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

Conclusion of Paper

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

   • Cell - Blood

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	ELISpot
   Cell count/volume	Flow cytometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(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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