The success of bone marrow or peripheral blood stem

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1 CD34 + Cell Enumeration by Flow Cytometry A Comparison of Systems and Methodologies Alain Ngoma, MD; Shunnichi Saito, MT; Hitoshi Ohto, MD, PhD; Kazuhiko Ikeda, MD, PhD; Hiroyasu Yasuda, PhD; Kinuyo Kawabata, MT; Takahiro Kanno, MD, PhD; Atsushi Kikuta, MD, PhD; Kazuhiro Mochizuki, MD, PhD; Kenneth E. Nollet, MD, PhD N Context. An increasing number of medical centers can collect bone marrow, peripheral blood, or umbilical cord stem cells. Pathology laboratories should accommodate this trend, but investment in additional equipment may be impractical. Objectives. To compare CD34 + cell counting results by using 2 widely available flow cytometry systems, with and without the use of a separate hematology analyzer (ie, single-platform versus dual-platform methodologies). Design. Whole blood and peripheral blood stem cell (PBSC) samples were analyzed from 13 healthy allogeneic PBSC donors and 46 autologous PBSC donors with various malignancies. The Cytomics FC500 (Beckman Coulter, The success of bone marrow or peripheral blood stem cell (PBSC) transplants is associated not only with phenotypic compatibility between donor and patient but also with the number of CD34 + white cells in the graft, relative to body weight or surface area of the recipient. 1 3 Flow cytometry is the method of choice to assess CD34 + cell content but protocols vary among institutions. 4 6 Moreover, CD34 expression is merely a surrogate measure of hematopoietic stem cell or progenitor cell content. Nevertheless, enumeration of CD34 + cells is an essential tool of PBSC harvest and for comparing different mobilization or collection schemes For successful enumeration of CD34 + cells, appropriate procedures are necessary to minimize pre-analytic, and postanalytic variables. Within the realm of analysis, the hardware and software of a flow cytometry system are responsible for collecting data on individual cells and aggregating that data into useful information. Thus, a system should be chosen that is appropriate to the task, and its operator(s) should be well versed in the nuances of its hardware, software, and the inputs (cells, antibodies, gating protocols, etc) that the hardware and software manipulate. Accepted for publication October 6, From the Department of Blood Transfusion and Transplantation Immunology, Fukushima Medical University, Fukushima City, Fukushima, Japan. The authors have no relevant financial interest in the products or companies described in this article. Reprints: Kenneth E. Nollet, MD, PhD, Department of Blood Transfusion and Transplantation Immunology, Fukushima Medical University, Hikarigaoka 1, Fukushima City, Fukushima , Japan ( nollet@fmu.ac.jp). Fullerton, California) was compared with the FACSCalibur (BD Biosciences, San Jose, California). Dual-platform CD34 + cell counting incorporated data from a KX-21 hematology analyzer (Sysmex, Kobe, Japan). Results. Subtle differences in CD34 + cell counting between 2 systems and 2 methods did not achieve statistical significance. Conclusion. Different systems and methods for CD34 + cell enumeration, properly validated, can support care for patients undergoing transplants and provide meaningful data for multicenter studies or meta-analyses. (Arch Pathol Lab Med. 2011;135: ) Single-platform methodologies, in which a known number of well-characterized fluorescent beads are added to the analyte, are intended to be more reliable and easier to use than dual-platform methods, which combine data from a conventional hematology analyzer and a flow cytometer. 