A new single-platform method for the enumeration of CD34+ cells

Guidelines for flow cytometric enumeration of CD34⁺ hematopoietic stem cells (HSC) recommend the use of a single-platform assay. The SCE kit has recently been commercialized by BD Biosciences. Results obtained with this newly available kit were compared with CD34⁺ cell enumerations obtained in parallel with already commercialized diagnostic kits; fresh peripheral blood, apheresis, cord blood (CB) and bone marrow (BM) samples, as well as thawed apheresis and CB samples, were assayed. The SCE kit produced data for CD34⁺ enumeration that correlate well with data produced with the older assays (r²≥0.9). Practical advantages were the ability to enumerate viable CD34 cells in all kinds of HSC products, the absence of bead pipetting (which decreases results precision) and a gating strategy complying with international recommendations. A major disadvantage was the absence of specific software for data analyses and presentation of results.     

Reduction of variation in T-cell subset enumeration among 55 laboratories using single-platform,three or four-color flow cytometry based on CD45 and SSC-based gating of lymphocytes

BACKGROUND: Enumeration of CD4(+) and CD8(+) T-cell subsets provides relevant information for diagnosis and monitoring of patients with cellular immunodeficiencies. As a result, an external quality assurance scheme was implemented in Belgium, The Netherlands, and Luxembourg in 1995. A workshop was held to train the participants in state-of-the art technology for assessment of absolute T-cell subset counts (i.e., a three or four-color, single-platform assay with lymphocyte gating based on CD45 and sideward light scatter) with the aim to achieve between-site coefficients of variation (CVs) <10% and within-site CVs <5% for > or =75% of the participants. METHODS: Three send-outs of stabilized blood from a healthy donor were distributed to 55 laboratories, each with the request to perform the standard assay on three occasions. For comparison, each laboratory performed its local technique in parallel. RESULTS: With the standard technique, between-site CVs of approximately 8% (CD3+ T cells), approximately 9% (CD4+ T cells), and approximately 10% (CD8+ T cells) were achieved. Within-site CVs were <5% for 82% (CD3+ T cells) and approximately 70% (CD4+ and CD8+ subsets) of the participants. Local techniques yielded between-site CVs of 13%-17% for CD3+, CD4+, and CD8+ T cells. CONCLUSIONS: The state-of-the-art technology for T-cell subset enumeration was implemented successfully among 55 Belgian-Dutch laboratories and resulted in significant reductions of between-site variation of absolute CD3+, CD4+, and CD8+ T-cell counts.     

Chemoattractant lymphokines specific for the helper/inducer T-lymphocyte subset

The cellular content of T-lymphocyte-rich inflammatory sites is dependent in part on the in situ elaboration of chemoattractant factors. We have previously described three T-lymphocyte-specific chemoattractant lymphokines; a chemokinetic factor, lymphocyte chemoattractant factor (LCF, MW 56,000), and two distinct lymphocyte migration inhibitory factors (LyMIF75K, MW 75,000; and LyMIF35K, MW 35,000). These factors are produced by human T cells in response to antigen, concanavalin A, or histamine stimulation. In this communication, we report that LCF and LyMIF35K are produced by OKT8+ (suppressor/cytotoxic) and OKT4+ (helper/inducer) lymphocytes, respectively, and are selectively chemoattractant for the OKT4+ lymphocyte subset. LyMIF75K is produced by OKT4+ cells and inhibits both OKT4+ and OKT8+ lymphocyte migration. Production of LCF and LyMIF35K by infiltrating lymphocyte subsets may be one mechanism whereby unactivated helper/inducer T lymphocytes are selectively recruited to sites of inflammation.     

True volumetric method for flow cytometric enumeration of CD34 + stem cells and its agreement with a standard bead-based single-platform protocol

Background aimsStem cells are commonly enumerated with bead-based methods in blood and marrow progenitor cell transplantation centers. We compared the International Society of Hematotherapy and Graft Engineering (ISHAGE) bead-based method with a true volumetric one that obviates the use of fluorescent beads for enumeration.MethodsFrom 31 samples, including 15 peripheral blood samples and 16 leukapheresis products, CD34 ⁺ cells were enumerated with the single-platform bead-based ISHAGE method and a true volumetric method. After exclusion of two outliers, one from the peripheral blood group and the other from the leukapheresis group, the results were compared.ResultsIn the peripheral blood category, no significant difference was observed. However, a proportional systematic error was seen in the leukapheresis group. The systematic error was corrected in the leukapheresis group using a regression line equation. The 95% confidence interval of differences was [–5.83, 2.18] for the peripheral blood and [–38.40, 38.77] for the leukapheresis group after correction of the systematic error.ConclusionsThe true volumetric method is a simple and reliable approach that can be used instead of the more popular bead-based procedures.     

