Irreversibly Sickled Cells Research Articles

The Calcium Homeostasis of Human Red Blood Cells in Health and Disease: Interactions of PIEZO1, the Plasma Membrane Calcium Pump, and Gardos Channels.

Calcium ions mediate the volume homeostasis of human red blood cells (RBCs) in the circulation. The mechanism by which calcium ions affect RBC hydration states always follows the same sequence. Deformation of RBCs traversing capillaries briefly activates mechanosensitive PIEZO1 channels, allowing Ca2+ influx down its steep inward gradient transiently overcoming the calcium pump and elevating [Ca2+]i. Elevated [Ca2+]i activates the Ca2+-sensitive Gardos channels, inducing KCl loss and cell dehydration, a sequence operated with infinite variations in vivo and under experimental conditions. The selected health and disease themes for this review focus on landmark experimental results that led to the development of highly constrained models of the circulatory changes in RBCs homeostasis. Based on model predictions, a new perspective emerged, pointing to PIEZO1 dysfunction as the main trigger in the formation of the profoundly dehydrated irreversible sickle cells, the main pathogenic participants in vaso-occlusion, the root cause of sickle cell disease.

Novel single-cell measurements suggest irreversibly sickled cells are neither dense nor dehydrated

In sickle cell anemia, deoxygenation causes erythrocytes to distort, while reoxygenation permits them to recover a normal biconcave disk shape. Irreversibly sickled cells (ISCs) remain distorted when reoxygenated and have been thought to have among the highest intracellular hemoglobin concentrations of the sickle red cell population and therefore the greatest vulnerability to vasoocclusion. Using a new optical method, which we describe, we have made precise measurements of the intracellular hemoglobin concentration, and intracellular O2 saturation, of ISCs, as well as oxygenated sickle cells with a normal biconcave disc shape, and cells with shapes distorted by the sickle fibers they contain. This method also provides good estimates of cell volumes, and hemoglobin per red cell. The concentration distribution of the ISCs is found to be similar to normal, discoid cells. Average ISC volumes exceed their discoid counterparts, with a much broader distribution, arguing against dehydration as their origin. The concentration distribution of the polymer-laden sickled cells is significantly higher in mean value, and their volume distributions indicate some dehydration. Previous assumptions about ISCs may have thus been colored by the presence of sickle cells that did contain polymer, and that true ISCs may be much more benign than once thought, which underscores the importance of accurate measurement on individual cells. This method could be used to follow changes in individual cell properties under various specific perturbations, and where characterization by flow cytometry is infeasible.

Pluronic F-68 Reduces the Endothelial Adherence and Improves the Rheology of Liganded Sickle Erythrocytes

A perfluorochemical blood substitute emulsified with Pluronic F-68 has been shown to improve the filterability of deoxygenated sickle red cells. We questioned whether some of the effect was independent of oxygen loading and studied the influence of Fluosol DA (Green Cross, Osaka, Japan) and Pluronic on the rheology and adhesion of sickle red cells saturated with oxygen and carbon monoxide. A 5-vol/vol% concentration of Fluosol or equivalent concentration of Pluronic was equally effective at improving the filtration of washed sickle cells through 5-micron-diameter pores at wall shear stresses approximating 1,000 dyne/cm2. The same concentration of Pluronic reduced the extensional static rigidity of irreversibly sickled cells (ISC) by 25% and also abolished the adherence of gravity-sedimented sickle cells to endothelial monolayers in the presence of saline or plasma. The inhibition of adherence was not reversible by washing and was accomplished with equal ease by isolated treatment of sickle cells or endothelium. Pluronic had no effect on the rheology or adhesion of normal adult red cells. Neither Fluosol nor Pluronic changed sickle or normal cell shape, mean cell volume, mean cell density, or cell density distribution. A lubricating effect of Pluronic on cell surfaces could explain all of the rheological observations and offers another direction of inquiry in the search for therapy for sickle cell disease.

New Insights into F-Cells, F-Reticulocytes and the Role of Fetal Hemoglobin in Protecting Sickle Red Cells from Hemolysis upon Mechanical Stress

