title: Effect of storage conditions on subpopulations of peripheral blood T lymphocytes isolated from naïve cattle and cattle infected with foot-and-mouth disease virus.
abstract: 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.
authors.affiliation: Plum Island Animal Disease Center (PIADC), Foreign Animal Disease Research Unit (FADRU), Agricultural Research Service (ARS), United States Department of Agriculture (USDA), Greenport, NY, USA.|Oak Ridge Institute for Science and Education, PIADC Research Participation Program, Oak Ridge, TN, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Eschbaumer
authors.affiliation: Plum Island Animal Disease Center (PIADC), Foreign Animal Disease Research Unit (FADRU), Agricultural Research Service (ARS), United States Department of Agriculture (USDA), Greenport, NY, USA.|Oak Ridge Institute for Science and Education, PIADC Research Participation Program, Oak Ridge, TN, USA.
authors.forename: Carolina
authors.initials: C
authors.lastname: Stenfeldt
authors.affiliation: Plum Island Animal Disease Center (PIADC), Foreign Animal Disease Research Unit (FADRU), Agricultural Research Service (ARS), United States Department of Agriculture (USDA), Greenport, NY, USA.
authors.forename: Juan M
authors.initials: JM
authors.lastname: Pacheco
authors.affiliation: Plum Island Animal Disease Center (PIADC), Foreign Animal Disease Research Unit (FADRU), Agricultural Research Service (ARS), United States Department of Agriculture (USDA), Greenport, NY, USA.|Oak Ridge Institute for Science and Education, PIADC Research Participation Program, Oak Ridge, TN, USA.
authors.forename: Steven I
authors.initials: SI
authors.lastname: Rekant
authors.affiliation: Plum Island Animal Disease Center (PIADC), Foreign Animal Disease Research Unit (FADRU), Agricultural Research Service (ARS), United States Department of Agriculture (USDA), Greenport, NY, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Arzt
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002417:Cattle / Q000097:blood*; D002418:Cattle Diseases / Q000097:blood* / Q000276:immunology; D015925:Cryopreservation / Q000379:methods / Q000592:standards / Q000662:veterinary*; D005240:Feasibility Studies; D005434:Flow Cytometry / Q000662:veterinary; D005536:Foot-and-Mouth Disease / Q000097:blood*; D029544:Foot-and-Mouth Disease Virus / Q000276:immunology; D005615:Freezing; D016130:Immunophenotyping / Q000662:veterinary; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008137:Longitudinal Studies; D008297:Male; D013048:Specimen Handling / Q000662:veterinary; D013601:T-Lymphocytes / Q000276:immunology*
keywords: Cryopreservation; flow cytometry; immunophenotyping; peripheral blood mononuclear cells; storage