Rheologic predictors of the severity of the painful sickle cell crisis

Deformable sickle erythrocytes have been reported by Mohandas and Evans to be more adherent to vascular endothelium than rigid irreversibly sickled cells (ISC). To define the clinical implications of this finding we have determined genetic, hematological, clinical, and rheological characteristics of sickle erythrocytes obtained from 65 patients with sickle cell anemia and fetal hemoglobin (Hb F) levels less than 15%. The alpha-globin gene number had a significant effect on the hematological parameters, the percentage of dense cells, ISC number, and HB A2 levels. The presence or absence of alpha thalassemia, however, had no effect on the frequency and severity of the sickle cell painful crisis (r = 0.06, P greater than .05). RBC deformability, determined by an ektacytometer, showed great heterogeneity among patients with three or four alpha-globin genes. Linear regression analyses of the data showed significant positive correlation of the frequency and severity of the painful crisis with RBC deformability (r = 0.49, P less than .001), and negative correlations with the percentage of dense cells (r = -0.37, P = .002), and the percentage of ISC (r = -0.46, P less than .001). We propose that the more deformable the sickle RBC are, the greater their adherence to vascular endothelium, and the more they cause vaso-occlusive crises, RBC deformability and the percentage of dense cells (or ISC) seem to have a predictive value of the frequency and severity of painful crises in sickle cell anemia.     

Sickle cell anemia with few painful crises is characterized by decreased red cell deformability and increased number of dense cells.

The frequency and severity of the painful sickle cell crisis vary greatly among affected patients. Aside from a high level of Hb F(greater than 20%) there is no established parameter which may modulate the clinical severity of the disease. In this paper we describe two groups of adult patients with homozygous SS and present their characteristics. The division into these two groups was on the basis of relatively low RBC deformability (less than or equal to 37% of control) and high RBC deformability (greater than 65% of control) in the steady state. None of the patients had alpha-gene deletion and all had Hb F level less than 6.0%. Each patient was followed for a minimum of 3 years. The number of dense cells was quantitated by centrifugation on discontinuous Stractan gradient. RBC deformability index in isotonic medium (DI 290) was determined by ektacytometry and expressed as % of control. The patients with low RBC deformability had significantly less painful crises and more leg ulcers than those patients with high RBC deformability. The average number of dense cells was 22.2% and 9.8% of total circulating cells in the first and second group respectively. Moreover, the group with high red cell deformability had 33% mortality during the study period whereas no deaths occurred in the group with low RBC deformability. The data indicate that there is a subset of patients with SS who have relatively few painful crises despite low Hb F level. We wish to designate these by the acronym MIDDD syndrome: Mild disease as far as painful crises are concerned, increased number of Dense cells, and Decreased red cell Deformability. In addition these patients have high incidence of leg ulcers, have low incidence of urinary tract infection, and less mortality. Cellular factors seem to contribute to the incidence of painful crises.     

The percentage of dense red cells does not predict incidence of sickle cell painful crisis

To test the hypothesis that the tendency of hemoglobin S (HbS) to polymerize within cells is the major determinant of the incidence of vaso-occlusive episodes, we have examined the effect of the percentage of dense cells (as measured by Percoll-Stractan continuous density gradient centrifugation) on the frequency of painful crises in a group of 36 patients with sickle cell disease. No correlation was found between the percentage of dense cells and admissions for crisis. Among the patients with known alpha-gene status (n = 25), the strong correlation between decreased dense cells and alpha-thalassemia (- alpha/alpha alpha) reported previously was confirmed (P less than .001). In addition, in this small subset, patients with alpha- thalassemia (-alpha/alpha alpha) appeared to have a marginally increased number of admissions for sickle cell crisis (t = 2.1910, P less than .05), which was independent of the percentage of dense cells. We conclude that the percentage of dense sickle cells cannot predict the incidence of painful crisis, suggesting that other factors (microcirculatory regulation or other humoral and cellular factors) are more important in the generation maintenance of painful crises than the necessary, but not sufficient, tendency of HbS-containing red cells to sickle.     

Red blood cell changes during the evolution of the sickle cell painful crisis.

