Although measurements of the optic disc and retinal nerve

Longitudinal Variability of Optic Disc and Retinal Nerve Fiber Layer Measurements Christopher Kai-shun Leung, 1,2 Carol Yim-lui Cheung, 1 Dusheng Lin, 1,3 Chi Pui Pang, 1 Dennis S. C. Lam, 1 and Robert N. Weinreb 2 PURPOSE. To evaluate the longitudinal variability of optic disc and retinal nerve fiber layer (RNFL) measurements obtained from optical coherence tomography (OCT), scanning laser polarimetry (SLP), and confocal scanning laser ophthalmoscopy (CSLO). METHODS. Forty-five normal and 43 glaucomatous eyes of 88 subjects were analyzed in this longitudinal study. RNFL thickness was measured by OCT (StatusOCT; Carl Zeiss Meditec, Dublin, CA) and SLP (GDx VCC; Carl Zeiss Meditec). Neuroretinal rim area was measured by CSLO (HRT 3; Heidelberg Engineering, GmbH, Dossenheim, Germany). Three separate measurements taken over an average period of 8.8 1.2 months were used to evaluate measurement variability. Reproducibility coefficient, coefficient of variation, intraclass correlation coefficient (ICC), and sensitivity to change [(97.5 percentile value 2.5 percentile value)/2 within-subject standard deviation (Sw)] of the global measures were calculated. The association between RNFL and rim area measurement variability and visual field MD (mean deviation) was evaluated with regression analysis. RESULTS. Low variability was found for global and sectorial rim area and RNFL measurements. The reproducibility coefficient, ICC, and sensitivity to change for OCT average RNFL thickness, GDx VCC TSNIT average, and HRT global rim area were 11.7 m (95% confidence interval [CI]: 10.5 12.9 m), 0.97 (0.96 0.98), 10.2 (9.2 11.4); 4.7 m (4.2 5.1 m), 0.98 (0.97 0.99), 11.3 (10.2 12.6); and 0.22 mm 2 (0.19 0.24 mm 2 ), 0.97 (0.95 0.98), 9.3 (8.4 10.4), respectively. No association was found between OCT (r 0.010 [95% CI: 0.200 0.219], P 0.924) and SLP (r 0.034 [95% CI: 0.241 0.177], P 0.756) RNFL thickness variances and visual field MD. The association between CSLO rim area variance and visual field MD became insignificant after adjustment for reference height variance. CONCLUSIONS. Longitudinal RNFL and neuroretinal rim measurements obtained with OCT, SLP, and CSLO have low variability. As the measurement variability does not change with the severity of glaucoma, these parameters are useful for assessment of glaucoma progression. (Invest Ophthalmol Vis Sci. 2008;49: 4886 4892) DOI:10.1167/iovs.07-1187 Although measurements of the optic disc and retinal nerve fiber layer (RNFL) with optical coherence tomography (OCT), scanning laser polarimetry (SLP), and confocal scanning laser ophthalmoscope (CSLO) have been shown to have high diagnostic performance for detection of glaucomatous damage, 1 5 their roles in analyzing glaucoma progression are still evolving. Distinguishing genuine change from measurement variability is a challenge in studying glaucoma progression with these imaging instruments. Repeatability and reproducibility are two commonly used indices to indicate intra- and intervisit measurement variability, respectively. 6,7 Intervisit variability is generally higher than intravisit variability, probably because of the potential differences in the scanning techniques among different operators and the different scanning angles and locations during different visits. 8,9 Most studies on RNFL imaging have been focused on repeatability with measurements taken in a single visit, 10 13 or reproducibility over a few days to a few weeks. 14 16 However, in the usual clinical setting, imaging is seldom repeated within a day or a month. Determining reproducibility over a longer period with multiple measurements would be clinically more useful and relevant to reflect the long-term measurement variability of these imaging instruments. The StratusOCT (Carl Zeiss Meditec, Dublin, CA), HRT 3 (Heidelberg Engineering, GmbH, Dossenheim, Germany), and GDx VCC (Carl Zeiss Meditec) are the latest commercially available versions of OCT, CSLO, and SLP, respectively. Although no difference has been found in their diagnostic sensitivity for glaucoma detection, 4,17 it is uncertain whether the results that they obtain are equally reproducible during longitudinal assessment. The purpose of this