A simple microassay for erythropoietin based on 3H-thymidine incorporation into spleen cells from phenylhydrazine treated mice.

Spleens taken from B6C3F1 mice on the third day following the second of 2 daily injections of phenylhydrazine HCl (60 mg/kg) contain approximately 10 times the number of cells (i.e., 10(9) cells) found in spleens from normal mice. More than 90% of these spleen cells are recognizable erythroid and form the basis of a new in vitro microassay for erythropoietin (Ep) which uses 3H-thymidine incorporation as an endpoint. This assay takes 24 h and can be carried out in the presence of either 20% FCS or a well-defined serum substitute. Under the conditions used, Ep levels from 30 mU/well down to as little as 0.2 mU/well can be accurately measured with a corresponding variation in counts of 50-100-fold. One spleen is sufficient for 1000 triplicate Ep determinations, and the use of this microassay procedure requires only very small (1-60) microliters) samples for evaluation. Preliminary studies with human plasma suggest that this assay may be more specific for Ep than established in vitro 59Fe bioassay methods.