ABO typing of a patient with an ABO incompatible bone marrow transplant was performed by serological and molecular standard tests. Because of ambiguous results a rare case of three ABO alleles was suspected. Further analysis by extended blood group sequencing with an inhouse NGS-assay in combination with serological methods was performed to clarify the serological and molecular background of the patients ABO alleles

The ABO phenotype was determined by standard blood group serology (gel- and tube-test BioRad, DiaMed GmbH, Cressier, Switzerland and CE-Immundiagnostika GmbH, Neckargemünd, Germany). Genotyping was carried out by PCR-SSP (RBC-Ready Gene ABO subtype, Inno-Train, Kronberg, Germany). DNA-sequencing by NGS was performed by custom primers specific for ABO in an inhouse NGS-Assay for amplification and sequencing reactions of all 7 exons including intron regions between exon 2 and 7 (MiSeq, Illumina, San Diego, USA). Analysis of the resulting sequences was performed by Geneious Prime Software (Dotmatics, Boston, USA) with manual assignment to known ABO-alleles deposited in the ISBT tables (v1.1 171023).

The patient"s blood group had been determined as B prior to transplantation, the donor was registered as blood group A. In the patient"s plasma, no isoagglutinin could be detected, the erythrocytes were typed as A. In the added tube test a weak B-Antigen was detected. Analysis by PCR-SSP revealed the presence of an ABO*c.261delG, typical for blood group O and *c.1061delG, typical for A2 and further SNPs that could not be unambiguously assigned to either ABO*O or *B variants. NGS sequencing revealed the complete genotype and the estimated percentage of each allele. The genotype was composed of ABO*A2.01, *O.01.01 and *B.01 in a ratio of approximately 35%, 44% and 17%, respectively, showing presence of the patients own B blood group.

Extended blood group serology and molecular typing identified three ABO alleles in a patient after haploident bone marrow transplantation. This case illustrates the importance of extended molecular typing in such complex cases, where standard serology typing and conventional PCR-SSP methods reach their limitations. Only a conscientious in-depth analysis in such particular cases enables identification the patient"s correct blood groups.

No conflict of interest.