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Background: There is a marked discrepancy between SARS-CoV-2 seroprevalence and COVID-19 cases and deaths in Africa. MAIN: SARS-CoV-2 stimulates humoral and cellular immunity systems, as well as mitogen-activated protein kinase (MAPK) and nuclear NF-kB signalling pathways, which regulate inflammatory gene expression and immune cell differentiation. The result is pro-inflammatory cytokines release, hyperinflammatory condition, and cytokine storm, which provoke severe lung alterations that can lead to multi-organ failure in COVID-19. Multiple genetic and immunologic factors may contribute to the severity of COVID-19 in African individuals when compared to the rest of the global population. In this article, the role of malaria, NF-kB and MAPK pathways, caspase-12 expression, high level of LAIR-1-containing antibodies, and differential glycophorins (GYPA/B) expression in COVID-19 are discussed., Conclusion: Understanding pathophysiological mechanisms can help identify target points for drugs and vaccines development against COVID-19. To our knowledge, this is the first study that explores this link and proposes a biological and molecular answer to the epidemiologic discrepancy in COVID-19 in Africa., (Copyright © 2021. Published by Elsevier B.V.)

Background: GP.Mur belongs to the GP(B-A-B) hybrid glycophorin family, which is the most common hybrid glycophorin in Southeast Asia. Antibodies against GP.Mur may cause a clinically significant haemolytic disease of the foetus and newborn (HDFN) although, so far, not many cases have been reported in mainland China., Materials and Methods: Two Chinese women with a history of severe hydrops foetalis were seen in our centre. Alloantibody identification and GYP.Mur genotyping analysis were used for prenatal evaluation. Intrauterine transfusion was performed in two pregnancies in case 1. The features of these two women are described and literature-reported cases of HDFN related to antibodies against GP.Mur are summarised., Results: The phenotype of both mothers was Mi a - Mur-, while the fathers' was Mi a + Mur+ with a heterozygous GYP.Mur hybrid gene as determined by a high-resolution melting method of genotyping. In case 1, the antibodies against GP.Mur were detected in the mother's serum and the cord blood of two foetuses. Fortunately, the latest foetus was successfully saved after intrauterine transfusion. In case 2, hydrops foetalis occurred in the first two pregnancies, but the risk of HDFN was excluded for the third foetus because of the GP.Mur negative phenotype. The literature review showed that 68.8% (11/16) of the reported cases of HDFN related to antibodies against GP.Mur occurred in the Chinese population, and that 37.5% (6/16) of them were cases of severe HDFN., Discussion: More cases of severe HDFN caused by antibodies against GP.Mur are presumably undetected as GP.Mur cells are not included in the panel of obligatory screening tests in most Southeast Asian countries including mainland China. The high-resolution melting method for GYP.Mur genotyping and zygosity detection is helpful in prenatal management.

Background: MNS is one of the highly polymorphic blood groups comprising many antigens generated by genomic recombination among the GYPA, GYPB, and GYPE genes as well as by single-nucleotide changes. We report a patient with red blood cell (RBC) antibody against an unknown low-frequency antigen, tentatively named SUMI, and investigated its carrier molecule and causal gene., Study Design and Methods: Standard serologic tests, including enzyme tests, were performed. Monoclonal anti-SUMI-producing cells (HIRO-305) were established by transformation and hybridization methods using lymphocytes from a donor having anti-SUMI. SUMI+ RBCs were examined by immunocomplex capture fluorescence analysis (ICFA) using HIRO-305 and murine monoclonal antibodies against RBC membrane proteins carrying blood group antigens. Genomic DNA was extracted from whole blood, and the GYPA gene was analyzed by polymerase chain reactions and Sanger sequencing., Results: Serologic screening revealed that 23 of the 541,522 individuals (0.0042%) were SUMI+, whereas 1351 of the 10,392 individuals (13.0%) had alloanti-SUMI. SUMI antigen was sensitive to ficin, trypsin, pronase, and neuraminidase, but resistant to α-chymotrypsin and sulfydryl-reducing agents. ICFA revealed that the SUMI antigen was carried on glycophorin A (GPA). According to Sanger sequencing and cloning, the SUMI+ individuals had a GYPA*M allele with c.91A>C (p.Thr31Pro), which may abolish the O-glycan attachment site., Conclusions: The new low-frequency antigen SUMI is carried on GPA encoded by the GYPA*M allele with c.91A>C (p.Thr31Pro). Neuraminidase sensitivity suggests that glycophorin around Pro31 are involved in the SUMI determinant., (© 2020 AABB.)

Protein "AND-gate" systems, in which a ligand acts only on cells with two different receptors, direct signaling activity to a particular cell type and avoid action on other cells. In a bifunctional AND-gate protein, the molecular geometry of the protein domains is crucial. Here we constructed a tissue-targeted erythropoietin (EPO) that stimulates red blood cell (RBC) production without triggering thrombosis. The EPO was directed to RBC precursors and mature RBCs by fusion to an anti-glycophorin A antibody V region. Many such constructs activated EPO receptors in vitro and stimulated RBC and not platelet production in mice but nonetheless enhanced thrombosis in mice and caused adhesion between RBCs and EPO-receptor-bearing cells. On the basis of a protein-structural model of the RBC surface, we rationally designed an anti-glycophorin-EPO fusion that does not induce cell adhesion in vitro or enhance thrombosis in vivo . Thus, mesoscale geometry can inform the design of synthetic-biological systems.

GP.Mur antigen belongs to the MNSs system and the corresponding antibody is called as anti-Mia antibody. Anti-Mia antibody is a clinically significant antibody capable of causing haemolytic disease of the new born (HDFN) and intravascular haemolytic transfusion reactions. Literature on anti-Mia antibody from India is very limited. We report here a case of anti-Mia antibody in a multi-transfused patient from India., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The separation of plasma from blood cells is critical for the accuracy of blood tests because cellular fractions can create discrepancies in analysis. The most common method to separate blood cells from the liquid part of the blood is centrifugation, which is not always applicable in resource-constrained areas and countries. In this study, we describe the generation of monoclonal antibodies (mAbs) against glycophorin A (GPA) of human erythrocytes. BALB/c mice were immunized with human erythrocytes followed by purified GPA. The splenocytes of the immunized mice were fused with Sp2/0 myeloma cells by hybridoma technique. Hybridoma clones were screened by hemagglutination assay and enzyme-linked immunosorbent assay (ELISA). Six hybridoma clones were obtained and subcloned. The characterization of the purified mAbs demonstrates that they are able to bind and retain erythrocytes in hemagglutination assay. Furthermore, one of the mAbs 1A9 recognizes purified GPA in ELISA, whereas the other mAb 1G7 is able to immunoprecipitate GPA from human erythrocyte lysates, and a band of 38 kDa is detected. In conclusion, the anti-GPA mAbs are useful tools in developing a quick and easy way to separate blood plasma from whole blood for clinical tests, and in developing bi-specific antibodies for other clinical applications.

