Search

Objectives: Southeast Asian ovalocytosis (SAO) is an inherited red blood cell (RBC) membrane disorder, whereas hemoglobinopathies are inherited globin gene disorders. In an area where both diseases are prevalent, the interaction between them resulting in variable hematologic parameters can be encountered. However, little is known about the genetic interaction of SAO and thalassemia. We investigated the prevalence of SAO and hemoglobinopathy genotypes among newborns in southern Thailand., Patients and Methods: This study was carried out on 297 newborns recruited consecutively at Naradhiwas Rajanagarindra Hospital in the south of Thailand. The SAO was identified on blood smear examination and polymerase chain reaction analysis. Thalassemia genotypes were defined. Hematologic parameters and hemoglobin (Hb) profiles were recorded and analyzed., Results: Among 297 newborns, 15 (5.1%) carried SAO, whereas 70 (23.6%) had thalassemia with 15 different thalassemia genotypes. Abnormal Hb including Hb C, Hb Q-Thailand, and Hb D-Punjab were observed in 5 newborns. It was found in the nonthalassemic newborns that RBC count, Hb, and hematocrit of the nonthalassemic newborns with SAO were significantly lower than those without SAO. The same finding was also observed in the thalassemic newborns; RBC count, Hb, and hematocrit of the thalassemic newborns with SAO were significantly lower than those without SAO. However, the mean corpuscular volume, mean corpuscular Hb, and RBC distribution width of the SAO-newborns were significantly higher., Conclusions: Both SAO and hemoglobinopathy genotypes are common in southern Thailand. One should take this into consideration when evaluating neonatal anemia and other hematologic abnormalities. Identification of both genetic defects and long-term monitoring on the clinical outcome of this genetic interaction should be essential to understand the pathogenesis of these common genetic disorders in the region., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Dizygotic twin males, born at 34 weeks gestation, had prolonged jaundice, microcytic, hypochromic anemia, FABarts hemoglobin, elevated end-tidal CO, and blood films consistent with hereditary pyropoikilocytosis. DNA sequencing revealed both had a heterozygous alpha spectrin (SPTA1) mutation (c.460_462dup) inherited from their asymptomatic mother, plus a 3-base pair duplication in alpha globin (HBA2) (c.364_366dupGTG) inherited from their asymptomatic father., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Familial distal renal tubular acidosis (dRTA) associated with mutations of solute carrier family 4 membrane - 1 (SLC4A1) gene could co-exist with red cell membrane abnormality, Southeast Asian ovalocytosis (SAO). Although this association is well described in Southeast Asian countries, it is less frequently found in Sri Lanka., Case Presentation: We describe six patients who had dRTA co-existing with SAO. All of them initially presented with severe hypokalemia and paralysis. They presented within a period of six months to the Teaching Hospital Anuradhapura, Sri Lanka. All had metabolic acidosis indicated by low serum bicarbonate. Three of them were having underlying chronic kidney disease as well. Those three patients had mixed high and normal anion gap metabolic acidosis indicated by low delta ratio. In all dRTA was confirmed by presence of normal anion gap, hyperchloraemia, high urine pH and positive urine anion gap. Examination of blood films of all of them revealed presence of stomatocytes and macro-ovalocytosis compatible with SAO. In relation to complications of dRTA, two patients had medullary nephrocalcinosis. Three patients had biochemical evidence of osteomalacia, with two of them having radiological evidence of diffuse osteosclerosis. One patient had secondary hyperparathyroidism and a pathological fracture., Conclusions: Erythrocyte in SAO is exceptionally rigid and this abnormality is said to be evolved as it protects against Plasmodium vivax malaria and cerebral malaria cause by Plasmodium falciparum. Although two families of SAO was described earlier, SAO and dRTA combination was reported only once in a patient from Anuradhapura district. Distal renal tubular acidosis, SAO combination and its related complications including nephrocalcinosis, chronic kidney disease and metabolic bone disease was not described in Sri-Lankan literature. This case series emphasize the importance of investigating recurrent/ chronic hypokalemia to diagnose dRTA and its associations, as early correction of acidosis could prevent development of chronic kidney disease and metabolic bone disease.

Systematic screening for congenital erythrocyte disorders is not a common practice, due to a lack in the accuracy of the methods and to the costs of the analyses. As a consequence, the diagnosis is usually made when a severe complication occurs. This study introduces an innovative method to perform the screening of a hereditary disease characterized by erythrocyte membrane defects such as hereditary spherocytosis (HS) and hereditary elliptocytosis (HE). Blood samples from healthy donors and patients affected by HS and HE were processed by thermogravimetric analysis (TGA) and the resulting curves were analyzed by chemometrics in order to develop a model of prediction. A novel test was developed and validated by chemometrics and results were found to be in accordance with the genetic diagnosis, confirming the prediction ability of the optimized model that uses few microliters of whole non pretreated blood to perform the diagnosis of HS/HE in less than one hour.