study was to evaluate the longitudinal variability of optic disc and RNFL measurements in normal subjects and in patients with glaucoma, with the StratusOCT, HRT 3, and GDx VCC. From the 1 Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong, Peoples Republic of China; the 2 Department of Ophthalmology, Hamilton Glaucoma Center, University of California, San Diego, California; and 3 The STU/ CUHK Joint Shantou International Eye Centre (JSIEC), Shantou University Medical College, Peoples Republic of China. Submitted for publication September 11, 2007; revised November 23, 2007, and January 14, February 19, April 5, and May 8, 2008; accepted September 5, 2008. Disclosure: C.K. Leung, Carl Zeiss Meditec (F); C.Y. Cheung, None; D. Lin, None; C.P. Pang, None; D.S.C. Lam, None; R.N. Weinreb, Carl Zeiss Meditec (C, F) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked advertisement in accordance with 18 U.S.C. 1734 solely to indicate this fact. Corresponding author: Christopher Kai-shun Leung, Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong Eye Hospital, 147K Argyle Street, Kowloon, Hong Kong, Peoples Republic of China; tlims00@hotmail.com. METHODS Subjects Forty-five normal subjects and 43 patients with glaucoma followed up during the period from February 2006 to October 2007 at the University Eye Center, The Chinese University of Hong Kong, were included in the analysis. These subjects were enrolled in a glaucoma imaging study designed to evaluate test retest variability longitudinally and glaucoma progression and had had at least three serial measurements obtained before October 2007. All subjects underwent a full ophthalmic examination including visual acuity, refraction, intraocular pressure measurement with Goldmann tonometry, gonioscopy, and fundus examination. The inclusion criteria were best corrected visual acuity of not worse than 20/40, spherical refractive error within the range of 6.0 to 6.0 D, and 6.0 D of cylinder. Individuals were excluded if they had a history of any retinal diseases, surgery or laser procedures, diabetes mellitus, or neurologic diseases. Normal subjects were indi- Investigative Ophthalmology & Visual Science, November 2008, Vol. 49, No. 11 4886 Copyright Association for Research in Vision and Ophthalmology

IOVS, November 2008, Vol. 49, No. 11 Longitudinal Variability of Optic Disc and RNFL Measurements 4887 viduals with no ocular diseases. In particular, they had no visual field defects, no structural optic disc abnormalities, no history of intraocular pressure higher than 21 mm Hg, and no history of ocular or neurologic diseases. The patients with glaucoma were defined based on the presence of visual field defects (described later). Only one eye was selected randomly in each subject for analysis. Each included eye underwent OCT, CSLO, and SLP imaging in three separate visits over an average period of 8.8 1.2 months, with a between-visit duration of at least 3 months. This study was conducted in accordance with the standards stated in the Declaration of Helsinki and approved by Clinical Research Ethics Committee of Hong Kong Hospital Authority Kowloon West Cluster, with informed consent obtained. Visual Field Testing Standard visual field testing was performed using static automated white-on-white threshold perimetry (SITA Standard 24-2, Humphrey Field Analyzer II; Carl Zeiss Meditec). Visual field testing was repeated at each follow-up visit in patients with glaucoma. A visual field was defined as reliable when fixation loss was 20% and false-positive and -negative rates were 25%. Visual field sensitivity was expressed as MD (mean deviation) and PSD (pattern standard deviation), as calculated by the perimetry software. None of the normal subjects had a visual field defect. A field defect was defined as having three or more significant (P 0.05) non edge-contiguous points with at least one at the P 0.01 level on the same side of the horizontal meridian in the PSD plot and as having results outside normal limits in the glaucoma hemifield test, confirmed with at least two visual field tests. Visual field progression was evaluated with the Collaborative Initial Glaucoma Treatment Study (CIGTS) visual field scoring system. 