RH genotypes and red cell alloimmunization rates in chronically transfused patients with sickle cell disease: A multisite study in the USA.

BACKGROUND: Red cell alloimmunization remains a challenge for individuals with sickle cell disease (SCD) and contributes to increased risk of hemolytic transfusion reactions and associated comorbidities. Despite prophylactic serological matching for ABO, Rh, and K, red cell alloimmunization persists, in part, due to a high frequency of variant RH alleles in patients with SCD and Black blood donors. STUDY DESIGN AND METHODS: We compared RH genotypes and rates of alloimmunization in 342 pediatric and young adult patients with SCD on chronic transfusion therapy exposed to >90,000 red cell units at five sites across the USA. Genotyping was performed with RHD and RHCE BeadChip arrays and targeted assays. RESULTS: Prevalence of overall and Rh-specific alloimmunization varied among institutions, ranging from 5% to 41% (p = .0035) and 5%-33% (p = .0002), respectively. RH genotyping demonstrated that 33% RHD and 57% RHCE alleles were variant in this cohort. Patients with RHCE alleles encoding partial e antigens had higher rates of anti-e identified than those encoding at least one conventional e antigen (p = .0007). There was no difference in anti-D, anti-C, or anti-E formation among patients with predicted partial or altered antigen expression compared to those with conventional antigens, suggesting that variant Rh on donor cells may also stimulate alloimmunization to these antigens. DISCUSSION: These results highlight variability in alloimmunization rates and suggest that a molecular approach to Rh antigen matching may be necessary for optimal prevention of alloimmunization given the high prevalence of variant RH alleles among both patients and Black donors.

Blood Group Serology and "Race": Looking Back to Move Forward.

Less than a decade after the discovery of the ABO antigens as a Mendelian inherited trait, blood group antigen frequencies were first used to define racial groups. This approach, known as seroanthropology, was the basis for collecting large amounts of blood group frequency data in different populations and was also sometimes used for racist purposes. Ultimately, population geneticists used these data to disprove race as a biological construct. Through understanding the history of seroanthropology, and recognizing the harms of its lingering presence, healthcare providers can better practice race-conscious, as opposed to race-based, transfusion medicine.

Evidence that donors with variant RH genotypes are associated with unexpected Rh antibodies.

BACKGROUND: We previously reported unexpected Rh antibodies in the plasma of patients with sickle cell disease (SCD) that demonstrated common Rh specificities in the absence of transfusion of RBCs positive for that antigen. We hypothesize that these antibodies might result from transfusion of antigen-negative donor units with variant RH genotypes. METHODS: Plasma testing by tube and IgG gel, extended RBC phenotyping, and HEA and RH genotyping were by standard methods. CASE: A 6-year-old female with SCD, phenotype D + C-c + E-e + K- undergoing exchange transfusion with CEK- and Fy(a-) units, presented with anti-C in the plasma, a + DAT and warm autoantibody (WAA) in the eluate. Her RH genotype was unremarkable: RHD*D/DAU0 and RHCE*ce/ce(48C). Units (n = 10) transfused over the prior 6 months were confirmed CEK- by serology and DNA testing. Most (n = 7) were Rh-negative. A unit with variant RH, RHD*DIIIa/weak partial 4.0, RHCE*ceVS.03/ceVS.02, was transfused 5 weeks prior. Anti-C and + DAT continued to demonstrate for 25 weeks. Total hemoglobin and % Hgb S did not deviate from her established baseline. CONCLUSION: We show direct association of plasma anti-C with transfusion of a C-negative unit with variant RH encoding partial D and uncommon V/VS+ hrB - phenotype. The antibody was transient, without evidence of compromised survival of transfused RBCs. The +DAT and WAA complicated workups and selection of units, and it is uncertain whether donors of the same genotype should be avoided. Minority donors are important for CEK-matching to avoid depleting Rh-negative supplies. Consideration of patient and donor RH genotypes may avoid unexpected antibodies and improve allocation of rare donations.

Variant RHD alleles and Rh immunization in patients with sickle cell disease.

