Solution and cell studies were performed to ascertain why individuals with hemoglobin (Hb) SC have disease whereas those with Hb AS do not. The polymerization of deoxygenated mixtures containing sickle cell Hb (Hb S; a2P26GIu-VaI) and Hb C (a2f326GIu Lys) was investigated by measurements of delay times and solubilities. In mixtures containing more than 40% Hb S. polymerization takes place by the same mechanism as in solutions of Hb S alone, with no evidence for independent crystallization of Hb C. A detailed comparison of Hb S/Hb C and Hb S/Hb A mixtures with identical concentrations and proportions of Hb S show that there is no significant difference in the tendency of Hb C and Hb A to copolymerize with Hb S. In 50:50 Hb S/ Hb C mixtures, polymerization is about 15 times more rapid than in 40:60 Hb S/Hb A mixtures at the same total Hb concentration. Measurements on density-fractionated erythrocytes show that SC cells contain a higher total Hb concentration and a more uniform distribution, of reticulocytes compared to normal (AA) or sickle trait (AS) cells. The concentration distribution for C trait (AC) cells is much closer to that of SC cells than to AS or AA cells. It appears, therefore, that the presence of Hb C results in the SC cell beginning its life-with an abnormally high Hb concentration. From these findings we conclude that both the larger proportion of Hb S and the higher intracellular Hb concentration contribute to the pathogenesis of Hb SC disease.Hemoglobin (Hb) SC disease is one of the most prevalent ofthe sickling disorders. Patients with Hb SC disease are double (or compound) heterozygotes. They inherit a s35-globin gene from one parent and a I3C-globin gene from the other. The erythrocytes from these individuals usually contain about 50% sickle cell Hb (Hb S) (a2p26 Glu-Val), 50% Hb C (a2026 Glu-*Lys) and no Hb A (a2 326 Glu). In contrast, the most frequently observed composition in the more common heterozygous state, sickle trait (AS), is 40% Hb S and 60% Hb A. Another important difference is that the mean intracellular Hb concentration (MCHC) is higher for SC erythrocytes compared to either AS cells or cells from patients homozygous for the PS gene (1).SC patients have a life-long hemolytic anemia of moderate severity and develop vaso-occlusive episodes and organ damage similar to that encountered in homozygous (SS) sickle cell anemia (1, 2). Why do SC individuals have significant clinical abnormalities, while sickle trait is essentially benign? It is generally assumed that the clinical manifestations of SC disease reflect more extensive formation of intracellular polymer compared to AS erythrocytes subjected to a comparable degree of deoxygenation (2). This is based on early in vitro measurements ofcell morphology and blood viscosity (3, 4). More recent work, including kinetic (5-7) and NMR studies (8-10), is consistent with these findings.Three factors could contribute to the increased polymerization in SC cells in vitro-the higher proportion of Hb S, the higher total intracellula...

A defect in urine concentrating ability occurs in individuals with sickle cell trait (HbAS). This may result from intracellular polymerization of sickle hemoglobin (HbS) in erythrocytes, leading to microvascular occlusion, in the vasa recta of the renal medulla. To test the hypothesis that the severity ofthe concentrating defect is related to the percentage of sickle hemoglobin present in erythrocytes, urinary concentrating ability was examined after overnight water deprivation, and intranasal desmopressin acetate (dDAVP) in 27 individuals with HbAS. The HbAS individuals were separated into those who had a normal a-globin genotype (aa/aa), and those who were either heterozygous (-a/aa) or homozygous (-a/-a) for gene-deletion a-thalassemia, because a-thalassemia modulates the HbS concentration in HbAS. The urinary concentrating ability was less in the aa/aa genotype than in the -a/aa or -a/-a genotypes (P < 0.05). After dDAVP, the urine osmolality was greater in patients with the -a/-a genotype than with the -a/aa genotype (882±37 vs. 672±38 mOsm/kg H20) (P < 0.05); patients with the -a/aa genotype had greater concentrating ability than individuals with a normal a-globin gene arrangement. There was an inverse linear correlation between urinary osmolality after dDAVP and the percentage HbS in all patients studied (r = -0.654; P < 0.05). A linear correlation also existed for urine concentrating ability and the calculated polymerization tendencies for an oxygen saturation of 0.4 and 0 (r = -0.62 and 0.69, respectively). We conclude that the severity of hyposthenuria in HbAS is heterogeneous. It is determined by the amount of HbS polymer, that in turn is dependent upon the percentage HbS, which is itself related to the a-globin genotype. (J. Clin.

