Hereditary Spherocytosis Red Blood Cells Research Articles

Kinetics of Eosin-5-maleimide Binding to Red Blood Cells

Abstract Introduction/Objective The binding of eosin-5-maleimide (EMA) to red blood cells in different pH conditions has not been well-investigated. The current standard protocol uses phosphate buffered saline at pH 7.0 to 7.5 for the binding reaction. The reaction typically uses a sixty-minute incubation. However, the effect of pH on EMA binding remains to be determined, nor have the reaction times at different pH conditions. This study utilizes optimal pH conditions of 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 to characterize EMA binding. The two parameters, time, and pH were investigated to extensively describe the binding kinetics of eosin-5-maleimide. Methods/Case Report The working EMA solutions in phosphate buffered saline at different pHs (specifically 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5) were incubated in the dark with 1 µL of washed red blood cells and 25 µL of working EMA solution. The red blood cells and working EMA solution were vigorously mixed before the specified incubation time. The binding was recorded at specified time points, namely 1, 2, 4, 8, 16, 32, and 64 minutes for each pH condition. The binding reaction was terminated by washing three times with 0.1% bovine serum albumin (BSA) in PBS and centrifuged at 400 g for 5 minutes. The red blood cells were then resuspended with 500 µL of PBS and ran in the BD FACSLyric™ flow cytometer. Results (if a Case Study enter NA) The current EMA binding protocol utilizes a. neutral pH of 7.0 to 7.5 recorded at 64 min. Based on our data, better discrimination of hereditary spherocytosis red blood cells from normal red blood cells may be obtained at a more basic pH of 8.5. Thus, a statistical t-test was performed for the comparison between the current setup and the apparent optimal pH conditions at shorter reaction times of 16 min and 32min at pH 8.5. Conclusion The resulting data suggest that enhanced discrimination of HS red blood cells can be achieved in a more basic pH. This was evidently observed at pH 8.5, where the highest percentage decrease is recorded, and the most significant difference compared to the other pH conditions. In conclusion, the study identifies that better discrimination of hereditary spherocytosis red blood cells from normal red blood cells can be determined using pH 8.5 with a shorter incubation time of 32 min. The pH 8.5 at 16 min condition could also be considered if the clinical batch size allows precise incubation of 15 min.

Ektacytometry Analysis of Post-splenectomy Red Blood Cell Properties Identifies Cell Membrane Stability Test as a Novel Biomarker of Membrane Health in Hereditary Spherocytosis.

Hereditary spherocytosis (HS) is the most common form of hereditary chronic hemolytic anemia. It is caused by mutations in red blood cell (RBC) membrane and cytoskeletal proteins, which compromise membrane integrity, leading to vesiculation. Eventually, this leads to entrapment of poorly deformable spherocytes in the spleen. Splenectomy is a procedure often performed in HS. The clinical benefit results from removing the primary site of destruction, thereby improving RBC survival. But whether changes in RBC properties contribute to the clinical benefit of splenectomy is unknown. In this study we used ektacytometry to investigate the longitudinal effects of splenectomy on RBC properties in five well-characterized HS patients at four different time points and in a case-control cohort of 26 HS patients. Osmotic gradient ektacytometry showed that splenectomy resulted in improved intracellular viscosity (hydration state) whereas total surface area and surface-to-volume ratio remained essentially unchanged. The cell membrane stability test (CMST), which assesses the in vitro response to shear stress, showed that after splenectomy, HS RBCs had partly regained the ability to shed membrane, a property of healthy RBCs, which was confirmed in the case-control cohort. In particular the CMST holds promise as a novel biomarker in HS that reflects RBC membrane health and may be used to asses treatment response in HS.

