NASH: Concurrent Fibrosis and Fat Imaging and Quantification

In the particular case of NAFLD and NASH, we concurrently acquire SHG (collagen) and 2PE (tissue morphometrics and inflammation) images from the same unstained FFPE section, which is returned undamaged for further molecular pathology tests if needed.

See 2017 publications on our website page here.

NASh Workflow.JPG

Our image Analysis tools concurrently quantify fibrosis and Microvesicular Steatosis: Fibrosis is quantified using the Second Harmonic Generation (SHG) channel, both in terms of Content and structure. Steatosis is quantified using the Two-Photon excitation Fluorescence (2PE) channel: we extract and quantify the microvesicular and macrovesicular features. Vascular features (also "round and dark") are excluded as they are surrounded by collagen (identified by SHG) and often contain internal 2PE fluorescent from hemoglobin.

In 2018, both Fibrosis and the NAS-activity score can potentially become fully automated. Subscribe here if you are interested to stay informed of our progress.

 Rodent fed with Fatty Diet exhibit the development of fibrosis (in green, SHG imaging) and fat (dark circular vesicules on the 2PE red image) of increasing severity vs diet duration (above). Such images can be post-processed to extract a % Collagen Area (SHG image) and a % Fat Area % ( Extracted from the vesicules), showing good correlation with Hepatic Triglicerides (ref: W. Liang, 2015. PhD Dissertation, Establishment of a General NAFLD Scoring System for Rodents Models and Comparison to Human Liver Pathology - Chapter 2 -  Link here )

Rodent fed with Fatty Diet exhibit the development of fibrosis (in green, SHG imaging) and fat (dark circular vesicules on the 2PE red image) of increasing severity vs diet duration (above). Such images can be post-processed to extract a % Collagen Area (SHG image) and a % Fat Area % ( Extracted from the vesicules), showing good correlation with Hepatic Triglicerides (ref: W. Liang, 2015. PhD Dissertation, Establishment of a General NAFLD Scoring System for Rodents Models and Comparison to Human Liver Pathology - Chapter 2 - Link here)

Concordance with conventional Histopathology

In this case Study (Fat Diet NASH Mouse model N=4, vs Drug A and Drug B candidates, N=4) we highlight the combined efficacy of the drug candidates as anti-steatotic and and-fibrotic compounds. Dots are the group averages. Our method uses ONE FFPE slide to derive all the information. Turnaround time is les than 2 weeks for a typical 4 arms x 4 animals study.

Towards a fully automated, robust and continuous scoring of Fibrosis (in NASH)

 Quantitative Morphometric Analysis of Fibrosis features  (qFP: quantitative Fibrosis Parameters) enables the construction of a Fibrosis Composite Score that evolves continuously as fibrosis Progress,and can be used effectively as a Clinical Tool to assess Fibrosis  Ref: DUAL PHOTON MICROSCOPY BASED QUANTITATION OF FIBROSIS-RELATED PARAMETERS (Q-FP) TO MODEL DISEASE PROGRESSION IN STEATOHEPATITIS Yan Wang MD PhD, Hepatology doi: 10.1002/hep.29090

Quantitative Morphometric Analysis of Fibrosis features  (qFP: quantitative Fibrosis Parameters) enables the construction of a Fibrosis Composite Score that evolves continuously as fibrosis Progress,and can be used effectively as a Clinical Tool to assess Fibrosis

Ref: DUAL PHOTON MICROSCOPY BASED QUANTITATION OF FIBROSIS-RELATED PARAMETERS (Q-FP) TO MODEL DISEASE PROGRESSION IN STEATOHEPATITIS Yan Wang MD PhD, Hepatology doi: 10.1002/hep.29090

Upcoming Developments

We are developing image Analysis tools to automatically quantify Lobular Inflammation and Ballooning from the same images used to quantify fibrosis and Steatosis. While the the method is still experimental, we welcome investigator-initiated studies.

Second Harmonic Generation Reveals Subtle Fibrosis Differences in Adult and Pediatric Nonalcoholic Fatty Liver Disease. Liu F, Zhao JM, Rao HY, Yu WM, Zhang W, Theise ND, Wee A, Wei L.  Am J Clin Pathol. 2017 Nov 20;148(6):502-512

Paper here 

Modeling the epidemic of Nonalcoholic Fatty Liver Disease demonstrates an exponential increase in burden of disease C. Estes, H. Razavi, R. Loomba, Z. Younossi, A.sanyal, Hepatology (67) 1, 2018

Paper Here

The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from th American Association for the Study of Liver Diseases. N. Chalasani, Z. Younossi, J.E. Lavine, M. Charlton, K. Cusi, M. Rinella, S. Harrison, E. Brunt, A.J. Sanyal  in Hepatology ,Vol. 67, No.1. Jan 2018

Paper here

Current concepts in pediatric nonalcoholic fatty liver disease S. Fleet, J. Lefkowitch,  J. Lavine Gastroenterol Clin N Am 46 (2017) 217–231

Paper here

Nonalcoholic Fatty Liver Disease (NAFLD) Activity Score and the Histopathologic Diagnosis in NAFLD: Distinct Clinicopathologic Meanings, Elizabeth M. Brunt, David E. Kleiner, Laura A. Wilson, Patricia Belt, and Brent A. Neuschwander-Tetri for the NASH Clinical Research Network (CRN)

Paper Here

Liver biopsy in modern clinical practice: a pediatric point-of-view N. Ovchinsky, R. K.Moreira, J.H. Lefkowitch, J.E. Lavine Adv Anat Pathol. 2012 Jul;19(4):250-62

Paper Here

The intersection of nonalcoholic fatty Liver disease and obesity. J.A. Woo Baidal and J.E. Lavine Science Translational Medicine  27 January 2016 Vol 8 Issue 323

Paper here

Sampling variability of liver biopsy in nonalcoholic fatty liver disease. Ratziu et all. Gastroenterology 2005 Jun; 126(7): 1898-906 

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