Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT.
BACKGROUND
Cryopreservation of PBMC and/or overnight shipping of samples are required for many clinical trials, despite their potentially adverse effects upon immune monitoring assays such as MHC-peptide tetramer staining, cytokine flow cytometry (CFC), and ELISPOT. In this study, we compared the performance of these assays on leukapheresed PBMC shipped overnight in medium versus cryopreserved PBMC from matched donors.


RESULTS
Using CMV pp65 peptide pool stimulation or pp65 HLA-A2 tetramer staining, there was significant correlation between shipped and cryopreserved samples for each assay (p <or= 0.001). The differences in response magnitude between cryopreserved and shipped PBMC specimens were not significant for most antigens and assays. There was significant correlation between CFC and ELISPOT assay using pp65 peptide pool stimulation, in both shipped and cryopreserved samples (p <or= 0.001). Strong correlation was observed between CFC (using HLA-A2-restricted pp65 peptide stimulation) and tetramer staining (p < 0.001). Roughly similar sensitivity and specificity were observed between the three assays and between shipped and cryopreserved samples for each assay.


CONCLUSIONS
We conclude that all three assays show concordant results on shipped versus cryopreserved specimens, when using a peptide-based readout. The assays are also concordant with each other in pair wise comparisons using equivalent antigen systems.
Maecker
Holden T
HT
BD Biosciences, San Jose, USA. holden_maecker@bd.com
Moon
James
J
Bhatia
Sonny
S
Ghanekar
Smita A
SA
Maino
Vernon C
VC
Payne
Janice K
JK
Kuus-Reichel
Kristine
K
Chang
Jennie C
JC
Summers
Amanda
A
Clay
Timothy M
TM
Morse
Michael A
MA
Lyerly
H Kim
HK
DeLaRosa
Corazon
C
Ankerst
Donna P
DP
Disis
Mary L
ML
D017951:Antigen Presentation; D000956:Antigens, Viral / Q000276:immunology*; D015415:Biomarkers / Q000032:analysis; D015925:Cryopreservation; D003587:Cytomegalovirus / Q000276:immunology*; D003586:Cytomegalovirus Infections / Q000097:blood / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D015789:HLA-A2 Antigen / Q000032:analysis* / Q000276:immunology; D006801:Humans; D007111:Immunity, Cellular; D007753:Laboratories; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology; D010750:Phosphoproteins / Q000276:immunology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016037:Single-Blind Method; D013048:Specimen Handling; D014611:Vaccination; D014763:Viral Matrix Proteins / Q000276:immunology*
D000956:Antigens, Viral; D015415:Biomarkers; D015789:HLA-A2 Antigen; D010446:Peptide Fragments; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa