Steady-state pattern electroretinogram and shortduration transient visual evoked potentials in glaucomatous and healthy eyes

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1 Clinical and Experimental Ophthalmology 2017; 2018; : 46: doi: doi: /ceo Original Article Steady-state pattern electroretinogram and shortduration transient visual evoked potentials in glaucomatous and healthy eyes Dilru C Amarasekera BS, Arthur F Resende MD, Michael Waisbourd MD, Sanjeev Puri MD, Marlene R Moster MD, Lisa A Hark PhD, L Jay Katz MD, Scott J Fudemberg MD and Anand V Mantravadi MD 1 Glaucoma Research Center, Wills Eye Hospital, Philadelphia, Pennsylvania, USA ABSTRACT Importance: This study evaluates two rapid electrophysiological glaucoma diagnostic tests that may add a functional perspective to glaucoma diagnosis. Background: This study aimed to determine the ability of two office-based electrophysiological diagnostic tests, steady-state pattern electroretinogram and short-duration transient visual evoked potentials, to discern between glaucomatous and healthy eyes. Design: This is a cross-sectional study in a hospital setting. Participants: Forty-one patients with glaucoma and 41 healthy volunteers participated in the study. Methods: Steady-state pattern electroretinogram and short-duration transient visual evoked potential testing was conducted in glaucomatous and healthy eyes. A 64-bar-size stimulus with both a lowcontrast and high-contrast setting was used to compare steady-state pattern electroretinogram parameters in both groups. A low-contrast and high-contrast checkerboard stimulus was used to measure short-duration transient visual evoked potential parameters in both groups. Main Outcome Measures: Steady-state pattern electroretinogram parameters compared were MagnitudeD, MagnitudeD/Magnitude ratio, and the signal-to-noise ratio. Short-duration transient visual evoked potential parameters compared were amplitude and latency. Results: MagnitudeD was significantly lower in glaucoma patients when using a low-contrast (P = 0.001) and high-contrast (P < 0.001) 64- bar-size steady-state pattern electroretinogram stimulus. MagnitudeD/Magnitude ratio and SNR were significantly lower in the glaucoma group when using a high-contrast 64-bar-size stimulus (P < and P = 0.010, respectively). Short-duration transient visual evoked potential amplitude and latency were not significantly different between the two groups. Conclusions and relevance: Steady-state pattern electroretinogram was effectively able to discern between glaucomatous and healthy eyes. Steady-state pattern electroretinogram may thus have a role as a clinically useful electrophysiological diagnostic tool. Key words: glaucoma, short-duration transient visual evoked potentials, steady-state pattern electroretinogram. INTRODUCTION Glaucoma is characterized by progressive loss of retinal ganglion cells and their axons, resulting in visual field (VF) defects. 1 It is a leading cause of blindness worldwide, and those affected with glaucoma are projected to rise from 60 million to 80 million by j Correspondence: Dr Lisa Hark, Glaucoma Research Center, Wills Eye Hospital, 840 Walnut Street, Philadelphia, PA 19107, USA. lhark@willseye.org Received 22 October 2016; accepted 23 May Competing/conflicts of interest: The Wills Eye Glaucoma Research Center received funding from Diopsys Inc. Dr Arthur F. Resende and Dilru C. Amarasekera received travel support from Diopsys Inc. Funding sources: The Wills Eye Innovation grant funded this study. The ss-perg and SD-tVEP testing platform was provided by Diopsys Inc. Contributed equally

2 Electrophysiology 2 for Glaucoma Diagnosis Amarasekera et 55 al. the year Because early diagnosis is known to decrease the risk of eventual vision loss, there is a continuous search for objective diagnostic tests in order to detect glaucoma and its progression in its earlier stages. 5 Electrophysiological tests are able to evaluate the dysfunction of rods, cones, retinal bipolar cells and retinal ganglion cells. 6 9 The pattern electroretinogram (PERG) is an electrophysiological test that uses stimuli consisting of alternating white and black bars or checkerboard patterns. The PERG test depends on the integrity of the retinal ganglion cells, and thus, parameters may be altered even before VF defects appear in patients with glaucoma. Previous studies have found abnormal PERG values in patients with glaucoma suspect and ocular hypertension without optic nerve or VF changes. 7,10 18 The PERG test represents a well-established surrogate measure of the function of retinal ganglion cells in glaucoma with a good repeatability. 