Effect of alpha thalassaemia on the rheology of homozygous sickle cell disease

A study of rheological determinants (plasma viscosity, whole-blood viscosity, and erythrocyte deformability) was made in 24 matched pairs of patients with homozygous sickle cell disease, with and without homozygous alpha-thalassaemia 2. Patients with coexisting alpha-thalassaemia showed a significant increase in erythrocyte deformability measured as filtration of washed erythrocytes through 5 micron diameter pores and also as viscosity of whole blood at high shear rate (230s-1) and standard haematocrit (0.45). This rheological advantage may explain the beneficial effect of alpha-thalassaemia 2 on haematological parameters and clinical events in homozygous sickle cell disease.     

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.     

Effect of chronic kidney disease on red blood cell rheology

BACKGROUND: Anemia of renal failure is primarily a problem of decreased RBC production due to erythropoietin deficiency. RBC survival is also reduced, perhaps due to decreased RBC deformability. This study measured blood viscosity over a range of shear rates in erythropoietin-treated patients on hemodialysis (HD), and compared the findings to matched patients with chronic renal insufficiency (CRI) and healthy controls. METHODS: Four groups (control, CRI, non-diabetic HD, and diabetic HD) of 9 matched subjects were recruited. Blood viscosity was measured using a cone-plate viscometer over a variety of shear rates (11 to 225 s(-1)). RESULTS: Control subjects had lower viscosity values throughout all shear rates when compared to the 3 renal disease groups (P value=0.039). A trend was observed to higher levels of renal function being associated with decreased blood viscosity in patients with CRI. CONCLUSIONS: Patients with kidney disease have increased blood viscosity at all shear rates. This may be related to changes in RBC shape and decreased deformability in patients with kidney disease, independent of HD- or DM-status. This may have implications for strategies to treat anemia in these patients.     

Rate of deoxygenation and rheologic behavior of blood in sickle cell anemia

To understand the relationship between deoxygenation rate, rheologic behavior, and red blood cell (RBC) morphologic characteristics of blood in sickle (SS) cell anemia, washed oxy SS RBC suspensions (hematocrit, 40%) were subjected to relatively fast and gradual deoxygenation procedures. Relatively fast deoxygenation resulted in 50% decline in percent hemoglobin oxygen saturation (%HbO2) within 1 minute. The SS suspensions following relatively fast deoxygenation showed two distinct phases in viscosity profiles. First, there was a sharp increase in individual viscosities to a peak value at 7 minutes of deoxygenation. Second, prolonged deoxygenation resulted in a 27% to 37% decrease in individual viscosities at 30 minutes as compared with the respective peak values at 7 minutes. Most of the viscosity increase (ie, about fourfold) occurred within the first 3 minutes of relatively fast deoxygenation. Scanning electron microscopy and differential morphologic analysis of deoxy cells showed that at 7 minutes a majority of cells had a granular appearance that was characterized by a bumpy irregular surface and the presence of small spicule-like projections. Prolonged deoxygenation resulted in the appearance of a large percentage of elongated cells that were unlike typical sickle cells. Transmission electron microscopy showed that the elongated shape resulted from the alignment of HbS polymers into long projections. In contrast, gradual deoxygenation over a period of 30 minutes resulted in a progressive increase in viscosity and in the formation of typical sickle shapes and holly leaf cells. The results show that at matching %HbO2, the SS suspensions containing mainly granular shaped cells after 7 minutes of relatively fast deoxygenation are as viscous as the gradually deoxygenated suspensions that contain classic sickle shapes and holly leaf forms, while the suspensions having a large percentage of elongated cells (30 minutes after relatively fast deoxygenation) are the least viscous. The two distinct time-dependent viscosity phases observed after relatively fast deoxygenation probably result from differences in the RBC shape characteristics reflecting physical attributes of the polymer, which could affect cell orientation in the viscometric flow.     