18 Visual field testing was repeated when there was evidence suggestive of progression. One subject was not included in the analysis because of progressive visual field change. Optic Disc and RNFL Imaging The pupils were not routinely dilated with a pharmacologic agent during optic disc and RNFL imaging. However, dilation with tropicamide and phenylephrine (0.5% each) was performed when the pupil size was not large enough to obtain an image with the required quality (described later). Images with poor centration, poor focus, or missing data were detected by the operator at the time of imaging, with rescanning performed in the same session. Subjects were excluded if good-quality images could not be obtained after three consecutive scans in the same visit. Optical Coherence Tomography. OCT was performed with the StratusOCT and analyzed with software version 4.0.1(Carl Zeiss Meditec Inc.). Each subject underwent RNFL scanning with the fast RNFL (3.4 mm; 256 A-scans) protocol. The nerve fiber layer, with its high reflectivity signal, can be visualized as the first layer in red on the scan. Its thickness is determined by the difference in distance between the vitreoretinal interface and a posterior border based on a predefined reflectivity signal level. The RNFL thickness was reported in the analysis printout after averaging the results of three sequential circular scans. All the images obtained had a signal strength of at least 7. Three subjects were excluded because of suboptimal signal strength in one of the three visits. Confocal Scanning Laser Ophthalmoscopy. CSLO was performed with the HRT 3 (Heidelberg Engineering) and analyzed with Heidelberg Eye Explorer version 1.5.1.0. In brief, a three-dimensional topographic image consisting of 384 384 16 up to 384 384 64 pixels is constructed from multiple focal planes axially along the optic nerve head. An average of three consecutive scans is obtained and aligned to compose a single mean topography for analysis. An experienced examiner outlined the optic disc margin on the mean topographic image. Images with an image quality SD greater than 30 m were excluded. Once the optic disc margin was drawn, the software automatically transferred the disc margin to subsequent visits and calculated all the optic disc measurements. The reference plane was defined at 50 m posterior to the mean retinal height between 350 and 356 along the contour line. The area above the reference plane was defined as the rim and that below as the cup. In this study, we focused only on the neuroretinal rim area measurements. Two subjects with image quality SD more than 30 m in one of the three visits were excluded. Scanning Laser Polarimetry. SLP was performed with the GDx VCC and analyzed with software version 5.5.0 (Carl Zeiss Meditec). The GDx VCC quantifies the RNFL by first measuring the eyespecific corneal birefringence, consisting of the corneal polarization axis and magnitude. It is determined with a macular image acquired with the retardance of VCC set to 0. The Henle fiber layer and corneal retardation can then be measured from the macular retardation profile. Subsequent imaging sessions for the same individual used the previously obtained values of corneal polarization magnitude and axis. To ensure accurate measurement, the software provides an image quality check score (1 10) based on the correct alignment, fixation, and refraction of the scan. A minimum score of 8 was set to be the minimum standard for a good-quality scan. Ten subjects were excluded because of a poor image quality score in one or more visits. One normal subject and one with glaucoma were also excluded because of images with atypical birefringence patterns (defined as having variable and splotchy areas of high and low retardation not conforming to the normal anatomic profile of RNFL in the retardation map and with a typical scan score less than 60). 