RH diversity among patients and donors contributes to Rh immunization despite serologic Rh-matched red cell transfusions. Anti-D can occur in D+ patients with RHD variants that encode partial D antigens. Anti-D has also been reported in patients with conventional RHD transfused primarily with units from Black donors who frequently have variant RHD. We report 48 anti-D in 690 D+ transfused individuals with sickle cell disease, categorized here as expressing conventional D, partial D or D antigen encoded by RHD*DAU0. Anti-D formed in a greater proportion of individuals with partial D, occurred after fewer D+ unit exposures, and remained detectable for longer than for those in the other categories. Among all anti-D, 13 had clinical or laboratory evidence of poor transfused red cell survival. Most individuals with anti-D were chronically transfused, including 32 with conventional RHD who required an average of 62 D- units/year following anti-D. Our findings suggest that patients with partial D may benefit from prophylactic D- or RH genotype-matched transfusions to prevent anti-D. Future studies should investigate whether RH genotype-matched transfusions can improve use of valuable donations from Black donors, reduce D immunization and minimize transfusion of D- units to D+ individuals with conventional RHD or DAU0 alleles.

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.

ABO Genotyping finds more A2 to B kidney transplant opportunities than lectin-based subtyping.

ABO compatibility is important for kidney transplantation, with longer waitlist times for blood group B kidney transplant candidates. However, kidneys from non-A1 (eg, A2) subtype donors, which express less A antigen, can be safely transplanted into group B recipients. ABO subtyping is routinely performed using anti-A1 lectin, but DNA-based genotyping is also possible. Here, we compare lectin and genotyping testing. Lectin and genotype subtyping was performed on 554 group A deceased donor samples at 2 transplant laboratories. The findings were supported by 2 additional data sets of 210 group A living kidney donors and 124 samples with unclear lectin testing sent to a reference laboratory. In deceased donors, genotyping found 65% more A2 donors than lectin testing, most with weak lectin reactivity, a finding supported in living donors and samples sent for reference testing. DNA sequencing and flow cytometry showed that the discordances were because of several factors, including transfusion, small variability in A antigen levels, and rare ABO∗A2.06 and ABO∗A2.16 sequences. Although lectin testing is the current standard for transplantation subtyping, genotyping is accurate and could increase A2 kidney transplant opportunities for group B candidates, a difference that should reduce group B wait times and improve transplant equity.

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.

YTGT: A new high-prevalence antigen in the Yt blood group system in two unrelated Native Americans and transfusion management.

BACKGROUND: The Yt system consists of five antigens: antithetical Yta /Ytb and the high-prevalence antigens YTEG, YTLI, and YTOT. We investigated a sample from a Native American (NA) female with post-operative anemia and an unidentified antibody who developed rigors, tachycardia, and hypotension on transfusion of incompatible RBCs. METHODS AND MATERIALS: Serologic testing methods included LISS, PEG, and IgG gel. Test RBCs were treated with papain, trypsin, alpha-chymotrypsin, 2-amino-ethylisothiouronium, and dithiothreitol. Rare RBCs were tested, and inhibition studies were performed. DNA extracted from WBCs was used for Sanger sequencing. RESULTS: Initial testing showed strong 3-4+ plasma reactivity with all panel cells at LISS IAT; auto control was negative. Positive reactions were observed with numerous rare RBCs except for PNH-III, which lack GPI-linked DO, Yt, CROM, JMH, and Emm. Enzyme sensitivity patterns suggest Yt specificity, and soluble recombinant srYt neutralized reactivity. ACHE sequencing revealed YT*A/A genotype but with a homozygous change in exon 2, c.290A>G (p.Gln97Arg). Antibody reactivity was reminiscent of that seen in an unrelated NA male investigated previously. His RBCs were nonreactive with her plasma. ACHE carried the same c.290G/G change. CONCLUSION: Two unrelated NA patients were found to have an antibody to a new high-prevalence Yt antigen, designated YTGT (YT6), associated with a clinically significant transfusion reaction. Identification of the specificity relied on enzyme sensitivity, use of PNH-III RBCs, neutralization using soluble recombinant Yt, and the finding of a novel change in ACHE, c.290A>G (p.Gln97Arg), designated YT*01.-06. IVIG and steroids were used to mitigate further reactions to transfusion.

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.

Accurate long-read sequencing allows assembly of the duplicated RHD and RHCE genes harboring variants relevant to blood transfusion.

