Visual Psychophysics and Physiological Optics Microperimetry as an Outcome Measure in Trials: and Beyond

Ioannis S. Dimopoulos, Calvin Tseng, and Ian M. MacDonald

Department of Ophthalmology and Visual Sciences, University of Alberta, Edmonton, Alberta, Canada

Correspondence: Ioannis S. Dimo- PURPOSE. To determine test-retest repeatability of microperimetry testing (MP) in choroide- poulos, Clinical Fellow, remia (CHM) subjects using standard and personalized stimulus grids. Department of Ophthalmology and Visual Sciences, 7-030 Katz Building, METHODS. Fifteen CHM subjects (28 eyes) underwent consecutive repeat examinations with University of Alberta, Edmonton, the Macular Integrity Assessment (MAIA) microperimeter using a standard (108) and a Alberta, Canada, T6G 2E1; customized macular grid adapted to individual macular pathology. Repeatability of standard- [email protected]. grid mean (MS) and point-wise (PWS) sensitivity was determined and compared with age- Submitted: February 16, 2016 matched controls (seven eyes), with PWS separately analyzed for loci within and outside the Accepted: June 26, 2016 border of degeneration. Interpolated volumetric indices were used to estimate repeatability of customized grids and compare their performance to standard grids. Citation: Dimopoulos IS, Tseng C, MacDonald IM. Microperimetry as an RESULTS. Test-retest measures of standard-grid MS yielded higher coefficients of variation (CV) outcome measure in choroideremia in CHM subjects compared with controls (0.09 vs. 0.02). Volumetric indices from customized trials: reproducibility and beyond. grids improved repeatability by driving CV values to 0.05 and close to 0.02 for region-of- Invest Ophthalmol Vis Sci. interest (ROI) analysis. Variability of PWS was significantly higher in CHM, especially at the 2016;57:4151–4161. DOI:10.1167/ border of degeneration (10.68 vs. 4.74 dB at the central , < 0.001). iovs.16-19338 P CONCLUSIONS. Microperimetry testing in CHM shows high test-retest variation at the border of degeneration, which influences repeatability of MS measures. Volumetric measures from customized grids can improve reliability of both global and regional sensitivity assessment. Nevertheless, inherent test-retest variation of individual points needs to be taken into account when assessing potential functional decline and/or progression. Keywords: choroideremia, microperimetry, retina, clinical trials, gene therapy

horoideremia (CHM) is an X-linked disorder defined by extrafoveal fixation,5 such as ABCA4-associated retinopathies6 C progressive degeneration of the neuroretina, the retinal and age-related (AMD).7 pigment epithelium (RPE), and the . Choroideremia is However, recent studies examining the repeatability of caused by loss-of-function mutations in the CHM gene,1 which perimetric sensitivity measures have suggested a high degree of encodes Rab escort protein 1 (REP1), a protein involved in test-retest variability at the border of pathologic changes8,9 and prenylation of Rabs. Clinically, affected males experience night the edge of deep .10 For CHM and other conditions blindness in early adulthood followed by progressive peripheral with centripetally advancing degeneration, these findings may visual field loss, with central vision preserved until later in life. hamper reliability of microperimetry testing especially during Current investigational therapeutic approaches aim to modify later disease stages, when macular involvement is noted. At the natural history of the disease through viral-mediated gene these stages, which have been shown to provide the best 2 discriminatory power to determine a potential treatment transfer. Visual acuity remains the most widely used outcome 11–13 measure in these trials, providing functional assessment of benefit, mapping macular sensitivity becomes particularly challenging due to the highly variable retinal morphology. Most foveal integrity; however, psychophysical tests have revealed standard perimetry grids fail to sufficiently sample all areas of significant deficits in macular function of CHM patients, surviving retina within the macular region. The use of including those with normal visual acuity.3 Therefore, more personalized grids could potentially improve sensitivity map- sensitive clinical outcome measures are required to determine ping, but their adoption in clinical trials is currently limited due early efficacy of therapeutic interventions in CHM. to the difficulty in comparing examinations acquired with In recent years, fundus-driven perimetry, also known as different grid arrangements. Recently though, interpolation microperimetry, has emerged as a robust method for assessing methods have enabled the generation of indices that surpass 4 visual function in patients with macular disease. Its compar- these limitations.14 ative advantage to conventional perimetry stems from real-time In studies described herein, we sought to investigate test- fundus imaging combined with eye-tracking technology. retest repeatability of microperimetry testing in CHM, espe- Fundus viewing allows ‘‘locking’’ of stimuli at predefined cially for disease stages targeted by current investigational trials. retinal locations, which enables structural-functional correla- Our focus expanded beyond conventional measures from tions to be explored while adapting perimetry grids to standard grids to include repeatability of indices extracted individual retinal morphology. Eye-tracking provides high- from interpolation of personalized grids. Determining test- accuracy functional measures, even in cases of unstable or retest limits of such indices will allow the adoption of a single

