Flow Cytometric Analysis of Normal Human Megakaryocytes

Abstract

Megakaryocytes from normal routine human bone marrow aspirates were analyzed by flow cytometry for size, fine cell structure and granularity, membrane expression of glycoprotein (GP) IIb/IIIa and ploidy. Marrow cells were initially enriched for megakaryocytes by a Percoll density gradient and megakaryocytes were labeled with a fluoresceinated monoclonal antibody directed to the GPIIb/IIIa complex. The cells were fixed with paraformaldehyde and stained with propidium iodide (PI) for DNA quantitation. Using two-color flow cytometry, megakaryocytes were identified by their high membrane immunofluorescence and their ploidy was determined according to the relative fluorescence intensity of the PI. Forward light scatter (FSC), correlating with cell size, 90 degrees side light scatter (SSC), reflecting primarily cell internal fine structure and granularity, and total cell membrane fluorescence were examined. To evaluate independently the relationship between size and cell membrane fluorescence obtained by flow cytometry, megakaryocytes were sorted directly on slides and analyzed by a laser-based anchored cell analyzer (ACAS). There was a strong correlation among size, SSC, and the level of membrane fluorescence. The mean diameter of megakaryocytes was 28.1 +/- 12.3 micron. The modal ploidy distribution was 16N with approximately one-fifth of the cells less than or equal to 4N. The mean FSC and SSC levels increased with increasing ploidy. However, the marked overlap observed between the ranges of these parameters in adjacent ploidy classes suggested that size and SSC increase continuously rather than by discrete steps as is characteristic for ploidy. The total surface membrane fluorescence was correlated with cell size (r = 0.98) as measured by FSC or directly by the ACAS (r = 0.85), and with cell ploidy (r = 0.99) indicating an augmentation in total membrane GPIIb/IIIa expression with an increase in cell size and ploidy. However, estimated GPIIb/IIIa fluorescence density was inversely correlated with FSC suggesting that the GPIIb/IIIa surface epitope density is decreased with increasing cell maturity. We conclude that flow cytometry is a useful technique for the rapid analysis of human megakaryocytes obtained by marrow aspiration, and should be applicable to studies of pathologic states.