19 The superior average, inferior average, and TSNIT average reported in the standard printout were analyzed in this study. The engineers from Carl Zeiss Meditec and Heidelberg Engineering check the calibration of our OCT and CSLO on a regular basis, as recommended by the manufacturers (the calibration of both OCT and CSLO was checked during the study period). For SLP, there is a daily system self-test that verifies the scan field centration and retardation measurement reproducibility. If the results exceed the preset acceptance limits, an error message is displayed, and the instrument is sent to the company for calibration. Statistical Analysis Statistical analyses were performed with commercial software (SPSS ver. 15.0; SPSS Inc, Chicago, IL). Differences in age, refraction, visual field MD between the diagnostics groups were evaluated by independent t-test. By setting the confidence interval as 20% on either side of the estimate of within-subject standard deviation (Sw): n 1.96 2 /[2 0.2 2 (m 1)], where n is the number of subjects and m is the number of observations, 20 it could be estimated that a minimum of 25 subjects in each group would be necessary. The Sw was calculated as the square root of the within-subject mean square of error (the unbiased estimator of the component of variance due to random error) in a one-way random-effects model. 20 Intersession sectorial and global measurements were compared with repeated-measures analysis of variance. The intersession within-subject standard deviation (Sw), coefficient of variation, reproducibility coefficient (2.77 Sw), and intraclass correlation coefficient (ICC) were computed. Statistical comparison of ICC was performed based on work described by Fisher. 21 A sample size calculation estimated that 87 subjects would be necessary for 80% power at a 5% two-sided significance level for the statistical test to detect a mean difference in ICC of 0.10 between 0.80 and 0.90. 22 The relationships between visual field MD and each of the global measures variances (average RNFL thickness for OCT; TSNIT average for GDx VCC; and global rim area for HRT) and image quality score variances (signal strength for OCT, image quality SD for HRT, and quality score for GDx VCC) were evaluated with multiple linear regression models with visual field MD as a dependent variable, global measures variances, and image quality score variances as predictors. The association between the SD and the mean of the repeated measurements was evaluated with correlation analysis (Kendall s correlation coefficient). Sensitivity to change 23 was calculated as (97.5 percentile value 2.5 percentile value)/2 Sw. This parameter refers

4888 Leung et al. IOVS, November 2008, Vol. 49, No. 11 TABLE 1. Demographics of Normal Subjects and Patients with Glaucoma to the number of progression events that can be detected in a given cohort of patients. A higher number signifies a higher ability to detect change. In all the analyses, P 0.05 was considered statistically significant. RESULTS Normal (n 45) Glaucoma (n 43) P Male/female 18/27 27/16 0.054* Age (SD) (y) 47.2 (12.9) 52.0 (13.6) 0.091 Range 3.0 to 71.0 27.0 to 73.0 Refraction (SD) (D) 0.69 (2.31) 1.04 (3.07) 0.541 Range 4.75 to 4.25 6.00 to 4.25 Visual field MD (SD) (db) 0.27 (1.29) 9.24 (7.32) 0.001 Range 3.35 to 2.54 29.51 to 0.53 * 2 test. Independent samples t-test. Forty-five normal and 43 glaucomatous eyes of 88 subjects were included in the analysis after excluding 17 subjects with inadequate image quality. There was no difference in age and refraction between the normal and glaucoma groups (Table 1). The average period ( SD) between the first and second visits was 4.6 0.9 months, and that between the second and third visits was 4.2 0.6 months. No significant changes in RNFL thicknesses and rim area measures (all with P 0.239, repeatedmeasures analysis of variance) in the glaucoma group were detected within the study period (8.8 1.2 months). The overall image quality indices for OCT, HRT, and GDx VCC were 9.5 0.5 (signal strength), 15.7 3.8 (image quality SD) and 8.7 0.5 (quality score), respectively. The HRT global rim area variance was significantly correlated with the variance of image quality SD (r 0.284, P 0.007), whereas no association was found between the variances of OCT average RNFL thickness (P 0.837)/GDx VCC TSNIT average (P 0.756) and the variances of their respective image-quality indices. The mean, Sw, and reproducibility coefficient of each measured parameter are shown in Tables 2 (normal group) and 3 (glaucoma group). Low measurement variability (low values of reproducibility coefficient) was evident in most of the parameters in