11 A new testing platform for steady-state PERG (ss-perg; Diopsys Inc., Pine Brook, NJ, USA) allows for rapid administration of the ss-perg testing sequence and can be completed within a few minutes in an office-based setting. Visual evoked potentials (VEPs) are signals obtained from electroencephalographic activity in the visual cortex and measure the integrity of the entire visual pathway including the eye, retina, optic nerve, optic radiations and the occipital cortex. 19 VEP examination has been previously used for the detection of different ocular and optic nerve pathologies This test is usually performed in tertiary referral centres and until recently was very rarely used in an office-based setting. A newly developed method called the short-duration transient VEP test (SD-tVEP; Diopsys Inc.) is a modified VEP test that was recently introduced. 22,24 26 The SD-tVEP test has a reduced testing time of 38 s per eye and produces a larger signal-to-noise ratio (SNR) than the traditional VEP method with a good repeatability in a previous study. 26 Prata et al. have previously confirmed that SD-tVEP can detect visual dysfunction in glaucomatous eyes. 24 Electrophysiological techniques thus play a valuable role in a diagnostic environment dominated by highly effective tools such as OCT via the addition of an objective functional perspective to the diagnosis of glaucoma. Although the use of PERG and VEP as a measure of retinal ganglion cell and visual pathway dysfunction has been established, few studies have measured the potential clinical utility of the novel rapid testing platform of ss-perg and SD-tVEP in patients with glaucoma. The purpose of this study was to compare the results of these two unique electrophysiological objective diagnostic tests (ss-perg and SD-tVEP), in a glaucoma group compared with an age-matched control group. Findings may provide useful information about the diagnostic and followup utility of these electrophysiological tests for glaucoma patients in clinical practice. METHODS The study was approved by the International Review Board of the Wills Eye Hospital and carried out in accordance with the rules and principles of the Declaration of Helsinki. Signed informed consent was obtained from all subjects before their enrolment in this study. Subjects All eligible glaucoma subjects were recruited from the Wills Eye Hospital Glaucoma Service, and control volunteers were recruited from the Wills Eye Cataract and Primary Eye Care Service. All subjects underwent a slit lamp examination. VF testing was performed in all glaucoma subjects using a Humphrey Field Analyser (24-2 Swedish interactive threshold algorithm standard strategy; Carl Zeiss Meditec Inc., Dublin, CA, USA) if they did not have available VF testing results within 6 months prior to study enrolment. Subjects were included in the glaucoma group if they had a diagnosis of open-angle glaucoma of any type as per the (365.xx) ICD-9 code. Subjects were included in the control group if they had a normal eye exam without any previous diagnosis of glaucoma, ocular hypertension or other ophthalmic conditions. Control subjects were age-matched to subjects in the glaucoma group. Exclusion criteria common to both groups were incisional eye surgeries or laser therapy within 1 month prior to enrolment, any cause for visual reduction (best-corrected visual acuity worse than 6/12), history of eye trauma or neurological pathology. The Diopsys NOVA testing system (Diopsys Inc.) was used to conduct the ss-perg and SD-tVEP tests. Pattern electroretinogram Prior to ss-perg testing, the area under the eyelids of all subjects was cleaned using the OCuSOFT Lid Scrub (OCuSOFT, Inc., Rosenberg, TX, USA). The subject s central forehead area was cleaned using NuPrep Skin Prep Gel (Weaver and Co., Aurora, CO, USA). For ss-perg testing, electrodes were placed in three locations: Diopsys ERG Lid Electrodes (Diopsys Inc.) were placed under the left and right eyelids, and a disposable electroencephalogram electrode was placed on the forehead. The electrodes were secured using Ten20 Conductive Paste (Weaver and Co.). Artificial tears were placed in each eye, and subjects were refracted using trial lenses that matched their current prescription. Subjects were seated 24 inches away from the LCD screen that