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TABLE 1. Characteristics of CHM Subjects

Subject Age Mutation Exon Protein Change Eye logMAR BCVA MS, dB FAF Area, mm2

P1 42 c.315-?_1166þ?del 5-8 Deletion of exons 5-8 (in frame); OD 0.40 5.8 1.87 REP-1 absent OS 0.40 8.79 2.74 P2 38 c.1245-521A > G REP-1 negative (Splice defect) OD 0.30 8.08 1.79 OS 0.40 5.94 2.08 P3 30 c.1245-521A > G REP-1 negative (Splice defect) OD 0.17 11.55 7.16 OS 0.30 11.2 6.31 P4 33 c.1218C > A 9 p.Cys406* OD 0 10.21 2.77 OS 0.09 9.56 2.19 P5 28 c.1218C > A 9 p.Cys406* OD 0.09 13.75 2.99 OS 0.09 9.59 2.06 P6 65 c.757C > T 6 p.Arg253* OD 0.09 8.8 3.29 OS 0.17 4.4 2.05 P7 27 c.757C > T 6 p.Arg253* OD 0 15.4 12.05 OS 0.17 8.9 8.87 P8 31 c.1218C > A 9 p.Cys406* OD 0.09 8.24 2.77 OS 0.17 4.91 1.17 P9 53 c.525_526delAG 5 p.Glu177Lysfs*6 OD 0.49 0.17 0.3 OS 0.32 3.91 0.55 P10 31 c.757C > T 6 p.Arg253* OD 0 6.31 3.01 OS 0.20 5.9 2.79 P11 63 c.1218C > A 9 p.Cys406* OD 0.39 NA 0.75 OS 0.30 3.2 1.01 P12 43 c.-29-?_1510þ?del 1-12 Deletion of exons 1-12 (in frame); OD 0 11.50 3.0 REP-1 absent OS 0.20 8.1 2.5 P13 33 c.1218C > A 9 p.Cys406* OD 0 19.3 6.61 OS 0.2 15.1 6.23 P14 33 c.470_473del 5 p.Gln157Leufs*10 OD 0.2 9.28 3.60 OS 0 11.23 4.40 P15 36 c.117-?_940þ?del 3-7 Deletion of exons 3-7; REP-1 absent OD 0.3 6.7 2.23 OS 2 NA NA

estimate for all microperimetry examinations used in current testing. A near-infrared (NIR) superluminescent diode (850 and future CHM clinical trials. nm, 1024 3 1024 pixel resolution, 368 field of view) is used to visualize the fundus, with eye-tracking performed at a rate of 25 frames per second using the entire fundus as a reference. To METHODS obtain sensitivity thresholds, Goldmann-type size III stimuli (duration: 200 ms) were presented against a background 1.27 Subjects cd/m2, using a 4-2 staircase strategy. Minimum and maximum 2 The study population consisted of 28 eyes of 15 CHM subjects stimulus luminance achieved was 0 and 318 cd/m , respec- (age 39.1 6 11.7 years [mean 6 SD]; range, 21–65 years). All tively, covering a dynamic range of 36 dB. Reliability was subjects had genetic or molecular confirmation of their evaluated by the frequency of responses to 10-dB stimuli at the diagnosis (Table 1). Inclusion criteria consisted of best- physiological blind spot (false positives). Any examination with corrected visual acuity (BCVA) better than or equal to 20/62 greater than 25% false-positive responses was discarded and (0.50 logMAR), stability of fixation determined with micro- repeated. Fixation stability was assessed using the MAIA P1 perimetry (see the Microperimetry Examination section), and fixation stability index, which measures the proportion of presence of active degeneration within the clinical macula fixation points located within a 28 diameter circle centered on determined with spectal-domain optical coherence tomogra- the fovea. Stable fixation was defined by P1 values greater 75%. phy (SD-OCT) (Heidelberg Engineering, Heidelberg, Germany). None of the subjects had concurrent ocular disease that could Retinal Imaging affect visual performance. Normal data for microperimetry were collected from seven eyes of seven healthy subjects (age Before study enrollment, all CHM subjects had undergone 35.3 6 5.1 years [mean 6 SD]; range, 25–40 years). All central 3083308 blue laser fundus autofluorescence (k ¼ 488 procedures conformed to the Code of Ethics of the World nm) imaging (FAF) and SD-OCT line scans using the Spectralis Medical Association (Declaration of Helsinki) and were done SD-OCT unit (Heidelberg Engineering). For FAF, automatic real with the understanding and written consent of each partic- time was set to at least 30 frames. Spectral-domain OCT volume ipant. scans were acquired using a setting of 37 B-scans covering a 3083158 area, with high-resolution mode set on and 15 frames Microperimetry Examination averaged per B-scan. Fundus autofluorescence imaging delin- eated the area of remaining central RPE tissue, which appears Subjects were assessed with the MAIA microperimeter hyperfluorescent, from the atrophic, nonfluorescent and (Macular Integrity Assessment; CenterVue, Padova, Italy). This degenerated retina (Fig. 1C). The margin of RPE atrophy was instrument integrates scanning laser ophthalmoscopy (SLO) usually well defined and easy to identify. For purposes of this and real-time eye-tracking with computerized perimetry study, the border of degeneration was defined as the area

