Specific antibodies to human fetal hemoglobin were prepared and, after conjugation with a fluorescent dye, were used to determine the distribution of Hb F-containing cells in blood smears from normal adults and individuals with hereditary and acquired conditions associated with abnormal levels of Hb F. The mean proportion of F-cells in normal persons was 2.7% +/- 1.4%, with a range of 0.5%-7.0%. Proportions of F-cells were increased in persons with several acquired and inherited disorders that are associated with an increased percentage of Hb F in hemolysates. A strong linear correlation between the amount of Hb F and proportion of F-cells was observed. This technique may prove useful in studies of a variety of disorders associated with Hb F elevations and also in investigations of the mechanisms controlling the transition from fetal to adult hemoglobin during the course of human development.

Anti-haemoglobin S antibodies were raised in horses and purified by affinity chromatography. These antibodies recognize beta6 valine, while they fail to bind to haemoglobins with a glutamy1 (Hb A) or a lysy1 (Hb C) residue in this position. The purified anti-Hb S antibodies were composed of equine IgG(a, b) and IgG(T) subclasses and failed to cause precipitation with Hb S, evidently because of the bivalencey of both antibodies and antigen. Identification of Hb S in single erythrocytes was achieved by reacting the fluorescein isothiocyanate-conjungated anti-Hb S antibodies with the haemoglobin antigen in fixed blood smears. All the red cells from persons with sickle cell trait and sickle cell anaemia are labelled with the anti-Hb S-FITC, while red cells from persons with normal haemoglobin or other mutant haemoglobin genotypes are not. Erythrocytes from newborns with sickle cell trait bind the anti-Hb S antibody, although the intensity of labelling varies from cell to cell. This method of cellular identification of Hb S preserves the morphological characteristics of the reactive cells and permits observations of the pattern of haemoglobin synthesis among cells of various degrees of maturity. Fluorescent identification of Hb S in fixed blood smears may be applicable to prenatal diagnosis of haemglobinopathies.

Antibodies against hemoglobulin C (alpha2beta2 6Glu leads to Lys) were produced by immunizing horses and were purified by affinity chromatography. As expected from the bivalency of both the antibody and the antigen, the purified antibodies failed to produce immunoprecipitates upon reaction with the corresponding antigens. Identification of hemoglobin C in individual erythrocytes was achieved by reacting the fluorescein isothiocyanate-conjugated antibodies with the hemoglobin antigen in fixed smears of peripheral blood. Red cells from persons having a hemoglobin C gene were labeled strongly upon reaction with anti-Hb C-FITC; there was no labeling of red cells containing normal hemoglobins or Hb S, suggesting that the anti-Hb C antibodies recognize only the amino-terminal segment of the beta chains that contain lysine in position beta6.