normal subjects and in those with glaucoma. In the normal group, the reproducibility coefficient of RNFL measurements ranged from 12.55 to 22.22 m for OCT and from 3.60 to 6.41 m for GDx VCC. The reproducibility coefficient of HRT 3 rim area ranged from 0.0163 to 0.1317 mm 2. In the glaucoma group, the reproducibility coefficient of OCT and GDx VCC measurements were generally comparable with those in the normal group, whereas the reproducibility coefficients of HRT measurements were generally higher in the glaucoma group. To investigate the effect of glaucomatous damage on the measurements reproducibility, the association between within-subject variance of the global measures (average RNFL thickness for OCT, TSNIT average for GDx VCC, and global rim area for HRT) and visual field MD were examined with regression analyses. Although no association was found for OCT (r 0.010 [95% confidence interval (CI): 0.200 0.219], P 0.924) and GDx VCC (r 0.034 [95% CI: 0.241 0.177], P 0.756) average RNFL thicknesses, there was a significant association between within-subject variance of HRT global rim area and visual field MD (r 0.456 [95% CI: 0.608 to 0.273], P 0.001). Nevertheless, this association became insignificant (standardized coefficient 0.145 [95% CI: 0.305 0.015], P 0.079) when the variances of reference height (mean variance 0.88 1.77 m; 0.642 [95% CI: 0.481 0.803], P 0.001) and image quality SD (mean variance 9.42 13.11 m; 0.095 [95% CI: 0.052 0.242], P 0.211) were included in the multivariate regression model (R 2 0.562, P 0.001). The coefficient of variation, ICC, and sensitivity to change of the global measures are shown in Table 4. No significant difference in ICC was found (P 0.119). There was no association between the SD and the mean of the intersession measurements in each of the global parameters (Fig. 1). TABLE 2. The Overall Mean, Sw, and Reproducibility Coefficient of StratusOCT, HRT 3, and GDx VCC Measurements in the Normal Group Overall Mean (SD) Sw (95% CI) Reproducibility Coefficient (95% CI) StratusOCT RNFL Thickness Measurement ( m) Superior 131.82 (16.23) 6.77 (5.78 7.76) 18.74 (16.01 21.48) Nasal 76.64 (14.61) 6.55 (5.59 7.50) 18.13 (15.48 20.78) Inferior 139.35 (18.15) 8.02 (6.85 9.19) 22.22 (18.98 25.47) Temporal 84.19 (15.10) 6.22 (5.31 7.13) 17.24 (14.72 19.76) Average 107.99 (11.46) 4.53 (3.87 5.19) 12.55 (10.72 14.38) HRT 3 Rim Area Measurement (mm 2 ) Temporal 0.2423 (0.1099) 0.0203 (0.0174 0.0233) 0.0563 (0.0481 0.0645) Superotemporal 0.2006 (0.0445) 0.0084 (0.0072 0.0096) 0.0232 (0.0198 0.0266) Inferotemporal 0.2084 (0.0495) 0.0077 (0.0066 0.0089) 0.0215 (0.0183 0.0246) Nasal 0.4047 (0.1107) 0.0127 (0.0109 0.0146) 0.0352 (0.0301 0.0403) Superonasal 0.2297 (0.0544) 0.0059 (0.0050 0.0068) 0.0163 (0.0140 0.0187) Inferonasal 0.2315 (0.0528) 0.0071 (0.0061 0.0082) 0.0198 (0.0169 0.0227) Global 1.5268 (0.3561) 0.0476 (0.0406 0.0545) 0.1317 (0.1125 0.1510) GDx VCC RNFL Measurement ( m) Inferior average 68.31 (8.19) 2.31 (1.98 2.65) 6.41 (5.47 7.34) Superior average 69.26 (8.10) 2.24 (1.91 2.57) 6.21 (5.29 7.13) TSNIT average 56.13 (5.44) 1.30 (1.11 1.49) 3.60 (3.07 4.14) n 45.

IOVS, November 2008, Vol. 49, No. 11 Longitudinal Variability of Optic Disc and RNFL Measurements 4889 TABLE 3. The Overall Mean, Sw, and Reproducibility Coefficient of StratusOCT, HRT 3 and GDx VCC Measurements in the Glaucoma Group Overall Mean (SD) Sw (95% CI) Reproducibility Coefficient (95% CI) StratusOCT RNFL Measurement ( m) Superior 79.80 (23.16) 6.03 (5.13 6.93) 16.71 (14.21 19.20) Nasal 58.39 (15.90) 5.74 (4.88 6.60) 15.91 (13.53 18.28) Inferior 75.18 (27.62) 7.26 (6.18 8.35) 20.11 (17.11 23.12) Temporal 54.05 (16.13) 4.47 (3.81 5.14) 12.39 (10.54 14.25) Average 66.86 (16.44) 3.85 (3.27 4.43) 10.66 (9.07 12.26) HRT 3 Rim Area Measurement (mm 2 ) Temporal 0.1388 (0.0497) 0.0306 (0.0260 0.0352) 0.0847 (0.0721 0.0974) Superotemporal 0.1312 (0.0552) 0.0214 (0.0182 0.0246) 0.0594 (0.0505 0.0683) Inferotemporal 0.0981 (0.0503) 0.0177 (0.0150 0.0203) 0.0490 (0.0415 0.0563) Nasal 0.2990 (0.1338) 0.0324 (0.0275 0.0372) 0.0897 (0.0763 0.1031) Superonasal 0.1675 (0.0581) 0.0147 (0.0125 0.0169) 0.0408 (0.0347 0.0469) Inferonasal 0.1595 (0.0566) 0.0161 (0.0137 0.0185) 0.0445 (0.0379 0.0512) Global 0.9950 (0.2866) 0.1013 (0.0861 0.1164) 0.2805 (0.2386 0.3224) GDx VCC RNFL Measurement ( m) Inferior average 43.81 (10.92) 2.64 (2.25 3.03) 7.31 (6.22 8.40) Superior average 45.71 (12.86) 2.36 (2.01 2.71) 6.54 (5.56 7.51) TSNIT average 38.83 (8.25) 2.00 (1.70 2.30) 5.54 (4.72 6.37) n 43. DISCUSSION High reproducibility was observed in most of the parameters measured by OCT, GDx VCC, and HRT, as indicated by their relatively low reproducibility coefficients. For example, the difference in average RNFL thickness obtained in two separate visits would be expected to be less than 11.7 m in OCT and 4.7 m in GDx VCC (Table 4) for 95% of pairs of observations. Although the reproducibility coefficient is lower in GDx VCC, it does not suggest that GDx VCC measurement is more reproducible. It is notable that although both instruments express RNFL thickness in micrometers, they are in different scales. RNFL thickness measured by OCT was higher than that measured by GDx VCC 24 and thus resulted in the higher reproducibility coefficients. In fact, all the global measurements demonstrated high ICCs (all 0.97), indicating that they are reproducible. In studying progression, a parameter should be both reproducible and sensitive to change. Reproducibility refers to the ability of an instrument to provide consistent measurements. It is expressed as 2 1.96 Sw. Jampel et al. 23 introduced sensitivity to change, defined as [(97.5 percentile value 2.5 percentile value)/2 Sw], to measure the ability of a parameter to detect change. This parameter indicates the number of progression events that can be detected in a given cohort of patients (i.e., from completely healthy to end-stage glaucoma). In other words, this parameter reflects the ability of an instrument to be clinically useful for monitoring glaucoma progression. In this regard, there may be no significant difference among the three imaging instruments. We compared only the global parameters. As the proportion of sectorial distribution of the structural measures in each imaging test is different, sectorial measurements cannot be directly compared. Conflicting results have been reported regarding the effect of glaucomatous damage on optic disc/rnfl measurement repeatability. Jampel et al. 23 found no association between the degree of glaucomatous damage (both in visual field MD and HRT rim area) and the SE of measurement of rim area, cup shape, and retinal thickness. On the contrary, DeLeon Ortega et al. 4 showed significant correlation between visual field MD and variances of OCT average RNFL thickness, GDx VCC TSNIT average, and HRT global rim area. In the present study, there was no significant association between the degree of glaucomatous damage and the variances of global RNFL/rim area measurements suggesting that longitudinal variability does not change with the severity of glaucoma. This conclusion is also supported by the finding that the SD was unrelated to the magnitude of the measurements (Fig. 1). In fact, the Sw s of the OCT and GDx VCC measurements were very similar between the normal and glaucoma groups (Tables 2, 3). Of note, the apparent association between HRT global rim area variance and visual field MD became insignificant when it was adjusted for the variances of reference height and image quality SD. These two predictors were selected in the multivariate analysis, because they were found to be the most influential for rim area variability. 8,25 In a study by Strouthidis et al., 8 50% of intravisit rim area difference was explained by reference height difference. Reference height and its relationship to reference plane are crucial in defining the measurement of the rim and TABLE 4. Comparison of Reproducibility, Coefficient of Variation, and Sensitivity to Change of the Global Measures of StratusOCT, HRT 3, and GDx VCC Reproducibility Coefficient (99% CI) Coefficient of Variation (%) (95% CI) Intraclass Correlation Coefficient (95% CI) Sensitivity to Change (95% CI) StratusOCT average RNFL thickness ( m) 11.67 (10.45 12.88) 4.79 (4.29 5.29) 0.97 (0.96 0.98) 10.2 (9.2 11.4) HRT 3 global rim area (mm 2 ) 0.2175 (0.1948 0.2402) 6.22 (5.57 6.87) 0.97 (0.95 0.98) 9.3 (8.4 10.4) GDx VCC TSNIT average ( m) 4.65 (4.17 5.14) 3.52 (3.16 3.89) 0.98 (0.97 0.99) 11.3 (10.2 12.6) n 88.