Sickle cell disease (SCD) is an autosomal hereditary recessive disorder caused by a single point mutation in the first exon of the β-globin gene generating abnormal hemoglobin (HbS). Hypoxia drives HbS polymerization and fiber formation that are responsible for red blood cell (RBC) sickling. In SCD patients expression of fetal hemoglobin (HbF) is higher than in healthy individuals and varies considerably. Although clinical studies have shown that HbF levels were inversely correlated with disease severity, studies addressing the relationship between the expression level of HbF at the cellular level, i.e. in F-cells, and the impact on RBC morphology and survival are scarce.In this study, we investigated the distribution and the expression level of HbF at the cellular level in a large cohort of SCD patients (SS or Sβ°) using high throughput imaging techniques, functional sickling assays and a novel microfluidic device.We determined the percentage of HbF-expressing cells (F+RBCs) in 46 patient blood samples and found that it was highly variable (mean: 44% +/- 24%) and correlated with the percentage of HbF measured by HPLC (R2 = 0.86). To estimate the content of HbF in F+RBCs at the single cell level we developed a method based on imaging flow cytometry and defined three different populations according to the expression level of HbF: low, medium and high F+RBCs (Figure 1). We determined the percentage of each subpopulation in 30 SCD blood samples and found that the low F+RBCs were the most prevalent, with a median of 17.85% of total circulating RBCs. The high F+RBCs were the least abundant, with a median of 0.63%, while medium F+RBCs accounted for 4.03% of RBCs.Next, we investigated the relationship between F+RBCs and irreversibly sickled cells (ISCs) using imaging flow cytometry. The %ISCs was significantly higher in the low than in the medium subpopulation and no ISCs were found in the high F+RBC subpopulation. Applying Percoll density fractionation we found that F+RBCs were predominant in the low dense (LD) fraction and poorly present in the high dense (HD) fraction; this was associated with low and high %ISCs in the LD and HD fractions, respectively.To investigate the potential role of HbF in the survival of circulating sickle RBCs we determined the percentage of F-cells within the reticulocyte and mature RBC populations using flow cytometry. Analyzing 46 blood samples, we found that the percentage of F+ reticulocytes (%F+Retics) was significantly lower than the percentage of F+ mature RBCs (%F+RBCs) within the same blood sample of all patients, indicating selective advantage of the F+Retics during maturation. We hypothesized that HbF might exert a protective effect by preserving the deformability of RBCs, protecting them from mechanical destruction. To investigate our hypothesis, we designed a microfluidic biochip to evaluate the resistance of F+RBCs undergoing repetitive mechanical stress, flowing through 5 x 5 μm channels (Figure 2). We measured the %F+RBCs in cell suspensions before (input) and after (output) flowing in the chip. We observed a significant increase of %F+RBC population in the output suspensions, indicating that F+RBCs withstand mechanical stress better than non-F-cells. Moreover, we found that high but not low F+RBCs were protected against lysis, indicating that a minimal threshold is needed for HbF to exert its protective role. In line with this finding, we measured the HbF/HbS ratio in the plasma (free hemoglobin) and in RBCs of SCD patients and found that it was much lower in the plasma, providing direct evidence that intravascular hemolysis targets RBCs with no or low HbF.Finally, there was no correlation between the percentage of reticulocytes and the %HbF or the %F+RBCs. However, a negative correlation was found between the percentage of reticulocytes and the %F+Retics, indicating that this latter is a more relevant marker of hemolysis and medullar regeneration than %HbF or %F+RBCs.Our study gives new insights into the role of the mechanical dimension as a critical contributing factor to anemia in SCD. It reveals a cellular mechanism through which HbF exerts its protective role in vivo, showing evidence that high HbF levels protect RBCs from increased density and from hemolysis upon mechanical stress. Our findings pave the way to future clinical studies in which the relationship between HbF expression at the early reticulocyte stage and clinical manifestations could be finely addressed. [Display omitted] DisclosuresBrousse:Add Medica: Membership on an entity's Board of Directors or advisory committees.

PIEZO1 and the mechanism of the long circulatory longevity of human red blood cells

Human red blood cells (RBCs) have a circulatory lifespan of about four months. Under constant oxidative and mechanical stress, but devoid of organelles and deprived of biosynthetic capacity for protein renewal, RBCs undergo substantial homeostatic changes, progressive densification followed by late density reversal among others, changes assumed to have been harnessed by evolution to sustain the rheological competence of the RBCs for as long as possible. The unknown mechanisms by which this is achieved are the subject of this investigation. Each RBC traverses capillaries between 1000 and 2000 times per day, roughly one transit per minute. A dedicated Lifespan model of RBC homeostasis was developed as an extension of the RCM introduced in the previous paper to explore the cumulative patterns predicted for repetitive capillary transits over a standardized lifespan period of 120 days, using experimental data to constrain the range of acceptable model outcomes. Capillary transits were simulated by periods of elevated cell/medium volume ratios and by transient deformation-induced permeability changes attributed to PIEZO1 channel mediation as outlined in the previous paper. The first unexpected finding was that quantal density changes generated during single capillary transits cease accumulating after a few days and cannot account for the observed progressive densification of RBCs on their own, thus ruling out the quantal hypothesis. The second unexpected finding was that the documented patterns of RBC densification and late reversal could only be emulated by the implementation of a strict time-course of decay in the activities of the calcium and Na/K pumps, suggestive of a selective mechanism enabling the extended longevity of RBCs. The densification pattern over most of the circulatory lifespan was determined by calcium pump decay whereas late density reversal was shaped by the pattern of Na/K pump decay. A third finding was that both quantal changes and pump-decay regimes were necessary to account for the documented lifespan pattern, neither sufficient on their own. A fourth new finding revealed that RBCs exposed to levels of PIEZO1-medited calcium permeation above certain thresholds in the circulation could develop a pattern of early or late hyperdense collapse followed by delayed density reversal. When tested over much reduced lifespan periods the results reproduced the known circulatory fate of irreversible sickle cells, the cell subpopulation responsible for vaso-occlusion and for most of the clinical manifestations of sickle cell disease. Analysis of the results provided an insightful new understanding of the mechanisms driving the changes in RBC homeostasis during circulatory aging in health and disease.

Variable Deformability of Irreversibly Sickled Erythrocytes

The relationship of altered deformability of irreversibly sickled cells (ISC) to their morphological and physiologic characteristics was evaluated in this study. ISC obtained by differential centrifugation and density gradient sedimentation were separated and enriched into hard and soft populations on the basis of their ability to pass through Nuclepore filters with an average pore size of 3.0 mu. Measurement of deformability by micropipette and Nuclepore elastimetry documented marked differences of erythrocyte rigidity in the two populations. Yet ISC morphology, intracellular viscosity determinants (mean cell hemoglobin concentration, density profile gradient, hemoglobin composition), and surface area geometry (mean cell volume, osmotic fragility) were similar in populations of hard and soft ISC. The similarity in intracellular viscosity and surface area geometry suggests that an intrinsic membrane alteration is responsible for the difference in deformability of filtration separated populations of ISC. Hard ISC may be more important to the pathophysiology of sickle cell disease than soft ISC, though ISC counts on peripheral blood smears would not be of help in distinguishing the two populations.