Erythrocyte Binding Antigen of 175 kDa (EBA-175) has a well-defined role in binding to glycophorin A (GpA) during Plasmodium falciparum invasion of erythrocytes. However, EBA-175 is shed post invasion and a role for this shed protein has not been defined. We show that EBA-175 shed from parasites promotes clustering of RBCs, and EBA-175-dependent clusters occur in parasite culture. Region II of EBA-175 is sufficient for clustering RBCs in a GpA-dependent manner. These clusters are capable of forming under physiological flow conditions and across a range of concentrations. EBA-175-dependent RBC clustering provides daughter merozoites ready access to uninfected RBCs enhancing parasite growth. Clustering provides a general method to protect the invasion machinery from immune recognition and disruption as exemplified by protection from neutralizing antibodies that target AMA-1 and RH5. These findings provide a mechanistic framework for the role of shed proteins in RBC clustering, immune evasion, and malaria., Competing Interests: MP, NS, YA, AO, AJ, DG, NT No competing interests declared

Moderate anemia is associated with increased mortality and morbidity, including acute kidney injury (AKI), in surgical patients. A red blood cell (RBC)-specific antibody model was utilized to determine whether moderate subacute anemia could result in tissue hypoxia as a potential mechanism of injury. Cardiovascular and hypoxic cellular responses were measured in transgenic mice capable of expressing hypoxia-inducible factor-1α (HIF-1α)/luciferase activity in vivo. Antibody-mediated anemia was associated with mild intravascular hemolysis (6 h) and splenic RBC sequestration ( day 4), resulting in a nadir hemoglobin concentration of 89 ± 13 g/l on day 4. At this time point, renal tissue oxygen tension (P t O 2 ) was decreased in anemic mice relative to controls (13.1 ± 4.3 vs. 20.8 ± 3.7 mmHg, P < 0.001). Renal tissue hypoxia was associated with an increase in HIF/luciferase expression in vivo ( P = 0.04) and a 20-fold relative increase in renal erythropoietin mRNA transcription ( P < 0.001) but no increase in renal blood flow ( P = 0.67). By contrast, brain P t O 2 was maintained in anemic mice relative to controls (22.7 ± 5.2 vs. 23.4 ± 9.8 mmHg, P = 0.59) in part because of an increase in internal carotid artery blood flow (80%, P < 0.001) and preserved cerebrovascular reactivity. Despite these adaptive changes, an increase in brain HIF-dependent mRNA levels was observed (erythropoietin: P < 0.001; heme oxygenase-1: P = 0.01), providing evidence for subtle cerebral tissue hypoxia in anemic mice. These data demonstrate that moderate subacute anemia causes significant renal tissue hypoxia, whereas adaptive cerebrovascular responses limit the degree of cerebral tissue hypoxia. Further studies are required to assess whether hypoxia is a mechanism for acute kidney injury associated with anemia.

The MEK/ERK pathway is found to be important in regulating different biological processes such as proliferation, differentiation and survival in a wide variety of cells. However, its role in self-renewal of haematopoietic stem cells is controversial and remains to be clarified. The aim of this study was to understand the role of MEK/ERK pathway in ex vivo expansion of mononuclear cells (MNCs) and purified CD34 + cells, both derived from human umbilical cord blood (hUCB). Based on our results, culturing the cells in the presence of an inhibitor of MEK/ERK pathway-PD0325901 (PD)-significantly reduces the expansion of CD34 + and CD34 +  CD38 - cells, while there is no change in the expression of stemness-related genes (HOXB4, BMI1). Moreover, in vivo analysis demonstrates that PD reduces engraftment capacity of ex vivo expanded CD34 + cells. Notably, when ERK pathway is blocked in UCB-MNCs, spontaneous erythroid differentiation is promoted, found in concomitant with increasing number of burst-forming unit-erythroid colony (BFU-E) as well as enhancement of erythroid glycophorin-A marker. These results are in total conformity with up-regulation of some erythroid enhancer genes (TAL1, GATA2, LMO2) and down-regulation of some erythroid repressor genes (JUN, PU1) as well. Taken together, our results support the idea that MEK/ERK pathway has a critical role in achieving the correct balance between self-renewal and differentiation of UCB cells. Also, we suggest that inhibition of ERK signalling could likely be a new key for erythroid induction of UCB-haematopoietic progenitor cells., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: Autoimmune hemolytic anemia (AIHA) due to anti-En a has been previously reported in association with massive intravascular hemolysis, disseminated intravascular coagulation, and fatal outcomes. Here we report a case of successfully treated AIHA due to anti-En a ., Case Report: A 69-year-old male with a past medical history of cirrhosis due to nonalcoholic steatohepatitis status post-orthotopic liver transplant presented with 1-month history of progressive anemia. At presentation, his hemoglobin (Hb) was 5.6 g/dL, hematocrit (Hct) 16%, reticulocytes 0.3%, direct bilirubin (bili) 4 g/dL, lactate dehydrogenase 533 units/L (reference, 125-220 units/L), and haptoglobin 254 mg/dL (reference, 40-273 mg/dL). Blood bank testing revealed an autoantibody present in his plasma and a direct antiglobulin test positive for immunoglobulin G (IgC) but negative for complement. He received 1 unit of an incompatible blood group O phenotypically matched red blood cell unit., Results: Over the course of the next 5 days, the Hb and Hct decreased to 4.1 g/dL and 12%, respectively, direct bili increased to 12.3 mg/day, reticulocytes slightly increased to 0.9%, and haptoglobin decreased to less than 8 mg/dL. Marrow study showed a hypercellular marrow with erythroid hyperplasia. Additional workup performed at a reference laboratory identified an anti-En a autoantibody. He received prednisone and weekly rituximab infusions and was monitored weekly. At the 2-month visit, Hb and Hct were 10 g/dL and 32%, respectively., Conclusion: Unlike two of the previously reported fatal cases of AIHA with anti-En a specificity, this 69-year-old male treated with weekly rituximab infusion underwent clinical recovery and significant anemia improvement., (© 2017 AABB.)

Healthy blood neutrophils are functionally quiescent in the bloodstream, have a short lifespan, and exit the circulation to carry out innate immune functions, or undergo rapid apoptosis and macrophage-mediated clearance to mitigate host tissue damage. Limitation of unnecessary intravascular neutrophil activation is also important to prevent serious inflammatory pathologies. Because neutrophils become easily activated after purification, we carried out ex vivo comparisons with neutrophils maintained in whole blood. We found a difference in activation state, with purified neutrophils showing signs of increased reactivity: shedding of l-selectin, CD11b upregulation, increased oxidative burst, and faster progression to apoptosis. We discovered that erythrocytes suppressed neutrophil activation ex vivo and in vitro, including reduced l-selectin shedding, oxidative burst, chemotaxis, neutrophil extracellular trap formation, bacterial killing, and induction of apoptosis. Selective and specific modification of sialic acid side chains on erythrocyte surfaces with mild sodium metaperiodate oxidation followed by aldehyde quenching with 4-methyl-3-thiosemicarbazide reduced neutrophil binding to erythrocytes and restored neutrophil activation. By enzyme-linked immunosorbent assay and immunofluorescence, we found that glycophorin A, the most abundant sialoglycoprotein on erythrocytes, engaged neutrophil Siglec-9, a sialic acid-recognizing receptor known to dampen innate immune cell activation. These studies demonstrate a previously unsuspected role for erythrocytes in suppressing neutrophils ex vivo and in vitro and help explain why neutrophils become easily activated after separation from whole blood. We propose that a sialic acid-based "self-associated molecular pattern" on erythrocytes also helps maintain neutrophil quiescence in the bloodstream. Our findings may be relevant to some prior experimental and clinical studies of neutrophils., (© 2017 by The American Society of Hematology.)

