Influence of disc area on retinal nerve fiber layer thickness measurement by spectral domain optical coherence tomography.

BACKGROUND: To examine the effect of optic disc area on peripapillary RNFLT (retinal nerve fiber layer thickness) measurement at circle diameter of 3.4 mm around optic nerve head using spectral OCT/SLO (Optical coherence tomography/scanning laser ophthalmoscope).
MATERIALS AND METHODS: In this prospective, cross sectional study, one hundred and two eyes of 102 normal subjects underwent RNFLT and disc area measurement using spectral OCT/SLO. Based on disc area, subjects were divided into three groups i.e., <3 mm2 (32 eyes), 3-4 mm2 (36 eyes) and >4 mm2 (34 eyes). The effect of disc area on RNFLT parameters was analyzed using linear regression analysis.
RESULTS: The mean and quadrant RNFLT did not show significant correlation with disc area in subjects with disc area of <4 mm2, however in eyes with disc area >4 mm2, average RNFLT, superior and temporal quadrant RNFLT showed negative correlation with disc area, which was statistically significant (P = 0.004, P = 0.005 and P = 0.002, respectively).
CONCLUSION: In healthy eyes of disc area <4 mm2, disc size does not appear to affect peripapillary RNFLT measurement by spectral OCT/SLO. Average, superior and temporal quadrant RNFLT measurements were inversely proportional to disc area in eyes with disc area >4 mm2. Hence, RNFLT measurement by OCT in eyes with optic disc area of >4 mm2 should be interpreted carefully.

Retinal nerve fiber layer thickness (RNFLT) measurement using optical coherence tomography (OCT) is useful adjunct in detection and monitoring of glaucoma.[1] OCT is non-contact, non-invasive imaging technique which produces high-resolution, cross-sectional images of optic nerve head (ONH) and RNFLT. It provides objective and quantitative estimation of RNFLT by measuring echo time delay of backscattered light from various layers in retina using Michelson interferometer.

New generation spectral domain OCT (SD-OCT) has faster acquisition speed and an increased depth resolution compared to previous generation stratus OCT (Time domain OCT) because echo time delays of light are measured by taking the Fourier transform of the interference spectrum of the light signal. A Fourier transform is a mathematical procedure that extracts the frequency spectrum of a signal.[2] Spectral OCT/SLO (Spectral OCT/scanning laser ophthalmoscope, OPKO/OTI, international version, Miami, Florida) is commercially available SD-OCT that has axial resolution of <6 μm and a scan velocity of 27,000 axial scans per second.

In the measurement of peripapillary RNFLT, a circle diameter of 3.4 mm is used around ONH to avoid intersecting tissue within the ONH margin in large disc and to avoid areas with peripapillary atrophy. Also, it is close to disc to allow a dense sampling covering the entire distribution of RNFL and the measurements at this location have been shown to be most reproducible compared with 2 other scan circles of different diameters.[3]

Previous studies have demonstrated positive correlation between disc size and RNFLT in normal eyes,[456] however, others did not find such correlation using OCT.[789] As optic disc[10] and RNFLT[11] show large inter individual variation within and among populations, we measured RNFLT in optic disc of various sizes by OCT.

The purpose of this study was to examine whether different optic disc areas affects peripapillary RNFLT measurement using spectral OCT/SLO at fixed circle diameter of 3.4 mm around ONH.

Materials and Methods

For this prospective, cross-sectional study, normal volunteers of different age groups were recruited from patients with refractive error and the Institute staff. Informed consent was obtained from each subject. All subjects were Asian Indians. Each participant underwent complete ophthalmic examination including best corrected visual acuity (BCVA), refraction, slit lamp biomicroscopy, intraocular pressure (IOP) measurement by goldmann applanation tonometer, gonioscopy by Volk four mirror Sussman gonio lens, optic disc and stereoscopic fundus evaluation with +78 Diopter (D) lens, achromatic automated perimetry with Humphrey visual field analyzer HFA 750 II (Carl Zeiss Meditec, Dublin, California, USA) using Swedish Interactive Threshold Algorithm (SITA), Standard 24-2 strategy.

