Precision Histology: Steatosis – Quantitative Analysis of Abnormal Lipid Accumulation

Abnormal lipid accumulation in tissue, or steatosis, is associated with numerous pathological conditions. Hepatic steatosis is a hallmark of alcoholic liver disease (ALD) and is also an early marker of Nonalcoholic Fatty Liver Disease (NAFLD), which is now the most prevalent cause of liver dysfunction in the United States. Histological scoring of hepatic steatosis is therefore an essential clinical tool for the evaluation of liver disease.

In addition to the liver, abnormal lipid deposits can be observed in numerous other tissues in the context of disease. For example, Gaucher disease and Nieman-Pick disease are inherited lipid storage metabolic disorders characterized by the harmful accumulation of lipids in the kidneys, spleen, lungs, bone marrow and brain. Moreover, identification of liposarcomas, tumors derived from fat cells, requires evaluation of lipid accumulation in tissue samples. The clinical relevance of pathological lipid deposits in tissue mean there is a critical need for sensitive histological approaches to evaluate and quantify steatosis in both patients and animal models of human disease.

Lipid accumulation is traditionally assessed by a Board Certified Pathologist; however, recent advances in image analysis and machine learning have enabled more precise quantitative analysis to be performed independently or to assist the pathologist at scale. Abnormal lipid accumulation is commonly assessed in preclinical research and clinical trials using any of all of the following approaches.

Method One: Hematoxylin and Eosin (H&E)

Figure 1. A) Liver tissue stained with H&E. B) Mask applied to original image to isolate lipid droplets (Blue) for quantification. The scale bar represents 50 μm.

H&E staining on formalin-fixed, paraffin-embedded (FFPE) samples is an easy, low cost approach to identify lipid accumulation in many tissues. Morphologically, lipid droplets appear as circular voids in the tissue as the fat content is dissolved by solvent treatment during tissue processing (Figure 1A). H&E staining is therefore an indirect method for the quantification of lipid accumulation. Whole slide images of H&E staining, obtained using bright field microscopy, can be analyzed to determine the number and size of lipid droplets as well as the degree of steatosis as a percentage of the total area of the tissue (Figure 1B). While H&E is a clinically approved method to score steatosis, additional histological methods can directly detect lipid deposits in tissues.

Method Two: Oil Red O

Lipid accumulation can be evaluated directly in fresh frozen tissue sections by staining with Oil Red O, a fat-soluble dye that specifically stains triglycerides and neutral lipids a deep red color (Figure 2A). The use of fresh frozen tissues avoids the loss of lipid content that occurs during processing of formalin-fixed tissue.

Figure 2. A) Frozen liver tissue section stained with Oil Red O (Red) and hematoxylin nuclear counterstain (Blue). B) Mask applied to original image to isolate lipid droplets (Red) for quantification. The scale bar represents 10 μm.

Whole slide images of tissue sections stained with Oil Red O can be generated using bright field microscopy. ImageDx™ analysis software precisely measures a range of parameters to quantify Oil Red O staining, including percentage of positivity, average number of droplets per unit area and mean droplet size (Figure 2B).

Method Three: Perilipin Immunostaining

Perilipin, also called lipid droplet-associated protein, is a protein that specifically coats the periphery of lipid droplets. Perilipin can be detected in both FFPE and frozen tissue sections by immunohistochemistry (IHC), a technique that uses primary antibodies raised against their specific target antigens. These primary antibodies are then detected using a range of secondary reagents, and visualized with 3,3-Diaminobenzidine (DAB – brown). Primary antibodies can also be detected using a range of fluorescent markers in a process known as immunofluorescence (IF). Staining tissue sections with perilipin specifically labels the outline of fat vacuoles, as shown in Figure 3.

Figure 3: Perilipin staining visualized with DAB (brown) on FFPE liver section. Nuclei are conterstained with hematoxylin (blue). The scale bar represents 50 μm.

Whole slide imaging of perilipin immunostaining can be performed using either bright field or immunofluorescence microscopy, depending on the secondary detection strategy. A wide range of quantification parameters can be reported including the mean number of lipid droplets per unit area, droplet size, and lipid area. Perilipin staining is extremely sensitive as it detects multiple small lipid droplets that may be missed by H&E. In addition, use of perilipin as a marker of steatosis offers the considerable advantage that it can be multiplexed with other biomarkers of interest on the same section.

In conclusion, the precise measurement and analysis of lipid accumulation in tissue sections provides valuable data when evaluating steatosis in both humans and animal models of human disease. Selection of the most appropriate method for your research depends on several parameters including the format of available tissue and the data end points required. Contact us to discuss how precision histology can benefit your next study.

Precision Histology: Accurate, Quantitative, Reproducible, Scalable

For more information, contact us at [email protected]

By: Caroline Morel, PhD

Project Manager, Reveal Biosciences