The association of sickle cell anemia with heterozygous and homozygous α-thalassemia-2: In vitro HB chain synthesis

The in vitro synthesis of hemoglobin chains was investigated in 34 scikle cell anemia (SS) patients and five patients with Hb S-β^o-thalassemia. Incubations were made for 30 minutes and for 120 minutes. Hematological and family data were also obtained. Although the 30-minute α/non-α total activity ratios were more widely distributed than the ratios at 120 minutes, a distinct classification of SS patients into groups without an α-thalassemia, with a heterozygous α-thal-2, or with a homozygous α-thal-2 could not be made. Family studies indicated that four patients who had 30-minute α/non-α ratios below 0.82 and mean corpuscular volume (MCV) values below 70 fl had a homozygosity for both α-thal-2 and Hb S. They had mild hematological features of SS disease. Many SS patients with 30-minute α/non-α ratios between 0.8 and 1.0 and MCV values above 70 fl had an associated α-thal-2 heterozygosity. Their hematological features were similar to those of SS patients with four active α chain genes. It appears that an α-thal-2 heterozygosity (−α/αα; β^S/β^S) does not alter the hematological expression of SS disease. An α-thal-2 homozygosity −α/−α;β^S/β^S results in a microcytosis similar to that seen in Hb S-β^o-thalassemia patients. The diagnosis of these α chain deficiencies in association with SS disease (by in vitro chain synthesis analyses) leaves several uncertainties.     

Long survival in sickle cell anemia

Ten patients with sickle cell anemia surviving beyond the age of 40 were compared to 11 subjects with sickle cell anemia who died before that age. Hematologic and biochemical data as well as clinical and physical parameters of the two groups were compared. There was no statistically significant difference between the two groups with respect to the degree of anemia, severity of hemolysis, or hemoglobin A2 levels. A statistically significant difference was found between the two groups with respect to fetal hemoglobin, red cell zinc, and secondary sexual characteristics. Although the long survivors had fewer crises per year than the short survivors, the long-term complications such as leg ulcer, congestive heart failure, and aseptic necrosis of the hip were more common in the older patients. Cerebrovascular accidents were the cause of death in 9/11 short survivors and are absent in the long survivors. The alpha/beta chain synthesis ratio was normal in the long-survivor group and alpha gene mapping in five subject in that group revealed the genotype alpha alpha/alpha alpha in four and -alpha/alpha alpha in one. The older patients as a group had higher red cell zinc values. The secondary sexual characteristics were also better developed in the older subjects. The overall significance of zinc status and of a higher HbF on longevity of sickle cell anemia patients remains unknown.     

Effects of hydroxyurea in a population of Brazilian patients with sickle cell anemia

Fetal hemoglobin (HbF) inhibits the polymerization of sickle hemoglobin, modulating the clinical features of sickle cell anemia (SCA). Hydroxyurea (HU) therapy can increase the HbF level, although its production can be influenced by genetic determinants. Twenty-two Brazilian SCA patients were evaluated over 5 years before and after HU use. We analyzed (1) betaS haplotype; (2) patient characteristics; and (3) toxicity. No differences between age, sex, and HU response were observed. We found 40.9% of homozygous for Bantu haplotype, and, in contrasting to other trials, we observed HbF level increase in this group (3.84-9.08 g/dL, P=0.003). Adverse effects were rare. Labyrinthitis was observed in 2 (9.10%) patients after HU use, although this complication had not been described before.     

The relationship between fetal hemoglobin level and glycosylation in sickle cell disease

Glycosylated hemoglobin (glyco Hb) was determined by affinity chromatography and Hb S1 and Hb F by Bio-Rex 70 chromatography in patients with sickle cell anemia, SC disease, S beta+-thalassemia, and S beta 0-thalassemia. SC and S beta-thalassemia patients had normal levels of glyco Hb whereas SS patients had significantly lower levels. Within each group of patients a direct correlation existed between Hb F and glyco Hb or Hb S1 levels. A similar relationship was noticed when glyco Hb and Hb F levels were compared in red cell populations of various densities (ages). Hb F seems to influence glycosylation through its effect on red cell survival.     

