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An Introduction to Routine and Special Staining

Routine H&E staining and special stains play a critical role in tissue-based diagnosis or research. By colouring otherwise transparent tissue sections, these stains allow highly trained pathologists and researchers to view, under a microscope, tissue morphology (structure) or to look for the presence or prevalence of particular cell types, structures or even microorganisms such as bacteria.

In the histopathology laboratory, the term “routine staining” refers to the hematoxylin and eosin stain (H&E) that is used “routinely” with all tissue specimens to reveal the underlying tissue structures and conditions. The term “special stains” has long been used to refer to a large number of alternative staining techniques that are used when the H&E does not provide all the information the pathologist or researcher needs.

Preparing Tissue for Staining

Before tissue can be stained and viewed, it must be prepared so that a very thin section, only one cell thick, can be cut and placed onto a microscope slide. This involves fixing the tissue (so it does not decay) then hardening and supporting it so that it can be cut to the very thin sections  needed (typically 2–7 µm). There are two main techniques used for this, referred to as frozen sections and paraffin-embedded sections.

Frozen sections are used when answers are needed fast, typically during surgery where the surgeon needs to know the excision margin when removing a tumour. They are quick to produce, but typically do not create the same section quality of as the paraffin technique.. The process for frozen section preparation is as follows:

  1. Tissue is quickly frozen to preserve and harden it.
  2. The frozen tissue is sectioned in cryostat (a sectioning microtome in a freezing chamber) and placed on a microscope slide for staining.
  3. The section is fixed immediately before it begins to decay and is then stained.

When paraffin sections are to be prepared the specimen is first preserved with a fixative and then the tissue structure is supported by infiltrating the specimen with paraffin wax. The process is more time-consuming than creating frozen sections, but provides better quality staining in most cases and the resultant samples (referred to as blocks) can be stored almost indefinitely. The paraffin section process is as follows:

  1. Fixation preserves the tissue (typically using a formaldehyde- based solution).
  2. Grossing isolates the particular area of tissue to be sectioned.
  3. Tissue processing uses a sequence of reagents to replace an aqueous (water-based) environment with a hydrophobic one enabling tissue elements to be infiltrated with paraffin wax.
  4. Embedding allows specimen orientation and secures the specimen in a block of wax for section cutting and storage.
  5. Sectioning is done on a microtome that cuts very fine sections which are floated-out on a water bath then picked up and placed on microscope slides.
  6. The slides are then dried in an oven or on a hot plate to remove moisture and help the tissue adhere to the slide.
  7. The tissue on the slide is now ready for staining.
  8. The first staining step is de-waxing which uses a solvent to remove the wax from the slide prior to staining. This is always done as part of the staining process. When a stain is complete the section is covered with a coverglass that makes the preparation permanent.
Figure 1: A microtomist creating a “ribbon” of very thin sections for staining
A microtomist creating a “ribbon” of very thin sections for staining

Why H&E Staining is Routine

Hematoxylin and Eosin (H&E) staining is used routinely in histopathology laboratories as it provides the pathologist/researcher a very detailed view of the tissue. It achieves this by clearly staining cell structures including the cytoplasm, nucleus, and organelles and extra-cellular components. This information is often sufficient to allow a disease diagnosis based on the organization (or disorganization) of the cells and also shows any abnormalities or particular indicators in the actual cells (such as nuclear changes typically seen in cancer). Even when advanced staining methods are used, the H&E stain still forms a critical part of the diagnostic picture as it displays the underlying tissue morphology which allows the pathologist/researcher to correctly interpret the advanced stain.

In a clinical histology laboratory, all specimens are initially stained with H&E and special or advanced stains are only ordered if additional information is needed to provide a more detailed analysis, for example to differentiate between two morphologically similar cancer types.

Because of the volume of H&E staining needed, most clinical laboratories use fully automated systems and manual staining is now rare.

