Comparison of Methods for Determining ABO Blood Type in Cynomolgus Macaques (Macaca fascicularis)

Thorough examination of ABO blood type in cynomolgus monkeys is an essential experimental step to prevent humoral rejection during transplantation research. In the present study, we evaluated current methods of ABO blood-antigen typing in cynomolgus monkeys by comparing the outcomes obtained by reverse hemagglutination, single-nucleotide polymorphism (SNP) analysis, and buccal mucosal immunohistochemistry. Among 21 animals, 5 were type A regardless of the method. However, of 8 serologically type B animals, 3 had a heterozygous type AB SNP profile, among which 2 failed to express A antigen, as shown by immunohistochemical analysis. Among 8 serologically type AB animals, 2 appeared to be type A by SNP analysis and immunohistochemistry. None of the methods identified any type O subjects. We conclude that the expression of ABO blood-group antigens is regulated by an incompletely understood process and that using both SNP and immunohistochemistry might minimize the risk of incorrect results obtained from the conventional hemagglutination assay.Abbreviation: SNP, single nucleotide polymorphismCynomolgus macaques (Macaca fascicularis) have been recognized as an appropriate model for transplantation research because of their similarity to humans in immunologic, genetic, and physiologic aspects.^8,9 Identification of ABO blood-group antigens is a prerequisite not only for donor–recipient matching to prevent rejection but also for transfusion during emergency animal care. In humans, the forward hemagglutination test, in which blood type is determined by using the antigen–antibody reaction between antigens on RBC and antisera, is recognized as the ‘gold standard’ for ABO blood-antigen determination. However, such a system is infeasible in macaques because they lack ABO antigens on their RBC.⁷ Therefore, hemagglutination assays using surrogate human blood cells and the antisera of the test animal is used.Although the hemagglutination assay is simple, cost-effective, and reliable on many occasions, its results are prone to subjectivity, and it has several technical limitations. The largest drawback of the agglutination assay arises from age-associated differences in the levels of serum antibody secreted in each animal.²⁰ The gel test method, in which hemagglutination is examined by assessing the differential movement of nonagglutinated and agglutinated RBC in dextran acrylamide gel after centrifugation, was introduced as an alternative to conventional hemagglutination in cynomolgus monkeys.⁴ However, interpreting the grade (0 to +4) can be subjective; therefore other validation data, obtained by using methods, such as DNA analysis and immunohistochemistry, are required to determine the blood type of cynomolgus monkeys.It is well established in humans that the A and B allele encode distinct glycosyltransferases that convert the H antigen to the respective A and B antigens, whereas an O-type person expresses an unmodified H antigen.²² Although a specific single-nucleotide polymorphism (SNP) in the ABO locus controls this activity, the molecular mechanism of blood-group antigen expression in cynomolgus macaques is not fully delineated. A proposed new method for ABO typing in cynomolgus macaques is based on the results of sequence comparison between various nonhuman primates and uses quantitative real-time PCR coupled with the TaqMan probe assay.^16,17 Such genetic analysis can clearly show the antigenic characteristics on the DNA level with the strength of high-throughput analysis of multiple samples.⁶ Despite the tremendous potential of that approach in immunohematologic applications, the SNP that govern ABO antigen expression in cynomolgus monkeys remain unclear. For example, the null allele (O) in cynomolgus monkeys lacks the stop codon mutation found in the human ABO locus,^16,22 prompting questions regarding whether O-type animals exist and how the A- or B-antigen moiety is generated from H antigen in cynomolgus monkeys. Therefore, discrepant outcomes between hemagglutination and modern DNA analysis methods are still possible.ABO antigens in Old World monkeys are expressed in a variety of tissues, including the endothelium of the vasculature of most organs, the epithelium of the gastrointestinal tract, saliva, and exocrine secretions.^15,19 If appropriately set up, immunohistochemistry provides unequivocal results regarding blood-group antigen expression.² However, the level of antigen expression on the epithelium differs among subjects,²⁰ and the quality of cells collected from epithelial tissues (for example, buccal mucosa) can vary widely, depending on the salivation status and general health conditions (for example, hydration) of the tested animals.Given those viewpoints, we evaluated current ABO typing methods for cynomolgus macaques by comparing the results from hemagglutination assays with those of SNP analysis and immunohistochemistry.