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R. Reinoso, M. Calonge, E. Castellanos, Y. Diebold, H. Martínez-Osorio, I. Fernández, M. Martino, M. E. Stern, A. Corell; Flow Cytometry Analysis of Conjunctival Cells From Healthy Subjects and Dry Eye Disease Patients. Invest. Ophthalmol. Vis. Sci. 2009;50(13):517.
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To analyze conjunctival cells by flow cytometry (FC) obtained by minimally invasive means from patients diagnosed with mild-to-moderate evaporative-type dry eye disease (DED) caused by meibomian gland disease (MGD).
Inferior bulbar conjunctival cells were collected by brush cytology (BC) from 23 MGD patients who had never been treated. Recovered cells were characterized by 3 FC assays: 1) leukocyte lineage (CD45) and intraepithelial lymphocyte (IEL) subtypes: total T cells, citotoxic/suppressor T cells, helper T cells, B cells, NK cells, 2) viability and apoptotic stage; 3) cell cycle stage. Results were compared with previous data from 15 healthy controls.
FC analysis showed that 2.7% of recovered cells were CD45+ leukocytes. Of those, 60% were T lymphocytes (CD4: 27.7%, CD8: 32.1%; ratio 1.03), 39.9% were B cells, and 0.2% were NK-lymphocytes. In controls however, leukocytes were 4%, with higher T cell (CD4/CD8 ratio 1.98) and lower B cell proportions. Regarding cell viability, two different populations of cells were observed: 79.8% of larger, more complex and less viable (20.9%) cells and 16.8% of smaller, less complex and more viable (64,8%) cells; this smaller cell population was significantly more viable in healthy individuals. There was not significant difference in cell cycle stage (G0/G1: 80.9%, S-phase: 3.2%, G2/M: 8.6%) between patients and controls.
To our knowledge, this is the first time that conjunctival cell viability, cell cycle, apoptotic stage, and IELs obtained by BC from a patient population have been analyzed. In general, conjunctival cells were less viable, total IELs decreased, B cells increased, and T cells (mainly CD4+) decreased in DED patients. The clinical relevance of these findings needs to be further evaluated.
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