Hereditary Spherocytosis Red Cells Research Articles

Detection of hereditary pyropoikilocytosis by the eosin-5-maleimide (EMA)-binding test is attributable to a marked reduction in EMA-reactive transmembrane proteins

Hereditary spherocytosis (HS) and hereditary pyropoikilocytosis (HPP, severe form of hereditary elliptocytosis) are unrelated red cell disorders caused by defects in distinct regions of the red cell cytoskeleton. The high predictive value of the eosin-5-maleimide (EMA)-binding test for the diagnosis of HS is because of its interaction with transmembrane proteins band 3, Rh protein, Rh glycoprotein and CD47, which are reduced on HS red cells. Our study was undertaken to determine why EMA-labelled HPP red cells were previously found to give much lower fluorescence readings than HS. Flow cytometry was used to determine the relative amounts of monoclonal antibodies bound to red cells from normal adults, HS and HPP groups. Confocal microscopy was used to visualise the overall staining pattern of the red cells with selected antibodies. In flow cytometry, HPP red cells gave lower antibody binding to the four EMA-reactive membrane proteins than HS red cells and bound less antibody to glycophorins A and C, and CD59. Confocal images of Rh protein and band 3 immunostaining revealed a greater number of HPP red cells having partial or no fluorescence than in HS and normal controls. Lesser amounts of EMA-reactive membrane proteins were detected in HPP than HS red cells, thus confirming their lower fluorescence readings in the EMA-binding test. The concomitant reduction in glycophorins A and C, and CD59 in HPP could have caused cellular contraction, resulting in poikilocytosis.

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.

SI38 Characterisation of Membrane Proteins on Vesicles from Normal Red Cells and on Red Cells of Patients with Membranopathy

Background Red cell vesicles are formed either after prolonged blood storage at 4°C or during incubation in calcium‐loaded medium. Unlike mature red cells, these vesicles are depleted in spectrin but carry band 3, glycophorins, acetylcholinesterase and CD47. Absorption of human anti‐D by vesicles indicated the presence of Rh proteins.Study Design The aims were to identify the membrane components present on red cell vesicles, and to investigate whether red cells after vesiculation in vitro were similar to those from patients with inherited haemolytic anaemias: hereditary spherocytosis (HS) and pyropoikilocytosis (HPP). Vesicles released from fresh normal red cells in Ca2+ loaded medium were purified by filtration through 0.8 μm filters. Specific monoclonal antibodies (Mabs) were used to identify integral membrane components. The analytical techniques used were SDS‐PAGE, Western blotting, confocal microscropy, and rosette formation with magnetic beads (Dynabeads) conjugated with anti‐mouse immunoglobulins. Flow cytometry quantified antibody bound to red cells after vesiculation and from patients with HS and HPP.Results We confirmed depletion of spectrin and the presence of band 3, CD47 and glycophorins in vesicles released from normal red cells. Confocal microscopy demonstrated the coating of Rh(D+) vesicles with human Mab anti‐D. Other membrane molecules detected on the vesicles were Duffy glycoprotein, Kell glycoprotein, glucose transporter, protein 4.2, Rh glycoprotein (RhAG), CD59, and blood group A antigen. Flow cytometry detected reductions in binding of Mabs to band 3, Rh proteins and RhAG on vesiculated red cells as well as on HS red cells. CD47 content was lower on HS than on vesiculated red cells. Greater losses of these membrane components occurred on HPP red cells with a concomitant reduction in CD59, and glycophorins A and C. Confocal microscopy of dual labelled HS and HPP red cells showed that the loss of membrane proteins was not a uniform process but ranged from strong, moderate to dim (or no) fluorescence.Conclusion Shedding a large number of membrane proteins in red cell vesicles is probably a physiological process of cell ageing. The greater loss of band 3, CD47, Rh protein and RhAG on HS and HPP red cells probably reflects an accelerated vesiculation process secondary to their underlying cytoskeletal defect.

Eosin-5-maleimide binding to band 3 and Rh-related proteins forms the basis of a screening test for hereditary spherocytosis.

