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Whole Blood Samples (whole + blood_sample)
Selected AbstractsWhen monocytes and platelets compete: The effect of platelet count on the flow cytometric measurement of monocyte CD36,CYTOMETRY, Issue 2 2010W.H. Dzik Abstract Background: Flow cytometric measurement of monocyte surface CD36 is relevant to several conditions including diabetes, cardiovascular disease, lipid disorders, platelet isoimmunization, and susceptibility to P falciparum malaria. CD36 is also strongly expressed on platelets where it is also known as platelet glycoprotein IV. Methods: Whole blood samples, containing identical monocyte concentrations, were adjusted to contain platelets ranging from 20,000/uL to 600,000/uL, were stained with fluorescent-labeled anti-CD36, and analyzed by flow cytometry. Results: CD36 median fluorescent intensity (MFI) observed on monocytes decreased as the platelet concentration in the sample increased with more than a 50% decline in monocyte MFI over the normal range of platelet values. The effect was not abolished by using larger volumes of monoclonal antibody and was observed with different clones of reagent anti-CD36. The findings were most consistent with competition by platelets for the CD36 reagent. Similar findings were observed with antibody to class I HLA. Under defined assay conditions, monocyte CD36 MFI declined with rising platelet concentration in a predictable fashion following an inverse linear relationship. Conclusions: Measurement of CD36 expression on monocytes by flow cytometry in whole blood samples is affected by the sample platelet count. When comparing the monocyte CD36 expression among different individuals, our approach can be used to adjust measured monocyte CD36 expression for the effect of the platelet concentration in the sample. Competition by platelets for monoclonal reagents may occur in other settings when whole blood assays are used and when the target antigen is strongly expressed on both platelets and leukocytes. © 2009 Clinical Cytometry Society [source] Simultaneous quantification of 2,,2,-difluorodeoxycytidine and 2,,2,-difluorodeoxyuridine nucleosides and nucleotides in white blood cells using porous graphitic carbon chromatography coupled with tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 19 2009Robert S. Jansen A novel assay for the simultaneous quantification of the widely used anticancer agent 2,,2,-difluorodeoxycytidine (gemcitabine; dFdC), its deaminated metabolite 2,,2,-difluorodeoxyuridine (dFdU) and their mono-, di- and triphosphates (dFdCMP, dFdCDP, dFdCTP, dFdUMP, dFdUDP and dFdUTP) in peripheral blood mononuclear cells (PBMCs) is described. Separation of all eight compounds was achieved within 15,min using a porous graphitic carbon column (Hypercarb) with a gradient from 0 to 25,mM ammonium bicarbonate in acetonitrile/water (15:85, v/v). Calibration ranges in PBMC lysate from 4.29 to 429, 29.0 to 2900, 31.4 to 3140 and 36.9 to 3690,nM for dFdC, dFdCMP, dFdCDP and dFdCTP and from 42.1 to 4210, 25.4 to 2540, 43.2 to 4320 and 52.7 to 5270,nM for dFdU, dFdUMP, dFdUDP and dFdUTP, respectively, were validated. Accuracies were within 82.3,119% at the lower limit of quantification (LLOQ) and the precisions were less than 20.0%. At the other tested levels accuracies were within 91.4,114% and precisions less than 14.9%. Mixtures of 13C,15N2 -labeled dFdC and dFdU nucleotides were synthesized and used as internal standards. Whole blood samples showed extensive ongoing dFdC metabolism when stored at room temperature, but not on ice-water, which made the addition of enzyme inhibitors unnecessary. Stock solutions and samples were stable under all analytically relevant conditions. The method was successfully applied to clinical samples. Copyright © 2009 John Wiley & Sons, Ltd. [source] The proinflammatory activity of recombinant serum amyloid A is not shared by the endogenous protein in the circulationARTHRITIS & RHEUMATISM, Issue 6 2010Lena Björkman Objective Elevated serum levels of the acute-phase protein serum amyloid A (SAA) are a marker for active rheumatoid arthritis (RA), and SAA can also be found in the tissues of patients with active RA. Based on a number of studies with recombinant SAA (rSAA), the protein has been suggested to be a potent proinflammatory mediator that activates human neutrophils, but whether endogenous SAA shares these proinflammatory activities has not been directly addressed. The present study was undertaken to investigate whether SAA in the plasma of patients with RA possesses proinflammatory properties and activates neutrophils in a manner similar to that of the recombinant protein. Methods Neutrophil activation was monitored by flow cytometry, based on L-selectin shedding from cell surfaces. Whole blood samples from healthy subjects and from RA patients with highly elevated SAA levels were studied before and after stimulation with rSAA as well as purified endogenous SAA. Results Recombinant SAA potently induced cleavage of L-selectin from neutrophils and in whole blood samples. Despite highly elevated SAA levels, L-selectin was not down-regulated on RA patient neutrophils as compared with neutrophils from healthy controls. Spiking SAA-rich whole blood samples from RA patients with rSAA, however, resulted in L-selectin shedding. In addition, SAA purified from human plasma was completely devoid of neutrophil- or macrophage-activating capacity. Conclusion The present findings show that rSAA is proinflammatory but that this activity is not shared by endogenous SAA, either when present in the circulation of RA patients or when purified from plasma during an acute-phase response. [source] Lack of tacrolimus circadian pharmacokinetics and CYP3A5 pharmacogenetics in the early and maintenance stages in Japanese renal transplant recipientsBRITISH JOURNAL OF CLINICAL PHARMACOLOGY, Issue 2 2008Shigeru Satoh WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT , Circadian variations of tacrolimus pharmacokinetics are controversial. , Also, the pharmacokinetics has time-dependent variability, such as a decrease in oral clearance and increase in the dose-adjusted AUC after transplantation. , Although the CYP3A5 polymorphism is associated with tacrolimus pharmacokinetics, differences in the influence of this gene on the pharmacokinetics between the early and maintenance stages have not yet been clarified. WHAT THIS STUDY ADDS , Tacrolimus pharmacokinetics did not show circadian variation in either the early or maintenance stage with our designated-time administration strategy. , Based on previous results and our own findings, the interval between food consumption and tacrolimus administration might influence the interindividual and interinstitutional variability of tacrolimus chronopharmacokinetics. , The CYP3A5 polymorphism may be associated with the time-dependent changes in tacrolimus oral clearance. AIMS We investigated whether tacrolimus pharmacokinetics shows circadian variation and the influence of