This study aimed to evaluate the repeatability and agreement of CD measurements by an AS-OCT device, the Casia 2 and the Pentacam AXL Wave Scheimpflug camera in a group of healthy subjects with a minimal refractive error, and assess their correlation with age, CCT and gender. This was the first study to evaluate Casia 2 CD measurements repeatability, the AS-OCT device and the Scheimpflug camera system revealed high repeatability, with ICC values exceeding 0.9 in all the corneal zones. Previous reports have also demonstrated the high repeatability of the Pentacam CD measurements in normal eyes [9, 10, 30], in good agreement with our findings. When the repeatability of the two instruments was compared, the Pentacam consistently showed higher CoV values than the Casia 2 in all corneal zones, suggesting that the former provides lower repeatability compared to AS-OCT, with a larger difference towards the peripheral zones, as the Pentacam shows a sharper increase in CoV values in zones 3 and 4. Our results are in agreement with the ones provided by Ní Dhubhghaill et al., who warned against Pentacam CD measurements from the peripheral cornea, since they can be affected by scleral reflectivity and therefore demonstrate lower repeatability, especially if the corneal diameter is smaller than 12 mm [9].

Regarding agreement, the Bland–Altman plots showed a consistent bias through all the corneal zones, while, only, the 95% LoA of zone 1 were found to lie outside the range of ± 2 SD from the mean of the differences (± 2.6 SD for standardized values), indicating low agreement [29], moreover, it failed to show a statistically significant linear correlation. Despite the general goodcorrelation between measurements and narrow 95% LoA, CD measurements obtained from the two instruments exhibit differences in distribution and across the different corneal zones making their measurements not interchangeable. Specifically, CD values obtained with the Casia 2 were found to be higher in zone 1 compared to the other zones, while the Pentacam AXL Wave CD measurements were consistently higher in zone 4 and lower in zone 1, which is in line with previous literature findings in normal and myopic eyes (9, 22). These variations in CD measurements are likely attributable to the different technologies employed by the two instruments. The Pentacam AXL Wave utilizes a Scheimpflug camera system, which captures light backscattering with laterally positioned cameras while the emitting light surce is centered on the visual axis. The Casia 2, by contrast, registers reflected light along the same axis as the one of light emission, leading to enhanced measurements in the central zones where the light has a reflection angle close to zero and attenuated measurements in the peripheral zones. Notably, the central artefact line (Fig. 4) observed in the Casia 2 images represents the axis where the light strikes the corneal apex perpendicularly, resulting in a strong registration signal that may appear as an artefact. To address this issue, the Casia 2 software excludes the central 0.5 mm where this artefact line appears in the densitometry analysis.

Casia 2 B scan image showing the central artifact line

Our study aimed to analyze eyes with small refractive error to minimize the effect of highly ametropic eyes on CD [31]. Nevertheless, a recent study by Xu et al. [22] analyzed CD values between Pentacam and Casia 2 in myopic eyes including refractive errors higher than -6D. Compared to our study, their results revealed similar differences of distributions across the four corneal zones for both instruments and lower agreement in the central zone, albeit with no statistical significance. Notably, their study included subjects within the age range of 18–41 years. In contrast, our study encompassed participants aged 23–82 years. Consequently, their findings consistently revealed lower CD values compared to those collected in our study and can not be further compared.

Despite the lack of interchangeability of measurements between the devices due to their distinct imaging principles, both data sets demonstrated comparable trends and exhibited a significant positive linear relationship in most zones. Therefore, it can be said that Casia 2 can detect densitometry changes in healthy eyes with limited refractive error, comparably with the Pentacam, peripherally to the 2 mm from the corneal apex. Further investigation is warranted to determine whether the differences exhibited in zone 1 could affect the clinical value of densitometry measurements, and whether the exclusion of the central artifact line could potentially impact the agreement between the two instruments in the central corneal zone. While it is unlikely to significantly affect the reliability of CD measurements in healthy and uniform corneas, its potential impact on CD measurements in pathological corneas, such as those with keratitis, keratoconus, or Fuchs dystrophy, needs to be evaluated.

Our study revealed that CD was positively correlated with age using both instruments in all corneal zones, except for zone 1, where the Pentacam AXL Wave did not show a statistically significant correlation. This finding could be attributable to age-related alterations that occur in each corneal layer. These changes may include a decrease in keratocyte and endothelial cell density, impairment of the epithelial cell barrier function, alteration of collagen fibers, and the presence of cellular debris within the collagen lamellae [32]. However, as regards the relationship between central CD and age, conflicting results are to be found in the literature. Previous studies have reported no correlation between CD and age in central corneal zones measured with a Scheimpflug camera [4, 6, 8, 9], as well as with other methods [33,34,35]. In contrast, Karmiris et al. [36] reported a significant correlation between age and 0–2 mm CD in each corneal layer and throughout the total thickness using the Pentacam. However, the study is limited by the inclusion of both eyes of the same subject. Based on our study and the literature findings, it can be postulated that the Pentacam AXL Wave may lack the resolution required to detect subtle age-related changes in CD within the central corneal zone. The AS-OCT device may be more sensitive in this regard. On the other hand, there is a consensus in the literature regarding the effect of corneal aging in the more peripheral zones and this effect increases as one proceeds toward the limbus [7, 8]. This may account for the more pronounced involvement of the peripheral cornea in involutional changes such as arcus senilis and Vogt’s girdle [32].

In agreement with the literature, densitometry values did not show a correlation with CCT [8], nor was there any difference between genders [9].

Comparing two instruments with different mechanisms poses some inconveniences, especially when differences between subjects are less obvious, as it is in healthy subjects with clear corneas, the instrument’s capacity is put under a stress and small technical differences between protocols and environmental factors might play a role in defining repeated measurements. In our case, images were obtained in the same room and light conditions for each patient, and in random instrument order, to eliminate external influences.

This study has some limitations and further investigation is warranted, enrolling only healthy eyes with minimal refractive error prevents our results from being generalized to patients with any corneal disease or more pronounced refractive error. It was not possible to compare CDs from different corneal layers, since the Casia 2 software does not provide such information.

In conclusion, our data indicate high repeatability of CD measurements obtained by the Casia 2. However, due to the differing principles of image capturing, the absolute values were not interchangeable with those obtained by the Pentacam. Additionally, The Casia 2 revealed more sensibility to age-related changes in the central zone. Further studies are needed to confirm this finding and evaluate the potential clinical implication for understanding and managing corneal health and pathologies.