Correlation Between Bone Marrow Blasts Counts With Flow Cytometry and Morphological Analysis in Myelodysplastic Syndromes

Dear Editor, Determination of the percentage of blasts in the bone marrow (BM) is one of the critical factors in diagnosing MDS. Flow cytometry (FCM) of BM cells has been introduced as an important co-criterion for diagnosing MDS [1-3]. However, FCM has not been well accepted because of the lack of consensus of the criteria to define a phenotypic myeloblast and the appropriate denominator for calculation. Moreover, BM samples contain variable amounts of peripheral blood mixed with immature cells, thereby complicating interpretation. The present study was designed to choose reagent combinations to identify blasts by FCM in MDS patients, to determine which cell mass as the denominator in the process of counting the percentages of blasts, and to reveal whether the aspirates with high proportions of mature neutrophils should be normalized based on the proportion of dim CD16 maturing myeloid cells. Sixty-nine patients with primary MDS were enrolled; BM samples were obtained from 20 control subjects without any known hematological disease and with normal blood cell counts. This study was approved by our institutional review board, and written informed consent was obtained from all subjects. Cell morphology was analyzed by conventional light microscopy, and the same BM samples were analyzed by FCM within 6 hr. The panel included four-color combinations of markers: HLA-DR-fluorescein isothiocyanate (FITC)/CD117-phycoerythrin (PE)/CD34-allophycocyanin (APC)/CD45-peridinin-chlorophyll protein (PerCP), CD16FITC/CD13-PE/CD11bAPC/CD45-PerCP, anti-ckappa-FITC/antilambda-PE/CD138APC/CD45-PerCP, and IgG1-FITC/IgG1-PE/ IgG1-APC/IgG1-PerCP. For each criterion, the percentage of blasts was calculated by using both the total number of nucleated BM cells (A) and the number of non-erythroid nucleated cells (N) as the denominator. For the A1 and N1 methods, blasts were defined as events showing a CD34 and CD45 phenotype. The A2 and N2 strategies identified CD34 and/or CD117 cells showing CD45 expression as corresponding to blasts. For the A3 and N3 methods, blasts were defined as events showing a CD34 and/or CD117 HLA-DR and CD45 phenotype. The ratio of the percentage of dim CD16 neutrophils to the average percentage from BM biopsies (80%) was calculated as a dilution factor of excess mature segmented neutrophils in the sample. We used