostate cancer we compared the technology with a FDA-approved CTC device, CellSearch and found a higher degree of sensitivity with the Celsee instrument. In conclusion, the integrated Celsee system represents a promising CTC technology for enumeration and molecular characterization. PMID:26808060

  8. Printed microwells with highly stable thin-film enzyme coatings for point-of-care multiplex bioassay of blood samples.

    PubMed

    Zhang, Liting; Cao, Xiaodan; Wang, Lu; Zhao, Xueyan; Zhang, Songping; Wang, Ping

    2015-06-21

    A paper-based colorimetric biosensor suitable for point-of-care bioassay of blood samples is developed using highly stable enzyme thin-film coatings confined within inkjet printed polymeric microwells. The microwells are developed through a simple one-step inkjet printing of hydrophobic polystyrene on paper, with walls formed by the polymer that fills the gaps inside the paper body. The microwells can also be patterned to be interlinked with printed microchannels for multiplex bioassays. Thin film enzyme coatings confined within the microwells are then constructed, thereby constituting biosensors that work like traditional microwell plates, yet allow easy colorimetric readouts with naked eyes or portable devices, such as smart phones. The efficiency of the paper-based sensor was demonstrated for colorimetric assays of glucose and lactate, both as individual analytes or mixed, as well as samples with red blood cells. Such sensors showed good sensitivities within the concentration ranges of the analytes in human blood (0.5-10 mM), with a visible sensitivity of <0.5 mM detectable by naked eyes for a sample size as small as 1 μL. More accurate digital readouts were shown to be feasible with computerized scanners or smartphones. The thin-film coating format affords the paper biosensors an extended lifetime, and they could retain 100% performance over 6 months of storage at room temperature, or up to one month heated at 50 °C, which promises refrigeration-free storage of the sensor. The simple preparation, high enzyme stability and ease-of-use of the paper-based sensor promise low-cost and reliable point-of-care multiplex bioassay for biomedical diagnostics. PMID:25893863

  9. Ibuprofen analysis in blood samples by palladium particles-impregnated sodium montmorillonite electrodes: Validation using high performance liquid chromatography.

    PubMed

    Loudiki, A; Boumya, W; Hammani, H; Nasrellah, H; El Bouabi, Y; Zeroual, M; Farahi, A; Lahrich, S; Hnini, K; Achak, M; Bakasse, M; El Mhammedi, M A

    2016-12-01

    The electrochemical detection of ibuprofen has been studied on Palladium-Montmorillonite (Mt) modified carbon paste electrode using differential pulse voltammetry. The optimization of the modifier preparation and the instrumental parameters was investigated. The results indicate that ibuprofen oxidation was favored in the presence of Pd-PdO particles. The quantitative determination of ibuprofen was statistically analyzed and validated using HPLC method. The detection and quantification limits, specificity and precision were found to be acceptable. Finally, the developed method was successfully applied for ibuprofen determination in human blood samples. PMID:27612754

  10. Ibuprofen analysis in blood samples by palladium particles-impregnated sodium montmorillonite electrodes: Validation using high performance liquid chromatography.

    PubMed

    Loudiki, A; Boumya, W; Hammani, H; Nasrellah, H; El Bouabi, Y; Zeroual, M; Farahi, A; Lahrich, S; Hnini, K; Achak, M; Bakasse, M; El Mhammedi, M A

    2016-12-01

    The electrochemical detection of ibuprofen has been studied on Palladium-Montmorillonite (Mt) modified carbon paste electrode using differential pulse voltammetry. The optimization of the modifier preparation and the instrumental parameters was investigated. The results indicate that ibuprofen oxidation was favored in the presence of Pd-PdO particles. The quantitative determination of ibuprofen was statistically analyzed and validated using HPLC method. The detection and quantification limits, specificity and precision were found to be acceptable. Finally, the developed method was successfully applied for ibuprofen determination in human blood samples.

  11. Catecholamine blood test

    MedlinePlus

    Norepinephrine -- blood; Epinephrine -- blood; Adrenalin -- blood; Dopamine -- blood ... A blood sample is needed. ... the test. This is especially true if both blood and urine catecholamines are to be measured. You ...

  12. Carbon dioxide generated from carbonates and acids for sampling blood-feeding arthropods.

    PubMed

    Burkett-Cadena, Nathan D; Blosser, Erik M; Young, Ryan M; Toé, Laurent D; Unnasch, Thomas R

    2015-09-01

    Carbon dioxide (CO2) is utilized to attract mosquitoes and other blood-feeding arthropods to traps around the world. Commercial forms of CO2 (e.g., dry ice and compressed gas) are often unavailable or extremely expensive in developing nations, where vector surveillance is essential to make life-saving decisions. We developed and tested inexpensive and reproducible methods of CO2 production from the combination of acids and carbonates, ranging from very basic (crushed seashells and vinegar) to relatively elaborate (a device that controls the timing of the acid-carbonate reaction and extends the reaction over several hours). When utilized with mosquito traps in Florida, USA and black fly traps in Region des Cascades, Burkina Faso, these carbonate-acid CO2 sources attracted significantly greater numbers of both vector groups, than did unbaited traps. CO2 was generated for more than four hours at levels sufficient to attract vectors over the entire period. The utility of this simple methodology in developing nations should be further evaluated.

  13. Carbon dioxide generated from carbonates and acids for sampling blood-feeding arthropods.

    PubMed

    Burkett-Cadena, Nathan D; Blosser, Erik M; Young, Ryan M; Toé, Laurent D; Unnasch, Thomas R

    2015-09-01

    Carbon dioxide (CO2) is utilized to attract mosquitoes and other blood-feeding arthropods to traps around the world. Commercial forms of CO2 (e.g., dry ice and compressed gas) are often unavailable or extremely expensive in developing nations, where vector surveillance is essential to make life-saving decisions. We developed and tested inexpensive and reproducible methods of CO2 production from the combination of acids and carbonates, ranging from very basic (crushed seashells and vinegar) to relatively elaborate (a device that controls the timing of the acid-carbonate reaction and extends the reaction over several hours). When utilized with mosquito traps in Florida, USA and black fly traps in Region des Cascades, Burkina Faso, these carbonate-acid CO2 sources attracted significantly greater numbers of both vector groups, than did unbaited traps. CO2 was generated for more than four hours at levels sufficient to attract vectors over the entire period. The utility of this simple methodology in developing nations should be further evaluated. PMID:26103427

  14. Predictors of victim disclosure in child sexual abuse: Additional evidence from a sample of incarcerated adult sex offenders.

    PubMed

    Leclerc, Benoit; Wortley, Richard

    2015-05-01

    The under-reporting of child sexual abuse by victims is a serious problem that may prolong the suffering of victims and leave perpetrators free to continue offending. Yet empirical evidence indicates that victim disclosure rates are low. In this study, we perform regression analysis with a sample of 369 adult child sexual offenders to examine potential predictors of victim disclosure. Specifically, we extend the range of previously examined potential predictors of victim disclosure and investigate interaction effects in order to better capture under which circumstances victim disclosure is more likely. The current study differs from previous studies in that it examines the impact of victim and offense variables on victim disclosure from the perspective of the offender. In line with previous studies, we found that disclosure increased with the age of the victim and if penetration had occurred. In addition, we found that disclosure increased when the victim came from a non-dysfunctional family and resisted the abuse. The presence of an interaction effect highlighted the impact of the situation on victim disclosure. This effect indicated that as victims get older, they are more likely to disclose the abuse when they are not living with the offender at the time of abuse, but less likely to do so when they are living with the offender at the time of abuse. These findings are discussed in relation to previous studies and the need to facilitate victim disclosure.

  15. Metabonomics of newborn screening dried blood spot samples: a novel approach in the screening and diagnostics of inborn errors of metabolism.

    PubMed

    Dénes, Júlia; Szabó, Eszter; Robinette, Steven L; Szatmári, Ildikó; Szőnyi, László; Kreuder, Joachim G; Rauterberg, Ernst W; Takáts, Zoltán

    2012-11-20

    A novel, single stage high resolution mass spectrometry-based method is presented for the population level screening of inborn errors of metabolism. The approach proposed here extends traditional electrospray tandem mass spectrometry screening by introducing nanospray ionization and high resolution mass spectrometry, allowing the selective detection of more than 400 individual metabolic constituents of blood including acylcarnitines, amino acids, organic acids, fatty acids, carbohydrates, bile acids, and complex lipids. Dried blood spots were extracted using a methanolic solution of isotope labeled internal standards, and filtered extracts were electrosprayed using a fully automated chip-based nanospray ion source in both positive and negative ion mode. Ions were analyzed using an Orbitrap Fourier transformation mass spectrometer at nominal mass resolution of 100,000. Individual metabolic constituents were quantified using standard isotope dilution methods. Concentration threshold (cutoff) level-based analysis allows the identification of newborns with metabolic diseases, similarly to the traditional electrospray tandem mass spectrometry (ESI-MS/MS) method; however, the detection of additional known biomarkers (e.g., organic acids) results in improved sensitivity and selectivity. The broad range of detected analytes allowed the untargeted multivariate statistical analysis of spectra and identification of additional diseased states, therapeutic artifacts, and damaged samples, besides the metabolic disease panel.

  16. International Study to Evaluate PCR Methods for Detection of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

    PubMed Central

    Schijman, Alejandro G.; Bisio, Margarita; Orellana, Liliana; Sued, Mariela; Duffy, Tomás; Mejia Jaramillo, Ana M.; Cura, Carolina; Auter, Frederic; Veron, Vincent; Qvarnstrom, Yvonne; Deborggraeve, Stijn; Hijar, Gisely; Zulantay, Inés; Lucero, Raúl Horacio; Velazquez, Elsa; Tellez, Tatiana; Sanchez Leon, Zunilda; Galvão, Lucia; Nolder, Debbie; Monje Rumi, María; Levi, José E.; Ramirez, Juan D.; Zorrilla, Pilar; Flores, María; Jercic, Maria I.; Crisante, Gladys; Añez, Néstor; De Castro, Ana M.; Gonzalez, Clara I.; Acosta Viana, Karla; Yachelini, Pedro; Torrico, Faustino; Robello, Carlos; Diosque, Patricio; Triana Chavez, Omar; Aznar, Christine; Russomando, Graciela; Büscher, Philippe; Assal, Azzedine; Guhl, Felipe; Sosa Estani, Sergio; DaSilva, Alexandre; Britto, Constança; Luquetti, Alejandro; Ladzins, Janis

    2011-01-01

    Background A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. Methodology/Findings An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10−3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95

  17. Mercury in human hair and blood samples from people living in Wanshan mercury mine area, Guizhou, China: an XAS study.

    PubMed

    Li, Yu-Feng; Chen, Chunying; Li, Bai; Li, Wei; Qu, Liya; Dong, Zeqin; Nomura, Masaharu; Gao, Yuxi; Zhao, Jinxuan; Hu, Wei; Zhao, Yuliang; Chai, Zhifang

    2008-03-01

    Human hair and blood samples from persons living in the town of Wanshan, a mercury mine area in Guizhou Province of China, were collected and the quantitative speciation and structural information of Hg and S in hair samples and of Hg in erythrocyte and serum samples were studied using X-ray absorption spectroscopy. Least-squares fitting of the X-ray absorption near-edge spectra found that inorganic mercury is the major mercury species in hair samples (91.74%), while inorganic and methyl mercury are both about 50% of total mercury in RBC and serum samples, which is in agreement with the data obtained by acidic extraction, fractionation of Hg(2+) and CH(3)Hg(+) and quantification by ICP-MS. Curve-fitting analysis revealed that the Hg-S bond length and coordination number in hair were 0.248+/-0.002 nm and 3.10, respectively, while the S-Hg bond length and coordination number in hair were 0.236+/-0.002 nm and 4.05. The Hg-S bond length and coordination number in RBC were 0.251+/-0.003 nm and 4.09, respectively, while they were 0.228+/-0.002 nm and 4.08 in serum, respectively. The techniques for speciation, structural and binding information described in this study will find the potential application in similar studies of other elements.

  18. Two red blood cell units collected in SAG-M additive solution with the ALYX component collection system.

    PubMed

    Schooneman, F; Huart, J J; Dernis, D; Tunez, V; Aguettaz, D

    2005-06-01

    The first protocol available for the new ALYX component system (Baxter Healthcare Inc.) allows automated collection of two Red Blood Cell (RBC) units from one donor. The primary objective of our evaluation was to assess donor safety, comfort and to check the quality of blood products collected. 30 procedures were performed on eligible donors according to French best donation practices. Eligibility criteria were defined in order to ensure a post donation hemoglobin concentration of 11 g/dL minimum. Pre donation ferritin level was also checked. 360 ml of absolute RBC were collected from each donor. Donors physiological parameters and haematological profile were measured immediately before and after donation. Adverse events and donors were observed during the procedure and followed daily during 5 days after donation. Hemolysis in RBC was followed until of shelf life (<0.8% on 42 days storage). The evaluation of different parameters during storage show no difference if we compare with the manual technique. The concentration of hemoglobin is good and all ou concentrates are conform. No serious adverse effects were reported during and after donation. All donors confirmed they would agree to donate 2 RBC units again with this system. We have seen a good quality of RBC products. This evaluation indicates that 2 RBC donation is feasible on the ALYX system, comfortable and safe for eligible donors.

  19. The effects of different syringe volume, needle size and sample volume on blood gas analysis in syringes washed with heparin

    PubMed Central

    Küme, Tuncay; Şişman, Ali Rıza; Solak, Ahmet; Tuğlu, Birsen; Çinkooğlu, Burcu; Çoker, Canan

    2012-01-01

    Introductıon: We evaluated the effect of different syringe volume, needle size and sample volume on blood gas analysis in syringes washed with heparin. Materials and methods: In this multi-step experimental study, percent dilution ratios (PDRs) and final heparin concentrations (FHCs) were calculated by gravimetric method for determining the effect of syringe volume (1, 2, 5 and 10 mL), needle size (20, 21, 22, 25 and 26 G) and sample volume (0.5, 1, 2, 5 and 10 mL). The effect of different PDRs and FHCs on blood gas and electrolyte parameters were determined. The erroneous results from nonstandardized sampling were evaluated according to RiliBAK’s TEa. Results: The increase of PDRs and FHCs was associated with the decrease of syringe volume, the increase of needle size and the decrease of sample volume: from 2.0% and 100 IU/mL in 10 mL-syringe to 7.0% and 351 IU/mL in 1 mL-syringe; from 4.9% and 245 IU/mL in 26G to 7.6% and 380 IU/mL in 20 G with combined 1 mL syringe; from 2.0% and 100 IU/mL in full-filled sample to 34% and 1675 IU/mL in 0.5 mL suctioned sample into 10 mL-syringe. There was no statistical difference in pH; but the percent decreasing in pCO2, K+, iCa2+, iMg2+; the percent increasing in pO2 and Na+ were statistical significance compared to samples full-filled in syringes. The all changes in pH and pO2 were acceptable; but the changes in pCO2, Na+, K+ and iCa2+ were unacceptable according to TEa limits except fullfilled-syringes. Conclusions: The changes in PDRs and FHCs due nonstandardized sampling in syringe washed with liquid heparin give rise to erroneous test results for pCO2 and electrolytes. PMID:22838185

  20. Measurement of carboxyhemoglobin in forensic blood samples using UV-visible spectrometry and improved principal component regression

    SciTech Connect

    Egan, William; Morgan, Stephen L. Brewer, William E.

    1999-02-01

    The forensic determination of carboxyhemoglobin (COHb) in blood was performed by using an improved principal component regression (PCR) technique applied to UV-visible spectra. Calibration data were decomposed into principal components, and the principal components useful for prediction were selected by their correlation with calibration spectra. Cross-validation of prediction results was done by leverage-corrected residuals. Confidence and prediction intervals derived from classical regression theory were found to be reasonable in size. The results compared favorably to a comparison study conducted by using a CO Oximeter method. In analysis of forensic case study samples, the improved PCR method allowed detection of abnormal samples and successfully predicted percentages of COHb and methemoglobin (MetHb), and provided error estimates for those predictions. {copyright} {ital 1999} {ital Society for Applied Spectroscopy}

  1. Straightforward and rapid determination of sulfadoxine and sulfamethoxazole in capillary blood on sampling paper with liquid chromatography and UV detection.

    PubMed

    Lindkvist, J; Malm, M; Bergqvist, Y

    2009-04-01

    A method for the determination of sulfadoxine and sulfamethoxazole in capillary blood on sampling paper has been developed and validated. The method is straightforward with minimal sample preparation, and is suitable for rural settings. Separation of sulfadoxine, sulfamethoxazole and internal standard was performed using a Purospher STAR RP-18 endcapped LC column (150x4.6mm) with a mobile phase consisting of acetonitrile:sodium acetate buffer pH 5.2, I=0.1 (1:2, v/v). For sulfadoxine, the within-day precision was 5.3% at 15micromol/l and 3.7% at 600micromol/l, while for sulfamethoxazole it was 5.7% at 15micromol/l and 3.8% at 600micromol/l. The lower limit of quantification was determined to 5micromol/l and precision was 5.5% and 5.0% for sulfadoxine and sulfamethoxazole, respectively.

  2. Development and validation of a dried blood spot-LC-APCI-MS assay for estimation of canrenone in paediatric samples.

    PubMed

    Suyagh, Maysa Faisal; Kole, Prashant Laxman; Millership, Jeff; Collier, Paul; Halliday, Henry; McElnay, James C

    2010-03-15

    A selective and sensitive liquid chromatography (LC)-atmospheric pressure chemical ionisation (APCI)-mass spectroscopic (MS) assay of canrenone has been developed and validated employing Dried Blood Spots (DBS) as the sample collection medium. DBS samples were prepared by applying 30 microl of spiked whole blood onto Guthrie cards. A 6mm disc was punched from the each DBS and extracted with 2 ml of methanolic solution of 17alpha-methyltestosterone (Internal Standard). The methanolic extract was evaporated to dryness and reconstituted in acetonitrile:water (1:9, v/v). The reconstituted solution was further subjected to solid phase extraction using HLB cartridges. Chromatographic separation was achieved using Waters Sunfire C18 reversed-phase column using isocratic elution, followed by a high organic wash to clear late eluting/highly retained components. The mobile phase consisted of methanol:water (60:40, v/v) pumped at a flow rate of 0.3 ml/min. LC-APCI-MS detection was performed in the selected-ion monitoring (SIM) mode using target ions at m/z 341.1 and 303.3 for canrenone and internal standard respectively. The selectivity of the method was established by analysing DBS samples from 6 different sources (individuals). The calibration curve for canrenone was found to be linear over 25-1000 ng/ml (r>0.994). Accuracy (% RE) and precision (% CV) values for within and between day were <20% at the lower limit of quantification (LLQC) and <15% at all other concentrations tested. The LLOQ of the method was validated at 25 ng/ml. Clinical validation of the method was achieved by employing the validated method for analysis of 160 DBS samples from 37 neonatal and paediatric patients. PMID:20153705

  3. Accurate measurement of circulating mitochondrial DNA content from human blood samples using real-time quantitative PCR.

    PubMed

    Ajaz, Saima; Czajka, Anna; Malik, Afshan

    2015-01-01

    We describe a protocol to accurately measure the amount of human mitochondrial DNA (MtDNA) in peripheral blood samples which can be modified to quantify MtDNA from other body fluids, human cells, and tissues. This protocol is based on the use of real-time quantitative PCR (qPCR) to quantify the amount of MtDNA relative to nuclear DNA (designated the Mt/N ratio). In the last decade, there have been increasing numbers of studies describing altered MtDNA or Mt/N in circulation in common nongenetic diseases where mitochondrial dysfunction may play a role (for review see Malik and Czajka, Mitochondrion 13:481-492, 2013). These studies are distinct from those looking at genetic mitochondrial disease and are attempting to identify acquired changes in circulating MtDNA content as an indicator of mitochondrial function. However, the methodology being used is not always specific and reproducible. As more than 95 % of the human mitochondrial genome is duplicated in the human nuclear genome, it is important to avoid co-amplification of nuclear pseudogenes. Furthermore, template preparation protocols can also affect the results because of the size and structural differences between the mitochondrial and nuclear genomes. Here we describe how to (1) prepare DNA from blood samples; (2) pretreat the DNA to prevent dilution bias; (3) prepare dilution standards for absolute quantification using the unique primers human mitochondrial genome forward primer (hMitoF3) and human mitochondrial genome reverse primer(hMitoR3) for the mitochondrial genome, and human nuclear genome forward primer (hB2MF1) and human nuclear genome reverse primer (hB2MR1) primers for the human nuclear genome; (4) carry out qPCR for either relative or absolute quantification from test samples; (5) analyze qPCR data; and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high-throughput use.

