This cross-sectional observational study took place at the Glaucoma Center of the Eye Clinic, Department of Surgical Sciences, University of Turin, Italy. The methods conformed to the principles of the Declaration of Helsinki; we obtained the informed consent of all subjects and the approval of the Ethics Committee (University and Polyclinic San Giovanni Battista of Turin).
The inclusion criteria for admission of study subjects were: 18 ≤ age ≤ 80 years, best-corrected visual acuity (BCVA) ≥ 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 gonioscopic evidence of open angle. No subjects with previous ocular surgery, or with ocular, systemic or neurological pathologies causing perimetric defects were included. 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.
We performed a full eye examination, Fourier-Domain-OCT (FD-OCT RTVue-100 software version A4, 5, 0, 59; Optovue, Fremont, CA, USA) for peripapillary and macular imaging, and measurement of axial lenght with low-coherence interferometry system (Aladdin biometer, Topcon). Standard Automated Perimery was performed with program 24-2 of the Humphrey Field Analyzer (HFA; Carl Zeiss Meditec, Jena, Germany), using Swedish Interactive Threshold Algorithm (SITA) Standard strategy, with reliability criteria of fixation losses ≤ 20%, false positives and false negatives ≤ 33%. The study included three groups of subjects: preperimetric glaucomatous subjects (PPG), primary open-angle glaucoma (POAG) subjects, and a healthy group that was required to have negative family history of glaucoma and normal IOP (≤ 21 mm Hg), visual field (VF) test and optic nerve head (ONH) appearance. PPG subjects had IOP > 21 mm Hg, and changes in optic nerve (cup–disc ratio alteration/disc hemorrhages/rim notching/diffused or localized RNFL defects), but no defects on VF. POAG subjects had IOP > 21 mm Hg, and glaucomatous alterations in optic disc and VF, as stated by Hodapp-Parrish-Anderson criteria for diagnosing glaucomatous damage (14). For the evaluation of optic nerve head appearance, we used slit-lamp biomicroscopy of posterior segment with 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.
We used the Glaucoma Protocol of FD-OCT RTVue-100 to acquire RNFL thickness measurements. We detailed this protocol in a previous study (Rolle et al.) (16)
Estimate of Retinal and Macular Ganglion Cell Count
To estimate RGC number we used the model by Medeiros et al (9-11, 15) based on the empirical formulas of Harwerth et al (8).
We described this formula in detail in our previous article (Rolle et al.). (16)
We used Microsoft Excel 2016 worksheets and the SPSS statistical program for Windows (version 19.0, SPSS Inc, Chicago, IL) for the collection, processing and statistical analysis of the results. The analysis of variance (ANOVA) and χ2 test was used 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.