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FIGURE 1. Microperimetry examination and FAF image alignment. (A) Fundus photograph in a CHM subject (P2) showing diffuse chorioretinal atrophy at the posterior pole with preservation of the central macula. (B) Microperimetry examination in subject P2 using the MAIA standard macular grid. The grid consists of 37 stimuli covering the central 10 degrees of vision. Infrared (IR) imaging allows real-time grid projection onto the fundus. A color decibel scale is provided showing the stimulus intensity range (0–36 dB). Black dots correspond to points not seen at 0 dB. (C) Corresponding 308 blue laser FAF image. White arrows highlight two separate zones: (i) the hyperfluorescent area of remaining central RPE tissue; and (ii) the atrophic, nonfluorescent, degenerated retina. The border of degeneration between those two zones is outlined with a 28-wide segmented line (yellow) congruous to the RPE margin, with 18 spanning into zone (i) and 18 into zone (ii) (inset). (D) Manual alignment of the IR and FAF images using commercial image-editing software.

within 18 from the margin of RPE atrophy in all directions (Fig. images between the two examinations. After completion of the 1C; inset). Fundus autofluorescence images were manually second standard examination, CHM subjects were given the aligned to the NIR-SLO image acquired with the MAIA opportunity to rest (mean 6 SD; 10 6 2 minutes) and microperimeter (Fig. 1D), using a free image-editing software customized/personalized grids were created following a two- (GNU Image Manipulation Program, GIMP version 2.8.14; step process. First, an NIR-SLO image of the fundus was available in the public domain: http://www.gimp.org/). Optic captured and used as a reference for positioning a square grid disc and retinal vessels were used as anatomic landmarks. of 121 equidistant stimuli at the fovea or the central area of surviving retina. Depending on the area of the latter, a Testing sampling density of 18 or 28 was chosen (covering 10 or 20 degrees of retina, respectively). Next, test points within Testing was performed by a single experienced examiner (ISD) degenerated areas of the retina were removed using FAF in a dimly lit room with nondilated and according to the images as guidance. Test points one or two rows (equivalent to manufacturer’s instructions. A small, red circle of 18 diameter 18 to 28) from the border of the surviving retina were was used as a fixation target. Subjects were seen at two preserved. Examples of customized grids generated with this separate visits, within a 1-week interval. One eye was tested at method are provided in Figures 2E through 2H, with each visit, patching the other eye. Subjects were instructed and corresponding FAF images shown in insets. In some cases, given a training test to ensure they were confident and reliable additional test points were manually added at nonsampled in the use of the microperimeter. areas of the surviving retina confirmed by FAF. For instance, At each visit, subjects underwent a sequence of four FAF imaging in subject P11 demonstrated an extrafoveal RPE microperimetry tests. First, a standard 37-stimuli grid pattern island in the superior outer macular region that remained was used, subtending 10 degrees of central visual field. The unmapped with the standard 37-stimuli grid (Figs. 2D, 2H). The grid consisted of a single foveal response and three concentric sampling density for additional tests points was either 18 or 28, rings of retinal loci distanced 18,38, and 58 from the fovea (Figs. depending on the area sampled. 2A–D). Following a short resting period (mean 6 SD; 5 6 1 To limit patient fatigue, caution was given not to exceed a minutes), the ‘‘follow-up’’ protocol was used to obtain a repeat total number of 68 stimuli at any customized grid, which is the of the first standard grid examination. The ‘‘follow-up’’ number of test points used in the 10-2 program of the protocol ensured automatic alignment of the infrared fundus Humphrey automated perimeter. Customized grid examina-

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FIGURE 2. Generation of customized/personalized grids in CHM. (A–D) Representative examples of MAIA 37-stimuli grid examinations in four CHM subjects. The grid consists of a single foveal response and three concentric rings of retinal loci distanced 18,38, and 58 from the fovea. (E–H) Customized/personalized grids corresponding to the same CHM subjects. A square grid of 121 equidistant stimuli is centered at the fovea and then modified by removing excess test points within the degenerated retina, using FAF images as guidance (insets). Additional test points are added at non- or undersampled areas of the surviving retina, as shown in (H) for subject P11.

