Enumeration of CD34-positive hematopoietic progenitor cells by flow cytometry: comparison of a volumetric assay and the ISHAGE gating strategy

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1 Bone Marrow Transplantation, (1998) 22, Stockton Press All rights reserved /98 $12. Enumeration of CD34-positive hematopoietic progenitor cells by flow cytometry: comparison of a volumetric assay and the ISHAGE gating strategy S Leuner, M Arland, C Kahl, K Jentsch-Ullrich, A Franke and H-G Höffkes Division of Hematology/Oncology, Department of Medicine, Otto-von-Guericke University of Magdeburg, Magdeburg, Germany Summary: In order to optimize peripheral blood stem cell (PBSC) collection for transplantation, absolute CD34 counts are necessary to determine the exact time-point for sufficient leukapheresis. In an effort to establish and to validate a rapid and reproducible assay for PBSC enumeration, different recommendations for selection of monoclonal antibodies, lysis techniques, analysis parameters and gating strategies were developed. In this methodical study, two gating strategies for PBSC enumeration were compared, in order to validate the accuracy of PBSC counting in peripheral blood and apheresis products. Gating strategy I was performed using volumetric flow cytometry and reference beads whereas gating strategy II was done according to the ISHAGE guidelines. The highly standardized volumetric assay seems to be superior to the more expert-reliant ISH- AGE procedure requiring more manual work by the cytometrist. Keywords: progenitor cells; CD34; flow cytometry; stem cell transplantation High-dose chemotherapy with peripheral blood stem cell transplantation (PBSCT) is increasingly used for treatment of different hematological malignancies because remission induction seems to be superior in comparison to conventional treatment. 1,2 PBSC have to a large extent replaced bone marrow as source of marrow repopulating cells and are able to reconstitute hematopoiesis in a shorter time after myeloablative chemotherapy. 3,4 A number of methods are available for mobilization of PBSC from bone marrow into peripheral blood. 5,6 Since the identification and characterization of the CD34 antigen as a marker for self-replicating PBSC, flow cytometric analysis has been used for PBSC enumeration in peripheral blood and apheresis products. 7,8 Today, the decision to commence leukapheresis after PBSC mobilization depends on flow cytometric enumeration of PBSC in peripheral blood and the total number of PBSC in leukaph- Correspondence: Dr S Leuner, Division of Hematology/Oncology, Department of Medicine, Otto-van-Guericke, University of Magdeburg, Leipziger Strasse 44, D-3912, Magdeburg, Germany Received 23 February 1998; accepted 5 June 1998 eresis products often determines the time-point of a future PBSC collection. Exact quantification of PBSC in apheresis products is required to assess the quality of harvest for engraftment after PBSCT. 9 Therefore, it is essential to establish a reliable flow cytometric assay for the enumeration of PBSC. Although CFU-GM numbers correlate with the absolute number of PBSC in apheresis products, culture assays have several disadvantages as compared to flow cytometry Cell growth is subjected to the numerous inconsistencies of culture assays and results are not available until 1 to 3 weeks after collection. 13 Flow cytometric assays can be performed in less than 3 h and results are available directly after analysis. Therefore, flow cytometric procedures appear to be a more sensitive, reliable and rapid alternative to colony assays. 1,14 Different flow cytometric methods to enumerate PBSC in peripheral blood and apheresis products have been described. However, the lack of a standardized method has led to widely inconsistent data. 15,16 To validate the precision of different gating strategies in PBSC enumeration, PBSC counts were assessed using two different enumeration assays. The first assay uses a volumetric method with reference beads (ProCount-Kit), and the second assay was performed with the ISHAGE gating strategy. Patients and methods Patient samples In this study, 3 peripheral blood samples and 45 leukapheresis product samples of patients with hematological malignancies selected for high-dose chemotherapy were analyzed in three independent analyses within 2 h of collection. Peripheral blood samples were drawn into EDTAvacutainers (Becton Dickinson, Heidelberg, Germany) and leukapheresis samples, obtained after routine PBSC collection, were drawn into a 5 ml syringe. Mobilization was performed by daily subcutaneous injection of G-CSF after mobilization chemotherapy and PBSC counting started when the leucocyte nadir was passed and the absolute leukocyte concentration was greater than /l.

