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Lymphocyte and markers of inflammation detection in the conjunctival epithelium of patients with dry eye by enhanced flow cytometry
Author(s) -
BARABINO S,
MONTALDO E,
SOLIGNANI F,
MINGARI C,
ROLANDO M
Publication year - 2009
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2009.3135.x
Subject(s) - flow cytometry , paraformaldehyde , conjunctiva , pathology , cytometry , cell counting , hla dr , lymphocyte , cytology , cd3 , medicine , cell , biology , immune system , immunology , antigen , cd8 , cell cycle , genetics
Purpose 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 (IC) sampling of the conjunctiva in dry eye patients, and make the analysis of epithelial and lymphocyte cell populations with flow cytometry possible. Methods IC specimens were collected in 15 normal subject and 15 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 a group of 30 dry eye patients were placed in FCS and stained for the expression of CK19, CD45, CD3, HLA‐DR, and analyzed by flow cytometry. A control group of 10 subjects was used as control. Results 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. Conclusion 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.