title: Creating a robust framework for the analysis of cryopreserved samples in quantitative immunological experiments.
abstract: Longitudinal clinical or experimental immunological studies warrant the use of cryopreserved samples for flow cytometric phenotyping. Most notably CD62L+ and CD25+Foxp3+ counts were shown to be reduced by past studies. Here we are the first to compare the effects of cryopreservation on cell type calculations performed on a longitudinal dataset. We first compared lymphocyte subpopulation counts from fresh samples with those from samples frozen for either 5 or 6 months coming from 9 individuals. This way, we found that the cell counts obtained after basic lymphocyte differentiation in CD3+CD4+, CD3+CD8+, CD3-CD19+ and CD3-CD56+ were relatively robust for cryopreservation. However, when further subtyping CD4+ and CD8+ cells, we only found CCR7 and CD45RA to have a relation between fresh and cryopreserved counts, but we could not conclude the same for CD62L. Also, CD4+CD25+Foxp3+ were shown to be approximately 0.5 times less counted after cryopreservation. Next, we performed basic longitudinal calculations for which we either subtracted the cell counts at time 1 from the cell counts at time 2 or calculated the ratio between the cell counts at time 2 and time 1, for both fresh and cryopreserved samples. This way, we found that the use of absolute cell counts supported a good one-to-one relation between fresh and cryopreserved counts for all markers except CD62L and CD4+CD25+Foxp3+. In conclusion, we found no support for the use of CD62L and CD4+CD25+Foxp3+ as markers for calculations on flow cytometric counts from cryopreserved longitudinal datasets. However, all other basic lymphocyte markers proved to be relatively robust if absolute counts were used.
authors.affiliation: Centre for Health Economics Research and Modeling Infectious Diseases, Vaccine & Infectious Disease Institute, University of Antwerp, Antwerp, Belgium. benson.ogunjimi@ua.ac.be
authors.forename: B
authors.initials: B
authors.lastname: Ogunjimi
authors.forename: N
authors.initials: N
authors.lastname: Hens
authors.forename: R
authors.initials: R
authors.lastname: Malfait
authors.forename: V
authors.initials: V
authors.lastname: Van Tendeloo
authors.forename: E
authors.initials: E
authors.lastname: Smits
mesh_terms: D015703:Antigens, CD / Q000737:chemistry / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000737:chemistry / Q000276:immunology; D002454:Cell Differentiation / Q000276:immunology; D015925:Cryopreservation / Q000379:methods; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D017493:Leukocyte Common Antigens / Q000737:chemistry / Q000276:immunology; D008137:Longitudinal Studies; D018655:Lymphocyte Count / Q000379:methods; D016131:Lymphocyte Subsets / Q000737:chemistry* / Q000276:immunology*; D054400:Receptors, CCR7 / Q000737:chemistry / Q000276:immunology
chemical_list: D015703:Antigens, CD; C515370:CCR7 protein, human; D054400:Receptors, CCR7; D017493:Leukocyte Common Antigens