Molecular dynamics of the human RhD and RhAG blood group proteins.

Introduction: Blood group antigens of the RH system (formerly known as "Rhesus") play an important role in transfusion medicine because of the severe haemolytic consequences of antibodies to these antigens. No crystal structure is available for RhD proteins with its partner RhAG, and the precise stoichiometry of the trimer complex remains unknown. Methods: To analyse their structural properties, the trimers formed by RhD and/or RhAG subunits were generated by protein modelling and molecular dynamics simulations were performed. Results: No major differences in structural behaviour were found between trimers of different compositions. The conformation of the subunits is relatively constant during molecular dynamics simulations, except for three large disordered loops. Discussion: This work makes it possible to propose a reasonable stoichiometry and demonstrates the potential of studying the structural behaviour of these proteins to investigate the hundreds of genetic variants relevant to transfusion medicine.

Eleven years of alloimmunization in 6496 patients with sickle cell disease in France who received transfusion.

ABSTRACT: Red blood cell (RBC) transfusion is a major therapy for sickle cell disease (SCD). Patients are at risk of forming antibodies to RBC antigens, which can result in the impossibility to find compatible units and can cause hemolytic transfusion reactions. This retrospective study investigates the evolution of RBC consumption and the frequencies, specificities, and chronology of the appearance of antibodies in a population of patients consistently receiving RH (C, D, E, c, e) and K-matched RBC units (RBCus) from a predominantly European donor population. Over the 11-year period in the Paris area, 6496 patients received transfusion at least once for a total of 239 944 units. Antibodies were made by 1742 patients. The first antibodies of a patient were predictive of subsequent immunization. By the 17th RBCu transfused (by the 20th, excluding warm autoantibodies), 75% of the patients who would make antibodies had made their first. By the 16th, 90% who would make antibodies to a high frequency antigen had made their first antibody to these antigens. Females made their first antibodies slightly earlier than males. Patients who received multiple transfusions (>50 units) had a higher immunization prevalence than those who rarely received transfusion (<12 units) but fewer clinically significant antibodies. Patients with SCD and prophylactic RH-K matching not immunized by the 20th RBCu are likely to have a low alloimmunization risk (to antigens other than RH-K), that is, be low responders, especially relative to the most clinically significant antibodies. This number of 20 units is a point before which close monitoring of patients is most important but remains open to future adjustment.

Alloimmunisation against red blood cells in sickle cell disease: transfusion challenges in high-income and low-income countries.

Sickle cell disease is the most frequent inherited disorder in sub-Saharan Africa and in many high-income countries (HICs). Transfusion is a key element of treatment, but it results in high rates of alloimmunisation against red blood cell antigens and post-transfusion haemolysis, which can be life-threatening in severe cases. The prevention of alloimmunisation is, therefore, an important issue in both HICs and in low-income countries (LICs). In HICs, the main reason for high alloimmunisation rates is blood group disparity between blood donors, who are mostly of European descent, and the patients, who are mostly of African descent. However, alloimmunisation rates also remain high in sub-Saharan Africa despite the homogeneity of blood group antigen frequencies between donors and patients; this occurrence is probably due to matching strategies limited to ABO blood group and RhD. However, other possible underlying causes of alloimmunisation have also been suggested, with each cause affecting HICs and LICs in different ways-eg, the immunogenetic and inflammatory status of the patient and the characteristics of the red blood cell products. In this Viewpoint, we discuss the available data and hypotheses that potentially account for the association of sickle cell disease with high rates of alloimmunisation in both settings, HICs and LICs (focusing particularly on sub-Saharan Africa), and the challenges faced by HICs and LICs to improve prevention of alloimmunisation.

A novel high-prevalence antigen in the Lutheran system, LUGA (LU24), and an updated, full-length 3D BCAM model.