A longitudinal study of the red blood cell (RBC) deformability, percent of dense erythrocytes, and hematologic parameters has been conducted during 117 painful crises affecting 36 patients with sickle cell anemia between January, 1985 and December, 1990. RBC deformability was determined by osmotic gradient ektacytometry and the percentage of dense cells was quantitated by centrifugation on a discontinuous Stractan density gradient. The data indicate that the painful crisis is a process that follows a bimodal form of evolution. The first phase of the painful crisis is characterized by increase in the severity of pain, increase in the number of dense cells, and a decrease in RBC deformability. In some patients the changes in dense cells and RBC deformability are evident 1 to 3 days before the onset of pain. In addition, the hemoglobin level decreases and the reticulocyte count increases during this initial phase. The second phase of the crisis is characterized by reduction in pain intensity, decrease in the number of dense cells, and increase in RBC deformability to values higher than those seen in the steady state. Moreover, the improvement in RBC deformability and the decrease in the number of dense cells at the end of a crisis seem to constitute new risk factors that may incite a recurrence of the crisis within 1 month in about 50% of painful episodes. The pathophysiologic events responsible for this bimodal behavior of RBCs during painful episodes may represent the appearance of factors that induce (1) preferential trapping of deformable cells in the microcirculation during the first phase of the crisis, followed by a decrease of dense cells and the appearance of new deformable RBCs released from the bone marrow during the second phase of the crisis; or (2) variable sickling of all circulating RBCs during the first phase followed by disappearance of dense RBCs and their replenishment by deformable cells during the second phase.     

Red blood cell deformability as a predictor of anemia in severe falciparum malaria.

Decreased erythropoiesis and increased clearance of both parasitized and noninfected erythrocytes both contribute to the pathogenesis of anemia in falciparum malaria. Erythrocytes with reduced deformability are more likely to be cleared from the circulation by the spleen, a process that is augmented in acute malaria. Using a laser diffraction technique, we measured red blood cell (RBC) deformability over a range of shear stresses and related this to the severity of anemia in 36 adults with severe falciparum malaria. The RBC deformability at a high shear stress of 30 Pa, similar to that encountered in the splenic sinusoids, showed a significant positive correlation with the nadir in hemoglobin concentration during hospitalization (r = 0.49, P < 0.002). Exclusion of five patients with microcytic anemia strengthened this relationship (r = 0.64, P < 0.001). Reduction in RBC deformability resulted mainly from changes in unparasitized erythrocytes. Reduced deformability of uninfected erythrocytes at high shear stresses and subsequent splenic removal of these cells may be an important contributor to the anemia of severe malaria.     

Monozygotic twins with sickle cell anemia and discordant clinical courses: clinical and laboratory studies.

We describe a rare set of monozygotic twins with coexistent sickle cell anemia and alpha-/alpha alpha thalassemia who have asynchronous painful crises of different frequency and severity. Studies include measurements of cell deformability and other hemorheologic tests, cell density distribution, the percentage of irreversibly sickled cells, adherence of red cells to endothelial cells, membrane heme and membrane free iron, calcium containing internal vesicles and serum antioxidants. Results of these studies, including estimates of organ damage (bone, spleen, retina), were similar except for an increase in red cell membrane free iron in the patient with more frequent and severe painful crises. The study supports the concept that non-inherited factors are important contributors to the frequency and severity of painful crises in sickle cell anemia.     

In vivo studies of sickle red blood cells

The defining clinical feature of sickle cell anemia is periodic occurrence of painful vasoocclusive crisis. Factors that promote trapping and sickling of red cells in the microcirculation are likely to trigger vasoocclusion. The marked red cell heterogeneity in sickle blood and abnormal adhesion of sickle red cells to vascular endothelium would be major disruptive influences. Using ex vivo and in vivo models, the authors show how to dissect the relative contribution of heterogeneous sickle red cell classes to adhesive and obstructive events. These studies revealed that (1) both rheological abnormalities and adhesion of sickle red cells contribute to their abnormal hemodynamic behavior, (2) venules are the sites of sickle cell adhesion, and (3) sickle red cell deformability plays an important role in adhesive and obstructive events. Preferential adhesion of deformable sickle red cells in postcapillary venules followed by selective trapping of dense sickle red cells could result in vasoocclusion. An updated version of this 2-step model is presented. The multifactorial nature of sickle red cell adhesion needs to be considered in designing antiadhesive therapy in vivo.     

