Simultaneous Assessment of i-Antigenic Expression and Fetal Hemoglobin in Single Red Cells by Immunofluorescence

Abstract

The expression of i-antigenic determinants in the red cells of normal adults, newborns, and a variety of anemic patients was examined using an immunofluorescent approach permitting i-antigen detection in single erythrocytes. In 29 normal adult individuals, an average of 8.7% of red cells were found to be positive for i (i-cells). Frequencies of i-cells in neonatal blood ranged from 40% to 66%, but intensity of fluorescent labeling was considerably stronger than that of adult i-cells. Twenty-three of 24 patients with sustained anemia and acquired elevation of HbF had increased levels of i-cells (from 15.4% to 92.9%). In order to investigate the relationship between HbF and i expression in the adult red cells, a double immunofluorescent method, permitting the detection of both i-antigen and HbF in the same red cell, was applied. Frequencies of i-cells, F-cells, and i-positive F-cells (iF-cells) were determined, and the observed frequencies of iF-cells were compared to the expected ones, calculated from the individual i-cell and F-cell measurements. In normal adults and the patients with chronic anemias, the observed frequencies of iF-cells were similar to the ones expected, under the assumption that the expression of the F and the i characters in a red cell is random (ratio of observed/expected iF-cell frequencies: in normal persons, 1.14; in patients with chronic anemia, 1.18). These findings suggest that there is no close association between i and F expression in the adult red cells; the F-cells express the i-characteristic as frequently as the erythrocytes that fail to show fetal hemoglobin. In contrast to the findings in the patients with chronic anemia, a significant degree of coexpression of HbF and i was noted with sequential studies of red cells from six patients who had received bone marrow transplantation and four patients recovering from erythroid aplasia. Initially, the observed frequencies of iF-cells were from 2.5 to 8 times higher than the expected ones and gradually declined, suggesting that during acute marrow expansion, a consistent but transient coexpression of i and HbF characterizes the newly produced red cells. The randomness of i and F expression in the red cell of the normal and chronically anemic adult is interpreted under the assumption that the expression of the two red cell phenotypes is determined at different levels of the erythroid cell differentiation-maturation process: the F-cell is presumably the product of premature terminal differentiation of early erythroid progenitors (BFU-E), while the increased i reactivity is the product of faster maturation of terminally differentiated erythroid cells. Thus, F-programmed cells and A-programmed cells have equal chance to be submitted to the maturational changes that allow i expression. The transient coexpression of i and HbF during the exponential increase of erythropoiesis is probably circumstantial, and it is attributed to the preferential production of F-cells during the exponential phase of erythropoietic recovery.