This cross-sectional observational study was conducted at the Glaucoma Center of the Eye Clinic, Department of Surgical Sciences, University of Torino, Italy. The methods were applied in accordance to the tenets of the Declaration of Helsinki; informed consent was obtained from all subjects and the Ethics Committee (University & General Hospital San Giovanni Battista of Torino) gave approval.
To be included in the study, participants had to meet the following criteria: age between 18 and 80 years, best-corrected visual acuity (BCVA) greater than or equal to 20/30, spherical equivalent between +1.00D and -1.00 D for emmetropic subjects, and between -3.00D and -7.00D for myopic subjects, and open angle on gonioscopy. Subjects with ocular surgery, retinal or macular pathology, or systemic or neurologic conditions that could produce visual field defects, were excluded. Patients with macular pathologic changes related to myopia were excluded from the study, as myopia is frequently associated with macular changes and degenerations that may affect ganglion cell count.
All subjects underwent a comprehensive ophthalmic examination consisting of: BCVA, ultrasound pachymetry, slit-lamp biomicroscopy of anterior and posterior segment, Goldmann applanation tonometry (GAT), diurnal tonometric curve, gonioscopy, peripapillary, and macular imaging using Fourier Domain-OCT (FD-OCT RTVue-100 software version A4, 5, 0, 59; Optovue, Fremont, CA, USA), and measurement of axial lenght with low-coherence interferometry system (Aladdin biometer, Topcon). Standard Automated Perimery was performed with Swedish Interactive Threshold Algorithm (SITA) Standard strategy, program 24-2 of the Humphrey Field Analyzer (HFA; Carl Zeiss Meditec, Jena, Germany). Fixation losses less than or equal to 20%, false positives and false negatives less than or equal to 33% were established as the reliability criteria. The study included three groups of subjects: preperimetric glaucomatous (PPG), glaucomatous, and a control group recruited from the healthy population. The latter were required to have no family history of glaucoma, highest daily IOP less than 21 mm Hg, a normal visual field (VF) test and a normal optic nerve head (ONH) appearance. Patients affected by PPG had the highest daily IOP greater than 21 mm Hg, normal VF test, and ONH changes (cup–disc ratio alteration, disc hemorrhages, rim notching, diffused or localized RNFL defects). Primary open-angle glaucoma (POAG) was diagnosed based on the presence of highest daily IOP greater than 21 mm Hg, abnormal VF according to Hodapp-Parrish-Anderson criteria for diagnosing glaucomatous damage (14), and glaucomatous optic disc changes. Optic nerve head appearance was evaluated by slit-lamp biomicroscopy of the posterior segment using a 78-D lens.
Each group was further divided into three subgroups based on axial length: emmetropic, myopic with AL<25 mm and myopic with AL>25 mm, in order to analyze the effect of mild and moderate myopia.
The Glaucoma Protocol of FD-OCT RTVue-100 was used to acquire RNFL thickness measurements. The same operator repeated all scans three times. The RNFL thicknesses used for the analysis derived from the scan with the highest signal strength index (SSI). This scan was used to calculate RNFL thicknesses. Scans with motion artifacts and with signal strength index less than 45 were excluded.
Estimate of Retinal and Macular Ganglion Cell Count
The estimated of RGC count was obtained by applying the model developed and described in detail by Medeiros et al (9–11,15) based on the empirical formulas processed by Harwerth et al (8). We described this formula in detail in our previous article (Rolle et al.). (16)
The collection, processing and statistical analysis of the results were carried out through the Microsoft Excel 2016 worksheets and the SPSS statistical program for Windows, version 19.0, SPSS Inc, Chicago, IL. We used the analysis of variance (ANOVA) and χ2 test to assess the comparability of the groups for continuous and dichotomic variables respectively.
For each of the three groups ( controls, PPG and POAG) the Pearson’s linear correlation coefficient was calculated to evaluate how the measured parameters are influenced by the AL increasing. Then, each group was further subdivided into 3 subgroups (emmetropes, myopic eyes with AL<25 mm and myopic eyes with AL>25 mm) to analyze the effects of mild and moderate axial myopia by the Mann-Whitney U test. (Figure 1)
To investigate the ability of OCT parameters and RGCcount to diagnose glaucoma, we calculated areas under the receiver operating characteristic (AUROC). We compare the AUROCs between RGCcount and OCT parameters in all subgroups with different axial length (emmetropes, all myopic eyes, and myopes with AL<25mm and >25mm) using the method described by DeLong et al (17). For the calculation of AUROC and for all the comparisons between AUROCs we used a statistical software package (MedCalc v. 12.0; MedCalc Statistical software, Marakierke, Belgium).
For all statistical analysis, a p value <0.05 was considered statistically significant.