tions were repeated after a final short resting period (mean 6 USA). Color-scaled contour plots and three-dimensional ren- SD; 5 6 2 minutes) using the ‘‘follow-up’’ option. Control dering of the interpolated surface were used for visualization. subjects underwent the same testing protocol, but with a 10-2 Custom-sized region-of-interest (ROI) rectangles were created grid pattern of 68 stimuli for examinations three and four to perform integration within a specific ROI of the contour (similar to the Humphrey 10-2 test). plot. The two-dimensional integration gadget (OriginPro 2015) was used to define the position and size of the ROI rectangle by Interpolated Surface Analysis of Microperimetry entering the desired visual field XY coordinates for the left/ Data right and top/bottom lines, respectively. Region-of-interest coordinates corresponded to the opposite of retinal XY Microperimetry data were first exported as xyz triplets, with z coordinates in NIR-SLO images. The ROI rectangle was representing the sensitivity of each tested point and x, y its prevented from moving in volume integration of repeat Cartesian coordinates with respect to fixation. An interpolated examinations. two-dimensional matrix surface was then generated from xyz microperimetry data of customized examinations using the Statistical Analysis thin plate spline (TPS) function. The TPS algorithm appends a thin, elastic ‘‘spline’’ (or surface) through all data points, which Dense scotomas were assigned a value of 1dBsoastobe is then deformed to find the best fit that minimizes the bending included in the calculation of mean sensitivity (MS). The energy function.15 Two-dimensional integration can then be distribution of sensitivity values was substantially positively applied to calculate the volume between any given z-plane and skewed by the large number of loci with dense scotomas. For 16 the matrix surface using a numerical integral method. In our that reason, the Tukey’s trimean sensitivity (TmS) was also case, the central macular region was considered a relatively computed as a more appropriate measure of central tendency; ‘‘flat,’’ continuous surface z ¼ f(x,y),(x,y) r, with r being the TmS is a weighted average of the median sensitivity (Smedian) domain of interpolated points. Therefore, the volume under- and top and bottom quartile values (Q1 ¼ 25th percentile and neath the macular surface was computed as: Q3 ¼ 75th percentile), defined by the following equation: RR mX1 Xn1 Q þð2S ÞþQ Trimean Sensitivity ðT SÞ¼ 1 median 3 f ðx; yÞ dx dy ¼ lim lim f ðxi; yiÞDxDy m ðrÞ Dx0 Dy0 4 i¼0 j¼0

mX1 Xn1 For point-wise analysis (PWS), all points recorded as not » f ðxi; yiÞDxDy seen (1 dB) or having a sensitivity value of 0 dB in both i¼0 j¼0 microperimetry tests were excluded to minimize floor effects. No spatial averaging was applied to the raw data. Changes in where m, n are the number of rows and columns of the MS, TmS and volumetric measures between repeated examina- interpolated matrix. Volumetric units are reported in dBdeg2. tions were explored using the Wilcoxon signed-rank test. For All interpolation and volumetric analyses were performed PWS, a linear mixed effects model was constructed (SPSS; IBM, using OriginPro 2015 (OriginLab Corp., Northampton, MA, Armonk, New York, NY, USA), with test number used as a fixed

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FIGURE 3. Repeatability of MS and relationship with patient-specific characteristics. (A, B) Bland-Altman plots illustrating test-retest agreement for controls (A) and CHM subjects (B). (C) Comparison of MS CoR between CHM and control eyes. No significant difference was observed (61.33 dB and 61.51 dB, respectively). (D) Root mean square CV for MS is significantly higher in CHM compared with control eyes (0.09 vs. 0.02, P ¼ 0.003). 2 (E) Correlation of within-subject SD (Sw) with MS. A positive correlation can be noted (r ¼ 0.32, P ¼ 0.007). Choroideremia Sw values do not exceed at any point those in controls (gray-shaded area). (F) Correlation of within-subject CV (CVw) and FAF area. Although no correlation can be noted for 2 the range of FAF areas tested, CVw values were significantly higher than expected from controls (gray-shaded area) for FAF areas <5mm . Asterisk denotes significance at P < 0.05. Error bars represent 95% confidence intervals.

