Diurnal Variation, Topographical Distribution and Day-to-Day Repeatability of Ocular Surface Epithelial Immune Cells in Individuals with Dry Eye Disease

Objectives: To assess diurnal changes, topographical differences, and day-to-day repeatability of ocular surface epithelial immune cell (EIC) density and morphology in dry eye disease (DED). Methods: Sixteen participants with moderate-to-severe DED (mean (SE) age 49.4 (4.2) years) underwent in vivo confocal microscopy at three timepoints (day-1 morning and evening and day-2 morning) at six locations: central cornea, inferior whorl, inferior cornea, and temporal cornea, limbus and conjunctiva. Diurnal and topographical variation in EIC density and morphology were analyzed using linear mixed-effects models with adjusted pairwise comparisons. Day-to-day repeatability was assessed using the coefficient of repeatability (CoR) for density and Cohen’s kappa for morphology. Results: EIC density and morphology varied by location (p < 0.001) but not by timepoint at any location (p = 0.59–0.90). Density was highest at the inferior cornea (model-estimated mean: 101.2 (SE: 21.7) cells/mm2) and temporal limbus (104.3 (22.7) cells/mm2), and lowest at the central cornea (26.8 [5.1] cells/mm2 and inferior whorl (38.3 [8.2] cells/mm2; all pairwise, p < 0.001). EICs with large bodies were more frequent in conjunctiva (100%), inferior cornea (94%), and temporal cornea (87%), than in central cornea (34%) and whorl (19%) (all p ≤ 0.007). EICs with dendrites, and with long dendrites were similarly distributed (p < 0.001). Bland–Altman analysis showed low mean bias and EIC density was more repeatable at the central (CoR ± 23.8 cells/mm2) and temporal cornea (±27.5 cells/mm2) than the inferior cornea (±47.9 cells/mm2) or temporal conjunctiva (±42.3 cells/mm2). Morphology agreement was substantial to near-perfect (κ = 0.71–0.97). Conclusions: In DED, EIC density and morphology are diurnally stable and maintain topographic distribution patterns similar to healthy eyes. Day-to-day repeatability show location dependent patterns. The study provides feasibility data for using IVCM for EIC metrics in disease monitoring.