Dendritic cells (DCs) are increasingly being used for multiple applications and are useful tools for many immunotherapeutic strategies. The understanding of the possible impact of the DCs-generation methods on the biological capacities of these cells is therefore essential. Although the immunomagnetic separation is regarded as a fast and accurate method yielding cells with the high purity and efficiency, still little is known about its impact on the properties of the generated DCs. The aim of this study was to compare the morphology of the monocyte derived dendritic cells (MoDCs), generated from monocytes selected with anti-CD14 mAbs (positive separation) and treated with anti-CD3, -CD7, -CD16, -CD19, -CD56, -CD123, glycophorin A (negative separation), using laser scanning microscopy. We found that the type of the immunomagnetic separation method used strongly influences the shape and cell dimension of the MoDCs. We observed that the height of both immature and LPS-matured DCs generated from monocytes isolated by negative separation was significantly higher compared to the cells obtained by positive separation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Alloimmunization against high-frequency erythrocyte antigens is a problematic situation in terms of laboratory diagnosis, transfusion and obstetrical management. We report the case of a pregnant woman alloimmunized against public Ag. We detail the difficulties of alloantibody (Ab) identification and transfusion management of the deliveries. A 29-year-old pregnant woman was hospitalized in gynecology and obstetrics departments at 36 weeks of gestation for assessment of hydrops fetalis. Antibody identification test revealed the presence of a pan-reactive antibody. Investigations realized in CNRGS (Paris) concluded in anti-GE2+anti-RH3+autoantibody. The red cell phenotype was GE: -2,3. A therapeutic interruption of the pregnancy was indicated. A program of autologous transfusion was organized with withdrawal of 2 units of blood. The 2nd pregnancy took place normally. Before delivery, an autologous blood reserve consisting of 2 red cell packs and 2 fresh frozen plasma was withdrawn and transfused after delivery. The management of anti-public alloimmunization poses several problems. The first one is of diagnostic nature with, on the one hand, the difficulty of Ab identification by the available red cell panels and, on the other hand, the possible presence of alloantibodies of transfusional or obstetric interest masked by anti-public Ab. The second is represented by transfusional care of these patients. In the absence of a national reserve of frozen rare blood, the autologous transfusion remains the only alternative. However, it can answer only a limited number of indications and only in case of moderate blood loss., (Copyright © 2016. Published by Elsevier SAS.)

Rosetting phenomenon has been linked to malaria pathogenesis. Although rosetting occurs in all causes of human malaria, most data on this subject has been derived from Plasmodium falciparum. Here, we investigate the function and factors affecting rosette formation in Plasmodium vivax. To achieve this, we used a range of novel ex vivo protocols to study fresh and cryopreserved P vivax (n = 135) and P falciparum (n = 77) isolates from Thailand. Rosetting is more common in vivax than falciparum malaria, both in terms of incidence in patient samples and percentage of infected erythrocytes forming rosettes. Rosetting to P vivax asexual and sexual stages was evident 20 hours postreticulocyte invasion, reaching a plateau after 30 hours. Host ABO blood group, reticulocyte count, and parasitemia were not correlated with P vivax rosetting. Importantly, mature erythrocytes (normocytes), rather than reticulocytes, preferentially form rosetting complexes, indicating that this process is unlikely to directly facilitate merozoite invasion. Although antibodies against host erythrocyte receptors CD235a and CD35 had no effect, Ag-binding fragment against the BRIC 4 region of CD236R significantly inhibited rosette formation. Rosetting assays using CD236R knockdown normocytes derived from hematopoietic stem cells further supports the role of glycophorin C as a receptor in P vivax rosette formation.

Background: Invasion of red blood cells (RBCs) is one of the critical points in the lifecycle of Babesia. The parasite does not invade other host cells. Earlier work has shown that GPA and GPB function as putative receptors during parasite invasion. The primary focus of this study was the delineation of parasite-binding domains on GPA and GPB., Study Design and Methods: The assay of choice to validate molecules that participate in invasion is an inhibition of invasion assay, in which changes in parasitemia are assessed relative to a wild-type assay (no inhibitors). Inhibition of invasion can be achieved by modification of different components of the assay or by the addition of competitors of the molecules that participate in invasion. In this study purified antibody fragments to various domains on GPA and GPB were tested for magnitude of inhibition of parasite invasion. Effects on invasion were monitored by assessment of Giemsa-stained smears every 24 hours., Results: Among 10 selected antibodies directed at various epitopes on GPA and GPB, antibodies directed against GPA(M) epitopes had the most severe effect (up to 35%) on inhibition of invasion, followed by antibodies directed against GPB(S) epitope (up to 24%)., Conclusion: This study confirms the role of RBC glycophorins A and B in Babesia divergens invasion and shows that the GPA(M) and GPB(S) epitopes are likely to play an important role in the entry process., (© 2013 American Association of Blood Banks.)

Background: Therapy for patients with autoimmune hemolytic anemia (AHA) remains a major challenge. Patients with glycophorin A (GPA)-specific immunoglobulin G antibodies can have severe hemolysis, which may occur by mechanisms independent from traditional macrophage-dependent Fcγ receptor (FcγR)-mediated extravascular hemolysis. As intravenous immune globulin (IVIG) is known to display its beneficial effects in FcγR-mediated cytopenias, and IVIG responses in AHA are inconsistent at best, we sought to gain insight into the mechanism of anemia by a GPA complex-specific monoclonal antibody (TER119) in a mouse model of immune hemolytic anemia and evaluate the therapeutic effect of IVIG., Study Design and Methods: The anemic effect of the TER119 antibody was studied in vitro by incubation of mouse RBC with the antibody and in vivo by infusing the antibody into normal mice versus mice genetically deficient for the Fc receptor γ chain (Fcγ), complement C3, mice naturally deficient in complement C5, and splenectomized mice. IVIG efficacy in anemia was determined by treating mice with an intensive IVIG dosing regimen., Results: The TER119-mediated anemia was independent of classical FcγR-, C3-, and C5-dependent mechanisms, but occurred by a mechanism consistent with RBC agglutination. In accordance with agglutination, the presence of the spleen accelerated the anemia observed but anemia could still occur in splenectomized mice. IVIG did not significantly affect the induction of anemia by TER119., Conclusion: The mechanism of anemia induced by AHA-causing antibodies may be an important factor to consider in the response to therapy with IVIG., (© 2013 American Association of Blood Banks.)

PfEBA175 has an important role in the invasion of human erythrocytes by Plasmodium falciparum and is therefore considered a high priority blood-stage malaria vaccine candidate. PfEBA175 mediates adhesion to erythrocytes through binding of the Duffy-binding-like (DBL) domains in its extracellular domain to Neu5Acα2-3Gal displayed on the O-linked glycans of glycophorin-A (GYPA). Because of the difficulties in expressing active full-length (FL) P. falciparum proteins in a recombinant form, previous analyses of the PfEBA175-GYPA interaction have largely focused on the DBL domains alone, and therefore they have not been performed in the context of the native protein sequence. Here, we express the entire ectodomain of PfEBA175 (PfEBA175 FL) in soluble form, allowing us to compare the biochemical and immunological properties with a fragment containing only the tandem DBL domains ("region II," PfEBA175 RII). Recombinant PfEBA175 FL bound human erythrocytes in a trypsin and neuraminidase-sensitive manner and recognized Neu5Acα2-3Gal-containing glycans, confirming its biochemical activity. A quantitative binding analysis showed that PfEBA175 FL interacted with native GYPA with a KD ∼0.26 μM and is capable of self-association. By comparison, the RII fragment alone bound GYPA with a lower affinity demonstrating that regions outside of the DBL domains are important for interactions with GYPA; antibodies directed to these other regions also contributed to the inhibition of parasite invasion. These data demonstrate the importance of PfEBA175 regions other than the DBL domains in the interaction with GYPA and merit their inclusion in an EBA175-based vaccine.