3 56 Electrophysiology for Glaucoma Diagnosis displayed the stimulus. An occluding lens was fit into the trial lens to cover the eye that was not being tested. Figure 1(a) displays electrode placement on a subject tested with ss-perg. Pattern electroretinogram, conducted prior to the SD-tVEP tests, displayed two patterns to subjects in both the glaucoma and control groups. The first pattern consisted of alternating white and black bars, with white bars of cd/m2 and black bars of 9.6 cd/m2, resulting in a Michelson contrast of 85% and a mean luminance of 66.3 cd/m2. The second pattern consisted of white bars of cd/m2 and grey bars of cd/m2. This resulted in a Michelson contrast of 70% and a mean luminance of cd/m2. Figure 1(b) displays the PERG stimulus used for testing. During ss-perg testing, the spatial frequency of the stimulus was a 64 bar size. Both the right and left eyes were tested, and the contrast sensitivity testing feature of the Diopsys Nova-LX testing system (Diopsys Inc.) was used within the described parameters. The test lasted approximately 50 s per eye (25 s for high contrast [Hc] of 85% and 25 s for low contrast [Lc] of 70%). While the tests for each eye were performed, the artefacts of the testing sequence were examined. Artefacts consisted of extraorbital or intraorbital eye movements with the potential to interfere with test results. Results with artefacts above a threshold of 5 experienced more interference from extraneous signals and were deemed unreliable. Amarasekera et al. 3 Thus, only those results with artefacts 5 were included in this study. If the artefacts were 5, the ss-perg testing was complete. If the artefacts were >5, then the eye was excluded from the study. Pattern electroretinogram parameters measured in both groups during testing included MagnitudeD, MagnitudeD/Magnitude ratio and SNR. MagnitudeD represents the amplitude of the ss-perg signal and is highly influenced by its phase consistency. MagnitudeD/Magnitude ratio is a ratio that is a within-subject representation of the phase consistency of ss-perg. SNR is the level of electrical noise compared with the level of the ss-perg signal at 15 Hz. Figure 1c,d displays ss-perg readings used to calculate each of these parameters in control and glaucoma subjects. Short-duration transient visual evoked potential Following the ss-perg testing, the electrodes were removed and the SD-tVEP electrodes were placed. The skin on subjects foreheads, temples and occipital lobes was scrubbed using NuPrep Skin Prep Gel (Weaver and Co.) to clear away debris and dead skin. Three disposable electroencephalogram electrodes were attached to each of these locations using Ten20 Conductive paste (Weaver and Co.). Artificial tears were instilled into both eyes. The subject was refracted using trial lenses that matched his or her Figure 1. (a) Electrode placement for steady-state pattern electroretinogram (ss-perg) testing. Electrodes are placed on the forehead and below the lower eyelids. (b) A 64-bar-size ss-perg stimulus is presented on a screen at a distance of 24 inches for 20 s. (c) ss-perg results from a healthy control showing higher amplitude and phase consistency. (d) ss-perg results from a glaucoma subject with decreased amplitude and phase consistency. Lc, low contrast Royal Australian and New Zealand College of Ophthalmologists 2017 Royal Australian and New Zealand College of Ophthalmologists

4 4Electrophysiology for Glaucoma Diagnosis Amarasekera et 57 al. best-corrected visual acuity, and the subject was seated 39 inches away from the LCD monitor that displayed the stimulus. The SD-tVEP examination used a stimulus previously described by Prata et al. 24 and Tello et al. 26 The stimulus consisted of a black and white checkerboard pattern generating low (Michelson contrast of 15%) and high (Michelson contrast of 85%) contrast patterns. For the SD-tVEP test, both eyes were tested for 20 s each. The pattern selected was checkerboard, with a spatial frequency of and a contrast of 15% for Lc and 85% for Hc. Subjects were instructed to fixate on the rotating red circle in the centre of the LCD monitor. The SD-tVEP parameters for both Lc and Hc stimuli required a reliability index of 80% for each eye, and if this was reached, the SD-tVEP testing was complete. Otherwise, the eye was excluded from the study. The reliability score is a measure of the ability of the test to elicit a response from the desired VEP waveform and is decreased with increased neural noise or external artefacts. The SD-tVEP parameters measured included amplitude and latency. The amplitude represents the strength of the signal arising from the visual cortex. The amplitude correlates with the nature of the stimulation, the number of axons conducting the signal and the integrity of the visual