DNA synthesis by erythroid precursors in a completely defined medium: a rapid, sensitive, and convenient bioassay for erythropoietin

Abstract

We detail a novel, sensitive, and reproducible in vitro bioassay for erythropoietin that can be performed conveniently in any laboratory and is well suited for analysis of large numbers of samples. The assay measures DNA synthesis by a cohort of highly erythropoietin-responsive red cell precursors appearing in bone marrow of anemic rabbits after treatment with a single dose of actinomycin D. The assay is conducted in a completely defined culture medium that totally dispenses not only with serum but also with serum-replacing factors. Under well-defined conditions, incorporation of [3H]thymidine by the cells depends specifically on erythropoietin. A stimulation index of up to 40-fold is obtained at 50 mU/ml of the hormone. The assay is linear in the range 0-50 mU/ml and not saturated before 1 U/ml of erythropoietin. Sample volumes of 1-30 microliter suffice for assay. Assay cells can be frozen in aliquots that retain their viability and ability to respond to erythropoietin over extended periods. Using microtiter-plate techniques, cells from one rabbit suffice for over 5000 triplicate erythropoietin determinations. Concentrations of 0.1-0.2 mU/well of erythropoietin can be detected. Erythropoietin values determined in sera from a variety of patients correlate extremely well with values obtained by the colony formation method. The ability to follow erythropoietin-dependent DNA synthesis and multiple cell divisions by a cohort of erythroid precursors in completely defined culture conditions may find application in controlled studies of red cell development.