Fetal hemoglobin reactivation in baboon and man: A short perspective

Present concepts of the mechanism of reactivation of synthesis of fetal hemoglobin (HbF) in the adult under conditions of erythropoietic stress are briefly reviewed. Since HbF can be considered an effective natural antisickling agent, the reactivation of its synthesis in patients with sickle cell anemia as a desirable therapeutic goal has been extensively explored since the discovery in 1982 that 5-azacytidine increases HbF levels in the baboon. Hydroxyurea (HU) has become the most widely used agent, although its effectiveness in increasing HbF levels and the number of F cells is highly variable. Recent investigations are cited showing that other agents such as butyrate, and the addition of recombinant hemopoietic growth factors, such as erythropoietin and stem cell factor, especially in combination with HU, offer important therapeutic possibilities. Transacting nuclear proteins are briefly discussed as possibly having a future role in the efforts of stimulating γ-chain synthesis. © 1993 Wiley-Liss, Inc.     

Relationship of burst-forming-unit-erythroid progenitors and their DNA-synthesis stage to fetal hemoglobin levels in hydroxyurea-treated patients with sickle cell anemia

DNA-synthesis stage and total number of circulating burst-forming-units-erythroid (BFU-E) have been inversely correlated with hemoglobin F levels in the peripheral blood, as well as in the cells from the BFU-E-derived colonies obtained from homozygous sickle cell anemia (SS) patients during steady state. Similar studies in SS patients treated with cytotoxic agents have not been reported. However, regeneration of the erythroid marrow that follows the cytoreduction phase of chemotherapy has been suggested as one of the mechanisms of stimulation of fetal hemoglobin synthesis. Therefore, a longitudinal study of hemopoiesis in hydroxyurea-treated SS patients was conducted. Thirty-two sets of hemopoietic studies, including total circulating BFU-E and S-phase BFU-E, were obtained from three patients treated with hydroxyurea. A dose-dependent decrease in total BFU-E colonies occurred in peripheral blood of all three patients (r = ?0.58, ?0.85, and ?0.97, respectively, with each P < 0.05). There was a strong positive correlation between hydroxyurea dose and fetal hemoglobin levels in two of the three patients who responded clinically (r = 0.89618 and 0.88632, respectively, with each P < 0.01). When data from all patients were combined (n = 32), there was a strong, inverse, linear relationship between total number of BFU-E and percentage S-phase BFU-E with fetal hemoglobin levels (r = ?0.6649 and ?0.7404, respectively, with each P < 0.0001). A stronger, curvilinear, multiple relationship was detected between total BFU-E and percentage S-phase BFU-E with fetal hemoglobin levels (R = 0.8351 and 0.8602 with each P < 0.0001). © 1994 Wiley-Liss, Inc.     

Sickle cell anemia and trait in southern India: further studies.

Population surveys and family studies among 568 members of nine ethnic groups in southern India identified 15 homozygotes for sickle hemoglobin (HbS)who had mild clinical and hematological manifestations with high levels of fetal hemoglobin (mean=20%, range 8-36%) in a heterogeneous red cell distribution. In one family, the heterozygous mother had a hemoglobin pattern consistent with a form of the heterocellular hereditary persistence of fetal hemoglobin. Sickle cell trait was found in 153(27%) of those studied. Chromatographic quantitation of the hemoglobin fractions in these heterozygotes showed a trimodal distribution of the proportion of HB Sexplicable by a genetic model postulating the presence of genotypes with two (-alpha/-alpha), three (-alpha/alpha alpha) and four (alpha alpha/alpha alpha) active alpha-globin genes. Globin synthesis studies in four heterozygotes believed to have two active alpha-globin genes demonstrated an alpha/non-alpha total activity ratio (0.57) consistent with this model.     

