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Seth P. Epstein, Peter G. Dentone, Maureen G. Maguire, Penny A. Asbell; Repeatability and 30-Day Storage Reproducibility of Impression Cytology Samples as Evaluated by HLA-DR Expression in Normal Subjects. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1936.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the repeatability and 30-day storage reproducibility of the expression of HLA-DR, as determined by cell cytology (FACS) analysis, in impression cytology (IC) samples.
Cells were collected via impression cytology after administration of topical anesthetic and stored at 4°- 8°C. Cells from the left side of each eye formed sample "A" and from the rights side of each eye formed sample "B". For 3 patients, A and B samples were split to assess repeatability between splits and between A and B samples. For 10 patients, one of the samples (A or B) was stained the next day and the other (B or A) was stained 30 days later. To stain, cells were mechanically separated from the filters and divided into four masked equivalents, stained with FITC conjugated anti-HLA-DR (+) or negative control (-), and analyzed with Flourescence Activated Cell Sorter (FACS) in a "masked" fashion to compare the relative amount of HLA-DR expressed. Prophylactic antibiotics were administered post collection. Differences in mean percent (%) HLA-DR values were assessed with the paired t-test. Bland-Altman limits of agreement were calculated and the variability in sampling areas (A, B) was compared to the variability in split samples through analysis of variance (ANOVA). The FACS analyses were performed by an independent observer in a masked fashion.
Repeatability: for split samples stained the same day, the mean difference was -0.12%, p = 0.23; the 95% limits of agreement between the two splits was (-0.82%, +0.58%). The mean difference between areas of the same subject (A and B) was 0.10%, p = 0.43. The variability between areas was not greater than the variability between splits (F1,19 = 0.21; p=0.65).30-Day Storage Analysis: The mean difference for HLA-DR% was 0.31% [ie, Day 30 values were on average higher by 0.31 %; p=0.11]. The 95% limits of agreement were ( -0.91%, +1.53%).
Our technique for impression cytology analysis of HLA-DR appears to be repeatable and reproducible. Sampling area (A and B) and storage of up to 30 days does not appear to have an effect on HLA-DR expression.
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