Trichrome staining

Last updated

Trichrome staining is a histological staining method that uses two or more acid dyes in conjunction with a polyacid. Staining differentiates tissues by tinting them in contrasting colours. It increases the contrast of microscopic features in cells and tissues, which makes them easier to see when viewed through a microscope.

Contents

The word trichrome means "three colours". The first staining protocol that was described as "trichrome" was Mallory's trichrome stain, which differentially stained erythrocytes to a red colour, muscle tissue to a red colour, and collagen to a blue colour. Some other trichrome staining protocols are the Masson's trichrome stain, Lillie's trichrome, and the Gömöri trichrome stain.

Purpose

Without trichrome staining, discerning one feature from another can be extremely difficult. Smooth muscle tissue, for example, is hard to differentiate from collagen. A trichrome stain can colour the muscle tissue red, and the collagen fibres green or blue. 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]

Procedure

Trichrome staining techniques employ two or more acid dyes. Normally acid dyes would stain the same basic proteins, but by applying them sequentially the staining pattern can be manipulated. A polyacid (such as phosphomolybdic acid or Phosphotungstic acid) is used to remove dye selectively. Polyacids are thought to behave as dyes with a high molecular weight: they displace easily removed dye from collagen.[ citation needed ]

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.[ citation needed ]

Trichrome stains in which dyes and a polyacid are applied sequentially are called multi-step trichromes. In "one-step" methods, all the dyes—with or without a polyacid—are combined in a single solution. One of the oldest single-step approaches to trichrome staining is van Gieson's method, which stains muscle and cytoplasm yellow, and collagen red. Another is the Gömöri trichrome stain, which closely mimics Masson's trichrome. In "yellowsolve" methods, 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, such as cellosolve (2-ethoxy-ethanol). The name yellowsolve is a blend of the terms yellow and cellosolve. Lendrum's phloxine-tartrazine for cell inclusions is one example of a yellowsolve stain.[ citation needed ]

Dyes

Among the dyes used for trichrome staining are:

Red
Acid fuchsin, xylidine ponceau, chromotrope 2R, Biebrich scarlet, ponceau 6R, phloxine
Blue and green
Light green SF yellowish, Fast Green FCF, methyl blue, water blue
Yellow
Picric acid, orange G, Martius yellow, tartrazine, milling yellow

See also

Notes

  1. Lamar Jones, M. "Special Stains Education Guide". Chapter 10: Mastering the Trichrome Stain. Dako. Retrieved 20 May 2011.

Related Research Articles

<span class="mw-page-title-main">Picric acid</span> Explosive chemical compound

Picric acid is an organic compound with the formula (O2N)3C6H2OH. Its IUPAC name is 2,4,6-trinitrophenol (TNP). The name "picric" comes from Greek: πικρός (pikros), meaning "bitter", due to its bitter taste. It is one of the most acidic phenols. Like other strongly nitrated organic compounds, picric acid is an explosive, which is its primary use. It has also been used as medicine (antiseptic, burn treatments) and as a dye.

<span class="mw-page-title-main">Haematoxylin</span> Natural stain derived from hearthwood and used in histology

Haematoxylin or hematoxylin, also called natural black 1 or C.I. 75290, is a compound extracted from heartwood of the logwood tree with a chemical formula of C
16
H
14
O
6
. This naturally derived dye has been used as a histologic stain, as an ink and as a dye in the textile and leather industry. As a dye, haematoxylin has been called palo de Campeche, logwood extract, bluewood and blackwood. In histology, haematoxylin staining is commonly followed by counterstaining with eosin. When paired, this staining procedure is known as H&E staining and is one of the most commonly used combinations in histology. In addition to its use in the H&E stain, haematoxylin is also a component of the Papanicolaou stain which is widely used in the study of cytology specimens.

<span class="mw-page-title-main">Staining</span> Technique used to enhance visual contrast of specimens observed under a microscope

Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology, in cytology, and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses of diseases at the microscopic level. Stains may be used to define biological tissues, cell populations, or organelles within individual cells.

