Primary immune-mediated hemolytic anemia (IMHA) is an important cause of serious morbidity and mortality in dogs. Despite numerous studies examining the demographics, treatment options, and prognostic indicators of disease, the mechanisms that underlie immune dysregulation remain poorly understood. The purpose of this study was to directly identify unique erythrocyte membrane antigens in dogs diagnosed with primary IMHA. Blood samples were obtained from dogs presented to the Ontario Veterinary College Teaching Hospital with primary IMHA prior to treatment, and also from control dogs (healthy dogs and dogs with non-immunologic anemia). Antibodies bound to erythrocyte membranes were eluted using xylene. Immunoblots using patient eluates reacted against pooled canine erythrocyte lysates, and autologous patient plasma reacted against xylene eluates, were performed. These results were compared to results of similar experiments using samples from control dogs. Bands appearing in patient but not control samples were considered potential autoantigens, and were submitted for identification by liquid chromatography followed by tandem mass spectrometry. Samples from 13 dogs with primary IMHA, 4 dogs with non-immunologic anemia, and 2 healthy dogs, were analyzed. Immunoblotting confirmed the presence of immunoglobulin in eluates from all dogs. Semi-quantitatively, eluates from IMHA patients contained more immunoglobulin than those of control dogs. Mass spectrometry identified complement C3 in patient but not in control dog samples. Additional peptides identified by mass spectrometry in patient but not control dog samples included peroxiredoxin 2 and calpain. The former comprises a cytosolic hydrogen peroxide scavenger, and has been associated with erythrocyte membranes under oxidative stress conditions inducing spherocytosis. Calpain is a calcium-dependent protease that may become activated with oxidative stress and induce erythrocyte apoptosis. These findings suggest that oxidative stress and apoptosis contribute to the pathogenesis of canine IMHA. / OVC Pet Trust Fund, American Kennel Club Canine Health Foundation