Discussion

The accumulation of TGs within hepatocytes is characteristic of HS.35 Raman spectroscopy has been validated to assess liver steatosis in animal models based on characteristic Raman peaks.20,21 While the calibration of the investigated Raman system was in keeping with previous reports,26the investigated Raman system demonstrated substantial positive correlations with both Raman (r = 0.82) and reflectance (r = 0.64) channels, indicating its capability to assess TG in liver specimens. This made it suitable for further evaluation in the histopathological validation stage. In the histopathological validation stage, the Raman channel was revealed to be able to classify MaS and global HS of human liver specimens. Combining information from the two channels, dual-variable predictions of 66 liver specimens reported good (AUROC = 0.80) classification of an ≥10% steatosis discrepancy between pathologist-estimated MaS and the algorithm-estimated HS (ORO-stained pixel area). These findings indicated that the investigated ambient light-compatible Raman system was capable of accurate ex-situ evaluation of global HS in human livers. The investigated Raman system has demonstrated preliminary efficacy in MaS assessments, indicating its potential as a valuable tool for the in-situ real-time surgical evaluation of donor liver steatosis. The primary strength of the present study lies in the multifaceted correlation of Raman assessments with a robust array of subjective and objective references. This included TG quantification, expert pathologist evaluation, and advanced the algorithm-based estimation, achieving validations from diverse and complementary perspectives. Moreover, the observed steatosis within the specimens in the histopathological validation stage exhibited a broad distribution of minimum-to-severe steatosis, facilitating a comprehensive evaluation of the HS classifications through the investigated Raman system. This wide-ranging analysis underscores the potential of our approach in advancing the understanding of integrated spectroscopic evaluation on HS and may pave the way for surgical diagnostic techniques. All specimens included in this study were initially snap-frozen. Although the specimens could not represent donor livers for recovery, their chemical compositions (to which Raman spectroscopy is sensitive) and morphology were to a great extent comparable to those of donor liver organs. All the specimens studied in the pathological validation stage were several times thicker than the 1-mm laser penetration depth reported in the previous clinical trial,26 enabling enough scattering interaction as a shone liver organ would provide. This study encountered several limitations in evaluating the Raman system for HS assessment. A significant hurdle was the mixed presence of MaS and MiS in most specimens, complicating the distinction between MaS and global HS. During the biochemical validation stage, the exclusion of 9 out of 25 specimens due to their size and weight markedly diminished the statistical power, and the reliance on duck fat-agar phantoms for setting up a “zero point” by calibration hampered the system’s accuracy and convenience. The research noted a promising dual-variable prediction based on Raman scattering and reflectance, capable of classifying specimens with a minimum 10% greater global HS than MaS. However, this finding, lacking a solid physicochemical basis, necessitates further exploration. van Staveren et al. (1991) accurately predicted the scattering coefficient of fat emulsion lntralipid-10% using Mie theory and the size distribution of lipid particles.36 A reasonable guess is that the reflectance channel (Mie scattering) contained information about both fat content and lipid droplet size; however, more studies according to the physical principles of Raman and Mie scattering, e.g., Monte Carlo simulations, are needed to probe the concept of the dual-variable prediction. Additionally, the untested performance of the Raman system in operating room conditions, despite its proven resistance to 10,000 lm LED light in laboratory settings, leaves a significant gap in understanding its comprehensive compatibility.26In this study, a single expert pathologist (AES) assessed all microscopic slide images and estimated the percentage of large droplet MaS and global HS. Despite the potential for diverse insights from multiple pathologists,37,38 we opted for a single expert to ensure consistent assessment methods and mitigate the risk of interobserver discordance. This approach also assured the quality of the assessment, leveraging the pathologist’s high expertise. The Positive Pixel Count algorithm, as an additional independent approach to quantify the fat content, validated the pathologist’s estimations and enabled finer quantitive discrepancy of global HS and MaS. To the best of the authors’ knowledge, the present study represents the inaugural trial of conventional Raman spectroscopy on human liver specimens for HS assessment. Advancing beyond the scope of preceding Raman studies, this research emphasized an ambient light-compatible methodology. The efficacy of the Raman spectroscopic approach in assessing the HS of human liver specimens was validated by correlating the fat content results provided by the examined Raman system with TG quantifications. Additionally, histopathological validation was conducted across a wide range of steatotic human liver specimens, culminating in the development of a dual-variable prediction for significant discrepancies (>10%) between global HS and MaS. The insights gained from this study may contribute to enhancing other spectroscopic surgical instruments.