Comparison of ABO genotyping methods: a study of two low-resolution polymerase chain reaction assays in a clinical testing laboratory

Abstract

The ABO blood group system is the most clinically significant system in transfusion medicine. Although serologic typing for ABO antigens is routine and reliable, molecular methods can be used to predict an ABO type in the absence of a blood specimen as well as to investigate ABO typing discrepancies often caused by ABO subgroups that cause weakened antigen expression, weak or missing serum reactivity, and/or extra red blood cell reactivity. By detecting single nucleotide variants that are hallmarks of the major ABO alleles, low-resolution genotyping methods can be used to make allele assignments and predict phenotypes. This approach has become a dependable tool, initially to resolve typing discrepancies identified in blood banks and donor centers and, more recently, to predict the ABO group in bone marrow transplant donors and in deceased donors of solid organs. The aim of this report is to compare two different low-resolution polymerase chain reaction (PCR)-based methods: a PCR-restriction fragment length polymorphism (RFLP) implemented based on a publication and a commercially available TaqMan-based sequence-specific primer-PCR for resolution of ABO typing discrepancies. Fifty-six peripheral blood samples from 31 patients and 25 blood donors were used to isolate genomic DNA and perform genotyping. Results of 49 of the 56 samples (87.5%) were concordant between methods, three samples yielded an unexpected banding pattern on the PCR-RFLP method, and four sample results were discordant between assays. The discordances all involved group A versus A2 discrepancies. Sanger sequencing was used as a high-resolution genotyping method to resolve discrepancies between the two low-resolution methods. This study demonstrates that, in the majority of cases, a low-resolution genotyping method can resolve an ABO discrepancy. Although there is no U.S. Food and Drug Administration–approved genotyping method for ABO determination, molecular testing for investigation of discrepancies is a useful tool for blood banks and transplant programs.

Rapid Identification of Seven Common <i>ABO</i> Alleles by Two-Dimensional Polymerase Chain Reaction Technology

“…ABO alleles could also be genotyped using PCR restriction fragment length polymorphism. But this approach requires enzyme digestion and electrophoresis in addition to PCR amplification [7, 22] and is easily affected by the effect of enzyme digestion. Certainly, PCR sequencing-based typing is the most accurate and reliable detection method that can detect new mutation points [9], but time and cost still remain the factors that need to be considered for large-scale clinical sample detection.…”

RhD detection in transfusion medicine

“…Progress was also seen with the discovery of the ABO, and Rh systems, as well as why however C, c, D, E, and e are the most prevalent and significant ones. The D antigen and its variants have also been described, and it is known that the D antigen is found in RhD-positive people, who make up around 85% of the population overall, but not in RhD-negative people, who cover up about 15% [73].…”