Hereditary erythrocyte membrane disorders are caused by mutations in genes encoding various transmembrane or cytoskeletal proteins of red blood cells. The main consequences of these genetic alterations are decreased cell deformability and shortened erythrocyte survival. Red blood cell membrane defects encompass a heterogeneous group of haemolytic anaemias caused by either (i) altered membrane structural organisation (hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikilocytosis and Southeast Asian ovalocytosis) or (ii) altered membrane transport function (overhydrated hereditary stomatocytosis, dehydrated hereditary stomatocytosis or xerocytosis, familial pseudohyperkalaemia and cryohydrocytosis). Herein we provide a comprehensive review of the recent literature on the molecular genetics of erythrocyte membrane defects and their reported clinical consequences. We also describe the effect of low-expression genetic variants on the high inter- and intra-familial phenotype variability of erythrocyte structural defects., (© 2019 British Society for Haematology and John Wiley & Sons Ltd.)

Introduction: South-East Asian ovalocytosis (SAO) is a common inherited red blood cell polymorphism in South-East Asian and Melanesian populations, coinciding with areas of malaria endemicity. Validation of light microscopy as a diagnostic alternative to molecular genotyping may allow for its cost-effective use either prospectively or retrospectively by analysis of archived blood smears., Methods: We assessed light microscopic diagnosis of SAO compared to standard PCR genotyping. Three trained microscopists each assessed the same 971 Giemsa-stained thin blood films for which SAO genotypic confirmation was available by PCR. Generalized mixed modeling was used to estimate the sensitivity, specificity, positive predictive value, and negative predictive value of light microscopy vs "gold standard" PCR., Results: Among red cell morphologic parameters evaluated, knizocytes, rather than ovalocytic morphology, proved the strongest predictor of SAO status (odds ratio [OR] = 19.2; 95% confidence interval [95% CI] = 14.6-25.3; P ≤ 0.0001). The diagnostic performance of a knizocyte-centric microscopic approach was microscopist dependent: two microscopists applied this approach with a sensitivity of 0.89 and a specificity of 0.93. Inter-rater reliability among the microscopists (κ = 0.20) as well as between gold standard and microscopist (κ = 0.36) underperformed due to misclassification of stomatocytes as knizocytes by one microscopist, but improved substantially when excluding the error-prone reader (κ = 0.65 and 0.74, respectively)., Conclusion: Light microscopic diagnosis of SAO by knizocyte visual cue performed comparable to time-consuming and costlier molecular methods, but requires specific training that includes successful differentiation of knizocytes from stomatocytes., (© 2018 John Wiley & Sons Ltd.)

In red blood cell (RBC) diseases, the spleen contributes to anemia by clearing the damaged RBCs, but its unique ability to mechanically challenge RBCs also poses the risk of inducing other pathogenic effects. We have analyzed RBCs in hereditary spherocytosis (HS) and hereditary elliptocytosis (HE), two typical examples of blood disorders that result in membrane protein defects in RBCs. We use a two-component protein-scale RBC model to simulate the traversal of the interendothelial slit (IES) in the human spleen, a stringent biomechanical challenge on healthy and diseased RBCs that cannot be directly observed in vivo. In HS, our results confirm that the RBC loses surface due to weakened cohesion between the lipid bilayer and the cytoskeleton and reveal that surface loss may result from vesiculation of the RBC as it crosses IES. In HE, traversing IES induces sustained elongation of the RBC with impaired elasticity and fragmentation in severe disease. Our simulations thus suggest that in inherited RBC disorders, the spleen not only filters out pathological RBCs but also directly contributes to RBC alterations. These results provide a mechanistic rationale for different clinical outcomes documented following splenectomy in HS patients with spectrin-deficient and ankyrin-deficient RBCs and offer insights into the pathogenic role of human spleen in RBC diseases., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Hereditaryelliptocytosis (HE) is a hereditary hemolytic disease, characterized by the presence of many elliptical erythrocytes in the peripheral blood that is caused by abnormal cytoskeletal proteins in the erythrocyte membrane. In the present study, a novel, causal HE mutation was reported. Routine blood examinations were performed on the proband and their family, and the fluorescence intensity of eosin‑5‑maleimide (EMA)‑labeled erythrocytes was determined via flow cytometry. Subsequently, DNA was extracted from the peripheral blood of the proband and their family members, and amplified by quantitative polymerase chain reaction. The Sanger sequencing approach was used to determine and identify gene mutations, which were verified by matrix‑assisted laser desorption‑ionization time of flight (MALDI‑TOF) mass spectrometry. To exclude genetic polymorphisms, newly identified mutations were subjected to large‑scale gene screening using high‑resolution melt analysis. Protein expression levels in the erythrocyte membrane of the proband were determined via SDS‑PAGE, which demonstrated that, compared with healthy controls, the proband exhibited a reduction in EMA‑labeled erythrocytes. In addition, DNA analysis demonstrated that the proband carried three mutations in the spectrin α chain erythrocytic 1 (SPTA1) gene: c.161A>C, c.5572C>G and 6531‑12C>T. The corresponding mutant polypeptides were also analyzed by MALDI‑TOF mass spectroscopy. SDS‑PAGE analysis indicated that the proband exhibited normal levels of erythrocyte membrane proteins. In the present study, a novel HE case with a His54Pro mutation in the SPTA1 gene was reported. The results suggested that the His54Pro mutation influenced the role of erythrocyte membrane proteins without reducing its level of expression.