11 Various platforms and methodologies have been compared, but prior results, regardless of outcome, should not discourage continued investigation and reporting of laboratory systems on which significant clinical outcomes depend. This article compares the Cytomics FC500 (Beckman Coulter, Fullerton, California) and the FACSCalibur (BD Biosciences, San Jose, California) flow cytometry systems, for the enumeration of CD34 + cells with both singleplatform and dual-platform methods. MATERIALS AND METHODS Research Subjects Participation of human research subjects conformed to institutional review board guidelines, applicable laws, and the World Medical Association Declaration of Helsinki. Sample Collection Samples were drawn from healthy allogeneic PBSC donors (n 5 13) and autologous PBSC patients (n 5 46) with malignant lymphoma (n 5 12), seminoma (n 5 9), multiple myeloma (n 5 8), rhabdomyosarcoma (n 5 4), neuroblastoma (n 5 3), and other conditions (n 5 10). Table 1 provides a demographic breakdown according to sample type. Peripheral blood stem cell (n 5 27) and whole blood (WB; n 5 32) samples were collected at a single institution from May 2005 through May 2009 and used for enumeration of CD34 + cells. Each WB sample was collected by venipuncture into an EDTA tube. Peripheral blood stem cell samples were collected by apheresis, Arch Pathol Lab Med Vol 135, July 2011 CD34 + Enumeration by Flow Cytometry Ngoma et al 909

2 Table 1. Demographic Characteristics Associated With 27 Peripheral Blood Stem Cell (PBSC) and 32 Whole Blood (WB) Samples Sample, type (No.) PBSC (27) WB (32) Age, median (range) 41.5 (1 65) 31 (1 62) Sex, male:female 13:14 13:19 Allogeneic PBSC donors, No. 6 7 Autologous PBSC patients, No Malignant lymphoma 6 6 Seminoma 4 5 Multiple myeloma 4 4 Rhabdomyosarcoma 1 3 Neuroblastoma 1 2 Amyloidosis 2 0 Ewing sarcoma 1 1 Wilms tumor 1 1 Ovarian cancer 1 1 Acute myeloid leukemia 0 1 Atherosclerosis obliterans 0 1 as described previously. 7,8 In all instances, sample collection immediately preceded sample preparation and analysis. Sample Preparation Freshly obtained WB and PBSC samples were used undiluted in experiments unless the white blood cell (WBC) count exceeded /mL. Samples with higher WBC concentrations were diluted to WBC/mL with phosphate-buffered saline. Immunofluorescence Staining, Cell Viability, and Erythrocyte Lysis Reagents Reagents (Stem-Kit, Beckman Coulter) for the Cytomics FC500 were as follows: phycoerythrin-conjugated anti-cd34 (CD34-PE, clone 581) and fluorescein isothiocyanate conjugated anti-cd45 (CD45-FITC, clone J33) for CD34 + WBC identification; PEconjugated isoclonic antibody combined with CD45-FITC (clone J33) as a control; 7-amino-actinomycin D (7-AAD) for cell viability assessment; ammonium chloride solution for erythrocyte lysis; and fluorescent bead reference standards of precisely known concentration (Stem-Count), for single-platform cell counting. Reagents (BD Biosciences) for the FACSCalibur were as follows: combined anti-cd45 and anti-cd34 (CD45-FITC/ CD34-PE, clones 2D1 and 8G12) for CD34 + WBC identification; 7-AAD for cell viability assessment; ammonium chloride solution (PharmLyse) for erythrocyte lysis; and test tubes preloaded with a precisely known quantity of lyophilized fluorescent bead reference standards (TruCount). Single-Platform Method Each manufacturer s procedure included red cell lysis without washing. 5,11 To assess white cell viability, 20 ml of 7-AAD was added to all tubes before any other reagents. For the Cytomics FC500, 20 ml of CD45-FITC/CD34-PE and 20 ml of control reagent were pipetted into different tubes and 100- ml samples were added to each tube. After 20 minutes of room temperature incubation in the dark, red cell lysis was initiated with 2 ml of buffered ammonium chloride (made fresh from 103 stock), followed by 8 to 10 minutes of incubation, without washing. Just before flow cytometry, 100 ml of Stem-Count fluorospheres was added and thoroughly mixed with the analyte. For the FACSCalibur, 20 ml of CD45-FITC/CD34-PE was pipetted into TruCount tubes, to which 50 ml ofsamplewas added. After 15 minutes of room-temperature incubation