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.

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.

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.