Evolution of laboratory parameters during sickle cell painful crisis: evidence compatible with dense red cell sequestration without thrombosis

We find that during 51 episodes of sickle cell painful crisis indirect bilirubin fell 52% from its steady state value of 2.3 +/- 1.9 mg% to a value of 1.1 +/- 0.37 mg% at the end of crisis (p less than .00000085). The indirect bilirubin decline correlates with a decrease in the dense sickle cells during crisis (r = .31, p less than .0009). During steady state, both indirect bilirubin and lactic acid dehydrogenase correlate significantly with number of dense red cells (r = .62, p less than .000002 and r = .32, p less than .02 respectively). Platelet counts, beta-thromboglobulin, Platelet Factor 4, and Fibrinopeptide A levels all were elevated during steady state and did not change during the evolution of crisis. These data demonstrate that elevated indices usually associated with platelet activation are a feature of the steady state of sickle cell disease but argue against thrombosis as a factor in the progression of a sickle cell painful crisis episode. The parallel decline of both dense cells and bilirubin during painful crises indicates that the disappearance of dense cells during crisis is not caused by hemolysis and supports the hypothesis that dense red cell sequestration, in the absence of evidence of thrombosis, is an intrinsic component of the evolution of sickle cell painful crisis.     

Nocturnal oxygen saturation and painful sickle cell crises in children

The pathogenesis of acute painful crisis in children with sickle cell disease is poorly understood; suggested risk factors include sickle cell type, severity of anemia, fetal hemoglobin concentration, and hypoxemia from upper airway obstruction. In a cohort study of 95 patients the relationship between clinical, laboratory, and sleep study data and frequency of painful crisis was investigated. Both univariate and multiple regression modeling showed that low nocturnal oxygen saturation was highly significantly associated with a higher rate of painful crisis in childhood (P <.0001). Screening and treatment for hypoxemia may reduce the frequency of this and other complications of the disease.     

Hydroxycarbamide-induced changes in E/beta thalassemia red blood cells

In thalassemia, fetal hemoglobin (HbF) augmentation with hydroxycarbamide (also known as hydroxyurea) is not always successful. The expected parallel effects on red cell (RBC) membrane deformability, cell hydration, and membrane phospholipid organization, all important for extending RBC life span and increasing Hb, have been infrequently examined. We analyzed these characteristics in 15 nontransfused E/beta(0) thalassemia patients treated with HU (mean 10.2 months). Membrane deformability and cell hydration mildly improved in association with increased HbF levels approaching statistical significance (r = 0.51, P = 0.06). All measures improved considerably in splenctomized patients. These findings underscore the disappointing results of hydroxyurea treatment in clinical trials and the importance of examining the effect on RBC characteristics for the development and understanding of HbF-enhancing agents.     

Red cell membrane stiffness in iron deficiency

The purpose of this study was to characterize red blood cell (RBC) deformability by iron deficiency. We measured RBC deformability to ektacytometry, a laser diffraction method for determining the elongation of suspended red cells subjected to shear stress. Isotonic deformability of RBC from iron-deficient human subjects was consistently and significantly lower than that of normal controls. In groups of rats with severe and moderate dietary iron deficiency, RBC deformability was also reduced in proportion to the severity of iron deficiency. At any given shear stress value, deformability of resealed RBC ghosts from both iron-deficient humans and rats was lower than that of control ghosts. However, increase of applied shear stress resulted in progressive increase in ghost deformation, indicating that ghost deformability was primarily limited by membrane stiffness rather than by reduced surface area-to-volume ratio. This was consistent with the finding that iron-deficient cells had a normal membrane surface area. In addition, the reduced mean corpuscular hemoglobin concentration (MCHC) and buoyant density of the iron-deficient rat cells indicated that a high hemoglobin concentration was not responsible for impaired whole cell deformability. Biochemical studies of rat RBC showed increased membrane lipid and protein crosslinking and reduced intracellular cation content, findings that are consistent with in vivo peroxidative damage. RBC from iron-deficient rats incubated in vitro with hydrogen peroxide showed increased generation of malonyldialdehyde, an end-product of lipid peroxidation, compared to control RBC. Taken together, these findings suggest that peroxidation could contribute in part to increased membrane stiffness in iron- deficient RBC. This reduced membrane deformability may in turn contribute to impaired red cell survival in iron deficiency.     

