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

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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

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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.

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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

Figure 3. Mitotic figures are sharply stained within the glandular epithelium in a section of small
intestine

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 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

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

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

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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

Figure 6: Hematoxylin chemical structure

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Figure 7: Eosin Y chemical structure

Figure 7: Eosin Y chemical structure

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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

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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.

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.

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.

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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.

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.

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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.

Figure 14: Alcian Blue and PAS (intestine). A stain that combines the properties of both Alcian Blue and Periodic Acid Schiff
staining.

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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.

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.

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