Southeast asian ovalocytosis (SAO) is characterized by macro-ovalocytes and ovalo-stomatocytes on blood smear. SAO is common in Malaisia and Papua-New-Guinea where upwards to 40 per cent of the population is affected in some coastal region. Inherited in an autosomal dominant way, illness results from deletion of codons 400-408 in SLC4A1 gene which encodes for band 3 erythrocyte membrane protein. This deletion is responsible for an unusual erythrocyte stiffness and oval shape of the cells on blood smear. Heterozygous carriers are usually asymptomatic whereas homozygous are not viable without an intensive antenatal care. Here, we describe 4 patients diagnosed incidentally by cytogram appearance of the Advia® 2120i (Siemens) representing hemoglobin concentration according to red blood mean cellular volume (GR/VCH).

The crystal structure of the dimeric membrane domain of human Band 3(1), the red cell chloride/bicarbonate anion exchanger 1 (AE1, SLC4A1), provides a structural context for over four decades of studies into this historic and important membrane glycoprotein. In this review, we highlight the key structural features responsible for anion binding and translocation and have integrated the following topological markers within the Band 3 structure: blood group antigens, N-glycosylation site, protease cleavage sites, inhibitor and chemical labeling sites, and the results of scanning cysteine and N-glycosylation mutagenesis. Locations of mutations linked to human disease, including those responsible for Southeast Asian ovalocytosis, hereditary stomatocytosis, hereditary spherocytosis, and distal renal tubular acidosis, provide molecular insights into their effect on Band 3 folding. Finally, molecular dynamics simulations of phosphatidylcholine self-assembled around Band 3 provide a view of this membrane protein within a lipid bilayer., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background and Aims: Erythropoiesis is closely related to iron metabolism in a balanced homeostasis. Analyses of diverse erythroid and iron metabolism disorders have shown that disrupted erythropoiesis negatively affects iron homeostasis and vice versa. The aim of this study was to characterize the relationship between erythropoietic activity and iron homeostasis in pediatric patients with erythrocyte membrane defects and thalassemia traits., Methods: Selected markers of erythropoietic activity (erythropoietin, soluble transferrin receptor - sTfR and growth differentiation factor 15) and iron status parameters (serum iron, ferritin and hepcidin) were evaluated in pediatric patients with erythrocyte membrane defects and thalassemia traits., Results: The patients with erythrocyte membrane defects and thalassemia traits had altered iron homeostasis due to disturbed erythropoiesis. In comparison with healthy controls, they had a normal to low hepcidin/ferritin ratio and concomitantly elevated sTfR., Conclusion: The findings suggest that pediatric patients with erythrocyte membrane defects and thalassemia traits are more susceptible to iron overload than the general population and that the (hepcidin/ferritin)/sTfR ratio can be used to monitor any worsening of the disease.

We employ a two-component red blood cell (RBC) membrane model to simulate lateral diffusion of band-3 proteins in the normal RBC and in the RBC with defective membrane proteins. The defects reduce the connectivity between the lipid bilayer and the membrane skeleton (vertical connectivity), or the connectivity of the membrane skeleton itself (horizontal connectivity), and are associated with the blood disorders of hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) respectively. Initially, we demonstrate that the cytoskeleton limits band-3 lateral mobility by measuring the band-3 macroscopic diffusion coefficients in the normal RBC membrane and in a lipid bilayer without the cytoskeleton. Then, we study band-3 diffusion in the defective RBC membrane and quantify the relation between band-3 diffusion coefficients and percentage of protein defects in HE RBCs. In addition, we illustrate that at low spectrin network connectivity (horizontal connectivity) band-3 subdiffusion can be approximated as anomalous diffusion, while at high horizontal connectivity band-3 diffusion is characterized as confined diffusion. Our simulations show that the band-3 anomalous diffusion exponent depends on the percentage of protein defects in the membrane cytoskeleton. We also confirm that the introduction of attraction between the lipid bilayer and the spectrin network reduces band-3 diffusion, but we show that this reduction is lower than predicted by the percolation theory. Furthermore, we predict that the attractive force between the spectrin filament and the lipid bilayer is at least 20 times smaller than the binding forces at band-3 and glycophorin C, the two major membrane binding sites. Finally, we explore diffusion of band-3 particles in the RBC membrane with defects related to vertical connectivity. We demonstrate that in this case band-3 diffusion can be approximated as confined diffusion for all attraction levels between the spectrin network and the lipid bilayer. By comparing the diffusion coefficients measured in horizontal vs. vertical defects, we conclude that band-3 mobility is primarily controlled by the horizontal connectivity.

Genetic disorders of haemoglobin, particularly the sickle cell diseases and the alpha and beta thalassaemias, are the commonest inherited disorders worldwide. The majority of affected births occur in low-income and lower-middle income countries. Screening programmes are a vital tool to counter these haemoglobinopathies by: (i) identifying individual carriers and allowing them to make informed reproductive choices, and (ii) generating population level gene-frequency estimates, to help ensure the optimal allocation of public health resources. For both of these functions it is vital that the screen performed is suitably sensitive. One popular first-stage screening option to detect carriers of beta thalassaemia in low-income countries is the One Tube Osmotic Fragility Test (OTOFT). Here we introduce a population genetic framework within which to quantify the likely sensitivity and specificity of the OTOFT in different epidemiological contexts. We demonstrate that interactions between the carrier states for beta thalassaemia and alpha thalassaemia, glucose-6-phosphate dehydrogenase deficiency and Southeast Asian Ovalocytosis have the potential to reduce the sensitivity of OTOFTs for beta thalassaemia heterozygosity to below 70%. Our results therefore caution against the widespread application of OTOFTs in regions where these erythrocyte variants co-occur., (© 2014 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.)

