**Purpose.**:
To analyze the intra-user reliability and inter-user reproducibility of assessment of corneal epithelial thickness by Fourier domain optical coherence tomography.

**Methods.**:
In this consecutive cross-sectional case series performed at a tertiary ocular care institution, 210 eyes of 210 subjects underwent anterior segment Fourier domain optical coherence tomography (FDOCT). A caliper tool software was used to measure the corneal thickness. For the reproducibility measures, the examination was done by 2 examiners (user 1, user 2) within 30 minutes of each other. For the reliability measure, the retest was done by user 1 on the next day, within 30 minutes of the previous test's time. The total corneal thickness, epithelial thickness, and corneal thickness excluding the epithelium were measured.

**Results.**:
The mean corneal thickness of the population measured by user 1 was 519.5 ± 31.1 μm, 58.6 ± 4.2 μm, and 460.95 ± 31.4 μm for total cornea, epithelium, and non-epithelial cornea, respectively. The difference in results between user 2 and user 1 was 0.8 ± 7.2 μm, 0.23 ± 3.3 μm, and 0.7 ± 8.2 μm for total, epithelium, and non-epithelial cornea, respectively, and the difference in results between the repeated series by user 1 was 0.49 ± 5.7 μm, −0.13 ± 2.7 μm, 0.61 ± 5.4 μm total, epithelium, and non-epithelial cornea, respectively (paired *t*-test, *P* > 0.05). Intraclass correlations ranged from 0.87 to 0.99, coefficients of repeatability from 4.5 to 14.11, and coefficient of variation from 2.3% to 11.1%.

**Conclusions.**:
Fourier domain anterior segment optical coherence tomography is reproducible and reliable for the measurement of epithelial thickness at vertex.

^{ 1,2 }The thickness of the corneal epithelium variably contributes to the optical power of the cornea.

^{ 2 }After excimer laser ablation surgeries, the corneal epithelium might influence tear film instability and may be associated with local irregularities of corneal topography.

^{ 3 }Epithelial hyperplasia after excimer ablation can cause an undesired refractive shift.

^{ 4–5 }Epithelial thinning can occur on continued usage of hydrogel contact lenses, due to overnight wear as in orthokeratology, or in ectatic disorders like keratoconus.

^{ 6–8 }Due to these multiple factors, accurate assessment of epithelial thickness is very important. In the past, Reinstein et al.

^{ 9 }measured the corneal epithelium profile in normal eyes using a very high-frequency digital ultrasound device in normal eyes. In another study done on rabbit eyes, researchers used a confocal microscopy through-focusing (CMTF) methodology.

^{ 10 }Furthermore, anterior segment optical coherence tomography (ASOCT) has been used in recent years to evaluate the corneal epithelial thickness. In a repeatability study based on the older technology of time domain optical coherence tomography, the authors found clinically reasonable outcomes.

^{ 11 }Their small study of 18 subjects (32 eyes) showed intraclass correlation (ICC) was 0.98 for cornea and 0.73 for epithelium, and the coefficients of repeatability were ±10.64 μm for cornea and ±6.53 μm for epithelium.

^{ 11 }Fourier domain (spectral domain)-based optical coherence tomography was initially used for posterior segment evaluation.

^{ 12–16 }However, recently the same has been applied into anterior segment applications.

^{ 17–23 }

^{ 24 }Another recent study demonstrated that Fourier domain OCT can be used to measure the thickness of the epithelium and Bowman's membrane.

^{ 25 }However, to the best of the authors' knowledge, there is no study in published literature that has evaluated repeatability (inter user and intra user) of Fourier domain OCT to measure epithelial thickness. The current study looks at the reliability and reproducibility of a commercially-available Fourier domain platform for assessment of corneal epithelial thickness, and compares it with non-epithelial and total corneal thickness.

