Mixed-field agglutination observed in column agglutination testing is not always associated with the A3 subgroup

Mixed-field agglutination (MFA) can be observed in forward typing of samples from A3 individuals with serologic ABO typing methods. The results of column agglutination testing (CAT) and tube agglutination testing using different antibody clones can be discordant. In this report, we reveal our experience using polymerase chain reaction-sequence-based typing (PCR-SBT) of ABO exon 7 to clarify serologic method discordance of A subgroup blood typing in Northern Thai donors. A total of 21 group A blood donors with either MFA or weak agglutination on routine ABO CAT were recalled. CAT was repeated with human and monoclonal anti-A, and tube agglutination testing with monoclonal anti-A and PCR-SBT of ABO exon 7 was performed. A total of 13 of the 21 donors returned, and ABO CAT with human anti-A was repeated. Eleven samples showed MFA suspected to be the A3 subgroup, and two samples showed 2+ strength suspected to be the Aweak subgroup. When tube agglutination testing using monoclonal antibody was performed, MFA was not observed in 9 of 11 samples with previously observed MFA from routine CAT, which were then interpreted as A2. From PCR-SBT performed in only exon 7 of the ABO gene, 7 of 13 sample results were consistent with ABO*A2 or ABO*AW alleles. Two samples suspected to be A2 or A3 had an ABO*AW allele. In two samples suspected to be Aweak, no mutation was detected in ABO exon 7, suggesting genetic variation elsewhere in the gene. Although other coding exons were not examined, in the alleles that could be assigned, ABO*A3 alleles were found less frequently than would be predicted from the serologic findings. These findings suggest that when MFA in routine CAT is observed, an A3 subgroup cannot be presumed. Caution should be exercised when MFA is noted in routine CAT.