Multi-site evaluation of the BD Stem Cell Enumeration Kit for CD34+ cell enumeration on the BD FACSCanto II and BD FACSCalibur flow cytometers

Background aimsEvaluation of the BD Stem Cell Enumeration Kit was conducted at four clinical sites with flow cytometry CD34⁺ enumeration to assess agreement between two investigational methods: (i) the BD FACSCanto II and BD FACSCalibur systems and (ii) the predicate method (Beckman Coulter StemKit and StemTrol, Immunotech SAS, Beckman Coulter, Marseille Cedex 9, France).MethodsLeftover and delinked specimens (n = 1032) from clinical flow cytometry testing were analyzed on the BD FACSCanto II (n = 918) and BD FACSCalibur (n = 905) in normal and mobilized blood, frozen and thawed bone marrow and leucopheresis and cord blood anticoagulated with citrate phosphate dextrose, anticoagulant citrate dextrose—solution A, heparin and ethylenediaminetetraacetate, alone or in combination. Fresh leucopheresis analysis addressed site equivalency for sample preparation, testing and analysis.ResultsThe mean relative bias showed agreement within predefined parameters for the BD FACSCanto II (−2.81 to 4.31 ±7.1) and BD FACSCalibur (−2.69 to 5.2 ±7.9). Results are reported as absolute and relative differences compared with the predicate for viable CD34⁺, percentage of CD34⁺ in CD45⁺ and viable CD45⁺ populations (or gates). Bias analyses of the distribution of the predicate low, mid and high bin values were done using BD FACSCanto II optimal gating and BD FACSCalibur manual gating for viable CD34⁺, percentage of CD34⁺ in CD45⁺ and viable CD45⁺. Bias results from both investigational methods show agreement. Deming regression analyses showed a linear relationship with R² > 0.92 for both investigational methods.DiscussionIn conclusion, the results from both investigational methods demonstrated agreement and equivalence with the predicate method for enumeration of absolute viable CD34⁺, percentage of viable CD34⁺ in CD45⁺ and absolute viable CD45⁺ populations.     

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.     

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.     

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.     

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.     

Enumeration of CD34+ cells in cord blood: a variation on a single-platform flow cytometric method based on the ISHAGE gating strategy

Single-platform flow cytometric absolute cell counting protocols provide increased robustness for CD34+ cell enumeration by limiting potential sources of imprecision. However, samples with any cellular fragmentation or debris, such as cord blood samples, provide challenges for these assays. We describe a simple, robust absolute CD34+ cell counting protocol, suitable for cord blood, using TRUCOUNT absolute count tubes (BD Biosciences, San Jose, CA) and a modified ISHAGE (International Society for Hematotherapy and Graft Engineering) gating strategy. An advantage of TRUCOUNT tubes is that each tube is supplied with a known number of lyophilized fluorescent beads. The method includes no-wash fixative-free ammonium chloride red blood cell lysis and the viability dye, 7-amino actinomycin D, to exclude dead cells. The threshold was set on CD45 expression in the FL1 channel and an exclusion gate in the forward scatter channel reduced debris. No manual adjustment of the gating regions was required, even for samples in less than optimal condition. Comparison of the TRUCOUNT-ISHAGE protocol with the original dual-platform ISHAGE assay (n = 30) and the single-platform ISHAGE protocol using Flow-Count Fluorospheres (Beckman Coulter, Fullerton, CA; n = 22) showed high correlation (R(2) = 0.949 and 0.989, respectively) and no significant difference or bias for samples ranging from 22 to 600 CD34+ cells per microliter. Results are presented that demonstrate the detrimental effect of a fixative-containing lysis reagent when used in a lyse-and-wash procedure. The TRUCOUNT-ISHAGE protocol combines the attributes of TRUCOUNT tubes and the ISHAGE gating strategy to provide a single-platform protocol capable of achieving readily standardization of CD34+ cell enumeration.     

