Flow Cytometry Analysis of Conjunctival Cells From Healthy Subjects and Dry Eye Disease Patients

Abstract

Purpose: : 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).

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: flow cytometry • conjunctiva • cornea: tears/tear film/dry eye