Effects of oxidative stress on red blood cell rheology in sickle cell patients

Sickle cell anaemia (SS) and sickle cell‐haemoglobin C disease (SC) patients exhibit severe red blood cell (RBC) rheological alterations involved in the development of several complications. The contribution of oxidative stress in these haemorheological abnormalities is still unknown. We compared RBC reactive oxygen species (ROS) and glutathione (GSH) content, and the haemorheological profile of SS (n = 11), SC (n = 11) and healthy subjects (n = 12) at baseline and after in‐vitro treatment with t‐butyl hydroperoxide (TBHP). We showed: (i) higher RBC ROS content in SS and SC patients, with the highest level observed in SS patients; (ii) lower RBC GSH content in sickle syndrome patients, especially in SS patients; (iii) TBHP increased RBC ROS production and decreased RBC GSH content in all groups; (iv) TBHP decreased RBC aggregation and increased the strength of RBC aggregates in all groups but the increase in RBC aggregates strength was greater in sickle cell patients; (v) TBHP decreased RBC deformability in the three groups but with a higher magnitude in sickle cell patients. These data suggest that RBCs from sickle cell patients have an exaggerated response to oxidative stress, which is accompanied by a profound abnormal haemorheological profile, with greater alterations in SS than in SC patients.     

Blood transfusion in pregnancies complicated by maternal sickle cell disease. Effects on blood rheology and uteroplacental Doppler velocimetry

Summary. Doppler ultrasound gives a measure of downstream resistance to blood flow. In this study it has been used to assess the change in placental vascular resistance following blood transfusion in pregnancies complicated by sickle cell disease. Eight women with homozygous sickle cell disease were followed prospectively to assess the effect of transfusion on haemoglobin S percentage, blood and plasma viscosity and uteroplacental Doppler velocimetry. Measurements were made immediately pre and post-transfusion and after several weeks when the desired improvement in haemoglobin S had been achieved. Despite significant changes in haemoglobin S levels and consequent rheological change there was found to be no change in measurements of resistance in the uteroplacental circulation as assessed by Doppler ultrasound.     

The effect of deoxygenation on whole-cell conductance of red blood cells from healthy individuals and patients with sickle cell disease

Red blood cells from patients with sickle cell disease (SCD) exhibit increased electrogenic cation permeability, particularly following deoxygenation and hemoglobin (Hb) polymerisation. This cation permeability, termed P(sickle), contributes to cellular dehydration and sickling, and its inhibition remains a major goal for SCD treatment. Nevertheless, its characteristics remain poorly defined, its molecular identity is unknown, and effective inhibitors have not been established. Here, patch-clamp methodology was used to record whole-cell currents in single red blood cells from healthy individuals and patients with SCD. Oxygenated normal red blood cells had a low membrane conductance, unaffected by deoxygenation. Oxygenated HbS cells had significantly increased conductance and, on deoxygenation, showed a further rise in membrane conductance. The deoxygenation-induced pathway was variable in magnitude. It had equal permeability to Na(+) and K(+), but was less permeable to NMDG(+) and Cl(-). Conductance to Ca(2+) was also of a similar magnitude to that of monovalent cations. It was inhibited by DIDS (100 microM), Zn(2+) (100 microM), and by Gd(3+) (IC(50) of approximately 2 microM). It therefore shares some properties with P(sickle). These findings represent the first electrical recordings of single HbS cells and will facilitate progress in understanding altered red blood cell cation transport characteristics of SCD.     

Rapid and reproducible characterization of sickling during automated deoxygenation in sickle cell disease patients