Molecular characteristics of pediatric patients with sickle cell anemia and stroke

Cerebrovascular accidents (CVA) are serious complications of sickle cell anemia (SS) in children. Factors that predispose children to this complication are not well established. In an effort to elucidate the risk factors associated with CVA in SS, we have determined the alpha-globin genotype and the beta(S) haplotype of children with this complication. Among 700 children with SS followed at Children's Hospital of Michigan, 41 (6%) are on chronic transfusions because of stroke due to cerebral infarction. The mean age of patients with CVA at the time of stroke was 5.6 +/- 3.2 years (mean +/- SD). The male/female ratio was 2/3. Only 8 of 41 patients (19.5%) had one alpha-gene deletion, compared to the reported prevalence of 30% in African-Americans. None of the patients had two alpha-gene deletions, and two (5%) had five alpha-genes. These findings are different than those in our adult patients with SS, where the prevalence of -alpha/-alpha and alphaalphaalpha/alphaalpha is 4% and <2%, respectively. Ten different beta(S)-haplotypes were detected in the patients studied. The majority of the patients (31%) were doubly heterozygous for the Ben/CAR haplotypes followed by Ben/Ben, Ben/Sen, and CAR/CAR haplotypes, respectively. The prevalence of these haplotypes, with the exception of the CAR/CAR haplotype, was higher in females than males. All the patients with CAR/CAR haplotype were males, had four alpha-genes, and ranked third in prevalence. Three patients were heterozygous for the Cameron haplotype. The Cameron and atypical haplotypes were more prevalent than reported in patients with SS at large. The data suggest that CVA in children seems to occur more frequently in females and in patients with certain beta(S) haplotype. alpha-Gene deletion seems to offer a protective effect against this complication. Neonates with four or more alpha-genes whose beta(S) haplotype is Ben/CAR, atypical, or CAR/CAR seem to be at a higher risk for CAV than other patients. A prospective study on a larger group of patients with or without CVA may clarify this issue.     

Isobutyramide therapy in patients with sickle cell anemia

We have administered Isobutyramide as a suspension over a period of 3 months, from a starting dose of 50 mg/kg/day up to 150 mg/kg/day, to four adult sickle cell (SS) anemia patients. The maximum dose was maintained for 3 weeks. The blood counts remained stable and the Hb F levels decreased slightly. The ^Gγ levels increased at the end of the trial, suggesting activation of the ^Gγ gene at the highest dose of Isobutyramide. Three patients showed a stable rate of hemolysis, while in one patient, an increase of lactate dehydrogenase occurred. None of the patients experienced pain crisis or organ-specific crisis, but all four complained about mild epigastric burning and a bitter taste. After the first month of treatment one patient complained about intolerable epigastric discomfort which was relieved by Omeprazole. Another patient complained about increasing dyspepsia in the 12th week leading to the termination of the trial. Oral Isobutyramide administration does not qualify as an effective treatment of SS patients. © 1995 Wiley-Liss, Inc.     

Priapism in a non-black with sickle cell anemia associated with α-thalassemia

Sickle cell anemia, the most prevalent type of hemoglobinopathy, appears almost exclusively in blacks. Since the first report of sickle cell anemia in a Caucasian by Cooley and Lee in 1929 [1], additional cases have been reported, mainly from Mediterranean countries, the Persian Gulf, and India [2]. Several cases were also found among Arabs in Israel with a relatively benign clinical course [3, 4]. The present report describes a unique case of sickle cell anemia associated with α-thalassemia in a non-black young adult. The diagnosis was made at the age of 23 years. Severe priapism was almost the first manifestation of the disease and subsided following transfusion of five units of packed red blood cells.     

Erythroid colony studies on sickle cell anemia in hypoproliferative crisis

Bone marrow cells, peripheral blood lymphocytes, and sera from patients with sickle-cell anemia in hypoproliferative crisis were studied in the plasma clot culture system in the presence or absence of erythropoietin (Epo). Bone marrow cells from five patients demonstrated a marked ability to form erythroid colonies in the presence of Epo. These studies also suggested that bone marrow cells from some patients may have an increased sensitivity to Epo. The most outstanding observation in the present study was the marked erythroid colony inhibition by serum taken from one patient during crisis. Serum taken from the same patient two months after hypoproliferative crisis had no suppressive effect on erythroid colony formation. Lymphocytes taken from three patients in crisis had a stimulatory effect on erythroid colony formation when included in culture. The conclusion is that the defect of erythropoiesis in sickle-cell anemia during hypoproliferative crisis is not due to the absence of erythroid precursor cells or to the presence of suppressor lymphocytes, but may in some cases be associated with a circulating inhibitor of erythroid maturation.     

