IMMUNOCYTOCHEMISTRY AND FLOW-CYTOMETRY EVALUATION OF HUMAN MEGAKARYOCYTES IN FRESH SAMPLES AND CULTURES OF CD34(+) CELLS

Adhering platelets on the cell surface can give misleading results whe n doing now cytometry analysis of platelet/megakaryocyte-specific glyc oprotein (GP) antigens to enumerate megakaryocytes (MK) in mobilized p eripheral blood (PB), apheresis products, or normal bone marrow (BM). For adequate quantification and characterization of human MK, we exami ned samples with parallel now cytometry and immunocytochemistry. MK ex pression of GP IIb/IIIa (CD41a), GP Ib (CD42b), GP IIIa (CD61), CD45, CD33, and CD11b, and their light scatter properties were evaluated. Fr esh samples of low density mononuclear cells (MNC) or purified CD34(+) cells contained 10-45% of platelet-coated cells. Platelet-coated cell s decreased dramatically after several days of incubation in a serum-f ree medium supplemented with stem cell factor, IL-3, IL-6, and/or GM-C SF. Between d 9-12, now cytometry detected a distinct CD41a(+) MK popu lation, 8.3 +/- 1.3% in BM CD34 cell cultures (n = 7) and 13.1 +/- 2.1 % in PB CD34 cell cultures (n = 14), comparable to immunocytochemistry data (7.8 +/- 1.9% and 16.4 +/- 2.6%, respectively). CD41a stained a higher proportion of MK than CD42b or CD61, while CD42b(+) or CD61(+) cells contained more morphologically mature MK than CD41a(+) cells in cultures containing aplastic serum. When fluorescence emission of CD41 a was plotted against forward-light scatter (FSC), subpopulations of s mall and large MK were observed. Such subpopulations overlapped in CD4 1a intensity and side-light scatter (SSC) property. Most MK co-express ed CD45 (98.8% positive) but not CD33 (80.7% negative) or CD11b (88.9% negative). Our data indicate that flow cytometry can be used effectiv ely to identify MK. However, caution should be taken with samples cont aining adherent platelets. (C) 1996 Wiley-Liss, Inc.