In sickle cell disease (SCD), sickle hemoglobin (HbS) polymerizes upon deoxygenation, resulting in sickling of red blood cells (RBCs). These sickled RBCs have strongly reduced deformability, leading to vaso-occlusive crises and chronic hemolytic anemia. To date, there are no reliable laboratory parameters or assays capable of predicting disease severity or monitoring treatment effects. We here report on the oxygenscan, a newly developed method to measure RBC deformability (expressed as Elongation Index - EI) as a function of pO₂. Upon a standardized, 22 minute, automated cycle of deoxygenation (pO₂ median 16 mmHg ± 0.17) and reoxygenation, a number of clinically relevant parameters are produced in a highly reproducible manner (coefficients of variation <5%). In particular, physiological modulators of oxygen affinity, such as, pH and 2,3-diphosphoglycerate showed a significant correlation (respectively R = ‑0.993 and R = 0.980) with Point of Sickling (PoS_5%), which is defined as the pO₂ where a 5% decrease in EI is observed during deoxygenation. Furthermore, in vitro treatment with antisickling agents, including GBT440, which alter the oxygen affinity of hemoglobin, caused a reproducible left-shift of the PoS, indicating improved deformability at lower oxygen tensions. When RBCs from 21 SCD patients were analyzed, we observed a significantly higher PoS in untreated homozygous SCD patients compared to treated patients and other genotypes. We conclude that the oxygenscan is a state-of-the-art technique that allows for rapid analysis of sickling behavior in SCD patients. The method is promising for personalized treatment, development of new treatment strategies and could have potential in prediction of complications.     

Relationship of clinical severity to packed cell rheology in sickle cell anemia.

There is wide variation in the clinical manifestations of sickle cell disease (SCD) from one affected individual to another. Many investigators have sought to discern parameters that would explain this variability. In the present studies we have attempted to correlate the frequency of painful events and the extent of end organ failure in SCD with rheologic properties of packed suspensions of sickle cells, using a magneto-acoustic ball microrheometer developed in our laboratory. Using this device we have measured the steady-state viscosity, and the viscous and elastic moduli of cell suspensions in 16 individuals with hemoglobin SS disease who were untransfused and in their steady state. The rheologic parameters were then correlated with clinical parameters. The clinical parameters measured were emergency department visits, hospitalizations, hemoglobin, reticulocyte count, age, and end organ failure (nephropathy, avascular necrosis of bone, stroke, retinopathy, resting hypoxemia after acute chest syndrome(s), leg ulcer, and priapism with impotence). The P value for the correlation between the steady state viscosity and end organ failure was .001 with a correlation coefficient (R value) of .73. The P value for the correlation between the viscous modulus of viscosity and end organ failure was .00006 with an R value of .83. The P value for the correlation between the elastic modulus of viscosity and end organ failure was .0006 with an R value of .76. However, there was no significant correlation between any component of packed cell rheology and emergency department visits or hospitalizations for pain.     

Abnormal neutrophil adhesion in sickle cell anaemia and crisis: relationship to blood rheology

Defects in neutrophil adhesion and migration may contribute to the susceptibility to infection seen in sickle cell anaemia (SCA). These dynamic defects may be influenced by abnormalities in blood rheology found in this disorder. A whole blood model was used to study neutrophil adhesion in SCA patients: neutrophil adhesion to protein coated glass was quantitated by measuring the rate of disappearance of neutrophils from heparinized whole blood circulating through a perfusion chamber. Twenty-three adult patients (Hb SS) were studied in asymptomatic steady state, of whom nine were also studied during pain crisis, both before and 4-7 d after conventional therapy. Red cell and granulocyte filterability and whole blood and plasma viscosity were also measured. The half-time for disappearance from the perfusion system (t1/2) of neutrophils from patients in the steady-state was 93.5 +/- 8.4 min, compared to 49.1 +/- 2.8 min in normal age-matched controls (P = 0.001). In crisis t1/2 was further prolonged to 170.0 +/- 16.1 min (P = 0.01 v. steady state). After therapy, t1/2 decreased to 57.0 +/- 4.5 min (P = 0.001 v. pre-therapy state and P = 0.009 v. steady state) and was comparable to Hb AA controls. These findings reveal a neutrophil adhesion defect in SCA which worsens in crisis but is corrected following supportive therapy. Red cell filterability (expressed as average resistance to flow and pore-clogging particles) and white cell filterability (expressed as pore-clogging particles) were also abnormal in SCA and were found to correlate with neutrophil adhesion. Plasma viscosity also correlated with adhesion t1/2. The defect appears to be related to abnormal blood flow properties in SCA but the rheological factors cannot fully explain either the steady-state defect or the marked changes in neutrophil adhesion during crisis.     

