Special Stains in Histology

Mucin stains

There are a variety of mucin stains, all attempting to demonstrate one or more types of mucopolysaccharide substances in tissues. The types of mucopolysaccharides are as follows:

• Neutral - These can be found in glands of the GI tract and in prostate. They stain with PAS but not with Alcian blue, colloidal iron, mucicarmine, or metachromatic dyes.
• Acid (simple, or non-sulfated) - Are the typical mucins of epithelial cells containing sialic acid. They stain with PAS, Alcin blue at pH 2.5, colloidal iron, and metachromatic dyes. They resist hyaluronidase digestion.
• Acid (simple, mesenchymal) - These contain hyaluronic acid and are found in tissue stroma. They do not stain with PAS, but do stain with Alcian blue at pH 2.5, colloidal iron, and metachromatic dyes. They digest with hyaluronic acid. They can be found in sarcomas.
• Acid (complex, or sulfated, epithelial) - These are found in adenocarcinomas. PAS is usually positive. Alcian blue is positive at pH 1, and colloidal iron, mucicarmine, and metachromatic stains are also positive. They resist digestion with hyaluronidase.
• Acid (complex, connective tissue) - Found in tissue stroma, cartilage, and bone and include substances such as chondroitin sulfate or keratan sulfate. They are PAS negative but do stain selectively with Alcian blue at pH 0.5.

There are a variety of stains for mucin:

• Colloidal iron ("AMP") - Iron particles are stabilized in ammonia and glycerin and are attracted to acid mucopolysaccharides. It requires formalin fixation. Phospholipids and free nucleic acids may also stain. The actual blue color comes from a Prussian blue reaction. Tissue can be pre-digested with hyaluronidase to provide more specificity.
• Alcian blue - The pH of this stain can be adjusted to give more specificity.
• PAS (peroidic acid-Schiff) - Stains glycogen as well as mucins, but tissue can be pre-digested with diastase to remove glycogen.
• Mucicarmine - Very specific for epithelial mucins.

The mucin stain with the most specificity is mucicarmine, but it is very insensitive, so it is not really very useful. The stain that is the most sensitive is PAS, but you must learn how to interpret it in order to gain specificity. Colloidal iron stains are unpredictable. Alcian blue stains are simple, but have a lot of background staining.

Stains for biogenic amines

Cells that produce polypeptide hormones, active amines, or amine precursors (epinephrine, norepinephrine) can be found individually (Kulchitsky cell of GI tract) or as a group (adrenal medulla). The following is a traditional classification of the staining patterns based upon the ability of the cells to reduce ammoniacal silver nitrate to metallic silver (black deposit in tissue section):

• Chromaffin
• Argentaffin
• Argyrophil (pre-reduction step necessary)

The distinction between chromaffin and argentaffin is artificial, since this depends upon the fixative used. "Chromaffin" cells have cytoplasmic granules that appear brown when fixed with a dichromate solution. "Argentaffin" cells reduce a silver solution to metallic silver after formalin fixation. Either reaction can be produced depending upon which fixative was used. Traditionally, chromaffin reaction is associated with adrenal medulla or extraadrenal paraganglion tissues (pheochromocytomas) whereas argentaffin reaction is associated with carcinoid tumors of the gut. Using a pre-reduction step may get more cells to stain, but they are called "argyrophil" then.

Types of stains for argentaffin include:

1. Diazo (diazonium salts)
2. Fontana-Masson
3. Schmorl's
4. Autofluorescence

Types of stains for chromaffin include:

1. Modified Giemsa
2. Schmorl's
3. Wiesel's

Types of stains for argyrophil include:

1. Grimelius (Bouin's fixative preferred)
2. Pascual's

Melanin stains

Melanin is normally found in the skin, eye, and substantia nigra. It may also be found in melanomas.

The commonly used Fontana-Masson ("melanin stain") method relies upon the melanin granules to reduce ammoniacal silver nitrate (but argentaffin, chromaffin, and some lipochrome pigments also will stain black as well).

Schmorl's method uses the reducing properties of melanin to stain granules blue-green.

The most specific method of all is an enzyme histochemical method called DOPA-oxidase. It requires frozen sections for best results, but paraffin sections of well-fixed tissues may be used. The stain works because the DOPA substrate is acted upon by DOPA-oxidase in the melanin-producing cells to produce a brownish black deposit.

Bleaching techniques remove melanin in order to get a good look at cellular morphology. They make use of a strong oxidizing agent such as potassium permanganate or hydrogen peroxide. Ocular melanin takes hours to bleach, while that from skin takes minutes.

Formaldehyde-induced fluorescence can be used to highlight biogenic amines (chromaffin, argentaffin) and melanin in tissues. Formalin fixation imparts a strong yellow autofluorescence to unstained tissues with these substances.

