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.

International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Blood Group Terminology Report of Basel and three virtual business meetings: Update on blood group systems.

BACKGROUND AND OBJECTIVES: Under the ISBT, the Working Party (WP) for Red Cell Immunogenetics and Blood Group Terminology is charged with ratifying blood group systems, antigens and alleles. This report presents the outcomes from four WP business meetings, one located in Basel in 2019 and three held as virtual meetings during the COVID-19 pandemic in 2020 and 2021. MATERIALS AND METHODS: As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. New blood group systems and antigens were approved and named according to the serologic, genetic, biochemical and cell biological evidence presented. RESULTS: Seven new blood group systems, KANNO (defined numerically as ISBT 037), SID (038), CTL2 (039), PEL (040), MAM (041), EMM (042) and ABCC1 (043) were ratified. Two (039 and 043) were de novo discoveries, and the remainder comprised reported antigens where the causal genes were previously unknown. A further 15 blood group antigens were added to the existing blood group systems: MNS (002), RH (004), LU (005), DI (010), SC (013), GE (020), KN (022), JMH (026) and RHAG (030). CONCLUSION: The ISBT now recognizes 378 antigens, of which 345 are clustered within 43 blood group systems while 33 still have an unknown genetic basis. The ongoing discovery of new blood group systems and antigens underscores the diverse and complex biology of the red cell membrane. The WP continues to update the blood group antigen tables and the allele nomenclature tables. These can be found on the ISBT website (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/).

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.

Red Cell Antigens and Antibodies.

Autoimmune hemolytic anemia (AIHA) is caused by the production of "warm-" or "cold-" reactive autoantibodies directed against RBC antigens that may be of undefined specificity, reacting with all RBCs tested or may have an apparent specificity. Autoantibodies may be of IgG, IgM, or rarely IgA isotypes and their production can be triggered by disease, viral infection, or drugs; from breakdown in immune system tolerance to self-antigens; or from exposure to foreign antigens that induce antibodies that cross-react with self-RBC antigens. Increasingly, AIHA is being reported in patients following allogeneic hematopoietic stem cell transplantation and treatment with anti-cancer checkpoint inhibitors. Autoantibodies, whatever their etiology, interfere with pretransfusion testing of patients requiring RBCs transfusion making compatibility testing complex and labor-intensive. The availability of extended antigen typing by DNA-based assay has made transfusion of RBCs that are selected based on the patient's extended phenotype (e.g., D, C, E, e, K, Jka, Jkb, Fya, Fyb, S and s) a feasible option and this can provide a significant measure of safety as they avoid the patient being immunized to antigens absent from their RBCs.

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.

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.

Use of an in-house trypsin-based method to resolve the interference of daratumumab.

BACKGROUND: Daratumumab (DARA) is a monoclonal antibody for treatment of plasma cell myeloma targeting CD38, a surface molecule expressed on plasma cells and red blood cells (RBCs). This complicates blood bank testing, requiring dithiothreitol (DTT) to remove DARA interference. A simple in-house method of removing DARA interference without use of DTT, a potentially hazardous chemical, is desirable. We demonstrate a trypsin-based method to remove interference in antibody testing at a medical center (MC), with parallel testing at an immunohematology reference laboratory (IRL). STUDY DESIGN AND METHODS: Pre-DARA type and screen (T&S) samples were obtained from 61 patients for antibody testing and RBC phenotyping using untreated reagent RBCs. Subsequent post-DARA T&S testing was performed with untreated reagent RBCs to demonstrate interference and repeated after trypsin treatment. Positive trypsin-treated antibody screens were reflexed to antibody identification using trypsin-treated panel cells. Parallel testing was performed on the same post-DARA samples at IRL. RESULTS: DARA interference was detected in 61/61 (100%) samples by MC and IRL. After trypsin treatment, DARA interference was eliminated in 60/61 (98.4%) antibody screens by both institutions with an overall percent agreement of 96.7% (95% confidence interval [CI] 88.7%-99.6%). Identification of known alloantibodies was confirmed in 3/3 patients with 100% concordant results between MC and IRL. There were no false-negative results demonstrated by IRL's functionally CD38-negative controls. CONCLUSION: Our in-house trypsin-based method enables pretransfusion testing of patients receiving DARA in an accurate and cost-effective manner without missing clinically significant alloantibodies. This presents an additional testing option where DTT use is undesirable.