Next-generation sequencing (NGS) technologies have transformed medical genetics. However, short-read lengths pose a limitation on identification of structural variants, sequencing repetitive regions, phasing of distant nucleotide changes, and distinguishing highly homologous genomic regions. Long-read sequencing technologies may offer improvements in the characterization of genes that are currently difficult to assess. We used a combination of targeted DNA capture, long-read sequencing, and a customized bioinformatics pipeline to fully assemble the RH region, which harbors variation relevant to red cell donor-recipient mismatch, particularly among patients with sickle cell disease. RHD and RHCE are a pair of duplicated genes located within an ∼175 kb region on human chromosome 1 that have high sequence similarity and frequent structural variations. To achieve the assembly, we utilized palindrome repeats in PacBio SMRT reads to obtain consensus sequences of 2.1 to 2.9 kb average length with over 99% accuracy. We used these long consensus sequences to identify 771 assembly markers and to phase the RHD-RHCE region with high confidence. The dataset enabled direct linkage between coding and intronic variants, phasing of distant SNPs to determine RHD-RHCE haplotypes, and identification of known and novel structural variations along with the breakpoints. A limiting factor in phasing is the frequency of heterozygous assembly markers and therefore was most successful in samples from African Black individuals with increased heterogeneity at the RH locus. Overall, this approach allows RH genotyping and de novo assembly in an unbiased and comprehensive manner that is necessary to expand application of NGS technology to high-resolution RH typing.

Anti-Emm, a rare specificity to the high-incidence antigen Emm in an Indian patient defining the new blood group system EMM (ISBT042).

A transfusion recipient lacking a high-incidence antigen (HIA) and has corresponding alloantibody pose a problem in providing compatible blood unit. We encountered a patient with an antibody to an HIA that required identification to assess if compatible blood could be organized. A 65-year-old male was posted for coronary artery bypass grafting surgery. His blood specimen collected in EDTA was referred to the blood bank to provide blood for transfusion. The patient, grouped AB RhD+, had an antibody reacting in saline and antiglobulin phases. It agglutinated all the red blood cells (RBCs) of the 11-cell panel and random donors, indicating specificity to an HIA, though one of his siblings was compatible. After ruling out specificity to HIAs such as H, Inb, and INRA (IN5), the specimen was referred to the New York Blood Centre for further work-up. The antibody reacted with examples of red cells lacking HIA, except those with the Emm- phenotype. The patient's RBCs were typed as Emm-. Anti-Emm in the patient appeared to be naturally occurring as there was no history of transfusion. Naturally occurring alloantibody to an HIA, identified as anti-Emm in phenotype Emm-, is rare and the first of its kind to be reported from India. The case was instrumental in recognizing the Emm as the new blood group system assigned with the symbol ISBT042.

PIGG defines the Emm blood group system.

Emm is a high incidence red cell antigen with eight previously reported Emm- probands. Anti-Emm appears to be naturally occurring yet responsible for a clinically significant acute hemolytic transfusion reaction. Previous work suggests that Emm is located on a GPI-anchored protein, but the antigenic epitope and genetic basis have been elusive. We investigated samples from a South Asian Indian family with two Emm- brothers by whole genome sequencing (WGS). Additionally, samples from four unrelated Emm- individuals were investigated for variants in the candidate gene. Filtering for homozygous variants found in the Emm- brothers and by gnomAD frequency of < 0.001 resulted in 1818 variants with one of high impact; a 2-bp deletion causing a frameshift and premature stop codon in PIGG [NM_001127178.3:c.2624_2625delTA, p.(Leu875*), rs771819481]. PIGG encodes for a transferase, GPI-ethanolaminephosphate transferase II, which adds ethanolamine phosphate (EtNP) to the second mannose in a GPI-anchor. The four additional unrelated Emm- individuals had various PIGG mutations; deletion of Exons 2-3, deletion of Exons 7-9, insertion/deletion (indel) in Exon 3, and new stop codon in Exon 5. The Emm- phenotype is associated with a rare deficiency of PIGG, potentially defining a new Emm blood group system composed of EtNP bound to mannose, part of the GPI-anchor. The results are consistent with the known PI-linked association of the Emm antigen, and may explain the production of the antibody in the absence of RBC transfusion. Any association with neurologic phenotypes requires further research.