in the dark, red cell lysis was initiated with 1 ml of ammonium chloride, followed by 3 to 5 minutes of incubation before flow cytometry. Both instruments allowed absolute CD34 + cell enumeration when using the known fluorescent bead standards incorporated into each sample. A total of CD45 + events were collected per tube in both the Cytomics FC500 and the FACSCalibur; Figure 1 illustrates subsequent analysis with Cytomics software. Quantitation of viable CD34 + cells followed manufacturers instructions. For the Cytomics FC500: CD34 z cells per ml~ 2 3 number of cells in the CD34 z fraction of the analyte 6 { ðnumber of cells in the CD34 z fraction of the 7 4 negative control sample 5 ðtotal number of fluorescent beads in each tubeþ ðdilution factorþ= ðnumber of beads countedþ: For the FACSCalibur: CD34 z cells per ml~ ðnumber of cells in CD34 z fraction=number of beads countedþ ðtotal number of beads in each bead-coated tubeþ ðdilution factorþ: Nonviable cells, as determined by 7-AAD infiltration, were excluded, as illustrated in Figure 1, histogram 8. Viability was % for WB and % for PBSCs with the Cytomics FC500 and % for WB and % for PBSCs with the FACSCalibur. Dual-Platform Method In this approach, the fraction of CD34 + cells was obtained by flow cytometry, and absolute nucleated cell count was derived from a KX-21 automated hematology analyzer (Sysmex, Kobe, Japan). Percentage of CD34 + cells was determined by using double-color immunofluorescence staining (CD45-FITC/CD34- PE). Absolute number of CD34 + cells was obtained by the product of the CD34 + cells fraction and the nucleated cell count. Gating Strategies and Data Analysis Triple fluorophore gating was set up according to the International Society of Hematotherapy and Graft Engineering (ISHAGE) guidelines. 15 Briefly, CD34 +, 7-AAD 2, CD45 dim cells with low side scatter fraction were defined as CD34 + cells in hematopoietic stem cell/hematopoietic progenitor cell fractions (Figure 1, histogram 4). Statistical Analysis SPSS for Windows 16.0 (SPSS, Chicago, Illinois) was used for statistical analysis. Regression analysis was used to correlate results obtained by the 2 systems. Results are expressed as median (range). Statistical significance (P,.05) was determined with the nonparametric Wilcoxon test. RESULTS CD34 + Counts, Single-Platform Method The number of CD34 + cells detected was slightly higher with the Cytomics FC500 than with the FACSCalibur for both WB (median, 31.0/mL [range, /mL] versus median, 29.8/mL [ /mL]) and PBSCs (median, 1040/mL [range, /mL] versus median, 857.9/ ml [range, /mL]). These differences did not achieve statistical significance (Table 2). CD34 + Counts, Dual-Platform Method Differences in CD34 + cells detected with the Cytomics FC500 and with the FACSCalibur for WB (median, 31.0/ ml [range, /mL] versus median, 30.4/mL [range, 910 Arch Pathol Lab Med Vol 135, July 2011 CD34 + Enumeration by Flow Cytometry Ngoma et al

3 Figure 1. Flow cytometric analysis of a fresh peripheral blood stem cell sample analyzed on the Cytomics FC500 with Stem-Kit reagents (Beckman Coulter). Histogram 1, region A includes viable CD45+ leukocytes (excluding dead 7-AAD+ cells per histogram 8, region I); region E (lower right oval) is a lymphocyte-specific gate. Histogram 2 plots events from A and J; CD34+ cells are gated in B (polygon). Histogram 3, region C (polygon) gates CD34+, CD45+ cells from A, B, and J. Histogram 4, region D (lower right polygon) plots events sequentially gated from J, A, B, and C. Histogram 5 plots CD45 versus CD34 for all events. Histogram 6, region F (lower right polygon) shows live lymphocytes, from E and J. Histogram 7, region H tracks single StemCount beads for single-platform cell quantification. Histogram 8, as previously described, gates cell viability according to 7-AAD fluorescence. Abbreviations: CD45-FITC, fluorescein isothiocyanate-conjugated