Measurement of human erythroid burst-promoting activity by a specific cell culture assay

A two-stage cell culture assay specific for human erythroid burst-promoting activity (BPA) is described. Human peripheral blood mononuclear cells were cultured in suspension with or without a BPA test sample for two days, then transferred to methylcellulose medium with added erythropoietin (EPO) and incubated for ten more days, and finally BFU-E-derived colonies were scored. An increase in number of colonies due to the presence of BPA was observed that was proportional to the concentration of BPA in the test sample. This response was linear with respect to number of cells plated between 2 and 5 X 10(5)/ml. The system was standardized with a partially purified human urinary BPA preparation. Dose responses to urinary protein preparations, plasma, and serum were parallel. The assay system was found to be nonresponsive to highly purified EPO and to bacterial endotoxin. It was determined that BPA action was confined to the suspension culture stage of the assay, while EPO presence was an absolute requirement during methylcellulose culture. In the two-stage assay optimal amounts of BPA caused up to 358% increases of BFU-E-derived colonies; the same amounts of BPA added to conventional methylcellulose cultures caused only up to 54% increases over the number of colonies obtained with EPO alone. Plasma and serum BPA levels of hematologically normal and abnormal individuals showed no correlation with EPO levels and hemoglobin (Hb) concentrations. This seems to rule out the possibility that BPA elaboration is regulated by oxygen availability or the amount of EPO circulating in an organism.