pathway. The latency represents the time between phototransduction and the maximum positive signal elicited by the visual cortex. The latency correlates to the nature of the stimulation and the length and integrity of the visual pathway. Statistical analysis Demographics, such as age, gender and race, were summarized separately using means and standard deviations (SDs), or as frequencies and percentages when appropriate. The analysis of variance test was used for continuous variables, and Fisher s exact test was used for categorical variables. The sample sizes of 81 eyes in the glaucoma group and 82 eyes in the control group achieve 99% power to detect a difference between the group proportions of 35% by two-sided Fisher s exact test with type I error rate controlled at 5%. To compare the ss-perg and SD-tVEP parameters between glaucoma and control groups, we used linear mixed models to fit the data while adjusting the age (in decades) effect and the correlation between eyes from same subject. All tests with P 0.05, two-sided, were considered statistically significant. Data were analysed using SAS, System Version 9.4 (SAS Institute, Cary, NC, USA). RESULTS A total of 41 glaucoma subjects (81 eyes) and 41 controls (82 eyes) were enrolled in this study. One eye from a glaucoma subject was excluded because the visual acuity was unable to meet the criteria needed for inclusion. Demographics and clinical characteristics of all study subjects are summarized in Table 1. In the glaucoma group, the mean ± SD age was 65.4 ± 10.1 years and subjects were predominantly female (n = 23). In the control group, the mean ± SD age was 64.2 ± 8.9 years and subjects were predominantly female (n = 24). The glaucoma group included more African Americans when compared with the controls (P = 0.003). Results of ss-perg analysis in glaucoma and control eyes are summarized in Table 2. For ss-perg testing (64 bar size), 151/163 (92.6%) eyes had reliable results for Lc and 152/163 (93.2%) had reliable results for Hc. The parameters MagnitudeD, MagnitudeD/ Magnitude ratio and SNR were compared between the glaucoma and control groups when using both Lc and Hc stimuli. The MagnitudeD was significantly lower in glaucomatous eyes when using both Lc (P =0.001) and Hc (P =<0.001) stimuli. The ratio MagnitudeD/Magnitude ratio was significantly lower in the glaucoma group when using an Hc stimulus (P < 0.001). The SNR was significantly lower in the glaucoma group when using an Hc stimulus (P = 0.01). Figures 2 shows results from a receiver operator characteristics (ROC) curve analysis for ss-perg Lc and Hc parameters. When using the Hc ss-perg stimulus, MagnitudeD had an area under the ROC curve (AUC) of Using this same stimulus, the ratio MagnitudeD/Magnitude ratio had an AUC of Results of SD-tVEP analysis in glaucoma and control eyes are summarized in Table 2. A total of 65/163 (39.9%) eyes had reliable Lc data and 115/163 (70.6%) eyes had reliable Hc data. For SD-tVEP, the amplitude and latency were compared between the glaucoma and control groups when using both Lc and Hc stimuli. None of the parameters tested showed a significant difference between the two groups. Figure 3 displays results from an ROC curve analysis for Lc and Hc SD-tVEP parameters. AUC values for both amplitude and latency (Lc amplitude AUC = 0.50, Lc latency AUC = 0.52) were lower than AUC values of ss-perg parameters. AUC values for Hc SD-tVEP parameters (Hc amplitude AUC = 0.57, Hc latency AUC = 0.62) were lower than those for Hc ss-perg parameters. DISCUSSION The aim of this study was to determine the ability of ss-perg and SD-tVEP to discern between glaucomatous and healthy eyes. The ss-perg parameters differed significantly between glaucoma

5 58 Electrophysiology for Glaucoma Diagnosis Amarasekera et al. 5 Table 1. Demographics and clinical characteristics of the patients enrolled in the study Glaucoma (n = 41 patients) Control (n = 41 patients) P-value Age (years) Mean ± SD 65.4 ± ± Gender (n, %) Male 18 (43.9) 17 (41.5) Female 23 (56.1) 24 (58.5) Race (n, %) Caucasian 7 (17.1) 16 (39.0) African American 32 (78.1) 17 (41.5) Other 2 (4.9) 8 (19.5) BCVA logmar Mean (95% CI) 0.08 (0.05, 0.1) 0.06 (0.04, 0.08) Intraocular pressure Mean (95% CI) 14.7 (13.3, 15.5) 15.6 (14.7, 16.5) Vertical C/D ratio Mean (95% CI) 0.7 (0.7, 0.8) 0.3 (0.3, 0.4) <0.001 Mean deviation Mean (95% CI) 7.5 ( 9.1, 5.9) Pattern SD 5.7 (4.8, 6.6) ANOVA test. Fisher s exact test. Mixed model calculation. ANOVA, analysis of variance; BCVA, best-corrected visual acuity; CI, confidence interval; C/D ratio, cup to disc