Corneal epithelial permeability to fluorescein in humans by a multi-drop method

PURPOSE: The permeability of the corneal epithelium to fluorescein P(dc) is an indicator of the health of the ocular surface. It can be measured in a clinical setting by determining the accumulation of fluorescein in the stroma following administration of the dye on the ocular surface. Here we demonstrate a new multi-drop method for the measurement of P(dc) by a spot fluorometer. METHODS: Twenty-nine healthy participants were recruited for this study. First, a probe-drop of fluorescein (0.35%, 2 μL) was instilled on the conjunctiva. The clearance of the dye from the tears was immediately measured using the fluorometer. Following this, two loading drops (2%; 6 μL each) were administered 10 min apart. Fifteen minutes later, the ocular surface was washed and fluorescence from the stroma F(s) was measured. Permeability was calculated using P(dc) = (Q x F(s))/ (2 x AUC), where Q is the stromal thickness and AUC is the area under the fluorescence vs. time curve for the loading drops. RESULTS: After the probe drop, the tear fluorescence followed an exponential decay (elimination rate constant; k(d) = 0.41 ± 0.28 per min; 49 eyes of 29 subjects), but the increase in F(s) was negligible. However, after the loading drops, the measured F(s) was ~ 20-fold higher than the autofluorescence and could be recorded at a high signal to noise ratio (SNR > 40). The intra-subject variability of k(d) was insignificant. Since fluorescein undergoes concentration quenching at > 0.5%, the value of AUC for the loading drops was estimated by scaling the AUC of the probe drop. The calculated P(dc) was 0.54 ± 0.54 nm/sec (n = 49). A Monte Carlo simulation of the model for the multi-drop protocol confirmed the robustness of the estimated P(dc). CONCLUSIONS: The new multi-drop method can be used in place of the single-drop approach. It can overcome a lack of sensitivity in fluorometers of high axial resolution. The P(dc) estimated by the multi-drop method is ~ 11-fold higher than previously reported but closer to the value reported for other drugs with equivalent octanol/water partition coefficient.