Quantifying hepatic steatosis - more than meets the eye

Histopathology. 2012 May;60(6):971-81. doi: 10.1111/j.1365-2559.2012.04193.x. Epub 2012 Feb 28.


Aims: The non-alcoholic fatty liver disease (NAFLD) activity score (NAS) is the histological tool used to assess disease severity based on steatosis, inflammation and hepatocyte ballooning. As steatosis contributes up to three of a potential eight points to NAS, it is important to quantify steatosis accurately. We sought to determine the optimum histological technique for identifying fat in tissue.

Methods and results: Using tissue from a mouse model of NAFLD, with validation in human liver biopsies, the percentage steatosis and fat droplet size were assessed in haematoxylin and eosin (H&E)- and Oil Red-O (ORO)-stained sections by light microscopy and digital image analysis (DIA). Results were compared to biochemical tissue triglyceride content and MRI assessment of hepatic lipid content. H&E steatosis assessment correlated poorly with tissue triglyceride concentration. However, ORO DIA exhibited much higher sensitivity and specificity for steatosis and correlated very well with triglyceride concentration in mouse and human liver (R = 0.706, P = 0.001 and R = 0.894, P =0.041, respectively). MRI-based assessment of steatosis was inaccurate.

Conclusions: ORO DIA is the most accurate method for detecting and quantifying steatosis. Although H&E-based NAS remains clinically valid in both clinical research and experimental situations, ORO DIA is a more robust technique to assess liver steatosis accurately for NAS scoring.

MeSH terms

  • Adipose Tissue / chemistry
  • Adipose Tissue / metabolism
  • Adipose Tissue / pathology*
  • Animals
  • Azo Compounds / chemistry
  • Body Weight / physiology
  • Coloring Agents / chemistry
  • Disease Models, Animal
  • Fatty Liver / metabolism
  • Fatty Liver / pathology*
  • Humans
  • Image Processing, Computer-Assisted
  • Liver / chemistry
  • Liver / metabolism
  • Liver / pathology*
  • Magnetic Resonance Imaging
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Non-alcoholic Fatty Liver Disease
  • Predictive Value of Tests
  • Reproducibility of Results
  • Triglycerides / analysis


  • Azo Compounds
  • Coloring Agents
  • Triglycerides
  • oil red O