All patients with pterygium examined in our office between May 2017 and May 2018 were invited to participate. Surgical procedures were performed by the same surgeon (FSV) during the same period. The inclusion criteria were age over 25 years and primary or recurrent pterygium in eyes with an intact superior limbal-conjunctival donor area. No restrictions were placed in the length of the pterygium. Exclusion criteria were recurrent pterygium in which the superior limbal-conjunctival region had been used as a donor in a previous surgery; eyes with diseases other than pterygium; very small or asymptomatic pterygium; patients with diffuse or sectoral limbic failure involving the superior limbal area; chronic inflammation from other etiologies not related to the pterygium itself; patients who failed to attend all postoperative assessments. The study followed the principles of the 1964 Declaration of Helsinki and was approved by the Research Ethics Committee of Federal University of Minas Gerais (CAAE: 80402017.4.0000.5149). All participating patients read and signed the previously approved informed consent form.
Surgical technique
Patients underwent wide pterygium dissection, clearance of the limbus, and resection of the pterygium’s fibrovascular bundle (Figs. 1A, 1B, 1C, 1D and 1E). The tissue for ALCT was removed from the superior bulbar conjunctiva. This graft had the same dimensions as the clear limbal and scleral areas at the site of the pterygium resection, as verified using a millimeter compass (Figs. 1F & 1G). Only the conjunctiva and limbal epithelium were removed, avoiding manipulation and excision of the TC as much as possible (Figs. 1F & 1G). The donor area was neither sutured nor cauterized nor had its wound edges approximated (Fig. 1H). The clear limbal and nasal sclera were restored after pterygium removal with ALCT, which was fixed using a fibrin sealant (Tyssel®; Baxter AG, Vienna, Austria) [23] (Figs. 1H and 1I).
Clinical evaluation in slit lamp and digital photography
Patients were clinically evaluated pre- and postoperatively by the same surgeon (FSV) for the biomicroscopic variables in the superior part of their eyeballs. In the pre- and postoperative visits (1, 7, 30, and 180 days after surgery), photographs of donor area were taken without flash at 10-x and 16-x magnification using a camera (Nikon®; Nikon Imaging Japan Inc.) attached to a slit-lamp (HR Asapt;® São Paulo, Brazil). We assessed: (1) conjunctival hyperemia (present or absent); (2) vessel path alteration in the donor area (congestion, path distortion, and variation in the number of vessels; their presence or absence, only considered ≥7 days after surgery.
Assessment of conjunctival mobility
It was assessed by the surgeon (FSV) using a cotton swab after instilling eye drops of 0.5% proxymetacaine (Anestalcon®; Alcon®, São Paulo-SP, Brazil). Conjunctiva was slightly pressed and displaced in the vertical and horizontal directions. Mobility was present when tissue displacement was seen. Data was recorded in a specific research protocol.
Assessment by external examiners
Six months after surgery, another clinical slit-lamp assessment of patients was performed by two ophthalmologists, glaucoma specialists, with over ten years of experience in TRAB. They were unaware of the preoperative presentation of patients and the scarring evolution up to examination. They answered questions about (1) observation of blood vessels from the ocular surface towards the limbus (presence or absence of changes such as congestion, path distortion, and variation in the number of vessels), (2) tissue mobility in the donor and recipient areas (presence or absence, assessed by touching with a cotton swab, and (3) the viability of surgical reintervention at the site, including TRAB.
AS-OCT Evaluation
AS-OCT exams were performed one day before the pterygium excision and, 30 and 180 days afterwards, using the Optovue-Avanti® device (Optovue Inc., Fremont, California, USA) with the lens as the anterior segment. This device acquires images at a speed of 70,000 A scans/second and an axial resolution of 5µm [24]. Conjunctival thickness and TC measurements were assessed based on the study by Howlett et al. who evaluated the conjunctiva-Tenon’s capsule complex in the superior bulbar conjunctiva [25].
The following pre- and postoperative measurements were compared (Fig. 2): (1) the thickness of the conjunctival epithelium; (2) the thickness of the conjunctival stroma; (3) the thickness of the TC, and (4) the assessment of the presence or absence of the STS.
To determine the site where TRAB trans-scleral fistula normally drains the aqueous humor, usually 3 mm from the limbus, the beginning of the epithelial limbus was marked, coinciding with the end of Bowman's layer [26]. This point of reference is accurate, unlike the transition from the epithelium limbus to the conjunctival epithelium, which is not a specific anatomical marker reproducible in all eyes. Measurements were taken 4 mm from the beginning of the limbus, instead of 3 mm from the end of the limbus (Fig. 2*). These measurements were performed using the imaging software built into the device. Each structure of interest was determined, and the thickness was recorded. The inbuilt software also obtained comparative AS-OCT pre- and postoperative images (Fig. 3).
Sample calculation
Sample calculation was determined using the conjunctival epithelium and conjunctival/TC stromal thickness from our pilot study, which used data from the first 10 patients and had a normal distribution. Standard deviation values of the conjunctival epithelium and conjunctival/TC stromal thickness were 7.35µm and 32.61µm, respectively. These values were similar to those reported by Zhang et al. [27,28] involving AS-OCT of the bulbar conjunctiva. Thus, the standard deviation values considered for the sample calculation were those obtained by Zhang et al. of a conjunctival epithelial thickness of 7.4µm and a conjunctival/TC stromal set of 32.5µm. With a sample of at least 23 eyes, a statistical power of 90% would be obtained in detecting the differences when comparing the pre- and postoperative mean values of the conjunctival epithelial thickness, as well as of the stromal/TC complex.
Statistical Analysis
A descriptive analysis was performed by evaluating the mean and the standard deviation of variables with normal distributions. The Shapiro–Wilk test was used to verify the
hypothesis of normality for continuous variables in micrometers (µm). Paired Student's t-test was used to compare the mean pre- and postoperative measurements of conjunctival epithelium thickness and the conjunctiva/TC stromal complex thickness.
Analyses were performed using the SPSS® Software (IBM, Chicago, IL) version 18, with a level of statistical significance of P<0.05.