the CYP3A5 A6986G polymorphism on the pharmacokinetics in both the early and maintenance stages after renal transplantation. METHODS Tacrolimus was administered twice daily at specified times (09.00 and 21.00 h) throughout the pre- and post-transplant period according to the trough-targeting strategy. Fifty recipients with stable graft function were studied on day 28 and beyond 1-year post transplantation. Whole blood samples were collected prior to and 1, 2, 3, 4, 6, 9 and 12 h after both the morning and evening doses during hospitalization. RESULTS Tacrolimus pharmacokinetics did not show circadian variation in either the early or maintenance stage [AUC0,12 197.1 (95% confidence interval 182.9, 212.3) in daytime vs. 203.6 ng h ml,1 (189.8, 217.4) in the night-time at day 28, 102.0 (92.1, 111.9) vs. 107.7 (97.9, 117.5) at 1 year, respectively]. In CYP3A5 *1 allele carriers (CYP3A5 expressers), body weight-adjusted oral clearance was markedly decreased from the early stage to the maintenance stage [0.622 (0.534, 0.709) to 0.369 l h,1 kg,1 (0.314, 0425)] compared with a smaller decrease [0.368 (0.305, 0.430) to 0.305 (0.217, 0.393)] in CYP3A5 non-expressers; however, the CYP3A5 genetic variation did not influence tacrolimus chronopharmacokinetics. CONCLUSION Equivalent daytime and night-time tacrolimus pharmacokinetics were achieved during both the early and maintenance stages with our specified-time administration strategy. The CYP3A5 polymorphism may be associated with the time-dependent changes in the oral clearance of tacrolimus, suggesting that genotyping of this polymorphism is useful for determining the appropriate dose of tacrolimus in both the early and maintenance stages after renal transplantation. [source] On-line cell lysis and DNA extraction on a microfluidic biochip fabricated by microelectromechanical system technologyELECTROPHORESIS, Issue 9 2008Xing Chen Dr. Abstract Integrating cell lysis and DNA purification process into a micrototal analytical system (,TAS) is one critical step for the analysis of nucleic acids. On-chip cell lysis based on a chemical method is realized by sufficient blend of blood sample and the lyzing reagent. In this paper two mixing models, T-type mixing model and sandwich-type mixing model, are proposed and simulation of those models is conducted. Result of simulation shows that the sandwich-type mixing model with coiled channel performs best and this model is further used to construct the microfluidic biochip for on-line cell lysis and DNA extraction. The result of simulation is further verified by experiments. It asserts that more than 80% mixing of blood sample and lyzing reagent which guarantees that completed cell lysis can be achieved near the inlet location when the cell/buffer velocity ratio is less than 1:5. After cell lysis, DNA extraction by means of a solid-phase method is implemented by using porous silicon matrix which is integrated in the biochip. During continuous flow process in the microchip, rapid cell lysis and PCR-amplifiable genomic DNA purification can be achieved within 20,min. The potential of this microfluidic biochip is illustrated by pretreating a whole blood sample, which shows the possibility of integration of sample preparation, PCR, and separation on a single device to work as portable point-of-care medical diagnostic system. [source] Immune cell function testing: an adjunct to therapeutic drug monitoring in transplant patient managementCLINICAL TRANSPLANTATION, Issue 2 2003Richard Kowalski Abstract:, Each year, 55 000 organ transplants are performed worldwide. Cumulatively, the number of living organ recipients is now estimated to be over 300 000. Most of these transplant recipients will remain on immunosuppressive drugs for the remainder of their lives to prevent rejection episodes. Controlled doses of these drugs are required to prevent over-medication, which may leave the patient susceptible to opportunistic infection and drug toxicity effects, or under-dosing, which may lead to shortened graft survival because of rejection episodes. This paper describes the result of a multicenter study conducted at the Universities of Pittsburgh, Alabama and Maryland to evaluate an in vitro assay (CylexTM Immune Cell Function Assay) for the measurement of global immune response in transplant patients receiving immunosuppressive therapy. The assay uses a whole blood sample to maintain the presence of the drug during incubation. Following overnight incubation of blood with phytohemagglutinin (PHA), CD4 cells are selected using paramagnetic particles coated with a monoclonal antibody to the CD4 epitope. The CD4-positive cells are targeted as major immunosuppressive drugs are designed to specifically inhibit T-cell activation which has been implicated in rejection. The data generated at these three sites were submitted in support of an Food and Drug Association (FDA) application for the use of this assay in the detection of cell-mediated immunity in an immunosuppressed population. The assay was cleared by the FDA on April 2, 2002. This cross-sectional study was designed to establish ranges for reactivity of this bioassay in the assessment of functional immunity for an individual solid organ recipient at any point in time. [source] When monocytes and platelets compete: The effect of platelet count on the flow cytometric measurement of monocyte CD36,CYTOMETRY, Issue 2 2010W.H. Dzik Abstract Background: Flow cytometric measurement of monocyte surface CD36 is relevant to several conditions including diabetes, cardiovascular disease, lipid disorders, platelet isoimmunization, and susceptibility to P falciparum malaria. CD36 is also strongly expressed on platelets where it is also known as platelet glycoprotein IV. Methods: Whole blood samples, containing identical monocyte concentrations, were adjusted to contain platelets ranging from 20,000/uL to 600,000/uL, were stained with fluorescent-labeled anti-CD36, and analyzed by flow cytometry. Results: CD36 median fluorescent intensity (MFI) observed on monocytes decreased as the platelet concentration in the sample increased with more than a 50% decline in monocyte MFI over the normal range of platelet values. The effect was not abolished by using larger volumes of monoclonal antibody and was observed with different clones of reagent anti-CD36. The findings were most consistent with competition by platelets for the CD36 reagent. Similar findings were observed with antibody to class I HLA. Under defined assay conditions, monocyte CD36 MFI declined with rising platelet concentration in a predictable fashion following an inverse linear relationship. Conclusions: Measurement of CD36 expression on monocytes by flow cytometry in whole blood samples is affected by the sample platelet count. When comparing the monocyte CD36 expression among different individuals, our approach can be used to adjust measured monocyte CD36 expression for the effect of the platelet concentration in the sample. Competition by platelets for monoclonal