^{ 26 }: The machine takes 27,000 A-scans per second. The axial resolution in tissues is 5.0 μm, transverse resolution is 15 μm, and A-scan depth is 2 mm. The optical source is a super-luminescent diode (SLD) with a scan beam wavelength of 840 nm. The exposure power at the cornea is <720 μW. The patient was positioned on the headrest. The infrared (IR) image of the cornea was seen directly on the examination screen. The patient was asked to look in the fixation light in the device. All scans were performed with the patient's eye wide open with his/her own effort. No topical anesthesia or lubricating drops were used. The instrument's anterior segment 5-line raster mode was used. It has a scan angle of 0 degrees, spacing of 0.25 mm between the lines, and a line length (scan length) of 3 mm. We centered the image capture on the corneal vertex. It was determined by observation on the dynamic output screen. The concentricity of the limbus and the capture annulus helped in determining alignment. In the image acquire mode, 5 lines for 5 scans were displayed on the screen on the real-time image of the cornea. Use of X–Y and defocus adjustments was done to align the central line with the corneal vertex. On satisfactory alignment of the central line and the vertex, the scan was captured. A repeat scan was taken if the first scan was not of satisfactory nature. The scan can be unsatisfactory when it is decentered due to the patient's eye movement, poor corneal apex reflection, or head tilt, or when poor corneal apex reflection undermines the quality of the image, preventing a good resolution and demarcation of corneal layers. There are no subjective guidelines of the same currently in peer-reviewed literature. However, the authors were stringent in terms of the scan selection, and in case even a minor tilt from was noticed, the scan was discarded and a repeat scan was done. Some figures are included to emphasize of the types of scan that were discarded from the study (see Fig. 2). It would be interesting to see future studies/guidelines or incorporation of software that does analysis of scan quality, as in devices like the Zywave aberrometer (Bausch and Lomb, Rochester, NY).

**Figure 1.**

**Figure 1.**

**Figure 2.**

**Figure 2.**

**Figure 3.**

**Figure 3.**

**Figure 4.**

**Figure 4.**

*t*-tests were used to analyze the difference of mean. Correlation coefficients and best fit linear equations were computed to assess the correlation between measures from retesting. For the repeatability measures, intraclass correlations, 95% limits of agreement (LOA), coefficient of reliability, and coefficient of variation were computed.

^{ 26 }They were computed as:

*Upper limit:*mean of difference plus 2 standard deviation of difference

*Lower limit:*mean of difference minus 2 standard deviation of difference

*Width of 95% LOA*: upper minus lower limit

**Table 1.**

**Table 1.**

Mean ± SD | 95% Confidence Interval | ||

Total corneal thickness | User 1 Measurement 1 | 519.5 ± 31.1 μm | 515.3 to 523.8 μm |

User 2 | 518.6 ± 32.5 μm | 514.2 to 523.1 μm | |

User 1 Measurement 2 | 519.0 ± 31.5 μm | 514.7 to 523.3 μm | |

Epithelial thickness | User 1 Measurement 1 | 58.6 ± 4.2 μm | 58.0 to 59.2 μm |

User 2 | 58.4 ± 5.3 μm | 57.6 to 59.1 μm | |

User 1 Measurement 2 | 58.7 ± 4.8 μm | 58.1 to 59.4 μm | |

Non-epithelial thickness | User 1 Measurement 1 | 460.95 ± 31.4 μm | 456.7 to 465.2 μm |

User 2 | 460.3 ± 31.2 μm | 456.0 to 464.6 μm | |

User 1 Measurement 2 | 460.2 ± 33.0 μm | 455.8 to 464.8 μm |

*t*-test,

*P*= 0.3). There was a correlation of

*r*= 0.78 (

*P*= 9.4 × 10

^{−45}). The best fit line was b = 0.98a + 0.835 (where b = user 2 epithelial thickness, a = user 1 measurement 1 epithelial thickness), with an R-squared of 0.6. The difference in the non-epithelial thickness was 0.7 ± 8.2 μm (paired

*t*-test,

*P*= 0.2). There was a correlation of

*r*= 0.96 (

*P*= 8.8 × 10

^{−128}). The equation for the best fit line was b = 1.02a − 10.02 (where b = user 2 non-epithelial thickness, a = user 1 measurement 1 non-epithelial thickness), with an R-squared of 0.94. The difference in the corneal thickness was 0.8 ± 7.2 μm (paired

*t*-test,

*P*= 0.08). There was a correlation of

*r*= 0.97 (

*P*= 6.4 × 10

^{−138}). The equation for the best fit line was b = 1.02a − 12.8 (where b = user 2 corneal thickness, a = user 1 measurement 1 corneal thickness), with an R-squared of 0.6.

