NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT.

Author(s): Maecker HT, Moon J, Bhatia S, Ghanekar SA, Maino VC, Payne JK, Kuus-Reichel K, Chang JC, Summers A, Clay TM, Morse MA, Lyerly HK, DeLaRosa C, Ankerst DP, Disis ML

Publication: BMC Immunol, 2005, Vol. 6, Page 17

PubMed ID: 16026627 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine the effects of shipping fresh rather than cryopreserved peripheral blood mononuclear cells (PBMCs) on major histocompatibility complex (MHC) peptide tetramer staining, cytokine flow cytometry (CFC) and ELISpot assay results using cytomegalovirus (CMV) seronegative and seropositive specimens.

Conclusion of Paper

Correlations between fresh and cryopreserved PBMCs were very strong using tetramer and ELISpot assays but modest to strong using CFC, depending on the stimulation method. Sensitivity was higher using fresh specimens than cryopreserved specimen and the tetramer assay, ELISpot or CFC assay with pp65 mix stimulation were higher or the same, but with the same assays specificity was lower using fresh specimens than cryopreserved specimens. In contrast, sensitivity was lower and specificity higher using fresh instead of cryopreserved specimens and the CFC assay with pp65 A2 stimulation.

Studies

  1. Study Purpose

    The purpose of this study was to determine the effects of shipping fresh rather than cryopreserved PBMCs on MHC peptide tetramer staining, CFC and ELISpot assay results using CMV seronegative and seropositive specimens. Fresh specimens were mailed overnight in RPMI with 10% fetal bovine serum (FBS) in a foam container. Cryopreserved specimens were frozen in freezing media containing RPMI with 20% DMSO and 12.5% human serum albumin (HSA), stored at -80 degrees C for 24 h, stored in liquid nitrogen for 30 days, shipped on dry ice, stored at -80 degrees C, and thawed slowly before assay.

    Summary of Findings:

    Tetramer staining results were very strongly correlated (r2=0.9) between shipped fresh and cryopreserved specimens. Interferon (IFN)-gamma production, measured by CFC, in fresh specimens, after stimulation with pp65 mix, only showed a modest correlation to that in cryopreserved specimens (r2=0.6), but after stimulation with pp65 A2, a strong correlation between fresh and cryopreserved specimens was observed (r2=0.8). IFN-gamma production, measured by ELISpot after stimulation with pp65 mix, was very strongly correlated between fresh and cryopreserved specimens (r2=0.9), but correlation decreased when only CMV positive specimens were considered (r2=0.7), and there was a bias toward higher responses in cryopreserved specimens than fresh specimens (p=0.04). Tetramer assay and ELISpot sensitivity were higher while specificity of these two assays was lower using fresh specimens as opposed to cryopreserved specimens. After simulation with pp65 mix, CFC sensitivity was similar between fresh and cryopreserved specimens, but specificity of CFC was greater with cryopreseved specimens. After stimulation with pp65 A2, sensitivity was greater while specificity was lower using cryopreserved specimens as opposed to fresh specimens. CFC results were very strongly correlated with those from the tetramer assay for both cryopreserved and fresh specimens (both, r2=0.9), but CFC results were only modestly correlated with ELISpot results in fresh specimens (r2=0.5) and strongly correlated with ELISpot results in cryopreserved specimens (r2=0.7).

    Biospecimens
    Preservative Types
    • None (Fresh)
    • Frozen
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    Cell count/volume ELISpot
    Cell count/volume Flow cytometry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Storage Between site transportation method Mailed
    Storage Specimen transport duration/condition Dry ice
    Ambient temperature
    Biospecimen Preservation Type of fixation/preservation Frozen
    None (fresh)
    Preaquisition Diagnosis/ patient condition CMV positive
    CMV negative
    Flow cytometry Specific Technology platform Tetramer assay
    ELISpot
    Flow cytometry Specific Reaction solution CMV pp65 mix
    CMV pp65 A2

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