This was a prospective study conducted on healthy volunteers. A detailed ophthalmic history was obtained, and a complete eye examination was performed, including best corrected visual acuity assessment, intraocular pressure measurement by Goldmann applanation tonometry, slit lamp biomicroscopy, and fundus examination. The exclusion criteria included a refractive error greater than or equal to -3 and +3 diopters (D), a best corrected visual acuity lower than 20/20 (Snellen), clinical evidence or ocular history of any corneal disease, dry eye condition, ocular surface or anterior segment disease, previous history of corneal trauma, ocular surgery, and contact lens use during the last month prior to the examination. One eye of each subject was randomly selected for the analysis. The study was performed in accordance with the ethical standards of the Declaration of Helsinki and approved by the hospital ethics committee (protocol number 0002793) of the IRCCS Azienda Ospedaliero-Universitaria of Bologna, Policlinico Sant’Orsola, Italy. Patients were informed about the potential risks and benefits of the procedure, and written informed consent was obtained from all participants.
Instruments
The Casia2 (software version: 50.6A.02) is a swept-source anterior segment optical coherence tomography (AS-OCT) device that uses a 1310 nm wavelength laser source with a scan rate of 50,000 A-scans per second and a scan depth of 13 mm to obtain images with an axial and transverse resolution of 10 and 30 µm, respectively. The radial scan reaches a diameter of 16 mm, while the raster is a 12x12 mm square. To measure CD, the “corneal map” scan type was performed, it is a radial scan method with a scan range of 16 mm, in total, 16 different radial scans are performed.
The Pentacam AXL Wave (software version: 1.27r12) is a high-resolution (1.45 megapixel) rotating Scheimpflug camera system for the analysis of anterior segment parameters. It takes up to 100 images in two seconds using a 420 nm wavelength blue LED light source. To measure CD, the 3D Scan protocol with the “25 pictures” option was performed. This method obtains 25 standard quality pictures of the anterior chamber.
Procedures
Instruments calibration was carried out before the beginning the measurement session. All patients underwent examination using the two devices on the same day, in a darkened room, in random order, and without pupil dilation. No eye drops were instilled before the examination and subjects were asked to blink before each image acquisition. The automatic acquisition modality was selected in both instruments to decrease the human effect on the measurements. The same operator successfully acquired three repeated measurements with each instrument to assess repeatability. After each acquisition, volunteers were asked to move away from the machine and placed back in position for a new measurement after a few seconds. With both instruments, scans were repeated when an alert signal revealed an insufficient measurement quality.
The process of obtaining measurements did not encounter difficulties, all the subjects were able to undergone three consecutive procedures with both instruments without experiencing discomfort or the need to take prolonged pauses. The Casia 2 was faster with a 0.6 second time per exam against 1 second for the Pentacam AXL Wave.
Measured parameters
Four concentric zones were analyzed: the first was a round area measuring 2 mm in diameter (zone 1) and centered on the corneal apex, the other 3 zones were ring-shaped with a diameter ranging from 2 to 6 mm (zone 2), from 6 to 10 mm (zone 3), and from 10 to 12 mm (zone 4). The total corneal densitometry (TCD), referring to the measurement area comprised between 0 and 12 mm, was also evaluated. These topographical zones are set as default in the Pentacam software, whereas they need to be individually set in the Casia 2 software by clicking on “Settings” on the “DENSITOMETRY” page (Fig. 1). The 0.5-4 mm zone defined as “Pupil Area”, which is pre-set on the Casia 2, was used only for the evaluation of repeatability.
The output was expressed in gray scale units (GSUs) calibrated by each instrument’s software: in the case of the Pentacam AXL Wave, measurements were expressed as percent values ranged from 0% (minimum transparency) to 100% (maximum transparency), whilst for the Casia 2 they ranged from 0 GSU(minimum transparency) to 256 GSU(maximum transparency). Each measurement represents the CD measured from the epithelium to the endothelium. Densitometry values from each corneal zone are the mean of all the measurements of the respective zone.
Statistical analysis
The statistical analyses were performed using SPSS (Version 25, IBM, United States). The sample size for the repeatability analysis was calculated to yield a minimum of 10% confidence in the estimate. According to McAlinden et al., at least 96 eyes should be enrolled for this purpose (23).
For the assessment of repeatability, as defined by the International Organization for Standardization, the following parameters were calculated (24):
1. Intra-session test-retest variability, calculated by multiplying the pooled within-subject SD (sw) by 2.77. Based on repeatability, the difference between two measurements for the same subject should be less than 2.77 sw for 95% of pairs of observations (25).
2. CoV: calculated as the sw divided by the mean of the measurements and expressed as a percentage.
3. Intra-class correlation coefficient (ICC). This is defined as the ratio of the between-subjects variance to the sum of the pooled within-subject variance and the between-subjects variance (26,27). It was automatically calculated using SPSS software with the 2-way mixed model and absolute agreement. The range varies from 0 to 1, therefore, a value of 1.0 means no variance between repeated measurements, an ICC of 0.90 or more means high agreement, an ICC from 0.75 to less than 0.90 means moderate agreement, and an ICC of less than 0.75 means poor agreement (28).
To ensure comparability between the CD measurements of the two instruments, which are expressed in different scales (from 0 to 256 for Casia 2 and from 0 to 100 for Pentacam AXL Wave), Casia 2 densitometry results were converted to percentage, calculated as the densitometry results divided by the maximum value of densitometry readings for each instrument, multiplied by 100%. To detect differences in groups means or medians the t-test for paired samples was used for normally distributed variables; the non-parametric Wilcoxon signed-rank test was used for the variables that were not normally distributed. Pearson correlation analyses were used to quantify the linear correlation between the two sets of values obtained from the two devices. A p-value less than 0.05 was considered statistically significant.
To assess agreement, Z-score standardization was applied to all data. To apply Z-score standardization, the mean of the correspondent sample is subtracted from each measurement (X) and then divided by the standard deviation of the sample according to the formula:
The resulting Z score distribution for each group of measurements has a mean of 0 and a standard deviation of 1. Each measurement is then expressed as the number of standard deviations from the mean of its sample.
Only the first measurement of each eye with both devices was used to evaluate agreement. This was assessed, according to Bland and Altman, by plotting the differences between measurements (y-axis) against their mean (x-axis) on the Z-scores (29). The 95% limits of agreement (LoAs) were defined as the mean ±1.96 standard deviation (SD) of the differences between the measurements of the two devices.