This study was designed to investigate the 4 year old boy with Hereditary Elliptocitosis (HE). The diagnosis of this rare hemolytic anemia was based on detailed family history (positive in the 4-th generation), physical examination and Para-clinical data analyses. The vast majority of patients with HE are asymptomatic, severe forms are rare. The most important is examination of blood films, which is helpful to detect the morphology abnormalities of red cells. In case of HE a different approach is required. Positive family history and series of investigations should be conducted to determine the HE.

Primary abnormalities of the erythrocyte membrane are characterized by clinical, laboratory, and genetic heterogeneity. Among this group, hereditary spherocytosis patients are more likely to experience symptomatic anemia. Treatment of hereditary spherocytosis with splenectomy is curative in most patients. Growing recognition of the long-term risks of splenectomy has led to re-evaluation of the role of splenectomy. Management guidelines acknowledge these considerations and recommend discussion between health care providers, patient, and family. The hereditary elliptocytosis syndromes are the most common primary disorders of erythrocyte membrane proteins. However, most elliptocytosis patients are asymptomatic and do not require therapy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We have detected an ovalo-stomatocytosis in a military 24-years-old man. We have sent a blood specimen to haematology laboratory in Kremlin-Bicetre (France) to test ektacytometry. The test concludes to an asymptomatic Melanesian ovalocytosis.

Hereditary spherocytosis and elliptocytosis are the two most common inherited red cell membrane disorders resulting from mutations in genes encoding various red cell membrane and skeletal proteins. Red cell membrane, a composite structure composed of lipid bilayer linked to spectrin-based membrane skeleton is responsible for the unique features of flexibility and mechanical stability of the cell. Defects in various proteins involved in linking the lipid bilayer to membrane skeleton result in loss in membrane cohesion leading to surface area loss and hereditary spherocytosis while defects in proteins involved in lateral interactions of the spectrin-based skeleton lead to decreased mechanical stability, membrane fragmentation and hereditary elliptocytosis. The disease severity is primarily dependent on the extent of membrane surface area loss. Both these diseases can be readily diagnosed by various laboratory approaches that include red blood cell cytology, flow cytometry, ektacytometry, electrophoresis of the red cell membrane proteins, and mutational analysis of gene encoding red cell membrane proteins., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Red blood cells (RBCs) from individuals with Southeast Asian ovalocytosis (SAO) contain a mutant band 3 protein that causes the formation of unique linear oligomers in the RBC membrane. We used single-particle tracking to measure the lateral diffusion of individual glycophorin C (GPC), band 3, and CD58 proteins in membranes of intact SAO RBCs and normal RBCs (nRBCs). GPC, an integral protein that binds with high affinity to the RBC membrane skeleton, showed oscillatory motion within confinement areas that were smaller in SAO RBCs than in nRBCs. The additional confinement in SAO RBCs could be due to membrane stiffening associated with the SAO phenotype. Band 3 in both SAO RBCs and nRBCs also showed confined motion over short times (ms) and distances (nm), and the area of confinement was smaller in SAO RBCs than in nRBCs. These data presumably reflect the constraints imposed by band 3 oligomerization. Similarly, the glycosylphosphatidylinositol-linked protein CD58 showed loosely confined diffusion in nRBCs and a substantially higher degree of confinement in SAO RBCs. Restricted protein mobility could contribute to the altered adherence of parasite-infected RBCs to vascular endothelium that is thought to protect individuals with SAO from severe manifestations of malaria., (© 2011 Blackwell Publishing Ltd.)

Southeast Asian ovalocytosis (SAO), α(+)-thalassemia, and low expression of complement receptor 1 (CR1) have been associated with protection against severe Plasmodium falciparum malaria. In a cohort of children 5-14 years of age the effect of α(+)-thalassemia, SAO (SLC4A1Δ27), CR1 polymorphisms, and Gerbich negativity (GYPCΔex3) on risk of P. falciparum infections and uncomplicated illness were evaluated. The risk of acquiring polymerase chain reaction (PCR)-diagnosed P. falciparum infections was significantly lower for α(+)-thalassemia heterozygotes (hazard ratio [HR]: 0.56) and homozygotes (HR: 0.51) than wild-type children. No such differences were seen in light of microscopy diagnosed infections (P = 0.71) or were α(+)-thalassemia genotypes associated with a reduced risk of uncomplicated P. falciparum malaria. No significant associations between the risk of P. falciparum infection or illness were observed for any of the other red blood cell polymorphisms (P > 0.2). This suggests that these polymorphisms are not associated with significant protection against P. falciparum blood-stage infection or uncomplicated malaria in school-aged children.

