Wall-to-Lumen Ratio Estimation in Adaptive Optics Retinal Imaging: Evaluation and Comparison.

Purpose: Retinal arteriolar changes reflect systemic microvascular alterations associated with cardiovascular risk, including hypertension. The retinal arteriolar wall-to-lumen ratio (WLR), obtained by adaptive optics (AO) imaging, is a promising noninvasive biomarker of early vascular damage. Its clinical utility is limited by inter- and intra-grader variability, lack of standardized protocols, and dependence on image quality, boundary definition, and measurement method. Standardized, objective WLR quantification is therefore needed to minimize observer bias and improve reproducibility. Methods: We compared point-based and region-based manual WLR estimation using 60 high-resolution AO images from 35 healthy subjects. Two experienced graders performed five point-based measurements per image, yielding one median WLR, followed by manual artery wall and lumen segmentation, yielding one WLR value. Inter-grader variability, method agreement, and sampling-location requirements were evaluated using statistical and Bland-Altman analyses. Results: Point-based assessment showed good inter-grader agreement, with a relative mean difference of 4.5% (Wilcoxon test, P < 0.05). Region-based assessment produced significantly lower WLR values than the point-based method (P < 0.01), with relative mean differences of 19.9% and 26.1% for graders A and B, respectively. Inter-grader relative mean difference was 6.2%, comparable to the point-based method. Approximately 10 measurement positions reduced the estimation error to about 5%; fewer positions increased error. Image quality and vessel-border visibility affected WLR variability. Conclusions: WLR estimation is influenced by measurement approach, grader experience, image quality, and sampling density. Although region-based assessment is more comprehensive, it is time-consuming. For clinical point-based assessment, at least 10 clearly visible arterial locations are recommended to minimize estimation error. Standardized protocols are essential for reliable WLR quantification. Translational Relevance: For standardized manual WLR assessment in AO retinal images, we recommend measuring only clearly visible vessel segments, using double-sided measurements whenever possible, avoiding bifurcations, crossings, and adjacent vessels, obtaining ≥12 measurements per artery, excluding the upper and lower deciles, and reporting the median of ≥10 remaining measurements as the final WLR.