期刊
BMC IMMUNOLOGY
卷 9, 期 -, 页码 -出版社
BMC
DOI: 10.1186/1471-2172-9-9
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资金
- NCI NIH HHS [U54 CA090818] Funding Source: Medline
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 intraassay, inter-assay, and inter-operator precision, as well as linearity, of CD8+ T cell IFN gamma-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 R-2 values from 0.85 to 0.99 depending upon the assay and antigen. Conclusion: 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.
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