  4. Higher Physical Activity Is Associated With Lower Aortic Stiffness but Not With Central Blood Pressure: The ADDITION-Pro Study

    PubMed Central

    Laursen, Anne Sofie Dam; Hansen, Anne-Louise Smidt; Wiinberg, Niels; Brage, Søren; Sandbæk, Annelli; Lauritzen, Torsten; Witte, Daniel R.; Jørgensen, Marit Eika; Johansen, Nanna Borup

    2015-01-01

    Abstract Physical activity is associated with reduced cardiovascular disease risk. However, improvements in conventional risk factors due to physical activity do not explain its full benefit. Therefore, we examined associations of objectively measured physical activity energy expenditure and intensity with central hemodynamics to provide new insight into the link between physical activity and cardiovascular disease. We analyzed data from 1816 Danes (median age: 66 years) without cardiovascular disease. Physical activity was estimated using combined accelerometry and heart rate monitoring. Aortic stiffness was assessed by applanation tonometry, as aortic pulse wave velocity, and central blood pressure was estimated from radial waveforms. Associations between physical activity energy expenditure and central hemodynamics were examined by linear regression. Furthermore, the consequence of substituting 1 hour sedentary behavior with 1 hour light or moderate-to-vigorous physical activity on central hemodynamics was examined. Median physical activity energy expenditure was 28.0 kJ/kg/d (IQR: 19.8; 38.7). A 10 kJ/kg/d higher energy expenditure was associated with 0.75% lower aortic pulse wave velocity (CI: −1.47; −0.03). Associations with central systolic blood pressure and central pulse pressure were not statistically significant. We observed no difference in central hemodynamics when substituting 1 hour sedentary behavior with 1 hour light or moderate-to-vigorous physical activity. In this relatively inactive population, higher physical activity energy expenditure was associated with lower aortic stiffness, while there was no statistically significant association between substitution of activity intensity and central hemodynamics. This suggests that lower aortic stiffness is one of a number of health benefits attributed to higher habitual physical activity. PMID:25654392

  5. Genotoxic and cytotoxic effects of Sunset Yellow and Brilliant Blue, colorant food additives, on human blood lymphocytes.

    PubMed

    Kus, Esra; Eroglu, Halil Erhan

    2015-01-01

    The synthetic dyes over fifty are used in many areas including the food industry around the world. Sunset Yellow FCF and Brilliant Blue FCF are used as colorant food additives in many food products. The present study investigated the genotoxic and cytotoxic effects of Sunset Yellow and Brilliant Blue. Genotoxic and cytotoxic activities of the food additives were evaluated in lymphocyte cell cultures using mitotic index, replication index and micronucleus assay. Mitotic index frequencies and replication index values were decreased and micronucleus frequency was increased with increasing concentrations of Sunset Yellow and Brilliant Blue. The changes in mitotic index and micronucleus are statistically significant (p<0.05). The results show that the Sunset Yellow and Brilliant Blue can have cytotoxic and genotoxic potential. It care must be taken when using these materials as a food additive.

  6. Chronically indwelling venous cannula and automatic blood sampling system for use with nonhuman primates exposed to 60 Hz electric and magnetic fields

    SciTech Connect

    Rogers, W.R.; Lucas, J.H.; Smith, H.D.; Orr, J.L.; Mikiten, B.C.

    1995-12-31

    An automated blood sampling system was developed for use with tethered baboons (Papio cynocephalus) during concurrent exposure to 60 Hz 30 kV/m electric fields and 0.1 mT (1.0 G) magnetic fields. The system was controlled by a FORTH-based microcomputer, which operated a pump, a fraction collector, and two pinch valves. A swivel mechanism at the end of the tether allowed the baboons to move freely in their cages. The hardware and software were designed for fail-safe operation. Heparinized saline was infused at a rate of 0.5 ml/min until a sample cycle was initiated. Then, blood was drawn from the animal into a storage tube at a rate of 12.5 ml/min, a sample of undiluted blood was taken from the end of the storage tube near the baboon, and the blood remaining in the storage tube was then flushed back into the animal. Use of the storage tube prevented the peristaltic pump rollers from pressing on tubing containing blood, and return of the blood diluted with saline limited the blood wasted per sample to less than 0.5 ml. The system functioned reliably in three experiments, collecting samples as scheduled 97% of the time. Although it was initially designed for and used successfully with primates in an electric and magnetic field environment, this type of system could be employed in many areas of biomedical research or medical treatment.

  7. An extremely simple method for extraction of lysophospholipids and phospholipids from blood samples.

    PubMed

    Zhao, Zhenwen; Xu, Yan

    2010-03-01

    Lipids, lysophospholipids and phospholipids in particular, have been shown to be biomarkers and potential therapeutic targets for human diseases. While many extraction and analytical methods have been developed for quantitative analyses of these molecules, most of them are laborious and time-consuming, with associated issues of poor reproducibility. This becomes one of the critical bottle-necks to move lipid markers to clinics. In the current work, we have developed an extremely simple method for lysophospholipids and phospholipids extraction from human plasma or serum samples, which only utilizes a single methanol (MeOH) solvent and involves a single step of centrifugation. This method has been subjected to strict validation by comparing it with classical lipid extraction methods. This simple method will be extremely useful for the lipidomic, diseases marker, and lipid biochemistry fields not only for its potential wide applications associated with its simplicity and reproducibility, but also for its impact in moving lipid markers into clinics through high-throughput processing. PMID:19783525

  8. Development of an optomicrofluidic flow cytometer for the sorting of stem cells from blood samples

    NASA Astrophysics Data System (ADS)

    Islam, Md. Z.; Su, Xuantao; Kirkwood, Sean E.; Singh, Kirat; McMullin, James N.; Rozmus, Wojciech; Janowska-Wieczorek, Anna; Tsui, Ying Y.

    2009-06-01

    In this paper, we report the preliminary development of a fiber coupled microfluidic flow cytometer with its potential application of sorting the very small embryonic like (VSEL) stem cells out of a mixture of platelets and VSEL stem cells. The identification of a VSEL stem cell from a platelet is based on the large difference of their abilities to scatter light. A simple cytometer prototype was built by cutting the fluidic and other channels into a polymer sheet and bonding it with epoxy between two standard glass slides. Standard photolithography was used to expose an observation window over the upper coated glass to reduce background scattered light. Liquid sample containing micro-particles (such as cells) is injected into the microfluidic channel. Light from a 532-nm CW diode laser is coupled into the optical fiber that delivers the light to the detection region in the channel to interrogate the flowing-by micro-particles. The scattering light from the interrogated micro-particle is collected by a photodiode placed over the observation window. The device sorts the micro-particle into the sort or waste outlet depending on the level of the photodiode signal. We used fluorescent latex beads to test the detection and sorting functionalities of the device. It was found that the system could only detect about half of the beads but could sort almost all the beads it detected.

  9. Coupling passive sampling with in vitro bioassays and chemical analysis to understand combined effects of bioaccumulative chemicals in blood of marine turtles.

    PubMed

    Jin, Ling; Escher, Beate I; Limpus, Colin J; Gaus, Caroline

    2015-11-01

    Conventional target analysis of biological samples such as blood limits our ability to understand mixture effects of chemicals. This study aimed to establish a rapid passive sampling technique using the polymer polydimethylsiloxane (PDMS) for exhaustive extraction of mixtures of neutral organic chemicals accumulated in blood of green turtles, in preparation for screening in in vitro bioassays. We designed a PDMS-blood partitioning system based on the partition coefficients of chemicals between PDMS and major blood components. The sampling kinetics of hydrophobic test chemicals (polychlorinated dibenzo-p-dioxins; PCDDs) from blood into PDMS were reasonably fast reaching steady state in <96 h. The geometric mean of the measured PDMS-blood partition coefficients for PCDDs, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) was 14 L blood kg PDMS(-1) and showed little variability (95% confidence interval from 8.4 to 29) across a wide range of hydrophobicity (logKow 5.7-8.3). The mass transfer of these chemicals from 5 mL blood into 0.94 g PDMS was 62-84%, which is similar to analytical recoveries in conventional solvent extraction methods. The validated method was applied to 15 blood samples from green turtles with known concentrations of PCDD/Fs, dioxin-like PCBs, PBDEs and organochlorine pesticides. The quantified chemicals explained most of the dioxin-like activity (69-98%), but less than 0.4% of the oxidative stress response. The results demonstrate the applicability of PDMS-based passive sampling to extract bioaccumulative chemicals from blood as well as the value of in vitro bioassays for capturing the combined effects of unknown and known chemicals. PMID:26091870

  10. Coupling passive sampling with in vitro bioassays and chemical analysis to understand combined effects of bioaccumulative chemicals in blood of marine turtles.

    PubMed

    Jin, Ling; Escher, Beate I; Limpus, Colin J; Gaus, Caroline

    2015-11-01

    Conventional target analysis of biological samples such as blood limits our ability to understand mixture effects of chemicals. This study aimed to establish a rapid passive sampling technique using the polymer polydimethylsiloxane (PDMS) for exhaustive extraction of mixtures of neutral organic chemicals accumulated in blood of green turtles, in preparation for screening in in vitro bioassays. We designed a PDMS-blood partitioning system based on the partition coefficients of chemicals between PDMS and major blood components. The sampling kinetics of hydrophobic test chemicals (polychlorinated dibenzo-p-dioxins; PCDDs) from blood into PDMS were reasonably fast reaching steady state in <96 h. The geometric mean of the measured PDMS-blood partition coefficients for PCDDs, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) was 14 L blood kg PDMS(-1) and showed little variability (95% confidence interval from 8.4 to 29) across a wide range of hydrophobicity (logKow 5.7-8.3). The mass transfer of these chemicals from 5 mL blood into 0.94 g PDMS was 62-84%, which is similar to analytical recoveries in conventional solvent extraction methods. The validated method was applied to 15 blood samples from green turtles with known concentrations of PCDD/Fs, dioxin-like PCBs, PBDEs and organochlorine pesticides. The quantified chemicals explained most of the dioxin-like activity (69-98%), but less than 0.4% of the oxidative stress response. The results demonstrate the applicability of PDMS-based passive sampling to extract bioaccumulative chemicals from blood as well as the value of in vitro bioassays for capturing the combined effects of unknown and known chemicals.

  11. Stability of spironolactone in rat plasma: strict temperature control of blood and plasma samples is required in rat pharmacokinetic studies.

    PubMed

    Tokumura, Tadakazu; Muraoka, Atsushi; Masutomi, Takashi; Machida, Yoshiharu

    2005-06-01

    The stability of spironolactone (SPN) in rat plasma was studied and its degradation was found to be an apparent first-order reaction. The apparent first-order rate constants (k(obs)) at 37, 23.5 and 0 degrees C were 3.543+/-0.261 (h-1, mean+/-S.D., n=3), 6.278+/-0.045 (x10(-1) h-1), and 7.336+/-0.843 (x10(-2) h-1), respectively. The half-lives were 0.20 h, 1.10 h, and 9.53 h. The degradation rate of SPN in rat plasma was markedly decreased when NaF, an esterase inhibitor, was added to the plasma, and the degradation was catalyzed by esterase in the plasma. These results indicated that not only plasma but also blood and serum samples in rat pharmacokinetic studies should be cooled to 0 degrees C, the temperature maintained, and treated as soon as possible. In pharmacokinetic studies reported previously, the temperature control of plasma, blood, and serum samples was not described. The pharmacokinetic study in rats after intravenous administration of SPN at 20 mg/kg was performed with strict temperature control of plasma and blood samples. The AUC, MRT, CL and Vd(ss) values (mean+/-S.E. of 4 rats) for SPN were 4100.8+/-212.9 ng h/ml, 0.29+/-0.01 h, 4915.7+/-248.0 ml/h/kg, and 1435.4+/-48.4 ml/kg, respectively. The AUC value was much larger than that previously reported. The AUC, MRT, Cmax and Tmax values (mean+/-S.E. of 4 rats) of canrenone, an active metabolite of SPN, after the administration of SPN were 4196.1+/-787.5 ng h/ml, 1.99+/-0.13 h, 1546.3+/-436.4 ng/ml and 1.0+/-0.0 h, respectively. This AUC value was almost identical to the value previously reported. PMID:15930762

  12. Genetic profiling of tumours using both circulating free DNA and circulating tumour cells isolated from the same preserved whole blood sample.

    PubMed

    Rothwell, Dominic G; Smith, Nigel; Morris, Daniel; Leong, Hui Sun; Li, Yaoyong; Hollebecque, Antoine; Ayub, Mahmood; Carter, Louise; Antonello, Jenny; Franklin, Lynsey; Miller, Crispin; Blackhall, Fiona; Dive, Caroline; Brady, Ged

    2016-04-01

    Molecular information obtained from cancer patients' blood is an emerging and powerful research tool with immense potential as a companion diagnostic for patient stratification and monitoring. Blood, which can be sampled routinely, provides a means of inferring the current genetic status of patients' tumours via analysis of circulating tumour cells (CTCs) or circulating tumour DNA (ctDNA). However, accurate assessment of both CTCs and ctDNA requires all blood cells to be maintained intact until samples are processed. This dictates for ctDNA analysis EDTA blood samples must be processed with 4 h of draw, severely limiting the use of ctDNA in multi-site trials. Here we describe a blood collection protocol that is amenable for analysis of both CTCs and ctDNA up to four days after blood collection. We demonstrate that yields of circulating free DNA (cfDNA) obtained from whole blood CellSave samples are equivalent to those obtained from conventional EDTA plasma processed within 4 h of blood draw. Targeted and genome-wide NGS revealed comparable DNA quality and resultant sequence information from cfDNA within CellSave and EDTA samples. We also demonstrate that CTCs and ctDNA can be isolated from the same patient blood sample, and give the same patterns of CNA enabling direct analysis of the genetic status of patients' tumours. In summary, our results demonstrate the utility of a simple approach that enabling robust molecular analysis of CTCs and cfDNA for genotype-directed therapies in multi-site clinical trials and represent a significant methodological improvement for clinical benefit. PMID:26639657

  13. Differences between capillary and venous blood-alcohol concentrations as a function of time after drinking, with emphasis on sampling variations in left vs right arm.

    PubMed

    Jones, A W; Jönsson, K A; Jorfeldt, L

    1989-03-01

    Twelve healthy men drank 0.8 g of ethanol per kilogram of body weight during 30 min after an overnight (10 h) fast. At nine exactly timed intervals (30-390 min after the start of drinking), blood was sampled through indwelling catheters in cubital veins on the left and right arms. Immediately thereafter, capillary blood was sampled from fingertips on the left and right hands. The blood ethanol concentration (BAC) was determined by headspace gas chromatography. The SD for alcohol determinations in venous blood, including the left vs right arm sampling variation, was 30 mg/L (range 8.3-83 mg/L), whereas for capillary blood the SD was 35 mg/L (range 11-60 mg/L). This difference much exceeded the purely analytical errors: SD = 2.67 mg/L for venous blood and 14.2 mg/L for fingertip blood. During the first 60 min after the subjects started to drink, capillary BAC exceeded venous BAC, the mean difference at 30 min being 136 mg/L (range 36-216 mg/L). In the postabsorptive state later than 60 min after drinking, venous BAC exceeded capillary BAC [mean difference 58 mg/L (range 0.0-170 mg/L]), the values for venous and capillary BAC crossing 37 min (range 6-77 min) after the end of drinking. Apparently, the source of blood analyzed, venous or capillary, must be considered in clinical pharmacokinetic studies of ethanol.

  14. Addition of Sodium Pyruvate to Stored Red Blood Cells Attenuates Liver Injury in a Murine Transfusion Model

    PubMed Central

    2016-01-01

    RBCs undergo numerous changes during storage and stored RBCs may induce adverse effects, ultimately resulting in organ injury in transfusion recipients. We tested the hypothesis that the addition of SP to stored RBCs would improve the quality of the stored RBCs and mitigate liver injury after transfusion in a murine model. RBCs were harvested from C57BL/6J mice and stored for 14 days in CPDA-1 containing either a solution of SP in saline or saline alone. Haemolysis, the 24-hour posttransfusion recovery, the oxygen-carrying capacity, and the SOD activity of stored RBCs were evaluated. The plasma biochemistry, hepatic MDA level, MPO activity, IL-6, TNF-α concentrations, and histopathology were measured two hours after the transfusion of stored RBCs. Compared with RBCs stored in CPDA-1 and saline, the addition of SP to stored RBCs restored their oxygen-carrying capacity and SOD activity, reduced the AST activity, BUN concentrations, and LDH activity in the plasma, and decreased the MDA level, MPO activity, and concentrations of IL-6 and TNF-α in the liver. These data indicate that the addition of SP to RBCs during storage has a beneficial effect on storage lesions in vitro and subsequently alleviates liver injury after the transfusion of stored RBCs in vivo. PMID:27746589

  15. A nutrigenomic approach to detect nutritional stress from gene expression in blood samples drawn from Steller sea lions.

    PubMed

    Spitz, Jérôme; Becquet, Vanessa; Rosen, David A S; Trites, Andrew W

    2015-09-01

    Gene expression profiles are increasingly being used as biomarkers to detect the physiological responses of a number of species to disease, nutrition, and other stressors. However, little attention has been given to using gene expression to assess the stressors and physiological status of marine mammals. We sought to develop and validate a nutrigenomic approach to quantify nutritional stress in Steller sea lions (Eumetopias jubatus). We subjected 4 female Steller sea lions to 3 feeding regimes over 70-day trials (unrestricted food intake, acute nutritional stress, and chronic nutritional stress), and drew blood samples from each animal at the end of each feeding regime. We then extracted the RNA of white blood cells and measured the response of 8 genes known to react to diet restriction in terrestrial mammals. Overall, we found that the genomic response of Steller sea lions experiencing nutritional stress was consistent with how terrestrial mammals respond to dietary restrictions. Our nutritionally stressed sea lions down-regulated some cellular processes involved in immune response and oxidative stress, and up-regulated pro-inflammatory responses and metabolic processes. Nutrigenomics appears to be a promising means to monitor nutritional status and contribute to mitigation measures needed to assist in the recovery of Steller sea lions and other at-risk species of marine mammals.

  16. A nutrigenomic approach to detect nutritional stress from gene expression in blood samples drawn from Steller sea lions.

    PubMed

    Spitz, Jérôme; Becquet, Vanessa; Rosen, David A S; Trites, Andrew W

    2015-09-01

    Gene expression profiles are increasingly being used as biomarkers to detect the physiological responses of a number of species to disease, nutrition, and other stressors. However, little attention has been given to using gene expression to assess the stressors and physiological status of marine mammals. We sought to develop and validate a nutrigenomic approach to quantify nutritional stress in Steller sea lions (Eumetopias jubatus). We subjected 4 female Steller sea lions to 3 feeding regimes over 70-day trials (unrestricted food intake, acute nutritional stress, and chronic nutritional stress), and drew blood samples from each animal at the end of each feeding regime. We then extracted the RNA of white blood cells and measured the response of 8 genes known to react to diet restriction in terrestrial mammals. Overall, we found that the genomic response of Steller sea lions experiencing nutritional stress was consistent with how terrestrial mammals respond to dietary restrictions. Our nutritionally stressed sea lions down-regulated some cellular processes involved in immune response and oxidative stress, and up-regulated pro-inflammatory responses and metabolic processes. Nutrigenomics appears to be a promising means to monitor nutritional status and contribute to mitigation measures needed to assist in the recovery of Steller sea lions and other at-risk species of marine mammals. PMID:25700740

  17. Comparison of two molecular assays for detection of cytomegalovirus DNA in whole blood and plasma samples from transplant recipients.