Evaluation of a universal template for single-platform absolute T-lymphocyte subset enumeration

The single-platform absolute T-lymphocyte subset analysis was evaluated utilizing a universal protocol in a Canadian multicenter study with the collaboration of the members of the Canadian HIV Trials Network (CTN). Participants used flow cytometers and reagents of their choice for labeling and lysing whole blood. Over a 2-year period, CTN laboratories performed single-platform absolute T-lymphocyte subset enumerations on fresh and commercial stabilized blood products using commercially available microfluorospheres TruCount and Flow-Count. This multicenter evaluation demonstrated that the application of a universal template for single-platform analysis provides a generic approach that embraces a wide array of immunophenotyping settings available in clinical laboratory.     

Single- versus dual-platform assays for human CD34+ cell enumeration

We comparatively assessed CD34+ cell quantification by two of the recently available single platform assays, the IMAGN 2000 STELLer (Immucor, Lisbon, Portugal) microvolume fluorimetry and the ProCOUNT (BD-ENZIfarma, Lisbon, Portugal) flow cytometry, with our "in-house" dual-platform flow cytometric assay. The performance of the methods was evaluated by linearity and reproducibility tests. The linearity study, over a range of 0-1,200 CD34+ cell/microl, gave a good linear relationship for the three methods, with R(2) > 0.99. Precision tested at three different concentrations gave coefficients of variation ranging from 3.6-26.4% for the STELLertrade mark, 2.4-13.8% for the ProCOUNT, and 3.2-6.4% for flow cytometry. CD34+ cells were quantified in umbilical cord blood (UCB), UCB enriched-leukocyte buffy-coat (BC), mobilized peripheral blood (PB) and mobilized peripheral blood progenitor cells (PBPC) collected by leucapheresis, from a total of 72 samples. Flow cytometric results showed good linear correlation to the absolute counts obtained by the STELLer and ProCOUNT for all samples (R > 0.90 for all methods), with no differences when compared by paired tests (P > 0.05). Linear correlations between methods were also found when individually looking at the different cell sources: UCB or PB, BC, and PBPC, with low, intermediate and high CD34+ cell concentrations, respectively. Furthermore, with the exception of a significant difference between the ProCOUNT and STELLer results for UCB (P < 0.05), no other difference between methods was found for each of the individual populations (P > 0.05). To our knowledge, this is the first report in which the results are presented and analyzed according to each source of CD34+ cells. Our results show that the STELLer and the ProCOUNT are equally efficient for the dual-platform flow cytometric assay in CD34+ cell quantification.     

An internal positive control for the enumeration of CD45(+) and CD34(+) cells by flow cytometry allows monitoring of reagent and operator performance

BACKGROUND: Three-color flow cytometry assays are used to determine CD34(+) cell doses prior to stem cell transplantation. These assays require high-quality reagents that are dispensed accurately to ensure reproducible results. We have developed a flow cytometry assay for CD34(+) cells with an integral positive control (KG1a cells) for monitoring reagent and operator performance. METHODS: The method was validated using samples from 127 allogeneic donations (42 BM, 85 PBSC) from healthy donors and 195 autologous donations (46 BM, 149 PBSC) from patients in remission from hematologic malignancies. The mean, SD and range of CD45(+) and CD34(+)cell counts were determined for each donation type. An internal control was used to assess performance of reagents and operators by comparison with a predetermined target value and an experienced operator. RESULTS: Replicate studies showed the method to be accurate and precise, with KG1a cells at 97.7+/-3.9% of the target value and a CV of 4.0%. In routine use over 322 samples, the accuracy was 91.7+/-17.7% of the target value, with a CV of 19.3%. Investigations into the cause of the reduced precision showed that reagents performed consistently well but operator performance was variable, with two of six operators significantly under-dispensing KG1a cells. DISCUSSION: This study validates our three-color flow cytometry assay and demonstrates that KG1a cells may be used to monitor test performance in the routine working environment. In addition to monitoring performance within a single laboratory, its wider use in multicenter studies may be helpful regarding standardization of methods.     

Noise, sensitivity, and resolution of flow cytometers.

The sensitivity and resolution of flow cytometers are functions of the signal produced by a given particle as well as by the noise in the presence of which the signal is detected. The noise is primarily due to the fact that emission of light as well as its detection by photoelectric devises are stochastic processes. This fact leads to equations describing how resolution and sensitivity are limited by the magnitude of the signal, the background, and the photoelectron quantum yield of the detector. The equations are pointing to a method by which the signal and noise of a flow cytometer can be measured in absolute terms, as well as a way to determine fluorescence sensitivity without having to extrapolate to the noise level. The equations appear to be validated when applied to measuring data obtained with two different flow cytometers.     

