Purpose
To determine cutoffs for interocular differences in ganglion cell–inner plexiform layer thickness in normal healthy eyes and to evaluate the diagnostic performance of these values for differentiating between normal subjects and glaucoma patients.
Design
Observational, cross-sectional study.
Methods
Macular and optic disc scanning were performed in 275 normal subjects, 58 glaucoma patients, and 58 normal controls by high-definition optical coherence tomography. The ganglion cell–inner plexiform layer thickness was calculated, and the normal ranges of the interocular differences were determined as 2.5th and 97.5th percentiles. The signed and absolute interocular differences were compared between normal subjects and glaucoma patients.
Results
The mean ± standard deviation interocular difference in the average ganglion cell–inner plexiform layer thickness thickness was 0.10 ± 2.31 μm, which was not statistically significant ( P = .466). The 2.5th and 97.5th percentiles of the interocular difference were −4.10 μm and +5.00 μm, respectively. On multiple regression analysis, the interocular difference in axial length was correlated with the interocular difference in average ganglion cell–inner plexiform layer thickness thickness ( β = 2.044, P = .003). The signed and absolute interocular differences in ganglion cell–inner plexiform layer thickness were higher in glaucoma patients than in normal subjects (all P < .001). Sensitivity and specificity of absolute interocular differences ranged from 25.9% to 51.7% and from 93.1% to 100.0%, respectively.
Conclusions
Ganglion cell–inner plexiform layer thickness shows significant interocular symmetry in normal subjects. An absolute interocular difference exceeding normal limits may be indicative of glaucoma.
Glaucoma is an optic neuropathy characterized by loss of retinal ganglion cells and their axons. Because of the chronic, progressive, and potentially blinding nature of this disease, timely detection and evaluation of glaucomatous damage through structural and functional analysis are important. Most previous studies investigating the relationship between structure and function in glaucoma have shown that structural changes precede visual function loss as tested by automated perimetry.
Although organ pairs are not always perfectly symmetric, healthy organ pairs mostly show symmetric anatomic features. However, the occurrence and progression of clinical disease can be different between organ pairs. From this perspective, studies have previously been performed to assess whether differences between organ pairs can reveal or influence the localization of diseases. In glaucoma studies, this method has been used to evaluate the cup-to-disc ratio, intraocular pressure (IOP), and retinal nerve fiber layer (RNFL) thickness. It has been demonstrated that interocular asymmetry in the cup-to-disc ratio in the same person is related to early glaucomatous damage. Further, interocular asymmetry in IOP is known to be associated with glaucomatous visual field loss. Because the loss of retinal ganglion cells and their axons is the key pathologic feature of glaucoma, previous investigations have focused on the quantitative analysis of peripapillary RNFL thickness. Several studies have suggested that an interocular difference in RNFL thickness is indicative of early glaucomatous damage.
Recent studies have shown that the measurement of retinal ganglion cell loss in the macula area can be a direct and powerful method. Previous studies revealed that assessment of the macular ganglion cell layer could be a surrogate method for the evaluation of glaucomatous damage. Moreover, measurement of the thickness of the macular ganglion cell–inner plexiform layer thickness by using a ganglion cell analysis algorithm by spectral-domain optical coherence tomography (OCT) was shown to be effective for the diagnosis and evaluation of glaucoma progression. However, limited information is available regarding the interocular symmetry of ganglion cell–inner plexiform layer thickness.
The current study was designed to determine the normal tolerance limits for the amount of asymmetry in the ganglion cell–inner plexiform layer thickness when the values in each eye are within normal limits. Additionally, we investigated the possible utility of interocular asymmetry beyond the normal tolerance limits for distinguishing glaucoma patients from normal healthy subjects. Determining tolerance limits for interocular symmetry of the ganglion cell–inner plexiform layer may help clinicians identify patients with glaucomatous damage, as is the case with RNFL thickness.
