Two-color flow cytometric measurement of DNA distributions of rat megakaryocytes in unfixed, unfractionated marrow cell suspensions

Abstract

The ploidy distribution of megakaryocytes shifts in response to platelet demand and thus provides a sensitive index of megakaryocytopoiesis. Flow cytometry (FCM) is a potentially valuable method for rapid determination of ploidy distributions of megakaryocyte populations; however, because megakaryocytes constitute only a very small proportion of the cells in unfractionated marrow, other rare events, such as cell clumping, complicate FCM analysis. We describe the measurement of cellular DNA distributions of megakaryocytes by two- color FCM in unfixed, unfractionated marrow--a method based on the resistance of megakaryocytes to hypotonic lysis in the cold for at least 2 days. Specific platelet antiserum was used to label megakaryocytes by indirect immunofluorescence with fluorescein (green fluorescence), and DNA was stained with propidium iodide (red fluorescence) in hypotonic citrate solution. The ploidy distribution of megakaryocytes was selectively determined with two-color, green-gated FCM, with which the red and green fluorescence of all cells is analyzed, but only the red fluorescence (DNA content) of cells that specifically bound the platelet antibody is recorded. We demonstrate that this method can readily detect changes in megakaryocyte DNA distributions due to experimental thrombocytopenia or platelet hypertransfusion and, therefore, should be useful for both experimental and clinical investigations of megakaryocytopoiesis.

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

“…In this investigation, we have tested the hypothesis that platelets should morphologically resemble the ma- ture megakaryocytes that are present in the bone marrow at the time of platelet production, using the murine model of experimental immune thrombocytopenia. Previous studies have indicated that acute thrombocytopenia results in an increased megakaryocyte maturation rate (Ebbe et al, 196813;Odell Jr. et al, 19691, increased megakaryocyte size (Ebbe et al, 1968a;Ebbe et al, 1988;Harker, 1968;Odell et al, 1976;Penington and Olsen, 19701, a ploidy shift in megakaryocytes (Corash et al, 1987;Ebbe et al, 1988;Jackson et al, 1984;Martin et al, 1983;Odell et a]., 1976;Penington and Olsen, 1970), and an increased mean platelet volume (Corash et al, 1987;Martin et al, 1983;Odell et al, 1976). Corash et al (1987) and Odell et al (1976) serially examined the megakaryocyte ploidy distributions and mean platelet volumes in thrombocytopenic animals.…”

“…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.…”

Neuraminidase‐induced thrombocytopenia in mice: Effects on thrombopoiesis

“…Almost all previous studies of the effects of experimental thrombocytopenia have utilized platelet antiserum to perturb thrombopoiesis. These investigations have reported multiple changes in megakaryocyte and platelet characteristics, notably; duration of megakaryocyte maturational stages, megakaryocyte ploidy distribution, platelet volume, and megakaryocyte cytoplasmic volume (Corash et al, 1987;Ebbe et al, 1968;Jackson et al, 1984;Martin et al, 1982;Martin et al, 1983;Ode11 et al, 1976;Penington and Olsen, 1970;Trowbridge and Martin, 1984). However, the possibility that administration of platelet antiserum may induce changes in megakaryocytopoiesis secondary to an adverse immunological reaction, rather than as a D 1991 WILEY-LISS, INC. direct response to thrombocytopenia, has been suggested (Burstein et al, 1982;Levin et al, 1980).…”

Thrombopoietin: Biology and Clinical Applications

“…The immature megakaryocytes then develop into larger, mature megakaryocytes that shed platelets into bone marrow sinusoids. In response to an increased demand for platelets, as occurs in idiopathic thrombocytopenic purpura (ITP), the number and size of megakaryocytes increase [11][12][13], the average megakaryocyte ploidy increases (from 16N to 32N) and platelet production rises up to a maximum of 11-fold [11]. The hematopoietic factor regulating this response has long been referred to as "thrombopoietin" [14].…”