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S. Barabino, F. Solignani, E. Montaldo, C. Mingari, M. Rolando; Epithelial and Immune Cells Study in Dry Eye by Enhanced Flow Cytometry. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4271.
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Flow cytometry has been used to study inflammatory markers on conjunctival cells in dry eye, but the low number of cells which can be analyzed has limited the application of this technique. The aim of our study was to test the hypothesis that the use of cell culture medium can increase the number of cells obtained by impression cytology sampling of the conjunctiva, and make the analysis of epithelial and lymphocyte cell populations possible.
Impression cytology specimens were collected by applying one filter paper in the non-exposed bulbar conjunctiva in 15 normal subject and 10 dry eye patients. Samples collected from the right eye were placed in cell culture medium containing 10% foetal calf serum (FCS), and samples from the left eye in Phosphate Bufferred Saline containing 0.05% paraformaldehyde (PFA). The number of cells was analyzed by flow cytometry in both groups. Samples collected from another group of 30 dry eye patients were placed in PFA (n=15) or FCS (n=15) and stained for the expression of CK19, CD45, CD3, HLA-DR, and analyzed by flow cytometry.
The number of cells counted in the FCS samples was statistically increased in the normal (12791 ± 11350 events per minute) and dry eye group (23468 ± 15596) when compared to PFA samples (2031 ± 2336, and 2608 ± 1814 respectively). In dry eyes the low number of cells in PFA samples made possible only the analysis of HLA-DR expression, while in FCS samples epithelial (CK19+), leukocyte (CD45+), and lymphocyte (CD3+) cell populations were characterized, other than analyzed for HLA-DR expression.
This study indicates that using this new method of preservation can enhance flow cytometry analysis of epithelial and immune cells of the conjunctiva in dry eye patients, opening new scenarios in the comprehension of its pathogenesis and in testing new therapies.
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