reagents may occur in other settings when whole blood assays are used and when the target antigen is strongly expressed on both platelets and leukocytes. © 2009 Clinical Cytometry Society [source] Discrepancy in measuring CD4 expression on T-lymphocytes using fluorescein conjugates in comparison with unimolar CD4-phycoerythrin conjugates,,CYTOMETRY, Issue 6 2007Lili Wang Abstract Background: Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on lymphocytes. However, the results from the use of these different QFC methods vary considerably in the literature. To better identify the causes of these discrepancies, we measured CD4 expression using FITC and phycoerythrin (PE) conjugates to stain CYTO-TROLÔ Control Cells and T-lymphocytes in whole blood and isolated cell preparations. We further examined pH of the cellular microenvironment as a cause of discordant results obtained with the FITC conjugate. Methods: Calibration with Quantibrite PE-labeled microspheres and the use of unimolar CD4-PE conjugates provided direct measurement of the antibody bound per cell value (ABC) for CD4 expression on normal T-lymphocytes. Calibration for CD4-FITC monoclonal antibody (Mab) labeled CYTO-TROL Control Cells and normal T-lymphocytes was based on molecules of equivalent soluble fluorochrome (MESF) as determined by FITC-labeled microspheres traceable to NIST RM 8640. The MESF value for CD4-FITC Mab was determined that enabled the conversion of the MESF values obtained for CYTO-TROL cells to ABC. We investigated the likely pH change in the fluorescein microenvironments within FITC-labeled Mab and cells stained with FITC-labeled Mab using a pH sensitive indicator. Results: The mean ABC value for T-lymphocytes prepared from fresh whole blood using CD4-PE conjugate (48,321) was consistent with previous results, and it was much higher than the mean ABC using CD4-FITC Mab (22,156). The mean ABC value for CYTO-TROL cells using CD4-PE conjugate (43,090) was also higher than that using CD4-FITC conjugate (34,734), although the discrepancy was not as great. Further studies suggested the discrepancy in CYTO-TROL results may be accounted for by the low pH of the membrane microenvironment, but the greater discrepancy in T-lymphocytes could not be fully explained. Conclusion: CD4 expression on fresh normal whole blood samples and CYTO-TROL cells can be consistently quantified in ABC units using Quantibrite PE quantification beads and unimolar CD4-PE conjugates. Quantification with CD4-FITC conjugate is not as consistent, but may be improved by the use of CD4 T-cells as biological calibrators. This approximation is valid only for surface receptors with consensus ABC values measured by different QFC methods serving as biological standards. Published 2007 Wiley-Liss, Inc. [source] Evaluation of a single-platform microcapillary flow cytometer for enumeration of absolute CD4+ T-lymphocyte counts in HIV-1 infected Thai patients,,CYTOMETRY, Issue 5 2007Kovit Pattanapanyasat Abstract Background: Various assays are used to enumerate peripheral blood absolute CD4+ T-lymphocytes. Flow cytometry is considered the gold standard for this purpose. However, the high cost of available flow cytometers and monoclonal antibody reagents make it difficult to implement such methods in the resource-poor settings. In this study, we evaluated a cheaper, recently developed single-platform microcapillary cytometer for CD4+ T-lymphocyte enumeration, the personal cell analyzer (PCA), from Guava® Technologies. Methods: CD4+ and CD8+ T-lymphocyte counts in whole blood samples from 250 HIV-1 infected Thais were determined, using a two-color reagent kit and the Guava PCA, and compared with the results obtained with two reference microbead-based methods from Becton Dickinson Biosciences: the three-color TruCOUNTÔ tube method and the two-color FACSCountÔ method. Statistical correlations and agreements were determined using linear correlation and Bland,Altman analysis. Results: Absolute CD4+ T-lymphocyte counts obtained using the Guava PCA method highly correlated with those obtained using TruCOUNT method (R2 = 0.95, mean bias +13.1 cells/,l, limit of agreement [LOA] ,101.8 to +168.3 cells/,l). Absolute CD8+ T-lymphocyte counts obtained using the Guava PCA method also highly correlated with those obtained with the two reference methods (R2 = 0.92 and 0.88, respectively). Conclusion: This study shows that the enumeration of CD4+ T-lymphocytes using the Guava microcapillary cytometer PCA method performed well when compared with the two reference bead-based methods. However, like the two reference methods, this new method needs substantial technical expertise. © 2007 Clinical Cytometry Society. [source] Statistical criteria to establish optimal antibody dilution in flow cytometry analysisCYTOMETRY, Issue 3 2007Cesar J. G. Collino Abstract Background: In direct techniques of flow cytometry, the optimal antibody dilution or titer point is established from the plateau area of the antibody titration curve. However, the plateau area is defined without any statistical criteria, which may lead to an incorrect selection of antibody dilution. Herein, we report statistical criteria to establish the optimal antibody dilution for CD14, CD8, CD4, and CD3 analysis by flow cytometry in peripheral whole blood. Methods: The unpaired t- test (two-tail P value) was used as statistical criteria to analyze the titration curve of the following monoclonal antibody panels: CD14-FITC, CD8-FITC, CD4-RD1, and CD3-PC5. Results: Using the unpaired t- test (two-tail P value), the plateau area from the antibody titration curve was fitted when two consecutive antibody volumes showed mean peak of channel fluorescence (MPCF) values not significantly different. When the antibody was used at volume corresponding to that of the antibody titration point, the flow cytometry analysis of whole blood samples with different density of cell antigens can be correctly discriminated. Conclusion: This statistical criteria allows the fitting of the plateau area of MPCF versus antibody volume and consequently, to define the optimal antibody dilution. © 2007 Clinical Cytometry Society [source] An optimized whole blood method for flow cytometric measurement of ZAP-70 protein expression in chronic lymphocytic leukemiaCYTOMETRY, Issue 4 2006T. Vincent Shankey Abstract Background: ZAP-70 protein expression has been proposed as a marker for immunoglobulin heavy chain mutational status, which some studies have correlated with disease course in B-cell chronic lymphocytic leukemia (CLL). Studies published to date measuring levels of expression of ZAP-70 intracellular protein using flow cytometry have demonstrated poor performance, as defined by the difference in signal in known positive and negative lymphocyte populations. Methods: A recently published method (Chow S, Hedley DW, Grom P, Magari R, Jacobberger JW, Shankey TV, Cytometry A 2005;67:4,17) to measure intracellular phospho-epitopes was optimized using a design of experiments (DOE) approach