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Microscopists: Have you Tried Trichrome?

Posted in: Microscopy and Imaging

Have You Tried Trichrome?

The trichrome stain is one of the most commonly used special stains in every histology lab.

The pedantic meaning of the word trichrome is “three-coloured”, referring to how the technique differentially stains tissue samples in three colors.

However, the term is now actually used to describe any staining method using two or more acid dyes of contrasting colors to selectively stain different basic tissue components.

What Does Trichrome Stain?

The technique uses three dyes to produce different coloration of different tissue types. Typically they are used to demonstrate collagen, often in contrast to smooth muscle, but can also be used to highlight fibrin in contrast to red blood cells. They can also selectively highlight other components.

Tissue TypeColor with trichrome stain
Collagen, BoneGreen-blue
Muscle, Fibrin, Cytoplasm, Red
Red blood cellsYellow or red
NucleiDark red-black

Who Uses Trichrome Stains?

These stains are widely used for both research and diagnostic purposes.  For example, researchers studying the progression of wound healing, or evaluating the efficacy of pharmacological products that accelerate it, will routinely examine trichrome-stained tissue sections to measure collagen deposition.

In diagnostic labs, pathologists often use this stain to help answer questions that may arise after examining routine haematoxylin and eosin-stained sections. In particular, this stain is routinely requested to evaluate disease in the liver. It can be especially valuable when examining liver samples from patients with cirrhosis where it can provide helpful information about the degree of fibrosis, and therefore the stage and progression of their disease.

General Principles of the Stain

During the procedure, Bouin’s solution (the ‘mordant’ which adheres dye to tissue) is applied to sections of tissue to intensify the final colour. Nuclei are stained with iron haematoxylin. Cytoplasm and muscle are then stained red with Biebrich scarlet-acid fuchsin. Following treatment with the polyacids phosphotungstic acid (PTA) and phosphomolybdic acid (PMA), collagen is stained with trichrome blue, containing aniline blue (or aniline light green which stains collagen green). Finally, an acetic acid solution is used to rinse the sections after staining which produces a more delicate shade of tissue coloration.

Different Trichrome Staining Techniques

Various trichrome recipes exist, all of which arose from pathologist Claude L. Pierre Masson’s original formulation from the early 1900s. Of the various types of trichrome stains available, those most commonly used today are:

  • Masson’s Trichrome: This is a multi-step staining technique. It uses the polyacids PTA and PMA. All of the mordant and staining steps (as described above) are performed individually.
  • Gomori’s Trichrome: This is a one-step method staining technique. This method does not use polyacids, and unlike its Masson counterpart, it combines all reagents into a single solution (except for the nuclear stain and the Bouin’s mordant) which is applied to tissue sections for a certain amount of time.

Which trichrome is most commonly used in your labs?

Do you have a favourite trichrome recipe?

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