Precision Histology: Quantitative Masson’s Trichrome

Masson’s Trichrome is one of the most common histological stains used to distinguish between muscle and collagen in tissue samples. While the original formulation has changed over the years, Masson’s Trichrome remains a three-dye based protocol that selectively stains muscle, erythrocytes and cytoplasm red, collagen blue and nuclei dark brown (Figure 1).

Figure 1: Liver and lung tissue stained with Masson’s Trichrome.

Fibrosis is characterized by excessive formation of fibrous connective tissue, generally initiated by inflammatory responses or tissue damage. Collagen is the main component of connective tissue; therefore Masson’s Trichrome staining of increased collagen accumulation in tissue is an accurate indicator of fibrosis. Numerous diseases affecting various tissues feature fibrosis, including cirrhosis, chronic kidney disease, Crohn’s disease, cystic fibrosis, cardiac fibrosis, muscular dystrophy and many others. Thus, Masson’s Trichrome stain has become the preferred method for histopathological identification of fibrosis in both patient biopsies and animal models of human fibrotic disease.

Although Masson’s Trichrome is routinely used to observe fibrosis, quantification of fibrosis in biological samples is largely a labor-intensive manual process that is prone to variability and bias. Therefore, there is a need for sensitive imaging tools to accurately measure fibrotic area in tissue samples. To address this need, our scientists at Reveal Biosciences have developed an automated quantification method using our proprietary Image Dx™ analysis software that specifically isolates the Trichrome positive region within a whole tissue section.

After whole slide scanning of the Trichrome stained tissue (Figure 2A), the image is processed using two different masks. The first mask excludes any airspace and background, as shown in figure 2B, while the second mask excludes the tissue structure (Figure 2C), resulting in the isolation of the Trichrome signal.

After automated tissue analysis is performed, the percentage of Trichrome positive tissue is calculated. As an example, the percentage of Trichrome positive tissue for the sample shown in figure 2 was 1.79%.

Figure 2: Image data of Masson’s Trichrome staining of mouse lung before and after automated tissue segmentation. (A) Bright field image of stained tissue before segmentation. (B) Image resulting from airspace and background masking. (C) Trichrome positive region after tissue structure masking.

Quantification of Masson’s Trichrome staining using ImageDx™ significantly reduces turnaround time and avoids the variability associated with traditional manual methods. You provide either whole slide images, stained slides, or fixed tissue, and our experienced team will generate your data.

For more information on how our quantitative Masson’s Trichrome technology can advance your projects, contact us at [email protected].

By: Caroline Morel, PhD

Project Manager, Reveal Biosciences