to provide the best separation of ZAP-70 expression in positive T- or NK-cells as compared to negative B-cells in peripheral blood samples. A number of commercially available anti-ZAP-70 antibody-conjugates were screened using this methodology, and the antibody-conjugate showing the best performance was chosen to develop a four-color, five antibody assays to measure ZAP-70 levels in whole blood specimens. Results: Using the optimized fixation and permeabilization method, improvement in assay performance (signal-to-noise, S/N) was seen in most of the antibodies tested. The custom SBZAP conjugate gave the best S/N when used in conjunction with this optimized fixation /permeabilization method. In conjunction with carefully standardized instrument set-up protocols, we obtained both intra- and interlaboratory reproducibility in the analysis of ZAP-70 expression in whole blood samples from normal and CLL patients. Conclusions: The development of a sensitive, specific and highly reproducible ZAP-70 assay represents only the first essential step for any clinical assay. The universal implementation of a validated data analysis method and the establishment of methodology-based cutoff points for clinical outcomes must next be established before ZAP-70 protein analysis can be routinely implemented in the clinical laboratory. © 2006 International Society for Analytical Cytology [source] Lectin-aided separation of circulating tumor cells and assay of their response to an anticancer drug in an integrated microfluidic deviceELECTROPHORESIS, Issue 18 2010Li Li Abstract Metastasis caused by the entry of circulating tumor cells (CTCs) into the bloodstream or lymphatic vessels is a major factor contributing to death in cancer patients. Separation of CTCs and studies on CTC,drug interactions are very important for prognostic and therapeutic implications of metastatic cancer. In this study, an integrated microfluidic device for CTC separation through the combination of lectin and microstructure is presented. This microfluidic device and lectin concanavalin A were utilized for the separation of K562 cells in whole blood samples. The results showed that the separation efficiency can reach 84%, which is much higher than that of an experiment without concanavalin A treatment. To further demonstrate the feasibility of this microfluidic device application in sequential studies after target cells were separated, the interactions of K562 cells and an anticancer drug, cytarabine, were also examined. After 6,h on-chip treatment with cytarabine, the viabilities of K562 cells were 85.29, 77.05, and 40% for drug concentration levels of 0.25, 0.5, and 1.0,g/L, respectively. This system can facilitate the rapid and efficient in vitro investigation of CTC separation and CTC-related studies. [source] Automated counting of white and red blood cells in the cerebrospinal fluidINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 4 2000M.W. Aune Summary The objective of this study was to examine to what extent the automated method of the Bayer H*2 instrument could replace the visual counting of white and red blood cells in cerebrospinal fluid. The number of white blood cells as well as the percentage of mononuclear and polymorphonuclear cells were counted in the ,Baso channel' (Research screen 3) whereas the number of red cells were registered as the ,R-count' (Research screen 1). All automated cell counts were compared to visual estimates. The automated count yielded reliable results down to 5 × 106 white blood cells/l and 5 × 108 red blood cells/l. In some samples ,noise' was present in the Baso channel. A correct white blood cell count could then be obtained by counting the cells directly as dots on the screen. It was possible to differentiate between polymorphnuclear cells and mononuclear cells at all WBC concentrations. The automated counting of cerebrospinal fluid can be performed without changing thresholds or sample volumes of the instrument. Thus, in the routine practice it will be possible to alternate between automated counting of whole blood samples and cerebrospinal fluid samples. [source] Triclosan inhibition of acute and chronic inflammatory gene pathwaysJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 5 2010Silvana P. Barros Barros SP, Wirojchanasak S, Barrow DA, Panagakos F, Devizio W, Offenbacher S. Triclosan inhibition of acute and chronic inflammatory gene pathways. J Clin Periodontol 2010; 37: 412,418. doi: 10.1111/j.1600-051X.2010.01548.x. Abstract Aim: We sought to determine whether triclosan (2,4,4,-trichloro-2,-hydroxydiphenylether), an extensively used anti-plaque agent with broad-spectrum anti-microbial activity, with reported anti-inflammatory effects via inhibition of prostaglandin E2 and interleukin 1 (IL-1),, could also more broadly suppress multiple inflammatory gene pathways responsible for the pathogenesis of gingivitis and periodontitis. Materials and Methods: As an exploratory study, the effects of triclosan on the inflammatory gene expression profile were assessed ex vivo using peripheral whole blood samples from eight periodontally healthy donors. Ten-millilitres whole blood aliquots were incubated 2 h with 0.3 ,g/ml Escherichia coli lipopolysaccharide (LPS) with or without 0.5 ,g/ml triclosan. Affymetrix microarray gene expression profiles from isolated leucocytes and pathway-specific quantitative polymerase chain reaction arrays were used to investigate changes in expression of target cytokines and cell signalling molecules. Results: Ex vivo human whole blood assays indicated that triclosan significantly down-regulated the LPS-stimulated expression of Toll-like receptor signalling molecules and other multiple inflammatory molecules including IL-1 and IL-6 and the dampening of signals that activate the T-helper type 1 acquired immune response via suppression of CD70 with concomitant up-regulation of growth factors related to bone morphogenetic protein (BMP)2 and BMP6 synthesis. Conclusions: Anti-inflammatory effects were found in this exploratory survey, including suppression of microbial-pathogen recognition pathway molecules and the suppression of acute and chronic mediators of inflammation. [source] Measurement of HbA1c from stored whole blood samples in the Atherosclerosis Risk in Communities studyJOURNAL OF DIABETES, Issue 2 2010Elizabeth SELVIN Abstract Background:, The aims of the present study were to demonstrate the reliability of HbA1c measurements during two time periods and to compare these measurements with HbA1c distribution in the general US population. Methods:, HbA1c was measured in 14 069 whole blood samples in the Atherosclerosis Risk in Communities (ARIC) study using different HPLC instruments across two time periods, namely 2003,2004 and 2007,2008. At the time of measurement, samples had been in storage at ,70°C for up to 18 years. To assess differences in values, HbA1c measurements were repeated in 383 samples at both periods. Indirect comparisons were made by comparing our measurements against