effect and stimulus points nested within subjects as a random difference between the two groups of eyes (Fig. 3C; 2-tailed effect to account for multiple measurements from same Mann-Whitney U test; P ¼ 0.85). However, the CV was 5-fold subjects and intereye correlations.7 Bland-Altman plots were greater in CHM (0.09) compared with control eyes (0.02) used to evaluate test-retest agreement of all parameters. For (Fig. 3D). Similar results were obtained for the TmS,withCV those parameters that did not exhibit a significant test-retest values of 0.08 and 0.02, respectively (Supplementary Fig. change, the coefficient of repeatability (CoR) was computed S1B). using the following equation17: pffiffiffi CoR ¼ 1:96 3 2 3 Sw; Relationship With Patient-Specific Characteristics To explore any dependence of test-retest variability of global where Sw was the within-subject SD. Differences in estimated CoR between controls and CHM measures on patient-specific characteristics, we correlated patients were investigated using the Mann-Whitney U test and individual test-retest SD (Sw) with the magnitude of sensitivity among different regions using the Kruskal-Wallis test. The and the area of residual RPE on FAF. As shown in Figure 3E, Sw was proportional to the magnitude of MS, without ever relationship of Sw with patient-specific characteristics was explored using Spearman correlations. The root mean square exceeding control values (r2 ¼ 0.32, P ¼ 0.007). Because of 18 coefficient of variation (CV) was used to compare reliability that observation, we converted Sw to within-subject CV (CVw) of volumetric against conventional sensitivity measures. and investigated its dependence to FAF area (Fig. 3F). For most 2 Significance was set at P < 0.05. All statistical analyses were CHM patients with areas less than 5 mm , higher CVw values performed using GraphPad Prism 6 (GraphPad Software, La (0.05–0.10) were noted compared with controls (0.02); CVw Jolla, CA, USA). values approximated those from controls in patients with larger areas. A similar trend was observed for TmS (Supplementary Fig. S1C). RESULTS Repeatability of Global Sensitivity Measures Changes in PWS We first examined trend changes in test-retest measures of Next, we sought to determine CoR for PWS. Using a linear MS from standard grid examinations to exclude a learning mixed effects model, we found that PWS from all subjects effect. No significant test-retest differences were noted for remained unchanged between the two tests, excluding a either control (P ¼ 0.24) or choroideremia eyes (P ¼ 0.81). learning effect. Bland-Altman plots were constructed for Bland-Altman plots were used to illustrate the agreement control and choroideremia eyes (Figs. 4A, 4B) and PWS CoR between the tests (Figs. 3A, 3B). The CoR for MS was calculated to be 64.5 and 68.7 dB, respectively. These CoRs estimated to be 61.51 dB and 61.33 dB for control and were significantly different between the two groups of eyes (P choroideremia eyes, respectively. There was no significant < 0.001; Fig. 4C).

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FIGURE 4. Repeatability of PWS. (A, B) Bland-Altman plots illustrating test-retest agreement for controls (A) and CHM subjects (B). (C) Comparison of PWS CoR between CHM and control eyes. Point-wise sensitivity CoRs were significantly higher in CHM eyes (68.7 vs. 64.5 dB; P < 0.001). Asterisk denotes significance at P < 0.05. Error bars represent 95% confidence intervals.

Sensitivity Changes of Loci at the Border and in average PWS for all of the three groups of points, the PWS Central Retina CoRs were computed to be, on average, 610.68 dB, 64.74 dB, and 64.78 dB for points at the border, central, and degenerated To further investigate the origin of the high PWS variability in retina, respectively. Points close to the border of degeneration choroideremia eyes, we aligned and registered microperimetry had significantly greater CoR compared with points at the SLO images to FAF images and determined the precise location central or degenerated retina (P < 0.001; Fig. 5E). of each tested sensitivity locus relative to the margins of the residual RPE island. A total of 441 points were analyzed and subdivided into three distinct groups: n ¼ 192 points at the Repeatability of Interpolated Surface Sensitivity border of degeneration or within 18 from the margin of RPE Indices atrophy (in all directions), n ¼ 160 points within the central ‘‘healthier’’ retina, and n ¼ 89 points within the degenerated Can the reproducibility of global sensitivity measures be retina (Fig. 5A). After excluding a systematic test-retest change improved with the use of customized microperimetry grids

FIGURE 5. Point-wise sensitivity repeatability at different regions. (A) Representative segregation of tested points into three groups based on their relationship with the border of degeneration (highlighted in dark green): blue loci (with the blue square symbol) correspond to test points within the degenerated retina; green loci (with the green square symbol) correspond to test points within the central, ‘‘healthier’’retina; and red loci (with the red square symbol) correspond to test points at the border of atrophy. (B–D) Bland-Altman plots illustrating PWS test-retest agreement for border loci (B), central loci (C), and degenerated loci (D). (E) One-way ANOVA comparing CoR from the three regions. Coefficient of repeatability for loci at the border were significantly higher compared with the other two regions (10.68 vs. 4.74 dB, P < 0.001). Asterisk denotes significance at P < 0.05. Error bars represent 95% confidence intervals.

