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C. N. Ghobrial, S. P. Epstein, M. Vallabhajosyula, M. L. Massingale, D. Chen, P. A. Asbell; Impression Cytology With HLA-DR Quantification for the Evaluation of Dry Eye Disease. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5855. doi: https://doi.org/.
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Dry Eye Disease (DED) is a disorder of decreased tear production and/or excessive evaporation characterized by symptoms and complaints of eye irritation/pain with fluctuation in vision or even loss of vision. It is most usually characterized by ocular surface inflammation, decreased tear production and symptomatologic complaints by the patient. The clinical criteria lack specificity and do not usually allow a precise diagnosis. HLA-DR is an inflammatory marker expressed on the surface of antigen presenting cells. To determine whether HLA-DR titers are increased in DED, cells were collected from dry eye and normal patients, stained for HLA-DR and compared.
Cytologic specimens were taken by impression from 3 patients with chronic dry eyes and 8 normal individuals with healthy eyes per ophthalmologic examination. Clinical diagnoses were made on the basis of subjective symptoms, Schirmer's test, and slit lamp examination. Impression cytology specimens were collected at least 15 min after instillation of the last staining eye drop and obtained with patients under topical anesthesia, using 0.20 µm polyethersulfone filters divided in half without exerting pressure. After collection, membranes were placed into 2 mL of phosphate-buffered saline (PBS) with fixative (0.05% paraformaldehyde) and stored at 4°C until immediately before use. Bovine Serum Albumin in PBS (0.5%: 2 mL) was then added, the cells dislodged from the filters by vortexing for 10 sec, then collected by centrifugation (1600 rpms, 4°C, 5 min). After removing the filters and decanting, the cells were stained for HLA-DR with monoclonal antibodies via standard, indirect immunocytochemical techniques. Conjunctival impression was analyzed by a masked observer via validated flow cytometry methods. Percentage of positive cells and fluorescence intensities was quantified with flow cytometry (FACSCalibur, BD Biosciences) and fluorospheres (Dako).
At least 1000 cells per sample were analyzed (mean ± st dev: DED: 2395 ± 871; normal: 3571 ± 2775). Elevated expression of HLA-DR by conjunctival cells was found in dry eyes, whereas specimens remained almost negative (<5% of cells were positive) in normal eyes. HLA-DR were 7.55% - 15.57% (10.61% ± 4.34%) in dry eyes and 0.39% - 4.42% (mean: 1.47% ± 1.20%) in normals (p = 0.07).
Results of this preliminary study indicate that conjunctival epithelial cells may express elevated quantities of HLA-DR in DED. Development of flow cytometry in analysis of cytologic specimens provides a new, sensitive, and objective tool for diagnosing dry eye pathology.
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