Methods

Study Population

In this retrospective longitudinal study, patients with MacTel enrolled in the MacTel Natural History and Observation Registry Study22 at the Department of Ophthalmology, University Hospital Bonn, Germany, were analyzed.

The study was approved by the Medical Faculty, University of Bonn ethics committee (reference number 124/05) and adhered to the tenets of the Declaration of Helsinki. All participants provided written informed consent before study inclusion.

The inclusion criteria were a confirmed diagnosis of MacTel and clear optic media to allow high-resolution retinal imaging. The exclusion criteria were any confounding conditions, such as but not limited to age-related macular degeneration, central serous chorioretinopathy, uveitic diseases, severe cataracts impacting retinal imaging, and refractive errors higher or lower than ± 6 diopters. If both eyes of a participant were eligible, both eyes were included. In addition, 10 eyes of 10 healthy participants were included to allow a graphical comparison with early MacTel patients.

Image Acquisition

All patients underwent a complete ophthalmologic examination, including slit lamp biomicroscopy and funduscopy after pupil dilatation with 0.5% tropicamide and 2.5% phenylephrine eye drops. Retinal imaging was performed using an investigational Spectralis HRA2 + OCT device (Heidelberg Engineering). The imaging protocol included combined near-infrared reflectance imaging and spectral-domain OCT (15° × 10°, 97 B-scans, high-resolution mode, automatic real-time averaging >9) and 2WAF (excitation wavelengths 488 nm and 514 nm; 30° × 30° scan field centered on the fovea). Color fundus photographs were obtained using the Zeiss Clarus 700 (Carl Zeiss Meditec).

Disease Progression

Disease stages for all eyes were assessed using the multimodal imaging-based staging system (stages 0-6) by Chew et al23 by one experienced reading center–trained grader (L.G.). Disease progression was defined as an increase in MacTel stage according to the classification by Chew et al during follow-up. Macular pigment optical density was assessed at baseline only, and its association with disease progression was evaluated. Eyes were classified as either stable or progressing based on the presence or absence of stage increase during follow-up.

Image Processing and Map Creation for MPOD Analysis

All OCT and 2WAF data were exported using the .XML format and were postprocessed using custom FiJi tools (freely available at https://sites.imagej.net/CreativeComputation/).24 For each OCT volume, the fovea was manually defined as the highest point of the inward rise of the external limiting membrane, where cone photoreceptors are at their longest,25 using the “Find_Fovea_OCT” plug-in. In cases in which the rise of the external limiting membrane was not visible, the lowest point of the foveal valley was selected.26 Two-wavelength autofluorescence images were postprocessed using “MPOD_XML_Reader,” which creates one 30° × 30° en face MPOD image per eye. Subsequently, MPOD levels were averaged within an ETDRS grid subfields per eye using “Grids_OCT.”27 To create en face MPOD maps, images of each disease group and healthy participants separately were averaged, using the foveal center and the optic nerve head as references to create stage-specific MPOD maps (“BatchStandardRetina”). Using the same approach, en face maps for stable and progressing eyes were created. In addition, to compare stable and progressing eyes more closely, we calculated pixel-level z-score maps for each group, using the following equation:where z is the z-score, x is the value of the group being evaluated, μ is the mean of the entire population, and σ the standard deviation of the entire population.

z=(x–μ)/σ

Statistical Analysis

Statistical analysis was performed using MatLab 2023b (The MathWorks, Inc). Parameters were summarized using means and standard deviations for normally distributed continuous variables and frequency distributions with percentages for categorical variables. Spatial MPOD differences across MacTel disease stages were analyzed using linear mixed-effects models to account for age and repeated measurements within eyes and patients. Eyes staged 0 to 5 were included in the progression analysis. Univariate regression was used to compare MPOD between stable and progressing eyes on an ETDRS subfield level. Subfields with significant differences were included in a multivariable regression model to estimate odds ratios (ORs) for disease progression per 0.01-unit increase in MPOD. Baseline disease stage and follow-up duration were included as covariates to adjust for differences in initial disease severity and observation time. A P value < 0.05 (two-sided) was considered significant. The subgroup of 10 healthy participants was not included in the statistical analysis, but is shown for graphical comparison.