Second harmonic generation microscopy provides accurate automated staging of liver fibrosis in patients with non-alcoholic fatty liver disease

<div><p>Background</p><p>Assessment of severity of liver fibrosis is essential in the management of non-alcoholic fatty liver disease (NAFLD). Second Harmonic Generation (SHG) microscopy is a novel optical tissue imaging system that provides automated quantification of fibrosis based on unique architectural features of collagen. This study aims to develop and validate a SHG-based index for automated staging of liver fibrosis in patients with NAFLD.</p><p>Methods</p><p>SHG microscopy was performed on archived liver biopsy specimens from 83 patients with NAFLD. A unique algorithm was developed to identify specific SHG parameters that correlated with fibrosis stage. The accuracy of the algorithm was compared against clinical assessment by experienced liver histopathologists using the Brunt fibrosis staging and further validated using the leave-one-out cross-validation method.</p><p>Results</p><p>Mean age of the study cohort was 51.8 ± 11.7 years, with 41% males. A fibrosis index (SHG B-index) was developed comprising 14 unique SHG-based collagen parameters that correlated with severity of NAFLD fibrosis in a continuous fashion. The SHG B-index had excellent correlation with Brunt fibrosis stage (Spearman’s correlation 0.820, p<0.001). AUROCs for prediction of Brunt fibrosis stages 1, 2, 3 and 4 were 0.853, 0.967, 0.985 and 0.941 respectively. In the cross-validation analysis, the SHG B-index demonstrated high specificity for diagnosis of all grades of fibrosis. A SHG B-index score of >1.76 had an overall diagnostic accuracy of 98.5% for prediction of presence of bridging fibrosis (Brunt stage ≥3) with sensitivity of 87.5%, specificity 98.0%, positive predictive value 96.6% and negative predictive value 92.6%.</p><p>Conclusion</p><p>The SHG B-index is a unique SHG-based index that provides accurate automated assessment of fibrosis stage in NAFLD patients.</p></div

Boxplot of SHG B-index and Brunt fibrosis stage (n = 83).

<p>Boxplot of SHG B-index and Brunt fibrosis stage (n = 83).</p

AUROCs of SHG B-index for prediction of Brunt fibrosis.

<p>AUROCs of SHG B-index for prediction of Brunt fibrosis.</p

Cross-validation of SHG B-index to predict Brunt fibrosis stage.

<p>Cross-validation of SHG B-index to predict Brunt fibrosis stage.</p

Illustration of collagen parameters on SHG microscopy.

<p>A) The RAW SHG/TPEF image. (B)The high magnification imaging performed in the area delimited by the blue square in picture A. (C) The aggregated and distributed strings of picture B. (D) The high magnification imaging performed in the area delimited by the blue square in picture B, and illustration of the width, length and area of a string. The thick string is by definition length-width ratio > 0.25.</p

Illustration of specific collagen distribution at central vein, portal tract and perisinusoidal regions in early versus advanced fibrosis.

<p>Illustration of specific collagen distribution at central vein, portal tract and perisinusoidal regions in early versus advanced fibrosis.</p

Demographics of patients (SGH cohort).

<p>Demographics of patients (SGH cohort).</p

Correlation between serum markers estimated by Spearman’s rank correlation coefficient, rho (n = 241).

*<p>The upper part are the Spearman’s rank correlation coefficients, rho; the lower part are the p-values of the Spearman’s rank correlation test.</p

Comparison of the Adequacy Index and Log Likelihood ratios of diagnostic models.

<p>Comparison of the Adequacy Index and Log Likelihood ratios of diagnostic models.</p

Comparison of serum MCP-1, prolactin and AFP levels in HCC and non-HCC patients.

<p>Serum concentrations of (A) MCP-1, (B) prolactin and (C) AFP in non-HCC chronic hepatitis B carriers (NC group, n = 115) and HCC patients (HCC group, n = 126) in the SGH cohort of patients were analyzed by multiplex sandwich ELISA (Quantibody Array). Serum MCP-1 concentrations in asymptomatic HBV/HCV carriers (AC group, n = 100), chronic hepatitis patients with evidence of transaminitis (CH group, n = 101) and HCC patients (HCC group, n = 98) in the MRIN cohort (A) were analyzed by sandwich ELISA. The boxes represent the central 50% of the data, spanning between the 25^th and 75^th percentiles and the horizontal line within each box indicates the median. The cut-off points were: 1.5×IQR above 75^th percentile (upper limit) and 1.5×IQR below 25^th percentile (lower limit). Values beyond the cut-off points were considered as outliers and are represented by the dots. Comparison of biomarker values between groups was performed using the Mann-Whitney <i>U</i> test.</p