The classical function of 4.1R in red blood cells is to contribute to the mechanochemical properties of the membrane by promoting the interaction between spectrin and actin. More recently, it has been recognized that 4.1R is required for the stable cell surface accumulation of a number of erythrocyte membrane proteins. 4.1R is one member of the mammalian 4.1 family - the others being 4.1N, 4.1G and 4.1B - and is expressed in many cell types other than erythrocytes. Recently we have examined the phenotype of hearts from 4.1R knockout mice. Although they had a generally normal morphology, these hearts exhibited bradycardia, and prolongation of both action potentials and QT intervals. Electrophysiological analysis revealed anomalies in a range of ion channel activities. In addition, the immunoreactivity of voltage-gated Na(+) channel NaV1.5 was reduced, indicating a role for 4.1R in the cellular accumulation of this ion channel. 4.1 proteins also have roles in the accumulation of at least two other classes of ion channel. In epithelia, 4.1 interacts with the store-operated channel TRPC4. In neurons, the ligand-gated channels GluR1 and GluR4 require 4.1 proteins for cell surface accumulation. The spectrum of transmembrane proteins that bind to 4.1 proteins overlaps with that of ankyrin. A hypothesis to investigate in the future is that differential regulation of 4.1 and ankyrins (e.g. by PIP(2)) allows highly selective control of cell surface accumulation and transport activity of a specific range of ion channels.

The Duffy antigen receptor for chemokines (DARC or Fy glycoprotein) carries antigens that are important in blood transfusion and is the main receptor used by Plasmodium vivax to invade reticulocytes. Southeast Asian ovalocytosis (SAO) results from an alteration in RBC membrane protein band 3 and is thought to mitigate susceptibility to falciparum malaria. Expression of some RBC antigens is suppressed by SAO, and we hypothesized that SAO may also reduce Fy expression, potentiallyleading to reduced susceptibility to vivax malaria. Blood samples were collected from individuals living in the Madang Province of Papua New Guinea. Samples were assayed using a flow cytometry assay for expression of Fy on the surface of RBC and reticulocytes by measuring the attachment of a phycoerythrin-labeled Fy6 antibody. Reticulocytes were detected using thiazole orange. The presence of the SAO mutation was confirmed by PCR. There was a small (approximately 10%) but statistically significant (p=0.049, Mann-Whitney U test) increase in Fy expression on SAO RBC compared with RBC from individuals without this polymorphism: mean Fy expression (mean fluorescence intensity [MFI]) was 10.12 +/- 1.22 for SAO heterozygotes versus an MFI of 8.95 +/- 1.1 for individuals without SAO. For reticulocytes the MFI values were 27.61 +/- 19.12 for SAO heterozygotes and 16.47 +/- 3.81 for controls. SAO is associated with increased and not decreased Fy6 expression so that susceptibility to P. vivax infection is unlikely to be affected.

Here are three images from peripheral blood smear from a patient with hereditary elliptocytosis. The patient presented for a general check up when his CBC showed only mild anemia with slightly increased reticulocytes. The diagnosis was made incidentally when the blood smear was examined.

Malaria is a major cause of childhood death throughout much of the tropical world. As a result, it has exerted a powerful force for the evolutionary selection of genes that confer a survival advantage. Identifying which genes are involved, and how they affect malaria risk, is a potentially useful way of exploring the host-parasite relationship. To date, some of the best-described malaria-protective polymorphisms relate to genes that affect the structure or function of red blood cells (RBC). Recent years have seen significant advances in our understanding of the importance of some of these genes, including glycophorin C (GYPC); complement receptor 1 (CR1); band 3 (SLC4A1); pyruvate kinase (Pklr); and the genes for alpha-(HBA) and beta-globin (HBB). The challenge for the future must be to convert these advances into fresh approaches to the prevention and treatment of malaria.

We report a case of hereditary elliptocytosis in an infant diagnosed a few months after the birth, in a context of regenerative normocytic normochromic anaemia. The investigations, including incubated osmotic fragility, erythrocytic enzymes study and haemoglobin electrophoresis, were not contributive. Only the persistence of elongated (or cigar-shaped) erythrocytes on blood smears was noted. Hereditary elliptocytosis was confirmed by specialized investigations (rheological study and erythrocytic membrane proteins electrophoresis). Investigations in the mother were realized and led to the discovery of a similar biological pattern. Hereditary elliptocytosis is a red blood cell membrane disorder due to the defect in cytoskeleton proteins (spectrin or 4.1), leading to the loss of deformability properties of erythrocytes. This disorder is considered as rare; however, its incidence is probably underestimated because most cases are pauci- or asymptomatic and the discovery is often fortuitous. The absence of detection of this defect by incubated osmotic fragility should not discard the hypothesis of erythrocytes membrane disorders. The persistent observation of elongated erythrocytes on blood smear must encourage the biologist to evocate a hereditary elliptocytosis.

