In vitro exposure to hydroxyurea reduces sickle red blood cell deformability

Hydroxyurea is a drug that is used to treat some patients with sickle cell disease. We have measured the deformability of sickle erythrocytes incubated in hydroxyurea in vitro and found that hydroxyurea acts to decrease the deformability of these cells. The deformability of normal erythrocytes was not significantly affected by hydroxyurea except at very high concentrations. Hydroxyurea also did not consistently reduce the deformability of sickle erythrocyte ghosts. We propose that the decreased deformability, observed in vitro, is due to the formation of methemoglobin and other oxidative processes resulting from the reaction of hydroxyurea and oxyhemoglobin. Although the reaction with normal hemoglobin is similar to that of sickle hemoglobin, the sickle erythrocytes are affected more. We propose that the sickle erythrocyte membrane is more susceptible to the reaction products of the reaction of hemoglobin and hydroxyurea. An earlier report has shown that hydroxyurea increases the deformability of erythrocytes in patients on hydroxyurea. Taken together, these data suggest that the improved rheological properties of sickle erythrocytes in vivo are due to the elevated numbers of F cells [cells with fetal hemoglobin]. The presence of the nitrosyl hemoglobin or methemoglobin from the reaction with hydroxyurea may also benefit patients in vivo by reducing sickling.     

Red blood cell aggregation, aggregate strength and oxygen transport potential of blood are abnormal in both homozygous sickle cell anemia and sickle-hemoglobin C disease

Background

Recent evidence suggests that red blood cell aggregation and the ratio of hematocrit to blood viscosity (HVR), an index of the oxygen transport potential of blood, might considerably modulate blood flow dynamics in the microcirculation. It thus seems likely that these factors could play a role in sickle cell disease.

Design and Methods

We compared red blood cell aggregation characteristics, blood viscosity and HVR at different shear rates between sickle cell anemia and sickle cell hemoglobin C disease (SCC) patients, sickle cell trait carriers (AS) and control individuals (AA).

Results

Blood viscosity determined at high shear rate was lower in sickle cell anemia (n=21) than in AA (n=52), AS (n=33) or SCC (n=21), and was markedly increased in both SCC and AS. Despite differences in blood viscosity, both sickle cell anemia and SCC had similar low HVR values compared to both AA and AS. Sickle cell anemia (n=21) and SCC (n=19) subjects had a lower red blood cell aggregation index and longer time for red blood cell aggregates formation than AA (n=16) and AS (n=15), and a 2 to 3 fold greater shear rate required to disperse red blood cell aggregates.

Conclusions

The low HVR levels found in sickle cell anemia and SCC indicates a comparable low oxygen transport potential of blood in both genotypes. Red blood cell aggregation properties are likely to be involved in the pathophysiology of sickle cell disease: the increased shear forces needed to disperse red blood cell aggregates may disturb blood flow, especially at the microcirculatory level, since red blood cell are only able to pass through narrow capillaries as single cells rather than as aggregates.     

Elastic properties of stored red blood cells from sickle trait donor units

BACKGROUND AND OBJECTIVES: Red blood cells (RBCs) from patients with sickle cell disease present reduced deformability. The aim of this study was to analyse the elasticity of stored RBCs from patients with the sickle cell trait (AS). MATERIALS AND METHODS: The cell elasticity was studied, using laser optical tweezers, on storage days 1, 14, 21, 28 and 35. RESULTS: The elasticity of RBC from AS units stored for 1, 14 and 21 days was significantly greater compared with that of control RBC cells stored for the same time-period. More than 30% of the cells from AS units stored for 28 or 35 days were very rigid and escaped from the optical trap. CONCLUSIONS: RBCs became rigid during storage, suggesting that haemoglobin S might compromise the cell elasticity.     

Automated red cell exchange in sickle cell disease

We used multiple optical trapping to study the mechanism of red cell (dis)aggregation. Two sets of optical 'tweezers' were used to bring two red blood cells together to form a two-cell aggregate and then to pull them apart, to study the interaction between the cells.
We found that cross-bridging occurred in normal reversible aggregation as we observed binding and the occurrence of small tethers between opposite cell membranes. Furthermore, the cells could only be parted by sliding them side by side with a maximum velocity in the order of μm/s indicating accumulation of the cross-bridges.     

Effects of Poloxamer 188 on red blood cell membrane properties in sickle cell anaemia

Vaso‐occlusive crisis (VOC) is the main acute complication in sickle cell anaemia (SS) and several clinical trials are investigating different drugs to improve the clinical severity of SS patients. A phase III study is currently exploring the profit of Velopoloxamer in SS during VOCs. We analysed, in‐vitro, the effect of poloxamer (P188) on red blood cell (RBC) properties by investigating haemorheology, mechanical and adhesion functions using ektacytometry, microfluidics and dynamic adhesion approaches, respectively. We show that poloxamer significantly reduces blood viscosity, RBC aggregation and adhesion to endothelial cells, supporting the beneficial use of this molecule in SS therapy.     

