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
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).
- None (Fresh)
Analyte Technology Platform Cell count/volume ELISpot Cell count/volume Flow cytometry
Classification Pre-analytical Factor Value(s) Storage Between site transportation method Mailed
Storage Specimen transport duration/condition Dry ice
Biospecimen Preservation Type of fixation/preservation Frozen
Preaquisition Diagnosis/ patient condition CMV positive
Flow cytometry Specific Technology platform Tetramer assay
Flow cytometry Specific Reaction solution CMV pp65 mix
CMV pp65 A2