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FIGURE 6. Repeatability of volumetric measures from different stimulus grids. Representative examples of microperimetry examinations and corresponding interpolated contour plots generated from the same subject (P6) using a standard 37-stimuli macular grid (A); a 10-2 grid of 68 stimuli (B); an equally spaced (18) custom grid of 121 stimuli (C); and a customized grid of 40 stimuli (D). Sensitivity levels have been color-coded at 4-dB intervals. Black dots correspond to points not seen at 0 dB. Although MS values are noncomparable, volumetric measures yield consistent measurements across the three grids.

in CHM? To investigate this, one should first create visual levels (20, 16, and 12 dB) yielded CoR of 62.70 deg2, function indices that are independent of the sampling density irrespective of isopter analyzed (Figs. 8B, 8C). This translates of test points. Such indices can be derived from interpolated into a CV value of 0.07 for the 16-dB and 12-dB isopters, but in surface maps of perimetry data as illustrated in Figure 6. a considerably low reliability index for the 20-dB isopter (0.27). Subject P6 underwent consecutive examinations using four These findings highlight that certain measures in customized different stimulus arrangements: a standard 37-stimuli grid, a grids continue to remain susceptible to border variation. 10-2 grid of 68 stimuli, an equally spaced (18) grid of 121 A comparative advantage of volumetric indices over stimuli, and a customized grid of 40 stimuli. Although MS traditional global measures is the ability to perform ROI-based values were noncomparable (4.4, 1.2, 0.2, and 5.6 dB), even analyses of the interpolated surfaces. Based on our previous when including seeing points only (14.16, 12.14, 7.75, and regional PWS analysis, we hypothesized that limiting sensitivity 12.89 dB), measurement of the volume underneath the assessment in areas within the central ‘‘healthier’’ retina would interpolated surface yielded consistent measures across the minimize any potential variation from the transitional zone and three grids, with values of 184.18, 177.15, 182.65, and 186.23 increase reproducibility. We thus explored repeatability of ROI dBdeg2, respectively. volume integration for areas well within the residual island of For that reason, we relied on volumetric measures of RPE. To achieve this, we first positioned an ROI rectangle interpolated data to extract a single estimate of test-retest within the central hyperfluorescent area of the FAF image, as repeatability for customized MP examinations. Table 2 summa- outlined in Figure 9A (area within the red rectangle), and rizes the parameters used for each patient’s customized grid. estimated its xy coordinates by adopting the scale of the The number of test points was on average 57.0 6 9.1 (mean 6 coregistrered microperimetry SLO image (microperimetry data SD), with the area of retina covered ranging from 68 to 208. on the NIR-SLO image have known xy retinal coordinates). Time to complete a customized examination was not Next, the opposite values were applied as xy constraints on the statistically different from the time required for control two-dimensional interpolated contour plot to perform volume subjects to undertake a 10-2 68-loci examination (8:12 minutes integration within the specified ROI. Central retina ROI 6 58 seconds vs. 8:52 minutes 6 32 seconds, P ¼ 0.11). After analysis was able to yield significantly lower CoR (6 27.34 excluding a significant test-retest change in volumetric dBdeg2) and CV values that were comparable to controls measures (P ¼ 0.67), the CoR for customized grids was (0.02). Table 3 summarizes repeatability for all volumetric 2 computed to be 662.15 dBdeg ; this translates into an overall indices used in this study. improved CV value (0.05) than that obtained from standard- grid MS measures (0.09), as shown in Figure 7D. Nevertheless, volumetric measures from interpolated 10-2 grid examinations DISCUSSION in control subjects continued to demonstrate lower overall test-retest error (CoR ¼ 6249.50 dBdeg2 and CV ¼ 0.02). Our study investigated the reproducibility of microperimetry Slightly lower reproducibility was encountered in the testing in CHM and its potential to serve as a reliable outcome construction of isopter contours from customized grids, which measure, in light of gene therapy clinical trials under way.19 are lines defining borders of regions with equal retinal The degenerative process in CHM resembles that of other sensitivity (Fig. 8A). Isopter area calculation at three threshold inherited retinal disorders, such as pigmentosa, in that

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TABLE 2. Customized Grid Parameters

Average Standard Average Customized Number of Loci in Examination Examination Customized Sampling Density of Retinal Area Customized Subject Eye Duration, min:s Duration, min:s Examination* Customized Grid Grid, deg