Figure 2. This section from the mucosa of small intestine shows well-defined heterochromatin and nucleoli in epithelial cells and plasma cells within the lamina propria
This section from the mucosa of small intestine shows well-defined heterochromatin and nucleoli in epithelial cells and plasma cells within the lamina propria
Figure 3. Mitotic figures are sharply stained within the glandular epithelium in a section of small intestine
Mitotic figures are sharply stained within the glandular epithelium in a section of small intestine
Figure 4. In this field from the lamina propria of small intestine, the cytoplasm of plasma cells has stained with hematoxylin except for the pale peri-nuclear area, which corresponds with a well-developed Golgi apparatus
In this field from the lamina propria of small intestine, the cytoplasm of plasma cells has stained with hematoxylin except for the pale peri-nuclear area, which corresponds with a well-developed Golgi apparatus
Figure 5. This autonomic ganglion from the myenteric plexus, located between the smooth muscle layers of the muscularis externa of the small intestine, contains ganglionic nurones that show well-defined basophilic Nissl substance (aggregations of endoplasmic reticulum and ribosomal RNA) in their cytoplasm
This autonomic ganglion from the myenteric plexus, located between the smooth muscle layers of the muscularis externa of the small intestine, contains ganglionic nurones that show well-defined basophilic Nissl substance (aggregations of endoplasmic reticu

H&E Chemistry

The H&E stain uses two dyes, hematoxylin and eosin. This combination is used as the dyes stain different tissue elements.

Hematoxylin reacts like a basic dye with a purplish blue colour. It stains acidic, or basophilic, structure including the cell nucleous (which contains DNA and nucleoprotein), and organelles that contain RNA such as ribosomes and the rough endoplasmic reticulum.

Eosin is an acidic dye that is typically reddish or pink. It stains basic, or acidophilic, structures which includes the cytoplasm, cell walls, and extracellular fibres.

Dye origins

Hematoxylin is extracted from the logwood tree and purified. It is then oxidized and combined with a mordant (typically aluminium) to allow it to bind to the cell structures. Of the many hematoxylin preparations used in histology Gill’s hematoxylin, Harris's hematoxylin and Mayer's hematoxylin are the most popular.

Eosin is formed by a reaction between bromine and fluorescein. There are two eosin variants typically used in histology: eosin Y which is slightly yellowish and eosin B which is slightly bluish. Eosin Y is most popular.

Figure 6: Hematoxylin chemical structure
Hematoxylin chemical structure
Figure 6: Hematoxylin chemical structure
Hematoxylin chemical structure
Figure 7: Eosin Y chemical structure
Eosin Y chemical structure
Figure 7: Eosin Y chemical structure
Eosin Y chemical structure

Special Stains

The term special stains traditionally referred to any staining other than an H&E. It covers a wide variety of methods that may be used to visualize particular tissue structures, elements, or even microorganisms not identified by H&E staining.

Other methods of staining use immunohistochemistry or in situ hybridization to target specific proteins or DNA/RNA sequences. These methods were sometimes also included as members of the “special stains” family. However they are quite different in method and purpose and are now typically separated into a third category know as “advanced stains”.

While there are literally hundreds of special stains for all manner of purposes, only a few are used with any regularity in clinical histology. The variety of stains also means that special staining is not as automated as H&E staining. While many larger laboratories do use automated instruments for the more common stains, they still have an area for hand staining. The complexity of some stains also works against the uses of automation.

Some Common Special Stains

The images below illustrate some of the common special stains and their applications.

