Optical Coherence Tomography Technique

Both eyes of each subject were scanned on each of 4 different OCT instruments in random order. For each subject, all 4 scans were completed on the same day, within a 2-hour period, by a single experienced ophthalmic technician. All 4 scans were repeated in a similar fashion on each eye 2 to 8 weeks later by the same technician in random order. Timing of subject reexamination was dependent on convenience for each subject.

For Stratus OCT (software version 4.0.2), the Fast RNFL mode was used for analysis. This protocol consists of 3 consecutive circumpapillary scans of 3.4 mm in diameter. Each circle scan is composed of 256 A-scans acquired consecutively. The computer algorithm automatically delineates the anterior and posterior margins of the RNFL and thickness is calculated by assessing 768 data points along the borders of RNFL. Signal intensity is listed on a scale from 1 to 10. Only scans with signal strength of ≥6 out of a possible 10 were included for analysis.

The Cirrus OCT (software version 4.0.0.29) scans were obtained using the Optic Disc Cube 200 × 200 protocol. Under this protocol, a 3D cube of data is generated over a 6-mm-square grid of 200 horizontal scan lines, each composed of 200 A-scans. A cirrus software algorithm automatically detects the center of the optic disc and positions a 3.46-mm-diameter calculation circle over this point. From the 256 A-scans along this circle, the borders of the RNFL are delineated and thickness is calculated at each point along the circle. All scans were reviewed to ensure signal strength of ≥6 for inclusion in analysis.

For RTVue OCT (software version 4.0.5.39), the NHM4 and 3D Disc protocols were used for analysis. Using the 3D Disc protocol, a 4 × 4-mm raster scan is acquired of 101 B-scans, each composed of 512 A-scans. The resulting en-face image is then used to manually draw a contour line along the disc margin. The NHM4 protocol is composed of 12 radial scans 3.4 mm in length and composed of 452 A-scans each. Using the previously defined contour line, 6 concentric ring scans are taken ranging from 2.5 to 4.0 mm in diameter, centered on the optic disc. This generates 9510 A-scans in approximately 0.39 seconds. The ring scan with diameter of 3.4 mm was used for comparison analysis. All scans were reviewed to ensure a signal strength indicator (SSI) of ≥40 out of 100.

The Spectralis OCT (software version 4.0) uses dual-beam simultaneous imaging with SD-OCT and confocal scanning laser ophthalmoscope to generate an en-face image on screen. The operator then manually centers a 3.4-mm-diameter circle on the optic disc. The eye-tracking system was activated for all scans including repeat scans, and the AutoRescan feature was implemented, allowing accurate placement of the scan circle in the same position each time. Fifteen images were then acquired at the scan circle under high-resolution settings (1536 A-scans) and averaged automatically by the software. RNFL boundaries were automatically delineated according to software algorithms underneath the circumpapillary circle. All scans were reviewed to ensure a signal strength >15 dB.

For each instrument, the subject was seated and properly aligned in typical fashion as is done in everyday practice. If the subject blinked or moved, resulting in an unreliable scan, the test was repeated. Any scans of poor quality were discarded. All scans were reviewed at study completion to evaluate for accurate computer detection of inner and outer boundaries of RNFL. If a scan possessed an error in segmentation, the information was not included in the statistical analyses.

Statistical Analysis

All statistical analyses were performed with STATA software (StataCorp, College Station, Texas, USA). The average and 4-quadrant RNFL thickness measurements from each SD-OCT device were compared with those of Stratus by use of Student paired t tests. For the comparison of RNFL thicknesses between machines, only the RNFL thicknesses from the first examination were used for each eye. Since in certain patients both eyes were used in the analysis, calculated standard errors were clustered at the patient level to account for the possibility that observations from the right and left eye of the same patient may not be independent. Scatter plots were generated for each SD-OCT RNFL thickness against Stratus RNFL thickness. Pearson correlation coefficients were also calculated from these plots. Bland-Altman plots were generated to assess agreement of measurements between each SD-OCT and Stratus. In these graphs, the differences in thickness between instruments were plotted against the mean RNFL measurement.

Mean intervisit differences were calculated for each subject on all machines for average and quadrant RNFL thickness measurements. The differences were compared with the Student paired t test. Intervisit reproducibility values were calculated using the following formula: 2.77 × the intervisit within-eye standard deviation (Sw). The coefficient of variation was also calculated for all measurements (100 × Sw/Overall mean). The intraclass correlation coefficient (ICC) was calculated as a ratio of between-eye variance to total variance (between-eye variance + within-eye variance). The within-eye variance of average and quadrant RNFL thickness was compared between each SD-OCT and Stratus by using the Student paired t test on the log-transformed variance (log Sw ² ). The difference in log-transformed within-eye variance between each machine will then be positive if the SD-OCT variance is greater than Stratus and negative if the variance is less. Bland-Altman plots were used to assess the relationship between the within-eye variance and RNFL thickness. P < .05 was considered to be statistically significant.

