Trichrome

Trichrome, meaning "three colour" was used as the name of a staining method (Mallory's trichrome) which differentially coloured erythrocytes orange, muscle red and collagen blue.

It now has become a general term to describe any histological staining method which uses two or more acid dyes in conjunction with a "polyacid" to differentially stain two basic materials in contrasting colours. The most common application is to stain muscle fibres red and collagen fibres green or blue, Masson's trichrome being the most commonly used method. This is necessary since smooth muscle fibres may be difficult to differentiate from collagen. In addition, liver biopsies may have fine collagen fibres between the liver cells, and the amount of collagen may be estimated based on the staining method. Trichrome methods are now used for differentiating muscle from collagen, pituitary alpha cells from beta cells, fibrin from collagen, and mitochondria in fresh frozen muscle sections, among other applications. It helps in identifying increases in collagenous tissue (i.e. fibrotic changes) such as in liver cirrhosis and distinguishing tumours arising from muscle cells and fibroblasts.^[1]

The techniques employ two or more acid dyes. Normally it would be expected that acid dyes would all stain the same basic proteins, but by applying them sequentially the staining pattern may be manipulated by removing dye from less intensely stained components with a "polyacid", usually molybdophosphoric or tungstophosphoric acids. These are thought to behave as very large molecular weight dyes and displace easily removed dye from collagen.

Usually a red dye in dilute acetic acid is applied first to overstain all components. Then a polyacid is applied to remove the red dye from collagen and some other components by displacement. A second acid dye (blue or green) in dilute acetic acid is applied which, in turn, displaces the polyacid, resulting in collagen stained in a contrasting colour to the initial dye used. If erythrocytes are to be stained, a small molecular weight yellow or orange dye is applied before staining with the red dye. It is usually applied from a saturated solution in 80% ethanol and often in conjunction with picric acid (itself a dye) and a polyacid. The methods exploit minor differences in tissue reaction to dyes, density, accessibility and so on.

The type of trichrome in which dyes and a polyacid are applied sequentially are called "multi-step trichromes". There are two other ways that these methods may be carried out. In "one-step" methods, all the dyes, sometimes with and sometimes without a polyacid, are combined in a single solution. One of the oldest is van Gieson's method which stains muscle and cytoplasm yellow, with red collagen. Another is Gömöri's method which closely mimics Masson's trichrome.

A third type are the yellowsolve methods in which a red dye in dilute acetic acid is first applied, then the section is very thoroughly dehydrated to ensure that no moisture remains. The red dye is then displaced by a yellow dye in a solvent, often cellosolve (2-ethoxy-ethanol), from which they get their name: yellow dye in cellosolve = yellowsolve. Lendrum's phloxine-tartrazine for cell inclusions is an example.

Red Dyes used: acid fuchsin, xylidine ponceau, chromotrope 2R, biebrich scarlet, ponceau 6R, phloxine.

Blue and green dyes used: light green SF yellowish, fast green FCF, aniline blue, methyl blue, water blue.

Yellow dyes used: picric acid, orange G, martius yellow, tartrazine, milling yellow.

References

Baker JR (1958) Principles of biological Microtechnique. London: Methuen.

Horobin RW (1982) Histochemistry. Stuttgart: Gustav Fischer.

Horobin RW (1988) Understanding Histochemistry. Chichester: Ellis Horwood.

Kiernan JA (2008) Histological and Histochemical Methods, 3rd ed. Bloxham: Scion.

Prento P (2009)Staining of macromolecules: possible mechanisms and examples. Biotech. Histochem. 84: 139-158.

Puchtler H, Isler H (1958) The effect of phosphomolybdic acid on the stainability of connective tissues by various dyes. J. Histochem. Cytochem. 6: 265-270.

Reid PE, Iagallo M, Nehr S, Jankunis M, Morrow P (1993) Mechanism of connective tissue techniques. 1. The effect of dye concentration and staining time on anionic dye procedures. Histochem. J. 25: 821-829.