AB blood group antigens are the most significant in transfusion medicine and neonatal isoerythrolysis (NI) for cats (1–5). The feline AB blood group system is based on A and B antigens and contains type A, type B, and the rare type AB. Type A erythrocytes express N-glycolylneuraminic acid (Neu5Gc) and type B erythrocytes express N-acetylneuraminic acid (Neu5Ac) (6, 7). Cat serum contains naturally occurring antibodies against other erythrocyte antigens, with 95% of type A cats showing antibodies to type B antigen, and 35% of type B cats showing antibodies to type A antigen (2). Type AB erythrocytes express both Neu5Gc and Neu5Ac, and cats of this type show no naturally occurring antibodies to blood types A or B (2).

The enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase is encoded by the CMAH gene, which changes Neu5Ac to Neu5Gc (8, 9). Humans only have Neu5Ac, because a 92-bp in exon 6 of the CMAH gene renders the resulting protein inactive (10, 11). Conversely, many DNA polymorphisms have been described in the cat CMAH gene, including c.139C>T, c.142G>A, c.179G>T, c.187A>G, c.268T>A, c.327A>C, c.364C>T, c.374C>T, c.376G>A, c.593A>C, c.868A>C, c.898A>G, c.933delA, c.1322delT, c.1342G>A, and c.1603G>A in the coding region of the cat CMAH gene (12–16). In our previous report, we regard that double haploids selected from multiple recessive alleles carrying one or several of these SNPs are presumed to lead to a loss or reduction of enzyme activity for synthesizing Neu5Gc from Neu5Ac, and would thus be associated with expression of Neu5Ac on the erythrocyte membrane in type B and AB cats (15).

Blood typing and cross-matching based on pre-transfusion testing are crucial to determining blood compatibility and thus preventing hemolytic transfusion reactions. There are some discrepancies in results of blood typing among different blood-typing kits depending on the sensitivity and specificity for detection of antigen (17–21). In addition, the FeLV-related anemia, other anemic cases, autoagglutination or mixed-field agglutination could affect the results of blood typing (17, 19, 20). In the present study, we describe the detection of an incompatible cross-match case (subject K) in the feline AB blood group system, and another cat (subject R) with discordant results from the card agglutination (CARD) method and alloantibody agglutination test for blood typing. We here describe our results from serological and genetic investigations to characterize blood samples from these two cats. We then characterized the low-frequency haplotypes carried according to six mutations and a novel haplotype with a new SNP in the feline CMAH genes of these cats.

Blood typing and cross-match testing are the most common procedures applied in effective and safe transfusion medicine. Normally, identification of matching blood types between donors and recipients prevents major and minor transfusion reactions in the feline AB blood group system. Classification of the blood type is thus very important. This study is the first to report CMAH variants in cats with discordant blood types between CARD and TUBE methods in the feline AB blood group system.

Type AB erythrocytes show decreased expression of both A and B antigens compared with type A or type B erythrocytes (6). Griot-Wenk et.al showed that the mean fluorescence of AB cells was half that seen with A or B cells alone (7). We here identified two cats (subjects K and R) that were blood type B according to the CARD method, but were blood type AB according to the TUBE method. Seth et al. also reported discordant results from several commercially available serological tests for blood typing as compared with results from the TUBE method (17–21). Among six of 58 discordant cases, three cats showed type B with the CARD method and type AB with not only the TUBE method, but also with immunochromatographic cartridge (CHROM), gel-based (GEL), and conventional slide (SLIDE) methods. These results suggested that the CARD method did not detect expression of A antigen in cases of blood type AB cat with extremely low levels of A antigen (17). Type AB erythrocytes have approximately half as much A and B antigen as A and B erythrocyte, respectively (6, 7). We consider that type AB cats with a weak A antigen (henceforth A_wB) exist at low frequency among cats categorized as type B by the CARD method. We recommend both CARD method and TUBE method to determine the blood type for incompatible cross-match cases.

Many DNA polymorphisms in the cat CMAH gene have been described (12–16). Subjects K and R with type AB did not carry the critical genotype for blood type B (c.268:AA) and type AB (c.364:TT) cats in the CMAH gene (12, 14–16). The diplotype of subject K corresponded to Dip.9, which we identified in a type B cat in a previous report (15). That previous cat with Dip.9 was not blood typed using the TUBE method, so the possibility remains that Dip.9 cats are uniformly A_wB cats. The Dip.9 type B cat in that previous study (15) was originally planned to be bred as a donor cat for type B cats in our Veterinary Medical Teaching Hospital. However, the cat was determined to be unsuitable as a donor cat and was transferred to a foster family. The present subject R with new SNPs and a new diplotype not only showed that further CMAH variants exist in cat populations, but also that type A_wB cats can show genetic variation. This study suggested that not only blood typing methods and cross-matching methods but also CMAH variant analysis are useful in identifying appropriate blood sources for AB blood transfusion medicine.

The original contributions presented in the study are included in the article/supplementary files, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by the Experimental Animal Ethics Committee at NVLU. Written informed consent was obtained from the owners for the participation of their animals in this study.

YU, MY, TK, KY, and TO: conceived and designed the experiments. YU, MY, TK, EO, KO, CN, and TO: performed the experiments. TO and MY: analyzed the data. YU, MY, CU, and TO: wrote the paper. All authors read and approved the final manuscript.

This work was supported by KAKENHI scientific research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Nos. 16K08062 and 19K06392).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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