Populations of human immature megakaryocytes (ImMk) are to be studied in hematologically normal marrow donors and hematologic neoplasias. Other than descriptive data, no information exists on this subpopulation of megakaryocytes. The first phase of this grant will be the functional and physical characterization of these cells. This population of cells will be enriched using combinations of physical cell separation techniques (velocity sedimentation, density centrifugation, counter-current centrifugal elutriation). Following enrichment, ImMk will be functionally characterized in short-term tissue culture assays. Responsiveness to various thrombopoietic stimuli will be tested as well as cell kinetics and relationships to megakaryocyte progenitor cells and mature megakaryocytes (Mk). Alternative characterizations of Mk/ImMk subpopulations will be studied using multiparameter flow cytometry (e.g. combinations of membrane antigen, DNA, and light scatter properties). Populations thus defined will be sorted directly into microtiter wells for in vitro analysis. The second phase of the study will be the analysis of alterations of ImMk in megakaryocytic dyscrasias. These studies will examine ImMk in an identical manner and be contrasted with the results on normal ImMk to define the nature of the defect in megakaryocytopoiesis. Concomitant animal studies will focus on questions which cannot be readily answered by examining changes in human megakaryocytopoiesis. Irradiated animal studies are designed to examine the in vivo differentiation process of immature megakaryocytes. Flow studies are to be performed to examine the DNA content of the murine ImMk.
