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

Author
item ESCHBAUMER, MICHAEL - Oak Ridge Institute For Science And Education (ORISE)
item STENFELDT, CAROLINA - Oak Ridge Institute For Science And Education (ORISE)
item Pacheco Tobin, Juan
item REKANT, STEVEN - Oak Ridge Institute For Science And Education (ORISE)
item Arzt, Jonathan

Submitted to: Veterinary Clinical Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/19/2015
Publication Date: 1/23/2016
Citation: Eschbaumer, M., Stenfeldt, C., Pacheco Tobin, J., Rekant, S.I., Arzt, J. 2016. Effect of storage conditions on subpopulations of peripheral blood T lymphocytes from naive cattle and cattle infected with foot-and-mouth disease virus. Veterinary Clinical Pathology. 45:110-115. https://doi.org/10.1111/vcp.12327.
DOI: https://doi.org/10.1111/vcp.12327

Interpretive Summary: Flow cytometry is a technique that is used to count and identify cells in blood. In this study, researchers were particularly interested in one kind of white blood cell, the T lymphocytes. With flow cytometry, T lymphocytes can be divided and counted as the subsets T-helper, T-killer and gamma-delta-T cells. How many of a given number of T lymphocytes belong to each group can help us to better understand the immune response to infections such as foot-and-mouth disease in cattle. This makes flow cytometry a powerful, yet very time-consuming tool, and it is often not possible to complete the procedure on the same day that a sample is taken in an animal experiment. Our study compared the reliability of flow cytometry measurements of samples collected and stored under different conditions. Researchers compared storage in a refrigerator, a low-temperature freezer, and a liquid-nitrogen tank for up to 3 weeks. While refrigerated cells did not yield good results, frozen cells were better, but there still were differences when compared to fresh cells. We propose freezing samples over the course of an animal experiment and analyzing them all together at the end may be an acceptable practice.

Technical Abstract: Immunophenotyping of peripheral-blood lymphocytes by flow cytometry is an important tool for infectious disease research. In many live-animal experiments and other longitudinal studies, the processing, prompt staining, and analysis of fresh samples is a logistical challenge and daily variation can confound data interpretation. To examine the feasibility of cryopreservation and deferred analysis of bovine T cells, peripheral-blood mononuclear cells (PBMC) were collected from 4 Holstein steers infected with foot-and-mouth-disease virus serotype Asia1. Identical aliquots were either stained and analyzed immediately, stained for deferred analysis, or stored under different conditions (at 4 degrees C, negative 70 degrees C, or negative 196 degrees C over liquid nitrogen) for up to 3 weeks before staining. Initial freezing induced statistically significant changes of phenotypic parameters. Specifically, the gamma delta T cell and CD8 positive alpha Beta T cell populations grew by 28 and 32 percent, while total CD3 positive cells and CD4 positive alpha Beta T cells shrank by 16 and 12 percent, yet subsequent storage of frozen cells for the duration of the study had no additional significant effect. Independent of time in frozen storage, there was less than 20 percent relative change in subpopulation sizes, and storage at negative 70 degrees C or over liquid nitrogen was found to be equivalent. Based on these data, deferred staining of PBMC collected in longitudinal studies is a viable option. Frozen cells can be maintained in storage until all samples in a given experiment have been collected and can be analyzed together.