Monovalent cation leaks in human red cells caused by single amino-acid substitutions in the transport domain of the band 3 chloride-bicarbonate exchanger, AE1.

We identified 11 human pedigrees with dominantly inherited hemolytic anemias in both the hereditary stomatocytosis and spherocytosis classes. Affected individuals in these families had an increase in membrane permeability to Na and K that is particularly marked at 0 degrees C. We found that disease in these pedigrees was associated with a series of single amino-acid substitutions in the intramembrane domain of the erythrocyte band 3 anion exchanger, AE1. Anion movements were reduced in the abnormal red cells. The 'leak' cation fluxes were inhibited by SITS, dipyridamole and NS1652, chemically diverse inhibitors of band 3. Expression of the mutated genes in Xenopus laevis oocytes induced abnormal Na and K fluxes in the oocytes, and the induced Cl transport was low. These data are consistent with the suggestion that the substitutions convert the protein from an anion exchanger into an unregulated cation channel.

Guidelines for the diagnosis and management of hereditary spherocytosis--2011 update.

Guidelines on hereditary spherocytosis (HS) published in 2004 (Bolton-Maggs et al, 2004) are here replaced to reflect changes in current opinion on the surgical management, (particularly the indications for concomitant splenectomy with cholecystectomy in children with mild HS, and concomitant cholecystectomy with splenectomy in those with asymptomatic gallstones). Further potential long term hazards of splenectomy are now recognised. Advances have been made in our understanding of the biochemistry of the red cell membrane which underpins the choice of tests. Biochemical assays of membranes proteins and genetic analysis may be indicated (rarely) to diagnose atypical cases. The diagnostic value of the eosin-5-maleimide (EMA) binding test has been validated in a number of studies with understanding of its limitations.

Toward the harmonization of result presentation for the eosin-5'-maleimide binding test in the diagnosis of hereditary spherocytosis.

BACKGROUND: The eosin-5'-maleimide (EMA) Binding test measures reduced mean channel fluorescence (MCF) reading of EMA-labeled red cells (EMA-RBCs) from patients with hereditary spherocytosis (HS). Reporting test results can be either in the actual MCF reading or as a ratio by normalization of the test MCF result to the mean MCF value of six normal controls. The latter format has potential for universal reporting. METHODS: We analyzed three years' archival MCF data from HS and non-HS patient groups for establishment of reference ranges of ratios for normal adults and HS. A prospective study used FC500 and FACS Canto II cytometers to analyze contemporaneously EMA-RBCs from several patient groups and normal donors. Statistical analyses of the prospective data determined the cut-off values, and the sensitivity and specificity for HS respectively for the MCF and the ratio result presentations. The effect of using fewer than six normal controls for the ratio denominator was explored. RESULTS: The FC500 gave a mean ratio of 0.782 (SD=0.086) in HS patients with an optimal cut-off ratio of 0.918 (98.7% specificity, 95.6% sensitivity), and gray area ratio of 0.868-0.918. The Canto II gave a mean ratio of 0.774 (SD=0.085) with an optimal cut-off ratio of 0.925 (97.1% specificity and 100% sensitivity), and gray area ratio of 0.859-0.925. CONCLUSION: Harmonization of result presentation is feasible with no apparent constraint by instrument design. Interpretation of gray-area data requires an assessment of patient's clinical presentation and family history or performing a family study.

The diagnostic challenge of congenital dyserythropoietic anaemia: two cases of 'CDA type II'.

The congenital dyserythropoietic anaemias (CDAs) are a group of rare hereditary disorders characterised by ineffective erythropoiesis and morphological abnormalities in the erythroblasts. Patients may present with jaundice or with symptoms of anaemia, gall stones or iron overload. The diagnosis can be challenging and cases have been confused with haemolytic anaemia, haemochromatosis or a haemoglobinopathy. A delayed diagnosis can lead to inappropriate treatment or delayed management of iron overload. We present two patients previously diagnosed as CDA type II in whom the diagnosis was revised to CDA type I and to hereditary spherocytosis. The conditions are compared and the approach to diagnosis is discussed.

Towards the harmonization of result presentation for the eosin-5'- maleimide (EMA) binding test in the diagnosis of hereditary spherocytosis.