Comparison of two single-platform ISHAGE-based CD34 enumeration protocols on BD FACSCalibur and FACSCanto flow cytometers

Background aimsEnumeration of viable CD34⁺ cells provides critical information for the bone marrow (BM) transplant physician. The single-platform ISHAGE protocol is the most reliable method currently available to quantitate accurately this important subset of cells. Previous studies have shown that 5 CD34⁺ cells/µL blood predicts the collection of at least 0.5 × 10⁶ CD34⁺ cells/kg patient weight. From the apheresis product, infusion of 2.5 × 10⁶ viable CD34⁺ cells (measured pre-cryopreservation)/kg patient weight will reliably permit engraftment of the hematopoietic system (as measured by the time to 20000 platelets/µL) by day 12–14 post-infusion.MethodsWe compared the CD34⁺ cell numbers derived from Flow Count-based Stem-Kit?; (Beckman Coulter) and Trucount? tube-based stem cell enumeration (SCE) kit (BD Biosciences) ISHAGE templates on BD FACSCalibur? and BD FACSCanto? cytometers on 12 granulocyte–colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) and 10 peripheral blood stem cell (PBSC) samples.ResultsComparison of results showed that there was no statistical difference between samples run with Stem-Kit on the FACSCalibur versus SCE kit-based assays on either the FACSCalibur or FACSCanto. Mean results for the Stem-Kit/Calibur combination were 137, for SCE kit/Calibur 140 and for SCE kit/Canto 137 cells/µL. Pair-wise comparison of data based on rank order showed no statistically significant difference and all correlation coefficients had an R²>0.98.ConclusionsThe two kits generated very similar data on a range of fresh samples regardless of instrument platform. These results confirm and extend the utility of the single-platform ISHAGE protocols with a variety of reagent kits and instrument platforms.     

Australasian CD34+ quality assurance program and rationale for the clinical utility of the single-platform method for CD34+ cell enumeration

BACKGROUND: Enumeration of CD34(+) cells should be accurate and comparable between institutions, particularly when making clinical decisions, evaluating data, and in clinical trials. An Australasian CD34(+) quality assurance program (QAP) has been established to compare CD34(+) cell results and method (Part 1). Unexpected variation in WBCCs led to Part 2 of this report. METHODS: Part 1: Methods reagents and results were evaluated for 12 QAP samples analyzed by 36-43 centers. Part 2: The effects of different anticoagulants on WBCC of 12 peripheral blood samples (PBs) were compared using three cell counters. To test the validity of applying the conclusions to clinical samples, the WBCCs of leukapheresed products and BM harvest were also compared. RESULTS: Part 1: In some samples, WBCCs determined by certain cell-counter groups were significantly different. Results for percentage of CD34(+) and CD34(+)/microL suggest that standardization on the lyse-no-wash and single platform (SP) method reduces variation of results between institutions. Part 2: Using different counters, PB WBCC in ACD-A showed greater variation than the same PB in EDTA. For PB in different anticoagulants, the extent of difference in WBCC for the same PB is dependent on the counter used. DISCUSSION: This CD34 QAP has identified ACD-A as an additional factor that contributes to the disparate WBCCs, which may further compromise the accuracy of CD34(+) cell counts obtained by the dual platform (DP) method, especially for leukapheresed products. In order to achieve greater accuracy within individual institutions, as well as permitting more reliable inter-institutional comparisons, our data supports the adoption of the SP as the standard method for CD34(+) cell enumeration.     

Evaluation of four methods for enumeration of Vibrio parahaemolyticus

Two membrane filter (MF) and two most-probable-number methods for enumerating Vibrio parahaemolyticus were compared. The MF methods used elevated-temperature incubations (41 and 42 degrees C) and were more specific than the most-probable-number methods (conducted at 35 degrees C). The MF method with a hydrophobic grid and a repair step was most effective.     

Evaluation of methods for enumeration of Vibrio parahaemolyticus from seafood

The efficiency of several enrichment broths in recovering Vibrio parahaemolyticus inoculated into fish homogenates was studied. Recovery by the most probable number technique was very low in all the broths, while direct plating on thiosulfate citrate bile salt sucrose agar yielded better recovery. A decrease in the enrichment time to 8 from 18 h did not improve recovery. At concentrations exceeding 2.5 micrograms/ml, polymyxin was inhibitory to V. parahaemolyticus.     

Evaluation of four methods for enumeration of Vibrio parahaemolyticus.

Two membrane filter (MF) and two most-probable-number methods for enumerating Vibrio parahaemolyticus were compared. The MF methods used elevated-temperature incubations (41 and 42 degrees C) and were more specific than the most-probable-number methods (conducted at 35 degrees C). The MF method with a hydrophobic grid and a repair step was most effective.     