P1 OD 5:18 8:51 68 18 128 OS 5:28 8:44 61 18 108 P2 OD 5:02 9:40 64 18 88 OS 5:31 8:26 49 18 88 P3 OD 6:04 8:57 61 28 158 OS 6:24 7:39 65 28 158 P4 OD 4:59 7:25 58 18 108 OS 5:50 7:08 57 18 108 P5 OD 4:58 8:43 51 18 108 OS 5:05 9:06 65 18 108 P6 OD 5:00 6:29 42 18 88 OS 4:54 8:26 49 18 88 P7 OD 5:46 8:34 49 28 208 OS 5:34 7:17 52 28 208 P8 OD 4:46 8:13 43 18 108 OS 5:20 8:31 47 18 108 P9 OD 4:15 5:24 45 18 68 OS 4:58 7:09 40 18 68 P10 OD 5:00 6:26 47 18 108 OS 5:45 8:02 42 18 108 P11 OD NA NA NA NA NA OS 4:11 7:29 54 18 and 28 88 P12 OD 4:50 7:14 53 28 and 18 208 OS 5:10 8:48 57 18 108 P13 OD 5:52 9:29 61 28 and 18 208 OS 6:04 9:36 64 28 and 18 208 P14 OD 5:34 9:54 63 18 108 OS 4:39 8:47 56 18 128 P15 OD 5:31 9:22 60 18 108 OS NA NA NA NA NA C1 OD 4:09 8:51 68 28 208 C2 OS 5:53 9:01 68 28 208 C3 OD 4:44 7:58 68 28 208 C4 OS 5:02 8:49 68 28 208 C5 OD 4:58 8:56 68 28 208 C6 OD 5:31 9:49 68 28 208 C7 OS 5:12 8:43 68 28 208 * For control subjects (C1–C7), customized examinations were performed using a 10-2 grid pattern of 68 stimuli similar to the Humphrey 10-2 test.

FIGURE 7. Interpolated surface analysis of customized grids. (A) Representative example of a CHM microperimetry examination using a customized grid. (B) Corresponding three-dimensional rendering of the interpolated surface generated from the customized examination in (A). The x- and y- axes of the Cartesian coordinate system represent degrees of visual field (deg) and the z-axis the measured sensitivity threshold in decibels. Numerical integration of the volume underneath the interpolated surface was used to compute volumetric indices. (C) Bland-Altman plots illustrating test-retest agreement for total volume calculation. (D) Comparison of repeatability between MS from 37-stimuli grid examinations and total volume from customized grids. Total volume yields significantly lower CV values than MS (0.05 vs. 0.09, P ¼ 0.03).

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FIGURE 8. Repeatability in calculation of isopter areas from customized grids. (A) Representative color-coded contour plot generated from the customized examination in Figure 7A. Isopter threshold levels have been color-coded at 4-dB intervals. (B) Bland-Altman plots illustrating test-retest agreement for isopter area calculation at three threshold levels: 20 dB, 16 dB, 12 dB. (C) Isopter area calculation yields similar CoR, irrespective of threshold level.

the chorioretinal atrophy is progressing in a centripetal fashion for all points, independent of their relative distance from a with foveal involvement occurring only at the very late stages deep . Apart from the smaller dynamic stimulus range of the disease.3 During the course of the disease, a transitional (0-20 dB) of the microperimeter (MP1; Nidek, Inc., Fremont, zone can be delineated between degenerated and relatively CA, USA) used in this study, reasons explaining the observed healthy retina, which is indicative of disease activity.20 discrepancy pertain mainly to the noninclusion of retinal loci Monitoring sensitivity changes at this zone could constitute at the immediate boundaries of deep scotomas and the three- an attractive functional marker of CHM progression. Our study point spatial averaging applied to the microperimetry data. Wu demonstrated that reproducible microperimetry measures et al. elegantly showed that by applying the same spatial cannot be obtained for that region in CHM; points close to averaging to their data, similar PWS CoR could be obtained the border of degeneration had significantly greater CoR, across all regions. compared with points at the central retina. Although our What could explain the higher test-retest variability reported CoR for central retinal points (64.74 dB) are observed at the border of degeneration? A plausible origin comparable to that obtained in patients with ABCA4-associated should be sought at the microsaccades occurring during the (64.21 dB),6 juvenile (65.4 dB),21 short stimulus presentation, which cannot be fully compen- and other macular disorders (65.6 dB),5 the issue of higher sated with the current fundus tracking frequency (25 Hz). variability at the border of degeneration has been inconsis- Therefore, loci at the retinal border may shift into the tently reported by other studies. degenerated retina demonstrating greater intrasession variabil- Wu et al.10 investigated the variability of microperimetric ity. This interpretation is supported by the work of Wyatt et sensitivity measures at the border of the head, in an al.,9 who showed a substantial contribution of small fixational effort to model deep scotomas, and compared it with other eye movements to test-retest variability by correlating the latter areas of normal retina. Coefficient of repeatability for points at with the gradient, the rate at which sensitivity changes with the border of the optic nerve head were significantly higher location. Higher variability at the margin of RPE atrophy also than points at the macular region, in agreement with the can be attributed to the presence of degenerating photorecep- higher CoR estimated in our study at the border of atrophy in tors, which have been shown to form outer retinal tubulations CHM. Similar observations have been made for automated in CHM and other disorders.22 These structures may contain perimetry measures at the border of glaucomatous defects in highly dysfunctional photoreceptors that exhibit inconsistency patients and the border of the blind spot in healthy between responses. Although less likely in cases of photore- subjects.8,9 However, a microperimetry study by Cideciyan et ceptor loss, variability may also originate from dysfunctional al.6 in patients with ABCA4-associated suggested ganglion cells subserving the transitional zone, as previously that a single estimate of test-retest repeatability can be adopted shown for glaucoma.9