Steady-state sVCAM-1 serum levels in adults with sickle cell disease

Cytokines and adhesion molecules play an important role in the pathophysiology of vaso-occlusion in sickle cell disease (SCD), and their in vivo profiles are potential tools for assessing SCD severity. We compared steady-state soluble vascular cell adhesion molecule-1 (sVCAM-1) serum levels to clinical (painful crisis frequency, occurrence of acute chest syndrome, leg ulcers, and cerebrovascular disease) and related hematological parameters of SCD severity (such as HbF%, hemoglobin levels, and leukocyte counts) in 29 HbSS adults. Serum sVCAM-1 levels were not related to clinical severity, but an inverse correlation was demonstrated between sVCAM-1 and hemoglobin levels (r=-0.71, p<0.001) with a positive correlation to serum lactate dehydrogenase levels (r=0.59, p=0.008). Based upon these results, steady-state serum sVCAM-1 levels do not seem to reflect clinical disease severity. However, as VCAM-1 is involved in hematopoiesis, sVCAM-1 levels might reflect bone marrow activity in SCD. This underlies the pleiotropic nature of adhesion molecules in vivo and the need for further research in this area, especially since therapies targeting (cellular) adhesive interactions involving the endothelium are emerging for SCD.     

Fetal hemorheology in normal pregnancy and severe preeclampsia

Hemorheological parameters were determined in 45 pairs of mothers with severe preeclampsia and their newborns in comparison with 45 women with uncomplicated pregnancies and their newborns. In both groups we investigated red cell deformability, the plasma viscosity, the red cell aggregation (during stasis and low flow), the macromolecules fibrinogen and factor VIIIR:Ag (VWF), and the blood count parameters hemoglobin, hematocrit, white cells, platelets, reticulocytes, MCV, MCHC. Cholesterol and triglycerides were correlated to the parameter of red cell deformability measured as red cell elongation. We found a significant lower plasma viscosity, red cell aggregation, fibrinogen, cholesterol, triglycerides and VWF in cord blood with a close association between plasma viscosity and fibrinogen (r = 0.56, p = 0.001). The red cell deformability measured as red cell elongation was statistically higher in the cord blood compared to the mothers and associated with a higher MCV. In contrast the MCHC values remained unchanged. Hematocrit and hemoglobin in the cord blood were higher than in the mothers. The incidence of fetal hyperviscosity-polycytemia syndrome in women with severe preeclampsia was between 4.7% and 4.9%. An elevated red cell aggregation was found in 2.8% (stasis) and 4.8% (low flow state), respectively. We conclude that in fetal blood the higher hematocrit and the presence of larger red cells do not cause impaired fetal hemorheology.     

The clinical significance of serum transferrin receptor levels in sickle cell disease

Summary. Serum transferrin receptor (TfR) levels were measured in 182 children with homozygous sickle cell (SS) disease, 47 with sickle cell–haemoglobin C (SC) disease and 41 normal (AA) controls on their eighth birthday. Highly significant elevations occurred in SS compared to SC disease and in SC disease compared to AA controls. Females had higher levels than males in controls but lower levels than males in SS and SC disease. In SS disease, serum TfR levels tended to rise with age from 2 to 8 years, the change within individuals correlating with a change in reticulocyte count ( r ,=0·38, P =0·017) and fall in fetal haemoglobin levels ( r ,=−0·51, P = 0·004). Serum TfR levels did not change with infection or painful crisis but were markedly elevated in hypersplenism and fell following splenectomy in these subjects. In the aplastic crisis, serum TfR levels tended to rise following clinical presentation and then fall, reflecting the reticulocyte counts. These observations are consistent with serum TfR levels being a useful indicator of the degree of erythropoietic expansion in sickle cell disease.     

The red cell distribution width in sickle cell disease--is it of clinical value?