    PubMed

    Costa, Cristina; Sidoti, Francesca; Mantovani, Samantha; Gregori, Gabriella; Proietti, Alex; Ghisetti, Valeria; Cavallo, Rossana

    2016-09-01

    In immunosuppressed patients, pre-emptive therapy and a strict follow-up of CMV infection are the standard of care for the prevention of CMV disease. Several real-time PCR assays for CMV DNA quantification on whole blood (WB) and plasma (PL) are commercially available. This study compared and correlated CMV viral loads obtained by the Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) platform on plasma specimens with those obtained on corresponding whole blood specimens by the real-time PCR assay (ELITe MGB-CMV) in 185 sequential samples from 41 immunosuppressed patients. Correlation between the two assays was good. Kinetics of CMV DNA within the same patient was similar, but PL viral load was constantly 1 log lower than WB. In patients under antiviral therapy, low level of CMV DNA persisted in WB, while it was absent in PL. The good correlation between CMV DNA detected on both PL and WB supports the reliability of the two matrices for viral monitoring and the therapeutic management of CMV infection. Nevertheless, due to significant quantification differences between PL and WB CMV DNA, the same biological specimen should be used for a sequential and reliable follow-up of patients at high risk of CMV infection. PMID:27602416

  18. Dextramer Reagents are Effective Tools for Quantifying CMV Antigen-Specific T Cells from Peripheral Blood Samples

    PubMed Central

    Tario, Joseph D.; Chen, George L.; Hahn, Theresa E.; Pan, Dalin; Furlage, Rosemary L.; Zhang, Yali; Brix, Liselotte; Halgreen, Charlotte; Jacobsen, Kivin; McCarthy, Philip L.; Wallace, Paul K.

    2015-01-01

    The enumeration of antigen-specific T cells is increasingly relevant in clinical and research settings. This information is useful for evaluating immune responses to treatment, monitoring the efficacy of anticancer vaccines, and for detecting self-reactive T cells in autoimmune disorders. Quantifying antigen-specific T cells can be accomplished via IFNγ ELISpot assay, the measurement of intracellular cytokine production by flow cytometry, or by lymphocyte proliferation assays in response to antigen. While robust, these technologies are labor-intensive and can take several days to obtain results. New technology has led to more powerful tools for quickly and accurately measuring antigen-specific T cells by flow cytometry via fluorescently-labeled TCR-specific multimers. In this study, we evaluated the use of an assay based on Dextramer reagents for enumerating cytomegalovirus (CMV) antigen-specific T cells (CASTs). Assay performance characteristics were assessed by establishing Dextramers’ sensitivity (median= 0.4; range=0.1–1.4 CASTs µl−1), determining their specificity (100%), evaluating assay robustness with different leukocyte sources and assay reproducibility via interlaboratory and interinstrument investigations. Furthermore, the levels of CASTs in 95 peripheral blood samples from 62 unique blood and marrow transplants recipients correlated well between Dextramers and Tetramers (R2 = 0.9042). PMID:25338522

  19. Software development for estimating the concentration of radioactive cesium in the skeletal muscles of cattle from blood samples.

    PubMed

    Fukuda, Tomokazu; Hiji, Masahiro; Kino, Yasushi; Abe, Yasuyuki; Yamashiro, Hideaki; Kobayashi, Jin; Shimizu, Yoshinaka; Takahashi, Atsushi; Suzuki, Toshihiko; Chiba, Mirei; Inoue, Kazuya; Kuwahara, Yoshikazu; Morimoto, Motoko; Katayama, Masafumi; Donai, Kenichiro; Shinoda, Hisashi; Sekine, Tsutomu; Fukumoto, Manabu; Isogai, Emiko

    2016-06-01

    The 2011 earthquake severely damaged the Fukushima Daiichi Nuclear Power Plant (FNPP), resulting in the release of large quantities of radioactive material into the environment. The deposition of these radionuclides in rice straw as livestock feed led to the circulation of contaminated beef in the market. Based on the safety concern of the consumers, a reliable method for estimating concentrations of radioactive cesium in muscle tissue is needed. In this study, we analyzed the concentrations of radioactive cesium in the blood and skeletal muscle of 88 cattle, and detected a linear correlation between them. We then developed software that can be used to estimate radioactive cesium concentrations in muscle tissue from blood samples. Distribution of this software to the livestock production field would allow us to easily identify high-risk cattle, which would be beyond the safety regulation, before shipping out to the market. This software is planned to be released as freeware. This software would contribute to food safety, and aid the recovery of the livestock industry from the damage creacted by the 2011 Tohoku earthquake and tsunami. PMID:26420060

  20. Facile synthesis of copper(II)-decorated magnetic particles for selective removal of hemoglobin from blood samples.

    PubMed

    Ding, Chun; Ma, Xiangdong; Yao, Xin; Jia, Li

    2015-12-11

    In this report, the Cu(2+)-immobilized magnetic particles were prepared by a facile route and they were used as adsorbents for removal of high abundance of hemoglobin in blood based on immobilized metal affinity chromatography. Ethylenediaminetetraacetic acid modified magnetic particles (EDTA-Fe3O4) were first synthesized through a one-pot solvothermal method and then charged with copper ions. The as-prepared Cu(2+)-EDTA-Fe3O4 particles were characterized by Fourier transform infrared spectrometry, scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray spectroscopy, vibrating sample magnetometry and zeta potential. Factors affecting the adsorption of bovine hemoglobin on Cu(2+)-EDTA-Fe3O4 particles (including contact time, solution pH, ionic strength and initial concentration of protein) were investigated. The adsorption process followed a pseudo-second-order kinetic model and the adsorption equilibrium could be achieved in 60min. The adsorption isotherm data could be well described by a Langmuir model and the maximum adsorption capacity was 1250mgg(-1). The as-prepared particles showed high efficiency and excellent selectivity for removal of hemoglobin from bovine and human blood. The removal process integrated the selectivity of immobilized metal affinity chromatography and the convenience of magnetic separation. The results demonstrated that Cu(2+)-EDTA-Fe3O4 particles had potential application in removal of abundant histidine-rich proteins in biomedical diagnosis analysis.

  1. Facile synthesis of copper(II)-decorated magnetic particles for selective removal of hemoglobin from blood samples.

    PubMed

    Ding, Chun; Ma, Xiangdong; Yao, Xin; Jia, Li

    2015-12-11

    In this report, the Cu(2+)-immobilized magnetic particles were prepared by a facile route and they were used as adsorbents for removal of high abundance of hemoglobin in blood based on immobilized metal affinity chromatography. Ethylenediaminetetraacetic acid modified magnetic particles (EDTA-Fe3O4) were first synthesized through a one-pot solvothermal method and then charged with copper ions. The as-prepared Cu(2+)-EDTA-Fe3O4 particles were characterized by Fourier transform infrared spectrometry, scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray spectroscopy, vibrating sample magnetometry and zeta potential. Factors affecting the adsorption of bovine hemoglobin on Cu(2+)-EDTA-Fe3O4 particles (including contact time, solution pH, ionic strength and initial concentration of protein) were investigated. The adsorption process followed a pseudo-second-order kinetic model and the adsorption equilibrium could be achieved in 60min. The adsorption isotherm data could be well described by a Langmuir model and the maximum adsorption capacity was 1250mgg(-1). The as-prepared particles showed high efficiency and excellent selectivity for removal of hemoglobin from bovine and human blood. The removal process integrated the selectivity of immobilized metal affinity chromatography and the convenience of magnetic separation. The results demonstrated that Cu(2+)-EDTA-Fe3O4 particles had potential application in removal of abundant histidine-rich proteins in biomedical diagnosis analysis. PMID:26596870

  2. Simple methodology to directly genotype Trypanosoma cruzi discrete typing units in single and mixed infections from human blood samples.

    PubMed

    Bontempi, Iván A; Bizai, María L; Ortiz, Sylvia; Manattini, Silvia; Fabbro, Diana; Solari, Aldo; Diez, Cristina

    2016-09-01

    Different DNA markers to genotype Trypanosoma cruzi are now available. However, due to the low quantity of parasites present in biological samples, DNA markers with high copy number like kinetoplast minicircles are needed. The aim of this study was to complete a DNA assay called minicircle lineage specific-PCR (MLS-PCR) previously developed to genotype the T. cruzi DTUs TcV and TcVI, in order to genotype DTUs TcI and TcII and to improve TcVI detection. We screened kinetoplast minicircle hypervariable sequences from cloned PCR products from reference strains belonging to the mentioned DTUs using specific kDNA probes. With the four highly specific sequences selected, we designed primers to be used in the MLS-PCR to directly genotype T. cruzi from biological samples. High specificity and sensitivity were obtained when we evaluated the new approach for TcI, TcII, TcV and TcVI genotyping in twenty two T. cruzi reference strains. Afterward, we compared it with hybridization tests using specific kDNA probes in 32 blood samples from chronic chagasic patients from North Eastern Argentina. With both tests we were able to genotype 94% of the samples and the concordance between them was very good (kappa=0.855). The most frequent T. cruzi DTUs detected were TcV and TcVI, followed by TcII and much lower TcI. A unique T. cruzi DTU was detected in 18 samples meantime more than one in the remaining; being TcV and TcVI the most frequent association. A high percentage of mixed detections were obtained with both assays and its impact was discussed.

  3. Validation of the QIAsymphony RGQ system for DNA quantitation of different BK virus genotypes in whole blood samples.

    PubMed

    Burrel, Sonia; Brunet, Christel; Hamm, Nathalie; Gits-Muselli, Maud; Hermet, Laurence; Aimé, Catherine; Agut, Henri; Boutolleau, David

    2014-02-01

    Human polyomavirus BK (BKV) is increasingly recognized as an opportunistic pathogen in transplant recipients. The aim of this work was to evaluate the artus(®) BK Virus QS-RGQ assay on the QIAsymphony RGQ system in whole blood (WB) samples (tests performed in an off-label capacity) according to different BKV genotypes by comparison with a laboratory-developed assay. BKV loads were measured in 111 WB samples and BKV genotype was determined by sequencing the full-length VP1 gene. The artus(®) assay exhibited a limit of detection of 77copies/mL, a linearity range from 3.0 to 6.0log10copies/mL, intra-assay and inter-assay coefficients of variation ranging from 0.65% to 5.18%. Regarding BKV quantitation, artus(®) and laboratory-developed assays were highly correlated (Spearman correlation coefficient Rho=0.79; P<0.0001) with an excellent overall agreement (96.4%) and no significant quantitative difference according to Bland-Altman analysis (mean difference: -0.34log10copies/mL). The results did not show any influence of BKV genotype on BKV quantitation by the artus(®) assay, except a potential underquantitation of BKV subtype Ia which deserves further confirmation. In conclusion, the QIAsymphony RGQ system appears to be appropriate for the quantitation of BKV load in WB samples.

  4. Sensitivity testing of trypanosome detection by PCR from whole blood samples using manual and automated DNA extraction methods.

    PubMed

    Dunlop, J; Thompson, C K; Godfrey, S S; Thompson, R C A

    2014-11-01

    Automated extraction of DNA for testing of laboratory samples is an attractive alternative to labour-intensive manual methods when higher throughput is required. However, it is important to maintain the maximum detection sensitivity possible to reduce the occurrence of type II errors (false negatives; failure to detect the target when it is present), especially in the biomedical field, where PCR is used for diagnosis. We used blood infected with known concentrations of Trypanosoma copemani to test the impact of analysis techniques on trypanosome detection sensitivity by PCR. We compared combinations of a manual and an automated DNA extraction method and two different PCR primer sets to investigate the impact of each on detection levels. Both extraction techniques and specificity of primer sets had a significant impact on detection sensitivity. Samples extracted using the same DNA extraction technique performed substantially differently for each of the separate primer sets. Type I errors (false positives; detection of the target when it is not present), produced by contaminants, were avoided with both extraction methods. This study highlights the importance of testing laboratory techniques with known samples to optimise accuracy of test results.

  5. Mining the Dynamic Genome: A Method for Identifying Multiple Disease Signatures Using Quantitative RNA Expression Analysis of a Single Blood Sample

    PubMed Central

    Chao, Samuel; Cheng, Changming; Liew, Choong-Chin

    2015-01-01

    Background: Blood has advantages over tissue samples as a diagnostic tool, and blood mRNA transcriptomics is an exciting research field. To realize the full potential of blood transcriptomic investigations requires improved methods for gene expression measurement and data interpretation able to detect biological signatures within the “noisy” variability of whole blood. Methods: We demonstrate collection tube bias compensation during the process of identifying a liver cancer-specific gene signature. The candidate probe set list of liver cancer was filtered, based on previous repeatability performance obtained from technical replicates. We built a prediction model using differential pairs to reduce the impact of confounding factors. We compared prediction performance on an independent test set against prediction on an alternative model derived by Weka. The method was applied to an independent set of 157 blood samples collected in PAXgene tubes. Results: The model discriminated liver cancer equally well in both EDTA and PAXgene collected samples, whereas the Weka-derived model (using default settings) was not able to compensate for collection tube bias. Cross-validation results show our procedure predicted membership of each sample within the disease groups and healthy controls. Conclusion: Our versatile method for blood transcriptomic investigation overcomes several limitations hampering research in blood-based gene tests. PMID:27600246

  6. The Role of Patients’ Age on Their Preferences for Choosing Additional Blood Pressure-Lowering Drugs: A Discrete Choice Experiment in Patients with Diabetes

    PubMed Central

    de Vries, Sieta T.; de Vries, Folgerdiena M.; Dekker, Thijs; Haaijer-Ruskamp, Flora M.; de Zeeuw, Dick; Ranchor, Adelita V.; Denig, Petra

    2015-01-01

    Objectives To assess whether patients’ willingness to add a blood pressure-lowering drug and the importance they attach to specific treatment characteristics differ among age groups in patients with type 2 diabetes. Materials and Methods Patients being prescribed at least an oral glucose-lowering and a blood pressure-lowering drug completed a questionnaire including a discrete choice experiment. This experiment contained choice sets with hypothetical blood pressure-lowering drugs and a no additional drug alternative, which differed in their characteristics (i.e. effects and intake moments). Differences in willingness to add a drug were compared between patients <75 years (non-aged) and ≥75 years (aged) using Pearson χ2-tests. Multinomial logit models were used to assess and compare the importance attached to the characteristics. Results Of the 161 patients who completed the questionnaire, 151 (72%) could be included in the analyses (mean age 68 years; 42% female). Aged patients were less willing to add a drug than non-aged patients (67% versus 84% respectively; P = 0.017). In both age groups, the effect on blood pressure was most important for choosing a drug, followed by the risk of adverse drug events and the risk of death. The effect on limitations due to stroke was only significant in the non-aged group. The effect on blood pressure was slightly more important in the non-aged than the aged group (P = 0.043). Conclusions Aged patients appear less willing to add a preventive drug than non-aged patients. The importance attached to various treatment characteristics does not seem to differ much among age groups. PMID:26445349

  7. 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.

  8. An integrative pharmacological approach to radio telemetry and blood sampling in pharmaceutical drug discovery and safety assessment

    PubMed Central

    2011-01-01

    Background A successful integration of the automated blood sampling (ABS) and telemetry (ABST) system is described. The new ABST system facilitates concomitant collection of physiological variables with blood and urine samples for determination of drug concentrations and other biochemical measures in the same rat without handling artifact. Method Integration was achieved by designing a 13 inch circular receiving antenna that operates as a plug-in replacement for the existing pair of DSI's orthogonal antennas which is compatible with the rotating cage and open floor design of the BASi Culex® ABS system. The circular receiving antenna's electrical configuration consists of a pair of electrically orthogonal half-toroids that reinforce reception of a dipole transmitter operating within the coil's interior while reducing both external noise pickup and interference from other adjacent dipole transmitters. Results For validation, measured baclofen concentration (ABST vs. satellite (μM): 69.6 ± 23.8 vs. 76.6 ± 19.5, p = NS) and mean arterial pressure (ABST vs. traditional DSI telemetry (mm Hg): 150 ± 5 vs.147 ± 4, p = NS) variables were quantitatively and qualitatively similar between rats housed in the ABST system and traditional home cage approaches. Conclusion The ABST system offers unique advantages over traditional between-group study paradigms that include improved data quality and significantly reduced animal use. The superior within-group model facilitates assessment of multiple physiological and biochemical responses to test compounds in the same animal. The ABST also provides opportunities to evaluate temporal relations between parameters and to investigate anomalous outlier events because drug concentrations, physiological and biochemical measures for each animal are available for comparisons. PMID:21244682

  9. Comparison of zopiclone concentrations in oral fluid sampled with Intercept(®) oral specimen collection device and Statsure Saliva Sampler™ and concentrations in blood.

    PubMed

    Gjerde, Hallvard; Øiestad, Elisabeth L; Øiestad, Åse Marit L; Langødegård, Marit; Gustavsen, Ingebjørg; Hjelmeland, Knut; Bernard, Jean-Paul; Christophersen, Asbjørg S

    2010-11-01

    A clinical study of zopiclone was performed using doses of 5 and 10 mg. Samples of oral fluid were collected using the Statsure and Intercept devices, and blood samples were collected simultaneously. Concentrations of zopiclone in samples of oral fluid and blood were determined with liquid chromatography-mass spectrometry, and concentrations in undiluted oral fluid were calculated. The concentrations of zopiclone in oral fluid were generally higher when using the Intercept compared to the Statsure device; the median oral fluid/whole blood concentration ratios were 3.8 (range 1.5-15.9) and 1.9 (range 1.2-4.6), respectively (n = 21). The correlation between zopiclone concentrations in oral fluid collected with the two devices was fairly poor, r(2) = 0.35. The results indicate that the type of sampling device may significantly affect the analytical result for zopiclone in sampled oral fluid.

  10. Detection of African swine fever virus DNA in blood samples stored on FTA cards from asymptomatic pigs in Mbeya region, Tanzania.

    PubMed

    Braae, U C; Johansen, M V; Ngowi, H A; Rasmussen, T B; Nielsen, J; Uttenthal, Å

    2015-02-01

    The aim of the study was to assess whether blood samples collected onto FTA(®) cards could be used in combination with real-time PCR for the detection of African swine fever virus (ASFV) DNA in samples from resource-poor settings under the assumption that asymptomatically (sub-clinically) infected pigs may be present. Blood samples were collected from clinically healthy pigs from Mbeya Region, Tanzania. The blood samples were stored on FTA(®) cards and analysed by real-time PCR assays in duplicate; three pigs had high levels of viral DNA (Ct values of 27-29), and three pigs had a low level of viral DNA (Ct 36-45). Four pigs were positive in one of the duplicate samples only, but clear products of the expected size were obtained when the reactions were analysed by gel electrophoresis. For comparison, blood samples from pigs experimentally infected with either a pathogenic (OURT T88/1) or a non-pathogenic (OURT T88/3) isolate of ASFV were collected, stored on FTA(®) cards and analysed in the same way. The blood from pigs infected with the OURT T88/1 isolate showed high levels of viral DNA (Ct 22-33), whereas infection with non-pathogenic OURT T88/3 isolate resulted in only low levels of viral DNA (Ct 39) in samples collected at 10-14 days after inoculation.

  11. Loop Mediated Isothermal Amplification (LAMP) Accurately Detects Malaria DNA from Filter Paper Blood Samples of Low Density Parasitaemias

    PubMed Central

    González, Iveth J.; Polley, Spencer D.; Bell, David; Shakely, Delér; Msellem, Mwinyi I.; Björkman, Anders; Mårtensson, Andreas

    2014-01-01

    Background Loop mediated isothermal amplification (LAMP) provides an opportunity for improved, field-friendly detection of malaria infections in endemic areas. However data on the diagnostic accuracy of LAMP for active case detection, particularly low-density parasitaemias, are lacking. We therefore evaluated the performance of a new LAMP kit compared with PCR using DNA from filter paper blood spots. Methods and Findings Samples from 865 fever patients and 465 asymptomatic individuals collected in Zanzibar were analysed for Pan (all species) and Pf (P. falciparum) DNA with the Loopamp MALARIA Pan/Pf kit. Samples were amplified at 65°C for 40 minutes in a real-time turbidimeter and results were compared with nested PCR. Samples with discordant results between LAMP and nested PCR were analysed with real-time PCR. The real-time PCR corrected nested PCR result was defined as gold standard. Among the 117 (13.5%) PCR detected P. falciparum infections from fever patients (mean parasite density 7491/µL, range 6–782,400) 115, 115 and 111 were positive by Pan-LAMP, Pf-LAMP and nested PCR, respectively. The sensitivities were 98.3% (95%CI 94–99.8) for both Pan and Pf-LAMP. Among the 54 (11.6%) PCR positive samples from asymptomatic individuals (mean parasite density 10/µL, range 0–4972) Pf-LAMP had a sensitivity of 92.7% (95%CI 80.1–98.5) for detection of the 41 P. falciparum infections. Pan-LAMP had sensitivities of 97% (95%CI 84.2–99.9) and 76.9% (95%CI 46.2–95) for detection of P. falciparum and P. malariae, respectively. The specificities for both Pan and Pf-LAMP were 100% (95%CI 99.1–100) in both study groups. Conclusion Both components of the Loopamp MALARIA Pan/Pf detection kit revealed high diagnostic accuracy for parasite detection among fever patients and importantly also among asymptomatic individuals of low parasite densities from minute blood volumes preserved on filter paper. These data support LAMPs potential role for improved detection of low

  12. Effects of the Dietary Addition of Amaranth (Amaranthus mantegazzianus) Protein Isolate on Antioxidant Status, Lipid Profiles and Blood Pressure of Rats.

