Quantitation of human in vitro megakaryocytopoiesis by radioimmunoassay

The isolation and characterization of human megakaryocyte growth factors has been hampered because evaluation of megakaryocyte growth in semisolid medium requires both lengthy incubation and visual quantitation. In addition, colony formation requires cell division, while most regulation of platelet production may involve individual, nonproliferating differentiating megakaryocytes. We have developed a radioimmunoassay (RIA) that makes use of an iodinated murine monoclonal antibody (MoAb) specific for platelet/megakaryocyte glycoprotein IIb/IIIa (GPIIb/IIIa) to measure megakaryocyte production in liquid marrow culture. This assay is sensitive to 3 X 10(3) platelets (roughly 30 megakaryocytes) and linear up to 1 X 10(6) platelets, and thus it provides a useful range for quantitating megakaryocyte production in in vitro marrow culture. Significant differences (threefold to fivefold) in megakaryocyte/platelet-specific GPIIb/IIIa complex are detected between stimulated and unstimulated marrow cultures by day 7, although antigen accrual in stimulated cultures continues through at least day 16. Conditions that promote megakaryocyte growth in semisolid medium (ie, aplastic plasma and PHA-LCM) have also been facilitory in liquid culture. This rapid and sensitive assay for cell-bound GPIIb/IIIa should facilitate recognition and isolation of megakaryocyte and platelet growth factors.