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. 2003 Jul;23(7):369-77.
doi: 10.1089/107999003322226023.

Intracellular staining for HIV-specific IFN-gamma production: statistical analyses establish reproducibility and criteria for distinguishing positive responses

Wendy L Trigona  1 , James H Clair, Natasha Persaud, Kara Punt, Margaret Bachinsky, Usha Sadasivan-Nair, Sheri Dubey, Lynda Tussey, Tong-Ming Fu, John Shiver
Affiliations

Intracellular staining for HIV-specific IFN-gamma production: statistical analyses establish reproducibility and criteria for distinguishing positive responses

Wendy L Trigona et al. J Interferon Cytokine Res. 2003 Jul.

Abstract

The cellular immune response plays a pivotal role in controlling the spread of HIV-1 infection by lysing virally infected cells and producing potent antiviral cytokines, such as interferon-gamma (IFN-gamma). Flow cytometric methods have been established to evaluate the contribution of both CD4 and CD8 subsets of T lymphocytes to the immune response to HIV by measuring their production of intracellular IFN-gamma following brief antigenic stimulation. We present a statistical treatment of intracellular cytokine staining (ICS) data that is aimed at establishing the reproducibility and robustness of this assay for use in HIV clinical trials. Comparisons of responses from HIV-seronegative and seropositive individuals were used to establish a 2-fold criterion for distinguishing positive responses with a low probability of false positives (<1%). Additional comparisons established that the reproducibility of the assay is between 1.4 and 2.0-fold depending on the magnitude of the response. Little variability was demonstrated between multiple operators for both the execution and analysis components of these experiments (<10% difference with 95% confidence). We conclude that the statistical criteria established by these analyses allow for the accurate detection and comparison of positive responses. Using these statistical criteria, the ICS assay is sufficiently robust for use in HIV-specific vaccine trials.

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