    PubMed

    Lado, María B; Burini, Julieta; Rinaldi, Gustavo; Añón, María C; Tironi, Valeria A

    2015-12-01

    The effects of the dietary addition of 2.5% (w/w) Amaranthus mantegazzianus protein isolate (AI) on blood pressure, lipid profiles and antioxidative status of Wistar rats were evaluated. Six diets were used to feed animals during 28 days: (base (AIN93G), Chol (cholesterol 1%, w/w), CE (α-tocopherol 0.005%, w/w), CholE (cholesterol 1% (w/w) + α-tocopherol 0.005%, w/w), CAI (AI 2.5% w/w), CholAI (cholesterol 1% (w/w) + AI 2.5%, w/w). Lipid profiles of plasma and liver and faecal cholesterol content were analyzed. Antioxidant status was evaluated by the ferric reducing activity of plasma (FRAP), the 2-thiobarbituric acid (TBA) assay and superoxide dismutase (SOD) activity in plasma and liver. Blood pressure was measured in the tail artery of rats. CholA group presented a significant (α < 0.05) reduction (16%) in the plasma total cholesterol. In liver, the intake of cholesterol (Chol group) induced a significant increment in cholesterol and triglycerides (2.5 and 2.3 times, respectively), which could be decreased (18% and 47%, respectively) by the addition of AI (CholA group). This last group also showed an increased faecal cholesterol excretion (20%). Increment (50%) in FRAP values, diminution of TBA value in plasma and liver (70% and 38%, respectively) and diminution of SOD activity (20%) in plasma of CholA group suggest an antioxidant effect because of the intake of AI. In addition, CA and CholA groups presented a diminution (18%) of blood pressure after 28 days.

  13. Effects of the Dietary Addition of Amaranth (Amaranthus mantegazzianus) Protein Isolate on Antioxidant Status, Lipid Profiles and Blood Pressure of Rats.

    PubMed

    Lado, María B; Burini, Julieta; Rinaldi, Gustavo; Añón, María C; Tironi, Valeria A

    2015-12-01

    The effects of the dietary addition of 2.5% (w/w) Amaranthus mantegazzianus protein isolate (AI) on blood pressure, lipid profiles and antioxidative status of Wistar rats were evaluated. Six diets were used to feed animals during 28 days: (base (AIN93G), Chol (cholesterol 1%, w/w), CE (α-tocopherol 0.005%, w/w), CholE (cholesterol 1% (w/w) + α-tocopherol 0.005%, w/w), CAI (AI 2.5% w/w), CholAI (cholesterol 1% (w/w) + AI 2.5%, w/w). Lipid profiles of plasma and liver and faecal cholesterol content were analyzed. Antioxidant status was evaluated by the ferric reducing activity of plasma (FRAP), the 2-thiobarbituric acid (TBA) assay and superoxide dismutase (SOD) activity in plasma and liver. Blood pressure was measured in the tail artery of rats. CholA group presented a significant (α < 0.05) reduction (16%) in the plasma total cholesterol. In liver, the intake of cholesterol (Chol group) induced a significant increment in cholesterol and triglycerides (2.5 and 2.3 times, respectively), which could be decreased (18% and 47%, respectively) by the addition of AI (CholA group). This last group also showed an increased faecal cholesterol excretion (20%). Increment (50%) in FRAP values, diminution of TBA value in plasma and liver (70% and 38%, respectively) and diminution of SOD activity (20%) in plasma of CholA group suggest an antioxidant effect because of the intake of AI. In addition, CA and CholA groups presented a diminution (18%) of blood pressure after 28 days. PMID:26497504

  14. The best practice for preparation of samples from FTA®cards for diagnosis of blood borne infections using African trypanosomes as a model system

    PubMed Central

    2011-01-01

    Background Diagnosis of blood borne infectious diseases relies primarily on the detection of the causative agent in the blood sample. Molecular techniques offer sensitive and specific tools for this although considerable difficulties exist when using these approaches in the field environment. In large scale epidemiological studies, FTA®cards are becoming increasingly popular for the rapid collection and archiving of a large number of samples. However, there are some difficulties in the downstream processing of these cards which is essential for the accurate diagnosis of infection. Here we describe recommendations for the best practice approach for sample processing from FTA®cards for the molecular diagnosis of trypanosomiasis using PCR. Results A comparison of five techniques was made. Detection from directly applied whole blood was less sensitive (35.6%) than whole blood which was subsequently eluted from the cards using Chelex®100 (56.4%). Better apparent sensitivity was achieved when blood was lysed prior to application on the FTA cards (73.3%) although this was not significant. This did not improve with subsequent elution using Chelex®100 (73.3%) and was not significantly different from direct DNA extraction from blood in the field (68.3%). Conclusions Based on these results, the degree of effort required for each of these techniques and the difficulty of DNA extraction under field conditions, we recommend that blood is transferred onto FTA cards whole followed by elution in Chelex®100 as the best approach. PMID:21548975

  15. Field confirmation of modified pressurized solvent extraction vessels for the analysis of polychlorinated biphenyl congeners in blood samples from Great Lakes Mallards (Anas platyrhynchos).

    PubMed

    Haskins, Stacey D; Kelly, David G; Weir, Ron D

    2013-06-01

    Miniaturized pressurized solvent extraction vessels were used to examine polychlorinated biphenyl congener (PCB) concentrations in 0.2 g sample sizes of whole blood, liver, heart and breast tissue sampled from twelve Great Lakes Mallard ducks (Anas platyrhynchos). This study successfully supported the blood extraction method, previously validated only using laboratory prepared blood samples, using field samples. In situ clean-up offered excellent sample throughput without degradation of GC-MS performance; using this method, extraction, instrument analysis and data interpretation for 100 samples could be accomplished within a one to two week time period. Results indicated contamination in the blood (∑PCB = 1.9-13 ng g(-1) ww), liver (∑PCB = 0.8-11 ng g(-1) ww), breast (∑PCB = <0.1-9 ng g(-1) ww) and heart tissue (∑PCB = <0.1-6 ng g(-1) ww). Quality control included the analysis of blank samples, NIST SRM 1589a and a duplicate of each sample type (blood or tissue). All blank samples were below the method detection limit, SRM values were within 70% of their certified values and duplicates were within 70% of each other. Correlations were examined for the suite of analysed congeners between blood and various tissues; within select individuals a strong and significant correlation was observed. TEQs were calculated and compared against known toxicity data for bird species. Based on the PCB levels found in this study, no adverse health effects are expected in the birds themselves. ∑PCB concentrations in the breast tissue were also compared against both the Canadian and American guidelines for the consumption of edible poultry and based on these values, the Mallards used in this research would be safe for human consumption.

  16. Prevalence of Mycobacterium avium subsp. paratuberculosis and Escherichia coli in blood samples from patients with inflammatory bowel disease.

    PubMed

    Nazareth, Nair; Magro, Fernando; Machado, Elisabete; Ribeiro, Teresa Gonçalves; Martinho, António; Rodrigues, Pedro; Alves, Rita; Macedo, Gonçalo Nuno; Gracio, Daniela; Coelho, Rosa; Abreu, Candida; Appelberg, Rui; Dias, Camila; Macedo, Guilherme; Bull, Tim; Sarmento, Amélia

    2015-12-01

    Mycobacterium avium subsp. paratuberculosis (MAP) and adherent-invasive Escherichia coli (AIEC) have been implicated as primary triggers in Crohn's disease (CD). In this study, we evaluated the prevalence of MAP and E. coli (EC) DNA in peripheral blood from 202 inflammatory bowel disease (IBD) patients at various disease periods and compared against 24 cirrhotic patients with ascites (CIR) (non-IBD controls) and 29 healthy controls (HC). MAP DNA was detected by IS900-specific nested PCR, EC DNA by malB-specific nested PCR and AIEC identity, in selected samples, by sequencing of fimH gene. CD patients with active disease showed the highest MAP DNA prevalence among IBD patients (68 %). Infliximab treatment resulted in decreased MAP detection. CIR patients had high individual and coinfection rates (75 % MAP, 88 % EC and 67 % MAP and EC), whilst HC controls had lower MAP prevalence (38 %) and EC was undetectable in this control group. EC DNA prevalence in IBD patients was highly associated with CD, and 80 % of EC from the selected samples of CD patients analyzed carried the fimH30 allele, with a mutation strongly associated with AIEC. Our results show that coinfection with MAP and AIEC is common and persistent in CD, although the high MAP and EC detection in CIR patients suggested that colonization is, at least, partially dependent on increased gut permeability. Nevertheless, facilitative mechanisms between a susceptible host and these two potential human pathogens may allow their implication in CD pathogenesis.

  17. Blood biochemical markers of bone turnover: pre-analytical and technical aspects of sample collection and handling.

    PubMed

    Lombardi, Giovanni; Lanteri, Patrizia; Colombini, Alessandra; Banfi, Giuseppe

    2012-02-03

    Casual or systematic errors occurring in pre-analytical, analytical or post-analytical phases influence laboratory test results. The areas where pre-analytical phase errors most often arise are: timing of specimen collection; selection of specimen type; and time and temperature of storage/transport. Bone turnover markers are clinically useful in evaluating bone metabolism. Although unquestionably valuable tools, little is known about the pre-analytical precautions for their correct use and there is no consensus on kind of sample, or storage time and temperature before analysis. Moreover, biological variability, because of uncontrollable and controllable factors, will affect pre-analytical variability. Serum should be preferred to simplify blood drawing; therefore, only one tube should be used for the analysis of all bone markers. Short-term storage at 4°C may be advisable to preserve stability, immediate storage at -70°C is recommended for longer periods, while avoiding repeated freeze-thawing cycles. Sampling should be performed in the morning in fasting subjects who have abstained from physical exercise for 24 h. This review aimed to give a knowledge update on pre-analytical phase precautions in performing bone turnover marker measurement.

  18. Validated UPLC-MS/MS assay for the determination of synthetic phosphodiesterase type-5 inhibitors in postmortem blood samples.

    PubMed

    Proença, Paula; Mustra, Carla; Marcos, Mariana; Franco, João Miguel; Corte-Real, Francisco; Vieira, Duarte Nuno

    2013-08-01

    The use of synthetic phosphodiesterase type 5 (PDE-5) inhibitors for the treatment of erectile dysfunction: sildenafil citrate (Viagra(®)), tadalafil (Cialis(®)) and vardenafil hydrochloride (Levitra(®)) has increased dramatically over the past 2 years. These substances are prescription drugs and must be used under medical supervision. However, they can easily be obtained over the internet from illegal sites, being a potential for a threat to public health. The development of an electrospray ionisation (ESI) ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) procedure for the simultaneous identification and quantification of three PDE5 inhibitors in blood samples was desired. Samples were prepared using Oasis(®) HLB solid-phase cartridges (3 cc, 60 mg) and chromatographic separation was achieved on an Acquity UPLC(®) HSS T3 (100 × 2.1 mm i.d., 1.8 μm particles) column with a gradient mobile phase of 0.1% formic acid and acetonitrile at a 0.5 mL/min flow rate. Quantification was achieved by multiple reaction monitoring (MRM) of two transitions per compound: m/z 475.1 > 58 e m/z 475.1 > 311.1 for sildenafil; m/z 389.9 > 267.9 e m/z 389.9 > 134.8 for tadalafil and m/z 489 > 71.9 e m/z 489 > 150.9 for vardenafil. Zolpidem-d6 (m/z 314.5 > 235.3) was used as the internal standard. Calibration curves were linear over the concentration range of 5-1000 ng/mL, with a coefficient of determination better than 0.997. The lower limits of detection and quantification for these substances were ≤ 3 ng/mL and ≤ 8 ng/mL, respectively. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity. A rapid, selective and sensitive UPLC-MS/MS method using solid-phase extraction was developed for the simultaneous determination and quantification of sildenafil, vardenafil and tadalafil in blood samples. PMID:23910856

  19. Novel system using microliter order sample volume for measuring arterial radioactivity concentrations in whole blood and plasma for mouse PET dynamic study

    NASA Astrophysics Data System (ADS)

    Kimura, Yuichi; Seki, Chie; Hashizume, Nobuya; Yamada, Takashi; Wakizaka, Hidekatsu; Nishimoto, Takahiro; Hatano, Kentaro; Kitamura, Keishi; Toyama, Hiroshi; Kanno, Iwao

    2013-11-01

    This study aimed to develop a new system, named CD-Well, for mouse PET dynamic study. CD-Well allows the determination of time-activity curves (TACs) for arterial whole blood and plasma using 2-3 µL of blood per sample; the minute sample size is ideal for studies in small animals. The system has the following merits: (1) measures volume and radioactivity of whole blood and plasma separately; (2) allows measurements at 10 s intervals to capture initial rapid changes in the TAC; and (3) is compact and easy to handle, minimizes blood loss from sampling, and delay and dispersion of the TAC. CD-Well has 36 U-shaped channels. A drop of blood is sampled into the opening of the channel and stored there. After serial sampling is completed, CD-Well is centrifuged and scanned using a flatbed scanner to define the regions of plasma and blood cells. The length measured is converted to volume because the channels have a precise and uniform cross section. Then, CD-Well is exposed to an imaging plate to measure radioactivity. Finally, radioactivity concentrations are computed. We evaluated the performance of CD-Well in in vitro measurement and in vivo 18F-fluorodeoxyglucose and [11C]2-carbomethoxy-3β-(4-fluorophenyl) tropane studies. In in vitro evaluation, per cent differences (mean±SE) from manual measurement were 4.4±3.6% for whole blood and 4.0±3.5% for plasma across the typical range of radioactivity measured in mouse dynamic study. In in vivo studies, reasonable TACs were obtained. The peaks were captured well, and the time courses coincided well with the TAC derived from PET imaging of the heart chamber. The total blood loss was less than 200 µL, which had no physiological effect on the mice. CD-Well demonstrates satisfactory performance, and is useful for mouse PET dynamic study.

  20. Branched perfluorooctane sulfonate isomer quantification and characterization in blood serum samples by HPLC/ESI-MS(/MS).

    PubMed

    Riddell, Nicole; Arsenault, Gilles; Benskin, Jonathan P; Chittim, Brock; Martin, Jonathan W; McAlees, Alan; McCrindle, Robert

    2009-10-15

    Perfluorooctane sulfonate (PFOS) is a global contaminant and is currently among the most prominent contaminants in human blood and wildlife samples. Although "total PFOS" (SigmaPFOS) analytical methods continue to be the most commonly used for quantification, recent analytical method developments have made it possible to resolve the various isomers of PFOS by HPLC-MS/MS. Characterized technical PFOS standards (i.e., containing a mixture of PFOS isomers) are now available that enable isomer specific quantification of PFOS, however the advantages of such an analysis have notyet been examined systematically. Herein, PFOS isomers have been individually quantified for the first time in real samples and the results are compared to a traditional SigmaPFOS method; the influence of analytical standards and isomer specific electrospray and MS/ MS behavior were also investigated. The two human serum standard reference materials chosen for analysis contained dramaticallydifferent PFOS isomer profiles (approximately 30-50% total branched isomers) emphasizing that isomer patterns should not be ignored and may provide useful information on exposure sources (i.e., direct exposure to PFOS vs indirect exposure from PFOS-precursors). Depending on the sample and the particular MS/MS transition chosen for SigmaPFOS analysis (i.e., 499-->80 or 499-->99), SigmaPFOS concentrations may be over- or underestimated compared to the isomer specific analysis. Differences in the extent of in-source fragmentation and MS/MS dissociation contributed to the systematic analytical bias. It was also shown that SigmaPFOS data are prone to interlaboratory variation due to various choices of PFOS standards and instrumental conditions used. In the future, for either SigmaPFOS or isomer specific PFOS analyses, we suggest that accuracy can be maximized and interlaboratory discrepancies minimized by using a common chemically pure technical PFOS standard characterized by 19F NMR.

  1. Solid sampling determination of lithium and sodium additives in microsamples of yttrium oxyorthosilicate by high-resolution continuum source graphite furnace atomic absorption spectrometry

    NASA Astrophysics Data System (ADS)

    Laczai, Nikoletta; Kovács, László; Péter, Ágnes; Bencs, László

    2016-03-01

    Solid sampling high resolution continuum source graphite furnace atomic absorption spectrometry (SS-HR-CS-GFAAS) methods were developed and studied for the fast and sensitive quantitation of Li and Na additives in microsamples of cerium-doped yttrium oxyorthosilicate (Y2SiO5:Ce) scintillator materials. The methods were optimized for solid samples by studying a set of GFAAS conditions (i.e., the sample mass, sensitivity of the analytical lines, and graphite furnace heating programs). Powdered samples in the mass range of 0.099-0.422 mg were dispensed onto graphite sample insertion boats, weighed and analyzed. Pyrolysis and atomization temperatures were optimized by the use of single-element standard solutions of Li and Na (acidified with 0.144 mol/L HNO3) at the Li I 610.353 nm and Na I 285.3013 nm analytical lines. For calibration purposes, the method of standard addition with Li and Na solutions was applied. The correlation coefficients (R values) of the calibration graphs were not worse than 0.9678. The limit of detection for oxyorthosilicate samples was 20 μg/g and 80 μg/g for Li and Na, respectively. The alkaline content of the solid samples were found to be in the range of 0.89 and 8.4 mg/g, respectively. The accuracy of the results was verified by means of analyzing certified reference samples, using methods of standard (solution) addition calibration.

  2. Blood cholesterol screening in several environments using a portable, dry-chemistry analyzer and fingerstick blood samples. Lipid Research Clinics Cholesterol Screening Study Group.

    PubMed

    Bradford, R H; Bachorik, P S; Roberts, K; Williams, O D; Gotto, A M

    1990-01-01

    A multicenter study of blood cholesterol screening was performed in several typical environments, such as community sites (shopping malls and a supermarket), health care sites, work sites, a blood bank and a school. Cholesterol was measured with a portable, dry-chemistry analyzer using capillary blood obtained by fingerstick. Data are reported from a total of 13,824 participants, spanning the entire age spectrum. Overall, 25% of screened subjects had blood cholesterol levels above the age-specific cutpoints used in the current study. Although in the aggregate this screening experience very closely approximates the expected level of referrals, the proportion of referred screened subjects differed significantly among the 5 types of screening environments and by gender. Follow-up telephone interviews indicated that 53% of referrals had initiated a physician contact. More than 75% of those who had seen a physician reported that the diagnosis of hypercholesterolemia had been confirmed, and almost 72% had been prescribed a diet. A large proportion of referred screened subjects reported having modified their diet, particularly when recommended to do so by a physician. This study has yielded encouraging evidence that physicians gave referred screened subjects appropriate initial advice for managing hypercholesterolemia. The new technology for blood cholesterol measurement evaluated in the current study has proven to be a feasible and reliable means for measuring blood cholesterol in typical screening settings.

  3. Human Erythrocyte PIG-A Assay: An Easily Monitored Index of Gene Mutation Requiring Low Volume Blood Samples

    PubMed Central

    Dertinger, Stephen D.; Avlasevich, Svetlana L.; Bemis, Jeffrey C.; Chen, Yuhchyau; MacGregor, James T.