Myocardial infarction in sickle cell anemia

A review of the electrocardiograms (ECG) of 108 patients with sickle cell anemia found only 3 with patterns consistent with myocardial infarction. Two of the 3 patients with ECG infarct patterns had postmortem examination confirmation of the infarction. These two patients had no significant coronary atherosclerosis nor did the other six autopsied patients in the present series. Literature reports of postmortem examinations on patients with sickle cell anemia confirm the scarcity of coronary atherosclerosis and myocardial infarction in these patients. Forty of the 108 ECGs showed signs of left ventricular hypertrophy and 20 others had nondiagnostic ST and T wave abnormalities. Nine showed first degree AV block and four right bundle branch block.     

Provoked priapism in sickle cell anemia

Two patients with sickle cell anemia are reported who developed relative impotence after repeated episodes of priapism. They learned that excessive ethanol ingestion produced an erection and utilized this mechanism to satisfy sexual partners. Hospitalization and transfusion therapy were required for repeated episodes of priapism. Extensive interviews were required in order to learn the sequence of events. This synergistic relationship between ethanol and priapism should be sought in patients with repeated hospitalizations for this complication of sickle cell anemia because it is preventable with appropriate counseling.     

Serum ionized magnesium levels and ionized calcium-to-magnesium ratios in adult patients with sickle cell anemia

Low levels of total magnesium in sickle cell erythrocytes have been linked to increased sickling due to cell dehydration. We tested the null hypothesis that adult sickle cell anemia (SCA) patients have the same serum level of ionized Mg (Mg(2+)) and Ca(2+)/Mg(2+) ratio as healthy African Americans (AA) and healthy Caucasians (CAUC). We measured serum Mg(2+) and ionized calcium (Ca(2+)) with ion-selective electrodes and calculated the serum Ca(2+)/Mg(2+) ratios in patients with SCA and control groups (AA and CAUC). Seventy-four SCA patients and 61 controls were compared. SCA patients had significantly (P < 0.001) lower levels of serum Mg(2+) (0.52 +/- 0.05) compared to healthy AA (0.57 +/- 0.04) and CAUC (0.62 +/- 0.03). Eighty-six percent of the adult SCA patients had serum Mg(2+) levels below the mean for the AA group, and 96% of SCA patients were above the AA group's mean serum Ca(2+)/Mg(2+). Of the SCA patients studied, 25.6% (95% CI, 16.2-37.2%) had serum Mg(2+) levels below the racially adjusted lower limit of normal and 50% (95% CI, 38.1-61.9%) were above the upper limit of serum Ca(2+)/Mg(2+) for AA controls. By measuring serum Mg(2+) and Ca(2+), we were able to define a subset of SCA patients with hypomagnesemia and elevated Ca(2+)/Mg(2+) ratios, who may benefit from magnesium supplementation.     

Quantitative analysis of erythrocytes containing fetal hemoglobin (F cells) in children with sickle cell disease

Variation in the level of fetal hemoglobin (HbF) accounts for much of the clinical heterogeneity observed in patients with sickle cell disease (SCD). The HbF level has emerged as an important prognostic factor in both sickle cell pain and mortality, and a % HbF of 10–20% has been suggested as a threshold level for diminished clinical severity. The number of erythrocytes that contain HbF (termed F cells) may also be critically important, as F cells resist intravascular sickling and have preferential in vivo survival. Since F cells can be enumerated with high accuracy using flow cytometry methods, we prospectively studied a cohort of 242 children with SCD. Children with HbS and hereditary persistence of fetal hemoglobin (S/HPFH) had essentially 100% F cells. In contrast, children with homozygous sickle cell anemia (HbSS), HbS/β⁰ thalassemia, or HbS/β⁺ thalassemia had significantly lower mean % F cell values (55.9, 61.6, and 51.3%, respectively; P < 0.001), and children with HbSC had even fewer F cells (27.0%; P < 0.001). There was a highly significant correlation between the % F cells and the log (% HbF), which was observed for the total population of children (r = 0.95, P < 0.001), as well as for each of the individual subgroups of children with HbSS (r = 0.94, P < 0.001), HbSC (r = 0.89, P < 0.001), or HbS/β⁰ thalassemia and HbS/β⁺ thalassemia (r = 0.95, P <0.001). This logarithmic correlation between % F cells and % HbF has not been previously described and has important implications for the pharmacologic manipulation of HbF in patients with SCD. Am. J. Hematol. 54:40–46, 1997 © 1997 Wiley-Liss, Inc.     

