Erin McDonald ^* Eric Kehoe ^* Darcy Deines Mary McCarthy Brie Wright Susan Huse

• Zoetis, Fort Collins, United States

Immunophenotyping is a powerful tool for grading disease severity, aiding in diagnosis, predicting clinical response, and guiding the development of novel therapeutics. This pilot study employs high parameter immunophenotyping panels (15 markers for dog, 12 for cat) and leverages unsupervised clustering to identify immune cell populations. Our analysis uses machine learning and statistical algorithms to perform unsupervised clustering, multiple visualizations, and statistical analysis of high parameter flow cytometry data. This method reduces user bias and precisely identifies cell populations, demonstrating its potential to detect variations and differentiate populations effectively.To enhance our understanding of cat and dog biology and test the unsupervised clustering approach on real-world samples, we performed in-depth profiling of immune cell populations in blood collected from client-owned and laboratory animals (dogs (n=55) and cats (n=68)). These animals were categorized based on pruritic behavior or routine check-ups (non-pruritic controls). Unsupervised clustering revealed various immune cell populations, including T-cell subsets distinguished by CD62L expression and distinct monocyte subsets. Notably, there were significant differences in monocyte subsets between pruritic and non-pruritic animals. Pruritic dogs and cats showed significant shifts in CD62LHi T-cell subsets compared to non-pruritic controls, with opposite trends observed between pruritic cats and dogs. These findings underscore the importance of advancing veterinary immunophenotyping, expanding our knowledge about marker expression on circulating immune cells and driving progress in understanding veterinary-specific biology and uncovering new insights into various conditions and diseases.