title: Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT.
abstract: 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.
authors: BD Biosciences, San Jose, USA. holden_maecker@bd.com
authors: Holden T
authors: HT
authors: Maecker
authors: James
authors: J
authors: Moon
authors: Sonny
authors: S
authors: Bhatia
authors: Smita A
authors: SA
authors: Ghanekar
authors: Vernon C
authors: VC
authors: Maino
authors: Janice K
authors: JK
authors: Payne
authors: Kristine
authors: K
authors: Kuus-Reichel
authors: Jennie C
authors: JC
authors: Chang
authors: Amanda
authors: A
authors: Summers
authors: Timothy M
authors: TM
authors: Clay
authors: Michael A
authors: MA
authors: Morse
authors: H Kim
authors: HK
authors: Lyerly
authors: Corazon
authors: C
authors: DeLaRosa
authors: Donna P
authors: DP
authors: Ankerst
authors: Mary L
authors: ML
authors: Disis
mesh_terms: 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*
chemical_list: D000956:Antigens, Viral; D015415:Biomarkers; D015789:HLA-A2 Antigen; D010446:Peptide Fragments; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa