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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.

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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 (
 

Steps to Better Special Stains

At Leica Biosystems our vision is to advance cancer diagnostics and improve lives. One way we can achieve this vision is by helping improve staining quality. As we recognize that IHC and ISH quality doesn‘t begin at the stainer, this series looks at many different aspects of staining quality, and considers how future tests will influence improved diagnosis.

Step 64 - Understand the Stain

Know what you are trying to demonstrate with the stain you are performing.

Just “following the method” and not really knowing what should be seen in the finished section will lead to poor results.

A section of liver stained with PAS. Lipofuscin and glycogen are PAS positive while traces of bile and hemosiderin are PAS negative and appear in their natural colors (yellow and brown respectively).
B This section shows an opportunistic fungal infection in lung (Aspergillus) stained with the Grocott-Gomori method. Fungal hyphae are black as is unstained carbon, a common feature in the lungs of smokers and most city dwellers.
Understand the Stain

Step 65 - Use a Positive Control

Always use a control slide known to contain the structure/ substance you are trying to demonstrate.

 “If the structure/substance we are staining for is not visible in a slide we assume it is not present.”

A section of cirrhotic liver stained with Perl’s method to demonstrate iron-containing hemosider in (blue). This would make a satisfactory control block for iron stains.
Use a Positive Control

Step 66 - Use Accurate Timing

 Use accurate timing.

Timing is always approximate. Inaccurate timing produces inconsistent results.

Both these sections of skin from the same block have been stained with the PAS method. Section A was treated with periodic acid (oxidation step) for 5 minutes where as section B had only 30 seconds (a mistake). Note that the basement membrane is very poorly stained in section B as a consequence.
Use Accurate Timing

Step 67 - Consider Reagent Stability

Be aware of the shelf life of the reagents you are using. Some reagents or dye solutions deteriorate slowly while others are very unstable and must be made up fresh and used immediately. Others have to be left for some time to oxidize (ripen) before they can be used at all.

We assume all reagents can be used for an indefinite period.

Muddy Weigert’s hematoxylin due to overoxidation. Note the brown staining of collagen.
Consider Reagent Stability

Step 68 - Store Reagents Correctly

Store reagents correctly. Some require refrigeration because they are inclined to support the growth of fungi or molds. Others are light sensitive and require storage in the dark.

 “All our reagents are stored on the shelf above the staining bench. Sometimes we see stray organisms in our sections.”

This section shows large deposits of extraneous microorganisms which have grown in the staining solution (in this case hematoxylin) then been deposited on top of the section.
Store Reagents Correctly

Step 69 - Adhere to the Method

Follow the protocol exactly.

Staff members achieve different results when supposedly using the same protocol.

These sections of formalin-fixed submucosa have been stained with Masson trichrome stain. Section A shows red smooth muscle. In this case the stain was performed correctly following the lab protocol and including a preliminary chromic acid step (sensitization or secondary mordanting). This step was overlooked when section B was being stained. Note the lack of differential coloration of muscle in section B (intestine).
Adhere to the Method

Step 70 - Record Any Changes

Document any departure from the method you are using.

Sometimes when results are poor it is difficult or impossible to work out why because protocol changes have not been recorded.

In this silver impregnation stain for reticulin the fibers are poorly demonstrated and there is a background scum (precipitate) on the slide. It is very difficult to determine the cause of such a problem if the method has not been followed exactly (Gordon & Sweets method, kidney).
Record Any Changes

Step 71 - Standardize Washing Steps

Take particular care with washing steps. Standardize them as far as possible as they are frequently the cause of variable results.

Lab staff members use different washing techniques – some use vigorous agitation, others are much more gentle.

These liver sections were stained by the same method. The only difference between them was the technique by which they were rinsed between impregnation and reduction. The reticulin fibers are black and better defined in section A (Gordon & Sweets method).
Standardize Washing Steps

Step 72 - Set Up Microscope Carefully

Use microscopic control at crucial stages such as differentiation steps. Be aware of the effect of the microscope setup on the appearance of un-coverslipped (wet) sections; it can produce the appearance of false background staining.

For all methods the level of staining is assessed by looking at the slide with the naked eye.

 

 

 

 

A Wet section (no coverslip) viewed under a microscope with closed condenser diaphragm. Note the false background.
B Wet section (no coverslip) viewed under a microscope with open condenser diaphragm. Note the clear background.
Set Up Microscope Carefully

Click here to download the full 101 Steps to Better Histology

This reference document is presented as a service to health care professionals by Leica Biosystems and has been compiled from available literature. Although every effort has been made to report faithfully the information, Leia Biosystems cannot be held responsible for the correctness. This document is not intended to be, and should not be construed as medical advice. For any use, the product information guides, inserts and operation manuals of the various drugs and devices should be consulted. Leica Biosystems and the editors disclaim any liability arising directly or indirectly from the use of drugs, devices, techniques or procedures described in this reference document.

Copyright © 2016 Leica Biosystems, a division of Leica Microsystems Inc.
All rights reserved. LEICA and the Leica Logo are registered trademarks of Leica Microsystems IR GmbH.

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