We examined the extent to which the intracellular polymerization of sickle hemoglobin (HbS) can account for the severity of anemia and of vaso-occlusive manifestations in the various sickling syndromes. Polymer formation in sickle cell disease depends principally on the intraerythrocytic hemoglobin composition and concentration. In our studies, the polymer fraction in sickle red cells was determined from reported mean values for hemoglobin composition and mean corpuscular hemoglobin concentration (MCHC) in 12 groups of patients with sickle hemoglobinopathies (homozygotes for HbS, with and without coexistent alpha-thalassemia or various forms of the hereditary persistence of fetal hemoglobin [HPFH], beta+-, beta 0-, and delta beta-thalassemia, and heterozygotes for HbS with HbA). The calculated HbS polymer fractions at full deoxygenation and at physiologic oxygen saturation values were closely correlated with mean blood hemoglobin concentrations. In addition, polymer fraction correlated with the ranking of the sickling syndromes by vaso-occlusive severity. We find that polymer fraction accounts for about 80% of the variability in hemolytic and clinical severity. The method of analysis presented here provides a quantitative and systematic means of assessing the role of polymer formation in the pathophysiologic manifestations of the sickling syndromes. Our results support the hypothesis that the intracellular polymerization of HbS is the primary determinant of the severity of both anemia and clinical symptomatology in the sickle hemoglobinopathies.

Intracellular hemoglobin S (HbS) polymerization is most likely to be the primary determinant of the clinical and biologic manifestations of sickle cell disease (SCD). Fetal hemoglobin (HbF) does not enter the HbS polymer and its intracellular expression in sickle erythrocytes inhibits polymerization. HbF levels, high at birth but decreasing thereafter, protect the newborn from the clinical manifestations of this hemoglobinopathy. We have measured the sequential changes in HbF, F reticulocytes, and F cells in the first 2 years of life in 25 children with SCD and compared the results with those obtained in 30 normal children (AA). We have also calculated HbF per F cell (F/F cell), the preferential survival of F cells versus non-F cells, as measured by the ratio F cells versus F reticulocytes (FC/FR) and polymer tendency at 40% and 70% oxygen saturation. HbF levels decreased from about 80.4% +/- 4.0% at birth to 9.2% +/- 2.9% at 24 months. During this time, we observed a regular decrease of the F reticulocytes and the F cells. The kinetics of the decline of F/F cell was comparable with the decline of HbF, rapid from birth (mean, 27.0 +/- 3.6 pg) to 12 months of age (mean, 8.5 +/- 1.5 pg) and then slower from 12 to 24 months of age (mean, 6.2 +/- 1.0 pg) in the SCD children. In the AA children, the decrease in HbF, due to changes in both numbers of F cells and F/F cell, was more precipitous, reaching steady-state levels by 10 months of age. Calculated values for mean polymer tendency in the F-cell population showed that polymerization should begin to occur at 40% oxygen saturation at about 3 months and increase progressively with age, whereas polymerization at 70% oxygen saturation would not occur until about 24 months. These values correspond to HbF levels of 50.8% +/- 10.8% and 9.2% +/- 2.9%, respectively, and F/F cell levels of 15.6 +/- 4.5 pg and 6.2 +/- 1.0 pg, respectively. In the non--F-cell population, polymerization was expected at birth at both oxygen saturation values. Three individuals had significantly greater predicted polymerization tendency than the remainder of the group because of early decreases in HbF. These individuals in particular, the remainder of the cohort, as well as other recruited newborns, will be studied prospectively to ascertain the relationship among hematologic parameters, which determine polymerization tendency and the various clinical manifestations of SCD.

Hydroxyurea (HU), an inhibitor of DNA synthesis, has been shown to increase fetal hemoglobin (HbF) levels in patients with sickle cell anemia and in some patients with beta-thalassemia. However, until now there have not been good in vitro model systems that simulate this effect for study of the molecular and cellular mechanism(s) involved in perturbing the normal ontogeny of the globin genes. We analyzed the cellular effects of HU using a two-phase liquid culture procedure (Fibach et al: Blood 73:100, 1989) in which human peripheral blood- derived progenitor cells undergo proliferation and differentiation. HU was found to have multiple effects on these cultured cells: (1) an increase in the proportion of HbF produced; (2) a decrease in cell number due to inhibition of cell proliferation; (3) an increase in hemoglobin content per cell (mean corpuscular hemoglobin [MCH]); and (4) an increase in cell size (mean corpuscular volume). The extent of these effects was related to the HU dose and time of addition. When added to cell cultures from normal individuals, 4 days following their exposure to erythropoietin (EPO), 100 mumol/L HU caused a 1.3- to 3.5- fold increase in the proportion of HbF, from 0.4% to 5.2% (mean 1.6) in untreated to 1.5% to 8.2% (mean 3.1) in HU-treated cultures and a 45% +/- 10% increase in MCH but only a 25% +/- 7% decrease in cell number on day 13. Cultures of cells derived from five patients with sickle cell anemia have shown a twofold to fivefold increase in the percentage of Hb F following addition of HU while four patients with beta- thalassemia showed a 1.3- to 6.2-fold increase. We believe that this primary cell culture procedure should prove useful in studying the cellular and molecular mechanisms of pharmacologic induction of HbF and might provide a valuable predictive assay system for evaluation of the response of individual patients with hemoglobinopathies to HU and similar agents.