The red cell distribution width (RDW) has been studied during the clinical steady state in 1121 patients with homozygous sickle cell (SS) disease, 344 with sickle cell-haemoglobin C (SC) disease, 68 with sickle cell-beta+ thalassaemia, 49 with sickle cell beta 0 thalassaemia and in 130 control subjects with a normal (AA) genotype. The mean RDW was moderately increased in S beta + thalassaemia and SC disease and markedly increased in S beta 0 thalassaemia and SS disease. In SS, SC and S beta 0 thalassaemia genotypes, lower RDW values occurred in females and with alpha thalassaemia. The RDW correlated negatively with total haemoglobin, mean cell haemoglobin concentration, mean cell volume, and fetal haemoglobin (HbF) and positively with reticulocyte count in SS disease. A low RDW was associated with higher weight and less frequent dactylitis, painful crisis, acute chest syndrome, acute splenic sequestration, and hospital admissions. A low RDW in SS disease is consistent with a high total haemoglobin, high HbF, low reticulocyte count, alpha thalassaemia, and a more mild clinical course.     

Levels of endothelial, neutrophil and platelet-specific factors in sickle cell anemia patients during hydroxyurea therapy.

It has been shown that the clinical course of sickle cell (SS) patients can be ameliorated by administration of hydroxyurea (HU). Induction of hemoglobin F (HbF) is thought to be the mechanism responsible for clinical improvement in some patients. However, HU has a variable effect on HbF production and there exists no good correlation between the extent of HbF increase and clinical response. On the other hand, the degree of adherence of SS to vascular endothelium and neutrophil counts correlate well with clinical severity. Being a cytotoxic drug, used in myeloproliferative diseases, HU may alter proliferation among various cell lines. Moreover, HU has been reported to reduce red blood cell (RBC) adhesion receptor expression in young SS individuals and induces changes in endothelial cells in vitro. It should be conceived that in addition to its effects on HbF production, HU may change the clinical symptoms of SS patients by affecting the degree of adherence of different blood cells, by influencing the activity of endothelium as well as the activity of white blood cells (WBC) and platelets. To analyze whether several of the determinants of adhesion are modulated by HU treatment we studied the levels of endothelial activity (soluble vascular adhesion molecule-1, (sVCAM-1), interleukin-8 (IL-8), fibronectin, neutrophil activity (sL-selectin, sIL-6 receptor-alpha, myeloperoxidase) and platelet activity (von Willebrand factor) in relation to clinical symptoms, hematological data and HbF levels in 8 SS patients before and during 5 months of HU therapy. Steady state sVCAM-1 levels are increased compared to normal controls and a significant decrease is noted during HU treatment, suggesting a decrease in the interactions between RBC and vascular endothelium. The IL-8 levels are comparable to those in normal controls and remain unaffected by HU therapy. Intercurrent infection and crises reveal striking increases in IL-8 which are accompanied by leukocytosis, but otherwise the IL-8 levels do not correlate with hematological data. HU has no demonstrable effect on fibronectin or soluble neutrophil adhesion molecules, but the levels of myeloperoxidase decrease significantly while WBC counts do not, implying a reduction in neutrophil activity which may help attenuate the propagation phase of a vasoocclusive crisis.     

Accurate and independent measurement of volume and hemoglobin concentration of individual red cells by laser light scattering

Cell volume (MCV) and hemoglobin concentration (MCHC) are the red cell indices used to characterize the blood of patients with anemia. Since the introduction of flow cytometric methods for the measurement of these indices, it has generally been assumed that the values derived by these instruments are accurate. However, it has recently been shown that a number of cellular factors, including alterations in cellular deformability, can lead to inaccurate measurement of cell volume by these automated instruments. Because cell hemoglobin concentration and hematocrit are computed from the measured values of cell volume, accuracy of these indices is also compromised by inaccurate determination of cell volume. A recently developed experimental flow cytometric method based on laser light scattering, which can independently measure volume and hemoglobin concentration, has been used in the present study to measure MCV and MCHC of density- fractionated normal and sickle red cells, hydrated and dehydrated normal red cells, and various pathologic cells. We found that the new method accurately measures both volume and hemoglobin concentrations over a wide range of MCV (30 to 120 fL) and MCHC (27 to 45 g/dL) values. This is in contrast to currently available methods in which hemoglobin concentration values are accurately measured over a more limited range (27 to 35 g/dL). In addition, as the experimental method independently measures volume and hemoglobin concentration of individual red cells, it allowed us to generate histograms of volume and hemoglobin concentration distribution and derive coefficient of variation for volume distribution and standard deviation of hemoglobin concentration distribution. We have been able to document that volume and hemoglobin concentration distributions can vary independently of each other in pathologic red cell samples.     