anti-cd45; CD34-PE, phycoerythrin-conjugated anti-cd34; FS, forward scatter; SS, side scatter; 7-AAD, 7-amino-actinomycin D /mL]) and PBSCs (median, 906.7/mL [range, /mL] versus 912.3/mL [range, /mL]) were not statistically significant (Table 3). Assessment of Single-Platform and Dual-Platform Measurements No statistically significant differences were observed between the number of CD34+ cells when using either Arch Pathol Lab Med Vol 135, July 2011 single-platform or dual-platform methods, regardless of the cytometer (Tables 4 and 5). CD34+ cell fraction (%) correlated well by linear regression when comparing FACSCalibur and Cytomics FC500 data for both WB (r ) and PBSCs (r ), as shown in Figures 2 and 3, respectively. Similarly, CD34+ absolute counts correlated well when comparing single-platform and dual-platform analysis of CD34+ Enumeration by Flow Cytometry Ngoma et al 911

4 Table 2. CD34 + Cell Quantitation of 27 Peripheral Blood Stem Cell (PBSC) and 32 Whole Blood (WB) Samples With Single-Platform Methodology (Cytomics FC500 Versus FACSCalibur) Cytomics FC500 FACSCalibur P Value WB, median (range), ml ( ) 29.8 ( ).88 PBSC, median (range), ml ( ) 857 ( ).72 WB with the FACSCalibur (r , Figure 4), WB with the Cytomics FC500 (r , Figure 5), PBSCs with the FACSCalibur (r , Figure 6), and PBSCs with the Cytomics FC500 (r , Figure 7). Error Analysis Surveillance for preanalytic variables included a comparison of results from 1 senior operator processing the same sample 4 times versus 4 operators individually processing the same sample. For this purpose, WB was diluted by a factor of 4 and PBSCs were diluted by a factor of 30. Single-operator results were not statistically different from results aggregated from 4 operators (data not shown). COMMENT It is now well established that engraftment success correlates with CD34 + cell count, independent of the degree of phenotypic similarity between donors and recipients. We owe this insight to the development of accurate cell counting methodologies, which are useful not only for individual patients but also for aggregating treatment and outcome data from many facilities. Flow cytometric quantification of CD34 + cells is now the most widely used tool to optimize mobilization and harvest of hematopoietic progenitor/stem cells for PBSC or bone marrow transplant. 14 The first flow cytometric gold standard for the care of patients undergoing transplants was, in fact, a synthesis of data from 2 antecedent gold standards: the hematology analyzer for absolute leukocyte counting and the flow cytometer for the relative proportion of progenitor cells, as estimated by a surrogate marker such as CD34. Since the emergence of single-platform flow cytometric measurements, the original dual-platform standard has been criticized for being labor-intensive. Furthermore, historical data have linked the use of hematology analyzers to high coefficients of variation in CD4 + T-lymphocyte enumeration. 16 Thus, the motivation for developing single-platform methods, using beads or volumetric techniques to generate absolute cell counts, is well founded. 17,18 Keeney et al 11 adapted ISHAGE dual-platform guidelines for single-platform use, with favorable results. Several studies 19,20 have demonstrated that the single-platform approach achieves the highest possible reliability to quantify viable CD34 + cells, owing to reduced intralaboratory and interlaboratory variation. Therefore, this approach has been included in a variety of national 21 and international 6 guidelines and a number of laboratory technical manuals. 