The "next" standard for ABO genotyping

ABO histocompatibility plays a critical role in red blood cell (RBC) transfusions and is of great importance in solid organ and hematopoietic stem cell transplantation. ABO is primarily typed from red blood cell samples using serological reagents. However, stem cell donors are often screened using buccal swabs which cannot be used for serologic testing. Furthermore, serologic testing can be complicated by recent transfusions, weak reactions, or unusual reaction patterns, which require resolution. ABO genotyping can help in these circumstances. Here we show the development and validation of a targeted Next Generation Sequencing (NGS) ABO genotyping assay and companion interpretive software.

Short-read NGS was performed on 237 samples (blood n = 237 and buccal swab n = 42) obtained for routine transplant work-up testing and 10 samples with complex and rare ABO with previous reference laboratory workups. Three samples selected to represent common heterozygous ABO allele combinations underwent long-read sequencing as a proof of principle. All data was analyzed by our bloodTyper software.

The targeted NGS results for the solid organ and stem cell donor sample set was 99.3% (n = 287/289) concordant with serology with the two discordants likely being due to an unrecognized A_el and natural tissue-specific chimerism. In the complex and rare ABO samples NGS improved characterization of the ABO gene and uncovered important changes not initially found in the previous workups. Long-read sequencing was able to fully phase the three samples tested.

Through use of standard NGS protocols and automated interpretive software, targeted NGS allowed for reliable genotyping of ABO allele haplotypes without subject matter expert intervention. As such, targeted ABO NGS should become the standard for ABO genotyping.