Analyzing immune cell profiles in dogs is gaining popularity due to the importance of immune status and its effect on tumors or other infectious and immune-mediated diseases. In this study, the frequencies of immune cell populations in 43 healthy companion dogs were investigated. The impact of pre-analytical delay on the biological integrity of blood samples was evaluated. Blood samples from 43 healthy companion dogs were used to isolate peripheral blood mononuclear cells (PBMCs) and white blood cells (WBCs). Flow cytometry analyzed immune cell frequencies and viability at fresh (~2h), 12h, and 24h after collection. RNA from inflammatory cytokines and apoptosis-associated genes was extracted by TRIzol-based and column-based methods and analyzed at fresh (~2h), 6h, and 12h. The immune cell frequencies in PBMCs were observed as follows: T-cells 30.93±17.62%; helper T-cells (Th) 18.70±12.04%; cytotoxic T-cells (Tc) 7.19±4.20%; B-cells 7.22±5.77%; monocytes 3.10±2.87%, and NK cells 0.60±0.68% of lymphocytes, and regulatory T-cells (Treg) 8.74±4.27% of CD4⁺ cells. Cell viability was maintained up to 12h in both PBMCs and WBCs, while monocyte, neutrophil, and Treg in WBCs significantly decreased at 24h (P<0.05). Cell frequencies remained stable in PBMCs up to 24h, but the frequencies of T-cell subsets were decreased in 12h (P<0.05). Although RNA yield was higher with the TRIzol-based extraction compared to the column-based method, mRNA extracted by column-based method was more stable. The mRNA levels varied with each target transcript and storing time, indicating a need for standardized protocols. This study provides preliminary data on immune cell profiles in canine PBMCs from companion dogs and throws light on experimental integrity during transportation of canine blood for multi-center research.

To Cite This Article: Oh EY, Kim Y, Lee JY, Seok J, Lee SM and Lee S, 2025. A descriptive survey of immune cell frequencies and an evaluation of pre-analytical delay effects on blood samples from healthy companion dogs. Pak Vet J. http://dx.doi.org/10.29261/pakvetj/2025.290