Flow cytometric analysis of eosin-5-maleimide (EMA) binding to red cells is a screening test for the diagnosis of hereditary spherocytosis (HS). The present study used chemical modifications to determine the integral membrane proteins that react with EMA. The predominant interaction of EMA, contributing c. 80% of fluorescence, was with the epsilon-NH2 group of lysine in band 3 protein, as previously reported. The remainder of the EMA fluorescence was attributable to labelling of accessible sulfhydryl groups on intact red cells. This reaction was heat labile. Three molecules containing sulfhydryl groups were shown to be associated with the Rh blood group protein complex by sodium dodecyl sulphate polyacrylamide gel electrophoresis. These were CD47 and the Rh-associated glycoprotein, both in the Mr 40-60 kD region, and the Rh blood group proteins in the 30-32 kD region. Immunoprecipitation, using specific monoclonal antibodies and antibody binding studies by flow cytometry, showed that the relative content of these three membrane proteins was lower in HS than normal red cells. Thus, the high predictive value of the EMA binding test for HS reflects changes in the relative amounts of the Rh-related integral membrane proteins as well as band 3 in HS red cells.

William Bosworth Castle: pioneer of haematological clinical investigation.

William Bosworth Castle (1897-1990) spent his entire haematological career at the Harvard Medical Unit and Thorndike Memorial Laboratory at Boston City Hospital during the golden age of haematology. Castle's experiments in solving the puzzle of pernicious anaemia by identifying the intrinsic factor are models of clinical investigation and marked a new era in haematology. Castle's insatiable curiosity about the mechanisms of disease, his ability to design and conduct simple experiments to test hypotheses, and his ability to attract clinical investigators to the Thorndike Laboratory, and inspire them and make them feel like intellectual equals, fuelled the dramatic output of seminal work on nutritional anaemias, haemolysis, splenic function, haemoglobin physiology and coagulation. Indeed it was Castle's breadth of scientific achievements in haematology and his leadership of the Thorndike Memorial Laboratory that contributed to the notion that modern haematology was born in Boston but nurtured at the Thorndike Memorial Laboratory.

Temporal differences in membrane loss lead to distinct reticulocyte features in hereditary spherocytosis and in immune hemolytic anemia

AbstractSpherocytic red cells with reduced membrane surface area are a feature of hereditary spherocytosis (HS) and some forms of autoimmune hemolytic anemia (AIHA). It is generally assumed that membrane loss in spherocytic red cells occurs during their sojourn in circulation. The structural basis for membrane loss in HS is improper assembly of membrane proteins, whereas in AIHA it is due to partial phagocytosis of circulating red cells by macrophages. A hypothesis was formed that these different mechanisms should lead to temporal differences in surface area loss during red cell genesis and during sojourn in circulation in these 2 spherocytic syndromes. It was proposed that cell surface loss could begin at the reticulocyte stage in HS, whereas surface area loss in AIHA involves only circulating mature red cells. The validity of this hypothesis was established by documenting differences in cellular features of reticulocytes in HS and AIHA. Using a novel technique to quantitate cell surface area, the decreased membrane surface area of both reticulocytes and mature red cells in HS compared with normal cells was documented. In contrast, in AIHA only mature red cells but not reticulocytes exhibited decreased membrane surface area. These data imply that surface area loss in HS, but not in AIHA, is already present at the circulating reticulocyte stage. These findings imply that loss of cell surface area is an early event during genesis of HS red cells and challenge the existing concepts that surface area loss in HS occurs predominantly during the sojourn of mature red cells in circulation.

The GEN.S: a fortuitous finding of a routine screening test for hereditary spherocytosis.

As part of the evaluation of the GEN.S (Coulter), we compared the Mean Corpuscular Volume (MCV) to the Mean Spherized Corpuscular Volume (MSCV) assessed during the reticulocyte count procedure under hypo-osmotic conditions. A sub-group of patients with hereditary spherocytosis (HS) was singled out: in all of them, the MSCV became smaller than the MCV. As the cell volume normally increases in red cells derived from other patients in the same conditions, we decided to further study the reason for this particular behaviour of HS red cells. Whereas normal red cells are able to undergo an osmotic expansion, the spherocytes reach a critical osmotic volume leading to cell fragmentation consistent with the decrease of MSCV. This fortuitous finding is likely to be a reliable improvement for the routine screening of HS.