<span class="mw-page-title-main">Methyl blue</span> Chemical compound

Methyl blue is a chemical compound with the molecular formula C37H27N3Na2O9S3. It is used as a stain in histology, and stains collagen blue in tissue sections. It can be used in some differential staining techniques such as Mallory's connective tissue stain and Gömöri trichrome stain, and can be used to mediate electron transfer in microbial fuel cells. Fungal cell walls are also stained by methyl blue.

<span class="mw-page-title-main">Ponceau S</span> Chemical compound

Ponceau S, Acid Red 112, or C.I. 27195 is a sodium salt of a diazo dye of a light red color, that may be used to prepare a stain for rapid reversible detection of protein bands on nitrocellulose or polyvinylidene fluoride (PVDF) membranes, as well as on cellulose acetate membranes. A Ponceau S stain is useful because it does not appear to have a deleterious effect on the sequencing of blotted polypeptides and is therefore one method of choice for locating polypeptides on western blots for blot-sequencing. It is also easily reversed with water washes, facilitating subsequent immunological detection. The stain can be completely removed from the protein bands by continued washing. Common stain formulations include 0.1% (w/v) Ponceau S in 5% acetic acid or 2% (w/v) Ponceau S in 30% trichloroacetic acid and 30% sulfosalicylic acid.

<span class="mw-page-title-main">Hematein</span> Chemical compound

Hematein or haematein is an oxidized derivative of haematoxylin, used in staining. Haematein should not be confused with haematin, which is a brown to black iron-containing pigment formed by decomposition of haemoglobin. In the Colour Index, haematein is called haematine.

<span class="mw-page-title-main">Masson's trichrome stain</span> Biological staining procedure used in study of tissues

Masson's trichrome is a three-colour staining procedure used in histology. The recipes emerged from Claude L. Pierre Masson's (1880–1959) original formulation have different specific applications, but all are suited for distinguishing cells from surrounding connective tissue.

<span class="mw-page-title-main">Aniline Blue WS</span> Chemical compound

Aniline Blue WS, also called aniline blue, diphenylamine blue, China blue, or Soluble blue, is a mixture of methyl blue and water blue. It may also be either one of them. It is a soluble dye used as a biological dye, in fluorescence microscopy, appearing a yellow-green colour after excitation with violet light. It is a mixture of the trisulfonates of triphenyl rosaniline and of diphenyl rosaniline.

<span class="mw-page-title-main">Water blue</span> Chemical compound

Water blue, also known as aniline blue, Acid blue 22, Soluble Blue 3M, Marine Blue V, or C.I. 42755, is a chemical compound used as a stain in histology. Water blue stains collagen blue in tissue sections. It is soluble in water and slightly soluble in ethanol.

<span class="mw-page-title-main">H&E stain</span> Histological stain method

Hematoxylin and eosin stain is one of the principal tissue stains used in histology. It is the most widely used stain in medical diagnosis and is often the gold standard. For example, when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E.

<span class="mw-page-title-main">Phosphotungstic acid</span> Chemical compound

Phosphotungstic acid (PTA) or tungstophosphoric acid (TPA), is a heteropoly acid with the chemical formula H3PW12O40]. It forms hydrates H3[PW12O40nH2O. It is normally isolated as the n = 24 hydrate but can be desiccated to the hexahydrate (n = 6). EPTA is the name of ethanolic phosphotungstic acid, its alcohol solution used in biology. It has the appearance of small, colorless-grayish or slightly yellow-green crystals, with melting point 89 °C (24 H2O hydrate). It is odorless and soluble in water (200 g/100 ml). It is not especially toxic, but is a mild acidic irritant. The compound is known by a variety of names and acronyms (see 'other names' section of infobox).

<span class="mw-page-title-main">Acid fuchsin</span> Chemical compound

Acid fuchsin or fuchsine acid, (also called Acid Violet 19 and C.I. 42685) is an acidic magenta dye with the chemical formula C20H17N3Na2O9S3. It is a sodium sulfonate derivative of fuchsine. Acid fuchsin has wide use in histology, and is one of the dyes used in Masson's trichrome stain. This method is commonly used to stain cytoplasm and nuclei of tissue sections in the histology laboratory in order to distinguish muscle from collagen. The muscle stains red with the acid fuchsin, and the collagen is stained green or blue with Light Green SF yellowish or methyl blue. It can also be used to identify growing bacteria.

