Flow cytometric analysis of megakaryocyte differentiation

Abstract

Megakaryocytes were isolated quantitatively from rat bone marrow by centrifugal elutriation (CE). CE-enriched megakaryocytes were stained supravitally for either (1) DNA content with Hoechst 33342, (2) surface membrane immunofluorescence with fluorescein isothiocyanate (FITC)-conjugated antiplatelet antibody, or (3) both. The cells were then measured using a Becton Dickinson FACS IV flow cytometer. The following correlations were analyzed: (1) DNA content and light scatter, (2) light scatter and antiplatelet immunofluorescence, and (3) 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 (SC, 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 nonMegakaryocytes constitute a unique system of hematopoietic cell differentiation characterized by high nuclear ploidy and development of a cytoplasmic mass that at end-stage differentiation disintegrates into anucleate blood platelets. The two most striking morphological features of megakaryocytes, size and nuclear structure, have served as qualitative indices of megakaryocyte differentiation. Light microscopy and tinctorial techniques have been used previously to identify megakaryocytes and classify them into maturation stages on a qualitative basis; with both these techniques DNA content is measured and correlated to presumptive levels of differentiation terminating in cytoplasmic platelet formation (12-14,17). However, our understanding of this unique cell line remains limited by the lack of an objective, quantitative approach that is based on large numbers of these rare cells and that is applied at the single-cell level to define characteristics of megakaryocytes for correlation with degrees of differentiation.Megakaryocytes are difficult cells to study, partly be- (20) that megakaryocytes are able to synthesize thromboxane B2, which is also a component of a major metabolic pathway in platelet hemostatic function, has provided evidence that the thromboxane pathway may serve as a marker of differentiation in these cells (21). Thus, both shared metabolic and antigenic characteristics should be of potential use in studying megakaryocyte differentiation.The application of laser-based flow cytometry to the analysis of megakaryocytes is an exciting new approach, since the ability to perform correlated, multiparameter analyses on large numbers of these cells (measured at rates in excess of 6 x lo4 celldmin) is well suited to the type of multivectorial analysis inherent in models of megakaryocyte differentiation that have been proposed (10,161. Flow cytometry has been applied successfully to the analysis of the DNA content of rodent (9) as well as guinea pig and monkey megakaryocytes (5) using both unfractionated marrow and fractions en...

Ultrastructural analysis of acute immune thrombocytopenia in mice: Dissociation between alterations in megakaryocytes and platelets

“…There was a marked dissociation between alterations in platelets and MK, since a statistically significant increase in platelet sectional area occurred 40 hr before the shift in modal ploidy class of MK, and platelet size subsequently decreased toward normal during the period that has been shown to be associated with the maximum shift in MK ploidy. These results strongly suggest that the characteristics of platelet release do not depend on the ploidy or cytoplasmic characteristics of MK.The process of thrombopoiesis remains controversial, particularly with respect to the normal megakaryocyte ploidy distribution (Bessman, 1984;Corash et al, 1987;Jackson et al, 1984;Levine et al, 1980;Worthington et al, 1984) and the determinants of platelet volume (Corash, 1985; Martin and Trowbridge, 1985). Recently, Corash et al (1987) used a murine model in which experimental immune thrombocytopenia was induced to evaluate the relationships between rnegakaryocyte ploidy and mean platelet volume.…”

Human marrow megakaryocyte differentiation: multiparameter correlative analysis identifies von Willebrand factor as a sensitive and distinctive marker for early (2N and 4N) megakaryocytes

“…Studies in experimental mammals showed similar ploidy distribution. [16][17][18][19] It is of interest that, whereas immunofluorescence, size, and granularity increase continuously with cell maturation, ploidy increases in discrete steps (Figure 2). Thus, MK ploidy level may be useful for discrimination between cell maturation subsets.…”

“…Following initial studies in experimental animals, [16][17][18][19] the flow cytometric method was adapted for the analysis of human MKs derived from routine marrow aspirates, 9-11 thus permitting sequential studies of MKs in normal and pathologic states. Moreover, with the use of fluorescence-activated cell sorting of bone marrow aspirates, it becomes possible to isolate highly pure (Ն 98%) viable human MKs capable of synthesis of both DNA and protein in vitro and responsive to hematopoietic growth factors.…”

Comparison of the DNA content of megakaryocytes identified immunologically with that identified morphologically

“…The DNA content of megakaryocytes has been commonly determined by a microphotometric method using Feulgen staining (Garcia 1964;Queisser et al 1971Queisser et al , 1972Queisser et al , 1974Nomura et al 1983), a microfluorometric method using DAPI staining (Kobayashi et al 1991) or flow cytometry using propidium iodide staining (Nakeff et al 1979;Worthington et al 1984;Tomer et al 1988). Each method has advantages and disadvantages.…”