PMID: 6159933Dec 1, 1980Paper

Quantitation of hemoglobins within individual red cells: asynchronous biosynthesis of fetal and adult hemoglobin during erythroid maturation in normal subjects.

Blood
G J Dover, S H Boyer

Abstract

We outline a method for estimating either HbF or HbA content in single erythrocytes and their precursors. Our 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, 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 logarithm of picograms per cell in samples with known HbF or HbA content. The following features emerged from such analyses. First, 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. Second, these detection thresholds depends on antihemoglobin affinity constants but are little influenced by antibody concentration. Third, our procedure is equally valid for persons with normal HbF constant (mean +/- SD = 4.4 +/- 0.3 pg per cell, 15 subjects) and for those with much higher levels. Fourth, like the percentage of HbF-bearing cells, HbF content is usually unchanging in serial samples. Fifth, the u...Continue Reading

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