Methods
Subjects
This was an observational, cross-sectional study based on the Macular Ganglion Cell Imaging Study, an ongoing prospective study of glaucoma patients and healthy volunteers at the Glaucoma Clinic of Seoul National University Hospital. Each subject was informed of the nature of the study, and written informed consent was obtained from each participant after study approval by the institutional review board of Seoul National University Hospital. The study protocol complied with the Declaration of Helsinki.
For estimating the normal tolerance limits of interocular difference of ganglion cell–inner plexiform layer thickness, eyes were selected from a database of healthy subjects. Eligibility was determined for each subject through a complete ophthalmologic examination, including measurement of visual acuity, slit-lamp examination, IOP measurement by Goldmann applanation tonometer, dilated fundus examination, red-free fundus photography, standard automated perimetry, and Cirrus high-definition OCT (Cirrus; Carl Zeiss Meditec, Dublin, California, USA). The inclusion criteria were as follows: age between 20 and 79 years; best-corrected visual acuity of ≥20/40; refractive error within ±6.00 diopters equivalent sphere and ±3.00 diopters astigmatism; less than 2.00 diopters anisometropia; an open anterior chamber angle at the initial examination; good-quality fundus images; absence of glaucomatous optic neuropathy; absence of RNFL defect in red-free fundus photography; and a normal standard automated perimetry result. The absence of glaucomatous optic neuropathy was defined as a cup-to-disc ratio less than 0.7 and an intact neuroretinal rim without optic disc hemorrhages, notches, or localized pallor.
Exclusion criteria were contraindication to dilation or intolerance to topical anesthetics or mydriatics; IOP of 22 mm Hg or more in either eye; any type of glaucoma in either eye; intraocular surgery in the study eye (except uncomplicated cataract surgery performed more than 1 year before enrollment); evidence of diabetic retinopathy, macular edema, or other vitreoretinal disease; evidence of optic nerve abnormality; or family history of glaucoma.
To evaluate the clinical efficacy of the normal tolerance limits of interocular difference, normal healthy subjects and glaucoma patients were enrolled from a database of subjects in the Macular Ganglion Cell Imaging Study. Glaucomatous eyes were defined as those with a glaucomatous visual field defect confirmed by 2 reliable standard automated perimetry measurements and by the presence of glaucomatous optic disc cupping irrespective of IOP. Glaucomatous optic disc cupping was defined as neuroretinal rim thinning, notching, excavation, or RNFL defect with a corresponding visual field deficit. Color disc and red-free RNFL images were evaluated independently by 2 observers in a random order and in a masked fashion, without knowledge of clinical information. The presence of glaucomatous optic disc cupping was determined by a consensus agreement between the 2 observers. The exclusion criteria were contraindication to dilation or intolerance to topical anesthetics or mydriatics; intraocular surgery in the study eye (except uncomplicated cataract surgery performed more than 1 year before enrollment); evidence of diabetic retinopathy, macular edema, or other vitreoretinal disease; or evidence of nonglaucomatous optic nerve abnormality.
Optical Coherence Tomography Scanning Procedure
Pupils were dilated with tropicamide 1% and phenylephrine 2.5% drops. All imaging procedures were performed using Cirrus high-definition OCT (software version 6.0; Carl Zeiss Meditec). Two scans, including 1 macular scan centered on the fovea (macula cube 200 × 200 protocol) and 1 peripapillary scan centered on the optic disc (optic disc cube 200 × 200 protocol), were acquired from both eyes of the same subject. The macular ganglion cell–inner plexiform layer, peripapillary RNFL thicknesses, and optic nerve head parameters were measured automatically using the internal Cirrus OCT algorithms. Only good-quality scans, which were defined as scans with signal strength ≥6 and the absence of involuntary saccade or blinking artifacts, were used for analysis. The ganglion cell analysis algorithm detects and measures the thickness of the macular ganglion cell–inner plexiform layer within a 6 × 6 × 2-mm elliptical annulus area centered on the fovea. The following ganglion cell–inner plexiform layer thickness measurements were analyzed: average, minimum, and 6-sector (superior, superonasal, inferonasal, inferior, inferotemporal, and superotemporal). The average, quadrants, and clock hours were included in the analyses for the peripapillary RNFL thickness measurements. For the clock hour RNFL thickness, 12 sectors of 30 degrees were defined in clockwise order for both eyes. Clock hour 1 in the right eye corresponded to clock hour 11 in the left eye, clock hour 2 in the right eye corresponded to clock hour 10 in the left eye, etc. The optic nerve head parameters included the disc area, rim area, cup-to-disc area ratio, vertical cup-to-disc diameter ratio, and cup volume.
