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  • Correlation analysis of intracellular and secreted cytokines via the generalized integrated mean fluorescence intensity.

    Cytometry A. 77(9):873-80. doi: 10.1002/cyto.a.20943. September 2010. View on PubMed.
  • Authors

    Shooshtari P, Fortuno ES 3rd, Blimkie D, Yu M, Gupta A, Kollmann TR, and Brinkman RR
  • Abstract

    The immune response in humans is usually assessed using immunogenicity assays to provide biomarkers as correlates of protection (CoP). Flow cytometry is the assay of choice to measure intracellular cytokine staining (ICS) of cell-mediated immune (CMI) biomarkers. For CMI analysis, the integrated mean fluorescence intensity (iMFI) was introduced as a metric to represent the total functional CMI response as a CoP. iMFI is computed by multiplying the relative frequency (percent positive) of cells expressing a particular cytokine with the MFI of that population, and correlates better with protection in challenge models than either the percentage or the MFI of the cytokine-positive population. While determination of the iMFI as a CoP can readily be accomplished in animal models that allow challenge/protection experiments, this is not feasible in humans for ethical reasons. As a first step toward extending the iMFI concept to humans, we investigated the correlation of the iMFI derived from a human innate immune response ICS assay with functional cytokine release into the culture supernatant, as innate cytokines need to be released to have a functional impact. Next, we developed a quantitatively more correlative mathematical approach for calculating the functional response of cytokine-producing cells by incorporating the assignment of different weights to the magnitude (frequency of cytokine-positive cells) and the quality (the MFI) of the observed innate immune response. We refer to this model as generalized iMFI.

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