JOURNAL OF HEMATOTHERAPY & STEM CELL RESEARCH 9:11–12 (2000)Mary Ann Liebert, Inc.

Correspondence

The Influence of Flow Cytometric Gating Strategy and CellPreparation Procedures on CD34 1 Cell Quantification

MUSTAFA N. YENEREL, TÜLIN BUDAK-ALPDOGAN, SEVGI KALAYOGLU-BESISIK,MELEK YANASIK, and DENIZ SARGIN

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BECAU SE THE SM ALL PO PU LATION of cells that expressthe CD34 antigen is known to correlate with multi-

lineage engraftment, CD34 1 cell quantification by flowcytometric analysis is of great importance in determin-ing the adequate number of hematopoietic stem cells(HSC) for both autologous and allogeneic peripheral stemcell transplantation (1). Flow cytometric enumeration ofCD34 1 stem cells varies according to the differences inthe method of cell preparation, the reagents used, and theflow cytometric analysis (2). The proposed Milano andISHAGE guidelines define different gating strategies forCD34 1 cell analysis by flow cytometry (3,4).

We analyzed 11 bone marrow (BM) and 11 peripheralblood (PB) samples obtained from healthy donors and 11leukapheresed stem cell (LSC) harvest samples to com-pare the influence of cell preparation procedures on thegating strategies. Briefly, 100 m l of sample (LSC and BMdiluted to 10 3 103 cells/ m l) was incubated with 10 m lof anti-CD34-PE (BIRMA-K3, Dako, Carpenteria, CA)and 10 m l of anti-CD45-FITC (T29-33, Dako), followed

by isotypic controls for 30 min at 4°C in the dark. Redcells were lysed with lysing solution (FACSLyse, Bec-ton Dickinson, Mountain View, CA), and specimens wereanalyzed without washing or after one wash with PBS orone wash with PBS 1 2% human albumin or two washeswith PBS.

Following the ISHAGE guidelines and the Milano pro-tocol, listmode data of each sample were analyzed by twodifferent gating strategies. In the Milano protocol, for-ward scatter (FSC) and side scatter (SSC) were used togate the nucleated cells and to exclude the RBC, debris,and cell aggregates (live gate) (3). The gated nucleatedcell population was plotted on CD34 versus SSC. OnlyCD34 1 events with low SSC were used to calculate thenumber of CD34 1 cells, expressed as a percentage of thegated nucleated cells.

In the second gating strategy, proposed by ISHAGE,the first gate selected the CD45 1 events, and a secondgate chose the CD34 1 events. CD34 1 cells were selectedby their CD34 expression, their characteristic low to in-

Department of Hematology, BMT Unit, Istanbul University, Istanbul Medical School, Istanbul, Turkey.

TA BL E 1. RESU LTS OF DIFFERE NT CEL L PREPA RA TIO N PROCED URES

COM PARING TH E MILA NO AND ISHAGE GA TIN G STR ATEG IES

Mean DifferenceMilano ISHAGE (Milano 1 ISHAGE)/2 Milano 2 ISHAGE

PBa Mean 0.33 0.13733 0.23367 0.19267

SD 0.28224 0.09917 0.1416 0.31429

BM Mean 0.63 0.5285 0.57925 0.1015

SD 0.62444 0.36983 0.48697 0.32377

LSC Mean 2.49917 1.82167 2.16042 0.6775

SD 0.79247 0.99501 0.7251 1.06443

Total Mean 1.011489 0.73383 0.87266 0.27766

SD 1.061861 0.859827 0.912667 0.633919

aPB, peripheral blood; BM, bone marrow; LSC, leukapheresed stem cell.

termediate CD45 antigen expression, and their low SSCproperties. From the CD45 1 /CD34 1 events, the third andfourth gates were defined to exclude events of high SSC,bright CD45 1 , and low FSC. The CD34 1 cells were de-fined as a percentage of CD45 1 cells (4). We summarizethe percentage means of CD34 1 cell determination withfour different cell preparations comparing the two dif-ferent gating strategies in Table 1. The mean differenceand SD of the difference between the two protocols arealso shown in Table 1.

Agreement between these two gating strategies wasevaluated by analyzing the relation between the differ-ences and the means, as proposed by Bland and Altman(5). The results are shown in Figure 1 as plots of the dif-ference against means. The percentage means of CD34 1

cells were more pronounced in the samples analyzedwithout any washing step after erythrocyte lysis. We alsoobserved that the variation among the different cell prepa-ration procedures was statistically significant with theMilano protocol (p , 0.0001), whereas these proceduresseemed hardly to affect CD34 1 cell enumeration usingthe ISHAGE protocol (p . 0.05). The percentage meansof CD34 1 cells were found to be 1.01% and 0.73% inthe Milano and ISHAGE protocols, respectively.

The plot of the differences against the means displaysconsiderable lack of agreement between the Milano andISHAGE protocols, with discrepancies of up to 1.53% inenumeration of CD34 1 cells (Fig. 2). This disagreementwas more obvious in PB and LSC samples (Fig. 1).

We consider the ISHAGE protocol to be more reliablein the assessment of CD34 1 cells, as this gating strategyappeared not to be influenced by the type of sample orthe type of cell preparation procedure.

REFERENCES

1. Serke S, S Sauberlich, and D Huhn. (1991). Multiparamete rflow cytom etrical quantitation of circulating CD34 ( 1 ) cells:correlation to the quantitation of circulating hematopoieticprogenitor cells by in vitro colony assay. Br J Haematol77:453–459.

2. Chang A, and DDF Ma. (1996). The influence of flow cy-tometric gating strategy on the standardization of CD34 1

cell quantitation: an Australian multicenter study. J Hema-tother 5:605–616.

3. Johnsen HE, and LM Knudsen. (1996). Nordic flow cytom -etry standards for CD34 1 cell enumeration in blood andleukapheresis products: report from the Second NordicWorkshop. J Hematother 5:237–245.

4. Sutherland DR, L Anderson, M Keeney, R Nayar, and IChin-Yee. (1996). The ISHAGE guidelines for CD34 1 celldetermination by flow cytometry. International Society ofHematotherapy and Graft Engineering. J Hematother5:213–226.

5. Bland JM, and DG Altman. (1996). Statistical methods forassessing agreem ent between two methods of clinical mea-surem ent. Lancet 1:307–310.

Address reprint requests to:Mustafa N. YenerelIstanbul University

Istanbul Medical SchoolDepartment of Hematology, BMT Unit

Istanbul, Turkey

YENEREL ET AL.

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FIG. 1. Results of the four cell preparation procedures com-paring two gating strategies.

FIG. 2. Difference against means for CD34 positivity.