Vesiculation of healthy and defective red blood cells

Vesiculation of mature red blood cells (RBCs) contributes to removal of defective patches of the erythrocyte membrane. In blood disorders, which are related to defects in proteins of the RBC membrane, vesiculation of the plasma membrane is intensified. Several hypotheses have been proposed to explain RBC vesiculation but the exact underlying mechanisms and what determines the sizes of the vesicles are still not completely understood. In this work, we apply a two-component coarse-grained molecular dynamics RBC membrane model to study how RBC vesiculation is controlled by the membrane spontaneous curvature and by lateral compression of the membrane. Our simulation results show that the formation of small homogeneous vesicles with a diameter less than 40 nm can be attributed to a large spontaneous curvature of membrane domains. On the other hand, compression on the membrane can cause the formation of vesicles with heterogeneous composition and with sizes comparable with the size of the cytoskeleton corral. When spontaneous curvature and lateral compression are simultaneously considered, the compression on the membrane tends to facilitate formation of vesicles originating from curved membrane domains. We also simulate vesiculation of RBCs with membrane defects connected to hereditary elliptocytosis (HE) and to hereditary spherocytosis (HS). When the vertical connectivity between the lipid bilayer and the membrane skeleton is elevated, as in normal RBCs, multiple vesicles are shed from the compressed membrane with diameters similar to the cytoskeleton corral size. In HS RBCs, where the connectivity between the lipid bilayer and the cytoskeleton is reduced, larger-size vesicles are released under the same compression ratio as in normal RBCs. Lastly, we find that vesicles released from HE RBCs can contain cytoskeletal filaments due to fragmentation of the membrane skeleton while vesicles released from the HS RBCs are depleted of cytoskeletal filaments.

Modeling of Vesiculation in Healthy and Defective Human Erythrocyte Membrane

We apply a two-component coarse-grained molecular dynamics (CGMD) red blood cell (RBC) membrane model which includes an explicit representation of the spectrin network, the lipid bilayer, and the band-3 proteins, to simulate vesiculation in the RBC membrane. We investigate vesiculation induced by the spontaneous curvature of the membrane domain and vesiculation induced by a stiffened cytoskeleton causing compression on the lipid bilayer. In addition, we model vesiculation in RBCs from patients suffering from the blood disorders of hereditary spherocytosis (HS) and elliptocytosis (HE). Our simulation results show that the spontaneous curvature of a membrane domain induces vesicles with a diameter less than 50 nm. We also found that compression on the membrane can cause the formation of vesicles having heterogeneous composition with a size similar to the size of the cytoskeleton corral. When both effects are taken into consideration, the compression on the membrane can facilitate the formation of vesicles originated from the membrane domain with the same spontaneous curvature. While the size of the vesicles induced by the compression in the normal RBC membrane is similar to the cytoskeleton corral size, the vesicle sizes become more diverse in HS RBCs because the constrain from the cytoskeleton on the lipid bilayer is reduced. When the vertical connectivity between the lipid bilayer and the cytoskeleton is elevated, multiple vesicles, with sizes similar to the cytoskeleton corral dimension, are generated from the compressed membrane. However, membrane with low vertical connectivity tends to produce larger vesicles under the same compression ratio as above. In HE RBCs, the reduced cytoskeleton connectivity facilitates the membrane vesiculation. It is noted that vesicles released from the HE RBCs could contain spectrin filaments while vesicles released from the HS RBCs are depleted of cytoskeleton components.

A new paradigm in the diagnosis of hereditary hemolytic anemia

A new paradigm in the diagnosis of hereditary hemolytic anemia

Role of Lu/BCAM glycoproteins in red cell diseases

Lu/BCAM glycoproteins (gps) are the unique erythroid receptors of laminin α5 chain, a major component of the extracellular matrix. They interact with the membrane skeleton by binding directly to spectrin via the Lu/BCAM RK573-574 motif. Lu/BCAM gps are involved in abnormal sickle red blood cell (RBC) adhesion to components of the vascular wall. This adhesion is activated by the phosphorylation of the Lu/BCAM long isoform Lu in a protein kinase A-dependent manner. A similar high adhesion to laminin was also observed with RBCs from Hereditary Spherocytosis (HS) patients suffering from haemolytic anaemia subsequent to spectrin deficiencies. We investigated the molecular mechanisms responsible for the Lu/BCAM-mediated abnormal RBC adhesion to laminin in sickle cell disease (SCD) and HS. We showed that SCD patients treated with hydroxycarbamide (HC) had a diminished RBC adhesion to laminin that was associated with reduced levels of the PKA upstream effector cAMP and a severe decrease in Lu isoform phosphorylation. On the other hand, we showed that increased Lu/BCAM-mediated HS RBC adhesion to laminin was independent of Lu/BCAM phosphorylation. A cellular model expressing the RK573-574AA Lu/BCAM mutant, which is unable to bind to spectrin, showed increased Lu/BCAM detergent extractability and enhanced cell adhesion to laminin. Similar results were obtained with HS RBCs, strongly suggesting that their increased adhesion could result from alteration of the Lu/BCAM-spectrin interaction following the severe spectrin deficiency.

