Proteomic characterization of DEA1 positive and negative RBC ghosts reveals specific molecular features to improve dog blood typing

Blood typing in dogs is a growing concern in veterinary science, considering the importance that pet healthcare has gained in the last years. In fact, a correct characterization can avoid immune-mediated transfusional reactions that typically occur at the second exposition with the non-self blood antigens after the first sensitization. In dogs, there are seven internationally recognized blood groups in the Dog Erythrocyte Antigen (DEA) classification, plus the recently discovered DaI group, determined by the presence of specific markers (proteins, lipids, or glycoconjugates) on red blood cells (RBC) membrane, which are little characterized so far. These antigens are inherited through Mendelian laws but independently, so a dog can co-express a different combination of these markers, being positive for some and negative for others. Currently, the most studied and clinically relevant antigen is DEA1, responsible for most of the transfusional reactions in DEA1-negative sensitized dogs.Worldwide prevalence of DEA1-positive dogs has been estimated to be around 50–65%, depending on the breed and the geographic area. Moreover, the DEA1 can be expressed in different quantities on the erythrocyte surface, giving dogs that are weakly or strongly positive at different levels [1]. For DEA1 blood typing, several tests have been developed through the years and the most widely used for routine analysis in veterinary clinics are the card agglutination kit and the immunochromatographic strip assay, which are based on the reaction between DEA1 and polyclonal or monoclonal murine antibodies. These techniques are easy, rapid and quite accurate, with specificity and sensitivity near or above 90%. However, some important drawbacks remain, such as the possibility of autoagglutination, the difficulty of having a clear result in dogs with anemia or other blood-related pathologies, the presence of a recent previous transfusion, and the subjectivity of the result interpretation, especially for the card agglutination test in which there can be different levels of the agglutination reaction based on DEA1 quantity [2]. For all these reasons, this study aimed to characterize the proteomic profiles of DEA1 positive and negative dogs’ RBC ghosts (erythrocyte membranes), in order to investigate the differences between the two types at protein level and to identify some possible differential markers that can provide novel targets for monoclonal antibody research and new diagnostic techniques.