    2015-01-01

    This laboratory has previously described a method for scoring the incidence of rodent blood Pig-a mutant phenotype erythrocytes using immunomag-netic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). The current work extends this approach to human blood. The frequencies of CD59- and CD55-negative reticulo-cytes (RETCD59−/CD55−) and erythrocytes (RBCCD59−/CD55−) seve as phenotypic reporters of PIG-A gene mutation. Immunomagnetic separation was found to provide an effective means of increasing the number of reticulocytes and erythro-cytes evaluated. Technical replicates were utilized to provide a sufficient number of cells for precise scoring while at the same time controlling for procedural accuracy by allowing comparison of replicate values. Cold whole blood samples could be held for at least one week without affecting reticulo-cyte, RETCD59−/CD55− or RBCCD59−/CD55− frequencies. Specimens from a total of 52 nonsmoking, self-reported healthy adult subjects were evaluated. The mean frequency of RETCD59−/CD55− and RBCCD592−/CD55− were 6.0 × 10−6 and 2.9 × 10−6, respectively. The difference is consistent with a modest selective pressure against mutant phenotype erythrocytes in the circulation, and suggests advantages of studying both populations of erythrocytes. Whereas intra-subject variability was low, inter-subject variability was relatively high, with RETCD59−/CD55− frequencies differing by more than 30-fold. There was an apparent correlation between age and mutant cell frequencies. Taken together, the results indicate that the frequency of human PIG-A mutant phenotype cells can be efficiently and reliably estimated using a labeling and analysis protocol that is well established for rodent-based studies. The applicability of the assay across species, its simplicity and statistical power, and the relatively non-invasive nature of the assay should benefit myriad research areas involving DNA damage

  4. Additional Human Papillomavirus Types Detected by the Hybrid Capture Tube Test among Samples from Women with Cytological and Colposcopical Atypia

    PubMed Central

    Kónya, József; Veress, György; Juhász, Attila; Szarka, Krisztina; Sápy, Tamás; Hernádi, Zoltán; Gergely, Lajos

    2000-01-01

    The type specificity of the human papillomavirus (HPV) Hybrid Capture Tube (HCT) test was evaluated by using typing with PCR (MY09-MY11)-restriction fragment length polymorphism (RFLP) and sequencing. All samples HCT test positive for only low-risk HPV (n = 15) or only high-risk HPV (n = 102) were confirmed, whereas 9 of 12 HCT test double-positive samples contained only high-risk HPV types as determined by PCR-RFLP. Several high-risk HPV types (HPV-53, -58, -62, -66, -CP8304, and -MM4) not included in the HCT test were indeed detected, indicating a broader detection range with retained distinction between low-risk and high-risk HPV types. PMID:10618127

  5. Electrofishing and the effects of depletion sampling on fish health: A review and recommendations for additional study

    USGS Publications Warehouse

    Panek, F.M.; Densmore, Christine L.; Cipriano, R.C.; Bruckner, A.W.; Shchelkunov, I.S.

    2011-01-01

    Depletion sampling in combination with multiple-pass electrofishing is an important fisheries management tool for wadeable streams. This combination of techniques has been used routinely by federal and state fishery management agencies for several decades as a reliable means to obtain quantitative data on trout populations or to describe fish community structure. In this paper we review the effects of electrofishing on fish and discuss this within the context of depletion sampling and multiple exposures of fishes to electric fields. The multiple wave forms most commonly used in sampling (alternating current, direct current, and pulsed direct current) are discussed as well as electrofishing induced response, injury and physiological stress. Fish that survive electrofishing injuries are more likely to suffer short and long-term adverse effects to their behavior, health, growth, or reproduction. Of greatest concern are the native, non-target species that may be subjected to multiple electrical shocks during the course of a 3-pass depletion survey. These exposures and their effects on the non-target species warrant further study as do the overall effects of electrofishing on populations and community structure. 

  6. Determination of organic additives in mortars by near-IR spectroscopy. A novel approach to designing a sample set with high-variability components.

    PubMed

    Blanco, Marcelo; Peguero, Anna

    2007-02-01

    Industrial mortars consist primarily of a mixture of cement and an aggregate plus a small amount of additives that are used to modify specific properties. Using too high or too low additive rates usually results in the loss of desirable properties in the end product. This entails carefully controlling the amounts of additives added to mortar in order to ensure correct dosing and/or adequate homogeneity in the final mixture. Near-IR (NIR) spectroscopy has proved effective for this purpose as it requires no sample pretreatment and affords expeditious analyses. The purpose of this work was to determine two organic additives (viz. Ad1 and Ad2) in mortars by using partial least squares regression multivariate calibration models constructed from NIR spectroscopic data. The additives are used to expedite setting and increase cohesion between particles in the mortar. In order to ensure that the sample set contained natural variability in the samples, we used a methodology based on experimental design to construct a representative set of samples. This novel design is based on a hexagonal antiprism that encompasses the concentration ranges spanned by the analytes and the variability inherent in each additive. The D-optimality criterion was used to obtain various combinations between Ad1 and Ad2 additive classes. The partial least squares calibration models thus constructed for each additive provided accurate predictions: the intercept and the slope of the plots of predicted values versus reference values for each additive were close to 0 and 1, respectively, and their confidence ranges included the respective value. The ensuing analytical methods were validated by using an external sample set.

  7. [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.

  8. New findings about trichodysplasia spinulosa-associated polyomavirus (TSPyV)--novel qPCR detects TSPyV-DNA in blood samples.

    PubMed

    Urbano, Paulo R; Nali, Luiz H S; Bicalho, Camila S; Pierrotti, Ligia C; David-Neto, Elias; Pannuti, Cláudio S; Romano, Camila M

    2016-02-01

    A new real-time PCR assay for trichodysplasia spinulosa-associated polyomavirus (TSPyV) DNA detection was designed, and blood samples from kidney transplant recipients and healthy individuals were screened. TSPyV-DNA was not detected in blood from healthy individuals, but 26.8% of kidney recipients presented TSPyV-DNA. This is the first report of TSPyV viremia.

  9. Isotope ratio measurements of iron in blood samples by multi-collector ICP-MS to support nutritional investigations in humans.

    PubMed

    Benkhedda, Karima; Chen, Heidi; Dabeka, Robert; Cockell, Kevin

    2008-05-01

    With the perspective of embarking on a human study using a double iron (Fe) stable isotope tracer protocol to assess iron bioavailability, investigations were conducted on Fe isotope ratios in blood samples using a VG Axiom Multi-collector ICP-MS. The factors affecting the precision and accuracy of Fe isotopic ratios, such as spectral- and matrix-induced interferences and Fe recoveries from sample preparation, have been identified and optimized. Major polyatomic interferences (e.g., Ar-O, Ar-OH, and FeH) were significantly reduced by using an Aridus nebulizer and desolvating system. Isobaric metal (e.g., (54)Cr(+) on (54)Fe(+) and (58)Ni(+) on (58)Fe(+)) interferences and Ca-oxides and hydroxides were quantitatively removed during chemical purification of blood samples and selective isolation of Fe by anion-exchange resin, after mineralization of the blood samples by microwave digestion. Quantitative recoveries of Fe from different steps of sample preparation were verified using whole blood reference material. Fe isotopic compositions of the samples were corrected for instrumental mass bias by the standard-sample bracketing method using the certified reference standard IRMM-014. External precisions on the order of 0.008-0.05 (% RSD), 0.007-0.015 (% RSD), and 0.03-0.09 (% RSD) were obtained for (54)Fe/(56)Fe, (57)Fe/(56)Fe, and (58)Fe/(56)Fe, respectively, in the blood for three replicate measurements. The level of precision obtained in this work enables the detection of low enrichments of Fe in blood, which is highly desired in nutrition tracer studies.

  10. Miniaturized blood sampling techniques to benefit reduction in mice and refinement in nonhuman primates: applications to bioanalysis in toxicity studies with antibody-drug conjugates.

    PubMed

    Caron, Alexis; Lelong, Christine; Pascual, Marie-Hélène; Benning, Véronique

    2015-03-01

    Minimizing the number of animals in regulatory toxicity studies while achieving study objectives to support the development of future medicines contributes to good scientific and ethical practices. Recent advances in technology have enabled the development of miniaturized blood sampling methods (including microsampling and dried blood spots) applicable to toxicokinetic determinations of small-molecule drugs. Implementation of miniaturized blood sampling methods in the context of biotherapeutic drugs is desirable because a limitation to this type of medicine remains the total blood volume needed from a single animal to support toxicokinetic determinations of several analytes (parent drug, metabolites[s], antidrug antibodies, and so forth). We describe here the technical details, applicability, and relevance of new miniaturized blood sampling procedures in mice and nonhuman primates in the context of the toxicologic evaluation of biotherapeutic drugs consisting of antibody-drug conjugates developed for oncology indications. These examples illustrate how these techniques can benefit the reduction of animal usage in mouse toxicity studies by decreasing the number of animals dedicated to toxicokinetic determinations and the refinement of practices in nonhuman primate toxicity studies by decreasing the blood volume repeatedly drawn for toxicokinetic determinations.

  11. Simultaneous determination of CRP and D-dimer in human blood plasma samples with White Light Reflectance Spectroscopy.

    PubMed

    Koukouvinos, Georgios; Petrou, Panagiota; Misiakos, Konstantinos; Drygiannakis, Dimitris; Raptis, Ioannis; Stefanitsis, Gerasimos; Martini, Spyridoula; Nikita, Dimitra; Goustouridis, Dimitrios; Moser, Isabella; Jobst, Gerhard; Kakabakos, Sotirios

    2016-10-15

    A dual-analyte assay for the simultaneous determination of C-reactive protein (CRP) and D-dimer in human blood plasma based on a white light interference spectroscopy sensing platform is presented. Measurement is accomplished in real-time by scanning the sensing surface, on which distinct antibody areas have been created, with a reflection probe used both for illumination of the surface and collection of the reflected interference spectrum. The composition of the transducer, the sensing surface chemical activation and biofunctionalization procedures were optimized with respect to signal magnitude and repeatability. The assay format involved direct detection of CRP whereas for D-dimer a two-site immunoassay employing a biotinylated reporter antibody and reaction with streptavidin was selected. The assays were sensitive with detection limits of 25ng/mL for both analytes, precise with intra- and inter-assay CV values ranging from 3.6% to 7.7%, and from 4.8% to 9.5%, respectively, for both assays, and accurate with recovery values ranging from 88.5% to 108% for both analytes. Moreover, the values determined for the two analytes in 35 human plasma samples were in excellent agreement with those received for the same samples by standard diagnostic laboratory instrumentation employing commercial kits. The excellent agreement of the results supported the validity of the proposed system for clinical application for the detection of multiple analytes since it was demonstrated that up to seven antibody areas can be created on the sensing surface and successfully interrogated with the developed optical set-up. PMID:26675113

  12. Advanced deep reactive-ion etching technology for hollow microneedles for transdermal blood sampling and drug delivery.

    PubMed

    Liu, Yufei; Eng, Pay F; Guy, Owen J; Roberts, Kerry; Ashraf, Huma; Knight, Nick

    2013-06-01

    Using an SPTS Technologies Ltd. Pegasus deep reactive-ion etching (DRIE) system, an advanced two-step etching process has been developed for hollow microneedles in applications of transdermal blood sampling and drug delivery. Because of the different etching requirements of both narrow deep hollow and large open cavity, hollow etch and cavity etch steps have been achieved separately. This novel two-step etching process is assisted with a bi-layer etching mask. Results show that the etch rate of silicon during this hollow etch step was about 7.5 microm/min and the etch rate of silicon during this cavity etch step was about 8-10 microm/min, using the coil plasma etching power between 2.0 and 2.8 kW. Especially for the microneedle bores etch, the deeper it etched, the slower the etch rate was. The microneedle bores have successfully been obtained 75-150 microm in inner diametre and 700-1000 microm long with high aspect ratio DRIE, meanwhile, the vertical sidewall structures have been achieved with the high etch load exposed area over 70% for the cavity etch step. PMID:24046906

  13. Methylene chloride intoxication in a furniture refinisher. A comparison of exposure estimates utilizing workplace air sampling and blood carboxyhemoglobin measurements

    SciTech Connect

    Shusterman, D.; Quinlan, P.; Lowengart, R.; Cone, J. )

    1990-05-01

    A 35-year-old furniture refinisher came to the occupational medicine clinic with complaints of upper respiratory irritation, fatigue, and lightheadedness occurring on a daily basis after using a methylene chloride-containing paint stripper. Determinations of blood carboxyhemoglobin (COHb) on three occasions showed an apparently linear elevation of COHb as a function of hours worked on the day of sampling. COHb levels predicted from spot industrial hygiene measurements were in close concordance with those observed in the patient, indicating the potential usefulness of COHb monitoring in estimating airborne exposure levels. Methylene chloride (or dichloromethane) is an organic solvent that has found wide use as a degreaser, paint remover, aerosol propellant, and a blowing agent for polyurethane foams, and as a solvent in food processing, photographic film production, and plastics manufacturing. Discovery of its unusual metabolic fate--conversion to carbon monoxide in vivo--has earned the compound a special place in the solvent toxicology literature. Demonstration of oncogenicity in experimental animals has occasioned a reconsideration of exposure limits, with emphasis upon stricter controls. In some workplaces, conditions prevail in which controls are inadequate to prevent even acute toxicity, much less long-term exposure risks.

  14. Non-enzymatic amperometric detection of hydrogen peroxide in human blood serum samples using a modified silver nanowire electrode.

    PubMed

    Thirumalraj, Balamurugan; Zhao, Duo-Han; Chen, Shen-Ming; Palanisamy, Selvakumar

    2016-05-15

    In this paper, we report a highly sensitive amperometric H2O2 sensor based on silver nanowires (AgNWs) modified screen printed carbon electrode. The AgNWs were synthesized using polyol method. The synthesized AgNWs were characterized by scanning electron microscopy, UV-vis spectroscopy and X-ray diffraction techniques. The average diameter and length of the synthesized AgNWs were found as 86±5 and 385nm, respectively. Under optimum conditions, the AgNWs modified electrode shows a stable amperometric response for H2O2 and was linear over the concentrations ranging from 0.3 to 704.8μM. The non-enzymatic sensor showed a high sensitivity of 662.6μAmM(-1)cm(-2) with a detection limit of 29nM. The response time of the sensor was found as 2s. Furthermore, the AgNWs modified electrode exhibited a good recovery of H2O2 (94.3%) in the human blood serum samples.

  15. DDT, DDE, and 1-hydroxypyrene levels in children (in blood and urine samples) from Chiapas and Oaxaca, Mexico.

    PubMed

    Pérez-Maldonado, Iván N; Trejo-Acevedo, Antonio; Pruneda-Alvarez, Lucia Guadalupe; Gaspar-Ramirez, Octavio; Ruvalcaba-Aranda, Selene; Perez-Vazquez, Francisco Javier

    2013-11-01

    The aim of this study was to evaluate the DDT, DDE, and 1-hydroxypyrene exposure levels of children living in communities located in southeastern Mexico. The study communities were Lacanja and Victoria in Chiapas state and Ventanilla in Oaxaca state. Children living in Lacanja had total blood DDT levels (mean ± SD, 29,039.6 ± 11,261.4 ng/g lipid) that were significantly higher than those of children in Victoria (10,220.5 ± 7,893.1 ng/g lipid) and Ventanilla (11,659.7 ± 6,683.7 ng/g lipid). With respect to the 1-hydroxypyrene levels in urine samples, the levels in Lacanja (4.8 ± 4.1 μg/L or 4.5 ± 3.9 μmol/mol creatinine) and Victoria (4.6 ± 3.8 μg/L or 3.9 ± 3.0 μmol/mol Cr) were significantly higher than levels found in Ventanilla (3.6 ± 1.4 μg/L or 2.5 ± 0.5 μmol/mol Cr). In conclusion, our data indicate high levels of exposure in children living in the communities studied in this work. The evidence found in this study could be further used as a trigger to revisit local policies on environmental exposures.

  16. Evaluating the effect of sample type on American alligator (Alligator mississippiensis) analyte values in a point-of-care blood analyser.

    PubMed

    Hamilton, Matthew T; Finger, John W; Winzeler, Megan E; Tuberville, Tracey D

    2016-01-01

    The assessment of wildlife health has been enhanced by the ability of point-of-care (POC) blood analysers to provide biochemical analyses of non-domesticated animals in the field. However, environmental limitations (e.g. temperature, atmospheric humidity and rain) and lack of reference values may inhibit researchers from using such a device with certain wildlife species. Evaluating the use of alternative sample types, such as plasma, in a POC device may afford researchers the opportunity to delay sample analysis and the ability to use banked samples. In this study, we examined fresh whole blood, fresh plasma and frozen plasma (sample type) pH, partial pressure of carbon dioxide (PCO2), bicarbonate (HCO3 (-)), total carbon dioxide (TCO2), base excess (BE), partial pressure of oxygen (PO2), oxygen saturation (sO2) and lactate concentrations in 23 juvenile American alligators (Alligator mississippiensis) using an i-STAT CG4+ cartridge. Our results indicate that sample type had no effect on lactate concentration values (F 2,65 = 0.37, P = 0.963), suggesting that the i-STAT analyser can be used reliably to quantify lactate concentrations in fresh and frozen plasma samples. In contrast, the other seven blood parameters measured by the CG4+ cartridge were significantly affected by sample type. Lastly, we were able to collect blood samples from all alligators within 2 min of capture to establish preliminary reference ranges for juvenile alligators based on values obtained using fresh whole blood. PMID:27382469

  17. Evaluating the effect of sample type on American alligator (Alligator mississippiensis) analyte values in a point-of-care blood analyser

    SciTech Connect

    Hamilton, Matthew T.; Finger, John W.; Winzeler, Megan E.; Tuberville, Tracey D.

    2016-01-01

    The assessment of wildlife health has been enhanced by the ability of point-of-care (POC) blood analysers to provide biochemical analyses of non-domesticated animals in the field. However, environmental limitations (e.g. temperature, atmospheric humidity and rain) and lack of reference values may inhibit researchers from using such a device with certain wildlife species. Evaluating the use of alternative sample types, such as plasma, in a POC device may afford researchers the opportunity to delay sample analysis and the ability to use banked samples. In this study, we examined fresh whole blood, fresh plasma and frozen plasma (sample type) pH, partial pressure of carbon dioxide (PCO2), bicarbonate (HCO3₋), total carbon dioxide (TCO2), base excess (BE), partial pressure of oxygen (PO2), oxygen saturation (sO2) and lactate concentrations in 23 juvenile American alligators (Alligator mississippiensis) using an i-STAT CG4+ cartridge. Our results indicate that sample type had no effect on lactate concentration values (F2,65 = 0.37, P = 0.963), suggesting that the i-STAT analyser can be used reliably to quantify lactate concentrations in fresh and frozen plasma samples. In contrast, the other seven blood parameters measured by the CG4+ cartridge were significantly affected by sample type. In conclusion, we were able to collect blood samples from all alligators within 2 min of capture to establish preliminary reference ranges for juvenile alligators based on values obtained using fresh whole blood.

  18. Evaluating the effect of sample type on American alligator (Alligator mississippiensis) analyte values in a point-of-care blood analyser

    DOE PAGES

    Hamilton, Matthew T.; Finger, John W.; Winzeler, Megan E.; Tuberville, Tracey D.

    2016-01-01

    The assessment of wildlife health has been enhanced by the ability of point-of-care (POC) blood analysers to provide biochemical analyses of non-domesticated animals in the field. However, environmental limitations (e.g. temperature, atmospheric humidity and rain) and lack of reference values may inhibit researchers from using such a device with certain wildlife species. Evaluating the use of alternative sample types, such as plasma, in a POC device may afford researchers the opportunity to delay sample analysis and the ability to use banked samples. In this study, we examined fresh whole blood, fresh plasma and frozen plasma (sample type) pH, partial pressuremore » of carbon dioxide (PCO2), bicarbonate (HCO3₋), total carbon dioxide (TCO2), base excess (BE), partial pressure of oxygen (PO2), oxygen saturation (sO2) and lactate concentrations in 23 juvenile American alligators (Alligator mississippiensis) using an i-STAT CG4+ cartridge. Our results indicate that sample type had no effect on lactate concentration values (F2,65 = 0.37, P = 0.963), suggesting that the i-STAT analyser can be used reliably to quantify lactate concentrations in fresh and frozen plasma samples. In contrast, the other seven blood parameters measured by the CG4+ cartridge were significantly affected by sample type. In conclusion, we were able to collect blood samples from all alligators within 2 min of capture to establish preliminary reference ranges for juvenile alligators based on values obtained using fresh whole blood.« less

  19. Evaluating the effect of sample type on American alligator (Alligator mississippiensis) analyte values in a point-of-care blood analyser

    PubMed Central

    Hamilton, Matthew T.; Finger, John W.; Winzeler, Megan E.; Tuberville, Tracey D.