Background: The EMA Binding test measures reduced mean channel fluorescence (MCF) reading of EMA-labeled red cells (EMA-RBCs) from patients with hereditary spherocytosis (HS). Reporting test results can be either in the actual MCF reading or as a ratio by normalization of the test MCF result to the mean MCF value of 6 normal controls. The latter format has potential for universal reporting. Methods: We analyzed three years' archival MCF data from HS and non-HS patient groups for establishment of reference ranges of ratios for normal adults and HS. A prospective study used FC500 and FACS Canto II cytometers to analyze contemporaneously EMA-RBCs from several patient groups and normal donors. Statistical analyses of the prospective data determined the cut-off values, and the sensitivity and specificity for HS respectively for the MCF and the ratio result presentations. The effect of using fewer than six normal controls for the ratio denominator was explored. Results:. The FC500 gave a mean ratio of 0.782 (SD = 0.086) in HS patients with an optimal cut-off ratio of 0.918 (98.7% specificity, 95.6% sensitivity), and grey area ratio of 0.868 - 0.918. The Canto II gave a mean ratio of 0.774 (SD = 0.085) with an optimal cut-off ratio of 0.925 (97.1% specificity and 100% sensitivity), and grey area ratio of 0.859 - 0.925. Conclusion: Harmonization of result presentation is feasible with no apparent constraint by instrument design. Interpretation of grey-area data requires an assessment of patient's clinical presentation and family history or performing a family study. © 2014 Clinical Cytometry Society.

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

BACKGROUND: 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. METHODS: Flow cytometry was used to analyze EMA-labeled RBCs. Membrane protein defects and spectrin variants were identified by SDS-polyacrylamide gel electrophoresis. RESULTS: 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. CONCLUSIONS: 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.

Phenotype and mRNA expression of syncytiotrophoblast microparticles isolated from human placenta.

Fetal and neonatal alloimmune thromboctyopenia due to maternal human platelet antigen (HPA)-1a antibodies affects primigravidas. Immunization must occur early in pregnancy before fetal platelets enter maternal blood via fetomaternal hemorrhage. The HPA-1a antigen is located on platelet glycoprotein (GP)IIIa (CD61, beta3 integrin), which is also present on the placental syncytiotrophoblast (ST) and in direct contact with maternal blood. Since ST debris is shed into maternal blood during pregnancy, this material might be immunogenic in vivo. For experimental purposes, we prepared and characterized ST microparticles (STMPs) in vitro from term placentas. Phenotype analysis by flow cytometry and Western blotting showed that STMP expressed more placental alkaline phosphatase (PLAP) than GPIIIa. Quantitative real-time PCR demonstrated expression of human placental lactogen (HPL), human chorionic gonadotrophin (HCG), and GPIIIa by STMP, in the order HPL > HCG > GPIIIa. PLAP, HPL, and HCG are trophoblast-specific proteins. These STMPs may be a useful model for studying the natural ST debris in plasma of pregnant women.

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.

Absence of CD47 in protein 4.2-deficient hereditary spherocytosis in man: an interaction between the Rh complex and the band 3 complex.

We present data on a patient of South Asian origin with recessive hereditary spherocytosis (HS) due to absence of protein 4.2 [4.2 (-) HS]. Protein 4.2 cDNA sequence analysis showed the presence of a novel 41-bp frameshift deletion that predicts a truncated peptide designated protein 4.2 Hammersmith. Quantitative reverse transcription-polymerase chain reaction indicated that the mutant mRNA was unstable. Sequencing of protein 4.2 genomic DNA revealed that the deletion stems from aberrant splicing. The proband was homozygous for a G>T substitution at position 1747 (cDNA numbering) that activates a cryptic acceptor splice site within exon 11 of the protein 4.2 gene (EPB42). The proband's mother was found to be heterozygous for this substitution. Unlike protein 4.2 null mice, the proband's red cells showed no evidence for abnormal cation permeability. Quantitation of red cell membrane proteins was carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and flow cytometric measurement. CD47, a protein associated with the Rh complex, was markedly reduced to about 1% (in the proband) and 65% (in the mother) that found in healthy controls. The Rh-associated glycoprotein migrated with a higher than normal apparent molecular weight on SDS-PAGE. There was no obvious reduction in Rh polypeptides. These observations indicate that protein 4.2 and CD47 interact in the human red cell membrane. They provide further evidence for an association between the band 3 complex (band 3, ankyrin, protein 4.2, glycophorin A) and the Rh complex (Rh-associated glycoprotein, Rh polypeptides, glycophorin B, CD47, LW) and define a point of attachment between the Rh complex and the red cell cytoskeleton.