Role of spleen in hereditary spherocytosis: evidence for increased in vitro proteolysis of red cell membrane

In vitro proteolysis of red cell membranes has been studied by means of electrophoretic separation on SDS-polyacrylamide gel of solubilized ghost proteins and subsequent densitometry of separated, stained bands; the amounts of major membrane proteins were measured in ghosts either with inhibited or with allowed proteolysis in the following cases: 15 patients suffering from hereditary spherocytosis (HS) with variable degree of spleen enlargement, eight cirrhotic patients with spleen enlargement and 12 healthy blood donors as control group. Proteolysis was present to a greater extent in HS patients with larger splenomegaly, lesser in HS with smaller splenomegaly, and was comparable to healthy controls both in splenectomized HS and in patients with spleen enlargement due to liver cirrhosis. The results suggest the involvement of splenomegaly in the enhancement of in vitro proteolysis in HS red cell membrane; it is probably attributable to joint effects of the damage induced in red cells by prolonged retention within haemolysing spleen together with the abnormalities genetically affecting the structure of HS red cell membrane.

Hereditary spherocytosis associated with deletion of human erythrocyte ankyrin gene on chromosome 8

Hereditary spherocytosis (HS) is one of the most common hereditary haemolytic anaemias. HS red cells from both autosound dominant and recessive variants are spectrin-deficient, which correlates with the severity of the disease. Some patients with recessive HS have a mutation in the spectrin alpha-2 domain (S.L.M. et al., unpublished observations), and a few dominant HS patients have an unstable beta-spectrin that is easily oxidized, which damages the protein 4.1 binding site and weakens spectrin-actin interactions. In most patients, however, the cause of spectrin deficiency is unknown. The alpha- and beta-spectrin loci are on chromosomes 1 and 14 respectively. The only other genetic locus for HS is SPH2, on the short arm of chromosome 8 (8p11). This does not correspond to any of the known loci of genes for red cell membrane proteins including protein 4.1 (1p36.2-p34), the anion exchange protein (AE1, band 3; 17q21-qter), glycophorin C (2q14-q21), and beta-actin (7pter-q22). Human erythrocyte ankyrin, which links beta-spectrin to the anion exchange protein, has recently been cloned. We now show that the ankyrin gene maps to chromosome 8p11.2, and that one copy is missing from DNA of two unrelated children with severe HS and heterozygous deletions of chromosome 8 (del(8)(p11-p21.1)). Affected red cells are also ankyrin-deficient. The data suggest that defects or deficiency or ankyrin are responsible for HS at the SPH2 locus.

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.

A spin-label study of membrane proteins and internal microviscosity of erythrocytes in hereditary spherocytosis

Electron spin resonance (ESR) spectra of erythrocyte membranes of patients with hereditary spherocytosis (HS) and of healthy controls labeled with a maleimide spin label did not differ significantly both before and after prolonged incubation at 37°C. It suggests that the different behavior of spin-labeled HS erythrocyte membranes upon incubation at a higher temperature reported previously is due indeed to structural abnormalities of HS red cell membranes and not to alterations in their proteolytic activity. Measurements of the rotational correlation time of Tempamine spin probe demonstrated a significant elevation of internal microviscosity of erythrocytes in HS, more pronounced in nonsplenectomized patients.

Increased calcium uptake in the red cells of unsplenectomized patients with hereditary spherocytosis: significant contribution of reticulocytosis

The presence of abnormal calcium (Ca) metabolism in the red cells of hereditary spherocytosis (HS) patients has long been in dispute. Thus, to clarify the discrepancies of experimental results published previously, Ca metabolism in the red cells was investigated in 12 unsplenectomized and 6 splenectomized HS patients. A marked increase of Ca uptake was observed in the ATP-depleted red cells of the unsplenectomized HS patients. The increased red cell Ca uptake was normalized after splenectomy in the same HS patients. The extent of increased Ca uptake was well correlated to the degree of reticulocytosis ( p < 0.001). The observation was confirmed by the fractionation studies of the HS red cells. Ca uptake was not increased in the spherocyte-rich bottom fraction, if reticulocytosis was not present. No significant difference was observed in red cell calcium content between normal and HS patients, neither unsplenectomized nor splenectomized.

Disappearance of microspherocytes in peripheral circulation and normalization of decreased lipids in plasma and in red cells of patients with hereditary spherocytosis after splenectomy.