those from a nationally representative study. Results:, The coefficients of variation for quality control samples were 1.8% (n = 89) in 2003,2004 and 1.4% (n = 259) in 2007,2008. The correlation between measurements at the two time points was high (r = 0.99), but with a slight bias: 0.29% points higher in 2007,2008 vs 2003,2004 (n = 383; P < 0.0001). The comparison yielded the following Deming regression equation: y(2007,2008) = 0.073 + 1.034x(2003,2004). After alignment using this equation, the distribution of HbA1c in the ARIC study was similar to that in the national study using fresh samples. Conclusions:, Measurements of HbA1c from samples stored for up to 18 years are highly reliable when using state-of-the-art HPLC instruments, but with some bias introduced over time. The HbA1c data now available in the ARIC study should be invaluable for investigations into the clinical utility of HbA1c as a diagnostic test for diabetes. [source] LTR real-time PCR for HIV-1 DNA quantitation in blood cells for early diagnosis in infants born to seropositive mothers treated in HAART area (ANRS CO 01)JOURNAL OF MEDICAL VIROLOGY, Issue 2 2009Avettand-Fènoël Véronique Abstract HIV-1 diagnosis in babies born to seropositive mothers is one of the challenges of HIV epidemics in children. A simple, rapid protocol was developed for quantifying HIV-1 DNA in whole blood samples and was used in the ANRS French pediatric cohort in conditions of prevention of mother-to-child transmission. A quantitative HIV-1 DNA protocol (LTR real-time PCR) requiring small blood volumes was developed. First, analytical reproducibility was evaluated on 172 samples. Results obtained on blood cell pellets and Ficoll-Hypaque separated mononuclear cells were compared in 48 adult HIV-1 samples. Second, the protocol was applied to HIV-1 diagnosis in infants in parallel with plasma HIV-RNA quantitation. This prospective study was performed in children born between May 2005 and April 2007 included in the ANRS cohort. The assay showed good reproducibility. The 95% detection cut-off value was 6 copies/PCR, that is, 40 copies/106 leukocytes. HIV-DNA levels in whole blood were highly correlated with those obtained after Ficoll-Hypaque separation (r,=,0.900, P,<,0.0001). A total of 3,002 specimens from 1,135 infants were tested. The specificity of HIV-DNA and HIV-RNA assays was 100%. HIV-1 infection was diagnosed in nine infants before age 60 days. HIV-DNA levels were low, underlining the need for sensitive assays when highly active antiretroviral therapy (HAART) has been given. The performances of this HIV-DNA assay showed that it is adapted to early diagnosis in children. The results were equivalent to those of HIV-RNA assay. HIV-DNA may be used even in masked primary infection in newborns whose mothers have received HAART. J. Med. Virol. 81:217,223, 2009. © 2008 Wiley-Liss, Inc. [source] Antiretroviral genotypic resistance in plasma RNA and whole blood DNA in HIV-1 infected patients failing HAART,JOURNAL OF MEDICAL VIROLOGY, Issue 10 2008Annalisa Saracino Abstract The extent to which HIV-1 proviral DNA mutations cause clinically relevant antiretroviral resistance is still controversial. Paired plasma HIV-1 RNA and whole blood DNA were compared in patients failing HAART to investigate if the additional knowledge of archived mutations could improve the selection of potentially active drugs. Seventy-three HIV-1-infected patients with first/second HAART failure were studied before starting a new regimen based on RNA genotyping. Follow-up data after a 12-week therapy were available. DNA genotyping was retrospectively performed on stored whole blood samples and mutational profiles were compared to those from RNA. The mean number of IAS pol mutations was significantly higher in RNA (4.45,±,2.76) than in DNA (2.88,±,2.47) (P,<,0.001). DNA genotyping provided a 6% increase in detection of resistance-associated mutations. Among 64/73 patients showing discordant DNA/RNA profiles, 54 (84%) also differed for predicted active drugs. 16/73 (22%) patients had ,1 mutation revealed by DNA genotyping alone, probably affecting therapy success in 2/16. However, neither RNA/DNA discordance nor detection of isolated DNA mutations were statistically associated with outcome. In conclusion, plasma RNA remains the elective choice for HIV genotyping in patients with therapy failure, even if the detection of proviral resistance-associated mutations, not simultaneously found in RNA, is a frequent event. Therefore, in some cases DNA plus RNA genotyping might assist in choosing more accurately subsequent antiretroviral regimens. J. Med. Virol. 80:1695,1706, 2008. © 2008 Wiley-Liss, Inc. [source] Elevated serum levels of the apoptosis related molecules TNF- ,, Fas/Apo-1 and Bcl-2 in oral lichen planusJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 7 2004A. Sklavounou-Andrikopoulou Background:, The serum circulatory levels of apoptosis related molecules measured in patients with oral lichen planus (OLP) and healthy individuals in order to investigate possible alterations associated with the clinical forms of OLP. Methods:, Serum levels of tumor necrosis factor (TNF)- ,, soluble Fas (sFas) and Bcl-2 studied by enzyme-linked immunosorbent assay in whole blood samples in 13 OLP reticular, 13 OLP atrophic-erosive form patients and 26 healthy subjects. Results:, Significantly elevated levels of TNF- , and sFas detected in OLP patients as compared with controls. Serum concentrations of Bcl-2 although increased in 17/26 patients, they were not statistically significant. Reticular OLP exhibited slightly elevated TNF- , and significantly elevated Bcl-2 serum levels, compared with erosive OLP. Conclusions:, These data suggest that a putative dysfunction in the Fas/FasL mediated apoptosis might be involved in the OLP pathogenesis. A downregulation of Bcl-2 serum levels in the atrophic-erosive OLP may be associated with promotion of the disease activity. [source] Postoperative serum attenuates LPS-induced release of TNF-, in orthopaedic surgeryJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 10 2007Olav Reikerås Abstract Studies with ex vivo stimulation of whole blood samples from injured patients have revealed a diminished production capacity for a broad range of secretory products, including inflammatory cytokines. Recent interest has focused on the release of mediators in serum that depress the cell-mediated immune response following trauma. The involvement of the lipid mediator prostaglandin E2 (PGE2) has been assumed because it is a potent endogenous immunosuppressor. In the present study, we tested the hypothesis that inhibitory substances circulating in the patient's serum after a major musculoskeletal trauma might impair leukocyte function by evaluating the effect of such serum on cytokine release in a whole blood model. Six females and three males undergoing elective total hip replacement were included in the study. Ex vivo LPS-induced TNF-, and IL-10 were measured in whole blood sampled preoperatively and added serum taken before, at the end of operation, and at postoperative days 1 and 6 with saline as negative control. LPS induced significant releases of TNF-, and IL-10 in whole blood. Addition of preoperative, postoperative, and day-1 postoperative serum did not alter the LPS-induced release of TNF-, as compared to saline. In the presence of serum from postoperative day 6, however, the expression of TNF-, was significantly reduced as compared to saline and preoperative serum (p,=,0.021 and 0.008, respectively). Neither of the serum samples altered the release of IL-10. PGE2 was significantly (p,=,0.008) increased in serum at postoperative day 6 as compared to preoperative levels. In conclusion, these data show that at day 6 after major orthopaedic surgery, the patient serum contained activity that inhibited ex vivo LPS-induced TNF-, release. The potent TNF-, inhibitory activity found at day 6 after injury correlated with increased levels of PGE2 and indicates cell-mediated hyporesponsiveness to a second stimulus. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1395,1400, 2007 [source] Antibiotics modulate the stimulated cytokine response to endotoxin in a human ex vivo, in vitro modelACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 9 2006S. Ziegeler Background:, Sepsis may lead to the suppression of stimulated cytokine release after Gram-negative stimuli, correlating with a fatal outcome. Treatment of sepsis includes adequate therapy with antibiotics. The aim of this study was to investigate the role of antibiotics in the modulation of the lipopolysaccharide (LPS)-stimulated cytokine response of human monocytes. Methods:, In this ex vivo, in vitro study, whole blood samples were taken from 10 healthy volunteers, stimulated with LPS in the presence or absence of various antibiotics (penicillin, amoxicillin, cefuroxime, ceftazidime, cefotaxime, piperacillin/tazobactam, imipenem/cilastatin, gentamicin, netilmicin, ciprofloxacin, vancomycin) and cultured for 24 h. Thereafter, tumor necrosis factor-, (TNF-,) and interleukin-10 (IL-10) were measured in the supernatants by enzyme-linked immunosorbent assay (ELISA). Furthermore, CD14 and HLA-DR expression on monocytes was assessed using flow cytometry. Results:, All cephalosporins decreased LPS-stimulated IL-10 release. Cefuroxime and cefotaxime also decreased the expression density of the LPS recognition molecule CD14 on monocytes. An increase in LPS-stimulated IL-10 release was observed with vancomycin. A suppression of LPS-stimulated TNF-, and IL-10 release was observed in the presence of ciprofloxacin. Conclusion:, These results indicate a modulation of the expression density of CD14 on monocytes, together with a shift from a balanced to an inflammatory cytokine release pattern, by cefuroxime and cefotaxime. Vancomycin changes the response to an anti-inflammatory release pattern. After ciprofloxacin, a profound unresponsiveness of immune-competent cells to LPS stimulation is observed. Because of the critical role of a balanced innate immune response, these data may be of importance for the selection of antibiotics in septic patients. [source] Respiratory burst activity of polymorphonuclear cells is dependent on the cell preparation techniqueACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 6 2003J. Zhao Background: Controversial results have been reported regarding the effect of anaesthetics on superoxide anion production during the respiratory burst (RB) of polymorphonuclear cells (PMN). The differences could be caused by the cell preparation methods and the aim of this study was to compare two techniques. Methods: RB activity was measured in cell suspensions isolated with the single-step Ficoll procedure and in unfractionated whole blood. Two concentrations of propofol (therapeutic and 10-fold of this, 6 µg ml,1 or 60 µg ml,1) were investigated after cell preparation with both methods. RB was stimulated with Escherichia coli (E. coli), phorbol 12-myristate 13-acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) and measured by means of fluorescence intensity in a flow cytometer. Results: The percentage of PMNs in whole blood which generate superoxide anions in response to fMLP was significantly lower (2.5 ± 0.7%; mean ± SEM) than that in Ficoll isolated cell suspensions (15.1 ± 1.7%). Incubation with propofol led to a concentration-related decrease of RB activity in Ficoll separated PMNs after both PMA and fMLP stimulation. No significant effect of propofol was observed on the RB in PMA stimulated whole blood samples. Conclusion: The results suggest that the influence of cell preparation methods should be considered when the in vitro effects of anaesthetics on PMN functions are studied with flow cytometric methods. [source] Determination of residues of endosulfan in human blood by a negative ion chemical ionization gas chromatographic/mass spectrometric method: impact of long-term aerial spray exposurePEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 3 2003Atmakuru Ramesh Abstract A new and sensitive analytical method using negative ion chemical ionization gas chromatography/mass spectrometry in selective ion monitoring (SIM) mode has been developed for the determination of residues of endosulfan in the human blood. The residues of endosulfan are extracted from whole blood samples without separating the serum by the addition of 60% sulfuric acid at 10,°C followed by partition with hexane,+,acetone (9,+,1 by volume). The total endosulfan is quantified as the sum of alpha-endosulfan, beta-endosulfan and endosulfan sulfate in SIM mode. The mass-fragment ions used for this purpose that are monitored for in SIM mode include endosulfan diol: 95, 169, 214, 313, alpha-endosulfan: 99, 242, 270, 406, beta-endosulfan: 99, 242, 270, 406, and endosulfan sulfate: 97, 353, 386. Recovery experiments were conducted at the concentration range 1.0,100,pg,ml,1. Results showed 112,98% recovery of total endosulfan from the whole blood samples. The relative standard deviation was 1.49,2.68%. The method was found to be highly sensitive in quantifying endosulfan residues down to the 0.1,pg,ml,1 level. Conversion of endosulfan to endosulfan diol was found to be less than 0.1% under the conditions used. The results were compared with published data. The applications of the analytical method for the determination of endosulfan residues in real samples was tested by analyzing 106 human blood samples collected from a population living in Padre village, Kasargode District, Kerala, India, where aerial spraying of endosulfan has been a common agricultural practice over the years. The results showed that none of the blood samples contained residues of endosulfan (alpha-endosulfan,+,beta-endosulfan,+,endosulfan sulfate) or endosulfan diol. The results were confirmed by the detection of the appropriate amounts in a number of these samples which had subsequently been spiked with endosulfan. © 2003 Society of Chemical Industry [source] Biomarkers of Mn exposure in humansAMERICAN