2 7 Comparison of two gating strategies for PBSC enumeration Assay Flow cytometric analysis was performed directly after collecting three blood samples for each donor sample. Aliquots of peripheral blood in K 3 EDTA coated tubes and aliquots of leukapheresis products, anticoagulated with ACD-A during apheresis were counted (Bayer Technicon H3, Frankfurt, Germany) and adjusted to a cell count of approximately leukocytes/l with a solution containing PBS with gelatin and.1% sodium azide. Cell viability was examined by light microscopy using trypan blue staining (GIBCO BRL, Eggenstein, Germany). For each sample, 2 l ProCount CD34 reagent (BD) were pipetted in each of three 5 ml Trucount-Tubes (BD), and 2 l control reagent were pipetted in a control tube. The ProCount CD34 reagent contained a nucleic acid dye, PE-labeled CD34 (clone 8G12) and PerCP-labeled CD45 (clone 2D1) whereas the control reagent contained a nucleic acid dye, PE-labeled IgG 1 (clone X4) and PerCPlabeled CD45 (clone 2D1). Using a reverse pipetting technique, 5 l of the well mixed donor sample was pipetted to each of the tubes. After vortexing the tubes were incubated for 15 min at room temperature in the dark. Thereafter 45 l 1 FACS lysing solution was added to each tube. After vortexing the cells were incubated for another 3 min in the dark at room temperature. After this incubation the samples were directly analyzed on the flow cytometer. Flow cytometry Analysis was performed on a FACSCalibur (Becton Dickinson) analytical flow cytometer. List mode data were acquired by the CellQuest software (Becton Dickinson). The cellular forward (FSC) and side scatter (SSC) signals, as well as the fluorescence of cell membrane bound peridiniumchlorophyll-protein (PerCP)- and (Phycoerythrin) PE-labeled antibodies were determined following illumination of the cells in the focal spot of a 15 mw air cooled argon-ion laser at 488 nm in the sample beam of the flow cytometer. The photomultiplier tube (PMT) voltages and the fluorescence compensation were set according to the recommendations of the manufacturer with fluorescent reference beads (Calibrite; Becton Dickinson, San Jose, CA, USA). Monitoring of instrument set-up for intensity and color compensation was three-fold: (1) using lymphocytes of normal persons according to the FACSComp software (Becton Dickinson); (2) CD4 (SK3) FITC)/CD8 (SK1) PE, (Becton Dickinson) dual staining of peripheral blood from normal blood donors; and (3) standardized fluorescent beads (FluoroSpheres; Dako Diagnostika, Hamburg, Germany). Fluorescence was collected between nm and nm in the PE and PerCP fluorescence light channels. Fluorescence compensation was adjusted by hardware circuits. The amplification for FSC and SSC signals was linear while fluorescence signals were amplified by four decade logarithmic amplifiers. Using the CellQuest software, 6 events were acquired for each sample. After acquiring data, analysis was done for every sample in two ways. The first method (gating strategy I) was performed according to the recommendations in the ProCount Kit (Figure 1). Six dot plots were created and analyzed with CellQuest software (Becton Dickinson). An attractor set was created to utilize region settings in routine use. In the following text, this gating strategy is called Attractors. The first dot plot (plot 1) FSC vs SSC of all events was displayed to show an overview of the whole cell population and the beads in the sample. A second dot plot (plot 2) was created to display DNA dye vs SSC of all events. Gate region R1 was set around the lymphocyte population extending into the monocytes but excluding any debris. In a third dot plot (plot 3), vs SSC of all events was displayed. Gate region R2 was set around the dim CD45/low SSC population. Additive regions R1 and R2 were shown in the next dot plot (plot 4). In this dot plot DNA dye vs CD34PE was displayed and region R3 was set around the bright CD34 cells. The population of CD34-positive cells was defined in the gate list as G1 = R1 and R2 and R3. For the enumeration of the beads two dot plots of all events were displayed (plot 5, plot 6). Plot 5 showed FSC vs and plot 6 showed CD34 PE vs SSC. Regions R4 and R5 were set around the beads as shown in Figure 1. Beads were defined in the gate list as G2 = R4 and R5. Absolute CD34 count was calculated using the following equation (for each sample the count of the isotype-control was subtracted from the donor sample): CD34 cells (events) beads (events) beads per test dilution factor = CD34 cells/ l test volume The second method (gating strategy II) was done according to the ISHAGE guidelines (Figure 2). Five dot plots were created and also analyzed with CellQuest software. In the first dot plot (plot 1) FSC vs SSC of all events was displayed. This represented an overview of the whole cell population and the beads in the sample. To exclude the beads region R5 was set around the beads. In a second dot plot (plot 2) vs SSC of all events was displayed. Region R1 was set around the leukocyte population, excluding beads and debris. The next dot plot (plot 3) was created with CD34 PE vs SSC of gated cells from region R1 and not R5. In this plot region R2 was set around the CD34-positive cell population. In plot 4, vs SSC was displayed of cells gated from additive regions R1 and R2. Region R3 was set around the cluster with low SSC and low to intermediate CD45 fluorescence. The last dot plot (plot 5) displayed cells from additive regions R1, R2 and R3. After creating the axes FSC vs SSC for these regions R4 was set according to the ISHAGE guidelines. The CD34-positive cell population was defined in the gate list as region R4. Absolute CD34-count was calculated using the following equation: Total no. of CD34 + events leukocyte count ( 1 9 /l) 1 Total no. of CD45 + events without beads dilution factor = CD34 cells/ l Statistical analysis Comparison of the results for the two methods was performed using the paired t-test and Spearman correlation with