Background: Continuing efforts in development of non-invasive prenatal genetic tests have focused on the isolation of fetal nucleated red blood cells (NRBCs) from maternal blood for decades. Because no fetal cell-specific antibody has been described so far, the present study focused on the development of monoclonal antibodies (mAbs) to antigens that are expressed exclusively on fetal NRBCs., Methods: Mice were immunized with fetal erythroid cell membranes and hybridomas screened for Abs using a multi-parameter fluorescence-activated cell sorting (FACS). Selected mAbs were evaluated by comparative FACS analysis involving Abs known to bind erythroid cell surface markers (CD71, CD36, CD34), antigen-i, galactose, or glycophorin-A (GPA). Specificity was further confirmed by extensive immunohistological and immunocytological analyses of NRBCs from umbilical cord blood and fetal and adult cells from liver, bone marrow, peripheral blood, and lymphoid tissues., Results: Screening of 690 hybridomas yielded three clones of which Abs from 4B8 and 4B9 clones demonstrated the desired specificity for a novel antigenic structure expressed on fetal erythroblast cell membranes. The antigenic structure identified is different from known surface markers (CD36, CD71, GPA, antigen-i, and galactose), and is not present on circulating adult erythroid cells, except for occasional detectability in adult bone marrow cells., Conclusions: The new mAbs specifically bind the same or highly overlapping epitopes of a surface antigen that is almost exclusively expressed on fetal erythroid cells. The high specificity of the mAbs should facilitate development of simple methods for reliable isolation of fetal NRBCs and their use in non-invasive prenatal diagnosis of fetal genetic status., (© 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Glycophorin C (GPC) is necessary in the maintenance of red blood cell structure. Severe autoimmune hemolytic anemia and hemolytic disease of the fetus and newborn (HDFN) have been associated with Gerbich (Ge) blood group system antigens expressed on GPC. Previous in vitro studies with cord blood progenitor cells have shown that anti-Ge suppresses erythropoiesis., Study Design and Methods: Here, we evaluated the K562 erythroleukemic cell line to study the cellular effects of a murine anti-GPC. Cell proliferation was evaluated after treatment with anti-GPC. Flow cytometry was used to evaluate exofacial phosphatidylserine (PS) expression and cell viability (propidium iodide binding). Cell morphology was evaluated under light microscopy with cytospin preparations stained with May-Grünwald Giemsa., Results: Anti-GPC dramatically inhibited K562 proliferation and increased PS expression, consistent with cytoplasmic blebbing, suggesting evidence of apoptosis. Z-VAD-FMK, an inhibitor of classical apoptosis, was unable to reverse the suppressive effect of anti-GPC. However, hemin was able to attenuate growth suppression., Conclusion: Together, the data suggest that anti-GPC suppresses erythroid proliferation through the induction of nonclassical apoptosis., (© 2012 American Association of Blood Banks.)

The preparation of a V(H)H (nanobody) named IH4 that recognizes human glycophorin A (GPA) is described. IH4 was isolated by screening a library prepared from the lymphocytes of a dromedary immunized by human blood transfusion. Phage display and panning against GPA as the immobilized antigen allowed isolating this V(H)H. IH4, representing 67% of the retrieved V(H)H sequences, was expressed as a soluble correctly folded protein in SHuffle Escherichia coli cells, routinely yielding approximately 100 mg/L fermentation medium. Because IH4 recognizes GPA independently of the blood group antigens, it recognizes red cells of all humans with the possible exception of those with some extremely rare genetic background. The targeted linear epitope comprises the GPA Y52PPE55 sequence. Based on surface plasmon resonance results, the dissociation constant of the IH4-GPA equilibrium is 33 nM. IH4 is a stable protein with a transition melting temperature of 75.8 °C (measured by differential scanning calorimetry). As proof of concept, we fused HIV p24 to IH4 and used the purified construct expressed in E. coli to show that IH4 was amenable to the preparation of autologous erythrocyte agglutination reagents: reconstituted blood prepared with serum from an HIV-positive patient was readily agglutinated by the addition of the bifunctional reagent., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Alloimmunization to antigens on transfused red blood cells (RBCs) represents a major barrier to chronic transfusion. In extreme cases of multiple alloimmunization, clinicians may be faced with the decision of transfusing incompatible RBCs or risking death from lack of transfusion. The disastrous results of hemolytic transfusion reactions are well understood, and major pathways of clearance have been described. However, well described but poorly understood is the survival of a subset of incompatible donor RBCs during hemolysis, despite antibody binding., Study Design and Methods: We utilize a tractable murine model of incompatible transfusion in which RBCs from transgenic donor mice expressing human glycophorin A (hGPA) are transfused into recipients passively immunized with anti-hGPA., Results: As in humans, the majority of RBCs are cleared but a subset of incompatible donor RBCs persist in circulation, despite being bound by antibodies. Data contained herein reject the hypothesis that lack of clearance is due to insufficient antibody or overwhelming of phagocytic machinery; rather, we establish that surviving RBCs represent a distinct population resistant to clearance., Conclusions: These studies demonstrate that surviving RBCs during incompatible transfusion can represent a population that is resistant to clearance., (© 2012 American Association of Blood Banks.)

Disrupting erythrocyte invasion by Plasmodium falciparum is an attractive approach to combat malaria. P. falciparum EBA-175 (PfEBA-175) engages the host receptor Glycophorin A (GpA) during invasion and is a leading vaccine candidate. Antibodies that recognize PfEBA-175 can prevent parasite growth, although not all antibodies are inhibitory. Here, using x-ray crystallography, small-angle x-ray scattering and functional studies, we report the structural basis and mechanism for inhibition by two PfEBA-175 antibodies. Structures of each antibody in complex with the PfEBA-175 receptor binding domain reveal that the most potent inhibitory antibody, R217, engages critical GpA binding residues and the proposed dimer interface of PfEBA-175. A second weakly inhibitory antibody, R218, binds to an asparagine-rich surface loop. We show that the epitopes identified by structural studies are critical for antibody binding. Together, the structural and mapping studies reveal distinct mechanisms of action, with R217 directly preventing receptor binding while R218 allows for receptor binding. Using a direct receptor binding assay we show R217 directly blocks GpA engagement while R218 does not. Our studies elaborate on the complex interaction between PfEBA-175 and GpA and highlight new approaches to targeting the molecular mechanism of P. falciparum invasion of erythrocytes. The results suggest studies aiming to improve the efficacy of blood-stage vaccines, either by selecting single or combining multiple parasite antigens, should assess the antibody response to defined inhibitory epitopes as well as the response to the whole protein antigen. Finally, this work demonstrates the importance of identifying inhibitory-epitopes and avoiding decoy-epitopes in antibody-based therapies, vaccines and diagnostics.

Background: Antibody binding to red blood cells (RBCs) can induce potentially fatal outcomes, including hemolytic transfusion reactions (HTRs), hemolytic disease of the fetus and newborn, and autoimmune hemolytic anemia. The mechanism(s) of RBC destruction following antibody binding is typically thought to require complement activation and/or the involvement of Fcγ receptors (FcγRs). In the current report, we analyzed mechanisms of HTRs during incompatible transfusions of murine RBCs expressing human glycophorin A (hGPA) into mice with anti-hGPA., Study Design and Methods: C3 and Fcγ receptor knockout, splenectomized, Fcγ receptor blocking antibody-treated, and clodronate-treated mice were passively immunized with anti-hGPA (10F7 or 6A7) and transfused with RBCs expressing the hGPA antigen. Posttransfusion blood and serum were collected and analyzed via flow cytometry and confocal microscopy., Results: This HTR model results in both rapid clearance and cytokine storm. Neither complement nor FcγRs were required for RBC clearance; in contrast, FcγRs were required for cytokine storm. Circulating aggregates of hGPA RBCs were visible during the HTR. Splenectomy and phagocyte depletion by clodronate had no effect on acute RBC clearance; however, incompatible RBCs reentered over 24 hours in clodronate-treated mice., Conclusion: These data demonstrate a biphasic HTR, the first phase involving sequestration of incompatible hGPA RBCs and the second phase involving phagocytosis of sequestered RBCs. However, the mechanism(s) of phagocytosis in the second phase required neither C3 nor FcγRs. These findings demonstrate novel mechanistic biology of HTRs., (© 2012 American Association of Blood Banks.)