Statistical Analysis
Statistical analyses were performed using SPSS software version 21.0 (IBM Corp, Armonk, New York, USA). For all analyses, a P value <.05 was considered statistically significant.
Statistical analysis in normal healthy subjects
The values from the 2 eyes were compared using the Student t test. The interocular differences of the values were determined by subtracting the values of the left eyes from those of the right eyes. The normal ranges for interocular differences were established as the 2.5th and 97.5th percentiles for the ganglion cell–inner plexiform layer thickness. The relationships between the interocular difference in the ganglion cell–inner plexiform layer thickness and age, interocular differences in the IOP, rim area, disc area, cup-to-disc area ratio, vertical cup-to-disc diameter ratio, and cup volume were evaluated by univariate and multivariate linear regression analysis. These parameters were first fitted in a univariate model; subsequently, variables with P values less than .05 were entered into a multivariate analysis to determine the independence of the effects.
Statistical analysis of the comparisons between the normal control group and the glaucoma group
The average values of interocular differences and absolute interocular differences in the ganglion cell–inner plexiform layer thickness parameters were compared between normal and glaucomatous eyes using the Student t test. Sensitivity and specificity of the absolute interocular differences were tested by comparing them to the normal tolerance limits of the absolute interocular differences. The normal tolerance limits of the absolute interocular difference were determined by selecting the unsigned larger value between the 2.5th and 97.5th percentiles of the interocular difference in normal healthy subjects. Receiver operating characteristic (ROC) curves were used to describe the diagnostic ability of absolute interocular difference to differentiate glaucomatous eyes from normal eyes. An area under the ROC curve (AUROC) of 1.0 represents perfect discrimination, whereas an AUROC of 0.5 represents a chance of discrimination.
Results
Data from 275 normal subjects were included in the analysis for normal healthy subjects. The mean ± standard deviation age was 36.5 ± 17.0 years, and 65 subjects were female. The ocular characteristics of the right and left eyes are shown in Table 1 . The right and left eye were moderately myopic, and no statistically significant difference was found between both eyes for any ocular characteristic, including refractive error, axial length, central corneal thickness, or disc parameters. Further, the mean signal strengths of OCT scans between right eyes (8.14 ± 1.27) and left eyes (8.20 ± 1.23) were not significantly different.
| Parameters | Right Eye (SD) | Left Eye (SD) | P Value a |
|---|---|---|---|
| Spherical equivalent (diopter) | −2.27 (2.37) | −2.07 (2.39) | .422 |
| Axial length (mm) | 24.17 (1.27) | 24.13 (1.27) | .731 |
| CCT (μm) | 549.51 (30.60) | 550.45 (29.62) | .727 |
| IOP (mm Hg) | 14.15 (2.65) | 14.33 (2.71) | .426 |
| Disc area (mm 2 ) | 2.02 (0.48) | 2.04 (0.47) | .541 |
| Rim area (mm 2 ) | 1.28 (0.60) | 1.24 (0.22) | .338 |
| Cup-to-disc area ratio | 0.58 (0.16) | 0.58 (0.16) | .940 |
| Vertical cup-to-disc diameter ratio | 0.54 (0.16) | 0.54 (0.16) | .939 |
| Cup volume (mm 3 ) | 0.28 (0.24) | 0.27 (0.22) | .692 |
a Student t test, comparison between the right eyes and the left eyes.