Multi-laboratory assay for harmonization of enumeration of viable CD34+ and CD45+ cells in frozen cord blood units

Background aimsIn 2016, specifications for both pre-cryopreserved and post-thawed cord blood were defined in the sixth edition of NetCord Foundation for the Accreditation of Cellular Therapy (FACT) Standards for Cord Blood Banks. However, for several experts, harmonization regarding flow cytometry analysis performed on post-thawed samples is still a concern. A multicenter study led by Héma-Québec aimed to provide scientific data to support the cord blood accreditation bodies such as NetCord FACT in the revision of standards.MethodsTwelve cord blood units were processed for plasma and red cell reduction following standard operating procedures. Cord blood unit aliquots were shipped to eight participating centers under cryogenic conditions for analysis before and after standardization of protocol. Repeatability of stem cell count, measured pre- and post-intervention with the centers, was estimated using multilevel linear regression models with a heterogeneous compound symmetry correlation structure among repeated measures.ResultsExcellent inter-center repeatability was reported by each participant regarding the viable CD34+ cells concentration, and a successful improvement effect of protocol standardization was also observed. However, we observed that better control over the critical parameters of the protocol did not have a significant effect on improving homogeneity in the enumeration of CD45+ cells.ConclusionsThe current practice in cord blood selection should now also consider relying on post-thaw CD34+ concentration, providing that all cord blood banks or outsourcing laboratories in charge of the analysis of post-thaw CB samples take into account the consensual recommendations provided in this work and adhere to a good-quality management system.     

Interactive display and analysis of data from bivariate flow cytometers.

An interactive computer program, SWELL, displayss and analyzes bivariate distributions generated by flow cytometers. SWELL is modular with options available via a menu, is written in Fortran, and utilizes a video color display system. Data are accumulated as a bivariate distribution that is transferred to the computer as a 64 x 64 matrix. For ease of visualization, matrices are displayed in pseudocolor. The distribution values are broken into eight ranges and each range is represented by a color. Each element of the matrix is then displayed in its assigned color. To allow pooling and comparison, distributions are aligned, edited, and standardized. Unknown samples are pooled or analyzed singly and compared to the normal pool by subtraction. Differences are displayed as pseudocolor matrices of sign, magnitude, or statistical magnitude in units of standard deviation. This latter display, scaled to tolerance limits, readily reveals regions of significant difference between normal and abnormal samples. Counts within such regions can be compared to diagnose samples automatically.     

Comparison of two different cryopreservation protocols for freezing goat semen

In this study, two different semen cryopreservation protocols were compared to freeze goat semen. The ejaculates (n = 12) were collected by using electro-ejaculator from six mature bucks (two ejaculates per each buck). Each ejaculate was divided into two groups as Protocol 1 (P1) and Protocol 2 (P2). In P1, semen was diluted directly in an extender containing 15% egg yolk, 300 mM Tris, 28 mM glucose, 95 mM citric acid 5% glycerol to a concentration of 200 × 10⁶ sperm/mL. In P2, after the removal of seminal plasma by centrifugation, the semen sample was diluted with the first portion of milk extender consist of 100 mg/mL skimmed milk powder and 27.75 mM glucose (without glycerol) to a concentration of 400 × 10⁶ sperm/mL. The second portion of the milk extender containing 14% glycerol was added to semen gradually in order to achieve sperm concentration 200 × 10⁶ sperm/mL and 7% glycerol level in the final volume. Extended semen was loaded in 0.25 mL straws, held for 2 h at 4 °C, frozen in nitrogen vapor and stored in liquid nitrogen. Post-thaw motility and live sperm rate (mean ± SEM) were significantly lower (P < 0.05) in P1 as compared to P2 (47.50 ± 1.23% vs. 55.63 ± 1.72%; 80.04 ± 1.29% vs. 84.04 ± 1.08%, respectively). However, live intact, total intact, abnormal, reacted acrosome and DNA damaged sperm rates were similar (P > 0.05) in both protocols. It was concluded that both protocols used in this study provided reasonable post-thaw parameters; however, P2 yielded better motility and live sperm rate compared to P1.