ABO Genotyping under Construction: Three different typing methods, three different results

Body: Despite known limitations of ABO phenotyping, ABO genotyping challenges also remain. Next-generation sequencing (NGS) introduces novel challenges in organ allocation and recipient-donor matching. This case explores an individual with discordant results between red blood cell (RBC) serology, high resolution ABO genotype (NGS), low resolution genotype (qPCR), and the ABO antibody profile.
The ABO blood group was assigned by different methods as shown in Table 1. By serology phenotyping, non-A1B was the assigned blood group but LinkSeq qPCR (Thermo Fisher Scientific) assigned A1B as the most likely typing. Further testing was performed with AlloSeq™ Tx17 (CareDx, Inc. Brisbane CA, USA) including hybrid capture probes targeting ABO, FUT1, FUT2, and FUT3 genes. Captured libraries were sequenced (2x150bp) on an illumina MiniSeq; data were analyzed using AlloSeq Assign™ software.
The ABO*A blood group was assigned as A1.01 by qPCR while NGS identified a novel A1.01. Sequences of exons 1-7 matched A1.01, however 2 SNPs in intron 1 were identified. Interestingly these 2 SNPs g.776T>C (rs532436) and g.1207 T>C (rs507666) have been previously reported as discriminating ABO*A1/A2 alleles (Bugert et al 2023), indicating a non-A1 genotype in this sample.
The ABO antibody profile was assessed by single antigen beads; weak IgM anti-A-II antibodies were detected (Figure 1) despite being serologically typed as A; A-II being the subtype antigen on vascular endothelium.

Conclusion: This case demonstrates the need for continued parallel testing of ABO phenotyping with ABO genotyping. Genotyping will advance the science of ABO histocompatibility and this case and suggests that inclusion of exon 1 and intron 1 is required. Defining ABO allele phenotypes will require a multi-pronged approach that includes the ABO antibody profile, RBC flow cytometry (underway for this case), and tissue immunohistochemistry. Transplant candidate ABO subgrouping and ABO antibody testing may also uncover potentially relevant antibodies in presumed ABO-compatible transplantation. The ISBT 001 allele definition table does not meet current needs for ABO-incompatible transplantation and is a barrier to advancing ABO-histocompatibility and allocation of ABO-incompatible organs. A collaborative approach, as used by our histocompatibility community to define HLA genotypes and phenotypes, will be required to solve these issues.