Precision and linearity targets for validation of an IFNgamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides.
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.
Maecker
Holden T
HT
BD Biosciences, San Jose, CA, USA. holden_maecker@sbcglobal.net
Hassler
Jeffrey
J
Payne
Janice K
JK
Summers
Amanda
A
Comatas
Karrie
K
Ghanayem
Manar
M
Morse
Michael A
MA
Clay
Timothy M
TM
Lyerly
Herbert K
HK
Bhatia
Sonny
S
Ghanekar
Smita A
SA
Maino
Vernon C
VC
Delarosa
Corazon
C
Disis
Mary L
ML
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*
D010455:Peptides; D014764:Viral Proteins; D007371:Interferon-gamma