Subjects

The study was conducted according to the tenets of the Declaration of Helsinki and approved by the Baylor College of Medicine Institutional Review Board. The written informed consent form was obtained from all participants after the explanation of the purpose and some possible consequences of the study. A total of 50 patients with different types of DED (41 women and 9 men; mean age: 58.88 ± 12.17 years; range: 31-86 years) were enrolled in this single-center observational study. Fifteen age- and sex-matched control subjects (11 women and 4 men; mean age: 50.67 ± 16.58 years; range: 30-77 years) were also recruited. All control subjects were selected from those who had no complaints of eye irritation, no anterior segment abnormality on biomicroscopic examination, and normal tear and ocular surface tests. Subjects with DED were classified as ADDE or EDE based on the criteria as described in our previous study. These groups were combined for some analyses. The presence of conjunctivochalasis (CCh) based on the presence of lid parallel folds in the temporal and nasal conjunctiva above the lid margin and visualization in OCT images of the inferior tear meniscus were noted.

Exclusion criteria for both groups was as follows: age under 18 years old, a history of any previous ocular surgery or ocular trauma, contact lens wear, active ocular inflammation/infection, or corneal disease or dystrophies that might affect the results.

Clinical Evaluation

All patients were asked to complete the Ocular Surface Disease Index (Allergan Inc, Irvine, California, USA) questionnaire (range: 0-100) and SANDE visual analog scale questionnaire (5 grades in 100 mm). Then, topographic surface regularity index and surface asymmetric index were measured using the Tomey TMS-4N computerized videokeratoscopy (Tomey, Phoenix, AZ ph). Finally, an ocular surface examination was performed in the following order: tear film break-up time (TBUT), corneal and conjunctival dye staining, and Schirmer test without anesthesia. TBUT was measured by instilling fluorescein into the lower fornix with a wetted fluorescein strip (BioGlo; HUB, Rancho Cucamonga, California, USA). Corneal fluorescein staining was graded 0-6 in each of 5 zones (central, nasal, temporal, inferior, superior), as previously described. Conjunctival lissamine green staining was graded on a scale of 0-3 exposed in the nasal and temporal bulbar conjunctiva with a total maximum score of 6. Schirmer I test without anesthesia was used to measure tear production by inserting a dry Schirmer test strip over the outer third of the lower eyelid margin. The ocular surface clinical parameters were all measured by the same experienced investigator (S.C.P.).

Tear Osmolarity

Tear osmolarity was measured with the TearLab Osmolarity System (OcuSense, San Diego, California, USA). All measurements were performed by a single investigator (K.G.) in the same room at the same temperature prior to any invasive clinical tests. The instrument was calibrated regularly in accordance with manufacturer guidelines.

Anterior Segment Optical Coherence Tomography and Image Analysis

All subjects underwent imaging with Optovue RTVue XR Avanti (Optovue, Inc, Fremont, California, USA) by a single well-trained investigator (K.G.) in order to visualize and measure corneal and conjunctival epithelial thickness. Careful and maximum attention was paid to the standardization of OCT scans. All scans were performed in a standardized manner using the same instrument at the same room conditions. Subjects were asked to blink several times and OCT images were obtained immediately after the last blink (within 2 seconds). Corneal epithelial thickness was automatically processed by using the instrument’s corneal epithelial thickness mapping and pachymetry software, corresponding to a 9-mm-diameter area ( Figure 1 ). While pachymetric and epithelial thickness maps of 9 mm diameter are shown on the right side of test output, other details are shown on the left side, including pachymetric statistics such as SN-IT (superior temporal – inferior temporal) (2-5 mm), S-I (superior – inferior) (2-5 mm), Min (minimum), and Min-Max values, and epithelium statistics such as S (superior) (2-7 mm), I (inferior) (2-7 mm), Min, Max (maximum), and Min-Max ( Figure 1 ).

An example of pachymetry wide report screen for the Optovue RTVue XR Avanti (Optovue, Inc, Fremont, California, USA). Summary statistics based on the pachymetric and epithelial thickness maps are displayed on the left side. Pachymetry and the corneal epithelial maps divided into zones are displayed on the right side.

The temporal (exposed) and inferior (nonexposed) bulbar conjunctiva were imaged to measure epithelial thickness. Temporal corneal-scleral limbus and bulbar conjunctiva were imaged using the same length horizontal scan that was adjusted to pass through the middle of the cornea, as shown in Figure 2 a. Inferior bulbar conjunctiva was imaged using the same horizontal scan length that was taken 1-2 mm below the inferior limbus centered at 6 o’clock, as shown in Figure 2 b. The conjunctival epithelium thickness (CET) was measured with the digital caliper from the surface to the change in brightness of the substantia propria noted in OCT images ( Figure 3 ). Three epithelium thickness measurements were made at 1.5-mm intervals in the central 3 mm of the conjunctival images ( Figure 3 ). The average of the 3 calculated values was used for statistical analysis.

Spectral-domain optical coherence tomography images of ocular surface epithelium. The temporal (exposed) (a) and inferior (nonexposed) (b) bulbar conjunctiva were imaged to measure epithelial thickness.

The conjunctival epithelium thickness measurement with the digital caliper from the surface to the change in brightness of the substantia propria noted in optical coherence tomography images. Three epithelium thickness measurements were made at 1.5-mm intervals in the central 3 mm of the conjunctival images.

Statistical Analysis

Statistical analyses were performed with SPSS for Mac version 23.0 (SPSS Inc, Chicago, Illinois, USA). For each patient, computer-generated randomization was performed to select 1 eye for statistical analysis. Regarding study variables, comparisons between groups were performed using the nonparametric Mann-Whitney U test, and comparisons between several groups were performed using the Kruskal-Wallis test, because all data were not normally distributed. The Spearman correlation test was performed to evaluate bivariate correlations between epithelial thickness parameters and the dry eye clinical tests. The test was considered to be statistically significant at P < .05.