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Original research
Quantifying and monitoring fibrosis in non-alcoholic fatty liver disease using dual-photon microscopy
  1. Yan Wang1,
  2. Grace Lai-Hung Wong2,3,
  3. Fang-Ping He4,
  4. Jian Sun1,
  5. Anthony Wing-Hung Chan5,
  6. Jinlian Yang1,
  7. Sally She-Ting Shu2,3,
  8. Xieer Liang1,
  9. Yee Kit Tse2,3,
  10. Xiao-Tang Fan6,
  11. Jinlin Hou1,
  12. Henry Lik-Yuen Chan2,3,
  13. Vincent Wai-Sun Wong2,3
  1. 1 State Key Laboratory of Organ Failure Research, Guangdong Provincial Research Center for Liver Fibrosis, Department of Infectious Diseases and Hepatology Unit, Nanfang Hospital, Southern Medical University, Guangzhou, China
  2. 2 Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong, China
  3. 3 State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China
  4. 4 Department of Infectious Diseases, The Eighth Affiliated Hospital of Sun Yat-Sen University, Shenzhen, China
  5. 5 Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong, China
  6. 6 Department of Hepatology, The First Affliated Hospital of Xinjiang Medical University, Urumqi, China
  1. Correspondence to Dr Vincent Wai-Sun Wong, Department of Medicine and Therapeutics, Chinese University of Hong Kong, Hong Kong, Hong Kong; wongv{at}


Objective Fibrosis stage is strongly associated with liver-related outcomes and is a key surrogate endpoint in drug trials for non-alcoholic steatohepatitis. Dual-photon microscopy allows automated quantification of fibrosis-related parameters (q-FPs) and may facilitate large-scale histological studies. We aim to validate the performance of q-FPs in a large histological cohort.

Design 344 patients with non-alcoholic fatty liver disease (NAFLD) underwent 428 liver biopsies (240 had paired transient elastography examination). Fibrosis stage was scored using the NASH Clinical Research Network system, and q-FPs were measured by dual-photon microscopy using unstained slides. Patients were randomly assigned to the training and validation cohorts to test the performance of individual q-FPs and derive optimal cut-offs.

Results Over 25 q-FPs had area under the receiver-operating characteristics curves >0.90 for different fibrosis stages. Among them, the perimeter of collagen fibres and number of long collagen fibres had the highest accuracy. At the best cut-offs, the two q-FPs had 88.3%–96.2% sensitivity and 78.1%–91.1% specificity for different fibrosis stages in the validation cohort. q-FPs and histological scoring had nearly identical correlations with liver stiffness measurement, suggesting that the accuracy of q-FPs approached that of histological assessment. Among patients with paired liver biopsies, changes in the same q-FPs were associated with changes in fibrosis stage. At a median follow-up of 5.6 years, baseline q-FPs predicted liver-related events.

Conclusion q-FP is highly accurate in the assessment of fibrosis in NAFLD patients. This automated platform can be used in future studies as objective and reliable evaluation of histological fibrosis.

  • non-alcoholic steatohepatitis
  • liver fibrosis
  • cirrhosis
  • liver biopsy

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  • Contributors YW and VW-SW designed the study. GL-HL-HW, SS and VW-SW (HK cohort), and FH and X-TF (Urumqi cohort) collected the clinical data. YW and JY performed dual-photon microscopy. AWHC evaluated and scored all liver biopsy samples. YKT and VW-SW performed statistical analysis. YW, YKT and VW-SW drafted the manuscript. JS, XL, JH and HLYC provided administrative support. All authors contributed to and approved the final version of the manuscript.

  • Funding This study was partly supported by the National Natural Science Foundation of China (81670522) to YW and the Guangzhou Science and Technology Plan Project (201607020019) to JH.

  • Competing interests VW-SW has served as an advisor/consultant for AbbVie, Allergan, Echosens, Gilead Sciences, Janssen, Novo Nordisk, Perspectum Diagnostics, Pfizer and Terns; and a speaker for Bristol-Myers Squibb, Echosens, Gilead Sciences and Merck. GL-HL-HW and HLYC have served as speakers for Echosens. The other authors report no conflict of interests.

  • Patient consent for publication Not required.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement Data are available upon reasonable request.