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

Abstract

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.

Dual‐photon microscopy‐based quantitation of fibrosis‐related parameters (q‐FP) to model disease progression in steatohepatitis

“…There are thus technical distinctions between q‐FP analyses of collagen versus traditional histological methods. Interested readers are referred to previous publications on more technical details of measurement …”

“…Interested readers are referred to previous publications on more technical details of measurement. (14,15)…”

“…Recently, dual‐photon microscopy has been used for assessment of hepatic fibrosis . This method identifies individual collagen fibrils in unstained paraffin‐embedded tissue sections and quantifies their physical attributes—such as number, length, diameter, orientation, contour, and cross‐linkage with each other—within the context of hepatic architecture.…”

“…Recently, dual-photon microscopy has been used for assessment of hepatic fibrosis. (14,15) This method identifies individual collagen fibrils in unstained paraffinembedded tissue sections and quantifies their physical attributes-such as number, length, diameter, orientation, contour, and cross-linkage with each other-within the context of hepatic architecture. It is thus technically quite different from other quantitative methods, such as collagen morphometry, which measures the area stained by collagen-specific stains (e.g., Sirius Red) and reads it out as a function of the total area measured (collagen proportional area).…”

Quantification of Liver Fibrosis—A Comparative Study

“…As stated in Wang et al [54], small changes in liver fibrosis may be overlooked in clinical trials, leading to invalid diagnostic outcomes. For this reason, they developed a fully automated method for providing objective hepatitis B-related fibrosis quantitative measurements.…”