In this study, we report the first Korean case of an anti-Gerbich (Ge) alloantibody to a high-incidence antigen that belongs to the Ge blood group system. The alloantibody was detected in a middle-aged Korean woman who did not have a history of transfusion. Her blood type was B+, and findings from the antibody screening test revealed 1+ reactivity in all panels except the autocontrol. The cross-matching test showed incompatible results with all 5 packed red blood cells. Additional blood type antigen and antibody tests confirmed the anti-Ge alloantibody. While rare, cases of hemolytic transfusion reaction or hemolytic disease in newborns due to anti-Ge have been recently reported in the literature. Therefore, additional further studies on alloantibodies to high-incidence antigens, including anti-Ge, are necessary in the future.

α-Hemoglobin-stabilizing protein (AHSP) is an abundant erythroid-specific chaperone protein that facilitates incorporation of nascent α-globin into hemoglobin A. We characterized AHSP expression by immunohistochemistry in a panel of 100 neoplastic and reactive bone marrow biopsies and splenic tissue with extramedullary hematopoiesis and compared it with established erythroid markers CD71 and CD235a. In all cases, AHSP expression was limited to physiological nucleated erythroid precursors (EPs) and blasts in erythroid leukemias. Although CD71 also stained EPs, it additionally stained nonerythroid malignant cells to varying extents in acute leukemia, diffuse large B-cell lymphoma, metastatic carcinomas, and small round blue cell tumors. In contrast, CD235a staining was erythroid-specific but stained non-nucleated red blood cells in all specimens, limiting its utility. We conclude that AHSP is superior to CD71 and CD235a for detecting normal and neoplastic nucleated EPs.

Monoclonal antibodies (MAbs) against glycophorin-A (GPA) could be used in identifying MN blood groups, detecting specific markers of erythroid differentiation, and studying parasite interactions. Large-scale production of MAbs in bioreactors demands an efficient and rapid separation technology. The present study describes the production of a human anti-GPA monoclonal antibody and its purification using a pseudo-bioaffinity L-histidine-convective interaction media (CIM) monolithic column. Hybridomas were generated by fusion of mouse myeloma cell line (Sp2/0) and spleen cells from the mouse immunized with Triton X-100 solubilized RBC membrane proteins. Hybridomas producing antibodies specific to commercial glycophorin-A were screened by indirect enzyme-linked immunosorbent assay (ELISA). The antibodies produced by the stable clones were found to be IgG1 with kappa light chain. Purification of IgG1 MAbs from the cell culture supernatant carried out with a CIM-EDA-histidine disk resulted in high specific activity with purification fold of 8.3 in the fraction eluted with MOPS buffer containing 0.2 M NaCl. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA showed that the antibodies obtained were highly pure, with high antigen-binding efficiency. The results indicate that faster separation and efficient recovery of high-purity anti-GPA MAbs could be achieved by using CIM-EDA-histidine disk.

Background: S, s, and U antigens belong to the MNS system. They are carried by glycophorin B (GPB), encoded by GYPB. Black people with the low-prevalence S-s- phenotype, either U- or U+(var), can make a clinically significant anti-U. Anti-U-like, a cold immunoglobulin G autoantibody quite commonly observed in S-s+U+ black persons, was previously described to be nonreactive with ficin-, α-chymotrypsin-, and pronase-treated red blood cells (RBCs); nonreactive or weakly reactive with papain-treated RBCs; and reactive with trypsin-treated RBCs. Here we describe, in S-s- people from different molecular backgrounds, an alloantibody to a high-prevalence GPB antigen, which presents the same pattern of reactivity with proteases as autoanti-U-like., Study Design and Methods: Four S-s- patients with an alloantibody to a high-prevalence GPB antigen were investigated by serologic and molecular methods., Results: An alloantibody was observed in two S-s-U-/Del GYPB, one S-s-U+(var)/GYPB(P2), and one S-s-U+(var)/GYPB(NY) patients. As this alloantibody showed the same pattern of reactivity with proteases as autoanti-U-like, we decided to name it "anti-U-like." Anti-U-like made by the two S-s-U- patients was reactive with the S-s-U+(var) RBCs of the two other patients., Conclusion: S-s-U-/Del GYPB, S-s-U+(var)/GYPB(P2), and S-s-U+(var)/GYPB(NY) patients can make an alloanti-U-like. Anti-U-like made by S-s-U- people appears reactive with GYPB(P2) and GYPB(NY) RBCs, which both express a weak and partial U-like reactivity. We recommend transfusing S-s-U- RBCs in S-s-U- patients showing alloanti-U-like. Our study contributes to a better understanding of alloimmunization to GPB in black people and confirms importance of genotyping in S-s- patients, especially those with sickle cell disease to be frequently transfused., (© 2011 American Association of Blood Banks.)

Most human transfusion recipients fail to make detectable alloantibodies to foreign RBC antigens ("nonresponders"). Herein, we use a murine model to test the hypothesis that nonresponders may be immunologically tolerant. FVB mice transfused with RBCs expressing transgenic human glycophorin A (hGPA) antigen in the absence of inflammation produced undetectable levels of anti-hGPA immunoglobulins, unlike those transfused in the presence of polyinosinic:polycytidylic acid-induced inflammation. Mice in the nonresponder group failed to produce anti-hGPA after subsequent transfusions in the presence of polyinosinic:polycytidylic acid, whereas anti-hGPA levels increased in the responder group. This tolerance was antigen specific, because nonresponders to hGPA produced alloantibodies to RBCs that expressed a different transgenic antigen. This tolerance was not an idiosyncrasy of the hGPA antigen nor of the recipient strain, because B10.BR mice transfused with membrane-bound hen egg lysozyme antigen-transgenic RBCs also demonstrated induced nonresponsiveness. These data demonstrate that RBCs transfused in the absence of inflammation can induce tolerance.

To mask the antigenic sites of cells for cell therapies, especially for blood transfusion, we investigated the hemocompatibility of two poly(2-(dimethylamino)ethyl methacrylate-co-poly(ethyleneglycol) compared with that of the homopolymer without PEG. Our strategy relies on the potential ability of these copolymers to self-assemble at the erythrocyte surface. The cationic sequence of the copolymer should be able to interact with the glycocalyx by ionic interaction. The other sequence, based on a polyethyleneglycol moiety, should prevent both nonspecific interactions and specific recognition of the biological surface. The hemocompatibility of these copolymers was assessed by analyzing alterations in human erythrocyte membrane viscoelasticity, morphology, granularity, and aggregation. Their properties to mask ABO system and three erythrocyte glycophorin sites were investigated. No alterations in the erythrocyte morphology were observed by confocal microscopy. On the other hand, a partial masking of different specific glycophorin sites leads to future optimization of the macromolecular structures of these functionalized copolymers., (Copyright © 2011 Wiley Periodicals, Inc.)