Agreement with Stratus

Seventy-nine of 80 eyes (98.8%) of 40 subjects qualified for inclusion in the study. Of these 40 subjects, 16 (40%) were male and 24 (60%) female. The mean (± standard deviation) age of subjects was 37.1 ± 11.0 years (range 21 to 61 years). Mean best-corrected Snellen visual acuity was 20/20, with spherical equivalent of −0.8 ± 1.7 diopters. Mean IOP was 15.6 ± 2.3 mm Hg, and mean cup-to-disc ratio 0.3 ± 0.1. From visual field analysis, mean pattern standard deviation was 1.62 ± 0.47 decibels (dB), while mean deviation was 0.18 ± 1.25 dB. Among the 80 eyes undergoing baseline examination, 1 eye was excluded because of history of unilateral trauma with question of angle recession. Of the 79 qualifying eyes, 8 (10.1%) were withdrawn from the analysis of agreement between machines. Seven of the 8 were withdrawn because of poor scan quality and/or failure of the software to accurately delineate the RNFL/ILM boundaries, and 1 was attributable to technician failure to scan on all 4 machines. For the reproducibility analysis, an additional 4 eyes (total of 12/79; 15.2%) were withdrawn because of poor scan quality of the repeat scans. The majority of poor-quality scans withdrawn were produced by Spectralis (10/11; 91%), while only one was from Cirrus.

The mean average RNFL thickness was 110.1 ± 12.8 μm by Stratus, 106.6 ± 12.8 μm by Spectralis, 98.7 ± 10.9 μm by Cirrus, and 112.8 ± 13.2 μm by RTVue ( Table 1 ). Both Spectralis and Cirrus measured average RNFL thicknesses significantly lower than Stratus; however, the difference was more pronounced for Cirrus. RTVue average RNFL measurements were significantly higher than Stratus. The mean difference between each SD-OCT instrument and Stratus was −3.36 μm for Spectralis, −11.33 μm for Cirrus, and +2.81 μm for RTVue. The association of average RNFL thickness measurements from each SD-OCT to Stratus is demonstrated by the scatter plot in Figure 1 . Bland-Altman plots in Figure 2 demonstrate the relationship of average RNFL thickness measurements taken by each SD-OCT and Stratus. The difference in average RNFL thickness between each SD-OCT and Stratus is plotted against the mean RNFL measurement of both instruments. The difference between Cirrus and Stratus was noted to have an increasing trend with higher average RNFL measurements. Comparisons of Stratus to RTVue and to Spectralis showed a fairly consistent discrepancy in measurements at all thicknesses. Pearson correlation coefficients listed in Table 1 were found to be quite high between all 3 SD-OCT machines and Stratus.

Scatter plot of average retinal nerve fiber layer (RNFL) thickness measurements from all subjects. The average RNFL thickness measured by each spectral-domain optical coherence tomography (SD-OCT) instrument is plotted against the average RNFL thickness measured by Stratus. The solid lines represent linear regression lines for each SD-OCT instrument along with coefficient of determination (R ² ) values.

Bland-Altman plots demonstrating agreement between average retinal nerve fiber layer (RNFL) thickness measurements (in μm) between each spectral-domain optical coherence tomography (SD-OCT) instrument and Stratus. The SD-OCT represented in each plot is (Top) Cirrus, (Middle) Spectralis, and (Bottom) RTVue. All measurements are in μm. The linear regression line demonstrates any change in agreement with different RNFL thicknesses.

Significant differences in quadrant RNFL thickness were also found between each SD-OCT instrument and Stratus. The mean quadrant RNFL thickness measurements by each instrument and Pearson correlation coefficients to Stratus are recorded in Table 1 . Bland-Altman plots were produced to demonstrate the relationship of quadrant RNFL thickness measurements taken by each SD-OCT and Stratus (not shown because of space constraints). The difference in quadrant RNFL thickness between each SD-OCT and Stratus is plotted against the mean quadrant RNFL measurement of both instruments. For the superior and inferior quadrants, the difference between each SD-OCT and Stratus was consistent across all thicknesses. For the nasal quadrant, Cirrus and Spectralis measurements were consistently lower than Stratus, and the difference between machines became wider at thicker mean RNFL values. RTVue measurements in the nasal quadrant were greater than Stratus at thicker mean RNFL values and lower than Stratus at thinner mean RNFL values. In the temporal quadrant, RTVue and Spectralis measurements were consistently higher than Stratus, and the difference between machines became wider at thicker mean RNFL values. Cirrus measurements in the temporal quadrant were consistently lower than Stratus, but the difference between machines decreased as mean RNFL thickness increased.

