Establishment of a microplate assay for flow cytometric assessment and it is use for the evaluation of age-related phenotypic changes in canine whole blood leukocytes

The effectiveness of flow cytometric assays for canine use is still requiring standardization. Despite several studies using purified mononuclear cells, no methodology or reference ranges are available for immunophenotyping of whole blood leukocytes (WBL). Fresh and pre-fixed WBL were used to identify cell-subsets, (Thy-1+/CD5+/CD4+/CD8+/CD21+ and CD14+) and measure MHC-II, CD45RA/CD45RB expression. We described here an efficient method for fast quantification of canine-WBL, using pre-fix in a microplate assay, which allows long-term sample storage prior to phenotyping. Decreased percentage of CD5+-T-cells within the lymphocyte-gate and increased percentage of CD21+-B-cells were observed in young animals, which led to higher T/B cell ratios in middle-aged dogs. Lower numerical counts of Thy-1+, CD4+, CD8+ and CD21+ lymphocyte were observed when compared to young animals. In addition, we identified an age-related decline of MHC-II/CD45RA expression by lymphocytes. We proposed an improved method for phenotyping of canine peripheral blood mononuclear cells (PBMC) that has significant use for researchers and veterinary clinicians. The hematological changes of senescence previously identified on PBMC could be adequately reproduced on features identified by whole blood. Furthermore, this study supplies normal range references as baseline standards for clinical purposes, besides specific immunological parameters to monitor canine aging process.