22,23 Nevertheless, CD34 + counting methodologies have not been exhaustively studied across the range of systems that are likely to be found in modern clinical pathology laboratories. To this end, we have compared the Cytomics FC500 and FACSCalibur, using both single-platform and dual-platform methodologies. Statistically significant differences would most likely cast single-platform methods in the most favorable light, whereas statistically insignificant differences would offer some reassurance that data from a wider variety of laboratories might be intelligently assimilated and analyzed. Differences between platforms and methodologies in our laboratory did not achieve statistical significance. This is in accordance with Sutherland et al, 14 who recently compared the CD34 + cell numbers derived from Stem-Kit and TruCount systems and found that the 2 kits generated very similar data on a range of fresh samples with FACSCalibur and FACSCanto instruments (both, BD Biosciences). In contrast, we report single-platform and dual-platform methodologies with 2 instruments from 2 different manufacturers. Linear Table 3. CD34 + Cell Quantitation of 27 Peripheral Blood Stem Cell (PBSC) and 32 Whole Blood (WB) Samples With Dual-Platform Methodology (Cytomics FC500 Versus FACSCalibur) Cytomics FC500 FACSCalibur P Value WB, median (range), ml ( ) 30.4 ( ).99 PBSC, median (range), ml ( ) ( ).99 Table 4. CD34 + Cell Quantitation of 27 Peripheral Blood Stem Cell (PBSC) and 32 Whole Blood (WB) Samples With the Cytomics FC500 (Single-Platform Versus Dual-Platform Methodology) Single Platform Dual Platform P Value WB, median (range), ml ( ) 31 ( ).91 PBSC, median (range), ml ( ) ( ).73 Table 5. CD34 + Cell Quantitation of 27 Peripheral Blood Stem Cell (PBSC) and 32 Whole Blood (WB) Samples With the FACSCalibur (Single-Platform Versus Dual-Platform Methodology) Single Platform Dual Platform P Value WB, median (range), ml ( ) 30.4 ( ).98 PBSC, median (range), ml ( ) ( ) Arch Pathol Lab Med Vol 135, July 2011 CD34 + Enumeration by Flow Cytometry Ngoma et al

5 Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Correlation of CD34 + cell fraction (%) in 32 whole blood samples: FACSCalibur versus Cytomics FC500. Correlation of CD34 + cell fraction (%) in 27 peripheral blood stem cell samples: FACSCalibur versus Cytomics FC500. Correlation of CD34 + cell counts in 32 whole blood samples: single-platform versus dual-platform, FACSCalibur. Correlation of CD34 + cell counts in 32 whole blood samples: single-platform versus dual-platform, Cytomics FC500. Correlation of CD34 + cell counts in 27 peripheral blood stem cell samples: single-platform versus dual-platform, FACSCalibur. Correlation of CD34 + cell counts in 27 peripheral blood stem cell samples: single-platform versus dual-platform, Cytomics FC500. regression analysis of our data yielded high correlation coefficients when comparing Cytomics FC500 and FACS- Calibur CD34 + fractions, and single-platform versus dualplatform absolute CD34 + counts on either instrument (Figures 2 through 7). It can be argued that laboratories routinely using a single-platform method should be confident of alternative methods, not only for quality assurance but also for reliable patient care in the event that properly stored, in-date calibration beads are unavailable. Preliminary assessment Arch Pathol Lab Med Vol 135, July 2011 CD34 + Enumeration by Flow Cytometry Ngoma et al 913

6 of interoperator performance on replicate samples has been favorable in our laboratory but needs to be formalized as an ongoing assessment of intralaboratory variation for singleplatform and dual-platform methods; we would welcome the opportunity to cooperate with other institutions to quantify interlaboratory variations. Altogether, these observations suggest that differences among flow cytometry systems need not deter laboratories from participating in local and multicenter transplant efforts. Multicenter investigations might corroborate this, or, alternatively, shed light on some as-yet unaccounted variables. As our laboratory only provides support for PBSC transplant, it would be instructive to know whether centers supporting bone marrow and/or cord blood transplant would reach similar conclusions as ours. Negative results in regard to differences among systems, methods, and laboratories would in fact be a positive outcome, indicating that consistent enumeration of CD34 + cells is achievable among laboratories. References 1. Fritsch G, Stimpfl M, Kurz M, et al. Characterization of hematopoietic stem cells. Ann N Y Acad Sci. 1995;770: Fritsch G, Stimpfl M, Kurz M, et al. The composition of CD34 subpopulations differs between bone marrow, blood and cord blood. Bone Marrow Transplant. 