Disorders of the erythrocyte membrane, including hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikilocytosis, and hereditary stomatocytosis, comprise an important group of inherited hemolytic anemias. These syndromes are characterized by marked clinical and laboratory heterogeneity. Recent molecular studies have revealed that there is also significant genetic heterogeneity in these disorders. This is particularly true for the spherocytosis syndromes where each kindred has a private mutation in one of the spherocytosis genes. Treatment with splenectomy is curative in most patients. Splenectomy via a laparoscopic approach has become the surgical method of choice. Growing recognition and understanding of the long-term risks and complications of splenectomy, including cardiovascular disease, thrombotic disorders, and pulmonary hypertension, and the emergence of penicillin-resistant pneumococci, a concern for infection in overwhelming postsplenectomy infection, have led to reevaluation of the role of splenectomy. Recent management guidelines acknowledge these important considerations when entertaining splenectomy and recommend detailed discussion between health care providers, patient, and family.

Plasmodium falciparum, the causative agent of the most lethal form of human malaria, uses multiple ligand-receptor interactions to invade host red blood cells (RBCs). We studied the invasion of P falciparum into abnormal RBCs from humans carrying the Southeast Asian ovalocytosis (SAO) trait. One particular parasite line, 3D7-A, invaded these cells efficiently, whereas all other lines studied invaded SAO RBCs to only about 20% of the extent of normal (non-SAO) cells. This result is consistent with the clinical observation that SAO individuals can experience high-density P falciparum infections and provides an explanation for previous discrepant results on invasion of SAO RBCs. Characterization of the invasion phenotype of 3D7-A revealed that efficient invasion of SAO RBCs was paralleled by relatively efficient invasion of normal RBCs treated with either neuraminidase, trypsin, or chymotrypsin and a novel capacity to invade normal RBCs treated sequentially with both neuraminidase and trypsin. Our results suggest that only parasites able to use some particular invasion pathways can invade SAO RBCs efficiently in culture. A similar situation might occur in the field.

The native population of Bahrain has a high prevalence of hemoglobinopathies and G6PD deficiency, probably as a result of past malarial endemism. We used the Biorad-Variant hemoglobin testing system for primary screening of hemoglobinopathies in 20,000 individuals. Hemoglobin abnormalities were detected in 7,206 (36.3%) cases.

Disorders of the red blood cell membrane, such as hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis, are characterized by heterogeneity in their clinical and laboratory manifestations. Advances in molecular biology have allowed determination of the precise genetic defect in many cases of membrane-associated anemia and have revealed significant genetic heterogeneity. Six genetic loci have been identified and many defects, including gene deletions and insertions, missense and nonsense mutations, and splicing mutations, have been found. Analysis of these defects has provided a better understanding of the pathogenesis of these disorders and allowed a better understanding of the structure/function relationships of the proteins of the erythrocyte membrane.

Mice with disruptions of the red blood cell (RBC) cytoskeleton provide severe hemolytic anemia models in which to study multiorgan thrombosis and infarction. The incidence of cerebral infarction ranges from 70% to 100% in mice with alpha-spectrin deficiency. To determine whether mutant RBCs abnormally bind adhesive vascular components, we measured adhesion of mouse and human RBCs to immobilized human thrombospondin (TSP) and laminin (LM) under controlled flow conditions. Mutant RBCs had at least 10-fold higher adhesion to TSP compared with normal RBCs (P <.006). Mutant relative to unaffected RBC adhesion to LM was significantly (P <.01) increased as well. Treatment of RBCs with the anionic polysaccharide dextran sulfate inhibited mutant RBC adhesion to TSP (P <.001). Treatment of RBCs with antibodies to CD47 or the CD47-binding TSP peptide 4N1K did not inhibit TSP adhesion of RBCs. Previously, we have shown that infarcts in alpha-spectrin-deficient sph/sph mice become histologically evident beginning at 6 weeks of age. TSP adhesion of RBCs from 3- to 4- and 6- to 8-week-old sph/sph mice was significantly higher than RBCs from adult mice (> 12 weeks old; P <.005). While the mechanism of infarction in these mice is unknown, we speculate that changes in RBC adhesive characteristics contribute to this pathology.

The diagnosis of hereditary spherocytosis (HS) is based on red cell morphology and other conventional tests such as osmotic fragility, autohemolysis and acidified glycerol lysis. However, milder cases are at times difficult to diagnose. Confirmation by red blood cell (RBC) membrane protein analysis is not possible in most laboratories. Recently, a flow cytometric method has been described for quantitating the fluorescence intensity of intact red cells after incubation with the dye eosin-5'-maleimide (EMA), which binds specifically to the anion transport protein (band-3) at lysine-430. This has been shown to be an effective screening test for red cell membrane disorders. We evaluated the usefulness of this approach for screening membrane protein disorders such as HS and hereditary elliptocytosis (HE) and its value in discriminating this group from other hemolytic anemias, such as glucose-6-phosphate dehydrogenase (G6PD) deficiency, beta-thalassemia trait, sickle cell anemia and autoimmune hemolytic anemia. Fluorescence intensity, expressed in mean channel fluorescence (MCF) units, was determined using a Becton Dickinson FACS Caliber flow cytometer. Membrane protein analysis was carried out by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE). RBCs from patients with HS and HE gave significantly lower MCF values (P < 0.001) than the normal control group and other patient groups. The diagnosis of HS in four cases was confirmed by RBC membrane protein electrophoresis and all showed a deficiency of spectrin. The advantage of the EMA dye method are its specificity for membrane disorders, as well as being a simple, user-friendly and rapid method which is inexpensive, provided a flow cytometer is available.