Background: The clinical significance of the Gerbich antibodies has been described as variable and there are no well-documented reports of hemolytic transfusion reactions (HTRs)., Case Report: We present the case of a woman with a long history of documented anti-Ge3 alloantibody who received multiple units of Ge+ red blood cells (RBCs) uneventfully. During the first admission to our hospital she was transfused 8 units of Ge+ RBCs and had a negative monocyte monolayer assay (MMA) before receiving the units. Within 2 weeks of the transfusions, the anti-Ge3 became significantly stronger by indirect antiglobulin test, and the MMA increased from 2.2 to 79.5% reactivity. She returned 4.5 years later with an emergent need for blood and was transfused with 2 units of Ge+ RBCs after premedication with steroids and intravenous immunoglobulin., Results: The first unit was transfused without incident; however, she developed clinical and laboratory signs consistent with an acute HTR with the second unit., Conclusion: After a comprehensive review of the literature, we believe this to be the first well-documented case of acute HTR due to anti-Gerbich alloantibodies., (© 2011 American Association of Blood Banks.)

Glycophorins comprise the major sialoglycoproteins of the human erythrocyte membrane. Several years ago we described a murine monoclonal antibody (MAb), designated 124,D-7 (IgM), developed by in vitro immunization with human erythrocyte membranes as antigen. We found the MAb reacted with a neuraminidase-dependent epitope on glycophorin A. Recent findings using ELISA with various bacteria as coating antigens have demonstrated strong cross-reactions of MAb 124,D-7 with some bacteria like Legionella and no reaction with bacteria such as Streptococcus pneumoniae. A second MAb, 130,E-4 (IgM), generated by the in vitro immunization technique, agglutinated human red cells irrespective of blood groups. This MAb showed strong cross-reactions with bacteria different from those being positive with MAb 124,D-7. The broad cross-reactivities of the two MAbs suggested that they are polyreactive antibodies. Sequencing of the V(H) and V(L) genes of MAb 124,D-7 showed germ-like sequences characteristic of polyreactive antibodies. The nucleotide sequences of the V(H) and V(L) genes of MAb 124,D-7 matched sequences coding for antibodies against CD34 and cross-reacting streptococcal antibodies. For Legionella pneumophila, the main interacting band on immunoblots was identified as the major outer membrane protein by mass spectrometry after separation by isoelectric focusing followed by SDS-PAGE. Flow cytometry showed that the epitope for MAb 124,D-7 was not displayed on live L. pneumophila but became exposed after heat treatment. Studies with one of the control MAbs, 145,F-2, directed against phosphorylcholine, which is known to be present on erythrocytes and some bacteria, showed that the epitope is deeply buried in the human erythrocyte membrane as neither neuraminidase nor papain exposed the epitope. The positive control MAb 3/1 directed against an epitope on LPS of L. pneumophila revealed weak cross-reactions with Pseudomonas aeruginosa.

The distinction between antemortem and postmortem wounds is one of the most important medico-legal problems. In fresh cadavers the macroscopic examination of haemorrhagic infiltration can be sufficient to reveal the vitality of a wound but in more difficult cases (putrefied corpses) histological and histochemical analyses need to be performed. The scope of this study was to detect the vitality of soft tissue samples in an advanced state of putrefaction using a monoclonal anti-human Glycoforin A antibody in order to evaluate the presence of red blood cells or red blood cell residues. Samples of skin wounds were taken from cadavers with a known time survival between trauma and death, and then submitted to a simulated putrefaction procedure. The skin samples were left to decompose for 30 days in air and in water and analyzed at a time interval of 3-6-15-30 days. These samples were stained with haematoxylin eosin stain, trichrome stain and with immunohistochemical stains. Results showed that in the air red blood cells could be seen for up to six days and granular deposits of Glycophorin reactive material for up to 15 days whereas no red blood cells were recognizable after 30 days. In water red cell putrefaction (or non-detection) was faster than in the air: after six days only amorphous granular deposits slightly reactive to Glycophorin could be seen. Nonetheless results showed that extravasated red blood cell residues can still be detected in skin bruises left to decompose in air and water, for up to at least 15 days in the open air and one week in water.

Glycophorin A (GPA, CD235a) is a major membrane glycoprotein and marker of cells of the erythroid lineage. It is also the target of Plasmodium falciparum and of influenza virus. We describe a novel series of 10 antibodies towards GPA, recognizing four extra- and intracellular peptide epitopes of this molecule (defined by epitope mapping) and one mixed peptide/carbohydrate epitope. All antibodies bind better to the desialylated than to the fully sialylated molecule, including those specific for the intracellular epitope. For some of the antibodies (representing all five epitopes) functional binding constants were determined by Surface Plasmon Resonance. The new panel complements the already known anti-glycophorin antibodies and offers several potential applications for, e.g., differential diagnosis of erythroleukemias, lineage analyses of erythroid cells, isolation of senescent erythrocytes, or a highly sensitive neuraminidase assay., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Anti-glycophorin C (GPC), blood group antibodies of which cause hemolytic disease of the fetus and newborn (HDFN), is a potent inhibitor of erythroid progenitor cell growth. The cellular mechanism for growth inhibition has not been characterized., Study Design and Methods: K562 cells were incubated in the presence of either anti-GPC, an immunoglobulin G isotype control, an inhibitor of actin polymerization called cytochalasin D with anti-GPC, or cytochalasin D alone. The JC-1 cationic dye was used to detect mitochondrial depolarization and the activity of the mitogen-activated protein kinases was assessed by Western blotting., Results: Anti-GPC inhibits the activity of extracellular regulated kinase (ERK)1/2 within 10 minutes but does not alter the activity of p38 or c-Jun N-terminal kinase. After 24 hours there was a significant loss of mitochondrial membrane potential compared to isotype control–treated cells. Both the ERK1/2 inhibition and the loss of mitochondrial potential were prevented by pretreatment with cytochalasin D., Conclusion: A cell surface antibody can cause anemia by altering the signaling pathways in erythroid cells by promoting depolarization of mitochondria via cytoskeletal rearrangement. The observation that neonates with anti-GPC HDFN are unresponsive to erythropoietin can be explained by the antibody inhibiting a protein kinase through which this hematopoietic growth factor achieves its effects.

Background: Alloantibodies that define some high-prevalence MNS antigens are made by people with glycophorin A (GPA) altered by a single-amino-acid change or replacement of amino acids from part of the Pseudoexon 3 of GYP*B. The finding of a patient whose plasma contained a novel alloanti-En(a)FR prompted this study., Results: The patient's serum contained an alloantibody to a high-prevalence antigen, resistant to papain, ficin, trypsin, alpha-chymotrypsin, or dithiothreitol. The antibody was strongly reactive with all panel red blood cells (RBCs) tested, showed reduced reactivity with ENEP- and ENAV- RBCs, and was nonreactive with M(k)M(k), En(a-), GP.Hil/GP.Hil, and GP.JL/M(k) RBCs. The patient's RBCs typed M+N-S+s-, Wr(a-b+(w)), ENEP-, and ENAV-. These results indicated that the antibody recognized a new high-prevalence antigen in the MNS system. Sequencing of DNA prepared from the patient's white blood cells revealed a GYP*A nucleotide substitution of 242T>G (predicted to change Val62 of GPA to Gly). This change ablates an RsaI restriction enzyme site and polymerase chain reaction-restriction fragment length polymorphism confirmed that the proband was homozygous for Nucleotide 242G., Conclusions: We describe a novel high-prevalence MNS antigen, characterized by Val62 in GPA and named ENEV. The absence of the antigen is associated with Gly62. The change explains the weakened reactivity of the patient's serum with ENEP- and ENAV- RBCs and nonreactivity with anti-ENEP and anti-ENAV against her RBCs. The ENEV antigen has been assigned the ISBT number MNS45.

