Zonated quantification of immunohistochemistry in normal and steatotic livers

Immunohistochemical stains (IHC) reveal differences between liver lobule zones in health and disease, including nonalcoholic fatty liver disease (NAFLD). However, such differences are difficult to accurately quantify. In NAFLD, the presence of lipid vacuoles from macrovesicular steatosis further hampers interpretation by pathologists. To resolve this, we applied a zonal image analysis method to measure the distribution of hypoxia markers in the liver lobule of steatotic livers. The hypoxia marker pimonidazole was assessed with IHC in the livers of male C57BL/6 J mice on standard diet or choline-deficient L-amino acid-defined high-fat diet mimicking NAFLD. Another hypoxia marker, carbonic anhydrase IX, was evaluated by IHC in human liver tissue. Liver lobules were reconstructed in whole slide images, and staining positivity was quantified in different zones in hundreds of liver lobules. This method was able to quantify the physiological oxygen gradient along hepatic sinusoids in normal livers and panlobular spread of the hypoxia in NAFLD and to overcome the pronounced impact of macrovesicular steatosis on IHC. In a proof-of-concept study with an assessment of the parenchyma between centrilobular veins in human liver biopsies, carbonic anhydrase IX could be quantified correctly as well. The method of zonated quantification of IHC objectively quantifies the difference in zonal distribution of hypoxia markers (used as an example) between normal and NAFLD livers both in whole liver as well as in liver biopsy specimens. It constitutes a tool for liver pathologists to support visual interpretation and estimate the impact of steatosis on IHC results.