    2016-01-01

    The assessment of wildlife health has been enhanced by the ability of point-of-care (POC) blood analysers to provide biochemical analyses of non-domesticated animals in the field. However, environmental limitations (e.g. temperature, atmospheric humidity and rain) and lack of reference values may inhibit researchers from using such a device with certain wildlife species. Evaluating the use of alternative sample types, such as plasma, in a POC device may afford researchers the opportunity to delay sample analysis and the ability to use banked samples. In this study, we examined fresh whole blood, fresh plasma and frozen plasma (sample type) pH, partial pressure of carbon dioxide (PCO2), bicarbonate (HCO3−), total carbon dioxide (TCO2), base excess (BE), partial pressure of oxygen (PO2), oxygen saturation (sO2) and lactate concentrations in 23 juvenile American alligators (Alligator mississippiensis) using an i-STAT CG4+ cartridge. Our results indicate that sample type had no effect on lactate concentration values (F2,65 = 0.37, P = 0.963), suggesting that the i-STAT analyser can be used reliably to quantify lactate concentrations in fresh and frozen plasma samples. In contrast, the other seven blood parameters measured by the CG4+ cartridge were significantly affected by sample type. Lastly, we were able to collect blood samples from all alligators within 2 min of capture to establish preliminary reference ranges for juvenile alligators based on values obtained using fresh whole blood. PMID:27382469

  20. Expression and methylation data from SLE patient and healthy control blood samples subdivided with respect to ARID3a levels.

    PubMed

    Ward, Julie M; Ratliff, Michelle L; Dozmorov, Mikhail G; Wiley, Graham; Guthridge, Joel M; Gaffney, Patrick M; James, Judith A; Webb, Carol F

    2016-12-01

    Previously published studies revealed that variation in expression of the DNA-binding protein ARID3a in B lymphocytes from patients with systemic lupus erythematosus (SLE) correlated with levels of disease activity ("Disease activity in systemic lupus erythematosus correlates with expression of the transcription factor AT-rich-interactive domain 3A" (J.M. Ward, K. Rose, C. Montgomery, I. Adrianto, J.A. James, J.T. Merrill et al., 2014) [1]). The data presented here compare DNA methylation patterns from SLE peripheral blood mononuclear cells obtained from samples with high numbers of ARID3a expressing B cells (ARID3a(H)) versus SLE samples with normal numbers of ARID3a(+) B cells (ARID3a(N)). The methylation data is available at the gene expression omnibus (GEO) repository, "Gene Expression Omnibus: NCBI gene expression and hybridization array data repository" (R. Edgar, M. Domrachev, A.E. Lash, 2002) [2]. Isolated B cells from SLE ARID3a(H) and ARID3a(N) B samples were also evaluated via qRT-PCR for Type I interferon (IFN) signature and pathway gene expression levels by qRT-PCR. Similarly, healthy control B cells and B cells stimulated to express ARID3a with the TLR agonist, CpG, were also compared via qRT-PCR. Primers designed to detect 6 IFNa subtype mRNAs were tested in 4 IFNa, Epstein-Barr Virus-transformed B cell lines ("Reduced interferon-alpha production by Epstein-Barr virus transformed B-lymphoblastoid cell lines and lectin-stimulated lymphocytes in congenital dyserythropoietic anemia type I" (S.H. Wickramasinghe, R. Hasan, J. Smythe, 1997) [3]). The data in this article support the publication, "Human effector B lymphocytes express ARID3a and secrete interferon alpha" (J.M. Ward, M.L. Ratliff, M.G. Dozmorov, G. Wiley, J.M. Guthridge, P.M. Gaffney, J.A. James, C.F. Webb, 2016) [4]. PMID:27656675

  1. Cross-institute evaluations of inhibitor-resistant PCR reagents for direct testing of aerosol and blood samples containing biological warfare agent DNA.

    PubMed

    Minogue, Timothy D; Rachwal, Phillip A; Trombley Hall, Adrienne; Koehler, Jeffery W; Weller, Simon A

    2014-02-01

    Rapid pathogen detection is crucial for the timely introduction of therapeutics. Two groups (one in the United Kingdom and one in the United States) independently evaluated inhibitor-resistant PCR reagents for the direct testing of substrates. In the United Kingdom, a multiplexed Bacillus anthracis (target) and Bacillus subtilis (internal-control) PCR was used to evaluate 4 reagents against 5 PCR inhibitors and down-selected the TaqMan Fast Virus 1-Step master mix (Life Technologies Inc.). In the United States, four real-time PCR assays (targeting B. anthracis, Brucella melitensis, Venezuelan equine encephalitis virus [VEEV], and Orthopoxvirus spp.) were used to evaluate 5 reagents (plus the Fast Virus master mix) against buffer, blood, and soil samples and down-selected the KAPA Blood Direct master mix (KAPA Biosystems Inc.) with added Platinum Taq (Life Technologies). The down-selected reagents underwent further testing. In the United Kingdom experiments, both reagents were tested against seven contrived aerosol collector samples containing B. anthracis Ames DNA and B. subtilis spores from a commercial formulation (BioBall). In PCR assays with reaction mixtures containing 40% crude sample, an airfield-collected sample induced inhibition of the B. subtilis PCR with the KAPA reagent and complete failure of both PCRs with the Fast Virus reagent. However, both reagents allowed successful PCR for all other samples-which inhibited PCRs with a non-inhibitor-resistant reagent. In the United States, a cross-assay limit-of-detection (LoD) study in blood was conducted. The KAPA Blood Direct reagent allowed the detection of agent DNA (by four PCRs) at higher concentrations of blood in the reaction mixture (2.5%) than the Fast Virus reagent (0.5%), although LoDs differed between assays and reagent combinations. Across both groups, the KAPA Blood Direct reagent was determined to be the optimal reagent for inhibition relief in PCR.

  2. Application of mRNA Expression Analysis to Human Blood Identification in Degenerated Samples that were False-negative by Immunochromatography(,) (.).

    PubMed

    Matsumura, Shusaku; Matsusue, Aya; Waters, Brian; Kashiwagi, Masayuki; Hara, Kenji; Kubo, Shin-Ichi

    2016-07-01

    Forensic laboratories are often faced with cases in which methamphetamine hydrochloride-mixed blood is unable to be identified as human blood by immunochromatography against human hemoglobin A0. The application of mRNA expression analysis to samples that showed a false-negative with immunochromatography was investigated as an alternative approach that did not depend on the antigen-antibody reaction. Real-time PCR was used to examine the expression levels of blood markers such as glycophorin A, spectrin beta, and hemoglobin beta. Hemoglobin beta was the only marker that was specifically detected in blood, while glycophorin A was useful for determining human specificity. Hemoglobin beta showed good detection sensitivity and was detectable in 37-year-old blood stains. Hemoglobin beta was exclusively detectable in methamphetamine hydrochloride-mixed blood stains. Detergents and disinfectants did not significantly influence mRNA markers. The proposed mRNA expression analysis was suitable for human blood identification as an alternative method to immunochromatography. PMID:27364269

  3. Percent recoveries of anthropogenic organic compounds with and without the addition of ascorbic acid to preserve finished-water samples containing free chlorine, 2004-10

    USGS Publications Warehouse

    Valder, Joshua F.; Delzer, Gregory C.; Bender, David A.; Price, Curtis V.

    2011-01-01

    This report presents finished-water matrix-spike recoveries of 270 anthropogenic organic compounds with and without the addition of ascorbic acid to preserve water samples containing free chlorine. Percent recoveries were calculated using analytical results from a study conducted during 2004-10 for the National Water-Quality Assessment (NAWQA) Program of the U.S. Geological Survey (USGS). The study was intended to characterize the effect of quenching on finished-water matrix-spike recoveries and to better understand the potential oxidation and transformation of 270 anthropogenic organic compounds. The anthropogenic organic compounds studied include those on analytical schedules 1433, 2003, 2033, 2060, 2020, and 4024 of the USGS National Water Quality Laboratory. Three types of samples were collected from 34 NAWQA locations across the Nation: (1) quenched finished-water samples (not spiked), (2) quenched finished-water matrix-spike samples, and (3) nonquenched finished-water matrix-spike samples. Percent recoveries of anthropogenic organic compounds in quenched and nonquenched finished-water matrix-spike samples are presented. Comparisons of percent recoveries between quenched and nonquenched spiked samples can be used to show how quenching affects finished-water samples. A maximum of 18 surface-water and 34 groundwater quenched finished-water matrix-spike samples paired with nonquenched finished-water matrix-spike samples were analyzed. Percent recoveries for the study are presented in two ways: (1) finished-water matrix-spike samples supplied by surface-water or groundwater, and (2) by use (or source) group category for surface-water and groundwater supplies. Graphical representations of percent recoveries for the quenched and nonquenched finished-water matrix-spike samples also are presented.

  4. Development of a Fibrinogen-Specific Sandwich Enzyme-Linked Immunosorbent Assay Microarray Assay for Distinguishing Between Blood Plasma and Serum Samples

    SciTech Connect

    Gonzales, Rachel M.; Zhang, Qibin; Zangar, Richard C.; Smith, Richard D.; Metz, Thomas O.

    2011-07-01

    We have developed a fibrinogen-specific sandwich ELISA microarray assay for use in qualitatively distinguishing between blood plasma and serum samples. Three capture antibodies, 49D2, HPA001900, and F8512, were evaluated in conjunction with 1D6 as detection antibody, and the data show that 49D2 and, to a lesser extent, F8512 successfully identify previously unknown plasma and serum samples based upon a ~28-fold difference in signal intensity between the sample types. This assay has utility in rapidly identifying previously archived clinical samples with incomplete annotation in a high throughput manner prior to proteomics analyses.

  5. Study Design and Percent Recoveries of Anthropogenic Organic Compounds With and Without the Addition of Ascorbic Acid to Preserve Water Samples Containing Free Chlorine, 2004-06

    USGS Publications Warehouse

    Valder, Joshua F.; Delzer, Gregory C.; Price, Curtis V.; Sandstrom, Mark W.

    2008-01-01

    The National Water-Quality Assessment (NAWQA) Program of the U.S. Geological Survey (USGS) began implementing Source Water-Quality Assessments (SWQAs) in 2002 that focus on characterizing the quality of source water and finished water of aquifers and major rivers used by some of the larger community water systems in the United States. As used for SWQA studies, source water is the raw (ambient) water collected at the supply well prior to water treatment (for ground water) or the raw (ambient) water collected from the river near the intake (for surface water). Finished water is the water that is treated, which typically involves, in part, the addition of chlorine or other disinfection chemicals to remove pathogens, and is ready to be delivered to consumers. Finished water is collected before the water enters the distribution system. This report describes the study design and percent recoveries of anthropogenic organic compounds (AOCs) with and without the addition of ascorbic acid to preserve water samples containing free chlorine. The percent recoveries were determined by using analytical results from a laboratory study conducted in 2004 by the USGS's National Water Quality Laboratory (NWQL) and from data collected during 2004-06 for a field study currently (2008) being conducted by the USGS's NAWQA Program. The laboratory study was designed to determine if preserving samples with ascorbic acid (quenching samples) adversely affects analytical performance under controlled conditions. During the laboratory study, eight samples of reagent water were spiked for each of five analytical schedules evaluated. Percent recoveries from these samples were then compared in two ways: (1) four quenched reagent spiked samples analyzed on day 0 were compared with four quenched reagent spiked samples analyzed on day 7 or 14, and (2) the combined eight quenched reagent spiked samples analyzed on day 0, 7, or 14 were compared with eight laboratory reagent spikes (LRSs). Percent

  6. Chromosome aberrations induced in human lymphocytes by U-235 fission neutrons: I. Irradiation of human blood samples in the "dry cell" of the TRIGA Mark II nuclear reactor.

    PubMed

    Fajgelj, A; Lakoski, A; Horvat, D; Remec, I; Skrk, J; Stegnar, P

    1991-11-01

    A set-up for irradiation of biological samples in the TRIGA Mark II research reactor in Ljubljana is described. Threshold activation detectors were used for characterisation of the neutron flux, and the accompanying gamma dose was measured by TLDs. Human peripheral blood samples were irradiated "in vitro" and biological effects evaluated according to the unstable chromosomal aberrations induced. Biological effects of two types of cultivation of irradiated blood samples, the first immediately after irradiation and the second after 96 h storage, were studied. A significant difference in the incidence of chromosomal aberrations between these two types of samples was obtained, while our dose-response curve fitting coefficients alpha 1 = (7.71 +/- 0.09) x 10(-2) Gy-1 (immediate cultivation) and alpha 2 = (11.03 +/- 0.08) x 10(-2) Gy-1 (96 h delayed cultivation) are in both cases lower than could be found in the literature.

  7. Chromosome aberrations induced in human lymphocytes by U-235 fission neutrons: I. Irradiation of human blood samples in the "dry cell" of the TRIGA Mark II nuclear reactor.

    PubMed

    Fajgelj, A; Lakoski, A; Horvat, D; Remec, I; Skrk, J; Stegnar, P

    1991-11-01

    A set-up for irradiation of biological samples in the TRIGA Mark II research reactor in Ljubljana is described. Threshold activation detectors were used for characterisation of the neutron flux, and the accompanying gamma dose was measured by TLDs. Human peripheral blood samples were irradiated "in vitro" and biological effects evaluated according to the unstable chromosomal aberrations induced. Biological effects of two types of cultivation of irradiated blood samples, the first immediately after irradiation and the second after 96 h storage, were studied. A significant difference in the incidence of chromosomal aberrations between these two types of samples was obtained, while our dose-response curve fitting coefficients alpha 1 = (7.71 +/- 0.09) x 10(-2) Gy-1 (immediate cultivation) and alpha 2 = (11.03 +/- 0.08) x 10(-2) Gy-1 (96 h delayed cultivation) are in both cases lower than could be found in the literature. PMID:1962281

  8. Cross-Institute Evaluations of Inhibitor-Resistant PCR Reagents for Direct Testing of Aerosol and Blood Samples Containing Biological Warfare Agent DNA

    PubMed Central

    Minogue, Timothy D.; Rachwal, Phillip A.; Trombley Hall, Adrienne; Koehler, Jeffery W.

    2014-01-01

    Rapid pathogen detection is crucial for the timely introduction of therapeutics. Two groups (one in the United Kingdom and one in the United States) independently evaluated inhibitor-resistant PCR reagents for the direct testing of substrates. In the United Kingdom, a multiplexed Bacillus anthracis (target) and Bacillus subtilis (internal-control) PCR was used to evaluate 4 reagents against 5 PCR inhibitors and down-selected the TaqMan Fast Virus 1-Step master mix (Life Technologies Inc.). In the United States, four real-time PCR assays (targeting B. anthracis, Brucella melitensis, Venezuelan equine encephalitis virus [VEEV], and Orthopoxvirus spp.) were used to evaluate 5 reagents (plus the Fast Virus master mix) against buffer, blood, and soil samples and down-selected the KAPA Blood Direct master mix (KAPA Biosystems Inc.) with added Platinum Taq (Life Technologies). The down-selected reagents underwent further testing. In the United Kingdom experiments, both reagents were tested against seven contrived aerosol collector samples containing B. anthracis Ames DNA and B. subtilis spores from a commercial formulation (BioBall). In PCR assays with reaction mixtures containing 40% crude sample, an airfield-collected sample induced inhibition of the B. subtilis PCR with the KAPA reagent and complete failure of both PCRs with the Fast Virus reagent. However, both reagents allowed successful PCR for all other samples—which inhibited PCRs with a non-inhibitor-resistant reagent. In the United States, a cross-assay limit-of-detection (LoD) study in blood was conducted. The KAPA Blood Direct reagent allowed the detection of agent DNA (by four PCRs) at higher concentrations of blood in the reaction mixture (2.5%) than the Fast Virus reagent (0.5%), although LoDs differed between assays and reagent combinations. Across both groups, the KAPA Blood Direct reagent was determined to be the optimal reagent for inhibition relief in PCR. PMID:24334660

  9. Evaluation of carbon monoxide in blood samples from the second national Health and Nutrition Survey. Progress report, April 1, 1979-April 1, 1980

    SciTech Connect

    Radford, E.P.

    1980-01-01

    Progress is reported on research into the epidemiology of CO. CO levels in blood have been determined from 8052 samples. Smoking histories for these subjects are becoming available. The magnitude of the impact of environmental sources distinct from smoking is being evaluated. (ACR)

  10. Comparison of Measurements of 12 Analytes in Equine Blood Samples Using the In-Practice Falcor 350 and the Reference KoneLab 30i Analysers

    PubMed Central

    Papasouliotis, K.; Tennant, K. V.; Dodkin, S.; Mason, J.

    2012-01-01

    Falcor 350 is a wet-reagent biochemistry analyser that is available for in-house use. The aim of this study was to compare the results produced by this analyser with those obtained by the KoneLab 30i that served as the reference instrument. Blood samples from 60 clinical cases were analysed for urea, creatinine, total proteins, albumin, creatine kinase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, total calcium, phosphate, sodium, and potassium using both instruments. Good to excellent correlations (rs value) value) were identified for creatinine (0.88), total proteins (0.92), albumin (0.93), creatine kinase (0.98), aspartate aminotransferase (0.98), alkaline phosphatase (0.94), total bilirubin (0.98), phosphate (0.95), and potassium (0.97). The correlations for total calcium (0.71), sodium (0.68), and urea (0.64) were fair. For albumin, aspartate aminotransferase, creatine kinase, phosphate, potassium, total bilirubin, creatinine, and total proteins, the two instruments produce values that are closely related to each other and are sufficiently similar to allow them to be used interchangeably without the need for additional correction factor computations. Because of differences in the methodologies, the Falcor results for alkaline phosphatase, total calcium, and sodium cannot be used interchangeably and should be interpreted using reference intervals established from the Falcor analyser. PMID:23762583

  11. Sensitive and selective spectrophotometric assay of piroxicam in pure form, capsule and human blood serum samples via ion-pair complex formation

    NASA Astrophysics Data System (ADS)

    Alizadeh, Nina; Keyhanian, Fereshteh

    2014-09-01

    A simple, accurate and highly sensitive spectrophotometric method has been developed for the rapid determination of piroxicam (PX) in pure and pharmaceutical formulations. The proposed method involves formation of stable yellow colored ion-pair complexes of the amino derivative (basic nitrogen) of PX with three sulphonphthalein acid dyes namely; bromocresol green (BCG), bromothymol blue (BTB), bromophenol blue (BPB) in acidic medium. The colored species exhibited absorption maxima at 438, 429 and 432 nm with molar absorptivity values of 9.400 × 103, 1.218 × 103 and 1.02 × 104 L mol-1 cm-1 for PX-BCG, PX-BTB and PX-BPB complexes, respectively. The effect of optimum conditions via acidity, reagent concentration, time and solvent were studied. The reactions were extremely rapid at room temperature and the absorbance values remained constant for 48 h. Beer’s law was obeyed with a good correlation coefficient in the concentration ranges 1-100 μg mL-1 for BCG, BTB complexes and 1-95 μg mL-1 for BPB complex. The composition ratio of the ion-pair complexes were found to be 1:1 in all cases as established by Job’s method. No interference was observed from common additives and excipients which may be present in the pharmaceutical preparations. The proposed method was successfully applied for the determination of PX in capsule and human blood serum samples with good accuracy and precision.

  12. Ultra-fast local-haplotype variant calling using paired-end DNA-sequencing data reveals somatic mosaicism in tumor and normal blood samples

    PubMed Central

    Sengupta, Subhajit; Gulukota, Kamalakar; Zhu, Yitan; Ober, Carole; Naughton, Katherine; Wentworth-Sheilds, William; Ji, Yuan

    2016-01-01

    Somatic mosaicism refers to the existence of somatic mutations in a fraction of somatic cells in a single biological sample. Its importance has mainly been discussed in theory although experimental work has started to emerge linking somatic mosaicism to disease diagnosis. Through novel statistical modeling of paired-end DNA-sequencing data using blood-derived DNA from healthy donors as well as DNA from tumor samples, we present an ultra-fast computational pipeline, LocHap that searches for multiple single nucleotide variants (SNVs) that are scaffolded by the same reads. We refer to scaffolded SNVs as local haplotypes (LH). When an LH exhibits more than two genotypes, we call it a local haplotype variant (LHV). The presence of LHVs is considered evidence of somatic mosaicism because a genetically homogeneous cell population will not harbor LHVs. Applying LocHap to whole-genome and whole-exome sequence data in DNA from normal blood and tumor samples, we find wide-spread LHVs across the genome. Importantly, we find more LHVs in tumor samples than in normal samples, and more in older adults than in younger ones. We confirm the existence of LHVs and somatic mosaicism by validation studies in normal blood samples. LocHap is publicly available at http://www.compgenome.org/lochap. PMID:26420835

  13. Ultra-fast local-haplotype variant calling using paired-end DNA-sequencing data reveals somatic mosaicism in tumor and normal blood samples.