Sickle red cell microrheology and sickle blood rheology

The hallmark of the phenotypic expression of sickle cell disease is the remarkable degree of heterogeneity in the clinical manifestations. They vary latitudinally among patients and longitudinally in the same patient. The pathogenesis of sickle cell anemia centers on the sequence of events that occur between polymerization of deoxy hemoglobin S and increased red cell destruction, vasoocclusion, and end organ damage. Cellular dehydration, changes in sickle red blood cell rheology, adhesion of sickle red cells to vascular endothelium, inflammatory response, and tissue injury are some of the factors that contribute to hemolytic anemia, vasoocclusion, and eventual multiorgan damage. The focus of this review is on the rheology of sickle blood and microrheology of sickle RBC. Determinants of sickle RBC rheology and the factors that modulate its severity are discussed.     

GBT021601 improves red blood cell health and the pathophysiology of sickle cell disease in a murine model

The pathophysiologic mechanism of sickle cell disease (SCD) involves polymerization of deoxygenated haemoglobin S (HbS), leading to red blood cell (RBC) sickling, decreased RBC deformability, microvascular obstruction, haemolysis, anaemia and downstream clinical complications. Pharmacological increase in the concentration of oxygenated HbS in RBCs has been shown to be a novel approach to inhibit HbS polymerization and reduce RBC sickling and haemolysis. We report that GBT021601, a small molecule that increases HbS-oxygen affinity, inhibits HbS polymerization and prevents RBC sickling in blood from patients with SCD. Moreover, in a murine model of SCD (SS mice), GBT021601 reduces RBC sickling, improves RBC deformability, prolongs RBC half-life and restores haemoglobin levels to the normal range, while improving oxygen delivery and increasing tolerance to severe hypoxia. Notably, oral dosing of GBT021601 in animals results in higher levels of Hb occupancy than voxelotor and suggests the feasibility of once-daily dosing in humans. In summary, GBT021601 improves RBC health and normalizes haemoglobin in SS mice, suggesting that it may be useful for the treatment of SCD. These data are being used as a foundation for clinical research and development of GBT021601.     

Prevalence and risk factors for red blood cell alloimmunization in 175 children with sickle cell disease in a French university hospital reference centre

Patients with sickle cell disease (SCD) show a high prevalence of red blood cell (RBC) alloimmunization, but few studies have focused on children. We aimed to study the prevalence and risk factors of RBC alloimmunization in SCD children. We retrospectively analysed the medical and transfusion files for 245 SCD children hospitalized in our centre in 2014 and included 175 patients who had received at least one RBC unit in their lifetime. The main clinical and immuno‐haematological characteristics of alloimmunized and non‐alloimmunized patients were compared. The prevalence of alloimmunization was 13·7% [95% confidence interval (CI) (8·6–18·6)], and 7·4% [95% CI (3·5–11·3)] after excluding the probable irregular natural antibodies (anti‐M, anti‐Le^a, anti‐Le^b, anti‐Le^x). Main risk factors for alloimmunization were increased number of RBC units received (median of 65 vs. 10 units per patient; P = 0·01) and the presence of one or more red cell autoantibodies (46·2% vs. 4·7%; P < 0·0001). The alloimmunization rate was higher for episodically transfused than chronically transfused patients (1·43 vs. 0·24/100 units received; P < 0·001). The presence of red cell autoantibodies appears to be a major risk factor for alloimmunization in SCD children and could justify specific transfusion guidelines.     

Malignancy in patients with sickle cell disease

Malignancy in patients with sickle cell disease (SCD) has been previously reported, but the types of cancer and its incidence remain undefined. With the advent of hydroxyurea therapy, there is concern about increasing the cancer risk for patients with SCD. The International Association of Sickle Cell Nurses and Physician Assistants identified 52 cases of cancer (49 patients) among 16,613 patients with SCD followed at 52 institutions. The median age at malignancy diagnosis was 34 years (range, 14 months-62 years). Twenty-one cases (40%) occurred in pediatric patients, primarily leukemia (n = 7) or Wilms' tumor (n = 5), with 15 children surviving. Most adults had solid tumors, especially carcinomas, and only nine were known to be alive. Three patients received hydroxyurea before the diagnosis of malignancy. These data provide essential baseline information for the accurate interpretation of future reports of malignancy in patients with SCD, especially those receiving hydroxyurea therapy.     