Role of the interaction between Lu/BCAM and the spectrin‐based membrane skeleton in the increased adhesion of hereditary spherocytosis red cells to laminin

Lu/BCAM, the unique erythroid receptor for laminin 511/521, interacts with the erythrocyte membrane skeleton through spectrin binding. It has been reported that Hereditary Spherocytosis red blood cells (HS RBC) exhibit increased adhesion to laminin. We investigated the role of Lu/BCAM-spectrin interaction in the RBC adhesion properties of 2 splenectomised HS patients characterized by 40% spectrin deficiency. Under physiological flow conditions, HS RBC exhibited an exaggerated adhesion to laminin that was completely abolished by soluble Lu/BCAM. Triton extraction experiments revealed that a greater fraction of Lu/BCAM was unlinked to the membrane skeleton of HS RBC, as compared to normal RBC. Disruption of the spectrin interaction site in Lu/BCAM expressed in the transfected K562 cell line resulted in a weakened interaction to the skeleton and an enhanced interaction to laminin. These results demonstrated that the adhesion of HS RBC to laminin was mediated by Lu/BCAM and that its interaction with the spectrin-based skeleton negatively regulated cell adhesion to laminin. Finally, the results of this study strongly suggest that the reinforced adhesiveness of spectrin-deficient HS RBC to laminin is partly brought about by an impaired interaction between Lu/BCAM and the membrane skeleton.

Using the eosin‐5‐maleimide binding test in the differential diagnosis of hereditary spherocytosis and hereditary pyropoikilocytosis

Flow cytometric analysis of eosin-5-maleimide (EMA)-labeled red blood cells (RBCs) has been used as a screening test for the diagnosis of patients with hereditary spherocytosis (HS). We assessed the fluorescence profiles for patients having HS and hereditary pyropoikilocytosis (HPP) together with their red cell indices. Flow cytometry was used to analyze EMA-labeled RBCs. Membrane protein defects and spectrin variants were identified by SDS-polyacrylamide gel electrophoresis. An overlay of single fluorescence peaks for normal individuals, and those with HS and HPP revealed a graded fluorescence intensity (normal > HS > HPP). The area under each peak defined a specific RBC subpopulation; namely, normal RBCs, spherocytes, and microspherocytes. HS RBCs having a gross reduction in band 3 or spectrin content gave fluorescence readings almost as low as those for HPP. Complex fluorescence profiles were obtained for isolated HS and HPP cases. The mean cell volume is a useful discriminator for HS and HPP. We presented evidence that a mixed RBC population could occur in some HS and HPP patients, either in a transient manner or for a long-term period. A differential diagnostic scheme for detecting HPP and HS by flow cytometry is proposed.

Regulation of band 3 rotational mobility by ankyrin in intact human red cells.