Figure 8: Masson's Trichrome (skin). This stain is intended for use in histological observation of collagenous connective tissue fibers in tissue specimens. It is used to assist in differentiating collagen and smooth muscle in tumors and assists in the detection of diseases or changes in connective/muscle tissue.
The Masson’s Trichrome Stain Kit is intended for use in histological observation of collagenous connective tissue fibers in tissue specimens. It is used to assist in differentiating collagen and smooth muscle in tumors and assists in the detection of di
Figure 9: Modified GMS Silver Stain (Left: Pneumocystis, lung) (Right: Aspergillus infection, lung). The Modified GMS Silver stain is intended for use in histological observation of fungi, basement membrane and some opportunistic organisms such as pneumocystis carinii in tissue specimens.
The Modified GMS Silver Stain Kit is intended for use in histological observation of fungi, basement membrane and some opportunistic organisms such as pneumocystis carinii in tissue specimens.
Figure 10: Periodic Acid Schiff (kidney). PAS staining is mainly used for staining structures containing a high proportion of carbohydrates such as glycogen,glycoproteins, proteoglycans typically found in connective tissues, mucus and basement membranes. Often used to stain kidney biopsies, liver biopsies, certain glycogen storage diseases in striated muscles and suspected fungal infections.
Periodic Acid Schiff (PAS) staining is mainly used for staining structures containing a high proportion of carbohydrates such as glycogen,glycoproteins, proteoglycans typically found in connective tissues, mucus and basement membranes. Often used to stain
Figure 11: Perls’ Prussian Blue Iron (liver). This stain is used to detect and identify ferric (Fe3+) iron in tissue preparations, blood smears,or bone marrow smears. Minute amounts of ferric iron (haemosiderin) are commonly found in bone marrow and in the spleen. Abnormal amounts of iron can indicate hemochromatosis and hemosiderosis.
Perls’ Prussian Blue Iron stains are used to detect and identify ferric (Fe3+) iron in tissue preparations, blood smears,or bone marrow smears. Minute amounts of ferric acid are commonly found in bone marrow and in the spleen. Abnormal amounts of iron c
Figure 12: Ziehl Neelsen (Acid Fast Bacillus, lung). This stain is used to detect and identify acid fast bacilli in tissue. Bacilli are rod-shaped bacterial organisms. A primary function of this stain is to identify tuberculosis in lung tissue.
Acid Fast Bacillus stains are used to detect and identify acid fast bacilli in tissue. Bacilli are rod-shaped bacterial organisms. A primary function of this stain is to identify tuberculosis in lung tissue, as well as other bacterial bacillus infected ti
Figure 13: Alcian Blue (intestine). Alcian Blue is normally prepared at pH 2.5 and is used to identify acid mucopolysaccharides and acidic mucins. Excessive amounts of non-sulfated acidic mucosubstances are seen in mesotheliomas, certain amounts occur normally in blood vessel walls but increase in early lesions of atherosclerosis.
Alcian Blue is normally prepared at an acidic pH of 2.5 and is used to identify acid mucopolysaccharides and acetic mucins. Excessive amounts of non-sulfated acidic mucosubstances are seen in mesotheliomas, certain amounts occur normally in blood vessel w
Figure 14: Alcian Blue and PAS (intestine). A stain that combines the properties of both Alcian Blue and Periodic Acid Schiff staining.
Alcian Blue and PAS combines the properties of both Alcian Blue and Periodic Acid Schiff staining.
Figure 15: Gomori Trichrome (blue) (submucosa). Trichrome stains are used to stain and identify muscle fibers, collagen and nuclei. They can be used to contrast skeletal, cardiac or smooth muscle. The Gomori Trichrome is a simplification of the more elaborate Masson trichrome stain and combine the plasma stain (chromotrope 2R) and connective tissue stain to provide a brilliant contrasting picture.
Gomori Trichrome Blue is used to stain and identify muscle fibers, collagen and nuclei. They can be used to contrast skeletal, cardiac or smooth muscle. The Gomori Trichrome is a simplification of the more elaborate stains and combines the plasma stain (c
Figure 16: Gomori Trichrome (green) (submucosa). Trichrome stains are used to stain and identify muscle fibers, collagen and nuclei. They can be used to contrast skeletal ,cardiac or smooth muscle. The Gomori Trichrome is a simplification of the more elaborate Masson stain and combines the plasma stain (chromotrope 2R) with the connective tissue stain to provide a brilliant contrasting picture.
Gomori Trichrome Green is used to stain and identify muscle fibers, collagen and nuclei. They can be used to contrast skeletal ,cardiac or smooth muscle. The Gomori Trichrome is a simplification of the more elaborate stains and combines the plasma stain (