Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides
<p>Abstract</p> <p>Background</p> <p>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 d...
| Main Authors: | , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2008-03-01
|
| Series: | BMC Immunology |
| Online Access: | http://www.biomedcentral.com/1471-2172/9/9 |
| _version_ | 1818039922685116416 |
|---|---|
| author | Lyerly Herbert K Clay Timothy M Morse Michael A Ghanayem Manar Comatas Karrie Summers Amanda Payne Janice K Hassler Jeffrey Maecker Holden T Bhatia Sonny Ghanekar Smita A Maino Vernon C delaRosa Corazon Disis Mary L |
| author_facet | Lyerly Herbert K Clay Timothy M Morse Michael A Ghanayem Manar Comatas Karrie Summers Amanda Payne Janice K Hassler Jeffrey Maecker Holden T Bhatia Sonny Ghanekar Smita A Maino Vernon C delaRosa Corazon Disis Mary L |
| author_sort | Lyerly Herbert K |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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<sup>2 </sup>values from 0.85 to 0.99 depending upon the assay and antigen.</p> <p>Conclusion</p> <p>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.</p> |
| first_indexed | 2024-12-10T08:06:20Z |
| format | Article |
| id | doaj.art-3fe1cec427d24d7aac9d4686edfae812 |
| institution | Directory Open Access Journal |
| issn | 1471-2172 |
| language | English |
| last_indexed | 2024-12-10T08:06:20Z |
| publishDate | 2008-03-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Immunology |
| spelling | doaj.art-3fe1cec427d24d7aac9d4686edfae8122022-12-22T01:56:39ZengBMCBMC Immunology1471-21722008-03-0191910.1186/1471-2172-9-9Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptidesLyerly Herbert KClay Timothy MMorse Michael AGhanayem ManarComatas KarrieSummers AmandaPayne Janice KHassler JeffreyMaecker Holden TBhatia SonnyGhanekar Smita AMaino Vernon CdelaRosa CorazonDisis Mary L<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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<sup>2 </sup>values from 0.85 to 0.99 depending upon the assay and antigen.</p> <p>Conclusion</p> <p>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.</p>http://www.biomedcentral.com/1471-2172/9/9 |
| spellingShingle | Lyerly Herbert K Clay Timothy M Morse Michael A Ghanayem Manar Comatas Karrie Summers Amanda Payne Janice K Hassler Jeffrey Maecker Holden T Bhatia Sonny Ghanekar Smita A Maino Vernon C delaRosa Corazon Disis Mary L Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides BMC Immunology |
| title | Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides |
| title_full | Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides |
| title_fullStr | Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides |
| title_full_unstemmed | Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides |
| title_short | Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides |
| title_sort | precision and linearity targets for validation of an ifnγ elispot cytokine flow cytometry and tetramer assay using cmv peptides |
| url | http://www.biomedcentral.com/1471-2172/9/9 |
| work_keys_str_mv | AT lyerlyherbertk precisionandlinearitytargetsforvalidationofanifngelispotcytokineflowcytometryandtetramerassayusingcmvpeptides AT claytimothym precisionandlinearitytargetsforvalidationofanifngelispotcytokineflowcytometryandtetramerassayusingcmvpeptides AT morsemichaela precisionandlinearitytargetsforvalidationofanifngelispotcytokineflowcytometryandtetramerassayusingcmvpeptides AT ghanayemmanar precisionandlinearitytargetsforvalidationofanifngelispotcytokineflowcytometryandtetramerassayusingcmvpeptides AT comataskarrie precisionandlinearitytargetsforvalidationofanifngelispotcytokineflowcytometryandtetramerassayusingcmvpeptides AT summersamanda precisionandlinearitytargetsforvalidationofanifngelispotcytokineflowcytometryandtetramerassayusingcmvpeptides AT paynejanicek precisionandlinearitytargetsforvalidationofanifngelispotcytokineflowcytometryandtetramerassayusingcmvpeptides AT hasslerjeffrey precisionandlinearitytargetsforvalidationofanifngelispotcytokineflowcytometryandtetramerassayusingcmvpeptides AT maeckerholdent precisionandlinearitytargetsforvalidationofanifngelispotcytokineflowcytometryandtetramerassayusingcmvpeptides AT bhatiasonny precisionandlinearitytargetsforvalidationofanifngelispotcytokineflowcytometryandtetramerassayusingcmvpeptides AT ghanekarsmitaa precisionandlinearitytargetsforvalidationofanifngelispotcytokineflowcytometryandtetramerassayusingcmvpeptides AT mainovernonc precisionandlinearitytargetsforvalidationofanifngelispotcytokineflowcytometryandtetramerassayusingcmvpeptides AT delarosacorazon precisionandlinearitytargetsforvalidationofanifngelispotcytokineflowcytometryandtetramerassayusingcmvpeptides AT disismaryl precisionandlinearitytargetsforvalidationofanifngelispotcytokineflowcytometryandtetramerassayusingcmvpeptides |