FIGURE 9. Repeatability in central retina ROI volume integration. (A) Fundus autofluorescence–guided positioning of a rectangle in a region well within the hyperfluorescent RPE residual tissue. The coordinates of the rectangle are estimated by adopting the scale of the coregistrered microperimetry SLO image. (B) Region-of-interest volume integration performed on the two-dimensional contour plot of an interpolated customized examination using the xy constraints estimated in (A). (C) Bland-Altman plots illustrating test-retest agreement in central retina ROI volume integration.

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TABLE 3. Repeatability of Conventional and Volumetric Measures

CoR CV (95% Confidence Interval)

Controls CHM Controls CHM

MS, dB 1.51 1.33 0.02 (0.011–0.023) 0.09 (0.084–0.119) Trimean sensitivity, dB 1.44 1.53 0.02 (0.018–0.027) 0.08 (0.073–0.12) Customized grid total volume, dBdeg2 249.50* 62.15 0.02 (0.012–0.026)* 0.05 (0.047–0.052) Central retina ROI volume, dBdeg2 NA 27.34 NA 0.02 (0.02–0.024) Isopter area, deg2 NA 2.70 NA 0.07 (0.067–0.073) * Based on interpolated 10-2 grid examinations.

In later stages of CHM, the high variability of the residual yield similar values regardless of stimulus array. All approaches retina’s morphology warrants the use of individualized discussed herein can be expanded to other forms of inherited perimetric grids to sufficiently map visual sensitivity in retinal disorders, in which perimetric sensitivity assessment is every case. However, MS measures from these grids become used as an outcome measure. dependent on the parameters of the applied grid pattern, such as test point number, spacing, and local condensa- tion.14 Several methods have been used to interpolate Acknowledgments perimetric test grids and generate indices that are not grid The authors thank Rafael C. Caruso, MD, at Princeton University specific. These include neural network algorithms,23 TPS,14 for his insightful comments on this manuscript. 14 and nearest neighbor interpolations. Our study adapted Supported by Canadian Institutes of Health Research Grant 14 the TPS algorithm, previously shown by Weleber et al. to 119190, Alberta Innovation Health Services Grant 201201139, have good performance and accuracy for modeling full-field and Canadian Foundation for Innovation Grant 28916. perimetry data. Volumetric indices were chosen to quantify Disclosure: ,None; ,None; the visual sensitivity of the customized interpolated grid.14 I.S. Dimopoulos C. Tseng I.M. MacDonald, None In our case, volume integration was performed in Cartesian rather than spherical coordinates, because of the small extension of the central macular region, which was References considered a relatively ‘‘flat’’ surface. Customized grids were able to improve not only the efficiency but also the 1. McTaggart KE, Tran M, Mah DY, Lai SW, Nesslinger NJ, reliability of sensitivity mapping in CHM. Similar observa- MacDonald IM. Mutational analysis of patients with the tions have been made for the use of individually condensed diagnosis of choroideremia. Hum Mutat. 2002;20:189–196. test grids in detection of glaucomatous visual field defects.24 2. MacLaren RE, Groppe M, Barnard AR, et al. Retinal gene Regional analysis of those locally condensed areas of interest therapy in patients with choroideremia: initial findings from a can be performed with high levels of reproducibility in phase 1/2 . Lancet. 2014;383:1129–1137. CHM, a capability particularly useful for evaluating the 3. Jacobson SG, Cideciyan AV, Sumaroka A, et al. Remodeling of outcome of localized therapeutic interventions, such as the human retina in choroideremia: rab escort protein 1 (REP- subretinal gene delivery.2 1) mutations. Invest Ophthalmol Vis Sci. 2006;47:4113–4120. Nevertheless, there are certain limitations in our study that 4. Acton JH, Greenstein VC. Fundus-driven perimetry (micro- need to be considered. Apart from the small sample size, it is perimetry) compared to conventional static automated perim- important to keep in mind that most CHM subjects were etry: similarities, differences, and clinical applications. Can J evaluated at late disease stages. Low reliability in global and Ophthalmol. 2013;48:358–363. PWS might not be encountered at early or intermediate stages 5. Chen FK, Patel PJ, Xing W, et al. Test-retest variability of of CHM. In addition, the estimation of test-retest variability was microperimetry using the Nidek MP1 in patients with macular based on two repeat tests (within the same session) rather than disease. Invest Ophthalmol Vis Sci. 2009;50:3464–3472. three or four examinations across a short period of time. 6. Cideciyan AV, Swider M, Aleman TS, et al. 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