Comparison of a PCR assay using novel selective primers with current methods in terms of ABO blood phenotyping in rhesus macaques

Nonhuman primates are important animal models in transplantation. To prevent fatal transplantation-induced immune responses, it is necessary to accurately phenotype the monkey ABH antigens, which are the same as those in humans but (unlike in humans) are not expressed on red blood cells (RBCs). We compared the ability of two established ABO-typing methods, namely, serological testing and immunohistochemistry (IHC), and our novel polymerase chain reaction (PCR)-based assay to type 66 rhesus monkeys. The serological test assessed the ability of monkey sera to hemagglutinate human RBCs. The IHC assay measured the binding of murine anti-A and anti-B antibodies to monkey buccal mucosa cells. The whole blood-based PCR assay involved selective primers that were derived from the exon 7 sequences of A+, B+, and O+ monkeys. IHC and PCR unequivocally yielded the same types in all monkeys. Serological testing yielded inconsistent types in seven (10.6%). FACS analysis with monkey sera preabsorbed with O+ RBCs showed that the incorrect serological results related to nonspecific or xenoreactive binding of the human RBCs. Unlike previous PCR-based assay, our algorithm directly detected O+ monkeys and A and B homozygotes and heterozygotes. Given the logistical limitations of IHC, this PCR assay may be useful for typing rhesus monkeys.