ratio; SD, standard deviation. Table 2. ss-perg and SD-tVEPs in glaucomatous eyes and normal control eyes ss-perg Step 1 (64 bar size) SD-tVEP spatial frequency Glaucoma Control P-value Lc n =72 n =79 MagnitudeD ([μv] mean, 0.32 (0.25, 0.39) 0.48 (0.42, 0.55) MagnitudeD/Magnitude 0.45 (0.39, 0.51) 0.51 (0.45, 0.57) ratio (mean, SNR (mean, 2.29 (1.9, 2.8) 2.75 (2.29, 3.32) Hc n =72 n =80 MagnitudeD ([μv] mean 0.43 (0.32, 0.54) 0.77 (0.67, 0.88) <0.001 MagnitudeD/Magnitude 0.50 (0.44, 0.56) 0.65 (0.59, 0.71) <0.001 ratio (mean SNR (mean 2.77 (2.23, 3.42) 4.06 (3.32, 5) Lc n =26 n =39 Latency (ms) (mean (107.69, ) (106.09, ) Amplitude (μv) (mean (8.23, 11.8) (9.97, 13.41) Hc n =54 n =61 Latency (ms) (mean (109.71, ) (116.12, ) Amplitude (μv) (mean 8.48 (6.99, 9.97) 7.93 (6.53, 9.32) Unreliable test results were excluded. CI, confidence interval; Hc, high contrast; Lc, low contrast; SD-tVEPs, short-duration transient visual evoked potentials; SNR, signal-to-noise ratio; ss-perg, steady-state pattern electroretinogram. and control eyes. The results from an ss-perg stimulus, 64 bar size with Lc and Hc settings, were analysed. According to our results, all 64-bar-size Hc parameters were significantly lower in patients with glaucoma, and thus, this stimulus may be more accurately able to identify RGC dysfunction. Previous studies have shown that an Hc stimulus requires a much higher metabolic demand from active RGCs than an Lc stimulus. 27 Therefore, when presented with an Hc stimulus, patients with RGC dysfunction cannot fulfil this metabolic demand. This results in decreased ss-perg parameters when compared with healthy controls. Data also showed that SNR was significantly lower in glaucomatous eyes when using a 64-bar-size

6 Electrophysiology for Glaucoma Diagnosis 6 Amarasekera et 59 al. Figure 2. Receiver operated characteristics (ROC) curves for 64-bar-size steady-state pattern electroretinogram (ss-perg) low-contrast and high-contrast parameters. AUC, area under the ROC curve. Figure 3. Receiver operated characteristics (ROC) curves for short-duration transient visual evoked potentials (SD-tVEP) lowcontrast and high-contrast parameters. AUC, area under the ROC curve. Hc stimulus. The SNR is a marker of ss-perg amplitude, or the strength of the signal arising from the inner retinal cells that is markedly diminished when RGC dysfunction is present.28 A decreased SNR indicates that the amplitude of the ss-perg signal was not strong enough to overcome extraneous noise from eye movements or blinks.29 This is consistent with previous studies that have found PERG amplitude to be decreased in patients with glaucoma.10,13,21 MagnitudeD was the most accurate ss-perg parameter for discerning glaucomatous dysfunction across all stimuli. MagnitudeD represents the amplitude of the ss-perg signal as well but is also heavily influenced by the phase consistency of the signal. This phase is calculated by averaging measurements taken during each testing sequence. Phase consistency describes the SD of these measurements. A low phase consistency indicates a failure of the RGCs to adapt to the large energy demand of an ss-perg stimulus, leading to different phases as testing time goes on.14 This difference in phase consistency is demonstrated in Figures 1c,d. A low ss-perg phase consistency and decreased amplitude have been previously seen in patients with glaucoma.30 Accordingly, we found MagnitudeD to be significantly lower in patients with glaucoma than in healthy controls. The ratio MagnitudeD/Magnitude ratio represents the phase consistency alone, and the SNR represents the ss-perg amplitude alone. MagnitudeD is the only ss-perg parameter that represents the combined amplitude and phase consistency of the signal, thus explaining its increased accuracy in detecting patients with glaucoma. The AUCs for the ss-perg parameters used in this study (MagnitudeD and MagnitudeD/Magnitude ratio) agree with those of previous studies that have found a standard PERG amplitude to have an AUC ranging from 0.72 to ,31,32 Our similar values indicate that this new ss-perg testing platform is comparable with older PERG sequences in its diagnostic capabilities. AUCs for SD-tVEP data were generally much lower than those of ss-perg. The generally decreased values of MagnitudeD, MagnitudeD/Magnitude ratio and SNR in patients with glaucoma indicate dysfunction of the RGCs. Thus, it is possible that ss-perg could be used as a surrogate measure of RGC dysfunction that is known to appear prior to VF defects in early glaucoma. A traditional PERG testing system has been established to be an effective and early diagnostic tool for 2017 Royal Australian and New Zealand College of Ophthalmologists 2017 Royal Australian and New Zealand College of Ophthalmologists

