Automated biphasic morphological assessment of hepatitis B-related liver fibrosis using second harmonic generation microscopy

Liver fibrosis assessment by biopsy and conventional staining scores is based on histopathological criteria. Variations in sample preparation and the use of semi-quantitative histopathological methods commonly result in discrepancies between medical centers. Thus, minor changes in liver fibrosis might be overlooked in multi-center clinical trials, leading to statistically non-significant data. Here, we developed a computer-assisted, fully automated, staining-free method for hepatitis B-related liver fibrosis assessment. In total, 175 liver biopsies were divided into training (n = 105) and verification (n = 70) cohorts. Collagen was observed using second harmonic generation (SHG) microscopy without prior staining, and hepatocyte morphology was recorded using two-photon excitation fluorescence (TPEF) microscopy. The training cohort was utilized to establish a quantification algorithm. Eleven of 19 computer-recognizable SHG/TPEF microscopic morphological features were significantly correlated with the ISHAK fibrosis stages (P < 0.001). A biphasic scoring method was applied, combining support vector machine and multivariate generalized linear models to assess the early and late stages of fibrosis, respectively, based on these parameters. The verification cohort was used to verify the scoring method, and the area under the receiver operating characteristic curve was >0.82 for liver cirrhosis detection. Since no subjective gradings are needed, interobserver discrepancies could be avoided using this fully automated method.