The presence of erythropoietin (Epo) receptors on cells besides red blood cell precursors, such as cancer cells or megakaryocyte precursors, can lead to side effects during Epo therapy including enhanced tumor growth and platelet production. It would be ideal if the action of Epo could be limited to erythroid precursors. To address this issue, we constructed single-chain variable fragment (scFv)-Epo fusion proteins that used the anti-glycophorin 10F7 scFv amino-terminal to Epo analogues that would have minimal activity alone. We introduced the Epo mutations N147A, R150A and R150E, which progressively weakened receptor affinity in the context of Epo alone, as defined by cell proliferation assays using TF-1 or UT-7 cells. Fusion of these mutant proteins to the 10F7 scFv significantly rescued the activity of the mutant proteins, but had a relatively small effect on wild-type Epo. For example, fusion to the 10F7 scFv enhanced the activity of Epo(R150A) by 10- to 27-fold, while a corresponding fusion to wild-type Epo enhanced its activity only up to 2.7-fold. When glycophorin was blocked by antibody competition or reduced by siRNA-mediated inhibition of expression, the activity of 10F7 scFv-Epo(R150A) was correspondingly reduced, while such inhibition had essentially no effect on the activity of 10F7 scFv-Epo(wild-type). In addition, potent stimulation of Epo receptors by 10F7 scFv-Epo(R150A) was observed in long-term proliferation and viability assays. Taken together, these results indicate that a combination of targeting and affinity modulation can be used to engineer forms of Epo with enhanced cell-type specificity.

Chemical coupling to carrier red blood cells (RBCs) converts tissue type plasminogen activator (tPA) from a problematic therapeutic into a safe agent for thromboprophylaxis. The goal of this study was to develop a more clinically relevant recombinant biotherapeutic by fusing a mutant tPA with a single-chain antibody fragment (scFv) with specificity for glycophorin A (GPA) on mouse RBCs. The fusion construct (anti-GPA scFv/PA) bound specifically to mouse but not human RBCs and activated plasminogen; this led to rapid and stable attachment of up to 30,000 copies of anti-GPA scFv/PA per mouse RBC that were thereby endowed with high fibrinolytic activity. Binding of anti-GPA scFv/PA neither caused RBC aggregation, hemolysis, uptake in capillary-rich lungs or in the reticuloendothelial system nor otherwise altered the circulation of RBCs. Over 40% of labeled anti-GPA scFv/PA injected in mice bound to RBC, which markedly prolonged its intravascular circulation and fibrinolytic activity compared with its nontargeted PA counterpart, anti-GPA scFv/PA, but not its nontargeted PA analog, prevented thrombotic occlusion in FeCl(3) models of vascular injury. These results provide proof-of-principle for the development of a recombinant PA variant that binds to circulating RBC and provides thromboprophylaxis by use of a clinically relevant approach.

Background: The hemoglobin of a 29-year-old man fell below 35 g/L over 5 days, despite 14 units of red blood cells (RBCs), due to an anti-Pr cold agglutinin (CA). His hemolytic anemia necessitated respiratory support in intensive care for 4 weeks., Study Design and Methods: The hemolysis was investigated by the effects on blood group-compatible RBCs of this anti-Pr and an anti-I CA and of a rabbit anti-human glycophorin A (GPA) immunoglobulin G (IgG) antibody on Ca(2+) permeability and of phosphatidylethanolamine (PE) exposure. 1) The anti-Pr CA (in a plasmapheresis product from the patient) was absorbed and eluted from RBC ghosts and its immunophenotype was determined by agarose electrophoresis and immunofixation. 2) Ca(2+) permeability was measured by the response of Fluo-3-labeled RBCs to addition of external Ca(2+). 3) Exposed PE was measured with streptavidin-labeled biotinylated peptide Ro 09-0198 (cinnamycin)., Results: 1) The patient's anti-Pr CA was a polyclonal IgG. 2) The anti-Pr and the rabbit anti-human glycophorin IgG, but not an anti-I CA, rapidly increased Ca(2+)-dependent fluorescence upon addition of external Ca(2+) in a fraction (15%-25%) of RBCs that also became positive for cinnamycin. 3) Trypsin treatment of RBCs reduced the Ca(2+) influx due to the anti-Pr IgG, but neither trypsin nor neuraminidase changed the responses to the rabbit anti-human GPA IgG., Conclusions: The anti-Pr CA and rabbit anti-human GPA increased exposure of PE and increased membrane Ca(2+) permeability that may have caused hemolysis. The difference in the responses to these antibodies to enzyme treatment of RBCs suggests that they react with different epitopes on GPA.

Antigens in the Gebrich blood group system are expressed on glycophorin C (GPC) and glycophorin D (GPD), which are both encoded by a single gene, GYPC. The GYPC gene is located on the long arm of chromosome 2, and Gebrich antigens are inherited as autosomal dominant traits. There are 11 antigens in the Gebrich blood group system, six of high prevalence (Ge2, Ge3, Ge4, GEPL [Ge10*], GEAT [Ge11*], GETI [Ge12*]) and five of low prevalence (Wb [Ge5], Ls(a) [Ge6], An(a) [Ge7], Dh(a) [Ge8], GEIS [Ge9]). GPC and GPD interact with protein 4.1R, contributing stability to RBC membrane. Reduced levels of GPC and GPD are associated with hereditary elliptocytosis, and Gebrich antigens act as receptors for the malarial parasite Plasmodium falciparum. Anti-Ge2 and anti-Ge3 have caused hemolytic transfusion reactions, and anti-Ge3 has produced hemolytic disease of the fetus and newborn (HDFN).

Background: Isolation of adequate numbers of fetal cells circulating in the maternal circulation is the major hurdle in developing noninvasive prenatal diagnostic procedures. We used flow cytometry and a combination of different monoclonal antibodies to compare the yield and purity of the fetal nucleated red blood cells at different periods of gestation., Methods: Using a Percoll discontinuous gradient, the fetal nucleated erythrocytes were enriched from 7 ml maternal blood. In 100 samples, the enriched cells were stained with CD45, anti-fetal hemoglobin, and glycophorin A antibodies and in 30 samples they were stained with CD45, anti-fetal hemoglobin, and CD71 and then sorted and used for fetal diagnosis of hemoglobinopathies., Results: Using the first set of antibodies, although we were able to obtain a higher percentage of fetal nucleated red cells (0.07% +/- 0.2%) as compared to the second set which yielded comparatively smaller numbers (0.025% +/- 0.03%), there was some compromise in purity., Conclusion: Using CD45, anti-fetal hemoglobin and CD71 would be preferred as minimizing maternal contamination is more important than yield for prenatal diagnosis., ((c) 2008 Clinical Cytometry Society.)

Background: The blood group antigens S and s are defined by amino acids Met or Thr at position 29, respectively, on glycophorin B (GPB). Commercial anti-s reagents are expensive to produce because of the scarcity of human anti-s serum. Our aim was to develop hybridoma cell lines that secrete reagent-grade anti-s monoclonal antibodies (MoAbs) to supplement the supply of human anti-s reagents., Study Design and Methods: Mice were immunized with the GPB(s) peptide sequence TKSTISSQTNGETGQLVHRF. Hybridomas were produced by fusing mouse splenocytes with mouse myeloma cells (X63.Ag8.653). Screening for antibody production was done on microtiter plates by hemagglutination. Characterization of the MoAbs was done by hemagglutination, immunoblotting, and epitope mapping., Results: Eight immunoglobulin G MoAbs were identified. Five antibodies are specific by hemagglutination for s and two MoAbs, when diluted, are anti-S-like, but additional analyses shows a broad range of reactivity for GPB. Typing red blood cells (RBCs) for s from 35 donors was concordant with molecular analyses as were tests on RBCs with a positive direct antiglobulin test (DAT) from 15 patients. The anti-s MoAbs are most reactive with peptides containing the (31)QLVHRF(36) motif, with (29)Thr. By Pepscan analyses, the anti-S-like MoAbs reacted within the same regions as did anti-s, but independently of (29)Met. One antibody was defined serologically as anti-U; however, its epitope was identified as (21)ISSQT(25), a sequence common for both GPA and GPB., Conclusion: In addition to their value as typing reagents, these MoAbs can be used to phenotype RBCs with a positive DAT without pre-test chemical modification.