Measurements of the form and size of erythrocytes are needed in the diagnosis of a number of diseases. However, such measurements, if made manually, are a labor-consuming and often inaccurate method, which ensures the determination of a very limited number of parameters. Hardware/software unit MEKOS-C1 enables an automated examination of blood smear, thus speeding up significantly the analysis and ensuring a more complete and accurate information. The possibilities of unit MEKOS-C1 were evaluated for the diagnosis of ovalocytosis. Blood smears of 19 patients from the Russian Pediatric Clinical Hospital, including 8 patients with inherited ovalocytosis, 1 patient with spherocytic ovalocytosis and 10 patients without the disease, were made use of Erythrocytes were isolated in the images of preparations and the contour of each cell was approximated by ellipse. The ratio between ellipse semi-axes served as a measure of erythrocytes' ovality. The mean ratio of semi-axes (RS) and the index of ovalocytosis (IO), i.e. a ratio of the mean maximum diameter to the mean minimal diameter, were calculated for each smear. Manual IO measurements were made in all preparations as a control. Since an additional error can enter the result because of the irregular smear nature and impossibility to standardize completely the technology of smear preparation, the data, obtained from two different smear parts and from parallel preparations, were compared. The reliability, stability and good reproducibility of the automated measurement results were demonstrated. The mean erythrocytes' RS correlated well with IO, obtained manually, and did not virtually differ from RS, measured in the automated manner. The mean RS value of erythrocytes, obtained from patients with inherited ovalocytosis, significantly differed from the control values, which is indicative of a high information density of the discussed parameter. Therefore, RS, when measured automatically, is a reliable and convenient characteristic of erythrocytes' ovality under the conditions of using the ordinary technique of smear preparation.

We have developed a new and simple flow cytometric method to detect damaged red blood cells (RBCs) using anti-Hb in hypotonic solution. We studied a total of 200 patients, including 62 patients with schistocytosis, 8 postsplenectomy patients, and 108 healthy controls. Peripheral blood (2 microl) was stained with phycoerythrin-conjugated (PE) antihemoglobin antibody (anti-Hb) in 0.6% (w/v) NaCl solution, and analyzed by flow cytometry omitting the washing step. The proportion of RBCs stained by anti-Hb was 0.55% (SD +/-0.23%) in normal controls and was significantly higher in patients with schistocytosis (2.95+/-2.95%, p <0.001). Six of 108 blood samples from normal controls and 60 of 62 samples from schistocytosis patients showed > or =1.01% stained RBCs (ie, values > mean+2SD of normal controls). The number of schistocytes counted by microscopic examination correlated with the proportion of RBCs stained by anti-Hb (r = 0.637, p <0.001). The proportions of stained RBCs in blood samples with malaria, spherocytosis, and elliptocytosis were also significantly higher than in normal controls. However, the results in postsplenectomy and iron-deficiency anemia (IDA) patients were not significantly different from the normal controls; the number of schistocytes in postsplenectomy patients was not related to the proportion of RBCs stained by anti-Hb. Based on these findings, flow cytometry of damaged RBCs using anti-Hb in hypotonic solution is a simple, sensitive, and accurate method to detect active hemolysis.

Background: Red blood cells (RBCs) must deform to pass the smallest capillaries of the microcirculation. Available techniques for measuring RBC deformability often provide an indication of the mean deformability. The latter may be decreased either by a slight overall deformability reduction or by the presence of a small fraction of rigid cells. A distinction between these two cases can be made with a RBC-deformability distribution (RBC-DD)., Methods: This paper explores RBC-DDs of healthy individuals and of cells with anomalous mechanical properties (sickle cell disease, dialysis patients, elliptocytosis, and cultivated malaria tropica). The distributions were measured with an automated rheoscope, which uses advanced image analysis techniques to obtain the deformability index of a large number of individual cells subjected to simple shear flow., Results: The RBC-DD of healthy volunteers is close to a normal distribution. In the investigated patients, distributions were markedly different and yielded significant changes in the mean, in the standard deviation, or in both. The presence of hypodeformable and hyperdeformable cell fractions can qualitatively and quantitatively be assessed from the deformability distribution (DD). In elliptocytosis, cells orient differently with respect to the streamlines, compared to normal cells. This causes the DD to be biased., Conclusions: The RBC-DD is a powerful representation to establish subpopulations with anomalous deformability. Fractions of hypodeformable and hyperdeformable cells and the standard deviation of the DD are new and excellent quantitative parameters to assess alterations in RBC deformability.

We identified a Japanese family with a beta-thalassaemia trait and hereditary elliptocytosis (HE). We studied five members of this family. One was normal, one had only the beta-thalassaemia trait, one had heterozygous HE, and two had compound heterozygous beta-thalassaemia trait and HE. The last two had already undergone splenectomy. The molecular profile of beta-thalassaemia was consistent with that of Hb Gunma: codon 127/128CAGGCT(Gln-Ala)--> CCT(Pro). Analysis of erythrocyte membrane proteins revealed a partial deficiency of protein 4.1 in all those with HE, whereas the spectrin content was within the normal range. Each heterozygous family member with either the beta-thalassaemia trait or HE was asymptomatic, whereas the two with both beta-thalassaemia and HE had marked red blood cell deformities and haemolysis. The abnormalities of the red blood cells in patients with the beta-thalassaemia trait might be enhanced by association with HE owing to a protein 4.1 deficiency.