The interocular differences in the ganglion cell–inner plexiform layer thickness between the right and left eyes are shown in Table 2 . The average ganglion cell–inner plexiform layer thickness was 81.87 ± 5.95 μm in the right eye and 81.77 ± 6.02 μm in the left eye. The interocular difference between both eyes was 0.10 ± 2.31 μm, which was not statistically significant ( P = .842). The minimum ganglion cell–inner plexiform thickness was also not significantly different between both eyes ( P = .681). Similarly, all 6 sectoral measurements showed no statistically significant interocular differences in ganglion cell–inner plexiform layer thickness. The 2.5th and 97.5th percentiles of the interocular difference tolerance limits for the average ganglion cell–inner plexiform layer thickness were −4.10 μm and +5.00 μm. The tolerance ranges for the minimum and sector ganglion cell–inner plexiform layer thicknesses were wider than were those of the average ganglion cell–inner plexiform layer thickness.
| Division | Right Eye (SD) a | Left Eye (SD) a | Difference (SD) a | P Value b | Percentile Distribution | |
|---|---|---|---|---|---|---|
| 2.5% | 97.5% | |||||
| Average (μm) | 81.87 (5.95) | 81.77 (6.02) | 0.10 (2.31) | .842 | −4.10 | 5.00 |
| Minimum (μm) | 78.38 (10.01) | 78.03 (10.27) | 0.36 (6.71) | .681 | −13.20 | 16.10 |
| Superonasal (μm) | 82.79 (6.91) | 82.82 (6.60) | −0.03 (3.93) | .955 | −8.00 | 10.10 |
| Superior (μm) | 84.13 (6.70) | 83.97 (6.88) | −0.16 (3.57) | .782 | −7.00 | 7.10 |
| Superotemporal (μm) | 81.79 (7.17) | 82.12 (6.26) | −0.33 (4.56) | .564 | −10.10 | 6.00 |
| Inferotemporal (μm) | 79.25 (6.93) | 79.06 (7.70) | 0.19 (4.99) | .762 | −12.00 | 9.00 |
| Inferior (μm) | 81.74 (6.25) | 81.51 (6.67) | 0.23 (4.09) | .673 | −8.00 | 10.00 |
| Inferonasal (μm) | 81.31 (5.95) | 81.32 (6.27) | −0.02 (3.48) | .978 | −7.00 | 9.00 |
a Data are presented as mean (standard deviation).
b Student t test, comparison between the right eyes and left eyes.
Interocular differences in the RNFL thickness parameters, including the mean interocular differences with their respective P values, are listed in Table 3 . The average RNFL thickness in the right eye was 1.20 ± 5.86 μm thicker compared to in the left eye. The RNFL thickness showed statistically significant interocular differences in the areas of the superior, nasal and temporal quadrants, and in the clock hours 12/12, 1/11, 2/10, 9/3, 10/2, and 11/1. The RNFL thickness in the right eye was thicker than the RNFL thickness in the left eye in the nasal and temporal sectors, and in clock hours 2/10, 9/3, 10/2, and 11/1. In the superior sector, clock hour 12/12, and clock hour 1/11, the RNFL thickness in the left eye was thicker than in the right eye. The 2.5th to 97.5 percentile interocular difference tolerance limits of the average RNFL thickness were −11.10 μm and +14.00 μm, respectively.
