Utilizing Corneal Fluorescein and Conjunctival Lissamine Green Staining Combined with in vivo Confocal Microscopy for Grading the Severity of Dry Eye Disease

Objective: To explore the application of corneal fluorescein sodium (CFS)-conjunctival lissamine green (CLG) staining combined with in vivo confocal microscopy (IVCM) in clinical grading of the severity of dry eye disease (DED). Methods: Eleven normal persons (4 males and 7 females) and thirty-two mild, moderate, and severe DED patients (aged 22 to 56 years, and mean age 45.4 +/- 12.9 years;14 males and 18 females) were included for CFS-CLG staining. The stained positive areas were observed and recorded for the number and morphology of staining points. Subsequently, the morphology and cell density of corneal and conjunctival cells were observed and analyzed by IVCM. Results: CFS-CLG staining revealed that the number of CFS-stained points were not significantly increased in mild DED, and there was a small amount of CLG staining in the temporal bulbar conjunctiva; the CFS-stained points of moderate DED were increased compared with mild DED, and the nasal conjunctiva diffuse small flake CLG staining was observed; the cornea of severe DED had the most fluorescein-stained points, and the conjunctiva diffuse large CLG staining. IVCM examination showed that corneal epithelial basal cell density was significantly decreased, while activated corneal Langerhans cells were significantly increased in moderate and severe DED. Meanwhile, the morphology of superficial stromal cells of cornea became irregular and the cell density decreased significantly in moderate and severe DED. The density of conjunctival goblet cells was also significantly reduced in moderate and severe DED. Moreover, the density of activated conjunctival Langerhans cells increased significantly in mild, moderate, and severe DED. Conclusion: Observing and detecting the morphology and density of corneal epithelial basal cells, superficial stromal cells, corneal Langerhans cells, conjunctival goblet cells, and conjunctival Langerhans cells in positive CFS-CLG stained areas by IVCM, which may be a reliable basis for clinical grading of DED severity.