<span class="mw-page-title-main">Phosphomolybdic acid</span> Chemical compound

Phosphomolybdic acid is the heteropolymetalate with the formula H3[Mo12PO40]·12H2O. It is a yellow solid, although even slightly impure samples have a greenish coloration. It is also known as dodeca molybdophosphoric acid or PMA, is a yellow-green chemical compound that is freely soluble in water and polar organic solvents such as ethanol. It is used as a stain in histology and in organic synthesis.

<span class="mw-page-title-main">Biebrich scarlet</span> Chemical compound

Biebrich scarlet is a molecule used in Lillie's trichrome.

Lillie's trichrome is a combination of dyes used in histology.

Infantile digital fibromatosis (IDF), also termed inclusion body fibromatosis, Reye tumor, or Reye's tumor, usually occurs as a single, small, asymptomatic, nodule in the dermis on a finger or toe of infants and young children. IMF is a rare disorder with approximately 200 cases reported in the medical literature as of 2021. The World Health Organization in 2020 classified these nodules as a specific benign tumor type in the category of fibroblastic and myofibroblastic tumors. IDF was first described by the Australian pathologist, Douglas Reye, in 1965.

<span class="mw-page-title-main">Perls Prussian blue</span> Histologic method to stain for iron

In histology, histopathology, and clinical pathology, Perls Prussian blue is a commonly used method to detect the presence of iron in tissue or cell samples. Perls Prussian Blue derives its name from the German pathologist Max Perls (1843–1881), who described the technique in 1867. The method does not involve the application of a dye, but rather causes the pigment Prussian blue to form directly within the tissue. The method stains mostly iron in the ferric state which includes ferritin and hemosiderin, rather than iron in the ferrous state.

Trichrome stains are staining methods in which three anionic dyes are used, in conjunction with either phosphomolybdic acid (PMA), phosphotungstic acid (PTA), or a mixture of these heteropolyacids. Probably the first trichrome method was that of Frank B Mallory, an American pathologist, first published in 1900. Unfortunately, none of Mallory's publications provide any explanation of the rationales of either his trichrome or his phosphotungstic acid-haematoxylin (PTAH) method. Nobody knows why Mallory introduced heteropolyacids into microtechnique.

Bouin solution, or Bouin's solution, is a compound fixative used in histology. It was invented by French biologist Pol Bouin and is composed of picric acid, acetic acid and formaldehyde in an aqueous solution. Bouin's fluid is especially useful for fixation of gastrointestinal tract biopsies because this fixative allows crisper and better nuclear staining than 10% neutral-buffered formalin. It is not a good fixative when tissue ultrastructure must be preserved for electron microscopy. However, it is a good fixative when tissue structure with a soft and delicate texture must be preserved. The acetic acid in this fixative lyses red blood cells and dissolves small iron and calcium deposits in tissue. A variant in which the acetic acid is replaced with formic acid can be used for both fixation of tissue and decalcification. The effects of the three chemicals in Bouin solution balance each other. Formalin causes cytoplasm to become basophilic but this effect is balanced by the effect of the picric acid. This results in excellent nuclear and cytoplasmic H&E staining. The tissue hardening effect of formalin is balanced by the soft tissue fixation of picric and acetic acids. The tissue swelling effect of acetic acid is balanced by the tissue shrinking effect of picric acid.

Verhoeff's stain, also known as Verhoeff's elastic stain (VEG) or Verhoeff–Van Gieson stain (VVG), is a staining protocol used in histology, developed by American ophthalmic surgeon and pathologist Frederick Herman Verhoeff (1874–1968) in 1908. The formulation is used to demonstrate normal or pathologic elastic fibers.

References

Commons-logo.svg Media related to Trichrome staining at Wikimedia Commons