Quantitation of human megakaryocyte progenitors (CFU-M) in plasma clot culture by an indirect immunoperoxidase method

: We have developed an indirect immunoperoxidase method to detect human megakaryocytic colonies in plasma clot culture. A monoclonal antibody directed against platelet glycoprotein IIb/IIIa complex is incubated with plasma clots fixed to gelatin-coated slides with methanol. This antibody is subsequently linked to horseradish peroxidase by means of an avidin-biotin sandwich technique. Megakaryocytic colonies are identified by precipitation of benzidine by the horseradish peroxidase. This method detects growth of megakaryocytic colonies in culture, which is dependent on factors present in leukocyte-conditioned medium and is linear with respect to the concentration of human light-density nonadherent bone marrow cells plated at a concentration of 1.5-7 X 10(5) cells/ml. This method is simple to apply, uses objective criteria for recognition of megakaryocytes, and leaves cultures mounted and stained for permanent record.