Insights into determinants of spleen injury in sickle cell anemia

AbstractSpleen dysfunction is central to morbidity and mortality in children with sickle cell anemia (SCA). The initiation and determinants of spleen injury, including acute splenic sequestration (ASS) have not been established. We investigated splenic function longitudinally in a cohort of 57 infants with SCA enrolled at 3 to 6 months of age and followed up to 24 months of age and explored the respective contribution of decreased red blood cell (RBC) deformability and increased RBC adhesion on splenic injury, including ASS. Spleen function was evaluated by sequential 99mTc heated RBC spleen scintigraphy and high-throughput quantification of RBCs with Howell-Jolly bodies (HJBs). At 6 and 18 months of age, spleen filtration function was decreased in 32% and 50% of infants, respectively, whereas the median %HJB-RBCs rose significantly (from 0.3% to 0.74%). An excellent correlation was established between %HJB-RBCs and spleen scintigraphy results. RBC adhesion to laminin and endothelial cells increased with time. Adhesion to endothelial cells negatively correlated with splenic function. Irreversibly sickled cells (ISCs), used as a surrogate marker of impaired deformability, were detected at enrollment and increased significantly at 18 months. %ISCs correlated positively with %HJB-RBCs and negatively with splenic uptake, indicating a relationship between their presence in the circulation and spleen dysfunction. In the subgroup of 8 infants who subsequently experienced ASS, %ISCs at enrollment were significantly higher compared with the asymptomatic group, suggesting a major role of impaired deformability in ASS. Higher levels of %HJB-RBCs were observed after the occurrence of ASS, demonstrating its negative impact on splenic function.

Quantifying Shear-Induced Deformation and Detachment of Individual Adherent Sickle Red Blood Cells

Vaso-occlusive crisis, a common painful complication of sickle cell disease, is a complex process triggered by intercellular adhesive interactions among blood cells and the endothelium in all human organs (e.g., the oxygen-rich lung as well as hypoxic systems such as liver and kidneys). We present a combined experimental-computational study to quantify the adhesive characteristics of sickle mature erythrocytes (SMEs) and irreversibly sickled cells (ISCs) under flow conditions mimicking those in postcapillary venules. We employed an in vitro microfluidic cell adherence assay, which is coated uniformly with fibronectin. We investigated the adhesion dynamics of SMEs and ISCs in pulsatile flow under well-controlled hypoxic conditions, inferring the cell adhesion strength by increasing the flow rate (or wall shear stress (WSS)) until the onset of cell detachment. In parallel, we performed simulations of individual SMEs and ISCs under shear. We introduced two metrics to quantify the adhesion process, the cell aspect ratio (AR) as a function of WSS and its rate of change (the dynamic deformability index). We found that the AR of SMEs decreases significantly with the increase of WSS, consistent between the experiments and simulations. In contrast, the AR of ISCs remains constant in time and independent of the flow rate. The critical WSS value for detaching a single SME in oxygenated state is in the range of 3.9–5.5 Pa depending on the number of adhesion sites; the critical WSS value for ISCs is lower than that of SMEs. Our simulations show that the critical WSS value for SMEs in deoxygenated state is above 6.2 Pa (multiple adhesion sites), which is greater than their oxygenated counterparts. We investigated the effect of cell shear modulus on the detachment process; we found that for the same cell adhesion spring constant, the higher shear modulus leads to an earlier cell detachment from the functionalized surface. These findings may aid in the understanding of individual roles of sickle cell types in sickle cell disease vaso-occlusion.

Simultaneous polymerization and adhesion under hypoxia in sickle cell disease

Polymerization and adhesion, dynamic processes that are hallmarks of sickle cell disease (SCD), have thus far been studied in vitro only separately. Here, we present quantitative results of the simultaneous and synergistic effects of adhesion and polymerization of deoxygenated sickle hemoglobin (HbS) in the human red blood cell (RBC) on the mechanisms underlying vasoocclusive pain crisis. For this purpose, we employ a specially developed hypoxic microfluidic platform, which is capable of inducing sickling and unsickling of RBCs in vitro, to test blood samples from eight patients with SCD. We supplemented these experimental results with detailed molecular-level computational simulations of cytoadherence and biorheology using dissipative particle dynamics. By recourse to image analysis techniques, we characterize sickle RBC maturation stages in the following order of the degree of adhesion susceptibility under hypoxia: sickle reticulocytes in circulation (SRs) → sickle mature erythrocytes (SMEs) → irreversibly sickled cells (ISCs). We show that (i) hypoxia significantly enhances sickle RBC adherence; (ii) HbS polymerization enhances sickle cell adherence in SRs and SMEs, but not in ISCs; (iii) SRs exhibit unique adhesion dynamics where HbS fiber projections growing outward from the cell surface create multiple sites of adhesion; and (iv) polymerization stimulates adhesion and vice versa, thereby establishing the bidirectional coupling between the two processes. These findings offer insights into possible mechanistic pathways leading to vasoocclusion crisis. They also elucidate the processes underlying the onset of occlusion that may involve circulating reticulocytes, which are more abundant in hemolytic anemias due to robust compensatory erythropoiesis.

Complement factor H levels in steady state sickle cell anaemia

Objective : The red cell membrane of sickle cell anaemia is vulnerable to attack from the alternative complement pathway. The activation of the alternative complement pathway is initiated by externalization of phosphatidylserine on red cell membrane. Serum and cell bound regulators normally prevent amplification of the cascade. However, red blood cells in sickle cell anaemia appear to be exposed and the cell lysing membrane attack complex is ubiquitous on irreversible sickle red blood cells. It is possible that there are deficiencies (either functional or quantitative) of complement regulators. In this study the quantitative defects of the most abundant serum phase regulator, complement factor H in sickle cell anaemia was investigated. Methods : We compared the plasma levels of complement factor H (a serum phase regulator of the alternative pathway) in 61 steady state Hb SS with 60 healthy Hb AA using an enzyme linked immunosorbent assay to analyze complement factor H level in the plasma. The full blood count parameters were estimated using flow cytometry. Results : There was no significant difference in the serum complement factor H levels between the steady state Hb SS and healthy Hb AA. Significant inverse relationships existed between complement factor H, total white cell count, granulocyte cell count and platelet count as well as significant direct relationships between complement factor H, haematocrit, and the haemoglobin concentration. Conclusion : Complement factor H in patients with sickle cell anaemia who are in steady state is not significantly lower than in controls. Keywords : Complement factor H, sickle cell anaemia, alternative pathway