Ankyrin mutations and combined spectrin and ankyrin deficiency are prominent features of red blood cells (RBCs) in patients with hereditary spherocytosis (HS). Band 3 is the most abundant integral protein in the human RBC membrane. Previous studies have shown that the lateral mobility, but not the rotational mobility, of band 3 is increased in RBCs from patients with severe autosomal recessive HS and selective spectrin deficiency. These observations are consistent with the steric hindrance model of lateral mobility restriction. Here we use the fluorescence photobleaching recovery and polarized fluorescence depletion techniques to measure the lateral and rotational mobility of band 3 in intact RBCs from six patients with HS, ankyrin mutations, and combined spectrin and ankyrin deficiency. As predicted by the steric hindrance model, the lateral diffusion rate of band 3 is greater in spectrin- and ankyrin-deficient RBCs than in control cells, and the magnitude of the increase correlates with the degree of spectrin deficiency. Unlike RBCs from patients with HS and selective spectrin deficiency, however, HS RBCs with ankyrin mutations exhibit a marked increase in band 3 rotational diffusion. The magnitude of the increase correlates inversely with the ankyrin/band 3 ratio and with the fraction of band 3 retained in the membrane skeleton following detergent extraction. These data suggest that ankyrin deficiency relaxes rotational constraints on the major (slowly rotating) population of band 3 molecules. Increases in band 3 rotation could be due to release of band 3 from low-affinity binding sites on ankyrin.

Alteration of the Erythrocyte Membrane Skeletal Ultrastructure in Hereditary Spherocytosis, Hereditary Elliptocytosis, and Pyropoikilocytosis

Abstract The membrane skeleton of normal erythrocytes is largely organized into a hexagonal lattice of junctional complexes (JC) crosslinked by spectrin tetramers, and occasional double tetramers and hexamers. To explore possible skeletal alterations in hereditary spherocytosis (HS), elliptocytosis (HE), and pyropoikilocytosis (HPP), we have studied the ultrastructure of the spread membrane skeletons from a subpopulation of HS patients with a partial spectrin deficiency ranging from 43% to 86% of normal levels, and in patients with HPP who, in addition to a mild spectrin deficiency, also carried a mutant spectrin that was dysfunctional, thus reducing the ability of spectrin dimers to assemble into tetramers. Membrane skeletons derived from Triton-treated erythrocyte ghosts were examined by negative staining electron microscopy. HS membrane skeletons contained structural elements, consisting of JC and spectrin filaments similar to the normal skeleton. However, less spectrin filaments interconnected the JC, and the decrease of spectrin filaments attached to JC appeared to correlate with the severity of spectrin deficiency. Only in severe HS associated with severe spectrin deficiency was the loss of spectrin sufficient enough to disrupt the overall skeletal architecture. In contrast, membrane skeletons prepared from red blood cells (RBCs) of subjects with HPP were strikingly different from HS RBCs with a comparable degree of spectrin deficiency. Although HPP RBCs were only mildly deficient in spectrin, their skeletal lattice was grossly disrupted, in contrast to only mild ultrastructural abnormalities of HS membrane skeletons with a nearly identical degree of spectrin deficiency. Skeletons from patients with common mild HE or asymptomatic carriers, carrying the mutant spectrin but having normal spectrin content, exhibited a moderate disruption of the skeletal lattice. We propose that the above differences in skeletal ultrastructure may underlie differences in the biomechanical properties and morphology of HS, HE, and HPP RBCs.

Alteration of the erythrocyte membrane skeletal ultrastructure in hereditary spherocytosis, hereditary elliptocytosis, and pyropoikilocytosis

The membrane skeleton of normal erythrocytes is largely organized into a hexagonal lattice of junctional complexes (JC) crosslinked by spectrin tetramers, and occasional double tetramers and hexamers. To explore possible skeletal alterations in hereditary spherocytosis (HS), elliptocytosis (HE), and pyropoikilocytosis (HPP), we have studied the ultrastructure of the spread membrane skeletons from a subpopulation of HS patients with a partial spectrin deficiency ranging from 43% to 86% of normal levels, and in patients with HPP who, in addition to a mild spectrin deficiency, also carried a mutant spectrin that was dysfunctional, thus reducing the ability of spectrin dimers to assemble into tetramers. Membrane skeletons derived from Triton-treated erythrocyte ghosts were examined by negative staining electron microscopy. HS membrane skeletons contained structural elements, consisting of JC and spectrin filaments similar to the normal skeleton. However, less spectrin filaments interconnected the JC, and the decrease of spectrin filaments attached to JC appeared to correlate with the severity of spectrin deficiency. Only in severe HS associated with severe spectrin deficiency was the loss of spectrin sufficient enough to disrupt the overall skeletal architecture. In contrast, membrane skeletons prepared from red blood cells (RBCs) of subjects with HPP were strikingly different from HS RBCs with a comparable degree of spectrin deficiency. Although HPP RBCs were only mildly deficient in spectrin, their skeletal lattice was grossly disrupted, in contrast to only mild ultrastructural abnormalities of HS membrane skeletons with a nearly identical degree of spectrin deficiency. Skeletons from patients with common mild HE or asymptomatic carriers, carrying the mutant spectrin but having normal spectrin content, exhibited a moderate disruption of the skeletal lattice. We propose that the above differences in skeletal ultrastructure may underlie differences in the biomechanical properties and morphology of HS, HE, and HPP RBCs.

