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

Tong-Hong Wang,Chau-Ting Yeh,Xiao Teng,Kung-Hao Liang,Tse-Ching Chen

doi:10.1038/srep12962

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.

Highlights

Liver biopsy examinations are considered the “gold standard” for liver fibrosis diagnosis, by which clinical strategies for patient management are drafted
Advanced second harmonic generation (SHG)/two-photon excitation fluorescence (TPEF) microscopic quantification of fibrillar collagen was highly correlated with ISHAK fibrosis scores
In SHG/TPEF imaging, fibrillar collagen was detected by SHG and is shown in green, whereas hepatocyte morphology was captured by TPEF microscopy and is shown in red

Summary

Introduction

Liver biopsy examinations are considered the “gold standard” for liver fibrosis diagnosis, by which clinical strategies for patient management are drafted. Several studies have quantified liver fibrosis by employing image analysis systems[17,18,19] These computer-aided systems are used to provide objective quantitative measurements that might exclude observer discrepancies. Other pathological features play vital roles in liver fibrosis grading and staging[23], necessitating computer-based systems that can provide measurements in addition to the total fibrosis area. Collagen quantitation using SHG/TPEF microscopy has been evaluated in animal models but not in a large collection of human tissues with various stages of fibrosis[26,27] Such studies should be completed before this technique can be applied to patient samples. We measured and quantified nineteen morphological features of collagen using SHG and TPEF microscopy and developed two algorithms for assessing fibrosis using these nineteen parameters for both early and later fibrosis stages. The validation results showed that the multivariate generalized linear model was better for assessing the fibrosis progression pattern at late liver fibrosis stages, whereas a non-linear (support vector machine, SVM) approach was better for the assessment of early fibrosis stages

Methods

Results

Conclusion