Adherence of sickle erythrocytes to vascular endothelial cells: requirement for both cell membrane changes and plasma factors

Hebbel and colleagues have proposed that increased adherence of sickle red cells to vascular endothelium may initiate vasoocclusive events in sickle cell disease. We have developed a micropipette technique to obtain direct, quantitative measure of the adherence of individual red cells to vascular endothelial cells. Using this technique, we found that the vast majority of sickle cells suspended in autologous plasma were strongly adherent to endothelial cells, whereas only a small fraction of normal cells were weakly adherent. Influence of plasma factors on adherence was determined by measuring adherence of sickle cells suspended in normal plasma and normal cells suspended in sickle plasma. Although over 90% of sickle cells adhered to endothelial cells in autologous plasma, the percentage of adherent cells decreased dramatically to less than 20% when the same sickle cells were suspended in normal plasma. In contrast, adhesion of normal red cells suspended in sickle plasma was only modestly increased compared to adhesion in autologous normal plasma. Our results provide direct evidence for markedly enhanced adherence of sickle cells to endothelial cells. In addition, they suggest that both cell membrane changes and plasma factors contribute to this interaction. The requirement for sickle plasma further implies that temporal changes in plasma factors may play an important role in determining the onset of vasoocclusive crisis.     

Studies on abnormal hemoglobins. V. The distribution of type S,sickle cell,hemoglobin and type F,alkali resistant,hemoglobin within the red cell population in sickle cell anemia

1. By transfusing sickle cell anemia erythrocytes with a relatively high concentration of F hemoglobin into normal recipients, it was demonstrated that thedisappearance rates of the transfused cells and of their alkali resistant pigmentconsistently showed great discrepancies. These observations suggest an unequaldistribution of the F pigment within the erythrocyte population. A nonuniformdistribution of F hemoglobin could also be detected in vitro by exposing sicklecell anemia bloods to mechanical trauma for a longer period of time. The cellsmost resistant to trauma contained a higher percentage of F hemoglobin thanthe original blood specimen.

2. The red cell population of patients with sickle cell anemia seems to be composed of three main fractions: (1) cells containing S hemoglobin and no or littleF hemoglobin, (2) cells containing both pigments and (3) cells containing Fpigment with no or little S hemoglobin.

3. The erythrocytes carrying mostly S hemoglobin have the shortest life span,whereas the red cells containing mostly F hemoglobin have the longest survivaltime.

4. The significance of these findings in regard to clinical and genetic aspectsof sickle cell anemia is discussed. No direct correlation is demonstrable in anindividual patient between the absolute amounts of either type S or type Fhemoglobin and the severity of the anemia. The latter depends on the variablesize of the portion of red cells containing mostly S hemoglobin, and also on theability of the marrow to replace this particular fraction.

Submitted on August 5, 1952 Accepted on September 10, 1952     

Sickle cell vasoocclusion and rescue in a microfluidic device

The pathophysiology of sickle cell disease is complicated by the multiscale processes that link the molecular genotype to the organismal phenotype: hemoglobin polymerization occurring in milliseconds, microscopic cellular sickling in a few seconds or less [Eaton WA, Hofrichter J (1990) Adv Protein Chem 40:63-279], and macroscopic vessel occlusion over a time scale of minutes, the last of which is necessary for a crisis [Bunn HF (1997) N Engl J Med 337:762-769]. Using a minimal but robust artificial microfluidic environment, we show that it is possible to evoke, control, and inhibit the collective vasoocclusive or jamming event in sickle cell disease. We use a combination of geometric, physical, chemical, and biological means to quantify the phase space for the onset of a jamming event, as well as its dissolution, and find that oxygen-dependent sickle hemoglobin polymerization and melting alone are sufficient to recreate jamming and rescue. We further show that a key source of the heterogeneity in occlusion arises from the slow collective jamming of a confined, flowing suspension of soft cells that change their morphology and rheology relatively quickly. Finally, we quantify and investigate the effects of small-molecule inhibitors of polymerization and therapeutic red blood cell exchange on this dynamical process. Our experimental study integrates the dynamics of collective processes associated with occlusion at the molecular, polymer, cellular, and tissue level; lays the foundation for a quantitative understanding of the rate-limiting processes; and provides a potential tool for optimizing and individualizing treatment, and identifying new therapies.