Variation of Red Blood Cell Rheology and Adhesiveness during Vaso-Occlusive Crises in Sickle Cell Patients

Introduction: Impaired red blood cell (RBC) rheology, increased RBC adhesiveness to the vascular wall, enhanced inflammation and blunted vascular reactivity are involved in the pathophysiology of sickle cell anemia (SCA). Painful vaso-occlusive crisis (VOC) is the most frequent complication encountered by SCA patients. While several studies compared several biomarkers of severity between patients at steady state and others during VOC, very few works compared the same patients in the two conditions. It is therefore difficult to know what happens during VOC. The present study was devoted to compare several hematological, biochemical, and rheological parameters, as well as RBC adhesiveness at steady state and during VOC. Altogether, 36 SCA patients were studied.Methods: This prospective monocentric study was performed at the University Hospital of Pointe-a-Pitre (Guadeloupe, French West Indies), in accordance with the guidelines set by the declaration of Helsinki and was approved by the Regional Ethics Committee (CPP Sud/Ouest Outre Mer III, Bordeaux, France, registration number: 2012-A00701-42). After admission to the emergency department for a VOC episode, patients were informed about the purpose and procedures of the study and gave their written consent. Blood was sampled at the arrival of the patients at the emergency department before they received any medications. A visit to the Sickle Cell Center was then scheduled at least 3 months after the emergency department admission to collect routine blood samples at steady state. Steady state condition was defined as a period free of blood transfusion in the previous three months and without any acute SCA complications in the previous two months. RBC deformability was determined at 3 and 30 Pa by ektacytometry (LORRCA, Mechatronics), RBC aggregation properties (RBC aggregation and RBC disaggregation threshold) by syllectometry (LORRCA), blood viscosity by cone-plate viscosimetry (Brookfield, DVII+ model with CPE 40 spindle) at 225 s-1. Irreversible sickle cells (ISCs) were measured on an Imagestream ISX MkII flow cytometer (Amnis Corp, EMD Millipore). Lu/BCAM, ICAM-4/Lw and the alpha4-beta1 integrin were measured by flow cytometry at the RBC surface (FACSCanto II, BD Biosciences). RBC adhesion to monolayers of transformed human bone marrow endothelial cells (TrHBMECs) was studied in continuous flow conditions in Vena8 Endothelial+ Biochips (Cellix Ltd). Other hematological and biochemical parameters were measured by standard techniques.Results: Compared to steady state values, white blood cell (9.2 [6.7-10.6] versus 12.3 [10.1-16.2] 109/L, p < 0.001) and C-Reactive Protein (3.7 [3.3-6.0] versus 7.1 [3.3-17.5] mg/L, p < 0.05) levels increased during VOC (table 1). Lactate dehydrogenase level slightly increased during VOC (418 [351-564] versus 437 [370-727] IU/L, p < 0.03) but no change was observed for hemoglobin. RBC deformability slightly decreased during VOC (0.34 [0.26-0.44]) compared to steady state (0.38 [0.31-0.46], p < 0.02). RBC aggregation increased during VOC (55 [46-60 %] compared to steady state (51 [46-54] %, p < 0.05). No difference was detected for blood viscosity, RBC surface proteins, RBC adhesion, and RBC disaggregation threshold between the two conditions. During VOC, the percentage of ISCs was inversely correlated with deformability (p < 0.002 and p < 0.006 at 3 and 30 Pa, respectively), but positively correlated with RBC disaggregation threshold (p < 0.002) and with RBC adhesion to TrHBMECs (p < 0.008) (figure 1). At steady state, it was significantly correlated only with the disaggregation threshold (positive correlation, p < 0.03).Discussion: The most striking observation of this study is the positive correlation between ISCs (rigid cells) and RBC adhesion properties during overt VOC. This is in contrast with the general observation that the most deformable RBCs are those exhibiting the strongest adhesiveness and possibly involved in VOC initiation. Strengthened RBC aggregates may also disturb the blood flow into the microcirculation, hence participating to VOC progression and sustention. Drugs targeting RBC deformability (ISCs) and RBC aggregates might be helpful during established VOC in SCA. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Single Molecule Studies of the Diffusion of Band 3 in Sickle Cell Erythrocytes.

Sickle cell disease (SCD) is caused by an inherited mutation in hemoglobin that leads to sickle hemoglobin (HbS) polymerization and premature HbS denaturation. Previous publications have shown that HbS denaturation is followed by binding of denatured HbS (a.k.a. hemichromes) to band 3, the consequent clustering of band 3 in the plane of the erythrocyte membrane that in turn promotes binding of autologous antibodies to the clustered band 3, and removal of the antibody-coated erythrocytes from circulation. Although each step of the above process has been individually demonstrated, the fraction of band 3 that is altered by association with denatured HbS has never been determined. For this purpose, we evaluated the lateral diffusion of band 3 in normal cells, reversibly sickled cells (RSC), irreversibly sickled cells (ISC), and hemoglobin SC erythrocytes (HbSC) in order to estimate the fraction of band 3 that was diffusing more slowly due to hemichrome-induced clustering. We labeled fewer than ten band 3 molecules per intact erythrocyte with a quantum dot to avoid perturbing membrane structure and we then monitored band 3 lateral diffusion by single particle tracking. We report here that the size of the slowly diffusing population of band 3 increases in the sequence: normal cells<HbSC<RSC<ISC. We also demonstrate that the size of the compartment in which band 3 is free to diffuse decreases roughly in the same order, with band 3 diffusing in two compartments of sizes 35 and 71 nm in normal cells, but only a single compartment in HbSC cells (58 nm), RSC (45 nm) and ISC (36 nm). These data suggest that the mobility of band 3 is increasingly constrained during SCD progression, suggesting a global impact of the mutated hemoglobin on erythrocyte membrane properties.