Evaluation of methods for enumeration of Vibrio parahaemolyticus from seafood.

The efficiency of several enrichment broths in recovering Vibrio parahaemolyticus inoculated into fish homogenates was studied. Recovery by the most probable number technique was very low in all the broths, while direct plating on thiosulfate citrate bile salt sucrose agar yielded better recovery. A decrease in the enrichment time to 8 from 18 h did not improve recovery. At concentrations exceeding 2.5 micrograms/ml, polymyxin was inhibitory to V. parahaemolyticus.     

Evaluation of selective media for enumeration of yeasts during wine fermentation

The growth of individual species of yeasts during wine fermentations was measured by plating wine samples on malt extract, ethanol sulphite and lysine agars. Colonies of Saccharomyces cerevisiae dominated on plates of malt extract agar and sometimes masked the presence of other non- Saccharomyces species. Lysine agar suppressed the growth of S. cerevisiae and enabled the enumeration of non- Saccharomyces species such as Kloeckera apiculata, Candida stellata and Saccharomycodes ludwigii. The growth of non- Saccharomyces yeasts on ethanol sulphite agar was variable.     

Evaluation of Enterolert for enumeration of enterococci in recreational waters.

Enterolert (IDEXX Laboratories Inc., Westbrook, Maine), a semiautomated, most probable number method for enumeration of enterococci, was compared with the standard membrane filter method by parallel testing of 138 marine and freshwater recreational bathing water samples. No statistically significant difference and a strong linear correlation were found between methods. Culturing of 501 Enterolert test wells resulted in false-positive and false-negative rates of 5.1 and 0.4%, respectively. Less time for setup, incubation (24 versus 48 h), and reading of Enterolert permits more efficient monitoring of recreational bathing areas.     

Evaluation of a rapid,quantitative real-time PCR method for enumeration of pathogenic Candida cells in water

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5' nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.     

Flow cytometric enumeration of absolute lymphocyte number in peripheral blood using two parameters of light scatter

A method was developed to measure the absolute lymphocyte count (ALC) of whole blood using the Spectrum III automated flow cytometer. Ninety-nine samples of human peripheral blood were analysed on the Spectrum and the Coulter Counter S Plus II, to allow for comparison of the two machines. Regression analysis was used to test the extent of agreement between the sets of measurements on the two machines. The results demonstrated that the slope of the regression line was not significantly different from one, indicating a high level of correlation between Spectrum and Coulter ALC's. However, the mean difference between Coulter and Spectrum ALC's was not equal to zero, with the Spectrum giving counts approximately 10% lower than those of the Coulter machine. This is attributed to the different ways by which the two machines define a lymphocyte, the Spectrum III by two parameters of light scatter and the Coulter S Plus II by the single parameter of cell volume.     

Evaluation of a pour-plate system with a rabbit plasma-bovine fibrinogen agar for the enumeration of Staphylococcus aureus in food

Investigations were carried out concerning the selectivity and productivity of rabbit plasma fibrinogen (RPF) agar according to Beckers et al. (H. J. Beckers, F. M. van Leusden, W. M. Hogeboom, and E. H. M. Delfgou-van Asch. 1980. De Ware(n)-Chemicus, 10: 125-130). Its selectivity was compared with pork plasma fibrinogen (PPF) medium according to Hauschild et al. (A. H. W. Hauschild, C. E. Park, and R. Hilsheimer. 1979. Can. J. Microbiol. 25: 1052-1057) and its productivity was compared with PPF medium and Baird-Parker's egg yolk tellurite glycine pyruvate (ETGP) agar. In total 139 samples of naturally contaminated foodstuffs were examined. RPF agar scored higher than ETGP agar; although only small (mean value of differences 0.09 log units), the differences were statistically significant. While no significant differences in sensitivity between RPF agar and PPF medium were encountered, RPF agar was statistically more selective than PPF medium. It is concluded that RPF agar is very suitable for the enumeration of Staphyloccus aureus in foods.     

Measuring percent lymphocytes by flow cytometry to calculate absolute lymphocyte subset counts for HIV+ specimens.

A comparison was made of lymphocyte percentages from an automated hematology analyzer (ELT 15) vs. a fluorescence flow cytometer (Cytofluorograf). The hematology values were consistently higher than the flow (by greater than 10% for 13 of 50 HIV+ specimens). The findings were similar to three other pairings: H*1 vs. Cytofluorograf, Cell Dyn 3000 vs. Cytofluorograf, and Cell Dyn 3000 vs. EPICS Profile. In another comparison manual percent lymphocytes matched much better with flow values. Factors of sample preparation and instrument analysis as they relate to nonrandom cell loss and lymphocyte resolution are examined.