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E. S. Rosenberg, M. L. Massingale, S. P. Epstein, M. Vallabhajosyula, Y. Wei, M. Suzuki, C. Ghobrial, D. C. Brocks, P. A. Asbell; Dry Eye Disease: Correlating HLA-DR Expression with Clinical Severity. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4664.
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Dry Eye Disease (DED) is a disorder associated with inflammation of the ocular surface. This study evaluated the hypothesis that the percentage of conjunctival epithelial cells expressing HLA-DR correlates with clinical severity as measured by the Dry Eye Workshop (DEWS) grading scale and may offer a new, objective means of classifying DED.
49 patients with DED were included in this IRB approved study, undergoing clinical examination and impression cytology. Diagnosis was based on reported symptoms, Schirmer's test, and slit lamp examination (TBUT, corneal and conjunctival staining). Patients were assigned a DEWS grade by three masked observers. At least 15 minutes after administering fluorescein, using 0.20 µm polyethersulfone filters and under topical anesthesia, specimens were obtained via impression cytology and stored in 2 mL of paraformaldehyde [0.05% in phosphate-buffered saline (PBS), 4°C]. Bovine Serum Albumin in PBS (0.5%: 2 mL) was added before vortexing (10 sec) and centrifugation (1600 rpms, 4°C, 5 min). Cells were then stained for HLA-DR with monoclonal antibodies via standard, indirect immunocytochemical techniques. Conjunctival impression was analyzed by masked observer via validated flow cytometry methods and the percentage of positive cells were quantified with flow cytometry (FACSCalibur, BD Biosciences) and fluorospheres (Dako).
The percentage of conjunctival epithelial cells expressing HLA-DR positively correlates with DEWS severity grade (corr coeff=0.44). Conjunctival staining contributes most strongly to this correlation with corneal staining contributing less (corr coef=0.45 and 0.28 respectively). OSDI, TBUT and Schirmer’s test negatively correlate to HLA-DR levels (corr coeff= -0.01, -0.26 and -0.09 respectively). The percentage of cells, by severity grade, expressing HLA-DR were: grade 1 rank sum median: 3.00%; grade 2: 8.18%; grade 3: 10.88% and grade 4: 21.03%. The rank sum median values were significantly different between all grades [p<0.05 (Wilcoxson-Mann-Whitney Rank-Sum Test)] with the exception of grade 2 as compared to grade 3 (p=0.76).
This study demonstrates that expression of HLA-DR positive cells on the ocular surface correlates with DEWS clinical severity grade. Of the measures that comprise the DEWS grade, conjunctival staining is the most strongly correlated with HLA-DR expression. Measurement of the percentage of cells that are HLA-DR positive may be a new, minimally invasive, objective method for more precisely classifying patients suffering from DED and evaluating the efficacy of new treatments.
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