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Seth P Epstein, Penny A Asbell, Neha J Gadaria-Rathod, Yi Wei; Flow Cytometry Analysis of Inflammatory Biomarker HLA-DR for Multicenter Clinical Trials of Ocular Surface Disease. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5514.
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© ARVO (1962-2015); The Authors (2016-present)
To optimize the flow cytometric analysis of cells highly expressing HLA-DR antigen from impression cytology samples for the establishment of a standardized methodology for quantifying the relative amount of HLA-DR on the ocular surface cells objective metric for clinical trials and translational research studies of ocular surface disease.
To establish a Standard Operating Procedure (SOP) for utilizing HLA-DR expression as a biomarker of inflammation of the ocular surface, cells from 270 impression cytology samples of both normal (138) and dry eye (DE; 132) individuals were collected, then stained for HLA-DR utilizing direct immunostaining. The standardization and optimization of the relative quantification of these FITC-labeled HLA-DR expressing cells was evaluated comparing flow cytometry dot plot [fluorescence channel (FL-1) vs. cellular granularity (side scatter)] cytograms to histogram [fluorescence channel (FL-1) vs. event count] cytograms.
The slightly slanting nature of the fluorescence channel (FL)-1 of cell populations in the histogram leads to overlap of cells which highly express HLA-DR with those which do not. These groups are clearly differentiated when a dot plot is used allowing for easier, more precise gating. For this reason, the reported relative expression of HLA-DR varied greatly (statistically significant) between flow cytometry dot plots of FL-1 versus side scatter as compared to those of the histogram FL-1 versus event count for both normal and dry eye individuals (Normal: Dot: 0.47% ± 0.71, Histo: 3.24% ± 5.30, p ≤ 0.01; DED: Dot: 1.62% ± 3.27, Histo: 12.19% ± 17.08, p ≤ 0.01). Even overall, the reported relative expressions of HLA-DR antigen reported by the dot plot (1.13% ± 2.57) were statistically different than those of the histogram (7.61% ± 13.26; p ≤ 0.01).
Gating variations can result in significant differences in the determined percentage of cells expressing high levels of any immunolabeled target(s) such as biomarkers. For relative quantification, dot plots displaying FL-1 vs. cellular granularity (side scatter) are easier to consistently gate uniformly and reproducibly as compared to histograms (FL-1 vs. event count). Consistent methodology and using well trained personnel facilitates the establishment of an SOP to produce repeatability in gating techniques.
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