Reproducibility

Repeat scans for each eye were performed a mean of 21 ± 9 days after the initial scan. In addition to Stratus, all 3 SD-OCT instruments demonstrated a very low intervisit difference in RNFL thickness. The within-eye differences in average and quadrant RNFL thicknesses for each instrument are shown in Table 2 . All 4 machines demonstrated excellent reproducibility for both average (0.90–0.97) and quadrant (0.70–0.96) RNFL measurements according to ICC depicted in Table 2 . ICC for average RNFL measurements was higher than quadrant RNFL ICCs for every instrument except Spectralis, where the nasal quadrant was highest (0.95 vs 0.90). Coefficients of variation (CoV) were exceptionally low for all machines as well (2.09–9.28). RTVue again demonstrated the lowest CoV for average RNFL (2.09), followed by Stratus (2.86), Cirrus (3.03), and Spectralis (3.91). The inferior quadrant CoV was lowest among the 4 quadrants for all machines tested.

TABLE 2

Intervisit Reproducibility of Mean Retinal Nerve Fiber Layer Thickness Measurements in Normal Patients Using Time-Domain (Stratus) and Spectral-Domain (Cirrus, Spectralis, RTVue) Optical Coherence Tomography

	Mean Intervisit Difference	95% CI	P Value	Reproducibility	95% CI	CoV	95% CI	ICC	95% CI
Stratus
Average	0.37	−0.79 to 1.53	.536	8.83	7.21 to 10.45	2.86	2.17 to 3.42	0.94	0.91 to 0.97
Temporal	−0.27	−2.24 to 1.71	.791	15.96	11.44 to 20.47	7.86	4.35 to 10.23	0.85	0.76 to 0.95
Superior	−0.01	−2.34 to 2.31	.990	18.05	15.44 to 20.65	4.89	3.84 to 5.75	0.87	0.82 to 0.92
Nasal	1.09	−1.63 to 3.81	.437	23.73	19.31 to 28.15	9.28	7.38 to 10.85	0.78	0.72 to 0.84
Inferior	0.70	−1.73 to 3.14	.576	18.31	12.67 to 23.95	4.52	2.62 to 5.82	0.89	0.82 to 0.95
Cirrus
Average	0.19	−1.14 to 1.53	.777	8.89	5.75 to 12.02	3.03	1.47 to 4.03	0.92	0.87 to 0.97
Temporal	0.33	−1.18 to 1.83	.672	11.91	7.33 to 16.48	5.79	2.82 to 7.69	0.87	0.77 to 0.96
Superior	0.75	−3.15 to 4.64	.710	24.77	11.55 to 37.99	7.10	0.00 a − 10.74	0.74	0.55 to 0.93
Nasal	−1.61	−3.92 to 0.69	.178	17.29	12.84 to 21.73	7.78	5.24 to 9.68	0.70	0.58 to 0.83
Inferior	0.33	−2.29 to 2.94	.807	18.63	14.37 to 22.88	5.02	3.57 to 6.13	0.88	0.82 to 0.94
Spectralis
Average	−0.01	−1.36 to 1.33	.983	11.72	9.31 to 14.13	3.91	2.81 to 4.77	0.90	0.85 to 0.95
Temporal	−0.87	−3.53 to 1.80	.528	21.28	16.50 to 26.05	8.79	6.42 to 10.46	0.74	0.65 to 0.82
Superior	−0.43	−2.07 to 1.20	.607	14.14	10.44 to 17.84	3.97	1.91 to 5.28	0.93	0.89 to 0.97
Nasal	0.45	−0.59 to 1.48	.401	8.39	6.86 to 9.91	3.90	2.98 to 4.64	0.95	0.93 to 0.97
Inferior	0.36	−1.37 to 2.09	.687	14.68	9.80 to 19.57	3.68	1.85 − 4.86	0.93	0.88 to 0.98
RTVue
Average	−0.56	−1.42 to 0.30	.209	6.59	5.59 to 7.59	2.09	1.71 to 2.41	0.97	0.96 to 0.98
Temporal	2.84	0.20 to 5.47	.041	18.96	13.70 to 24.22	7.56	4.79 to 9.57	0.89	0.82 to 0.96
Superior	−1.24	−2.83 to 0.35	.134	13.09	11.14 to 15.05	3.51	2.79 to 4.11	0.93	0.91 to 0.96
Nasal	−0.16	−1.98 to 1.66	.861	15.31	12.24 to 18.38	6.04	4.51 to 7.25	0.86	0.80 to 0.93
Inferior	−0.60	−2.03 to 0.83	.418	12.02	10.00 to 14.03	2.99	2.31 to 3.54	0.96	0.94 to 0.98

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