3 123 Comparison of two gating strategies for PBSC enumeration FSC-Height R DNA dye Plot 1: FSC vs SSC of all events. Plot 2: DNA dye vs SSC of all events. Setting of region R R3 CD34 PE R Plot 3: vs SSC of all events. Setting of region R DNA dye Plot 4: DNA dye vs CD34PE of cells gated from additive regions R1 and R2. Setting of region R R5 R4 CD34 PE Plot 5: vs SSC of all events. Setting of region R4 around the beads DNA dye Plot 6: DNA dye vs CD34 PE of all events Setting of region R5 around the beads. Figure 1 Gating of the CD34-positive cell population in a leukapheresis sample with gating strategy (attractors). Regions were set according to the recommendations in the ProCount-Kit (Becton Dickinson).

4 Comparison of two gating strategies for PBSC enumeration R5 123 FSC-Height Plot 1: FSC vs SSC of all events. Setting of region R5 around the beads. R Plot 2: vs SSC of all events. Setting of region R R2 R Plot 3: CD34 PE vs SSC of the events from region R1 and not region R5. Setting of region R2. Plot 4: vs SSC of the events from additive regions R1 and R2. Setting of region R R4 123 FSC-Height Plot 5: FSC vs SSC of the events from additive regions R1, R2 and R3. Setting of region R4. Figure 2 Gating of the CD34-positive cell population in the same leukapheresis sample using gating strategy II (ISHAGE). Regions were set using an ISHAGE comformable gating strategy. For details, see text.

5 Comparison of two gating strategies for PBSC enumeration 6 73 Attractors 5 ISHAGE 4 CD34-count/ µ l Test number 1 75 Figure 3 Comparison of the two gating strategies. Results of 75 samples of peripheral blood and leukapheresis products. Assay for each sample was done three times. Light bars show means for each analysis following the ISHAGE-conformable method. Error bars represent standard deviation (s.d.). 6 CD34 count/ µ l Paired t-test: Attractors vs ISHAGE P =.554 Correlation coefficient: r =.9583 Attractors ISHAGE Figure 4 Overall comparison of the two methods shown by box-and-whiskers plots. Mean values of the two data sets. Boxes extend from the 25th to the 75th percentile with a horizontal line at the median for each data set. Whiskers extend from the smallest to the largest value. Correlation was significant. GraphPadPrism (GraphPad software, San Diego, CA, USA). Results In this study the efficiency of two flow cytometric gating strategies for PBSC enumeration was compared. For each of the 75 apheresis and peripheral blood samples three assays were done as described in Methods. After acquiring data on the flow cytometer every run was analyzed with gating strategy I (attractors) and afterwards with gating strategy II (ISHAGE). The distribution of samples covered a broad range of PBSC counts (ISHAGE: r = ), containing mostly low PBSC values but also a small number of samples with a very high PBSC content (ISHAGE: mean = 93.2, median = 43.7, s.d. = 113., s.e.m. = 12.96) (see Figure 3).

