Methodology article

Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides

Holden T Maecker¹ , Jeffrey Hassler² , Janice K Payne³ , Amanda Summers⁴ , Karrie Comatas⁴ , Manar Ghanayem⁴ , Michael A Morse⁴ , Timothy M Clay⁴ , Herbert K Lyerly⁴ , Sonny Bhatia¹ , Smita A Ghanekar¹ , Vernon C Maino¹ , Corazon delaRosa⁵ and Mary L Disis⁵

¹BD Biosciences, San Jose, CA, USA

²Cancer Research and Biostatistics, Seattle, WA, USA

³Beckman-Coulter, San Diego, CA, USA

⁴Departments of Surgery, Medicine, Pathology, and Immunology, and Duke Comprehensive Cancer Center, Duke University Medical Center, Durham, NC, USA

⁵Tumor Vaccine Group, Division of Oncology, University of Washington, Seattle, WA, USA

author email corresponding author email

BMC Immunology 2008, 9:9doi:10.1186/1471-2172-9-9

Published:	17 March 2008

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 IFNγ-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.

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.