A possible mechanism of the formation of microspherocytosis in unsplenectomized patients with hereditary spherocytosis (HS) was investigated in relation to lipid metabolism in plasma and in red cells of these patients. Plasma lipids (total cholesterol, free cholesterol, high density lipoprotein cholesterol, free fatty acids, and phospholipids) were markedly reduced in unsplenectomized HS patients with microspherocytosis. Red cell membrane lipids (free cholesterol and phospholipids such as phosphatidyl ethanolamine, sphingomyelin, phosphatidyl choline, and lysophosphatidyl choline) were also decreased in these unsplenectomized HS patients. After splenectomy, microspherocytosis disappeared concomitant to substantial normalization of plasma and red cell membrane lipids. These observations suggest that plasma lipid decrement in the unsplenectomized HS patients is at least one of the causative factors in pathogenesis of the formation of microspherocytosis. So-called "splenic conditioning" may not imply a physical "loss" of membrane by fragmentation or pitting of the once-formed HS red cells in the peripheral circulation. Instead, the decreased plasma lipids in the presence of the spleen may affect the de novo synthesis of red cell membrane lipids, resulting in the formation of microspherocytosis.

Effect of ouabain on osmotic resistance and monovalent cation transport of red cells in hereditary spherocytosis.

The effect of ouabain on the osmotic resistance of red cells from 17 splenectomized patients with congenital or hereditary spherocytosis (HS), from 5 of their relatives suspected of having a subclinical form of the disease and from unsplenectomized and splenectomized normal controls was studied. In red cells from the HS patients and the splenectomized controls a small ouabain-induced decrease in osmotic resistance was seen, whereas in the unsplenectomized controls no such change was observed. The osmotic resistance of red cells from the 5 relatives, on the other hand, was significantly decreased by ouabain. The usefulness of ouabain in unmasking HS in its subclinical form was further tested by studying monovalent cation influx into red cells. The active influx of 86Rb+ into red cells from HS suffers was significantly increased. A similar degree of ouabain-induced inhibition of 86Rb+ transport was seen in all groups studied. The passive influx of 22Na+, which was also increased in HS red cells, was not affected by ouabain in any of the four groups.

Heat-induced Electrophoretic Anomalies of Red Cell Membrane Nonsialoglycoproteins

AbstractThe effects of heating at 100° in the presence and absence of dithiothreitol has been examined in SDS-solubilized ghost membranes and isolated integral membrane proteins from human red cells. A significant increase in electrophoretic mobility and broadening of the major polypeptide band 3 was found. The anomalous behavior of band 3 alone could explain the apparent disappearance of band 4 which has occasionally been reported in hereditary spherocytosis red cells.

Effect of Pentoxifylline on Red Cell Flexibility and Cation Transport in Healthy Subjects and Patients with Hereditary Spherocytosis

The effect of pentoxifylline (3,7-dimethyl-1-(5-oxo-hexyl)-xanthine) on the flexibility of red cells was studied using a filtration method in which the red cells are forced, at a constant flow-rate, through a porous polycarbonate membrane. The filtration pressure reflects red cell rigidity and the amount of Hb released from the disrupted cells ('free Hb') red cell fragility. The advantage of this method is that it allows the two important determinants of red cell flexibility, rigidity and fragility, to be studied simultaneously. Pentoxifylline significantly improved normal red cell flexibility both in vivo and in vitro as judged by this method. The impaired flexibility of red cells from patients with congenital or hereditary spherocytosis (HS) was aggravated by pentoxifylline in vitro. A similar effect of pentoxifylline was also observed on red cells from relatives of HS patients. The effect of pentoxifylline on Ca2+ transport and Ca2+-Mg2+-ATPase activity in red cells from normal and HS patients was investigated. Under in vitro conditions pentoxifylline did not affect the Ca2+-Mg2+-ATPase activity of or Ca2+ efflux from normal and HS red cells. Neither the influx of monovalent cations (Na+, Rb+) or the osmotic resistance of normal or HS red cells was affected by pentoxifylline.

Kinetics of red cell membrane phosphorylation: altered affinity of HS membrane protein acceptors.