BACKGROUND: The basal cell adhesion molecule (BCAM) carries the antigens of the Lutheran (LU, ISBT005) system. We report a novel Lutheran antigen and propose an updated, full-length 3D model of BCAM. STUDY DESIGN AND METHODS: Red blood cell testing, antibody identification, and BCAM genomic DNA sequencing were done by standard methods. Multi-template homology modeling of BCAM used structural templates selected for coverage, highest sequence identity, and protein domain family. All variants causing the loss or gain of a Lutheran antigen were analyzed for residue accessibility and intraprotein interactions. RESULTS: An antibody to a high-prevalence antigen in the plasma of a pregnant woman was determined to be directed at a novel Lutheran antigen. Sequencing of BCAM found three homozygous changes: c.212G > A (p.Arg71His) and two silent, c.711C > T and c.714C > T. The model was built from the first two immunoglobulin crystallized domains of BCAM (D1, D2), three other templates (for D3, D4 and D5 with a higher sequence identity with the target than those used for the model proposed by Burton and Brady in 2008, and for the transmembrane region) and RaptorX (for the intracellular domain). All residues associated with a Lutheran antigen were found to be exposed in wild-type or variant proteins, except p.447 associated with loss of Lu13 expression. CONCLUSION: The c.212G > A change results in the loss of LUGA (LU24) antigen. Whole genome sequencing continues to reveal polymorphisms with uncertain immunogenicity. This model and demonstration that nearly all residues associated with the expression of a Lutheran antigen are exposed will help evaluate the significance of new polymorphisms.

Two new Scianna variants causing loss of high prevalence antigens: ERMAP model and 3D analysis of the antigens.

BACKGROUND: Scianna (Sc) antigens, seven high and two of low prevalence, are expressed on erythrocyte membrane-associated protein (ERMAP). We investigated SC (ERMAP) in individuals who made antibodies to high prevalence Scianna antigens, and propose a 3D model for ERMAP to precisely localize the residues associated with the known antigens. METHODS: Serological testing and DNA sequencing was performed by standard methods. A 3D structural model was built using a multi-template homology approach. Protein structures representing missense variants associated with the loss or gain of an antigen were generated. Residue accessibility and intraprotein interactions were compared with the wild-type protein. RESULTS: Two new SC alleles, one with c.349C > T (p.Arg117Cys) in a woman from South India with anti-Sc3 in her plasma, and a c.217_219delinsTGT (p.Arg73Cys) in an African-American woman with an antibody to a new high prevalence antigen, termed SCAC, were identified. Six structural templates were used to model ERMAP. 3D analysis showed that residues key for Scianna antigen expression were all exposed at the surface of the extracellular domain. The p.Arg117Cys change was predicted to abolish interactions between residues 93 and 117, with no compensating interactions. CONCLUSION: We confirm the extracellular location of Scianna residues responsible for antigen expression which predicts direct accessibility to antibodies. Loss of intraprotein interactions appear to be responsible for a Sc null and production of anti-Sc3 with p.117Cys, SC*01 N.03, and for loss of a high prevalence antigen with p.73Cys, termed SCAC for Sc Arg to Cys. Comparative modeling aids our understanding of new alleles and Scianna antigen expression.

Red blood cell alloimmunisation in sickle cell disease patients in the Democratic Republic of the Congo.