    PubMed

    Sengupta, Subhajit; Gulukota, Kamalakar; Zhu, Yitan; Ober, Carole; Naughton, Katherine; Wentworth-Sheilds, William; Ji, Yuan

    2016-02-18

    Somatic mosaicism refers to the existence of somatic mutations in a fraction of somatic cells in a single biological sample. Its importance has mainly been discussed in theory although experimental work has started to emerge linking somatic mosaicism to disease diagnosis. Through novel statistical modeling of paired-end DNA-sequencing data using blood-derived DNA from healthy donors as well as DNA from tumor samples, we present an ultra-fast computational pipeline, LocHap that searches for multiple single nucleotide variants (SNVs) that are scaffolded by the same reads. We refer to scaffolded SNVs as local haplotypes (LH). When an LH exhibits more than two genotypes, we call it a local haplotype variant (LHV). The presence of LHVs is considered evidence of somatic mosaicism because a genetically homogeneous cell population will not harbor LHVs. Applying LocHap to whole-genome and whole-exome sequence data in DNA from normal blood and tumor samples, we find wide-spread LHVs across the genome. Importantly, we find more LHVs in tumor samples than in normal samples, and more in older adults than in younger ones. We confirm the existence of LHVs and somatic mosaicism by validation studies in normal blood samples. LocHap is publicly available at http://www.compgenome.org/lochap. PMID:26420835

  14. Evaluation of Trapper-Collected Nobuto Filter-Paper Blood Samples for Distemper and Parvovirus Antibody Detection in Coyotes (Canis latrans) and Raccoons (Procyon lotor).

    PubMed

    Kamps, Amanda J; Dubay, Shelli A; Langenberg, Julie; Maes, Roger K

    2015-07-01

    Blood samples are often collected from free-ranging wildlife for antibody detection. However, filter-paper (FP) strips are more cost efficient and easy to collect and store. We evaluated trapper-collected FP strips and body-cavity blood for canine distemper (CDV) and parvovirus (CPV-2) antibody detection in raccoons (Procyon lotor) and coyotes (Canis latrans). From 2008 to 2010, licensed trappers near Madison and Milwaukee, Wisconsin, US collected paired samples from harvested animals. Canine distemper antibodies were detected using virus neutralization and parvovirus antibodies were detected using hemagglutination inhibition. Titers ≥ 1:32 for CDV and ≥ 1:25 for CPV-2 were considered evidence of exposure. Using Cohen's kappa test of agreement, FP strip titers agreed with sera for CDV in coyotes (n = 28, K = 0.772) and raccoons (n = 29, K = 0.858) and for CPV-2 in coyotes (n = 40, K = 0.775) and raccoons (n = 70, K = 0.646). However, raccoons determined to be exposed to CPV-2 from sera were unexposed by FP strips in 35% of the samples. Titer results may be affected by quality and volume of blood samples, interval between collection and processing, small sample sizes, and diagnostic testing procedures. Filter-paper strips can be useful for detecting CDV and CPV-2 exposure in coyotes and raccoons with correct field sample collection and appropriate diagnostic testing procedures.

  15. Evaluation of Trapper-Collected Nobuto Filter-Paper Blood Samples for Distemper and Parvovirus Antibody Detection in Coyotes (Canis latrans) and Raccoons (Procyon lotor).

    PubMed

    Kamps, Amanda J; Dubay, Shelli A; Langenberg, Julie; Maes, Roger K

    2015-07-01

    Blood samples are often collected from free-ranging wildlife for antibody detection. However, filter-paper (FP) strips are more cost efficient and easy to collect and store. We evaluated trapper-collected FP strips and body-cavity blood for canine distemper (CDV) and parvovirus (CPV-2) antibody detection in raccoons (Procyon lotor) and coyotes (Canis latrans). From 2008 to 2010, licensed trappers near Madison and Milwaukee, Wisconsin, US collected paired samples from harvested animals. Canine distemper antibodies were detected using virus neutralization and parvovirus antibodies were detected using hemagglutination inhibition. Titers ≥ 1:32 for CDV and ≥ 1:25 for CPV-2 were considered evidence of exposure. Using Cohen's kappa test of agreement, FP strip titers agreed with sera for CDV in coyotes (n = 28, K = 0.772) and raccoons (n = 29, K = 0.858) and for CPV-2 in coyotes (n = 40, K = 0.775) and raccoons (n = 70, K = 0.646). However, raccoons determined to be exposed to CPV-2 from sera were unexposed by FP strips in 35% of the samples. Titer results may be affected by quality and volume of blood samples, interval between collection and processing, small sample sizes, and diagnostic testing procedures. Filter-paper strips can be useful for detecting CDV and CPV-2 exposure in coyotes and raccoons with correct field sample collection and appropriate diagnostic testing procedures. PMID:25973631

  16. Novel system using microliter order sample volume for measuring arterial radioactivity concentrations in whole blood and plasma for mouse PET dynamic study.

    PubMed

    Kimura, Yuichi; Seki, Chie; Hashizume, Nobuya; Yamada, Takashi; Wakizaka, Hidekatsu; Nishimoto, Takahiro; Hatano, Kentaro; Kitamura, Keishi; Toyama, Hiroshi; Kanno, Iwao

    2013-11-21

    This study aimed to develop a new system, named CD-Well, for mouse PET dynamic study. CD-Well allows the determination of time-activity curves (TACs) for arterial whole blood and plasma using 2-3 µL of blood per sample; the minute sample size is ideal for studies in small animals. The system has the following merits: (1) measures volume and radioactivity of whole blood and plasma separately; (2) allows measurements at 10 s intervals to capture initial rapid changes in the TAC; and (3) is compact and easy to handle, minimizes blood loss from sampling, and delay and dispersion of the TAC. CD-Well has 36 U-shaped channels. A drop of blood is sampled into the opening of the channel and stored there. After serial sampling is completed, CD-Well is centrifuged and scanned using a flatbed scanner to define the regions of plasma and blood cells. The length measured is converted to volume because the channels have a precise and uniform cross section. Then, CD-Well is exposed to an imaging plate to measure radioactivity. Finally, radioactivity concentrations are computed. We evaluated the performance of CD-Well in in vitro measurement and in vivo (18)F-fluorodeoxyglucose and [(11)C]2-carbomethoxy-3β-(4-fluorophenyl) tropane studies. In in vitro evaluation, per cent differences (mean±SE) from manual measurement were 4.4±3.6% for whole blood and 4.0±3.5% for plasma across the typical range of radioactivity measured in mouse dynamic study. In in vivo studies, reasonable TACs were obtained. The peaks were captured well, and the time courses coincided well with the TAC derived from PET imaging of the heart chamber. The total blood loss was less than 200 µL, which had no physiological effect on the mice. CD-Well demonstrates satisfactory performance, and is useful for mouse PET dynamic study.

  17. Fast Screening of Whole Blood Samples and Pharmaceutical Compounds for Enantiorecognition of Free L-T3 , L-T4 , and D-T4.

    PubMed

    Mitrofan, Grigorina; Stefan-van Staden, Raluca-Ioana; Comnea-Stancu, Ionela Raluca; van Staden, Jacobus Frederick; Bazylak, Grzegorz; Kapnissi-Christodoulou, Constantina P; Aboul-Enein, Hassan Y

    2015-12-01

    A fast screening method of whole blood was proposed for enantiorecognition of free L-T3 , L-T4 , and D-T4 . Stochastic microsensors based on four inulins (IN, IQ, TEX, and HD) immobilized on diamond paste (DP) were used for recognition of free L-T3 , L-T4 , and D-T4 . For the enantiorecognition of free L-T4 and D-T4 in whole blood and pharmaceutical samples, the best microsensor was the one based on TEX/DP (wide linear concentration ranges, and low limits of quantification). The best limit of detection for the assay of free L-T3 (400 fmol/L) was recorded using the microsensors based on HD/DP, while for the assay of free L-T4, and D-T4 the best limit of determination (1 pmol/L) was recorded using the TX/DP-based microsensor. For the enantiorecognition of free L-T3 in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification - of pmol/L magnitude order). For the enantiorecognition of free L-T3 in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification - of pmol/L magnitude order). Free L-T3 , L-T4 , and D-T4 were recovered with high reliabilities in whole blood samples (recoveries higher than 99.00%, with RSD values lower than 1.00%) and pharmaceutical samples (recoveries higher than 95.00% with RSD values lower than 1.00%). PMID:26447904

  18. Fast Screening of Whole Blood Samples and Pharmaceutical Compounds for Enantiorecognition of Free L-T3 , L-T4 , and D-T4.

    PubMed

    Mitrofan, Grigorina; Stefan-van Staden, Raluca-Ioana; Comnea-Stancu, Ionela Raluca; van Staden, Jacobus Frederick; Bazylak, Grzegorz; Kapnissi-Christodoulou, Constantina P; Aboul-Enein, Hassan Y

    2015-12-01

    A fast screening method of whole blood was proposed for enantiorecognition of free L-T3 , L-T4 , and D-T4 . Stochastic microsensors based on four inulins (IN, IQ, TEX, and HD) immobilized on diamond paste (DP) were used for recognition of free L-T3 , L-T4 , and D-T4 . For the enantiorecognition of free L-T4 and D-T4 in whole blood and pharmaceutical samples, the best microsensor was the one based on TEX/DP (wide linear concentration ranges, and low limits of quantification). The best limit of detection for the assay of free L-T3 (400 fmol/L) was recorded using the microsensors based on HD/DP, while for the assay of free L-T4, and D-T4 the best limit of determination (1 pmol/L) was recorded using the TX/DP-based microsensor. For the enantiorecognition of free L-T3 in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification - of pmol/L magnitude order). For the enantiorecognition of free L-T3 in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification - of pmol/L magnitude order). Free L-T3 , L-T4 , and D-T4 were recovered with high reliabilities in whole blood samples (recoveries higher than 99.00%, with RSD values lower than 1.00%) and pharmaceutical samples (recoveries higher than 95.00% with RSD values lower than 1.00%).

  19. Evaluation of the Punch-it™ NA-Sample kit for detecting microbial DNA in blood culture bottles using PCR-reverse blot hybridization assay.

    PubMed

    Kim, Jungho; Wang, Hye-Young; Kim, Seoyong; Park, Soon Deok; Yu, Kwangmin; Kim, Hyo Youl; Uh, Young; Lee, Hyeyoung

    2016-09-01

    DNA extraction efficiency affects the success of PCR-based method applications. The Punch-it™ NA-Sample kit for extracting DNA by using paper chromatography is technically easy to use and requires just two reagents and only 10min to complete. The Punch-it™ NA-Sample kit could be offered as a rapid, accurate, and convenient method for extracting bacterial and fungal DNA from blood culture bottles. We compared the efficiencies of the commercial kit (Punch-it™ NA-Sample kit) and an in-house conventional boiling method with Chelex-100 resin for DNA extraction from blood culture bottles. The efficiency of the two DNA extraction methods was assessed by PCR-reverse blot hybridization assay (PCR-REBA, REBA Sepsis-ID) for detecting Gram positive (GP) bacteria, Gram negative (GN) bacteria, and Candida species with 196 positive and 200 negative blood culture bottles. The detection limits of the two DNA extraction methods were 10(3)CFU/mL for GP bacteria, 10(3)CFU/mL for GN bacteria, and 10(4)CFU/mL for Candida. The sensitivity and specificity of the Punch-it™ NA-Sample kit by REBA Sepsis-ID were 95.4% (187/196) and 100% (200/200), respectively. The overall agreement of the two DNA extraction methods was 98.9% (392/396). Three of four samples showing discrepant results between the two extraction methods were more accurately matched up with the Punch-it™ NA-Sample kit based on conventional culture methods. The results indicated that the Punch-it™ NA-Sample kit extracted bacterial and fungal DNA in blood culture bottles and allowed extracted DNA to be used in molecular assay. PMID:27263831

  20. The role of methanol addition to water samples in reducing analyte adsorption and matrix effects in liquid chromatography-tandem mass spectrometry.

    PubMed

    Li, Wei; Liu, Yucan; Duan, Jinming; Saint, Christopher P; Mulcahy, Dennis

    2015-04-10

    Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis coupled simply with water filtering before injection has proven to be a simple, economic and time-saving method for analyzing trace-level organic pollutants in aqueous environments. However, the linearity, precision and detection limits of such methods for late-eluting analytes were found to be much poorer than for early-eluting ones due to adsorption of the analytes in the operating system, such as sample vial, flow path and sample loop, creating problems in quantitative analysis. Addition of methanol (MeOH) into water samples as a modifier was shown to be effective in alleviating or even eliminating the negative effect on signal intensity for the late-eluting analytes and at the same time being able to reduce certain matrix effects for real water samples. Based on the maximum detection signal intensity obtained on desorption of the analytes with MeOH addition, the ratio of the detection signal intensity without addition of MeOH to the maximum intensity can be used to evaluate the effectiveness of methanol addition. Accordingly, the values of <50%, 50-80%, 80-120% could be used to indicate strong, medium and no effects, respectively. Based on this concept, an external matrix-matched calibration method with the addition of MeOH has been successfully established for analyzing fifteen pesticides with diverse physico-chemical properties in surface and groundwater with good linearity (r(2): 0.9929-0.9996), precision (intra-day relative standard deviation (RSD): 1.4-10.7%, inter-day RSD: 1.5-9.4%), accuracy (76.9-126.7%) and low limits of detection (0.003-0.028μg/L).

  1. Development and validation of an LC-MS/MS method for determination of p-phenylenediamine and its metabolites in blood samples.

    PubMed

    Mohamed, Khaled M; Cromarty, Duncan; Steenkamp, Vanessa

    2015-08-01

    In some developing countries, p-phenylenediamine (PPD) is used in combination with Henna as hair dye or skin decoration. A sensitive LC-MS/MS method was developed and validated for the simultaneous determination of p-phenylenediamine (PPD) and its metabolites N-acetyl-p-phenylenediamine (MAPPD) and N,N-diacetyl-p-phenylenediamine (DAPPD) in human blood. Acetanilide was used as an internal standard (IS). The LC-MS/MS was operated under multiple reaction-monitoring mode using the electrospray positive ionization technique. The transition ions m/z 109→92, m/z 151→92, m/z 193→92, and m/z 136→77 were selected for the quantification of PPD, MAPPD, DAPPD, and IS, respectively. The linear range was 10-2000ng/mL for all the compounds. The absolute recoveries were 51.94, 56.20 and 54.88% for PPD, MAPPD and DAPPD, respectively. Intra- and inter-assay imprecision were lower than 14% (RSD), and the bias of the assay was lower than 15% for all the compounds. The stability studies demonstrated that critical degradation for PPD in blood samples and autosampler occurred after 6h, while MAPPD and DAPPD were stable in blood samples and the autosampler up to 48h and 24h, respectively. This newly developed method allows for the detection of PPD and its metabolites in blood samples in the clinical and forensic setting. PMID:26073911

  2. Validation of self diagnosis of high blood pressure in a sample of the Spanish EPIC cohort: overall agreement and predictive values

    PubMed Central

    Tormo, M; Navarro, C; Chirlaque, M; Barber, X; the, E

    2000-01-01

    STUDY OBJECTIVE—High blood pressure is a variable related to several chronic conditions whose repeated measurement in large cohort studies is often not feasible having to rely on the self reporting of the subjects. The aim of the study is to validate such self diagnosis in a sample of members from the Spanish EPIC cohort study.
DESIGN—Comparison of high blood pressure self diagnosis with the information provided by the personal medical record drawn from the primary health centre of reference for such population.
SETTING—A small town near the EPIC-Murcia centre, one of five Spanish EPIC centres located in the south east, where inclusion in the cohort was offered to the general population.
PARTICIPANTS—The agreement between self reported high blood pressure status and data from medical records was measured in a representative sample of men and women (n= 248) aged 30-69 years. Medical records were studied for a diagnosis of high blood pressure, an anti-hypertensive pharmacological treatment or subject's inclusion in a hypertension control programme run in the medical centre only for hypertensive people (definite high blood pressure cases). As well, in the absence of such a diagnosis, medical annotations of systolic or diastolic high blood pressure⩾ 140/90 mm Hg (possible high blood pressure cases) were considered. Sensitivity, specificity, positive and negative predictive values and κ scores were calculated for all, definite and possible high blood pressure cases. Variables associated with the probability of having a true positive or negative self report of high blood pressure were also tested.
MAIN RESULTS—As expected, sensitivity was higher among definite cases (72.7%) than among possible cases (31.6%). Accordingly, the agreement between self report and medical record was higher for definite cases (κ = 0.65) than for possible (κ = 0.29) cases leading to a moderate overall agreement for all cases (κ = 0.58; 95% CI: 0.47, 0.69). Having

  3. Application of the Reverse Line Blot Assay for the Molecular Detection of Theileria and Babesia sp. in Sheep and Goat Blood Samples from Pakistan

    PubMed Central

    Iqbal, F; Khattak, RM; Ozubek, S; Khattak, MNK; Rasul, A; Aktas, M

    2013-01-01

    Background The present study was designed to detect the presence of tick-borne parasites (Theileria and Babesia spp.) in 196 blood samples collected from apparently healthy sheep and goats from two provinces, Punjab and Khyber Pukhtoon Khwa, in Pakistan. Methods Reverse line blot (RLB) assay was applied for the parasitic detection by the amplification of hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene. A membrane with covalently linked generic and species specific oligonucleotide probes was used for the hybridization of amplified PCR products. Results Parasites were detected in 16% of the ruminant blood samples under study. Two Theileria species, T. lestoquardi and T. ovis, were identified in samples. 25, of the total 32, infected animals were from Khyber Pukhtoon Khwa. Conclusion Sheep were more prone to tick borne haemoprotozans as 81% infected samples were sheep as compared to 19% goats (P > 0.001). Risk factor analysis revealed that male (P = 0.03), animals infested by ticks (P = 0.03) and herd composed of sheep only (P = 0.001) were more infected by blood parasites. PMID:23914243

  4. In vivo assessment of an industrial waste product as a feed additive in dairy cows: Effects of larch (Larix decidua L.) sawdust on blood parameters and milk composition.

    PubMed

    Tedesco, D; Garavaglia, L; Spagnuolo, M S; Pferschy-Wenzig, E M; Bauer, R; Franz, C

    2015-12-01

    When larch (Larix spp.) is processed in the wood industry, the sawdust is currently disposed of as waste or used as combustible material, even though it is rich in biologically active compounds. In this study the effect of larch sawdust supplementation on blood parameters as well as milk composition was examined in healthy mid-lactating dairy cows. Twenty-four multiparous Italian Friesian dairy cows were assigned to groups receiving either 300 g/day/cow of larch sawdust or a control diet, and treatments were continued for a 20 day period. Milk parameters were unaffected by treatment. A lower plasma total protein concentration was observed and can be attributed to a decrease in globulin concentration. A lower plasma urea concentration was also detected in the larch group. Moreover, biomarkers of liver function were influenced by the treatment. Total bilirubin was lower in larch-treated animals, and cholesterol tended to be lower. In addition, an interaction between day and treatment was observed for very low density lipoprotein. The concentration of other parameters, including reactive oxygen metabolites, superoxide dismutase, glutathione peroxidase and nitrotyrosine, did not differ between treatments. The observed benefits, together with the good palatability, make larch sawdust a promising candidate for the development of beneficial feed supplements for livestock. Further studies will be useful, particularly to evaluate its efficacy in different health conditions. PMID:26526868

  5. Gas chromatographic method using electron-capture detection for the determination of musk xylene in human blood samples. Biological monitoring of the general population.

