Development of Human Megakaryocyte Progenitor Assay

With the advent of cytokine therapy, thrombocytopenia is the major dose-limiting toxicity of anti-cancer and anti-AIDS drugs. Early screening of developmental agents for toxicity to hematopoietic, particularly megakaryocytic, progenitors would allow the selection of drugs that are effective against tumor or virus, but are not toxic. Current clonogenic assays for human megakaryocyte progenitors (CFU-Meg) are hampered by a non-specific staining procedure and a requirement for human AB plasma. We will attempt, firstly, changes in the staining methodology and secondly, development of serum- and protein-free conditions. Serum-and protein-free assays can better model in vivo conditions by addition of physiological concentrations of human drug-binding proteins (i. e., human albumin, y-globulin, etc.). An alternative to clonogenic assays will also be investigated. This approach will use flow cytometry to measure changes in the total number of megakaryocytes growing in liquid culture. The use of flow cytometry to measure progenitor differentiation would be of significant advantage over traditional clonogenic assays because of the simple labeling procedures, thequantitative and objective nature of the measurement and the potential for obtaining results sooner the 12-14 days required for a human CFU-Meg assay. Simple, standardized assays for human megakaryocyte progenitors may assist physicians in determining the number of progenitors necessary for successful bone marrow and/or peripheral blood transplants.