Current issues with blood transfusions in sickle cell disease

With increased recognition of the profound morbidity of sickle cell disease and with growing evidence of the efficacy of transfusion therapy in prevention and treatment of sickle cell complications, most patients now receive intermittent transfusion therapy. The purpose of this report is to review blood component therapy and its risks for sickle cell patients. Packed red cells are the preferred blood component. Leukocyte-reduced units should be standard because of their beneficial effects in reducing alloimmunization, transfusion reactions, platelet refractoriness, and infection transmission. The use of washed, frozen, or irradiated units is limited to specific problems. Sickle trait-positive units function normally, but because of difficulties with calculating hemoglobin S percentages and leukocyte filters, they are not routinely used. Transfusion-acquired infections have shown a marked decrease but still present a major risk. Viral hepatitis transmission is currently low, but at least 10% of adult sickle cell patients are hepatitis C positive, and they often have liver damage. Although bacterial infections are rare, they account for 16% of transfusion-related fatalities. Patients who are iron overloaded are particularly vulnerable to Yersina enterocolitica. Red cell alloimmunization is a serious problem that could potentially affect 50% of transfused patients. However, preventive phenotypic matching for common antigens can minimize alloimmunization; limited matching for at least E, C, and K has become the standard of care. Recently, more patients are being identified who have developed red cell autoantibodies, which can mask alloantibodies and occasionally are hemolytic. Careful laboratory evaluation of all cases is essential. Transfusions also may trigger sickle cell events, including pain crises, stroke, and acute pulmonary deterioration. In part, these are induced by blood viscosity and increased blood pressure. Diuretic therapy and close monitoring of transfusion volume and vital signs can minimize these events. In summary, transfusion therapy carries risks, but the routine use of leukocyte-reduced, phenotypically matched units in conjunction with close monitoring of patients can make transfusion therapy safer.     

Effect of blood transfusion on iron status in sickle cell anaemia

The iron status of 31 patients with sickle cell anaemia (Hb SS) and balanced globin chain synthesis was studied. Twelve patients (group I) had never been transfused; 14 had received up to 4 units of blood in the past (group II) and five had been hypertransfused for 6 months to 2 years (group III). The hypertransfused group had significantly higher MCV and MCH than the nontransfused one, and significantly lower total iron binding capacity and higher serum ferritin concentration than either groups I or II. The serum ferritin concentration was lower than normal in eight patients (five in group I and three in group II), and higher than normal in seven patients (four in group III and three in group II). The remaining 16 patients had normal serum ferritin concentrations. Our results indicate that iron overload is uncommon in adults with Hb SS who have not been transfused, and that a proportion of patients have lower than normal serum ferritin concentrations. Transfused patients, even 6 months after the last transfusion, show increased haemoglobin content of red cells, which may have an adverse effect on the frequency and severity of sickle crises.     

Determinants of resting cerebral blood flow in sickle cell disease

Stroke is common in children with sickle cell disease and results from an imbalance in oxygen supply and demand. Cerebral blood flow (CBF) is increased in patients with sickle cell disease to compensate for their anemia, but adequacy of their oxygen delivery has not been systematically demonstrated. This study examined the physiological determinants of CBF in 37 patients with sickle cell disease, 38 ethnicity matched control subjects and 16 patients with anemia of non‐sickle origin. Cerebral blood flow was measured using phase contrast MRI of the carotid and vertebral arteries. CBF increased inversely to oxygen content (r² = 0.69, P < 0.0001). Brain oxygen delivery, the product of CBF and oxygen content, was normal in all groups. Brain composition, specifically the relative amounts of grey and white matter, was the next strongest CBF predictor, presumably by influencing cerebral metabolic rate. Grey matter/white matter ratio and CBF declined monotonically until the age of 25 in all subjects, consistent with known maturational changes in brain composition. Further CBF reductions were observed with age in subjects older than 35 years of age, likely reflecting microvascular aging. On multivariate regression, CBF was independent of disease state, hemoglobin S, hemoglobin F, reticulocyte count and cell free hemoglobin, suggesting that it is regulated similarly in patients and control subjects. In conclusion, sickle cell disease patients had sufficient oxygen delivery at rest, but accomplish this only by marked increases in their resting CBF, potentially limiting their ability to further augment flow in response to stress. Am. J. Hematol. 91:912–917, 2016. © 2016 Wiley Periodicals, Inc.     

