QUANTITATION OF HEMOGLOBINS WITHIN INDIVIDUAL RED-CELLS - ASYNCHRONOUS BIOSYNTHESIS OF FETAL AND ADULT HEMOGLOBIN DURING ERYTHROID MATURATION IN NORMAL SUBJECTS

Abstract

A method for estimating HbF or HbA content in single [human] erythrocytes and their precursors is outlined. The method depends on microphotometric assay of darkfield reflectance arising from individual pericellular immunoprecipitates developed with anti-HbF or anti-HbA. When uniform-diameter latex microspheres were used to normalize comparisons between preparations, the mean coefficient of variation for HbF reflectance among separate preparations of the same sample was < 3%. Reflectance is a faithful (r = 0.99) linear function of the log of pg per cell in samples with known HbF or HbA content. Despite the use of antigenically-specific antihemoglobins from different sources, the least detectable quantity of HbF (3.2 pg) and HbA (14.8 pg) remained invariant. These detection thresholds depends on antihemoglobin affinity constants but are little influenced by antibody concentration. The procedure was equally valid for persons with normal HbF content (mean .+-. SD = 4.4 .+-. 0.3 pg per cell, 15 subjects) and for those with much higher levels. Like the percentage of HbF-bearing cells, HbF content was usually unchanging in serial samples. The utility of the method was evidenced in bone marrow analyses of 5 hematologically normal persons in whom HbF content, unlike HbA content, remained constant throughout maturation from erythroblasts to erythrocytes. In vivo HbF biosynthesis is normally completed long before HbA production.