Background: Anti-"Mi(a)" is one of the most important irregular red blood cell antibodies found in Taiwan. The aim of this study was to investigate whether specific HLA-DRB1 alleles are associated with anti-"Mi(a)" production., Study Design and Methods: A case-control retrospective study was performed on 68 patients showing presence of anti-"Mi(a)" and 219 unrelated control subjects from the Mackay Memorial Hospital. HLA-DRB1 genotyping was carried out using sequence-based typing method. Fisher's exact test using 2 x 2 contingency tables was used to analyze significance of the association between DRB1 polymorphisms and presence of anti-"Mi(a)" in patients., Results: HLA-DRB1*0901 allele frequency in the anti-"Mi(a)" group (30%) was significantly higher than in the control group (16%) with an odds ratio of 2.27 (95% confidence interval, 1.44-3.55; p = 0.0005; p(c) = 0.016)., Conclusion: HLA-DRB1*0901 is significantly more prevalent in the anti-"Mi(a)" patients group than in the control group. It is suggested that cells from DR9 individuals might present processed "Mi(a)" antigen-allospecific peptides more effectively than cells from individuals carrying other DR phenotypes. Finally, it was predicted that two epitopes, derived from the MiIII glycophorin amino acid sequence, were likely to bind preferentially with the DR9 molecule. Further work will be necessary to determine if these epitopes are responsible for anti-"Mi(a)" alloimmunization.

The MNS blood group system is second only to the Rh blood group system in its complexity. Many alloantibodies to antigens in the MNS system are not generally clinically significant although antibodies to low-prevalence and high-prevalence MNS antigens have caused hemolytic disease of the fetus and newborn. The MNS antigens are carried on glycophorin A (GPA), glycophorin B (GPB), or hybrids thereof, which arise from single-nucleotide substitution, unequal crossing over, or gene conversion between the glycophorin genes. Antigens in the MNS system are fully developed at birth. This review summarizes aspects of the MNS system, including the molecular basis of some antigens in the MNS blood group system. Readers are referred to existing excellent reviews for background information. Throughout this document, information given without references can be found in the reviews listed previously, and the reader is referred to these reviews for references to original reports.

Background: The glycophorin (GP) molecule associated with the GP.Dane phenotype is a GP(A-B-A) hybrid that contains some amino acids encoded by the Pseudoexon 3 of GYPB and Asn(45) of GPA and carries the low-prevalence MNS antigens DANE and Mur. Serum from a woman of English ancestry contained an immunoglobulin M alloantibody to a high-prevalence MNS antigen, and the purpose of this study was to identify the molecular basis of her phenotype., Study Design and Methods: Hemagglutination, Western blotting, and DNA analyses were performed by standard methods., Results: Tests of the proband's RBCs with monoclonal antibodies indicated a change of amino acids between positions 27 and 55 of GPA. Her RBCs expressed M, s, Mur, and DANE antigens and were M(g)-negative. The antigen recognized by her antibody was sensitive to treatment with papain, ficin, and trypsin and resistant to alpha-chymotrypsin and dithiothreitol. Sequencing of DNA from the proband revealed a sequence of nucleotides identical to the GYP(A-B-A) encoding GP.Dane but without the adenyl nucleotide substitution, which has been predicted to change Ile(46) of GPA to Asn(45). Testing of her immediate family revealed the presence of an M(k) gene., Conclusion: The proband had a novel GYP(A-B-A) encoding a DANE+ GP that is in cis to GYPB(s) and in trans to M(k). The high-prevalence antigen lacking from this GP.Dane phenotype and recognized by the proband's serum is called ENDA (ISBT Number MNS44). Our results indicate that the change of Ile(46) of GPA to Asn(45) of GP.Dane is not required for expression of the DANE antigen.

Glycophorins are the major sialoglycoproteins in red blood cell membranes, possessing various physiological and pathological roles. We examined membrane glycoproteins in canine red cells and cloned cDNAs for two major glycophorins, glycophorins A (GPA) and C (GPC) from bone marrow cells. Periodic acid-Schiff staining and immunoblotting analyses showed that canine red cell membranes contained several glycoproteins immunoreactive to an anti-bovine GPC antibody, whereas the most abundant sialoglycoproteins, the candidates for GPA, did not react with an anti-human GPA antibody. The amino acid sequences of the extracellular domains of GPA and GPC had no significant homology to those from other mammalian species, including humans, and had O-linked and/or N-linked glycosylation sites. On the other hand, the C-terminal cytoplasmic domain and/or the transmembrane helices of GPA and GPC were conserved among species, indicating some functional significance of those regions in red cell membranes that include dimerization of GPA in the membrane-spanning region, and association of GPC with membrane skeletal proteins through binding with protein 4.1 and p55 in the cytoplasmic domain. These findings provide insights for clinical studies to evaluate the involvement of GPA and GPC in the pathogenesis of red cell diseases.

Background: Alloantibodies (ALLOs) to red blood cells (RBCs) are frequently associated with autoantibodies (AABs). An association between ALLO specificity and AABs has not yet been described., Study Design and Methods: All patients and healthy blood donors screened for RBC antibodies between 2000 and 2006 were included. The odds ratio (OR) for ALLOs in patients with AABs compared to those without AABs was correlated with the OR of general ALLO prevalence in patients with AABs (normalized OR)., Results: ALLOs were found in 4,626 of 204,330 patients and healthy blood donors (2.3%). The ALLOs were associated with AABs in 413 cases (8.9%). Among the specificities, anti-S with a normalized OR of 2.9 was overrepresented. This was most evident in pregnant women who showed a normalized OR of 15.1 for anti-S and AABs. The normalized OR revealed an additional association between Rh antibodies and AABs. No association was found between ALLOs to the Kell glycoprotein, Duffy protein, Lewis, or glycophorin A (M/N) and AABs., Conclusion: The majority of associated ALLOs and AABs are directed against neighboring antigens of the Rh complex and glycophorin B.

Recent in vitro-based studies using several Babesia spp. have suggested that sialic acids and/or sialoglycoproteins on host red blood cells (RBCs) play an important role in their invasion of RBCs. In the present study, we analyzed the RBC characteristics of glycophorin A (GPA)-knockout mice and studied their in vivo susceptibility to lethal infection of Babesia rodhaini for the first time. In immunoblot and lectin blot analyses, glycoproteins containing O-linked oligosaccharides terminated with alpha2-3-linked sialic acids disappeared from the RBCs of GPA homozygous ((-/-)) mice. Flow cytometric analysis showed a remarkable reduction of Maackia amurensis lectin II binding to the surface of GPA(-/-) RBCs relative to control RBCs, indicating an appreciable loss of alpha2-3-linked sialic acids on the RBC surface of GPA(-/-) mice. Importantly, while B. rodhaini caused lethal infection in wild-type mice, the infected GPA(-/-) mice showed inhibition of parasite growth and eventually survived. These results indicate that RBC sialoglycoproteins lost in GPA(-/-) mice are involved in the in vivo growth of B. rodhaini, probably functioning as essential molecule(s) for the parasite invasion of host RBCs in the blood circulation.