In the erythrocyte, ankyrin is the major adapter protein linking tetramers of band 3 to the spectrin-actin cytoskeleton. This linkage involves a direct interaction between ankyrin and the 14th-15th repeat unit of beta-spectrin. The spectrin cytoskeleton itself is stabilized by the self-association of spectrin heterodimers into tetramers and larger oligomers, a process mediated by the 17th repeat unit of beta-spectrin and a short NH(2) -terminal sequence in alpha-spectrin. The self-association of spectrin and its ankyrin-mediated membrane binding have generally been considered independent events. We now demonstrate that spectrin self-association, the binding of spectrin to ankyrin, and the binding of ankyrin to the 43-kDa cytoplasmic domain of band 3 (cdb3) are coupled in a positively cooperative way. In solution, [(125)I]-labeled ankyrin was found by ND-PAGE3 to enhance the affinity of spectrin self-association by 10-fold. The reciprocal process was also true, in that spectrin tetramers and oligomers bound ankyrin with enhanced affinity relative to dimer spectrin. Saturation of the beta-spectrin self-association site by an NH(2) -terminal 80-kDa alpha-spectrin peptide enhanced the affinity of spectrin dimer for ankyrin, indicating a direct relationship between ankyrin binding and the occupancy of the beta-spectrin self-association site. cdb3 accentuated these cooperative interactions. Several inherited spectrin mutations that cause hemolytic disease but that do not directly destabilize the self-association or ankyrin-binding sites can be explained by these results. Three classes of mutations appear to disrupt cooperative coupling between self-association and ankyrin binding: (i) mutation of the linker sequences that join helices C and A in repeat units that intervene between the two functional sites, mutations that presumably block repeat-to-repeat transfer of conformational information; (ii) mutations in alpha-spectrin repeats 4 to 6 that disrupt the ability of this region to trans-regulate ankyrin binding by the adjacent beta-spectrin repeats 14-15; and (iii) exon-skipping mutations that shorten alpha-spectrin and force repeats 4 to 6 to fall out-of-register with the ankyrin-binding motif in beta-spectrin. Collectively, these results demonstrate a molecular mechanism whereby a membrane receptor can directly promote cytoskeletal assembly., (Copyright 2001 Academic Press.)

The membrane skeleton, a dynamic network of proteins associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Deficiencies or defects in membrane skeletal proteins are associated with inherited disorders of erythrocyte morphology and function. Adducin is one of the proteins localized at the spectrin-actin junction of the membrane skeleton. In this work we show that deficiency of beta-adducin produces an 80% decrease of alpha-adducin and a fourfold up-regulation of gamma-adducin in erythrocytes. beta-Adducin or any other isoform generated by translation of abnormally spliced messenger RNAs could not be detected by our antibodies either in ghosts or in cytoplasm of -/- erythrocytes. Actin levels were diminished in mutant mice, suggesting alterations in the actin-spectrin junctional complexes due to the absence of adducin. Elliptocytes, ovalocytes, and occasionally spherocytes were found in the blood film of -/- mice. Hematological values showed an increase in reticulocyte counts and mean corpuscular hemoglobin concentration, decreased mean corpuscular volume and hematocrit, and normal erythrocyte counts that, associated to splenomegaly, indicate that the mice suffer from mild anemia with compensated hemolysis. These modifications are due to a loss of membrane surface and dehydration that result in an increase in the osmotic fragility of red blood cells. The marked alteration in osmotic fragility together with the predominant presence of elliptocytes is reminiscent of the human disorder called spherocytic hereditary elliptocytosis. Our results suggest that the amount of adducin remaining in the mutant animals (presumably alphagamma adducin) could be functional and might account for the mild phenotype. (Blood. 2000;95:3978-3985)

During the past years many studies on erythrocyte membrane structure and function have allowed for exponential development of this field. Inborn errors of red cell membrane include a variety of disorders characterized by alterations of the membrane skeleton or other membrane proteins. Most recently, many studies have been published using methods from molecular biology allowing for clarification of genotype phenotype relationship. This review outlines in details clinical aspects and organisational consequences of defects molecules of membrane proteins and skeleton and their contributions to the pathophysiology of hemolytic anaemia.

This review describes some of the naturally occurring band 3 (AEI) variants and their association with disease. Southeast Asian Ovalocytic (SAO) band 3, an inactive and misfolded protein, is probably only maintained in certain populations because it provides protection against the cerebral form of malaria. Many mutations that cause instability of band 3, either at the mRNA or protein level, result in hereditary spherocytosis (HS). Some polymorphisms alter amino acid residues in the extracellular loops of band 3 and are associated with blood group antigens. A truncated form of AEI is expressed in kidney cells and certain AEI mutations are associated with distal renal tubular acidosis (dRTA). The molecular basis of these variants and their effect on the structure and function of band 3 are discussed. The association between band 3 and glycophorin A (GPA) and the structure/function changes of band 3 in the absence of GPA are also described.