Red blood cell alloimmunization is influenced by recipient inflammatory state at time of transfusion in patients with sickle cell disease

Sickle cell disease (SCD) patients are at increased risk of red blood cell (RBC) alloimmunization. Recipient inflammatory state at time of transfusion has been shown to regulate alloimmunization in murine models, but evidence is lacking in SCD patients. We retrospectively studied a cohort of alloimmunized SCD patients to determine the influence of pro‐inflammatory SCD‐related complications at time of transfusion on alloimmunization. For each transfusion, the presence of pro‐inflammatory state, degree of RBC antigen matching, unit age, storage solution and alloantibody detection date were ascertained. Transfusion‐associated pro‐inflammatory events were compared between transfusions resulting and not resulting in new alloantibodies. Univariate analysis and multivariate logistic regression were performed. Fifty‐two patients received 3166 pre‐storage leuco‐reduced transfusions of which 128 resulted in alloantibodies. Transfusions during inflammatory events were associated with increased alloantibody risk on univariate and multivariate analysis; acute chest syndrome and vaso‐occlusive crisis showed strongest associations with alloimmunization. Increased antigen matching demonstrated a protective effect on alloimmunization (univariate and multivariate analysis). Although an association was seen between citrate‐phosphate‐dextrose (adenine) stored units and alloimmunization on univariate analysis, no effect was found on multivariate analysis. Identifying recipient pro‐inflammatory states at time of transfusion that promote alloimmunization can impact RBC unit selection decisions for SCD patients at risk for alloimmunization.     

Follow-up of sickle cell disease patients with priapism treated by hydroxyurea

Hydroxyurea is one of the most successfully used therapies for sickle cell disease. Results of many clinical trials point to hydroxyurea administration for patients with frequent painful crises and acute chest syndrome. Priapism is one of the complications that could be prevented by hydroxyurea, but there are few reports demonstrating the results. Since November 1993, hydroxyurea has been used in our clinic for preventing priapism in patients with stuttering or major attacks who are still capable of achieving intercourse on demand. Five patients were enrolled in the study, and 4 cases benefited by this treatment. After the initial treatment for the acute attack, all five patients developed stuttering priapism. Hydroxyurea was then introduced at the initial dose of 10 mg/kg, and as the hydroxyurea dosage increased, the number or length of priapism episodes decreased. One to two months after the maximal dose (20-35 mg/kg) was introduced, the episodes disappeared. In two patients, we were forced to administer over 30 mg hydroxyurea/kg to abort the episodes, and, in another patient, 25 mg/kg was necessary. All patients present normal sexual activity. Hydroxyurea was discontinued in two patients, but stuttering priapism reappeared. Hydroxyurea was then re-introduced, and priapism disappeared. One patient, using 20 mg hydroxyurea/kg, had a 6-year remission of priapism after hydroxyurea administration; however, he experienced stuttering priapism, 1 month before a major attack, that progressed to impotence. During that month, he did not seek medical attention. In conclusion, the data here presented suggests that hydroxyurea may prevent priapism attacks in sickle cell disease, probably at higher doses than usually prescribed for painful crisis prevention.     

Hydroxyurea use during pregnancy: A case report in sickle cell disease and review of the literature

A patient being treated for sickle cell disease with hydroxyurea (1 g/d) conceived, and drug treatment was discontinued at nine weeks gestational age. The pregnancy and delivery were complicated by vaso-oclusive crises. A healthy male infant was born at 39 weeks with no evidence of congenital malformations. A literature review, including this case, suggests that the risk of hydroxyurea exposure during in pregnancy may have been overestimated. Further studies are required to determine its safety in pregnancy. Am. J. Hematol. 60:148–150, 1999. © 1999 Wiley-Liss, Inc.     

Higher resistance to Plasmodium falciparum infection in patients with homozygous sickle cell disease in western Kenya

Sickle haemoglobin (HbS) is considered to be protective against malaria. Malaria is fatal in homozygous sickle cell (HbSS) disease. In a cross‐sectional survey by alkaline Hb‐electrophoresis of 766 residents of Western Kenya near Lake Victoria, 20 were found to have HbSS disease, 120 sickle cell trait (HbAS) and 626 the normal genotype (HbAA). Blood slides for malarial parasites (MPs) were performed in 728 cases, i.e. 592 HbAAs, 116 HbASs and all 20 HbSSs. Malaria parasites were found in 261 (35.8%) HbAAs, 42 (5.8%) HbASs and 4 (0.5%) HbSSs. Malaria prevalences per genotypic group were 44.1% (261 out of 592) in HbAAs, 36.2% (42 out of 116) in HbASs, and 20% (4 out of 20) in HbSSs. The relative risk of malarial infection was 0.33 in the HbSSs compared to both HbAAs and HbASs. It seems that the protection conferred by HbS against malaria is more marked in HbSS disease than in HbAS and is HbS‐content related, and that the balanced polymorphism in the HbS–malaria relationship is maintained by higher mortality risk of HbAAs due to malaria and high mortality risk of HbSSs caused by complications of HbSS.