*t*-test,

*P*= 0.3). There was a correlation of

*r*= 0.88 (

*P*< 0.001). The equation for the best fit line was b = 1.002a + 0.014 (where b = user 1 measurement 2 epithelial thickness, a = user 1 measurement 1 epithelial thickness), with an R-squared of 0.78. The difference in the non-epithelial thickness was 0.61 ± 5.4 μm (paired

*t*-test,

*P*= 0.1). There was a correlation of

*r*= 0.98 (

*P*< 0.001). The equation for the best fit line was b = 0.99a + 2.9 (where b = user 1 measurement 2 non-epithelial thickness, b = user 1 measurement 1 eye non-epithelial thickness), with an R-squared of 0.97. The difference in the corneal thickness was 0.49 ± 5.7 μm (paired

*t*-test,

*P*= 0.3). There was a correlation of

*r*= 0.98 (

*P*< 0.001). The equation for the best fit line was b = 0.997a + 0.87 (where b = user 1 measurement 2 corneal thickness, a = user 1 measurement 1 eye corneal thickness), with an R-squared of 0.97.

**Table 2.**

**Table 2.**

Comparison | Intraclass Correlation Coefficients | 95% Confidence Interval | P Value | |

Lower | Upper | |||

Epithelial thickness – User 1 and User 1 repeat test (reliability) | 0.93 | 0.91 | 0.95 | P < 0.001 |

Epithelial thickness – User 1 and User 2 (reproducibility) | 0.87 | 0.83 | 0.90 | P < 0.001 |

Non-epithelial thickness – User 1 and User 1 repeat test (reliability) | 0.99 | 0.99 | 0.99 | P < 0.001 |

Non-epithelial thickness – User 1 and User 2 repeat test (reproducibility) | 0.98 | 0.99 | 0.99 | P < 0.001 |

Total corneal thickness – User 1 and User 1 repeat test (reliability) | 0.99 | 0.98 | 0.99 | P < 0.001 |

Total corneal thickness – User 1 and User 1 repeat test (reproducibility) | 0.98 | 0.98 | 0.99 | P < 0.001 |

**Table 3.**

**Table 3.**

Mean of Difference (μ) | Standard Deviation of Difference (μ) | 95% Limits of Agreement | ||||

Upper Limit (μ) | Lower Limit (μ) | Width (μ) | ||||

Inter-user reproducibility | Epithelial | 0.23 | 3.3 | 6.83 | −6.37 | 13.2 |

Non-epithelial | 0.7 | 8.2 | 17.1 | −15.7 | 32.8 | |

Total | 0.8 | 7.2 | 15.2 | −13.6 | 28.8 | |

Intra-user reliability | Epithelial | 0.13 | 2.7 | 5.53 | −5.27 | 10.8 |

Non-epithelial | 0.6 | 5.4 | 11.4 | −10.2 | 21.6 | |

Total | 0.49 | 5.7 | 11.89 | −10.91 | 22.8 |

^{ 11,24 }However, previous to this study, the reliability and reproducibility of Fourier domain-based anterior segment optical coherence tomography systems for epithelial thickness measurement had not been established. The mean epithelial thickness noted in the present cases was similar to that noted by Tao, et al. (52.5 ± 2.4 μm) on FDOCT.

^{ 25 }Sin and Simpson on time domain ASOCT (52 ± 3 μm.

^{ 11 }and that by Reinstein, et al. on a very high-frequency digital ultrasound (53.4 ± 4.6 μm).

^{ 9 }

^{ 24 }Another study by Ishibazaw.

^{ 17 }showed the mean central Fourier domain ASOCT-based pachymetry to be 530 ± 33 μm. Therefore, racial- and instrument algorithm-based differences may occur in pachymetric analysis in general, and therefore, the same can apply to isolated epithelium measurement. Researchers should keep this in mind when comparing results across different platforms. Fair reliability and reproducibility values were seen for epithelial thickness, as well as for the mean non-epithelial thickness and overall thickness.

^{ 2 }values suggested a good data fit in a linear fashion, suggesting that both types of repeat measurements (by the same user and by a different user) produced a good concordance with the initial values. The values for reliability were slightly better compared to those achieved for reproducibility. However, on paired tests for mean differences, this did not show a significant difference in the mean of 2 different users.

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