Modified Actriib-Fc Fusion Protein (ACE-536) Mitigates Sickling and Red Cell Pathology in a Murine Model of Sickle Cell Disease

Sickle cell disease (SCD) is a debilitating hereditary disorder caused by a single point mutation in the β-globin gene resulting in the production of sickle hemoglobin variant (HbS). In the deoxygenated state, HbS is labile and undergoes auto-oxidation and polymerizes to generate rigid and irreversibly sickled erythrocytes. The pathophysiology of SCD include increased red cell hemolysis, reactive oxygen species and phosphatidylserine (PS) exposure on RBC membranes which leads to a very short red cell half-life, increased reticulocytosis and splenomegaly. Sickled RBCs show enhanced adherence to activated endothelium causing chronic inflammation leading to frequent and acute painful vaso-occlusive crises in SCD patients. Hydroxyurea (HU) augments fetal hemoglobin production, decreases irreversible sickle cells and painful events, and is the only approved therapy for SCD patients. However, recent studies have shown dose limiting myelosupression with HU treatment. Approximately one-third of patients do not respond to HU therapy thereby highlighting the need for alternative treatment strategies.ACE-536 is a modified type IIB activin receptor-Fc fusion protein (ACE-536)1 which functions as a ligand trap for certain members of the TGFβ superfamily. In a murine model of β-thalassemia, RAP-536 (murine ortholog of ACE-536) treatment reduced hemichromes on RBC membranes, decreased reactive oxygen species, reduced hemolysis, improved red cell half-life and thus corrected anemia and mitigated disease complications of β-thalassemia syndrome2.In the present study, we evaluated RAP-536 as a monotherapy and combination therapy with HU in the murine model of sickle cell disease (βS/βS)3.SCD mice were dosed with RAP-536 (1 mg/kg, twice weekly, s.c.) or TBS vehicle (VEH) control (N=5/group) for 3 months. A combination treatment with HU (100mg/kg, i.p.) and RAP-536 (10mg/kg, s.c) twice weekly for 2 months was performed and compared with vehicle or HU monotherapy treated SCD mice. Non-symptomatic compound heterozygote (β/βS) littermates were treated similarly (N=5/group) and used as controls to confirm disease in SCD (βS/βS) mice.At study baseline, SCD mice had reduced RBC number (-28%, P<0.01) and hemoglobin (-14.5%, P<0.05) and increased reticulocytes (+50%, P<0.001) compared to compound heterozygote mice. Following one month of treatment, RAP-536 (1mg/kg) significantly reduced spleen weight (-20.5%, P<0.05), decreased serum bilirubin content (-17%, P<0.01) and cell free hemoglobin (-30.7%, P=0.06) compared to vehicle treated mice indicating decreased hemolysis. Most remarkably, blood smears from RAP-536 treated SCD mice displayed a decrease in number of irreversibly sickled erythrocytes (-66.5%, P<0.001) as well as reduced annexin V/PS exposure (-18.75%, N.S), suggesting improved membrane phospholipid asymmetry. RAP-536 treatment showed increased RBC number (+15.2%, P<0.01) and hemoglobin (+9.28%, P<0.05) compared to VEH treatment with concomitant decrease in reticulocytes (-13.5%, P< 0.05), suggestive of an increase in red cell half-life. Furthermore, histopathological analysis of spleen, kidneys and heart revealed a trend toward reduced intravascular congestion in RAP-536 treated SCD mice.Preliminary data from the combination treatment of HU and RAP-536 in SCD mice displayed additive beneficial effects as compared to HU alone. The combination of RAP-536 and HU produced a greater reduction in annexin V/PS exposure on peripheral blood cells than did HU alone compared to vehicle treatment (-35.6%, P<0.001 vs. -22.2%, N.S, respectively). Similarly, HU+RAP-536 showed a greater reduction in spleen size than HU alone (-50.7%, P<0.05 vs. -20.2%, N.S) respectively, compared to vehicle treated SCD mice. Additional analyses are in progress. Taken together, these data demonstrates that RAP-536 reduces the RBC sickling and red blood cell pathology in SCD and also shows its utility as monotherapy and in combination with HU to further mitigate the disease severity.ACE-536 is currently being tested in Phase 2 clinical trials in MDS and β-thalassemia patients, and merits evaluation as a therapy for SCD patients.

Exercise‐induced erythrocyte dysfunction in individuals with sickle cell trail (547.1)

Irreversibly sickled cells (ISC) remain unaggregated in the presence of oxygen and yet fail to function normally. We reported earlier that sickle cell trait (SCT) individuals are potentially at risk with microvascular complications due to physical exercise. We also reported that human RBC membrane is heterogeneous and during hemolysis of RBCs, the RBC membrane is sliced into two populations, dense and light. The objective of this study was to characterize whether physical stress causes changes in specific biochemical and physical properties of SCT erythrocytes under conditions of (a) physical stress or (b) deoxygenation. Fractionations of RBC demonstrated differences in the density or age profiles of RBC between AA, AS and SS thus indicating a higher clearance and shortened survival rate. Our studies indicated that the exposure of SCT subjects to treadmill resulted in an increase not only in the susceptibility of red blood cells to oxidation but also in the density of RBC membrane and loss of deformability and increased membrane rigidity in AS than AA. The increased susceptibility of red blood cells to oxidation as a consequence of exercise in AS was associated with relatively excessive production of H2O2 and lipid peroxidation, and increased ATPase’s activities. The damaging consequences on their performance in these conditions could be due to tissue oxygen delivery limitations.