Functional characteristics of red cells of sickle cell anemia patients with and without α-thalassemia-2

Oxygen equilibrium curves, red cell indices, 2,3-DPG levels, and the percentages of ISC and Hb F were determined for red cell fractions isolated by Dextran 40 density gradient centrifugation from blood of a sickle cell anemia (SS) patient, an SS patient with an additional homozygosity for α-thalassemia-2, and a normal control. In the SS patient, the MCHC and P₅₀ values increased and 2,3-DPG levels decreased with an increase in red cell density; the P₅₀ values were directly related to the MCHC values and inversely related to the 2,3-DPG levels. The bottom fraction contained about 80% ISC, and had the highest MCHC and P₅₀ values and the lowest 2,3-DPG level. In the SS α-thalassemia-2 patient, a decrease in the P₅₀ value corresponded with a decrease in 2,3-DPG levels because no change in MCHC value was observed. The bottom fraction contained about 10% ISC and had the lowest P₅₀ value. These data confirm that both MCHC and 2,3-DPG levels influence the oxygen affinity of SS red cells; an increase in MCHC decreases the oxygen affinity to a great extent while a concomitant decrease in 2,3-DPG level partially abolishes this effect.     

Hemoglobin synthesis in BFU-E colonies derived from transfused blood in a sickle cell anemia patient

In an in vitro progenitor cell culture system, a significant amount of normal hemoglobin (Hb A) synthesis was observed in a transfused sickle cell anemia (SS) patient, indicating the contribution of stem cells obtained from the normal donor's blood cells. As this study was done long after the last blood transfusion (greater than 100 days), the survival of the donor's stem cells would indicate their repopulating the recipient's bone marrow. On extending the study to a few other transfused SS patients who had received multiple blood transfusions at frequent intervals, no such synthesis of Hb A was observed. A loss of repopulating ability of the donor's stem cells with frequent transfusions at short intervals is suggested as a possible explanation, with a gradual development of immune response against these cells as a second possibility. The differences in the survival of the donor's stem cells would thus appear to have some reflection on the frequency of blood transfusions required by an individual patient.     

Circulating cytokines response and the level of erythropoiesis in sickle cell anemia