The kinetics of sickle blood viscosity changes following rapid deoxygenation and reoxygenation

A technique is described in which alterations in sickle blood viscosity can be measured following rapid oxygenation or deoxygenation. The results for two SS blood samples and one SC sample indicate that no measurable rheological changes occur for 21 to 33 sec after partial deoxygenation. Viscosity changes following reoxygenation were too rapid to be accurately measured with this technique. Possible evidence that oxygen exchange may be impaired in sickle cells is also presented. The results suggest that sickle cell crisis may be initiated when the sickling delay time, following exposure to low oxygen tension, becomes shorter than the time required for most cells to return to the lungs, rather than the time needed to traverse a systemic capillary.     

The lung in sickle cell disease.

Sickle cell disease is the most common inherited disorder in African-Americans. Although the primary defect is hematological, the changes in the erythrocytes lead to a vasculopathy with multiorgan injury. The pulmonary complications, i.e., acute chest syndrome and chronic sickle cell lung disease, are significant causes of morbidity and mortality. The pulmonary manifestations result from a unique constellation of factors which come into play in sickle cell disease. Based on the growing understanding of the molecular and cellular biology of sickle cell disease, new therapies are being developed that are likely to ameliorate the natural history of this disease and its complications.     

Liver involvement in sickle cell disease.

BACKGROUND/AIMS: Liver involvement in sickle cell disease may take place due to the primary disease itself or to secondary conditions such as iron overload, viral hepatitis and cholelithiasis. In the present study we have tried to evaluate the frequency of hepatic dysfunction and etiological factors in 48 patients with sickle cell disease. METHODS: Clinical and laboratory investigation including liver function tests, serological tests for viral hepatitis, and abdominal ultrasonography were performed in all of the patients. Additionally, liver biopsies were taken from 13 patients. RESULTS: Intrasinusoidal sickling and Kupffer cell hyperplasia were consistently seen in all of the biopsy specimens. Hepatomegaly was present in all patients, whereas liver function test abnormalities were seen in 27%. The prevalence of cholelithiasis was found as 35%. Serological tests demonstrated the presence of hepatitis B surface antigen in three, antibody to hepatitis B virus in 19 and antibody to hepatitis C virus in four of the patients. The most significant contributory finding was the presence of hemosiderosis in histological examination of liver specimens. CONCLUSION: Our data suggest that chronic liver injury in patients with sickle cell disease seems to be a multifactorial phenomenon depending mostly on overlapping factors such as iron overload and viral damage rather than primary disease itself.     

Impaired vasodilation by red blood cells in sickle cell disease

Red blood cells (RBCs) have been ascribed a unique role in dilating blood vessels, which requires O2-regulated binding and bioactivation of NO by Hb and transfer of NO equivalents to the RBC membrane. Vasoocclusion in hypoxic tissues is the hallmark of sickle cell anemia. Here we show that sickle cell Hb variant S (HbS) is deficient both in the intramolecular transfer of NO from heme iron (iron nitrosyl, FeNO) to cysteine thiol (S-nitrosothiol, SNO) that subserves bioactivation, and in transfer of the NO moiety from S-nitrosohemoglobin (SNO-HbS) to the RBC membrane. As a result, sickle RBCs are deficient in membrane SNO and impaired in their ability to mediate hypoxic vasodilation. Further, the magnitudes of these impairments correlate with the clinical severity of disease. Thus, our results suggest that abnormal RBC vasoactivity contributes to the vasoocclusive pathophysiology of sickle cell anemia, and that the phenotypic variation in expression of the sickle genotype may be explained, in part, by variable deficiency in RBC processing of NO. More generally, our findings raise the idea that defective NO processing may characterize a new class of hemoglobinopathy.