Retinal nerve fibre layer (RNFL) examination is important in diagnosing and monitoring the progress of glaucoma.  Damage to RFNL mostly precedes visual field loss. Hence, objective methods of measuring RNFL thickness may aid physicians in making an accurate and early diagnosis. Recent advances in various imaging technologies have made assessment of RFNL possible.  These methods utilise the optical property of RFNL for obtaining the quantitative RFNL thickness measurements.  These techniques are rapid, have good reproducibility and objectivity. 
Optical Coherence Tomography (OCT) is a non-contact, non-invasive diagnostic technique that provides detailed structural information of the posterior segment. It shows a cross-sectional living histology of retina with high resolution (of approximately 10 µ) than most of the conventional imaging systems.  It also has a high reproducibility.  Optical coherence tomo-graphy allows direct measurement of RNFL thickness by in vivo visualisation of retina and RNFL. A high reflectance layer located just under the inner surface of the retina that corresponds to the RFNL is measured using a computer-fed algorithm to generate the RFNL measurement. Optical coherence tomography generated morphologic findings in experimental animals have been shown to correspond very well with histological findings.
Retinal nerve fibre layer has been shown to have considerable inter-individual variation. This variation can be age or race related.  Retinal nerve fibre layer measurement varies with the technique used. The measurement may also differ with the population used as a database. It would therefore be preferable to use the values derived from a normative population as close as possible to the population for which the instrument is used. To the best of our knowledge there are no reported data for RFNL thickness in Indian eyes (Medline Search).
Material and Methods
One hundred and forty six healthy volunteers constituting patients or attendants of patients who were not blood relatives were included in this cross sectional study. Informed consent was obtained. All subjects underwent anterior segment slitlamp examination, Goldmann applanation tonometry, gonioscopy and fundus examination with plus 90D lens. Automated refraction (Retinomax 2 Autorefractor, Nikon Corp., Japan), axial length measurement (EchoScan US 3300, Nidek Corp., Japan) and automated visual field examination (Humphrey visual field analyser, HFA, Model 745, Humphrey Instruments; full threshold program 30-2) was performed.
Refractive error of <±4 Diopters, a normal optic nerve head, normal posterior segment, intraocular pressure - 21mmHg, mean deviation (MD) and corrected pattern standard deviation (CPSD) on HFA within 95% confidence interval, and normal glaucoma hemi-field test.
Family history of glaucoma, history of prior photocoagulation, history of prior ocular disease, history of intraocular surgery, previous ocular trauma, vertical asymmetry of cup: disc (C:D) ratio (>0.2) between the two eyes, high C: D ratio (>0.6), disc haemorrhages, disc pallor, and localized RNFL defects. Visual fields : Pattern deviation plot showing a cluster of 3 or more non-edge points that have sensitivities occurring in fewer than 5% of the normal population (P< 5%), with one of these points with a sensitivity occurring in less than 1% of the normal population (P< 1%), CPSD with P< 5%; and glaucoma hemifield test outside normal limits; and consistently unreliable visual fields (defined as false negative> 33%, false positive> 20%, fixation losses> 20%).
Normal contra-lateral eyes of the patients with incipient cataract or unilateral macular hole, fulfilling the above criteria were also included in the study.
Optical coherence tomography
OCT measurements were performed using OCT (OCT 3 STRATUS, Zeiss Humphrey, Dublin CA), with software version 3. The basic principle and technical characteristics of the OCT have been described previously.
Each eye was dilated with tropicamide 1% before recording the images, and scans were performed with a minimum pupillary diameter of 5 mm. The internal fixation target was used owing to its higher reproducibility. The fast retinal nerve fibre layer thickness protocol was used [Figure - 1]. This protocol provides better reproducibility than the single scan. It consists of three circular scans each of 3.46 mm in diameter centered on the optic disc. This diameter has been shown to be optimal and reproducible for RNFL thickness analysis. One of the authors (PS) acquired all the images. Mean RNFL thickness was calculated using the inbuilt RNFL thickness average analysis protocol. Retinal thickness was measured using the location of the vitreo-retinal interface and the retinal pigment epithelium defining the inner and outer boundaries of retina respectively. These are seen as sharp edges with high reflectivity. The boundaries of RNFL were defined by first determining the thickness of the neuro-sensory retina. The location of posterior boundary of RNFL was determined by evaluating each A-scan for a threshold value at 15dB greater than the filtered maximum reflectivity of the adjacent retina.
Various parameters were employed for evaluation of RNFL thickness. The important ones included RNFL average thickness over the entire cylindrical section and average RNFL thickness in each quadrant (superior, nasal, temporal and inferior). Eyes that fulfilled both exclusion and inclusion criteria were selected for analysis, if both eyes fulfilled the criteria only the eye with better image quality and higher Signal to Noise Ratio (SNR) was used for analysis.
The analysis was performed using SAS commercial statistical software package (SAS institute, Inc, Cary, NC). Unpaired 't' test was used to evaluate gender difference. One-way ANOVA was used to compare the different parameters amongst different age groups with post hoc test whenever applicable. Associations between age, axial length, refractive error and OCT parameters were evaluated by Pearson's coefficient of correlation. A 'P' value - 0.05 was considered statistically significant.
One hundred and forty six eyes of 146 patients were included in the study. The mean age was 44.55±16.14 years (range 20-70). There were 84 males and 62 females. The various RNFL thickness parameters measured during the study are presented in [Table - 1]. The average RNFL thickness in our sample population was 104.27±8.51µ (range 79.03-140.53 µ). The superior quadrant had an average thickness of 131.09±14.31µ (range 85-171µ), inferior quadrant 132.34±14.70µ (range 90-180µ), temporal quadrant 67.10±12.77µ (range 35-145µ) and nasal quadrant 85.93±17.85µ (range 44-150µ). The difference between inferior and superior quadrants was not statistically significant (p=0.48). The average RNFL thickness had a normal distribution in the sample population [Figure 2].