A hemoglobin F (HbF) level between eight and nine percent divides sickle cell anemia (SS) patients into two populations, according to the kinetics of circulating burst forming units-erythroid (BFU-E), long term culture-initialing cells (LTC-IC), and cytokine plasma concentrations. The SS patients with HbF levels lower than 8–9% are more anemic (LFSS patients) than those with HbF levels higher than 8–9% who have less severe anemia (HFSS patients). We report here that the level of erythropoiesis [evaluated by the levels of soluble transferin receptors (sTfR)] is not identical in these two patient populations, supporting the idea that a different set of regulatory mechanisms might be required to maintain the two levels of increased hematopoiesis. The plasma sTfR concentration was increased in all SS samples compared with controls (P < 0.002) and sTfR levels were negatively correlated with peripheral HbF%. (r = −0.574, P < 0.002). Furthermore, sTfR levels were higher in LFSS than in HFSS patients. Erythropoietin (Epo) levels were increased in the plasma of LFSS individuals (range = 34–215 ml U/ml), while the values in HFSS patients were in the normal range (3–20 ml U/ml). Furthermore, we identify here stem cell factor (SCF) and transforming growth factor-β (TGF-β) as regulatory factors specifically affected by the presence of SS genotype and its level of severity. The plasma concentrations of SCF and TGF-β were increased compared with normal controls and high levels of SCF (up to 7,000 pg/ml) were detected in LFSS patients. The latter also showed increased proportion of SCF⁺ CD³⁴ enriched circulating cells (49%). Low SCF in HFSS patients is associated with elevated TGF-β, suggesting a regulatory role of the latter on either SCF release or c-kit expression in progenitor cells. Occasional elevation of granulocyte macrophage-colony stimulating factor (G-CSF), interleukin (IL)-7, and macrophage inflammatory protein (MIP)-1α in plasma of SS patients is not specific because no relation to HbF could be demonstrated. All plasma tested for leukemia inhibitory factor (LIF) were negative. Data presented here, complementing previously published information, supports a model in which HFSS patients achieve a balance between inhibitory (TGF-β) and stimulatory (SCF, IL-3) factors, resulting in moderate erythropoietic response. In contrast, in LFSS patients, low levels of TGF-β and the increased release of GM-CSF and SCF maintain the intense erythropoiesis in response to higher erythropoietic stress, in these more severe patients. Am. J. Hematol. 60:105–115, 1999. © 1999 Wiley-Liss, Inc.     

Erythrophagocytosis in vivo in sickle cell anemia

Recent observations that the sickle RBC are excessively susceptible to phagocytosis by macrophages in vitro prompted me to look for evidence of in vivo erythrophagocytosis (Ep) in patients with sickle cell anemia (SS). Freshly prepared smears of unmanipulated blood of 27 patients with SS in steady state were examined for Ep by a 500-cell differential white blood cell (WBC) count performed in duplicate. Ten of 27 (37%) SS patients showed Ep (1-6/1,000 WBC or 1-10/100 monocytes). By contrast, no Ep was found in similarly prepared blood smears of 25 normal adult controls and nine splenectomized subjects. The mean hemotocrit value of the Ep(+) SS patients was significantly lower than that of the Ep(-) patients (21.0 +/- 1.7% vs 24.0 +/- 2.7% p less than 0.01). Considering the rarity of spontaneous Ep in unmanipulated blood from normal subjects and the relative insensitivity of the method used, the finding of Ep in over one third of SS patients indicates a significant membrane injury of the sickle RBC and serves to validate the in vitro observations. The possible role of the "senescence" mechanism in the induction of Ep is discussed.     

The effect of Hb F and alpha-thalassemia on the red cell indices in sickle cell anemia

This study examines the effect of different levels of fetal hemoglobin (Hb F) and the presence or absence of genes for alpha-thalassemia on the red cell indices and degree of anemia among 102 patients with homozygous sickle cell disease (S/S) between the ages of 15 and 62 years. Patients were divided into those with an average Hb F of less than 10 gm/L ("low" Hb F group) and those with greater than 10 gm/L ("high" Hb F group). alpha-Thalassemia was assessed by restriction enzyme analysis of DNA by the Southern blotting technique. Homozygosity for the beta(s) gene was confirmed by restriction enzyme analysis of DNA using the enzyme Mst II. There were 51 patients with four alpha-globin genes, 28 of whom had "high" and 23 "low" Hb F levels. Fifty-one patients had alpha-thalassemia, 38 of whom were heterozygous and 13 homozygous for the 3.7 kb alpha-thalassemia deletion. Nine had "high" and 31 had "low" Hb F. Irrespective of alpha-globin genotype, patients in the high Hb F group had a higher mean Hb, Hct, MCV, and MCH than those in the low HB F group. In patients without alpha-thalassemia Hb F was positively correlated with MCV and MCH (p less than 0.001), patients with high Hb F levels having macrocytosis confirmed by microhematocrit studies. Patients with alpha-thalassemia had a lower MCHC than patients with four alpha-globin genes and this was not significantly affected by the level of Hb F. The combination of alpha-thalassemia and high levels of Hb F appears to result in a distinctive S/S phenotype that is similar to the type of S/S disease described in Southern India.