Effect of storage conditions on subpopulations of peripheral blood T lymphocytes isolated from naïve cattle and cattle infected with foot-and-mouth disease virus

Background: Immunophenotyping of blood lymphocytes by flow cytometry is important in infectious disease research. In animal experiments and other longitudinal studies, the processing, prompt staining, and analysis of fresh samples is a logistical challenge and daily assay variation can confound data interpretation. Objective: This study examined the feasibility of cryopreservation and deferred analysis of bovine peripheral blood T lymphocytes from normal or infected animals. Methods: Peripheral blood mononuclear cells were collected from 4 naïve Holstein steers and 4 steers infected with foot-and-mouth-disease virus serotype Asia1. Identical aliquots were labeled and analyzed immediately, labeled for deferred analysis, or stored at -70°C or over liquid nitrogen for up to 3 weeks before labeling. Results: Freezing of unlabeled cells induced statistically significant changes in phenotypic recognition. In infected animals, the γδ T-cell population increased by 28% and CD8⁺ αβT cells by 32%, while total CD3⁺ cells decreased by 16%, and CD4⁺ αβT cells decreased by 12%. Subsequent storage of frozen cells for the duration of the study, however, had no significant effect. There was less than 20% relative change in subpopulation sizes, and storage at -70°C or over liquid nitrogen was equivalent. Conclusions: Depending on the objectives and practical limitations of a study, deferred labeling of peripheral blood lymphocytes can be a viable option. Although frozen storage of lymphocytes can introduce some artifactual distortion of relative cell populations, frozen cells can be maintained in storage until all samples in a longitudinal study can be analyzed in batch under standardized conditions and without introducing further bias.