Subjects were classified as having normal eyes if they had BCVA of ≥20/30, refractive error within ±3.5 D of sphere and ±2 D of cylinder, IOP <21 mm Hg, open angles on gonioscopy, normal appearing optic disc with healthy neuroretinal rim, no asymmetry in cup disc ratio between 2 eyes of >0.2 or ONH abnormality (evidence of peripapillary atrophy, tilted disc, disc hemorrhage etc.), normal and reliable visual field. Normal visual field indices were defined as Mean deviation and Pattern standard deviation within 95% confidence limits and Glaucoma hemifield test ‘within normal limits’. Reliability criteria for automated perimetry were fixation losses <20%, false positives and false negatives ≤33%. Subjects with family history of glaucoma, ocular trauma, neurological disease, intraocular surgery, ONH abnormality and ocular pathology were excluded.

OCT technique

After dilation with 1% tropicamide eye drop, one randomly selected eye of 102 normal subjects underwent RNFL scanning using spectral OCT/SLO. All images were acquired by single operator.

The subjects were asked to look at internal fixation target and a circular scan with a circle diameter of 3.4 mm was centered around optic disc and the location was observed on the SLO image to ensure proper positioning of scan in relation to the ONH. Average of three consecutive OCT image of the RNFL, within <5% thickness variation, was obtained. The RNFL analysis uses an automated OCT software algorithm to identify anterior and posterior margins of RNFL.

The following RNFL parameters were evaluated

Average peripapillary RNFLT (360°) and the four quadrant RNFLT (superior, nasal, inferior and temporal). The sectors were defined in clockwise order for right eye and counter clockwise order for the left eye. For ONH analysis, optic nerve topography scan mode was used. The topography stack covers an area of 5.5 × 5.5 mm with a depth of 2 mm. A three dimensional tomographic image of the optic nerve region was generated from a stack of sequential OCT and confocal SLO images. The operator made sure that the center of the optic disc was in the center of the SLO image. The software analyses the data and creates report on disc area and other ONH parameters. Criteria for determining scan quality were: Signal strength >7, a clear fundus image allowing optic disc and scan circle visibility prior to and during image acquisition, even and dense color saturation throughout all retinal layers with red color visible in the retinal pigment epithelium and RNFL, a continuous scan pattern without missing or blank areas (i.e., no algorithm failures) and automated detection of disc margin in ONH analysis.

Statistical analysis

Statistical analysis was performed using SPSS version 15 (SPSS Inc, Chicago, IL). One-way ANOVA with post hoc test of LSD was used to compare mean RNFLT among the 3 groups of different disc sizes. Correlation between disc area, average and quadrant RNFLT was analyzed using linear regression analysis. Level of significance was considered as P < 0.05.

Results

One hundred and two eyes of 102 subjects were enrolled for this study. The mean disc area was 3.66 ± 0.91 mm2. Based on disc area, subjects were divided into 3 groups i.e., <3 mm2 (32 eyes) 3-4 mm2 (36 eyes) and >4 mm2 (34 eyes). The mean age ± standard deviation (SD) was 38. 9 ± 16.76 years (range, 14-79). There was no significant difference of age among 3 groups (P = 0.06) [Table 1]. There were 46 males and 56 females.

Table 1

Baseline characteristics of sample population

There was no statistically significant mean difference between gender regarding mean disc area (males: 3.45 ± 0.95 mm2, females: 3.42 ± 0.8 mm2). The refractive error was similar in all three groups (P = 0.5) [Table 1].

Mean ± SD values for average and quadrant RNFLT for various disc areas is shown in Table 2. There was no statistically significant mean difference between average RNFLT (P = 0.97), superior quadrant (P = 0.49) and inferior quadrant (P = 0.6) among the 3 groups [Table 2]. There was statistically significant mean difference between temporal quadrant (P = 0.001) and nasal quadrant RNFLT (P < 0.04) between the 3 groups [Table 2].

Table 2

Descriptive analysis of retinal nerve fiber layer thickness among disc areas of various sizes

The mean and quadrant RNFLT did not show significant correlation with disc area among subjects in subgroups of disc area of <3 mm2 and that between 3-4 mm2 disc area, however in subgroup of disc area >4 mm2, average RNFLT, superior and temporal quadrant RNFLT showed negative correlation with disc area, which was statistically significant [Table 3].

