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Flow Cytometric Measurement (flow + cytometric_measurement)
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] Use of a blocking antibody method for the flow cytometric measurement of ZAP-70 in B-CLLCYTOMETRY, Issue 4 2006Mark Shenkin Abstract Background: In this study we developed a method to measure the amount of ZAP-70 [zeta accessory protein] in B-CLL cells without relying on the ZAP-70 expression of patient B or T cells to normalize fluorescence intensity. Methods: B-CLL cells were fixed with formaldehyde before surface staining with gating antibodies CD19PC5 and CD5FITC. The cells were permeabilized with saponin, and the ZAP-70 antigen was blocked in one tube with unlabeled antibody to ZAP-70 [clone 1E7.2]. Zap-70-PE was then added to this tube. ZAP-70-PE was added to a second tube without unlabeled antibody to ZAP-70. The mean fluorescence intensity of the ZAP-70 in the tube without unlabeled antibody divided by the mean fluorescence intensity of the ZAP-70 in the tube with unlabeled antibody equals the RATIO of total fluorescence to non-specific ZAP-70 fluorescence in the B-CLL cells. In a second method of analysis, a region is created in the histogram showing ZAP-70 fluorescence intensity in the tube with unlabeled antibody to ZAP-70. This region is set to 0.9% positive cells. This same region is then used to measure the % positive [%POS] ZAP-70 cells in the tube without unlabeled antibody to ZAP-70. The brighter the ZAP-70 fluorescence above the non-specific background, the higher the %POS. Results: Due to the varying amount of non-specific staining between patient B-CLL cells and other cells, the blocking antibody method yielded a more quantitative and reproducible measure of ZAP-70 in B-CLL cells than other methods, which use the ratio of B-CLL fluorescence to normal B or T-cell fluorescence. Using this improved method, ZAP-70 was determined to be negative if the RATIO was less than 2:1 and positive if the RATIO was greater than 2:1. ZAP-70 was determined to be negative if the %POS was less than 5% and positive if the %POS was greater than 5%, a cut-off value lower than previous values published, due to exclusion of non-specific staining. Both cut-offs were based upon patient specimen distribution profiling. Conclusions: Use of a blocking antibody resulted in a robust, reproducible clinical B-CLL assay that is not influenced by the need to measure the amount of ZAP-70 in other cells. ZAP-70 results segre gate patients into indolent and aggressive groups suggested by published clinical outcomes. © 2006 International Society for Analytical Cytology [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] DNA Index in childhood acute lymphoblastic leukaemia: a karyotypic method to validate the flow cytometric measurementINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 3 2010P. RACHIERU-SOURISSEAU Summary The DNA index (DI) is a prognostic factor in childhood acute lymphoblastic leukemia (ALL). The accuracy of DI measurement is important for treatment stratification: hyperdiploidy with DI , 1.16 is predictive of favorable prognosis whereas hypodiploidy is associated with poor prognosis. The aim of this study was to validate the accuracy of the DI measured by flow cytometry (FCM) by comparison with the karyotype. From samples of 112 childhood ALL, we created a formula to calculate a theoretical DNA index (tDI) based on the blast cell karyotype, taking into account the additional or missing chromosome material of the major clone. FCM DI correlated with tDI calculated from karyotype (R = 0.987) and with modal chromosome number (DI = 0.0202 × Modal NB + 0.0675 and R = 0.984). In three cases a hypodiploid blast cell population was detected by FCM, while only the duplicated clone was identified by the karyotype. The strong correlation between tDI and DI validates the accuracy of FCM quantification, which is technically fast on fresh or frozen samples. If the karyotype is essential to analyze chromosomal abnormalities, FCM provides complementary information in aneuploid ALLs, either by confirming the cytogenetic data or by detecting additional clones not identified when only using cytogenetic data. [source] Dilazep, a nucleoside transporter inhibitor, modulates cell cycle progression and DNA synthesis in rat mesangial cells in vitroCELL PROLIFERATION, Issue 1 2000T. Sakumura The direct effects of the nucleoside transporter inhibitor dilazep on the cell cycle of mesangial cells have not before been investigated. The purpose of this study was to elucidate whether dilazep can inhibit the proliferation of mesangial cells and how it interferes with the cell cycle of these cells. DNA histograms were used and BrdUrd uptake rate was measured by flow cytometry. There was no significant difference in the cell numbers among the untreated group and the 10,5M, 10,6M or 10,7M dilazep-treated groups at 24 h of incubation. However, at 48 and 72 h, the cell numbers in the dilazep-treated groups were significantly lower compared with that of the untreated group (P0.005). The DNA histograms of cultured rat mesangial cells at 12, 24, and 48 h of incubation with 10,5 M dilazep showed that the ratio of the S phase population in the dilazep-treated group decreased by 2.2% at 12 h, by 9.6% at 24 h, and by 18.9% at 48 h compared with the untreated group. The ratio of the G0/G1 phase population in the dilazep-treated group significantly increased: 6.8% at 12h (P 0.05), 13.9% at 24 h (P 0.001), and 76.5% at 48 h (P 0.001) compared with the untreated group. A flow cytometric measurement of bivariate DNA/BrdUrd distribution demonstrated that the DNA synthesis rate in the S phase decreased after 6 h (P 0.005) and 12 h (P 0.05) of incubation compared with the untreated group. These results suggest that dilazep inhibits the proliferation of cultured rat mesangial cells by suppressing the G1/S transition by prolonging G2/M and through decreasing the DNA synthesis rate [source] In Vitro and In Silico Analysis of Annexin V Binding to Lymphocytes as a Biomarker in Emergency Department Sepsis StudiesACADEMIC EMERGENCY MEDICINE, Issue 9 2007Colin F. Greineder Background: Peripheral blood lymphocyte apoptosis is a recognized feature of serious infection and sepsis and can be easily quantified by flow cytometric measurement of annexin V binding to the cell surface. Use of apoptosis as a biomarker in emergency department (ED) studies of sepsis is potentially difficult because of sample processing requirements and limited availability of a research cytometer with which to measure patient samples. Objectives: To assess, in vitro and in simulation, the relationship between sample stability, timing of patient enrollment, and diagnostic performance of a flow cytometric assay for sepsis in patients evaluated in EDs. Methods: Assuming any clinical trial would require daily sample batching, the authors measured the stability of lymphocyte samples over time, noting the rate at which annexin V,negative cells became positive as ED processing delays increased. With these data, they then optimized a study design that could evaluate lymphocyte apoptosis as a sepsis biomarker by using a series of Monte Carlo,based simulated clinical trials. Results: The authors found that annexin V,negative lymphocytes become positive during storage delays that would be encountered in an ED sepsis trial. The extent of this deterioration was least among cells left as whole blood at room temperature until just before analysis or when lymphocytes were isolated early and stored in culture media at 4°C until analysis. When the expected rate of sample deterioration was considered in simulated clinical trials, an inverse relationship was found between the rate at which patients are enrolled and the best achievable receiver operating characteristic curve a study could produce. Conclusions: Peripheral blood samples being analyzed for lymphocyte apoptosis degrade at a rate relevant to the design of ED trials of sepsis. Because of sample processing delays inherent in studying unscheduled septic patients, the performance of annexin V binding as a biomarker for sepsis can approach, but not be expected to exceed, its performance in a comparable intensive care unit,based study. [source] | |||||||||||||