Study design
The repeatability of our MP analysis workflow was evaluated on a cohort of GA patients from the OAKS study (NCT03525600) [7]. The OAKS study was a 24-month, multicenter, randomized, double-masked, sham-controlled, phase 3 study, which enrolled patients at 110 clinical sites. The study adhered to protocols approved by the institutional review board of each site and complied with the Declaration of Helsinki. The inclusion and exclusion criteria of the OAKS study are described elsewhere [8]. Patients were randomly assigned (2:2:1:1), i.e., pegcetacoplan monthly, pegcetacoplan every other month, sham injection monthly, or sham injection every other month, by a central web-based randomization system to intravitreal 15 mg per 0.1 mL.
MP testing
MP testing was conducted using the Macular Integrity Assessment (MAIA) device (iCare, Padova, Italy) at baseline and every 6 months for up to 24 months. All follow-up MP acquisitions were obtained using a ‘follow-up’ mode to allow registration to the baseline acquisition. MP testing was conducted in a dark room under pharmacologic pupil dilation while the contralateral eye was patched. MP testing was performed using a rectilinear 10 − 2 grid distribution (68 stimulus points; Goldman Size III (0.43º diameter)) centered on the anatomic fovea, with a 4 − 2 staircase threshold strategy, mesopic background luminance of 4 asb (1.27 cd/m2), and a 1º diameter red central fixation target. All MP testing was performed prior to any imaging to prevent photoreceptor bleaching.
Fundus autofluorescence imaging and baseline lesion tracing
Fundus autofluorescence (FAF) imaging was performed using the Spectralis HRA + OCT (Heidelberg Engineering, Heidelberg, Germany) at all study visits. In the high-speed mode, a 30° × 30° field centered on the fovea was imaged. FAF images consisted of 768 × 768 pixels, and average number of sampling frames set between 15 and 25. Based on FAF image tracings made as part of the OAKS Phase 3 GA study, baseline GA tracings for this study were performed by A. Y. A., with the minimum lesion size defined as 0.05 mm [2, 12, 13]. Importantly, all subsequently described analysis used the same baseline GA tracings, meaning that GA tracing was not a source of variability in this study. For analysis, each traced GA focus was represented as polygon, comprising a set 2-D vertices.
Junctional zone MP analysis
The workflow for GA junctional zone MP analysis, which follows the approach used by Hariri [14], is presented in Fig. 1. In brief, using custom MATLAB (MathWorks, Natick, Massachusetts) software, each grader (Grader 1 and Grader 2) registered baseline MP images (sensitivity maps superimposed on their respective scanning laser ophthalmoscopy (SLO) fundus images) to their corresponding baseline FAF images using fiducial markers manually positioned at corresponding bifurcations of the retinal vasculature. For registration, a similarity-type transformation (translation, rotation, and isotropic scaling) was used. This transformation type requires that a minimum of three corresponding points be selected, although the graders were free to select additional points. The estimated transformation was also used to transform the MP stimuli coordinates into the FAF image coordinate frame. For all analysis, MP stimuli were treated as 2-D points, with their coordinates corresponding to the centers of the stimulus. With the GA tracings and MP measurements in the same coordinate frame (i.e., the FAF coordinate frame), the signed Euclidean (i.e., straight-line) distance from each MP stimulus point to the closest point on the baseline GA margin was computed. Negative and positive distances represent stimuli that lie inside and outside the areas of atrophy, respectively. A junctional zone, defined as all fundus positions within 250 μm of the GA margin (including regions both inside and outside the region of atrophy), was automatically generated. The position and width of the junctional zone used in this study matched that used for the post-hoc MP analysis of the OAKS trial [15].
Workflow for longitudinal tracking of microperimetry (MP) sensitivities within the GA junctional zone. Baseline (visit 1) MP scanning laser ophthalmoscopy (SLO) images (panel A) are registered with baseline fundus autofluorescence (FAF) images (panel B), allowing the MP stimulus points to be overlaid on the FAF image (panels C, D). Each MP stimulus point is associated with a signed distance from the lesion margin, as defined by FAF lesion tracing; distances, in micrometers, are shown next to each MP stimulus point. Positive and negative distances correspond to points outside and inside regions of atrophy, respectively. The junctional zone comprising all points within 250 μm of the lesion boundary is shaded red; those MP stimulus positions within the junctional zone have filled markers, and those outside the junctional zone have unfilled markers
Statistical analysis
Two graders (A. Y. A. and E. M. M.), both experienced with ophthalmic image registration, performed registrations on images from a systematic sampling (every 20th eye) of the OAKS dataset. The repeatability of the mean sensitivity and number of scotomatous points (any stimulus with a -1 dB raw value or < 0 dB on the sensitivity map) within the junctional zone were assessed using Bland–Altman analysis [16], intraclass correlation coefficients (ICCs) and coefficients of repeatability (CoRs), also referred to as the smallest real difference [17]. For Bland-Altman analysis of the mean sensitivity within the junctional zone, individual confidence intervals for the limits of agreement were computed [18]. To assess the repeatability of our MP analysis workflow without considering a particular junctional zone (e.g., ± 250 μm), we considered two measures, both of which incorporate all 68 stimulus points: (1) the repeatability of the signed-distance from each MP stimulus point to the GA margin (“stimulus-to-margin distance”; distances are negative for points within the GA lesion margin and positive for points outside the GA lesion margin), and (2) the distances between the coordinates of corresponding MP stimulus points when transformed by the two readers into the FAF coordinate frame (“stimulus coordinate difference”). The repeatability of the stimulus-to-margin distance was assessed using Bland-Altman analysis, as well as ICC and CoR. Following Taylor et al. [19], linear mixed modeling was used to account for repeated measures. Stimulus coordinate differences were summarized with boxplots and descriptive statistics.
We emphasize that all repeatability analyses in this study assess only the repeatability of the MP analysis, which is determined by the repeatability of registering the MP SLO images to the FAF images. In particular, this study did not consider the repeatability of the MP acquisition (both graders used the same MP SLO images), which have been reported by other authors [20], or of the GA lesion tracing (both graders used the same GA lesion tracings).
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