Cryopreservation modulates the detection of regulatory T cell markers

Sebastian Sattui, Carolina De La Flor, Cesar Sanchez, Dorothy Lewis, Giovanni Lopez, Emiliana Rizo-Patrõn, A. Clinton White, Martin Montes

Research output: Contribution to journalArticle

28 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)54-58
Number of pages5
JournalCytometry Part B - Clinical Cytometry
Volume82 B
Issue number1
DOIs
StatePublished - Jan 2012
Externally publishedYes

Fingerprint

Cryopreservation
Regulatory T-Lymphocytes
HIV-1
Blood Cells
HIV
Peripheral Tolerance
Nonparametric Statistics
Dimethyl Sulfoxide
Communicable Diseases
Flow Cytometry
Nitrogen
Research

Keywords

  • cryopreservation
  • FoxP3
  • HIV
  • regulatory T cells

ASJC Scopus subject areas

  • Cell Biology
  • Histology
  • Pathology and Forensic Medicine

Cite this

Sattui, S., De La Flor, C., Sanchez, C., Lewis, D., Lopez, G., Rizo-Patrõn, E., ... Montes, M. (2012). Cryopreservation modulates the detection of regulatory T cell markers. Cytometry Part B - Clinical Cytometry, 82 B(1), 54-58. https://doi.org/10.1002/cyto.b.20621

Cryopreservation modulates the detection of regulatory T cell markers. / Sattui, Sebastian; De La Flor, Carolina; Sanchez, Cesar; Lewis, Dorothy; Lopez, Giovanni; Rizo-Patrõn, Emiliana; White, A. Clinton; Montes, Martin.

In: Cytometry Part B - Clinical Cytometry, Vol. 82 B, No. 1, 01.2012, p. 54-58.

Research output: Contribution to journalArticle

Sattui, S, De La Flor, C, Sanchez, C, Lewis, D, Lopez, G, Rizo-Patrõn, E, White, AC & Montes, M 2012, 'Cryopreservation modulates the detection of regulatory T cell markers', Cytometry Part B - Clinical Cytometry, vol. 82 B, no. 1, pp. 54-58. https://doi.org/10.1002/cyto.b.20621
Sattui S, De La Flor C, Sanchez C, Lewis D, Lopez G, Rizo-Patrõn E et al. Cryopreservation modulates the detection of regulatory T cell markers. Cytometry Part B - Clinical Cytometry. 2012 Jan;82 B(1):54-58. https://doi.org/10.1002/cyto.b.20621
Sattui, Sebastian ; De La Flor, Carolina ; Sanchez, Cesar ; Lewis, Dorothy ; Lopez, Giovanni ; Rizo-Patrõn, Emiliana ; White, A. Clinton ; Montes, Martin. / Cryopreservation modulates the detection of regulatory T cell markers. In: Cytometry Part B - Clinical Cytometry. 2012 ; Vol. 82 B, No. 1. pp. 54-58.
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AB - 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.

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