4890 Leung et al. IOVS, November 2008, Vol. 49, No. 11 FIGURE 1. Scatterplots of SD versus the (A) mean of OCT average RNFL thickness, (B) GDx VCC TSNIT average, and (C) HRT global rim area. the cup. Change in the orientation or tilting of the optic disc surface during repeated imaging may result in a shift in the level of reference plane, causing measurement variability of the rim. This variability may be more substantial in glaucomatous disc in which the surface of the disc could be lower than the parapapillary retinal surface, resulting in a more superficially located reference plane. Even slight shifts or tilts in the images could lead to significant changes in rim area measurement. Although rim area variability is unlikely to be related to the severity of glaucomatous damage, glaucomatous optic disc per se may result in higher reference height variation, accounting for the higher rim area variability.

IOVS, November 2008, Vol. 49, No. 11 Longitudinal Variability of Optic Disc and RNFL Measurements 4891 Determining the reproducibility of optic disc and RNFL measurements is fundamental in differentiating measurement variability from pathologic changes in the detection of glaucoma progression. Measurement variability could be intrinsic to the instrument, or it could be due to physiological change in the cornea, lens, or retina over time. Investigating longitudinal variability is thus clinically important in deciding whether the change observed is within the limits of physiological variation. In this study, the average follow-up time for imaging was 4.4 months, which is closely matched with the average follow-up schedule for patients with glaucoma in a glaucoma clinic, and a total of three visits, spanning a period of 8.8 months, were used in the calculation of reproducibility. The study was designed in a way to resemble clinical practice in which imaging tests are usually repeated over several months (and not in a day or a few weeks), and yet in a relatively short period (8.8 months), to minimize potential pathologic changes. The observations that the mean measurements remained unchanged (all with P 0.239, repeated-measures ANOVA) and the Sw s of RNFL thicknesses measured by OCT and GDx VCC were comparable between normal subjects and those with glaucoma support the assumption that no progression occurred over the study period. HRT defines rim area by the area bounded by the disc margin and the cup margin. Although the disc margin was automatically exported between visits, thus eliminating the variability of disc area measurement, cup area measurement varied between visits because of the variation of the HRT reference height. Given that the cup area is measured where the reference plane intersects the optic disc on the z-axis, any axial shifts between the optic disc and reference plane due to variations of optic disc orientation, scanning alignment, and centering of optic disc could lead to variability of cup and rim area measurements. Because each imaging device has its unique features and limitations, the current findings could provide a useful reference for clinicians to compare the measurement variability of OCT, CSLO, and SLP when the scan quality has been optimized. It is notable that although ICC may be useful to reflect the relative measurement reliability among different imaging instruments in the same study population, it cannot be compared in different studies because of population heterogeneity. Our study is limited in that we analyzed only quadrant and average RNFL thicknesses from the OCT, TSNIT and superior/ inferior RNFL average from the GDx VCC, and sectorial and global rim area measurements from the HRT. These parameters were selected because they represent the most commonly used indicators in the evaluation of glaucoma. The measurement of neuroretinal rim area to differentiate normal subjects from those with glaucoma has been advocated. 26 29 The data from the nerve fiber indicator (NFI) of the GDx VCC were not analyzed, because this parameter was calculated based on different variables and not designed for serial monitoring. All the images taken in this study were obtained by highly trained and experienced technicians in an imaging research unit with high image-quality scores and consistent image centration. Results obtained in this study may not be directly translated to other populations in different clinical settings. Although intraocular pressure reduction could affect topographical measurements of the optic disc, 30 it is controversial whether there is a significant change in RNFL thickness with the lowering of intraocular pressure. 31,32 In summary, longitudinal RNFL and rim measurements obtained from the StratusOCT, GDx VCC, and HRT 3 have high reproducibility. The observation that the measurement variability was independent of the severity of glaucoma suggests that serial monitoring of these measurements is useful to detect glaucoma progression. A longitudinal study with longer follow-up time is needed to compare the performance of these imaging instruments in detection of glaucoma progression. References 1. Schuman JS, Hee MR, Puliafito CA, et al. Quantification of nerve fiber layer thickness in normal and glaucomatous eyes using optical coherence tomography. Arch Ophthalmol. 1995;113:586 596. 2. Budenz DL, Michael A, Chang RT, et al. 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