title: Precision and linearity targets for validation of an IFNgamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides.
abstract: BACKGROUND
  Single-cell assays of immune function are increasingly used to monitor T cell responses in immunotherapy clinical trials. Standardization and validation of such assays are therefore important to interpretation of the clinical trial data. Here we assess the levels of intra-assay, inter-assay, and inter-operator precision, as well as linearity, of CD8+ T cell IFNgamma-based ELISPOT and cytokine flow cytometry (CFC), as well as tetramer assays.
  
  
  RESULTS
  Precision was measured in cryopreserved PBMC with a low, medium, or high response level to a CMV pp65 peptide or peptide mixture. Intra-assay precision was assessed using 6 replicates per assay; inter-assay precision was assessed by performing 8 assays on different days; and inter-operator precision was assessed using 3 different operators working on the same day. Percent CV values ranged from 4% to 133% depending upon the assay and response level. Linearity was measured by diluting PBMC from a high responder into PBMC from a non-responder, and yielded R2 values from 0.85 to 0.99 depending upon the assay and antigen.
  
  
  CONCLUSIONS
  These data provide target values for precision and linearity of single-cell assays for those wishing to validate these assays in their own laboratories. They also allow for comparison of the precision and linearity of ELISPOT, CFC, and tetramer across a range of response levels. There was a trend toward tetramer assays showing the highest precision, followed closely by CFC, and then ELISPOT; while all three assays had similar linearity. These findings are contingent upon the use of optimized protocols for each assay.
authors: BD Biosciences, San Jose, CA, USA. holden_maecker@sbcglobal.net
authors: Holden T
authors: HT
authors: Maecker
authors: Jeffrey
authors: J
authors: Hassler
authors: Janice K
authors: JK
authors: Payne
authors: Amanda
authors: A
authors: Summers
authors: Karrie
authors: K
authors: Comatas
authors: Manar
authors: M
authors: Ghanayem
authors: Michael A
authors: MA
authors: Morse
authors: Timothy M
authors: TM
authors: Clay
authors: Herbert K
authors: HK
authors: Lyerly
authors: Sonny
authors: S
authors: Bhatia
authors: Smita A
authors: SA
authors: Ghanekar
authors: Vernon C
authors: VC
authors: Maino
authors: Corazon
authors: C
authors: Delarosa
authors: Mary L
authors: ML
authors: Disis
mesh_terms: D003587:Cytomegalovirus / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis*; D010455:Peptides / Q000032:analysis*; D015203:Reproducibility of Results; D014019:Tissue Donors; D014764:Viral Proteins / Q000032:analysis*
chemical_list: D010455:Peptides; D014764:Viral Proteins; D007371:Interferon-gamma