Deformability analysis of sickle blood using ektacytometry.

Sickle cell disease (SCD) is characterized by decreased erythrocyte deformability, microvessel occlusion and severe painful infarctions of different organs. Ektacytometry of SCD red blood cells (RBC) is made difficult by the presence of rigid, poorly-deformable irreversibly sickled cells (ISC) that do not align with the fluid shear field and distort the elliptical diffraction pattern seen with normal RBC. In operation, the computer software fits an outline to the diffraction pattern, then reports an elongation index (EI) at each shear stress based on the length and width of the fitted ellipse: EI=(length-width)/(length+width). Using a commercial ektacytometer (LORCA, Mechatronics Instruments, The Netherlands) we have approached the problem of ellipse fitting in two ways: (1) altering the height of the diffraction image on a computer monitor using an aperture within the camera lens; (2) altering the light intensity level (gray level) used by the software to fit the image to an elliptical shape. Neither of these methods affected deformability results (elongation index-shear stress relations) for normal RBC but did markedly affect results for SCD erythrocytes: (1) decreasing image height by 15% and 30% increased EI at moderate to high stresses; (2) progressively increasing the light level increased EI over a wide range of stresses. Fitting data obtained at different image heights using the Lineweaver-Burke routine yielded percentage ISC results in good agreement with microscopic cell counting. We suggest that these two relatively simple approaches allow minimizing artifacts due to the presence of rigid discs or ISC and also suggest the need for additional studies to evaluate the physiological relevance of deformability data obtained via these methods.

Analysis of sickle hemoglobin

Background: Sickle Hemoglobin is the most common pathological hemoglobin mutation worldwide which forms sickle shape or elongated forms on deoxygenation. According to malaria hypothesis, there is resistance against malaria by the heterozygous carrier state. There is spread of Hb S allele from the highly malarial regions to most other regions worldwide. The study was conducted as Sickle Hemoglobin has been frequently seen in our institute. Materials and Methods: This study was conducted in the department of pathology, Maharajgunj medical campus, Tribhuwan University Teaching hospital, from January 2011 to January 2013. All the relevant data were obtained from the archives of the department. Hemoglobin electrophoresis was performed by Cellulose Acetate Electrophoresis at alkaline pH Method. Sickling test was done using sodium dithionite. Results: A total of 35 cases were diagnosed as sickle cell disorder. The male: female ratio was 2.5:1with the commonest age group 11-20 years (42.8%). Sixty percent showed hemoglobin between 6-8 gm/ dl. Tharu, Chaudhary and Tharu, Rana (91.3%) were the commonest ethnic group with both sickle cell anemia and trait. Lama, Neupane and Baral suffered only from trait. Mean corpuscular volume ranged from 82 to 94 fL. On peripheral blood smear reversible and irreversible sickle cells were present in 88.56 % of cases and 11.42% were devoid of sickle cells. Sickling test was positive in all cases. On cellulose acetate electrophoresis 22 (62.85%) cases had Hb S, Hb F and Hb A2 or HbS and Hb A2 where as 13(37.1%) showed Hb S, Hb A and Hb A2 variants. Conclusion: The commonest ethnic group of Tharus(91.3%), Chaudhary and Rana are from Terai region of Nepal which is a malarial zone. However 8.4% of cases were seen in other ethnic group who are from non-endemic region. DOI: http://dx.doi.org/10.3126/jpn.v3i6.8989 Journal of Pathology of Nepal (2013) Vol. 3, 437-440

Increased Erythrocyte Rigidity Is Sufficient to Cause Endothelial Dysfunction in Sickle Cell Disease

Abstract 818Endothelial dysfunction is a major component of sickle cell disease (SCD) pathophysiology. Interestingly, previous cardiovascular research has definitively shown that endothelial cells biologically respond to mechanical forces and aberrations in these forces cause endothelial dysfunction via pro-inflammatory pathways that are also involved in SCD. While endothelial dysfunction in SCD has been well characterized biologically, little research has focused on the direct biophysical effects of SCD blood on endothelium. As endothelial cells are in constant contact with flowing “stiffened” sickle erythrocytes, we propose that the direct mechanical interactions between the physically altered sickle erythrocytes and endothelial cells are an additional cause of endothelial dysfunction in SCD (Figure 1A).Endothelial dysfunction in SCD is thought to be caused by the downstream effects of vaso-occlusion and/or hemolysis. Our laboratory has recently developed and published a description of an in vitro microvasculature model comprised of endothelial cells that are cultured throughout the entire 3D inner surface of a microfluidic system designed for investigating cellular interactions in hematologic diseases (Tsai, et al, JCI, 2012), (Figure 1B-D). This microvasculature-on-a-chip recapitulates an ensemble of physiological processes and biophysical properties, including adhesion molecule expression, blood cell-endothelial cell interactions, cell deformability, cell size/shape, microvascular geometry, hemodynamics, and oxygen levels (Myers et al. JoVE, 2012), all of which may contribute to endothelial dysfunction in SCD. We hypothesize that the mechanical interactions between sickle erythrocytes and endothelial cells alone are sufficientto cause endothelial dysfunction in our microvasculature-on-a-chip.To test our hypothesis, we flowed different suspensions of healthy red blood cells (RBCs), and stiffened RBCs, through our microvasculature on a chip cultured with HUVECs. We suspended fresh human RBCs in media at a low hematocrit recapitulating the anemic conditions typically seen in SCD patients as a control. The experimental conditions used the same solution as the control, but also contained glutaraldehyde-stiffened RBCs, which are of the same stiffness as irreversibly sickled cells (ISCs), at approximately the same concentrations as ISCs in SCD patients. The stiffened RBC suspension was washed multiple times to eliminate all traces of glutaraldehyde and to ensure that any endothelial cell dysfunction in our system was due to mechanical effects between the endothelium and RBCs. After 4 hours of perfusion, the number of occlusions in our microsystem was counted and the cells were fixed and stained for Vascular Cell Adhesion Molecule 1 (VCAM-1). VCAM-1 been shown to be a marker of endothelial cell dysfunction and is a biomarker for severe vasculopathy in SCD (Dworkis, Am J Hematol, 2011). Immunofluorescence staining in our microsystem confirmed that VCAM1 is upregulated (Figure 2) in HUVECs when exposed to flowing stiffened RBCs compared to control RBCs. VCAM-1 upregulation appears to be diffuse throughout the length of the device. After experimentation, endothelial cells in our system can be isolated for further RT-PCR or microarray analysis. As such, ongoing work involves investigating and quantifying the expression of other pro-inflammatory molecules to elucidate the underlying mechanisms of this biomechanical process involving RBCs and endothelial cells. Additional experiments complementary experiments using endothelial cells from other anatomic areas, SCD patient samples, and murine SCD models are also underway.Our data indicates that purely physical interactions between endothelial cells and stiffened RBCs are sufficient to cause some degree of endothelial dysfunction, even in the absence of vaso-occlusion, ischemia, or oxidative stress due to hemolysis. As sickle RBCs and ISCs are constantly circulating in the blood of SCD patients, our results have profound implications for SCD pathophysiology and may help explain why SCD patients develop chronic diffuse vasculopathy over time. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Recurrent Acute Splenic Sequestration in Two Children with Coinheritance of Sickle Cell Trait and Hereditary Spherocytosis.

AbstractAbstract 2091 Background:Patients with sickle cell trait (AS) are generally asymptomatic, but they are known to be at risk of developing acute morbidity such as splenic infarction mainly occurring on exposure to high altitude. Much lesser known to most hematologists is that young children with AS who co-inherit hereditary spherocytosis (HS) seem to be at risk of developing acute splenic sequestration crisis (ASSC), which are undistinguishable from those observed in homozygous sickle cell anemia (SCA). Objective:The aim of this abstract is to report 2 patients with sickle cell trait and hereditary spherocytosis, presenting with recurrent episodes of severe hemolytic anemia with features characteristic of ASSC. Patients:Patient 1 is a 4-year-old African American female, whose birth history revealed hemoglobin (Hgb) AS phenotype on newborn screening and hyperbilirubinemia on the 4th day of life requiring phototherapy. At 3 years of age, she presented with severe hemolytic anemia (Hgb 4.6 g/dl, reticulocyte count 9.99%, indirect bilirubin 5 mg/dl, haptoglobin <15 mg/dl) and marked splenomegaly, associated with fever. Blood smear did not show microangiopathic changes or irreversible sickle cells. Hemoglobin electrophoresis confirmed diagnosis of sickle cell trait. Osmotic fragility test revealed increased red cell lysis; band 3 staining on flow cytometry was abnormal. G6PD and pyruvate kinase assays, as well as direct antiglobulin test, were all negative. Subsequently, 4 months apart, she had two similar episodes of severe anemia (Hgb 6.1 and 4.7 g/dl, respectively) and massive splenomegaly, associated with febrile respiratory illnesses. Each time, rapid resolution of splenomegaly and dramatic rise of Hgb following red cell transfusions were observed. On outpatient follow-ups, her baseline Hgb was determined to be 9–10 g/dl. Investigation on both parents disclosed AS phenotype on mother, and mild hemolytic anemia with microspherocytes on blood smear as well as normal Hgb phenotype on father. Due to the recurrent and life-threatening nature of her sequestration crisis, splenectomy was performed recently. Spleen was enlarged and weighed 92 grams; histologic examination showed a markedly congested red pulp and no evidence of recent or remote infarcts, findings felt to be consistent with the diagnosis of HS. Post-splenectomy, Hgb level was 11.8 g/dl.Patient 2 is a 5-year-old African American female with sickle cell trait confirmed by electrophoresis at age 2, when she first presented with fever, splenomegaly and severe hemolytic anemia (Hgb 5.7 g/dl, bilirubin 2.8 mg/dl). Diagnosis of HS was confirmed by abnormal osmotic fragility test. Direct antiglobulin test was negative. Thereafter, she was hospitalized 7 times in the span of 2 years for episodes of severe hemolytic anemia (Hgb ranging 2.6 to 6.3 g/dl and reticulocyte count 10.4 to 29.98%) with marked splenomegaly (as large as 12 cm below costal margin), associated with febrile illnesses. Her baseline Hgb concentration was 8–9 g/dl. She also experienced a dramatic rise in Hgb and rapid resolution of splenomegaly following red cell transfusions. Child underwent splenectomy recently. Her spleen weighed 307 grams; histologic findings were similar as for Patient 1. Conclusion:The above reported cases illustrate that children with combined heterozygosity of AS and HS clinically and hematologically manifest as classical HS (i.e. chronic hemolytic anemia, splenomegaly), but the sickle cell component adds recurrent ASSC to the picture. It has been suggested that the high MCHC characteristic of HS which results in elevated erythrocytic Hgb S concentration might be a predisposing factor for development of ASSC. In addition, we surmise that fever and dehydration during infections worsen the hypoxic and acidotic splenic milieu which predisposes to massive intrasplenic sickling. In a single previously reported experience on two other children with AS and HS whose clinical course was complicated by ASSC, splenectomy was performed. Based on our experience, we suggest that patients with sickle cell trait presenting with hemolytic anemia and splenomegaly should be thoroughly evaluated to exclude HS. Likewise, given the life-threatening nature of the splenic sequestrations observed in our patients, splenectomy (after due immunizations) appears to be the best treatment approach for these children. Disclosures:No relevant conflicts of interest to declare.

Optical measurement of biomechanical properties of individual erythrocytes from a sickle cell patient

Sickle cell disease (SCD) is characterized by the abnormal deformation of red blood cells (RBCs) in the deoxygenated condition, as their elongated shape leads to compromised circulation. The pathophysiology of SCD is influenced by both the biomechanical properties of RBCs and their hemodynamic properties in the microvasculature. A major challenge in the study of SCD involves accurate characterization of the biomechanical properties of individual RBCs with minimum sample perturbation. Here we report the biomechanical properties of individual RBCs from a SCD patient using a non-invasive laser interferometric technique. We optically measure the dynamic membrane fluctuations of RBCs. The measurements are analyzed with a previously validated membrane model to retrieve key mechanical properties of the cells: bending modulus; shear modulus; area expansion modulus; and cytoplasmic viscosity. We find that high cytoplasmic viscosity at ambient oxygen concentration is principally responsible for the significantly decreased dynamic membrane fluctuations in RBCs with SCD, and that the mechanical properties of the membrane cortex of irreversibly sickled cells (ISCs) are different from those of the other types of RBCs in SCD.

Phenotypic and Genetic Discordance in Monozygotic Twins with Sickle Anemia and ð-Thalassemia.

Abstract 5084 IntroductionDiscordant monozygotic twins with sickle cell anemia (SCA) are rare. In the present study we describe current clinical and laboratory data in a set of 49 year old discordant monozygotic twins with coexistent SCA and α-/αα thalassemia. We compare the current findings to their previously described data (Hemoglobin 1991;15:247-56). MethodsEvaluations of painful episode frequency, episode severity, frequency of the acute chest syndrome, standard blood counts and chemistries, hemolysis, pulmonary hypertension, irreversibly sickled cells (ISC) and RBC rheology were obtained. DNA from peripheral blood was genotyped using Illumina 610K SNP (single nucleotide polymorphism) beadchip allowing analysis of SNP genotypes and DNA copy number variations (CNVs). ResultsTwin I is right handed and Twin II is left handed. Frequency of hospitalized painful episodes examined over 34 years were similar (approximately 1 episode per year) and were asynchronous. Prior to approximately 25-27 years of age, the painful episodes in Twin I were moderately severe with decreasing severity as ageing occurred. In contrast, Twin II, who also had moderately severe painful episodes during early years, showed marked worsening of severity as ageing occurred. Episodes of acute chest syndrome were asynchronous with similar frequency (Twin I had 2 episodes in 18 years; Twin II had 2 episodes in 21 years). Systemic blood pressures were similar. Osteonecrosis, leg ulcers, priapism, stroke were absent.Laboratory results (Current)*Twin ITwin IIHb(gm/dl)9.238.76Reticulocytes (%)9.111.4Fetal Hb (%)5.04.5Plasma Hb(uM) (Normal <2uM)3.727.79Total GPA(+)CD71, RBC-derived vesicles x 105/l blood2.986.32Total Bilirubin(mg/dl)5.356.83- Indirect Bilirubin (mg/dl)4.635.7RBC survival(51Cr T1/2 days)(Hemoglobin1991;15:247-56)1312.3Irreversibly sickled cells(%)24.417.5Plasma nitrite (nM)(Normal 114 ± 11)84.52100.04Tricuspid regurgitant velocity(m/sec)2.512.51Rheology- Extent of RBC aggregation (Normal 16.3 ± 0.34)9.8811.58- Strength of RBC aggregation (Normal 78 ± 3.6)49134- T1/2 time for RBC aggregation (Normal 2.8 ± 0.09)4.755.33- RBC deformabilityIdentical maximum deformation indexCT scanGallstones, Aortic aneurysmGallstonesLDH-T1(u/l)254248LDH-1(%)3435Blood group phenotype, Chance of monozygosity(%)99.4005399.005*Levels of Hb, Reticulocytes, Hb F, Total and Indirect bilirubin are the Means of 3 samplesConcordance of SNP genotypes confirmed that the twins are monozygotic. Analysis of the entire genome for CNVs revealed a number of candidate loci that differ. Most differences in CNVs occurred in Twin II. ConclusionsClinical and laboratory findings in these twins include differences and similarities. Clinical differences have been observed in handedness, asynchronism of painful episodes and differences in episode severity with ageing. Similarity was observed in the frequency of the painful episodes. Laboratory differences indicate that the degree of intravascular hemolysis is greater in Twin II than in Twin I, as shown by the differences in plasma Hb and RBC-derived vesicle levels (Brit J Haematol 2008;142:126-35). In contrast, the current pulmonary pressures and the current and previous rheologic studies are similar. Ageing contributes to the extent of intravascular hemolysis, as noted when the current findings are compared to those observed in the earlier study. Levels of ISC,which are related to the life span of sickle RBC (Am J Med 1978;64:253-7, Brit J Haemat 1969;17:527-533), appear to be inversely associated with indicators of intravascular hemolysis, such as plasma Hb and RBC-derived vesicles (Twin I, ISC 24.4%, Pl Hb 3.72, RBC-vesicles 2.98; Twin II, ISC 17.5%, Pl Hb 7.79, RBC-vesicles 6.32). The difference in CNVs between the twins, which is greater in Twin II, may explain the phenotypic discordance (Amer J Human Genetics 2008;82:763-71). The loci showing the differences will require further study. To what extent genetic differences in handedness in the twins are associated with the phenotypic differences have not been examined (Mol Psychiatry 2007;12:1129-39). DisclosuresNo relevant conflicts of interest to declare.