Membrane ghosts were prepared from red blood cells of normal and hereditary spherocytosis (HS) subjects. Time dependent phosphorylation studies of the membrane (in the presence of gamma labelled AT32P) showed that the HS membrane appeared to incorporate less phosphate than the normal membrane but the results were not significantly different. Using the initial linear rate of phosphorylation and varying the ATP concentration, it was found that the Michaelis constant (Km) for the normal membrane was 24.8 microns ATP and Vmax was 0.120 nmol phosphate bound/mg membrane protein/min. These values for the HS membrane were found to be 39.0 and 0.118 respectively. In one family the affected mother and son each showed a positive cooperativity effect for similar reciprocal plots. This indicates that phosphorylation of various HS membrane proteins may be an ordered rather than random process and suggests biochemical heterogencity of the HS condition. The constants, Km and Vmax, however, were found to be similar to the rest of HS subjects studied. Crude extracts of the enzyme protein kinase, which catalyses the membrane phosphorylation, showed no significant difference in the Km and Vmax value in HS compared with that from normal red cells. The kinetic difference in phosphorylation is probably due to abnormalities in the membrane protein(s) which accept the phosphate from protein kinase in HS red cells.

Erythrocyte Cellular and Membrane Deformability in Hereditary Spherocytosis

AbstractIn order to determine whether the relative rigidity of the hereditary spherocytosis (HS) red cell is due to membrane rididity or merely to an altered surface/volume ratio, we investigated the deformability of resealed red cell membranes from patients with HS. Whereas the osmotic fragility of intact red cells of HS patients showed the expected increase, the osmotic fragility of resealed HS membranes was normal, thus indicating that their surface/volume ratio was normal. Measurements with an ektacytometer showed that deformability of intact HS cells was markedly diminished, whereas deformability of resealed HS membranes was normal. These findings indicate that the HS red cell membrane is not intrinsically abnormally rigid, as has been suggested, but that the lack of deformability of the erythrocyte is primarily a function of the altered surface/volume ratio.

Erythrocyte cellular and membrane deformability in hereditary spherocytosis

In order to determine whether the relative rigidity of the hereditary spherocytosis (HS) red cell is due to membrane rididity or merely to an altered surface/volume ratio, we investigated the deformability of resealed red cell membranes from patients with HS. Whereas the osmotic fragility of intact red cells of HS patients showed the expected increase, the osmotic fragility of resealed HS membranes was normal, thus indicating that their surface/volume ratio was normal. Measurements with an ektacytometer showed that deformability of intact HS cells was markedly diminished, whereas deformability of resealed HS membranes was normal. These findings indicate that the HS red cell membrane is not intrinsically abnormally rigid, as has been suggested, but that the lack of deformability of the erythrocyte is primarily a function of the altered surface/volume ratio.

The molecular lesion of hereditary spherocytosis (HS): a continuing enigma [Editorial

V ERY FEW, if any, abnormalities of the human erythrocyte have been studied with such diligence as those associated with hereditary spherocytosis (HS). Not only has the small, round, densely staining spherocyte provided a long-recognized morphologic benchmark, but its inordinate susceptibility to lysis in hypotonic saline has been clearly documented for 75 years.’ Forty years ago, Haden (cited above) and Castle and Daland2 related the latter to loss of the usual discoidal form of the erythrocyte, and to a probable structural abnormality resulting in a decreased surface area/volume ratio. The unique role ofthe spleen in recognizing, sequestering, and finally destroying the abnormal cell, and the effect of splenectomy in correcting clinical manifestations were investigated in great detail in the 1940’s and early l95O’s.�� The assumption that a genetically determined, usually dominantly transmitted7”#{176} abnormality of the red cell membrane lies at the heart of the disorder has pervaded studies of its pathophysiobogyand, in the past 20 years, has fostered many investigations of red cell membrane structure and function. While these studies have added much to our understanding of red cell physiology, the secret of the ultimate molecular lesion still remains locked within the osmotically fragile spherocyte. These investigations have defined a variety of abnormal properties in HS red cells. These cells are abnormally permeable to sodium and this “sodium leak”2 necessitates an increased glycolytic rate to support greater-thannormal adenosine triphosphate-adenosine triphosphatase (ATP-ATPase)dependent pumping of sodium from the cell’s interior.’2’4 While total lipids have been reported to be relatively somewhat decreased both before’5”6 and after’7 splenectomy, and abnormalities of phospholipid turnover have been suggested,’t no consistently demonstrable data thus far support specific abnormalities in membrane lipid structure. However, under conditions of glucose deprivation, HS membrane lipid is abnormally unstable, and is released by a budding process, thus contributing to a still greater deficit in surface area.’5”9’2#{176}