Minor hemoglobins in sickle cell anemia, β-thalassemia, and related conditions: A study of red cell fractions isolated by density gradient centrifugation

Abstract

Red cells from 6 patients with sickle cell anemia, 2 Hb-S heterozygotes, 3 β-thalassemia homozygotes, 2 β-thalassemia heterozygotes, 5 HPFH heterozygotes, 2 AA adults, and 2 normal newborns have been fractionated by a discontinuous Dextran 40 density gradient centrifugation. In the SS patients, from top to bottom fraction, the reticulocyte count, the MCV, and 2,3-DPG levels decreased and the MCHC values increased substantially. The bottom cell fraction of the SS patients contained nearly 90% irreversibly sickled cells (ISC). The percentages of this fraction varied from patient to patient, and were directly related to the percentages of ISC in circulation. Chromatographic analyses showed that the percentages of the hemoglobins F 0 and F 1 were lower and those of hemoglobins S 0 and A 2 higher in both the top and bottom fractions than in the middle fractions; thus, Hb-F was mostly present in the middle fractions. There was an increase in the percentage of Hb-S 1 with an increase in red cell density. The extent of heterogeneity of several hemoglobin types varied markedly in different SS patients. Analyses of whole blood from a larger group of SS patients showed a wide variation in hematological parameters, in the number of ISC(%), the red cell density distribution ( D 50), and in hemoglobin composition. A direct relationship appeared to exist between the percentage of ISC and D 50 value and an inverse relationship between the percentage of ISC and the level of Hb-F. Chromatographic analyses indicated that the levels of the hemoglobins A and A 2 in β-thalassemia homozygotes were high in the top fractions and low in the middle fractions whereas the levels of Hb-F were low in the top and high in the middle fractions. The levels of these hemoglobins in the bottom fractions were intermediate to those of the top and the middle fractions. Thus, the amounts of hemoglobins A 2, A, and F changed with red cell density but the extent of heterogeneity in the distribution of these hemoglobins varied from one patient to the other. The levels of each hemoglobin type were nearly the same in each cell fraction isolated from blood of β-thalassemia heterozygotes and HPFH heterozygotes.