Measurement of Total and Fetal Hemoglobin in Cultured Human Erythroid Cells by a Novel Micromethod

The present paper reports on the application of a simple micromethod for quantitation of total and fetal hemoglobin in small volumes of diluted erythroid cell hemolysates. A modified alkaline denaturation procedure was used to denature and precipitate non-fetal hemoglobins and the tetramethylbenzidine procedure was used for quantitation of the soluble hemoglobin. Using a combination of these procedures, total hemoglobin concentrations as low as 0.2 mg% (2 micrograms/ml) and a fetal hemoglobin proportion of 0.5% could be reliably determined. We have utilized this procedure to study two aspects of hemoglobin production in human erythroid cells grown in culture; the proportion of fetal hemoglobin of cells in culture as compared to that in the peripheral blood of the donors, and the kinetics of hemoglobin production during maturation of these cultured cells. The results show that the cultured erythroid cells have a hemoglobin phenotype similar to the individual from whom they are derived and that erythroid differentiation may be studied in vitro in these cultured cells by measurement of these and other parameters.