Improved microbioassay for plasma erythropoietin based on CFU-E colony formation

Abstract

We examined the conditions necessary for performing a reliable erythropoietin (EPO) assay based on CFU-E colony formation in fetal mouse liver cell (FMLC) microcultures using 96-well microtiter plates. Both linearity of colony numbers with the number of cells plated and comparison among the colony ratios at various densities of seeding cells indicated that the colonies originated from a single progenitor cell when 7500 or fewer cells were plated into individual microtiter wells. About a twofold CFU-E enrichment in 12- to 13-day FMLC was achieved by Ficoll-Paque centrifugation. Plasma treated with acid-boiling stimulated the colony formation most and contained no colony inhibitor. Dose-response curve for the plasma was parallel to the EPO standard curve. The "erythroid colony-stimulating activity" in the plasma was additive to that in the standard EPO, and was completely neutralized by a monoclonal antibody against recombinant human EPO. Using the assay procedure thus established, plasma EPO titer was determined in normal subjects, in patients with nonuremic anemia and polycythemia vera, and in dialysis patients with chronic renal failure. The use of different preparations of standard EPO resulted in a significant difference in the titers because their dose-response curves differed from one another. An inverse relationship was found between EPO titers and hemoglobin concentrations in the nonuremic anemic patients, but not in the dialysis patients with about one half the normal EPO level.