OBJECTIVES: To determine the prevalence of red blood cell (RBC) alloimmunisation and alloantibody specificity in sickle cell disease (SCD) patients in Kisangani, Democratic Republic of Congo (DRC) in comparison with those followed at the Centre Hospitalier Régional (CHR) de la Citadelle of Liège (Belgium). BACKGROUND: Data regarding RBC alloimmunisation (immune response of the organism to foreign erythrocyte antigens, antigens that lack on its own RBC) in SCD patients are scarce in sub-Saharan Africa. METHODS: We conducted a multi-site-based cross-sectional study among 125 SCD patients at Kisangani and 136 at the CHR de la Citadelle of Liège. The diagnosis of SCD was confirmed by high-performance liquid chromatography. Alloantibodies were screened using the agglutination technique on gel cards and their specificity determined using 11 and/or 16 cell panels. Statistical analyses were carried out using SPSS. RESULTS: The prevalence of RBC alloimmunisation was 9.6% among SCD patients in Kisangani versus 22.8% in those of Liège. At Kisangani as well as at Liège, the median age of alloimmunised patients was higher than that of non-alloimmunised patients, 15.5 years (IQR:4.8-19.8) and 24 years (IQR:14-31) versus 10 years (IQR: 6.5-17) and 17 years (IQR:12-24), respectively. The median number of blood units was higher in both Kisangani and Liège immunised patients compared to non-immunised patients, 8 (IQR:5-11) versus 5 (IQR:3-13) and 41(IQR:6-93) versus 6.5(3-37) respectively. At Kisangani (N = 14), the most frequent antibodies were anti-D (28.6%) and anti-C versus anti-E (13.6%), anti-S (13.6%) and anti-Lea (11.4%) at Liège (N = 44). CONCLUSIONS: These findings stated that alloimmunisation is a common complication in SCD patients in the DRC. In the resource-limited setting of this country, blood transfusion with minimal ABO, D, C and E antigen matching in addition to the use of compatibility test could significantly reduce the incidence of this complication.

3D analysis of CROMER (DAF) and a new antigen CRAG.

BACKGROUND: Cromer antigens are carried on decay accelerating factor (DAF, CD55), for which the crystal structure is available. We investigated two samples with an unidentified antibody to a high prevalence antigen and evaluate the location and characteristics of amino acids associated with antigens on the CD55 by 3D modelling. MATERIALS AND METHODS: Antigen typing and antibody identification were by standard methods. CD55 was sequenced, and Cromer variants were generated using the protein's crystal structure (1OK3, chain A). Antigen-associated residues and intraprotein interactions were investigated in 3D (Naccess, Protein Interactions Calculator). RESULTS: The antibody in the sample from a woman of Kashmiri descent was identified as anti-IFC (anti-CROM7). Her RBCs were negative for high-prevalence Cromer antigens including IFC. CD55 sequencing revealed a silent c.147G>A (p.Leu49=) and c.148G>T (p.Glu50Ter) changes, designated CROM*01N.05. The antibody in the sample from a woman of Greek ancestry was only compatible with IFC- RBCs but her RBCs were positive for known high-prevalence Cromer antigens. CD55 sequencing found she was homozygous for c.173A>G (p.Asp58Gly). The high prevalence antigen was named CRAG (ISBT CROM18 or 021018) and the allele designated CROM*01.-18. By 3D analysis, all known antigen-associated residues, including the new CRAG antigen, were exposed at the protein surface. Interactions between antigen-associated residues within the same CD55 domains were identified. DISCUSSION: Identification of antibodies to high prevalence Cromer antigens can be challenging. The surface exposure of antigen-associated residues likely accounts for their immunogenicity. 3D analysis of CD55 provides insight into previous serologic observations regarding the influence of some Cromer antigens on the expression of others.

How to avoid the problem of erythrocyte alloimmunization in sickle cell disease.

Erythrocyte alloimmunization is a major barrier to transfusion in sickle cell disease (SCD) because it can lead to transfusion deadlock and the development of life-threatening hemolytic transfusion reactions (HTRs). Several risk factors have been identified, such as blood group polymorphism in these patients of African ancestry frequently exposed to antigens they do not carry and an inflammatory clinical state of the disease. The most important preventive measure is prophylactic red blood cell antigen matching, and there is a consensus that matching for Rh (D, C, E, c, e) and K antigens should be performed for all SCD patients. However, some patients are high responders and more at risk of developing antibodies and HTRs. For these patients, the extension of matching to other blood groups, including variant antigens of the RH blood group, the use of genotyping rather than serology to characterize significant blood groups, and the prophylactic administration of immunosuppressive treatments remain a matter of debate due to low levels of certainty concerning their effects and the difficulty of determining which patients, other than those already immunized, are at high risk. These issues were recently addressed by a panel of experts established by the American Society of Hematology. Here, we review and stratify the various interventions for preventing alloimmunization, based on the literature and our experience and taking into account the obstacles to their implementation and any future developments required.