Table 3

Regression model of RNFL parameters (Y) with disc area (X)

Discussion

Previous histological studies on human and non-human primate eyes have shown that the optic nerve fiber count increases linearly with an increase in optic disc size.[1213] However, another histological study on human eye could not identify correlation between axon count and area of scleral canal.[14] Histological study on post-mortem human eyes has shown that the convergence of ganglion cellaxons from the retinal periphery towards the optic disc gives rise to increase in RNFLT as ONH is approached.[15] A study on healthy eyes using high speed, ultrahigh resolution OCT showed that RNFLT is inversely related to the distance from ONH center.[16] Blumenthal et al,[17] reported that RNFLT was found to be inversely related to the distance from the center of optic disc.

The clinical standard for peripapillary RNFLT measurement with OCT is with a circular scan of 3.4 mm scanning diameter centered around ONH, regardless of ONH size. Hence, using a fixed diameter circular scan in all eyes may result in RNFLT measurements performed at different distances from ONH margin.

A study from Savini et al.[4] in normal eyes found a positive correlation between optic disc size and mean RNFLT as measured by stratus OCT. They suggested such a correlation may be result of either an increased number of nerve fibers in the eyes with larger disc or the use of a fixed-diameter scan produced artifact. The latter hypothesis was derived from the notion that if a fixed diameter circular scan is used, the distance between the scan and the ONH margin will be reduced in the presence of a large ONH, which would lead to overestimation of RNFLT in patients with large ONH as the measurement would be made closer to the optic disc edge. In an another study, RNFLT was measured at 3.4 mm circle diameter and 2 customized-diameter scans (at 0.5 mm and 1 mm) from the ONH edge in 81 healthy subjects by stratus OCT.[5] It was found that when a fixed diameter circular scan is used, larger discs show higher values; conversely when the diameter is adjusted on the basis of ONH size, larger discs show lower values.

Funaki et al,[6] reported a significant positive correlation between optic disc size and total, as well as the regional integral of RNFLT with scanning laser polarimetry and suggested that larger optic disc have more optic nerve fibers.

In a study by Kaushik et al,[7] in 32 normal eyes, the peripapillary RNFL was scanned with the fast scanning protocol at 3.4 mm circle diameter using stratus OCT, the disc area did not affect RNFLT measurement. They suggested that RNFLT is dependent on the distance from the center of the optic disc rather than the point of exit from scleral canal and RNFL would be measured at similar distances from center of the optic disc, regardless of the size of scleral canal. Previous study by our group, using spectral OCT/SLO in 65 healthy eyes failed to demonstrate significant correlation between optic disc size and average and quadrant peripapillary RNFLT even after correction for age.[8] It was hypothesized that large inter individual variability for RNFLT and disc area within the population probably minimizes the effect of various ONH size on RNFLT measurement and hence, there is need to study correlation of RNFLT and disc area in eyes with small or large optic disc area or similar disc area to minimize optic disc size variation. To understand the clinical significance of RNFLT in vivo, we determined whether the peripapillary RNFLT was dependent on optic disc size in normal subjects. In this study, to reduce inter individual variability, we studied patients in upper and lower range of ONH size and average disc size. In eyes with disc area <4 mm2, disc area did not affect RNFLT measurement. Average, superior and temporal quadrant RNFLT measurements were inversely proportional to disc area in eyes with disc area >4 mm2. This may indicate that RNFLT is dependent on the distance from the center of the optic disc rather than the point of exit from the scleral canal as reported earlier and hence this negative correlation can be easily explained.[7] RNFL fibers emerging from a large ONH must be distributed over a wider circumference, as a consequence, the larger spatial distribution will result in thinner RNFLT when large ONHs are analyzed.

To conclude, in normal eyes, disc area <4 mm2 does not appear to affect peripapillary RNFLT measurement by spectral OCT/SLO. However, RNFLT measurement in eyes with optic disc area >4 mm2 by OCT should be interpreted carefully.