title: Cryopreservation modulates the detection of regulatory T cell markers.
abstract: BACKGROUND
  Regulatory T cells (Tregs) modulate the host response in infectious diseases and are key mediators of peripheral tolerance. Cryopreservation of peripheral blood mononuclear cells (PBMCs) is commonly used in immunological field studies where access to complex laboratory tests is not feasible. Our objective is to assess the effects of cryopreservation on the flow cytometric detection of surface and intracellular markers of Tregs.
  
  
  METHODS
  Heparinized venous blood was obtained from 36 healthy individuals and 15 HIV-1 infected subjects. PBMCs were isolated and stained for surface and intracellular markers of Tregs. PBMCs from each subject were cryopreserved in liquid nitrogen with DMSO; these cells were thawed and stained at a later date. All samples were analyzed by flow cytometry. The proportion of Tregs was compared using Wilcoxon signed-rank test.
  
  
  RESULTS
  Cryopreservation decreased the proportion of Tregs identified by surface and intracellular markers in healthy individuals and in HIV-1 patients. The proportion of CD4+CD25+FoxP3+ was decreased from 3.13 to 2.16% (P < 0.001) for non-HIV subjects and from 2.68 to 0.94% (P < 0.001) for HIV subjects, compared to fresh samples. Significant reduction was also observed for CD4+CD25+CD127lo-neg. However, the effect varied considerably between samples. The effect was similar among HIV and non-HIV patients (P = 0.38).
  
  
  CONCLUSIONS
  Cryopreservation modulates the detection of surface and intracellular markers of Tregs. These results confirm that research on Tregs, including studies of HIV-1 infected patients, should be carried out prospectively on fresh samples in order to obtain unbiased conclusions. Results using cryopreserved cells should be regarded as only preliminary.
authors.affiliation: Universidad Peruana Cayetano Heredia, Lima, Peru.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Sattui
authors.forename: Carolina
authors.initials: C
authors.lastname: de la Flor
authors.forename: Cesar
authors.initials: C
authors.lastname: Sanchez
authors.forename: Dorothy
authors.initials: D
authors.lastname: Lewis
authors.forename: Giovanni
authors.initials: G
authors.lastname: Lopez
authors.forename: Emiliana
authors.initials: E
authors.lastname: Rizo-Patrón
authors.forename: A Clinton
authors.initials: AC
authors.lastname: White
authors.forename: Martin
authors.initials: M
authors.lastname: Montes
mesh_terms: D000328:Adult; D016477:Artifacts; D015415:Biomarkers / Q000378:metabolism; D003080:Cold Temperature / Q000009:adverse effects*; D015925:Cryopreservation / Q000379:methods*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D051858:Forkhead Transcription Factors / Q000378:metabolism; D015658:HIV Infections / Q000097:blood / Q000276:immunology* / Q000473:pathology; D006801:Humans; D016130:Immunophenotyping; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism; D053699:Interleukin-7 Receptor alpha Subunit / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism; D008297:Male; D050378:T-Lymphocytes, Regulatory / Q000166:cytology* / Q000378:metabolism
chemical_list: D015415:Biomarkers; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D053645:Interleukin-2 Receptor alpha Subunit; D053699:Interleukin-7 Receptor alpha Subunit