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

BackgroundImmunophenotyping 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. ObjectiveThis study examined the feasibility of cryopreservation and deferred analysis of bovine peripheral blood T lymphocytes from normal or infected animals. MethodsPeripheral blood mononuclear cells were collected from 4 naive 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 degrees C or over liquid nitrogen for up to 3 weeks before labeling. ResultsFreezing of unlabeled cells induced statistically significant changes in phenotypic recognition. In infected animals, the T-cell population increased by 28% and CD8(+) T cells by 32%, while total CD3(+) cells decreased by 16%, and CD4(+) T 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 degrees C or over liquid nitrogen was equivalent. ConclusionsDepending 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.