| Division | Right Eye (SD) | Left Eye (SD) | Difference (SD) | P Value a |
|---|---|---|---|---|
| Average RNFLT (μm) | 94.35 (8.36) | 93.15 (8.86) | 1.20 (5.86) | .010 |
| Quadrant RNFLT (μm) | ||||
| Superior | 116.51 (14.84) | 120.55 (14.73) | −4.04 (12.19) | .001 |
| Nasal | 66.58 (8.98) | 63.66 (9.05) | 2.92 (7.33) | <.001 |
| Inferior | 121.81 (15.33) | 119.10 (17.18) | 2.71 (12.58) | .052 |
| Temporal | 72.57 (12.85) | 69.52 (12.29) | 3.05 (7.70) | .005 |
| Clock hours R/L (μm) | ||||
| Clock-hr 12/12 | 114.42 (25.19) | 121.54 (25.36) | −7.12 (20.92) | .001 |
| Clock-hr 1/11 | 100.63 (21.94) | 114.34 (20.94) | −13.71 (20.16) | <.001 |
| Clock-hr 2/10 | 82.03 (15.58) | 76.71 (15.37) | 5.32 (14.23) | <.001 |
| Clock-hr 3/9 | 56.24 (9.39) | 55.27 (9.28) | 0.96 (9.30) | .227 |
| Clock-hr 4/8 | 61.15 (10.79) | 59.57 (10.98) | 1.58 (9.66) | .089 |
| Clock-hr 5/7 | 93.89 (18.50) | 91.81 (19.56) | 2.08 (15.72) | .201 |
| Clock-hr 6/6 | 128.85 (24.86) | 128.12 (32.25) | 0.72 (27.72) | .768 |
| Clock-hr 7/5 | 142.71 (21.31) | 138.98 (30.04) | 3.73 (29.28) | .094 |
| Clock-hr 8/4 | 75.85 (16.75) | 75.50 (43.40) | 0.35 (42.40) | .901 |
| Clock-hr 9/3 | 56.99 (10.24) | 55.21 (9.85) | 1.78 (7.18) | .038 |
| Clock-hr 10/2 | 84.53 (17.48) | 79.98 (15.93) | 4.55 (12.73) | .002 |
| Clock-hr 11/1 | 133.21 (18.50) | 125.69 (20.85) | 7.52 (16.04) | <.001 |
a Student t test, comparison between the right eye and left eye.
Increasing age was associated with a decrease in average ganglion cell–inner plexiform layer thickness in both eyes (right eyes: R 2 = 0.047, P < .001; left eyes: R 2 = 0.059, P < .001). However, the signed interocular difference and absolute difference in the average ganglion cell–inner plexiform layer thickness was not associated with increasing age (signed difference: P = .160, absolute difference: P = .100). This trend is shown in the Figure . No significant difference was found in the interocular difference with respect to the average and minimum ganglion cell–inner plexiform layer thickness between male and female subjects (average: P = .651, minimum: P = .952).
On univariate regression analysis, the interocular difference in the average ganglion cell–inner plexiform layer thickness significantly correlated with the interocular difference of the refractive error (R 2 = 0.055, P = .001), axial length (R 2 = 0.042, P = .005), cup-to-disc area ratio (R 2 = 0.018, P = .028), vertical cup-to-disc diameter ratio (R 2 = 0.020, P = .019), and cup volume (R 2 = 0.019, P = .022), but not with age ( P = .160), sex ( P = .651), the interocular difference of the IOP ( P = .244), central corneal thickness ( P = .190), disc area ( P = .232), and rim area ( P = .153). Multiple regression analysis including 5 significant variables from the univariate analysis showed that the interocular difference in axial length ( β = 2.044, P = .003) was independently correlated with the interocular difference in average ganglion cell–inner plexiform layer thickness. These results are presented in Table 4 .
| Variables | Univariate Analysis | Multivariate Analysis a | ||
|---|---|---|---|---|
| β ± SE | P | β ± SE | P | |
| Age | 0.012 ± 0.008 | .160 | ||
| Sex (male = 1, female = 0) | −0.149 ± 0.328 | .651 | ||
| Interocular difference (right eye minus left eye) | ||||
| IOP (mm Hg) | −0.098 ± 0.084 | .244 | ||
| Refraction (diopters) | 0.721 ± 0.214 | .001 b | 0.149 ± 1.801 | .073 |
| Axial length (mm) | 1.927 ± 0.676 | .005 b | 2.044 ± 0.668 | .003 b |
| CCT (μm) | −0.031 ± 0.024 | .190 | ||
| Disc area (mm 2 ) | 0.598 ± 0.499 | .232 | ||
| Rim area (mm 2 ) | 0.335 ± 0.234 | .153 | ||
| Cup-to-disc area ratio | −4.938 ± 2.234 | .028 b | −0.067 ± 0.899 | .370 |
| Vertical cup-to-disc diameter ratio | −4.452 ± 1.892 | .019 b | −0.064 ± 0.852 | .395 |
| Cup volume (mm 3 ) | −2.652 ± 1.150 | .022 b | −0.051 ± 0.683 | .496 |
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