Decreased amount of the Rh antigen D in hereditary spherocytosis (HS)

This study was done to determine whether hereditary spherocytosis (HS) red blood cells (RBC) have decreased amounts of Rh antigens. Initially we studied the RBC of five members of one family, two of whom had HS. Using automated quantitative haemagglutination tests, we demonstrated that HS RBC agglutinated less with Rh antisera of four specificities than did normal RBC, indicating that Rh antigens are decreased on HS RBC. In this family, the strength of other blood group antigens on HS RBC was estimated by manual titres and agglutination scores. No appreciable differences in the agglutination of HS and normal RBC were observed with non-Rh antisera. To assess the strength of the D antigen more accurately, the number of D sites was quantitated on the RBC of 19 individuals with HS and 11 of their healthy relatives. HS RBC had 9209 +/- 4084 (mean +/- SD) D sites, whereas the normal RBC had 15 394 +/- 5763 D sites. These two means were significantly different (P less than 0.01). HS RBC were also compared to normal RBC of unrelated individuals who had the same Rh phenotype. These analyses showed that HS RBC had about half of the normal number of D sites. Our data indicate that HS red cells have decreased amount of the Rh antigen D and probably also of other Rh antigens.

Increased membrane binding of erythrocyte catalase in hereditary spherocytosis and in metabolically stressed normal cells

Normal red blood cell (RBC) membranes were compared with (1) RBC membranes from six patients with hereditary spherocytosis (HS), (2) normal membranes after hemolysis of the RBC in the presence of calcium, or (3) normal membranes after incubation of RBC for 24 hr in phosphate-buffered saline containing calcium without added glucose. When compared with normal controls, polyacrylamide gel electrophoresis with sodium dodecyl sulfate (PAGE SDS) of all three preparations showed an increase in membrane binding of globin and protein band 4.5 (60,000 molecular weight). In an attempt to identify band 4.5, 14 enzymes were assayed in the RBC membranes. Of these, catalase and lactate dehydrogenase were increased in membranes from HS RBC and from normal cells exposed to calcium. Only catalase, however, was present in sufficient quantity and had the correct subunit molecular weight on PAGE SDS and calcium-dependent membrane binding to account for an appreciable portion of 4.5. Caralase was further identified with a component of band 4.5 by double immunodiffusion using a specific anti-catalase antibody.

Increased Membrane Binding of Erythrocyte Catalase in Hereditary Spherocytosis and in Metabolically Stressed Normal Cells

Abstract Normal red blood cell (RBC) membranes were compared with (1) RBC membranes from six patients with hereditary spherocytosis (HS), (2) normal membranes after hemolysis of the RBC in the presence of calcium, or (3) normal membranes after incubation of RBC for 24 hr in phosphate- buffered saline containing calcium without added glucose. When compared with normal controls, polyacrylamide gel electrophoresis with sodium dodecyl sulfate (PAGE SDS) of all three preparations showed an increase in membrane binding of globin and protein band 4.5 (60,000 molecular weight). In an attempt to identify band 4.5, 14 enzymes were assayed in the RBC membranes. Of these, catalase and lactate dehydrogenase were increased in membranes from HS RBC and from normal cells exposed to calcium. Only catalase, however, was present in sufficient quantity and had the correct subunit molecular weight on PAGE SDS and calcium- dependent membrane binding to account for an appreciable portion of 4.5. Caralase was further identified with a component of band 4.5 by double immunodiffusion using a specific anti-catalase antibody.