The average RNFL thickness, average superior, nasal, inferior and temporal RNFL thickness showed a trend towards decrease with the advancing age. The age had significant negative correlation (Pearson's correlation coefficient) with average RNFL thickness (r=-0.321, P=0.000) and with average superior (r=-0.233, P=0.005) and average inferior RNFL thickness (r=-0.234, P=0.004). There was no significant correlation between age and average nasal or temporal RNFL thickness. The one-way analysis of variance (ANOVA) was used to compare the various parameters, amongst the various age groups (A=< 30 Years, B=31-50 Years, C=>51 years). The results are given in [Table - 2]. The differences in average RNFL thickness, average inferior and average superior RNFL thickness and maximum inferior quadrant thickness (IMAX) were significant. These parameters (Average RNFL, Inferior average, Superior average, IMAX) showed no significant difference amongst the group A and group B, but the difference was significant when group A and group C were compared. Most of the other parameters showed no significant differences amongst the three age groups.
Refractive error and axial length did not show significant correlation with any of the measured RNFL parameters. The various parameters obtained were compared between males and females but were not statistically different [Table - 3].
Retinal nerve fibre layer damage invariably occurs in glaucoma.  Various investigational modalities like, retinal nerve fibre layer analyser (NFA), scanning laser ophthalmoscope (GDx, and GDx with variable corneal compensation), and OCT are used to measure the RNFL changes.  OCT is a non-invasive, non-contact modality that can be used for measurement of peripapillary RNFL thickness. It is found to correlate with RNFL as measured with scanning laser ophthalmoscope (SLO) and the Heidelberg retinal tomography (HRT). OCT measured RNFL thickness is not affected by the corneal and lenticular birefringence, as is the case with confocal scanning laser polarimetry. No additional reference plane is required to calculate the RNFL thickness because OCT provides an absolute cross-sectional measurement of retina, from which RNFL thickness is calculated. Additionally, the measurement is unaffected by the refractive status and axial length of the eye.
A high level of correlation between OCT generated RNFL thickness and visual function has been reported in previous studies. The RNFL may show a racial variation and the various values may be specific to the population under study. The detection of RNFL loss also varies in accordance with the imaging technology used, and the normative RNFL data of the concerned population. RNFL thickness parameters are already studied in the western population.  To the best of our knowledge, this is the first report of RNFL thickness measurements with OCT in normal Indian eyes.
The mean RNFL thickness in our sample population was 104.27 ± 8.5 microns, and it is comparable to the RNFL thickness reported in the Chinese population. A summary of some of the previous reports on normal RNFL thickness parameters is presented in [Table - 4]. It shows a higher value of RNFL thickness in most of the studies in Caucasians (except those 1 reported by Bowd and Mistelberger when compared to Chinese eyes. Such a discrepancy has not been addressed earlier but might be related to the ethnicity of study group, or to the OCT model, and the analysis protocol used.
Previous studies have similarly shown that RFNL thickness decreases with advancing age. We also observed this in our study. The decrease in average inferior RNFL thickness with advancement of age was more as compared to decrease in average superior RNFL thickness; this is also seen as increase in the S Max/ I Max ratio with age [Table - 2]. However, the one-way analysis of variance amongst the three age groups failed to show statistically significant difference in other variables. Further study with a larger sample size with more number of individuals in each age group may highlight any possible difference.
On quadrant-wise analysis of the RNFL thickness, we observed that the RNFL was thickest in the inferior (132.34±14.70µ) and superior (131.09±14.13µ) quadrants. The thickness was lesser in nasal (85.93±17.89µ) and temporal (67.1±12.77µ) quadrants. This corresponds to the double hump pattern of RNFL as is previously described. The difference between inferior and superior quadrants was not statistically significant suggesting that the ISNT rule does not the apply to Indian eyes. Kanamori et al in their study of 160 normal eyes showed slightly higher values than ours. They found that superior thickness (145.5± 19.6µ), was maximum followed by inferior RNFL thickness (143.1±19.5µ), temporal (98.7±20.8µ) and lastly in nasal quadrant (92.6±20.4µ). Their observation also did not follow the previously described ISNT rule. Bowd et al (30 eyes) found lower values as compared to our study. They noted a highest inferior (107.6µ) followed by superior (105.7µ) quadrant RNFL thickness; the temporal (66.2µ) quadrant had a higher thickness as compared to the nasal quadrant (61.8µ). These variations in the quadrantic RNFL thickness can again be attributed to racial variation in the population studied.
We found I max/S max ratio close to one (1.05 ± 0.172), denoting a symmetrical RNFL superiorly and inferiorly. This again shows that ISNT rule is not followed. The ratios, S max/ T avg. (2.48±0.94) and I max / T avg. (2.48±0.45) were almost similar, though no comparative values of these ratios are available in literature on OCT generated RNFL thickness.
There was no effect of gender on the RNFL parameters measured in our study. A similar finding has been reported previously. Schuman et al showed nerve fibre layer of men were usually thinner than the females, but not statistically significant.
In conclusion, our study provides a normative database for the retinal nerve fibre layer thickness in normal Indian eyes by optical coherence tomography. This can serve as a useful guideline in diagnosis, management and research in glaucoma.
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Conflict of Interest:
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