1996;17(2): Allan DS, Keeney M, Howson-Jan K, et al. Number of viable CD34(+) cells reinfused predicts engraftment in autologous hematopoietic stem cell transplantation. Bone Marrow Transplant. 2002;29(12): Chin-Yee I, Anderson L, Keeney M, Sutherland DR. Quality assurance of stem cell enumeration by flow cytometry; Canadian QASE Study Group. Cytometry. 1997;30(6): Fritsch G, Printz D, Stimpfl M, et al. Quantification of CD34 + cells: comparison of methods. Transfusion. 1997;37(8): Gratama JW, Orfao A, Barnett D, et al. Flow cytometric enumeration of CD34 + hematopoietic stem and progenitor cells; European Working Group on Clinical Cell Analysis. Cytometry. 1998;34(3): Ikeda K, Ohto H, Kanno T, et al. Automated programs for collection of mononuclear cells and progenitor cells by two separators for peripheral blood progenitor cell transplantation: comparison by a randomized crossover study. Transfusion. 2007;47(7): Ikeda K, Ohto H, Nemoto K, et al. Collection of MNCs and progenitor cells by two separators for PBPC transplantation: a randomized crossover trial. Transfusion. 2003;43(6): Kessinger A, Sharp JG. Mobilization of blood stem cells. Stem Cells. 1998; 16(suppl 1): Kessinger A, Sharp JG. The whys and hows of hematopoietic progenitor and stem cell mobilization. Bone Marrow Transplant. 2003; 31(5): Keeney M, Chin-Yee I, Weir K, Popma J, Nayar R, Sutherland DR. Single platform flow cytometric absolute CD34 + cell counts based on the ISHAGE guidelines; International Society of Hematotherapy and Graft Engineering. Cytometry. 1998;34(2): Barbosa IL, Sousa ME, Godinho MI, Sousa F, Carvalhais A. Single- versus dual-platform assays for human CD34 + cell enumeration. Cytometry. 1999;38(6): Gajkowska A, Oldak T, Jastrzewska M, et al. Flow cytometric enumeration of CD34 + hematopoietic stem and progenitor cells in leukapheresis product and bone marrow for clinical transplantation: a comparison of three methods. Folia Histochem Cytobiol. 2006;44(1): Sutherland DR, Nayyar R, Acton E, Giftakis A, Dean S, Mosiman VL. Comparison of two single-platform ISHAGE-based CD34 enumeration protocols on BD FACSCalibur and FACSCanto flow cytometers. Cytotherapy. 2009;11(5): Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I. The ISHAGE guidelines for CD34 + cell determination by flow cytometry; International Society of Hematotherapy and Graft Engineering. 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Validation of the single-platform ISHAGE method for CD34(+) hematopoietic stem and progenitor cell enumeration in an international multicenter study. Cytotherapy. 2003;5(1): Barnett D, Janossy G, Lubenko A, Matutes E, Newland A, Reilly JT. Guideline for the flow cytometric enumeration of CD34 + haematopoietic stem cells: prepared by the CD34 + haematopoietic stem cell working party; General Haematology Task Force of the British Committee for Standards in Haematology. Clin Lab Haematol. 1999;21(5): Gratama JW, Kraan J, Keeney M, Mandy F, Sutherland DR, Wood BL. Enumeration of Immunologically Defined Cell Populations by Flow Cytometry; Approved Guideline. Vol 27 (16). 2nd ed. Wayne, Pennsylvania: Clinical and Laboratory Standards Institute; Sutherland DR, Keeney M, Gratama JW. Enumeration of CD34 + hematopoietic stem and progenitor cells. Curr Protoc Cytom. 2003;chapter 6:unit Arch Pathol Lab Med Vol 135, July 2011 CD34 + Enumeration by Flow Cytometry Ngoma et al