Flow cytometric analysis of megakaryocyte differentiation

Megakaryocytes were isolated quantitatively from rat bone marrow by centrifugal elutriation (CE). CE-enriched megakaryocytes were stained supravitally for either DNA content with Hoechst 33342, surface membrane immunofluorescence with fluorescein isothiocyanate (FITC)-conjugated antiplatelet antibody, or both. The cells were then measured using a Becton Dickinson FACS IV flow cytometer. The following correlations were analyzed: DNA content and light scatter, light scatter and antiplatelet immunofluorescence, and DNA content and antiplatelet immunofluorescence. Although the range of light scatter increased as a function of DNA content, discrete subpopulations of megakaryocytes with different light scatter properties were detected within each of the three principal ploidy classes (8C, 16C, and 32C). Other discrete megakaryocyte subpopulations were revealed in the analysis of antiplatelet surface immunofluorescence as a function of degree of light scatter. The nonlinear relationship between the latter suggested that the degree of membrane immunofluorescence did not bear a simple relationship to cell size as reflected in light scatter. Megakaryocyte DNA content, on the other hand, varied in a linear fashion with membrane immunofluorescence, supporting the conclusion that there may be a proportional increase in the expression of platelet antigens with DNA content. The use of multiple markers, correlated multiparameter flow cytometry and multivectorial analysis to define differentiation on a single cell basis have revealed new complexities in this process. Flow cytometric analysis holds promise as a useful method for further characterization of megakaryocyte differentiation.