Elsevier

Journal of Hepatology

Volume 52, Issue 3, March 2010, Pages 398-406
Journal of Hepatology

Research Article
Fibrillar collagen scoring by second harmonic microscopy: A new tool in the assessment of liver fibrosis

https://doi.org/10.1016/j.jhep.2009.12.009Get rights and content

Background & Aims

Imaging of supramolecular structures by multiphoton microscopy offers significant advantages for studying specific fibrillar compounds in biological tissues. In this study, we aimed to demonstrate the relevance of Second Harmonic Generation (SHG) for assessing and quantifying, without staining, fibrillar collagen in liver fibrosis.

Methods

We first showed the relationship between SHG signal and collagen forms over-produced and accumulated during fibrosis progression. Taking this property into consideration, we developed an innovative method to precisely quantify the fibrosis area in histological slices by scoring of fibrillar collagen deposits (Fibrosis-SHG index).

Results

The scoring method was routinely applied to 119 biopsies from patients with chronic liver disease allowing a fast and accurate measurement of fibrosis correlated with the Fibrosis-Metavir score (rho = 0.75, p < 0.0001). The technique allowed discriminating patients with advanced (moderate to severe) fibrosis (AUROC = 0.88, p < 0.0001) and cirrhosis (AUROC = 0.89, p < 0.0001). Taking advantage of its continuous gradation, the Fibrosis-SHG index also allowed the discrimination of several levels of fibrosis within the same F-Metavir stage. The SHG process presented several advantages such as a high reliability and sensitivity that lead to a standardized evaluation of hepatic fibrosis in liver biopsies without staining and pathological examination.

Conclusions

Second harmonic microscopy emerges as an original and powerful tool in the assessment of liver fibrosis and offers new possibilities for the evaluation of experimental protocols. We expect that this technology could easily be applicable in the study of other fibro-proliferative pathologies.

Introduction

Chronic liver diseases are generally associated with extra-cellular matrix (ECM) over-production. Hepatic fibrosis is the result of a dynamic process characterized by an imbalance between fibrogenesis and fibrolysis responsible for the accumulation of fibrillar components in the liver such as fibrillar collagen [1]. The progression of human fibrosis may lead to cirrhosis and cancer with clinical complications followed by high morbidity and mortality rates particularly in chronic hepatitis B or C virus (HBV, HCV) infections [2], [3]. In order to determine the degree of fibrosis, several methods are based on the pathological examination of liver biopsies by a trained pathologist using numerical systems with increasing severity stages like Scheuer [4], Ishak [5] or Metavir [6] scores after ECM staining [7], [8]. The Metavir grading system appears well adapted for assessing chronic liver damage related to viral hepatitis. An alternative to the numerical scoring systems relies on a direct measurement of the amount of fibrotic deposits by morphometric image analysis [9], [10]. To date, histomorphometric methodologies are not well standardized and appear to be time consuming procedures compared to classic numerical systems.

The aim of this study was to develop an innovative optical method for an accurate quantification of human liver fibrosis. This original process is based on an application of multiphoton microscopy enabling the observation of unstained samples using endogenous sources of nonlinear signals such as Two-Photon Excitation Fluorescence (TPEF) and Second Harmonic Generation (SHG). SHG makes the specific detection of supramolecules possible with high crystalline triple-helix structures deprived of centrosymmetry at microscopic and mesoscopic scales [11], [12], [13], [14]. In biological tissues, fibrillar collagen displays these properties. Like myosin fibers and microtubules, fibrillar collagen can generate a two-photon coherent process selectively collected and recorded at precisely half-wavelength of excitation which is characteristic of the SHG signature [15], [16], [17], [18]. SHG microscopy has been successfully employed in various models and has revealed its high potential for imaging and analyzing fibrillar collagen in unstained tissues [19], [20], [21], [22]. Recent data also suggested that SHG microscopy could be an interesting approach in the assessment of fibro-proliferative diseases [23], [24].

In this study, we show that multiphoton microscopy using SHG provides a powerful tool for evaluating human liver fibrosis. We established a reliable and standardized method for the quantification of fibrillar collagen and analyzed the correlation between the Fibrosis-SHG index and the Fibrosis-Metavir score currently considered to be the gold standard in the clinical assessment of liver fibrosis.

Section snippets

Biopsies and histological data

The study cohort included 119 patients who underwent percutaneous biopsies (n = 73) or large surgical biopsies (n = 46). Patients presented chronic liver disease with various degrees of severity related to HBV or HCV infection in 8.22% and 90.41% of cases, respectively; 1.37% presented co-infection with HBV and HCV. Exclusion criterion was inadequate specimen (length of biopsy <20 mm and/or portal tract <5). The mean biopsy length (percutaneous) was 39.6 ± 8.8 cm and mean number of fragments 2.4 ± 1.5.

Multiphoton microscopy in human liver fibrosis

The SHG phenomenon within molecules exhibiting non-centrosymmetric organization is characterized by a change of the excitation energy from two infrared incident photons into one emerging visible photon at exactly twice the energy (precisely half of the excitation wavelength) (Fig. 1A). In order to illustrate the high potential of multiphoton microscopy, we first imaged liver with moderate fibrosis (F2-Metavir). Liver cryosections (100 μm thick) were made and imaged without staining, at

Discussion

Second harmonic microscopy presented a major challenge in the assessment of liver fibrosis. Our results demonstrate that SHG provides an original and powerful tool for the quantitative evaluation of fibrillar collagen deposits. We first validated the selectivity of SHG signals for fibrotic deposits (fibrillar collagen type I and III) in human liver by establishing a strong correlation between SHG and immunochemistry assays. According to the literature, immunolabelings of non-fibrillar collagen

Acknowledgements

The authors thank the Histo-Pathology Platform (IFR 140), Pascale Bellaud for technical support in histologic preparation and staining, as well as Nina Cutro-Kelly for critical reading of the manuscript. We also thank Michel Samson, Claire Piquet-Pellorce and Mariette Lisbonne (EA SeRAIC), Anne Renault (UMR-CNRS 6251) and Pixel platform (UMR-CNRS 6026) for constructive comments on this study. We thank the CNRS project “Interface Physique, Chimie, Biologie: soutien à la prise de risque” for

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