JOURNAL OF INDUSTRIAL MEDICINE, Issue 11 2007Donald Smith PhD Abstract Background Studies have reported associations between manganese (Mn) exposures and Mn levels in blood and urine, though the suitability of these biological measures as biomarkers of exposure is not well known. Methods We evaluated whether whole blood, plasma, and urine Mn levels reflect exposures in occupationally exposed humans. Results In active ferroalloy workers, blood Mn was associated with total air Mn levels in subjects currently exposed to low (median,=,0.42 ,g/m3, P,=,0.009) and moderate (median,=,4.2 ,g/m3, P,=,0.007) air Mn levels, but not in workers exposed to the highest Mn levels (median,=,292 ,g/m3, P,=,0.31). In bridge welders blood Mn (P,<,0.01), but not plasma or urine Mn was significantly associated with their cumulative respiratory exposure index. In welders, ,6% (range ,3,9%) of whole blood Mn was contained in the plasma fraction, though there was no association between whole blood and plasma Mn levels (Pearson's R,=,0.258, P,=,0.12). In contrast, in fresh whole blood samples spiked with Mn ex vivo ,80% or more of added Mn partitioned in the plasma, while only ,20% or less partitioned in the cellular fraction. Conclusions These data suggest a complex and limited relationship between exposure and blood Mn levels that may depend upon exposure attributes and the latency of blood sampling relative to exposure; plasma and urine Mn appear to be of little utility as exposure biomarkers. This underscores the need to fully characterize and validate these or other biomarkers for use in constructing appropriate exposure metrics and determining exposure,effect relationships. Am. J. Ind. Med. 50:801,811, 2007. © 2007 Wiley-Liss, Inc. [source] PP13 stability in first trimester maternal serum and whole bloodPRENATAL DIAGNOSIS, Issue 6 2010N. J. Cowans Abstract Background Maternal serum placental protein 13 (PP13) has been proposed as a marker for prenatal screening of pre-eclampsia (PE) and other adverse pregnancy outcomes. This study aims to examine the stability of PP13 at different routine temperatures. Methods Maternal serum pools and whole blood samples were stored at ,30 °C', room temperature or refrigerator temperature. Further, serum pools were also subjected to repeated freeze,thaw cycles. PP13 was measured at set time points using an AutoDELFIA® research assay. Results Levels of PP13 are stable, defined as less than 10% change in concentration, in serum for 17.4 h at ,30 °C', 3.4 days at room temperature and for at least 34 days at refrigerator temperature. PP13 concentration is not altered following three ,20 °C to room temperature freeze,thaw cycles. PP13 is stable in whole blood for at least 3 days at all three temperatures studied. Conclusions PP13 is a suitably stable molecule and can be treated under routine laboratory and normal transport temperatures. Copyright © 2010 John Wiley & Sons, Ltd. [source] Derivated fetal haemoglobin as a marker for red cell age in the human fetus reflecting stimulated or impaired red blood cell productionPRENATAL DIAGNOSIS, Issue 7 2001Margriet Huisman Abstract We have determined whether derivated fetal haemoglobin (dHbF, consisting of glycated and acetylated HbF) can be used as a cell age marker for fetal red blood cells (RBCs). Cord blood was obtained between 19 and 39 weeks of gestation from 28 alloimmunised anaemic fetuses (23 RhD+ and 5,Kell) and from 20 non-anaemic fetuses and newborns (controls). Density gradient centrifugation was applied to 36 samples (20 RhD+, 15 controls and 1,Kell) to obtain fractions of increasing cell age. Blood samples were used for measurements of mean cellular volume (MCV), mean cell haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), pyruvate kinase activity (PK) and derivated fetal haemoglobin (dHbF) by cation-exchange HPLC. Reticulocytes were counted only in the whole blood samples. In all density gradient separated RBC fractions, the values for MCV, MCH and PK activity decreased and those of MCHC and dHbF increased with increasing density (equivalent to increasing cell age). The mean density was lower for RBCs of the anaemic RHD group (1.072±0.007,g/ml) than for the non-anaemic controls (1.077±0.005,g/ml) (p<0.05) The RBC density of the Kell sensitised fetus did not differ from those of the controls. In the control group, the values of the cell age markers in whole blood changed significantly with the gestational age, showing an increase of mean age of the erythrocyte population. The best linear relationship was found for dHbF (y=6.28+0.17*weeks; r=0.84; p<0.001). In the anaemic RhD+ fetuses, the RBC age markers did not change with gestational age; the dHbF percentages were lower, and the MCV, MCH, PK values and the reticulocyte counts were higher than in the controls (0.05 A semi-automated solid-phase extraction liquid chromatography/tandem mass spectrometry method for the analysis of tetrahydrocannabinol and metabolites in whole blood,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 17 2009Eshwar Jagerdeo Marijuana is one of the most commonly abused illicit substances in the USA, making cannabinoids important to detect in clinical and forensic toxicology laboratories. Historically, cannabinoids in biological fluids have been derivatized and analyzed by gas chromatography/mass spectrometry (GC/MS). There has been a gradual shift in many laboratories towards liquid chromatography/mass spectrometry (LC/MS) for this analysis due to its improved sensitivity and reduced sample preparation compared with GC/MS procedures. This paper reports a validated method for the analysis of ,9 -tetrahydrocannabinol (THC) and its two main metabolites, 11-nor-9-carboxy-,9 -tetrahydrocannabinol (THC-COOH) and 11-hydroxy-,9 -tetrahydrocannabinol (THC-OH), in whole blood samples. The method has also been validated for cannabinol (CBD) and cannabidiol (CDN), two cannabinoids that were shown not to interfere with the method. This method has been successfully applied to samples both from living people and from deceased individuals obtained during autopsy. This method utilizes online solid-phase extraction (SPE) with LC/MS. Pretreatment of samples involves protein precipitation, sample concentration, ultracentrifugation, and reconstitution. The online SPE procedure was developed using Hysphere C8-EC sorbent. A chromatographic gradient with an Xterra MS C18 column was used for the separation. Four multiple-reaction monitoring (MRM) transitions were monitored for each analyte and internal standard. Linearity generally fell between 2 and 200,ng/mL. The limits of detection (LODs) ranged from 0.5 to 3,ng/mL and the limits of quantitation (LOQs) ranged from 2 to 8,ng/mL. The bias and imprecision were determined using a simple analysis of variance (ANOVA: single factor). The results demonstrate bias as <7%, and imprecision as <9%, for all components at each quantity control level. Published in 2009 by John Wiley & Sons, Ltd. [source] Lifestyle incongruity, stress and immune function