Blood group genotype matching for transfusion.

The last decade has seen significant growth in the application of DNA-based methods for extended antigen typing, and the use of gene sequencing to consider variation in blood group genes to guide clinical care. The challenge for the field now lies in educating professionals, expanding accessibility and standardizing the use of genotyping for routine patient care. Here we discuss applications of genotyping when transfusion is not straightforward including when compatibility cannot be demonstrated by routine methods, when Rh type is unclear, when allo- and auto-antibodies are encountered in stem cell and organ transplantation, for prenatal testing to determine maternal and foetal risk for complications, and Group A subtyping for kidney and platelet donors. We summarize current commercial testing resources and new approaches to testing including high-density arrays and targeted next-generation sequencing (NGS).

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.

Insights into anti-D formation in carriers of RhD variants through studies of 3D intraprotein interactions.

BACKGROUND: Many RhD variants associated with anti-D formation (partial D) in carriers exposed to the conventional D antigen carry mutations affecting extracellular loop residues. Surprisingly, some carry mutations affecting transmembrane or intracellular domains, positions not thought likely to have a major impact on D epitopes. STUDY DESIGN AND METHODS: A wild-type Rh trimer (RhD1 RhAG2 ) was modeled by comparative modeling with the human RhCG structure. Taking trimer conformation, residue accessibility, and position relative to the lipid bilayer into account, we redefine the domains of the RhD protein. We generated models for RhD variants carrying one or two amino acid substitutions associated with anti-D formation in published articles (25 variants) or abstracts (12 variants) and for RHD*weak D type 38. We determined the extracellular substitutions and compared the interactions of the variants with those of the standard RhD. RESULTS: The findings of the three-dimensional (3D) analysis were correlated with anti-D formation for 76% of RhD variants: 15 substitutions associated with anti-D formation concerned extracellular residues, and structural differences in intraprotein interactions relative to standard RhD were observed in the others. We discuss the mechanisms by which D epitopes may be modified in variants in which the extracellular residues are identical to those of standard RhD and provide arguments for the benignity of p.T379M (RHD*DAU0) and p.G278D (RHD*weak D type 38) in transfusion medicine. CONCLUSION: The study of RhD intraprotein interactions and the precise redefinition of residue accessibility provide insight into the mechanisms through which RhD point mutations may lead to anti-D formation in carriers.

A structural entropy index to analyse local conformations in intrinsically disordered proteins.

Sequence - structure - function paradigm has been revolutionized by the discovery of disordered regions and disordered proteins more than two decades ago. While the definition of rigidity is simple with X-ray structures, the notion of flexibility is linked to high experimental B-factors. The definition of disordered regions is more complex as in these same X-ray structures; it is associated to the position of missing residues. Thus a continuum so seems to exist between rigidity, flexibility and disorder. However, it had not been precisely described. In this study, we used an ensemble of disordered proteins (or regions) and, we applied a structural alphabet to analyse their local conformation. This structural alphabet, namely Protein Blocks, had been efficiently used to highlight rigid local domains within flexible regions and so discriminates deformability and mobility concepts. Using an entropy index derived from this structural alphabet, we underlined its interest to measure these local dynamics, and to quantify, for the first time, continuum states from rigidity to flexibility and finally disorder. We also highlight non-disordered regions in the ensemble of disordered proteins in our study.

Alloimmunization risk associated with amino acid 223 substitution in the RhD protein: analysis in the light of molecular modeling.

BACKGROUND: Partial D status is a major concern for transfusion and pregnancy, due to the possibility of carriers becoming immunized. When known carriers of a D variant have never been exposed to complete D, they are assumed to have D partial status based on the position of the amino acid substituted. New approaches for predicting immunization risk are required. We built a three-dimensional (3D) structural model to investigate the consequences of substitutions of Amino Acid 223 involved in a large number of D variants. STUDY DESIGN AND METHODS: Homology modeling was performed with multiple templates. The model was evaluated by comparing the interactions of the known p.Phe223Val variant (RHD*08.01) and a new p.Phe223Ser variant (RHD*52) to RhD reference allele (p.Phe223). The consequences predicted by modeling the variants were compared with serologic data. RESULTS: The 3D structural model was generated from two related protein structures and assessed with state-of-the-art approaches. An analysis of the interactions of the variant Residue 223 in the proposed 3D model highlighted the importance of this position. Modeling predictions were consistent with the serologic and clinical data obtained for the D antigen with a substitution of Amino Acid 223. CONCLUSION: We used a 3D structural model to evaluate the effect of the p.Phe223 substitution on the conformation of the RhD protein. This model shed light on the influence of substitutions on the structure of the RhD protein and the associated alloimmunization risk. These initial findings indicate that the p.Phe223Ser variant can be considered partial.

High immunogenicity of red blood cell antigens restricted to the population of African descent in a cohort of sickle cell disease patients.

BACKGROUND: Sickle cell disease (SCD) patients undergo multiple red blood cell (RBC) transfusions and are regularly exposed to low-prevalence (LP) antigens specific to individuals of African descent. This study evaluated the prevalence of antibodies against LP antigens in SCD patients and the need to identify these antibodies in everyday practice. STUDY DESIGN AND METHODS: Plasma from 211 SCD patients was tested with RBCs expressing the following LP antigens: RH10 (V), RH20 (VS), RH23 (DW ), RH30 (Goa ), KEL6 (Jsa ), and MNS6 (He). RESULTS: Nine LP antibodies were found in eight patients (3.8%): five anti-RH23, two anti-RH30, and two anti-MNS6. The exposure risk, calculated for each LP antigen, was below 3% per RBC unit, for all antigens tested. Thus, in this cohort of transfused SCD patients, the prevalence of LP antibodies was similar to that of antibodies against antigens of the FY, JK, and MNS blood group systems. These findings also reveal the occurrence of anti-RH23 in SCD patients. No anti-RH20 or anti-KEL6 were found, despite the high frequency of mismatch situations. CONCLUSION: These results highlight the immunogenicity of these LP antigens, and the evanescence of antibodies against LP antigens. They also highlight the importance of appropriate pretransfusion testing for patients frequently transfused, who are likely to be exposed to multiple types of blood group antigens.

Genotyping in Sickle Cell Disease Patients: The French Strategy.

This review presents the French strategy for blood group genotyping in high-responder and newly diagnosed sickle cell disease (SCD) patients. In addition to FY, JK, and MNS genotyping, the RH blood group system is now explored in SCD patients in France. Molecular typing has been used for the deduction of partial RH2 (C) antigens since 2010, and the gradual implementation of systematic RHD and RHCE genotyping nationwide was initiated in late 2014. In our laboratory, 962 RH:2 (C-positive) SCD patients have been tested since 2010, and 1,148 SCD patients of all RH phenotypes have been genotyped for clinically relevant alleles of RHD and RHCE since late 2014.