Impact of Long-Term Cryopreservation on Blood Immune Cell Markers in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Implications for Biomarker Discovery.
Author(s): Gómez-Mora E, Carrillo J, Urrea V, Rigau J, Alegre J, Cabrera C, Oltra E, Castro-Marrero J, Blanco J
Publication: Front Immunol, 2020, Vol. 11, Page 582330
PubMed ID: 33329554 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to assess the stability of immune markers during prolonged frozen storage and cytokine production in subsets of immune cells after prolonged frozen storage. Established immune markers as well as potential markers of myalgic encephalomyelitis (ME) / chronic fatigue syndrome (CFS) were evaluated in peripheral blood mononuclear cells (PBMCs) collected from ME/CSF patients and healthy individuals immediately after collection and after ≥3 years of storage in liquid nitrogen.
Conclusion of Paper
Significant differences in the frequency of some but not all of the T cell subpopulations and natural killer (NK) cell subpopulations suggest that several of the cell markers evaluated are affected by thawing and/or frozen storage of PBMCs for ≥3 y. Frozen and thawed biobanked PBMCs from ME/CSF patients that were thawed once had a significantly lower percentage of natural Tregs but a significantly higher percentage of Ki-67+ CD4+ T cells than fresh PBMCs (p=0.0461), while no differences were observed in the percentage of CD56+ cells or effector CD8 T cells. When frozen and thawed biobanked PBMCs from ME/CSF patients and healthy individuals were compared no significant differences in the frequency of immune cell subsets were observed; however, a higher frequency of natural Treg cells and a lower frequency of effector cells in ME/CFS patients compared to healthy controls when fresh PBMCs were assessed in an earlier publication (Curriu et al. 2013, PMID 23514202). Compared to fresh PBMC’s, frozen and thawed biobanked PBMCs had a lower frequency of natural killer (NK) cell subpopulations that were CD69+ (p=0.0002) and CD25+ cells (p=0.0003) and a higher frequency of a NK p46+ cells (P<0.001).”Cytokine expression was stimulated in frozen and thawed biobanked PBMCs from ME/CFS patients and healthy individuals, although the levels of the cytokines measured did not differ between the two diagnoses and data on fresh PBMCs was not collected.
Studies
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Study Purpose
The purpose of this study was to assess the stability of immune markers during prolonged frozen storage and cytokine production in subsets of immune cells after prolonged frozen storage. Whole blood was collected from 22 patients with ME/CFS into tubes containing EDTA that also had case-matched biobanked PBMC specimens. Two different cohorts of healthy controls were used for freshly processed PBMCs and those that had been cryopreserved for ≥3 y (n=18 and 12, respectively); controls were age- and sex-matched but were not the same individuals. PBMCs were isolated by density gradient centrifugation with Ficoll, washed and either analyzed immediately or resuspended in a cryomedium that contained fetal bovine serum and dimethyl sulfoxide (DMSO). Aliquots (1 mL) containing 1x107 cells per mL were frozen gradually at -80°C in a Mr. Frosty device over 48 h, and then transferred to liquid nitrogen until analysis. Frozen PBMCs were thawed in a 37°C water bath for 5 min prior to cell viability analysis. Immune cell population analysis was limited to viable cells. Cell viability was defined as the percentage of living cells relative to total cell count and was determined by exclusion of the eFluor stain and analysis by flow cytometry. Viable cells (that did not uptake the eFluor stain) were washed and incubated with corresponding antibodies for immunophenotyping by cell cytometry. Cytokine production was measured by antibody staining and flow cytometry analysis after stimulation and induction of production of the following cytokines: IFN-ɣ, IL-17, IL-4, TGF-β1.
Summary of Findings:
The percentage (or frequency) of natural and classic Treg cells were significantly but modestly correlated between fresh and frozen and thawed biobanked PBMCs from ME/CSF patients (r=0.50, p=0.043 and r=0.54, p=0.032, respectively); while, the percentage of CD4+ Ki-67+ cells were not correlated between freshly isolated PBMCs and frozen and thawed biobanked PBMCs. When directly compared, frozen and thawed biobanked PBMCs from ME/CSF patients had a significantly lower percentage of natural Tregs anda significantly higher percentage of Ki-67+ CD4+ T cells than fresh PBMCs (p=0.0461), but no differences were observed in the percentage of CD56+ cells or effector CD8 T cells. Thawed biobanked PBMCs from ME/CSF patients and healthy individuals had comparable frequency of immune cell subsets, but fresh PBMCs from ME/CSF patients and healthy individuals differed from one another in the frequency of two T cell subpopulations; ME/CFS patients had a higher frequency of natural Treg cells and a lower frequency of effector T cells)(reported in an earlier publication, Curriu et al. 2013, PMID 23514202). When natural killer (NK) cell subpopulations were compared between fresh and frozen biobanked PBMCs from ME/CFS patients, weak and nonsignificant correlations were observed for CD69+ (r=0.027), CD25+ (r=0.049), and NKp46+ cells (r=0.268). A lower frequency of CD69+ (p=0.0002) and CD25+ cells (p=0.0003) was observed in thawed frozen biobanked PBMCs, while a higher frequency of NK p46+ cells was observed in frozen and thawed biobanked PBMCs compared to fresh PBMCs (p<0.001). Cytokine expression was stimulated in frozen and thawed biobanked PBMCs from ME/CFS patients and healthy individuals, although the levels of the cytokines measured did not differ between the two diagnoses and data on fresh PBMCs was not collected.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
Diagnoses:
- Other diagnoses
Platform:
Analyte Technology Platform Cell count/volume Flow cytometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation None (fresh)
Frozen
Storage Storage duration 0 y
≥3 y