    PubMed

    Angerer, J; Käfferlein, H U

    1997-05-23

    Musk xylene (2,4,6-trinitro-1,3-dimethyl-5-tert.-butylbenzene, MX), a synthetic musk often used in different fragrances and soaps to substitute the natural musk, is a potential contaminant of humans. In this publication, a specific and sensitive detection method for the determination of musk xylene in human blood samples is described. The clean-up of the blood samples includes an extraction step followed by a solid-phase adsorption to separate MX from other plasma components. Separation and detection was carried out by capillary gas chromatography and an electron capture detector (GC-ECD). The results were verified using qualitative capillary gas chromatography and a mass selective detector with electron impact ionisation (GC-EI-MS). epsilon-Hexachlorocyclohexane (epsilon-HCH) is used as internal standard. The reliability of the GC-ECD method has been proved. The relative standard deviations of the within-series imprecision were 12.7% for samples with a concentration of 0.5 microg/l and 2.1% for samples with a concentration of 5.0 microg/l, whereas the relative standard deviations for the between-day imprecision were 14.9% (0.5 microg/l samples) and 3.4% (5.0 microg/l samples). The losses during sample treatment were between 10.1% and 17.8%. No interfering peaks were observed. The absolute detection limit was 0.1 microg/l plasma. A total of 72 human blood samples were analysed to determine the MX concentrations within the general population. In 66 of the 72 human blood samples, the MX concentrations ranged from 0.10 to 1.12 microg/l plasma for the described method. In six samples no MX was detected. The median concentration was 0.24+/-0.23 microg MX/l plasma. The 95 percentile was 0.79 microg/l. No correlation could be found between MX concentrations and smoking habit, broca index, age, sex as well as fish consumption habits. Nevertheless, the results demonstrate the exposure of the general population to MX.

  6. Contemporary-use pesticides in personal air samples during pregnancy and blood samples at delivery among urban minority mothers and newborns.

    PubMed Central

    Whyatt, Robin M; Barr, Dana B; Camann, David E; Kinney, Patrick L; Barr, John R; Andrews, Howard F; Hoepner, Lori A; Garfinkel, Robin; Hazi, Yair; Reyes, Andria; Ramirez, Judyth; Cosme, Yesenia; Perera, Frederica P

    2003-01-01

    We have measured 29 pesticides in plasma samples collected at birth between 1998 and 2001 from 230 mother and newborn pairs enrolled in the Columbia Center for Children's Environmental Health prospective cohort study. Our prior research has shown widespread pesticide use during pregnancy among this urban minority cohort from New York City. We also measured eight pesticides in 48-hr personal air samples collected from the mothers during pregnancy. The following seven pesticides were detected in 48-83% of plasma samples (range, 1-270 pg/g): the organophosphates chlorpyrifos and diazinon, the carbamates bendiocarb and 2-isopropoxyphenol (metabolite of propoxur), and the fungicides dicloran, phthalimide (metabolite of folpet and captan), and tetrahydrophthalimide (metabolite of captan and captafol). Maternal and cord plasma levels were similar and, except for phthalimide, were highly correlated (p < 0.001). Chlorpyrifos, diazinon, and propoxur were detected in 100% of personal air samples (range, 0.7-6,010 ng/m(3)). Diazinon and propoxur levels were significantly higher in the personal air of women reporting use of an exterminator, can sprays, and/or pest bombs during pregnancy compared with women reporting no pesticide use or use of lower toxicity methods only. A significant correlation was seen between personal air level of chlorpyrifos, diazinon, and propoxur and levels of these insecticides or their metabolites in plasma samples (maternal and/or cord, p < 0.05). The fungicide ortho-phenylphenol was also detected in 100% of air samples but was not measured in plasma. The remaining 22 pesticides were detected in 0-45% of air or plasma samples. Chlorpyrifos, diazinon, propoxur, and bendiocarb levels in air and/or plasma decreased significantly between 1998 and 2001. Findings indicate that pesticide exposures are frequent but decreasing and that the pesticides are readily transferred to the developing fetus during pregnancy. PMID:12727605

  7. Characterization of the non-coding control region of polyomavirus KI isolated from nasopharyngeal samples from patients with respiratory symptoms or infection and from blood from healthy blood donors in Norway.

    PubMed

    Song, Xiaobo; Van Ghelue, Marijke; Ludvigsen, Maria; Nordbø, Svein Arne; Ehlers, Bernhard; Moens, Ugo

    2016-07-01

    Seroepidemiological studies showed that the human polyomavirus KI (KIPyV) is common in the human population, with age-specific seroprevalence ranging from 40-90 %. Genome epidemiological analyses demonstrated that KIPyV DNA is predominantly found in respiratory tract samples of immunocompromised individuals and children suffering from respiratory diseases, but viral sequences have also been detected in brain, tonsil, lymphoid tissue studies, plasma, blood and faeces. Little is known about the sequence variation in the non-coding control region of KIPyV variants residing in different sites of the human body and whether specific strains dominate in certain parts of the world. In this study, we sequenced the non-coding control region (NCCR) of naturally occurring KIPyV variants in nasopharyngeal samples from patients with respiratory symptoms or infection and in blood from healthy donors in Norway. In total 86 sequences were obtained, 44 of which were identical to the original isolated Stockholm 60 variant. The remaining NCCRs contained one or several mutations, none of them previously reported. The same mutations were detected in NCCRs amplified from blood and nasopharyngeal samples. Some patients had different variants in their specimens. Transient transfection studies in HEK293 cells with a luciferase reporter plasmid demonstrated that some single mutations had a significant effect on the relative early and late promoter strength compared with the Stockholm 60 promoter. The effect of the NCCR mutations on viral replication and possible virulence properties remains to be established. PMID:27031170

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

    PubMed

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

    2016-03-01

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

  9. Comparison of different blood sample processing methods for sensitive detection of low level chimerism by RHD real-time PCR assay.

    PubMed

    Javadi, Ahmad; Verduin, Esther P; Brand, Anneke; Schonewille, Henk

    2013-01-01

    The rhesus D blood group, which is expressed on the red blood cells (RBC) of 85% of the Caucasian population, is one of the most immunogenic RBC antigens, inducing D antibody formation in up to 20-80% of D-negative transfusion recipients and about 10% of pregnancies at risk. Pregnancy-induced D-antibodies can persist for many years, but the mechanisms underlying this persistence are unclear. The LOTUS study, a long-term follow-up study of mothers from severely affected children with hemolytic disease of the fetus and newborn investigates, among other endpoints, whether persistent feto-maternal chimerism is associated with long-term maternal anti-D persistence. We questioned which blood sample processing method should be used to detect low levels of RHD chimerism with the highest sensitivity and specificity using qPCR. After optimization of primer and probe concentrations for singleplex RHD exon 5 and 7 qPCR, sensitivity, specificity and efficiency of RHD and DYS1 qPCR were investigated in artificial chimeric samples. Sensitivity of DYS1 was one log higher (0.0001%) in enriched mononuclear cell fractions as compared with whole blood. Comparable linear sensitivity (0.007%) and mean efficiency (84-99%) for RHD qPCR were observed in all samples regardless whether whole blood or pre- or post-mixing of cellular fractions had been used. We conclude that RHD chimerism using singleplex exon 5 and 7 qPCR is linearly detectable down to 1.0 GE, without an advantage of fraction enrichment.

  10. Changes in haematology measurements with the Sysmex XT-2000iV during storage of feline blood sampled in EDTA or EDTA plus CTAD.

    PubMed

    Granat, Fanny; Geffré, Anne; Bourgès-Abella, Nathalie; Braun, Jean-Pierre; Trumel, Catherine

    2013-06-01

    In veterinary medicine a complete blood cell count (CBC) cannot always be performed within 24 h as usually recommended, particularly for specimens shipped to a reference laboratory. This raises the question of the stability of the variables, especially in ethylenediamine tetra-acetic acid (EDTA) feline blood specimens, known to be prone to in vitro platelet aggregation. Citrate, theophylline, adenosine and dipyridamole (CTAD) has been reported to limit platelet aggregation in feline blood specimens. The aim of this study was to measure the stability of the haematological variables and the platelet aggregation score in EDTA and EDTA plus CTAD (EDCT) feline blood specimens during 48 h of storage at room temperature. Forty-six feline EDTA and EDCT blood specimens were analysed with a Sysmex XT-2000iV analyser, and the platelet count and score of platelet aggregation were estimated immediately and after 24 and 48 h of storage. A significant increase in mean corpuscular volume, haematocrit, reticulocyte and eosinophil counts, and a significant decrease in mean corpuscular haemoglobin concentration and monocyte count were observed. Haemoglobin, mean corpuscular haemoglobin, and red blood cell, white blood cell, neutrophil and lymphocyte counts remained stable. Changes in reticulocyte indexes with time (low fluorescence ratio, medium fluorescence ratio, high fluorescence ratio and immature reticulocyte fraction) were not significant. Changes were generally more pronounced in EDTA than in EDCT. Platelet aggregation decreased markedly in initially highly aggregated EDTA specimens, and increased slightly in initially non- or mildly-aggregated EDTA or EDCT specimens. Platelet counts increased and decreased, or remained stable, respectively. CTAD can reduce storage-induced changes of the haematological variables in feline samples, thus improving the reliability of a CBC and limiting clinical misinterpretations.

  11. West Nile virus infection incidence based on donated blood samples and neuroinvasive disease reports, Northern Texas, USA, 2012.

    PubMed

    Cervantes, Diana T; Chen, Shande; Sutor, Laurie J; Stonecipher, Shelley; Janoski, Nicolette; Wright, David J; Busch, Michael P

    2015-04-01

    During the 2012 outbreak of West Nile virus in the United States, approximately one third of the cases were in Texas. Of those, about half occurred in northern Texas. Models based on infected blood donors and persons with neuroinvasive disease showed, respectively, that ≈0.72% and 1.98% of persons in northern Texas became infected.

  12. Detection of Anaplasma marginale and A. phagocytophilum in bovine peripheral blood samples by duplex real-time reverse transcriptase PCR assay.

    PubMed

    Reinbold, James B; Coetzee, Johann F; Sirigireddy, Kamesh R; Ganta, Roman R

    2010-07-01

    Insufficient diagnostic sensitivity and specificity coupled with the potential for cross-reactivity among closely related Anaplasma species has made the accurate determination of infection status problematic. A method for the development of simplex and duplex real-time quantitative reverse transcriptase PCR (qRT-PCR) assays for the detection of A. marginale and A. phagocytophilum 16S rRNA in plasma-free bovine peripheral blood samples is described. The duplex assay was able to detect as few as 100 copies of 16S rRNA of both A. marginale and A. phagocytophilum in the same reaction. The ratio of 16S rRNA to 16S DNA copies for A. marginale was determined to be 117.9:1 (95% confidence interval [95% CI], 100.7:1, 135.2:1). Therefore, the detection limit is the minimum infective unit of one A. marginale bacterium. The duplex assay detected nonequivalent molar ratios as high as 100-fold. Additionally, the duplex assay and a competitive enzyme-linked immunosorbent assay (cELISA) were used to screen 237 samples collected from herds in which anaplasmosis was endemic. When the cELISA was evaluated by the results of the qRT-PCR, its sensitivity and specificity for the detection of A. marginale infection were found to be 65.2% (95% CI, 55.3%, 75.1%) and 97.3% (95% CI, 94.7%, 99.9%), respectively. A. phagocytophilum infection was not detected in the samples analyzed. One- and two-way receiver operator characteristic curves were constructed in order to recommend the optimum negative cutoff value for the cELISA. Percentages of inhibition of 20 and 15.3% were recommended for the one- and two-way curves, respectively. In conclusion, the duplex real-time qRT-PCR assay is a highly sensitive and specific diagnostic tool for the accurate and precise detection of A. marginale and A. phagocytophilum infections in cattle.

  13. Selection of suitable reference genes for normalization of quantitative RT-PCR in peripheral blood samples of bottlenose dolphins (Tursiops truncatus)

    PubMed Central

    Chen, I-Hua; Chou, Lien-Siang; Chou, Shih-Jen; Wang, Jiann-Hsiung; Stott, Jeffrey; Blanchard, Myra; Jen, I-Fan; Yang, Wei-Cheng

    2015-01-01

    Quantitative RT-PCR is often used as a research tool directed at gene transcription. Selection of optimal housekeeping genes (HKGs) as reference genes is critical to establishing sensitive and reproducible qRT-PCR-based assays. The current study was designed to identify the appropriate reference genes in blood leukocytes of bottlenose dolphins (Tursiops truncatus) for gene transcription research. Seventy-five blood samples collected from 7 bottlenose dolphins were used to analyze 15 candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ, LDHA, SDHA). HKG stability in qRT-PCR was determined using geNorm, NormFinder, BestKeeper and comparative delta Ct algorithms. Utilization of RefFinder, which combined all 4 algorithms, suggested that PGK1, HPRT1 and RPL4 were the most stable HKGs in bottlenose dolphin blood. Gene transcription perturbations in blood can serve as an indication of health status in cetaceans as it occurs prior to alterations in hematology and chemistry. This study identified HKGs that could be used in gene transcript studies, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. PMID:26486099

  14. Selection of suitable reference genes for normalization of quantitative RT-PCR in peripheral blood samples of bottlenose dolphins (Tursiops truncatus).

    PubMed

    Chen, I-Hua; Chou, Lien-Siang; Chou, Shih-Jen; Wang, Jiann-Hsiung; Stott, Jeffrey; Blanchard, Myra; Jen, I-Fan; Yang, Wei-Cheng

    2015-10-21

    Quantitative RT-PCR is often used as a research tool directed at gene transcription. Selection of optimal housekeeping genes (HKGs) as reference genes is critical to establishing sensitive and reproducible qRT-PCR-based assays. The current study was designed to identify the appropriate reference genes in blood leukocytes of bottlenose dolphins (Tursiops truncatus) for gene transcription research. Seventy-five blood samples collected from 7 bottlenose dolphins were used to analyze 15 candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ, LDHA, SDHA). HKG stability in qRT-PCR was determined using geNorm, NormFinder, BestKeeper and comparative delta Ct algorithms. Utilization of RefFinder, which combined all 4 algorithms, suggested that PGK1, HPRT1 and RPL4 were the most stable HKGs in bottlenose dolphin blood. Gene transcription perturbations in blood can serve as an indication of health status in cetaceans as it occurs prior to alterations in hematology and chemistry. This study identified HKGs that could be used in gene transcript studies, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans.

  15. A non-organic and non-enzymatic extraction method gives higher yields of genomic DNA from whole-blood samples than do nine other methods tested.

    PubMed

    Lahiri, D K; Bye, S; Nurnberger, J I; Hodes, M E; Crisp, M

    1992-12-01

    We compared ten methods for extraction of DNA from whole blood. Nine methods require incubation with either enzymes or treatment of organic solvents or both. The 'Rapid Method' (RM) (Method 10) avoids the use of organic solvents (phenol/chloroform) and eliminates completely the use of proteinase K. Thus, the time and cost of DNA extraction are reduced significantly. This is accomplished by salting out and precipitation of the cellular proteins in saturated sodium chloride. This method takes less than an hour to completion, without compromising the yield or the quality of DNA. Using RM, we can make DNA from 0.1 ml of whole blood and as little as 0.5 ml of blood yields DNA sufficient to run a few Southern blots. The RM can also be applied to packed cells. The DNA is free of RNA, protein and degrading enzymes. The uncut DNA runs as a typical slow-migrating, high-molecular-weight and undegraded species in an agarose gel. The DNA is suitable for digestion by various restriction endonucleases. This procedure works equally well with fresh blood samples and with those that are stored at 4 degrees C and -70 degrees C. To our knowledge the RM reported here is the safest, fastest and most quantitative and economical method for preparation of DNA from whole blood and cells.

  16. Blood culture contaminants.

    PubMed

    Dawson, S

    2014-05-01

    Blood cultures are an essential diagnostic tool. However, contamination may impact on patients' care and lead to increased patient stay, additional tests, and inappropriate antibiotic use. The aim of this study was to review the literature for factors that influence the rate of blood culture contamination. A comprehensive literature search was performed using Medline and CINAHL on blood culture contamination. Hospitals/units should have in place a protocol for staff on how to take blood cultures, incorporating use of an aseptic technique. Studies have shown that several key factors in the process may lower contamination rates such as adherence to a protocol, sampling by peripheral venepuncture route rather than via an intravascular catheter, use of sterile gloves, cleaning tops of blood culture bottles with antiseptics and inoculating blood culture bottles before other blood tubes, samples being taken by a phlebotomy team, monitoring contamination rates, and providing individual feedback and retraining for those with contaminants. Although skin antisepsis is advocated there is still debate on which antiseptic is most effective, as there is no conclusive evidence, only that there is benefit from alcohol-containing preparations. In conclusion, hospitals should aim to minimize their blood culture contamination rates. They should monitor their rate regularly and aim for a rate of ≤3%. PMID:24768211

  17. Blood culture contaminants.

    PubMed

    Dawson, S

    2014-05-01

    Blood cultures are an essential diagnostic tool. However, contamination may impact on patients' care and lead to increased patient stay, additional tests, and inappropriate antibiotic use. The aim of this study was to review the literature for factors that influence the rate of blood culture contamination. A comprehensive literature search was performed using Medline and CINAHL on blood culture contamination. Hospitals/units should have in place a protocol for staff on how to take blood cultures, incorporating use of an aseptic technique. Studies have shown that several key factors in the process may lower contamination rates such as adherence to a protocol, sampling by peripheral venepuncture route rather than via an intravascular catheter, use of sterile gloves, cleaning tops of blood culture bottles with antiseptics and inoculating blood culture bottles before other blood tubes, samples being taken by a phlebotomy team, monitoring contamination rates, and providing individual feedback and retraining for those with contaminants. Although skin antisepsis is advocated there is still debate on which antiseptic is most effective, as there is no conclusive evidence, only that there is benefit from alcohol-containing preparations. In conclusion, hospitals should aim to minimize their blood culture contamination rates. They should monitor their rate regularly and aim for a rate of ≤3%.

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

    PubMed

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

    2014-10-01

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

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

    PubMed

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

    2014-10-01

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

  20. Effects of different fibre sources and fat addition on cholesterol and cholesterol-related lipids in blood serum, bile and body tissues of growing pigs.

    PubMed

    Kreuzer, M; Hanneken, H; Wittmann, M; Gerdemann, M M; Machmuller, A

    2002-04-01

    Knowledge is limited on the efficacy of hindgut-fermentable dietary fibre to reduce blood, bile and body tissue cholesterol levels. In three experiments with growing pigs the effects of different kinds and levels of bacterially fermentable fibre (BFS) on cholesterol metabolism were examined. Various diets calculated to have similar contents of metabolizable energy were supplied for complete fattening periods. In the first experiment, a stepwise increase from 12 to 20% BFS was performed by supplementing diets with fermentable fibre from sugar beet pulp (modelling hemicelluloses and pectin). Beet pulp, rye bran (modelling cellulose) and citrus pulp (pectin) were offered either independently or in a mixture in the second experiment. These diets were opposed to rations characterized in carbohydrate type by starch either mostly non-resistant (cassava) or partly resistant (maize) to small intestinal digestion. The third experiment was planned to explore the interactions of BFS from citrus pulp with fat either through additional coconut oil/palm kernel oil blend or full-fat soybeans. In all experiments the increase of the BFS content was associated with a constant (cellulose) or decreasing (hemicelluloses, pectin) dietary proportion of non-digestible fibre. In experiment 1 an inverse dose-response relationship between BFS content and cholesterol in blood serum and adipose tissue as well as bile acid concentration in bile was noted while muscle cholesterol did not respond. In experiment 2 the ingredients characterized by cellulose and hemicelluloses/pectin reduced cholesterol-related traits relative to the low-BFS-high-starch controls whereas, except in adipose tissue cholesterol content, the pectinous ingredient had the opposite effect. However, the changes in serum cholesterol mainly affected HDL and not LDL cholesterol. Adipose tissue cholesterol also was slightly lower with partly resistant starch compared to non-resistant starch in the diet. Experiment 3 showed that

  1. Storage conditions of blood samples and primer selection affect the yield of cDNA polymerase chain reaction products of hepatitis C virus.

    PubMed Central

    Cuypers, H T; Bresters, D; Winkel, I N; Reesink, H W; Weiner, A J; Houghton, M; van der Poel, C L; Lelie, P N

    1992-01-01

    We have noticed that suboptimal specimen processing and storage conditions may cause false-negative results in the detection of hepatitis C virus (HCV) RNA in plasma or serum. To establish the influence of specimen handling in a serological laboratory on the rate of detection of HCV RNA by the cDNA polymerase chain reaction (cDNA-PCR), we tested routine serum samples and fresh-frozen plasma samples from the same bleeding from confirmed anti-HCV-positive blood donors. When primers from the NS3/NS4 region were used, HCV RNA was detected in fresh-frozen plasma from 67% of the donors, whereas positive results were obtained with only 50% of the serum samples that had been subjected to routine serological procedures. Analysis of the same samples with primers from the highly conserved 5'-terminal region (5'-TR) revealed an HCV RNA detection rate of 92% for both the routine and the fresh-frozen s