Quantitation of erythroid differentiation in vitro using a sensitive colorimetric assay for hemoglobin

We have developed a new colorimetric method to detect early erythroid clonogenic progenitor cells by virtue of their hemoglobin-containing progeny in vitro. The method relies upon the specific oxidation of 2,7-diaminofluorene (DAF) by the pseudoperoxidase activity of hemoglobin (Hb). Generation of fluorene blue (FB), the chromophore generated upon oxidation of DAF, was a linear function of Hb and erythrocyte concentration. Measurement of FB absorbance at 610 nm was 80 times more sensitive than measurement of Hb absorbance at 530 nm. The limit of resolution was approximately 8 pmol of Hb corresponding to the Hb content of approximately 5000 murine peripheral blood erythrocytes. FB concentration closely paralleled eight-day BFU-E colony formation as a function of erythropoietin dose. However, the method was unable to detect a response for CFU-E to erythropoietin because of persistently high background levels of Hb in two-day cultures. Generation of FB by cells likely to contain myeloperoxidase was negligible, and FB production was not correlated with the number of CFU-GM colonies. DAF oxidation by hemoglobin showed a higher hydrogen peroxide optimum than oxidation by microperoxidase, so that, under the conditions used, FB generation by peroxidase was not favored. As a further confirmation of specificity, peritoneal exudate cells failed to show significant generation of FB. The DAF reagent was also used to stain both CFU-E and BFU-E colonies in situ, and was more sensitive than benzidine dihydrochloride in the histochemical detection of Hb. DAF can replace hazardous benzidine-related compounds for the staining erythroid colonies in culture dishes, and the method offers a new quantitative approach to the study of erythropoietin responsiveness in vitro.