6 Comparison of two gating strategies for PBSC enumeration 74 PBSC-count/ µ l (Attractor) Group Ι (PBSC count: 1/ µ l) 5 1 r =.893 P <.1 15 PBSC count/ µ l (ISHAGE) Group ΙΙ (PBSC count: 1 1/ µ l) 2 The observed correlation among the two methods was significant for the whole range of values (Figure 4). But higher values were better correlated than lower and very high values (Spearman correlation for (a) lower values (PBSC count 1/ l): r =.893; (b) higher values (PBSC count: 1 1/ l): r =.96; and (c) very high values (PBSC count 1/ l: r =.9343) (Figure 5). The reproducibility of the results showed high variation for low values but was comparable for both methods (mean coefficient of variation (CV) of the replicate samples: (a) lower values (PBSC count 1/ l): ISHAGE: mean CV = 25.43% attractor: mean CV = 24.1%; (b) higher values (PBSC count: 1 1/ l): ISHAGE: mean CV = 1.38% attractor: mean CV = 1.75%; and (c) very high values (PBSC count 1/ l): ISHAGE: mean CV = 8.32% attractor: mean CV = 7.79%) (Table 1). The paired t-test showed borderline significance in the PBSC estimation of the two techniques (P =.554 (twotailed), t = 1.946). The results, determined with the attractor method were slightly higher in comparison to the ISHAGE strategy (mean of the differences: PBSC/ l (95% confidence interval:.1821 to 14.75)). PBSC-count/ µ l (Attractor) PBSC-count/ µ l (Attractor) r =.96 P <.1 PBSC count/ µ l (ISHAGE) Group ΙΙΙ (PBSC count: >1/ µ l) 25 r =.9343 P <.1 5 PBSC count/ µ l (ISHAGE) Figure 5 Correlation of the ISHAGE values and the volumetric assay. Symbols show mean values of three analyses. Spearman correlation was significant for all groups of values Discussion Flow cytometric enumeration of PBSC is the standard procedure in PBSCT. Because of the paucity of these cells in either peripheral blood or PBSC harvests, minor methodical variations can significantly influence results. 8,12,14 Therefore, it is necessary to compare different methods between different transplant centers and within a single laboratory. In this study, the intralaboratory results for two PBSC enumeration methods were compared to assess their application in PBSCT. The results of both methods, as well as those of other groups demonstrated that different flow cytometric methods are applicable for CD34 enumeration and show comparable results for peripheral blood and apheresis products Although strong correlations among different methods were demonstrated in single center studies it is important to consider that interlaboratory reproducibility is still a problem. 15,21 In multicenter studies of PBSC counting a wide range of results was found although the same antibodies and lysis techniques were used. 22 The major source of variation was the gating strategy used for cytometric data acquisition and analysis. 9,23 Indeed, evaluation of flow cytometric results seems to be dependent on the marker settings used by the individual cytometrist, and standardization of antibody selection and lysis technique cannot compensate for the differences in individual region settings of operators in multicenter studies. As a consequence, a multicenter study to evaluate the precision and interlaboratory reproducibility of a new automated software has been initiated. 24 In summary, both methods are applicable to determine the point of time for sufficient leukapheresis and for enumeration of PBSC in leukapheresis products. The results for these specimens show no significant differences and provide comparable intralaboratory linearity and reproducibility. Their application is a more rapid alternative to

7 Comparison of two gating strategies for PBSC enumeration Table 1 Column statistics for the results of the two gating strategies Group I Group II Group III (PBSC count: 1/ l) (PBSC count: 1 1/ l) (PBSC count 1/ l) 75 ISHAGE Attractor ISHAGE Attractor ISHAGE Attractor Number of mean values (n) Range Mean Mean CV (%) Spearman correlation r =.893 r =.96 r =.9343 Mean values of three replicates for each group. Coefficients of variation (CV) were calculated for each patient sample and both methods. Mean CV shows the mean of all CV in each group. colony assays for quantifying PBSC content in apheresis products. 14 From the point of quality assurance, the more standardized volumetric assay using reference beads and an attractor-based gating strategy seems to be a good, rapid alternative to the ISHAGE strategy. Attractors software helps to automate gating and minimizes the major source of variation, the individual setting of the gates. 9,25 The recently announced new software for data acquisition and automated analysis should close the gap between the need for quality controlled measurement and the widely used list mode file analysis performed by experts. References 1 Barlogie B, Jagannath S, Vesole DH. Superiority of tandem autologous transplantation over standard therapy for previously untreated mutliple myeloma. Blood 1997; 89: Hryniuk W, Bush H. The importance of dose intensity in chemotherapy of metastatic breast cancer. J Clin Oncol 1984, 2: Tricot G, Jagannath S, Vesole DH et al. Peripheral blood stem cell transplants for multiple myeloma: identification of favourable variables for rapid engraftment in 225 patients. Blood 1995; 85: Kessinger A, Armitage JO. The evolving role of autologous peripheral stem cell transplantation following high-dose therapy for malignancies. 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