Three-Dimensional Imaging of Cleared Human Liver Tissue Reveals Extensive Spatial Heterogeneity of Fibrosis in Metabolic Dysfunction Associated-Steatotic Liver Disease

Abstract

Background: Precise assessment of liver fibrosis in patients is challenging. Liver biopsy is a well-established technique, however histopathological examination of such small samples is prone to staging inaccuracy, hence novel modalities are needed. We aimed to perform 3D imaging of optically transparent liver samples by light-sheet microscopy (LSM) to quantify extracellular matrix (ECM) proteins. Methods and Results: Fifty-seven MASLD, thirty-eight chronic hepatitis and twelve healthy subjects were included. Liver tissues were cleared with a CLARITY method. 3D imaging of ECM was performed via the newly developed LSM. Collagen Proportionate Volume (CPV) and Elastin Proportionate Volume (EPV) values were calculated by analysis of over 200 sections per sample through morphometry. We have optimised a method which achieves transparency of liver tissues in advanced fibrotic stages of MASLD and optimized non-destructive slide-free fibrosis pathology of whole fresh and FFPE liver biopsy samples. Cut-off values for CPV and EPV were determined for fibrotic stages. CPV and EPV analysis showed a considerable optical section heterogeneity resulting in a fibrosis stage of variance within the sample. Volumetric image analysis for fibrosis staging revealed that only 44% and 47% of optical sections would be staged the same for F3 and F4, respectively. Interpretation: For the first time, we have developed a method of analysing 3D digital pathology of liver fibrosis using in-house LSM. Volumetric imaging of whole liver biopsy samples showed that fibrosis heterogeneity occurs even in different sections.