7 60 Electrophysiology for Glaucoma Diagnosis Amarasekera et al. 7 glaucoma. 12,13,21 Our data indicate that that the new, easy to administer, office-based protocol of ss-perg may be of similar diagnostic value. Although some SD-tVEP parameters differed between patients with glaucoma and healthy controls, there was no statistical difference in test results between the two groups. These findings disagree with previous studies that have found a decreased VEP amplitude and increased latency in patients with glaucoma. A study by Parisi et al. found that an increased P100 latency of transient VEP had a sensitivity and specificity of 100% for detecting glaucoma. 24,25,32 The difference in SD-tVEP results between the two studies may be explained by differing study populations, study designs, levels of disease severity among tested patients or the reduced sample size after applying exclusion criteria. The superiority of ss-perg over SD-tVEP may be attributed to its physiological characteristics. The ss- PERG is known to specifically represent the function of the RGCs themselves, 33 and RGCs are damaged in the pathophysiology of glaucoma. 34 SD-tVEP is a measure of a function of the entire visual pathway from the photoreceptors to the visual cortex and therefore may not be as sensitive to dysfunction of the RGCs themselves. 24,25 The use of PERG and VEP to measure the functional status of the RGCs and visual pathway, respectively, has been well established. The value add of electrophysiological techniques in a diagnostic environment dominated by highly effective tools such as OCT is the addition of an objective functional perspective to the diagnosis of glaucoma. Previous studies have found that PERG can complement structural clues to glaucoma by offering insight into the current functional status of a patient s RGCs. Additionally, as RGC dysfunction has been found to precede loss of optic nerve tissue and RGC density, PERG has the ability to identify dysfunction prior to obvious structural changes present on other imaging modalities. 35 Despite its value, a major limitation in electrophysiological advancement and incorporation into the clinical setting thus far has been its invasiveness and hassle factor as described by Bach et al. in his 2013 review of the future challenges of electrophysiology. 6 ss-perg and SD-tVEP eliminate the corneal electrodes, anaesthesia, pupillary dilation and length of testing that older paradigms once required and thus represent significant advancements towards clinically useful electrophysiology. Numerous studies have confirmed the ability of older paradigms of PERG to detect the RGC dysfunction in patients with glaucoma prior to RGC loss detected by tests such as OCT. 36 In our study, the average (95% confidence interval) mean deviation of patients with glaucoma was 7.5 ( 9.1, 5.9), indicating that the patients in this study had moderate progression of their disease. This finding supports the ability of ss-perg to detect glaucoma in its moderate stages, but further prospective, longitudinal studies are needed to confirm the ability of ss-perg and SD-tVEP to identify dysfunction in RGCs or the visual pathway before other imaging modalities. Our study identifies the ss-perg parameters that are most sensitive for glaucoma detection and offers us early confirmation of a viable, clinically feasible electrophysiological tool with early potential for clinical incorporation and advancement. Our study had several limitatio ns. The ss-perg and SD-tVEP tests were not conducted by the same individual for all patients, and thus, inter-test variability may have affected the results. Additionally, it is unclear if these electrophysiological tests are able to distinguish glaucoma damage from other causes of vision loss, and therefore, results may not exclusively reflect damage because of glaucoma. Furthermore, the sample size of patients who underwent both ss-perg and SD-tVEP testing was significantly decreased because test results of some eyes could not achieve sufficient reliability. This lower sample size is more pronounced with SD-tVEP testing, which may have directly influenced test results. Glaucomatous eyes had more difficulty achieving reliability using SD-tVEP testing than control eyes, with 55/81 eyes excluded using the Lc stimulus and 27/81 eyes using the Hc stimulus. 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