Multivectorial analysis of megakaryocytic differentiation.
The definition of the cytokinetics of megakaryocytic differentiation has rested mainly on morphologic classifications that lack a quantitative data base. The approach that we have developed comprises the systematic combination of new techniques for quantitative megakaryocyte separation and correlated, multiparameter analysis of multiply labeled single cells by flow cytometry. We have coined this approach a multivectorial analysis of megakaryocytic differentiation. Modifications of combined centrifugal elutriation (CE) and density gradient centrifugation permit the rapid collection of large numbers of intact, essentially homogeneous, marrow megakaryocytes. These cells, labeled simultaneously with multiple markers of DNA content and surface membrane immunofluorescence have been subjected to correlated, dualparameter analysis by flow cytometry and cell sorting, in an attempt to relate polyploidization (as a unique megakaryocytic property) to cytoplasmic progress towards end-state platelet formation. This approach provides a reasonable data base for an objective appraisal of the interactive roles of nuclear endoreduplication and cytoplasmic development during differentiation. Furthermore, the potential usefulness of matrix models and other innovations inherent in their exploitation hold the promise of describing regulatory mechanisms that are poorly understood, both in normal megakaryocytes and those produced in disease.
Nakeff
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Department of Internal Medicine, Wayne State University School of Medicine, Detroit, Michigan 48201.
D000818:Animals; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000166:cytology; D002469:Cell Separation; D002499:Centrifugation, Density Gradient; D003593:Cytoplasm / Q000166:cytology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology* / Q000254:growth & development; D051379:Mice; D051381:Rats