in indigenous Siberians: The health impacts of rapid social and economic changeAMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY, Issue 1 2009Mark V. Sorensen Abstract The purpose of this study was to investigate the impact of economic and cultural change on immune function and psychosocial stress in an indigenous Siberian population. We examined Epstein-Barr virus antibodies (EBV), an indirect biomarker of cell-mediated immune function, in venous whole blood samples collected from 143 Yakut (Sakha) herders (45 men and 98 women) in six communities using a cross-sectional study design. We modeled economic change through the analysis of lifestyle incongruity (LI), calculated as the disparity between socioeconomic status and material lifestyle, computed with two orthogonal scales: market and subsistence lifestyle. EBV antibody level was significantly negatively associated with both a market and a subsistence lifestyle, indicating higher cell-mediated immune function associated with higher material lifestyle scores. In contrast, LI was significantly positively associated with EBV antibodies indicating lower immune function, and suggesting higher psychosocial stress, among individuals with economic status in excess of material lifestyle. Individuals with lower incongruity scores (i.e., economic status at parity with material resources, or with material resources in excess of economic status) had significantly lower EBV antibodies. The findings suggest significant health impacts of changes in material well-being and shifting status and prestige markers on health during the transition to a market economy in Siberia. The findings also suggest that relative, as opposed to absolute, level of economic status or material wealth is more strongly related to stress in the Siberian context. Am J Phys Anthropol, 2009. © 2008 Wiley-Liss, Inc. [source] Pharmacodynamics of Mycophenolate Mofetil after Heart Transplantation: New Mechanisms of Action and Correlations with Histologic Severity of Graft RejectionAMERICAN JOURNAL OF TRANSPLANTATION, Issue 8 2002Markus J. Barten The primary mechanism of action in vivo of mycophenolate mofetil (MMF) is believed to be inhibition of lymphocyte proliferation. We used novel assays of lymphocyte functions (pharmacodynamics, PD) in whole blood collected from rat heart allograft recipients treated with MMF to investigate the mechanisms of action of the active metabolite of MMF, mycophenolate acid (MPA) in vivo. Allograft recipients were treated orally once daily with 3 different doses of MMF. Seven days after transplantation, blood was collected 24 h after the penultimate dose and several timepoints after the last dose, after which grafts were removed for microscopic grading of rejection. Lymphocytes in whole blood samples were mitogen stimulated through calcium-dependent and -independent signaling pathways. Inhibition of PD was measured by lymphocyte proliferation and expression of several surface antigens on T cells, and was calculated as area under the time-inhibition of immune function effect curve (AUE0,24 h). We found that inhibition of lymphocyte proliferation and antigen expression by MPA correlated highly with MMF-dose, MPA level and with the histologic severities of graft rejection (p <,0.05). In summary, MPA suppressed lymphocyte proliferation and expression of T-cell surface antigens in whole blood collected from MMF-treated allograft recipients, thus demonstrating the multiple mechanisms of suppression of rejection on peripheral blood T cells after MMF treatment. [source] The proinflammatory activity of recombinant serum amyloid A is not shared by the endogenous protein in the circulationARTHRITIS & RHEUMATISM, Issue 6 2010Lena Björkman Objective Elevated serum levels of the acute-phase protein serum amyloid A (SAA) are a marker for active rheumatoid arthritis (RA), and SAA can also be found in the tissues of patients with active RA. Based on a number of studies with recombinant SAA (rSAA), the protein has been suggested to be a potent proinflammatory mediator that activates human neutrophils, but whether endogenous SAA shares these proinflammatory activities has not been directly addressed. The present study was undertaken to investigate whether SAA in the plasma of patients with RA possesses proinflammatory properties and activates neutrophils in a manner similar to that of the recombinant protein. Methods Neutrophil activation was monitored by flow cytometry, based on L-selectin shedding from cell surfaces. Whole blood samples from healthy subjects and from RA patients with highly elevated SAA levels were studied before and after stimulation with rSAA as well as purified endogenous SAA. Results Recombinant SAA potently induced cleavage of L-selectin from neutrophils and in whole blood samples. Despite highly elevated SAA levels, L-selectin was not down-regulated on RA patient neutrophils as compared with neutrophils from healthy controls. Spiking SAA-rich whole blood samples from RA patients with rSAA, however, resulted in L-selectin shedding. In addition, SAA purified from human plasma was completely devoid of neutrophil- or macrophage-activating capacity. Conclusion The present findings show that rSAA is proinflammatory but that this activity is not shared by endogenous SAA, either when present in the circulation of RA patients or when purified from plasma during an acute-phase response. [source] Dried blood spot sampling in combination with LC-MS/MS for quantitative analysis of small moleculesBIOMEDICAL CHROMATOGRAPHY, Issue 1 2010Wenkui Li Abstract The collection of whole blood samples on paper, known as dried blood spot (DBS), dates back to the early 1960s in newborn screening for inherited metabolic disorders. DBS offers a number of advantages over conventional blood collection. As a less invasive sampling method, DBS offers simpler sample collection and storage and easier transfer, with reduced infection risk of various pathogens, and requires a smaller blood volume. To date, DBS-LC-MS/MS has emerged as an important method for quantitative analysis of small molecules. Despite the increasing popularity of DBS-LC-MS/MS, the method has its limitations in assay sensitivity due to the small sample size. Sample quality is often a concern. Systematic assessment on the potential impact of various blood sample properties on accurate quantification of analyte of interest is necessary. Whereas most analytes may be stable on DBS, unstable compounds present another challenge for DBS as enzyme inhibitors cannot be conveniently mixed during sample collection. Improvements on the chemistry of DBS card are desirable. In addition to capturing many representative DBS-LS-MS/MS applications, this review highlights some important aspects of developing and validating a rugged DBS-LC-MS/MS method for quantitative analysis of small molecules along with DBS sample collection, processing and storage. Copyright © 2009 John Wiley & Sons, Ltd. [source]
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