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

PURPOSE: The permeability of the corneal epithelium to fluorescein Pdc 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 Pdc 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 Fs was measured. Permeability was calculated using Pdc = (Q x Fs)/ (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; kd = 0.41 ± 0.28 per min; 49 eyes of 29 subjects), but the increase in Fs was negligible. However, after the loading drops, the measured Fs 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 kd 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 Pdc 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 Pdc. 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 Pdc 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.

Ocular Spot Fluorometer Equipped With a Lock-In Amplifier for Measurement of Aqueous Flare.

PURPOSE: To evaluate a custom-made ocular fluorometer for detection of intensity of light scatter (ILS) from the anterior chamber (A/C) as an objective measure of aqueous flare. METHODS: The fluorometer, equipped with a lock-in amplifier, was employed in the scatter mode to detect ILS from A/C. Measurements were performed with two illumination slit widths of 0.5 and 0.25 mm. The axial resolution at these slit widths were 80 and 200 µm, respectively. Healthy and pseudophakic eyes, with grade 0 Standardization of Uveitis Nomenclature (SUN) score, were employed as control subjects. ILS was also recorded in a cohort of patients who had undergone phacoemulsification and showed grades 1+ or 2+ on postoperative days 1 and 4. RESULTS: The inter- and intraobserver variabilities in the measurement of ILS were not significant. In cataract patients, ILS was significantly higher on postoperative day 1 relative to healthy eyes. By day 4, ILS decreased significantly and was only marginally different from ILS in quiet pseudophakic eyes or healthy eyes. Eyes with higher SUN scores showed proportionately increased ILS. The receiver-operator characteristic analysis indicated no advantage in using the smaller slit width in discriminating ILS at different SUN scores although it provided higher axial resolution. CONCLUSIONS: The lock-in-based spot fluorometer is reliable for measurement of ILS with high precision and accuracy.The measured ILS correlates linearly with SUN scores and can be used to provide a higher granularity for recording aqueous flare. TRANSLATIONAL RELEVANCE: The instrument can be used in the clinical management of uveitis and drug development toward uveitis.