The pseudomelanin of melanosis coli is PAS positive whereas true melanin is not. Moreover, pseudomelanin pigment is usually found in macrophages.

Melanin pigment in cells of malignant melanoma, Fontana-Masson stain.

Lipochrome (lipofuschin) pigments

These are the breakdown products within cells from oxidation of lipids and lipoproteins. They are the wear-and-tear pigments found most commonly in heart, liver, CNS, and adrenal cortex (zona reticularis). The less highly oxidized "ceroid" pigment of testis interstitium and seminal vesicle is another form of lipochrome.

Lipochrome can be stained by Sudan black B, long Ziehl-Neelson acid fast, and Schmorl's methods. Lipochrome may also exihibit a strong orange autofluorescence in formalin-fixed, unstained paraffin sections.

Lipochrome in liver, H and E stain.

Iron (hemosiderin)

Hemosiderin (storage iron granules) may be present in areas of old hemorrhage or be deposited in tissues with iron overload (hemosiderosis is the term used if the iron does not interfere with organ function; hemochromatosis refers to a condition of iron overload associated with organ failure).

Perl's iron stain is the classic method for demonstrating iron in tissues. The section is treated with dilute hydrochloric acid to release ferric ions from binding proteins. These ions then react with potassium ferrocyanide to produce an insoluble blue compound (the Prussian blue reaction). Mercurial fixatives seem to do a better job of preserving iron in bone marrow than formalin.

Hemosiderin, liver, iron stain.

Calcium

Only calcium that is bound to an anion (such as PO4 or CO3) can be demonstrated. Calcium forms a blue-black lake with hematoxylin to give a blue color on H&E stain, usually with sharp edges.

VonKossa stain is a silver reduction method that demonstrates phosphates and carbonates, but these are usually present along with calcium. This stain is most useful when large amounts are present, as in bone.

Alizarin red S forms an orange-red lake with calcium at a pH of 4.2. It works best with small amounts of calcium (such as in Michaelis-Gutman bodies). The alizarin method is also used on the Dupont ACA analyzer to measure serum calcium photometrically.

Azan stain can be used to differentiate osteoid from mineralized bone.

Urates

Uric acid crystals are seen in acid urine. In tissue, urates are present as sodium urate. They are soluble in aqueous solutions and slightly soluble in weak alcoholic solutions. Therefore, tissues must be fixed in 95% or absolute alcohol to prevent leaching of urates.

Methenamine silver stains urates black. Sodium urate crystals are also birefringent on polarization. Using a red plate, the crystals show negative birefringence (yellow color) when the crystal's long axis is aligned in the direction of the slow wave. At 90 degrees to this, the crystals will be blue.

Uric acid crystals, polarized, with red plate.

Exogenous pigments and minerals

These come from industrial or environmental exposure by inhalation, ingestion, or contact. Sometimes exposure comes from work-related activities (miners). Sometimes they are planned (tattoo).

Carbon appears as anthracotic pigment in the lungs. It can be distinguished from melanin by doing a melanin bleach. Poorly fixed tissues may contain formalin-heme pigment, which is black and finely granular, but this is widely scattered in the tissues without regard to cellular detail. Formalin-heme pigment is also birefringent on polarization.

Asbestos is a special type of long-thin silica crystal, usually of the mineral group chrysotile. In tissue, these crystals are highly irritative and highly fibrogenic. The fibers become coated with a protein-iron-calcium matrix, giving them a shish-kebab appearance. These are called "ferruginous bodies" because they are highlighted with an iron stain.

Asbestos body, unstained.

Asbestos bodies, iron stain.

Silica is present in many minerals and building materials. Most forms are very inert and cannot be stained in tissue but can be demonstrated by white birefringence on polarization. It is most often present in lung, but can make its way into lymph node.

Silica crystals in silicosis of lung, polarized.

Street drugs for injection often are diluted with compounds containing minerals such as silica or talc. These crystals can be found throughout the body, but especially in lymphoreticular tissues.

Polarizable crystals in lung with intravenous drug use.

Tattoo pigment is usually black and is inert and non-polarizable. Red tattoo pigment often contains cinnabar (which has mercury in it).

Tattoo pigment in dermis of skin, H and E stain.

In general, minerals are best demonstrated by microincineration techniques or by scanning electron microscopy with energy dispersive analysis (SEM-EDA).

Another use for SEM-EDA in forensic pathology is for analysis of gunshot residue. The primer residue has a characteristic pattern because of the elemental composition which contains antimony, barium, and lead.

SEM-EDA pattern of gunshot residue.

Fat stains

The oil red O (ORO) stain can identify neutral lipids and fatty acids in smears and tissues. Fresh smears or cryostat sections of tissue are necessary because fixatives containing alcohols, or routine tissue processing with clearing, will remove lipids. The ORO is a rapid and simple stain. It can be useful in identifying fat emboli in lung tissue or clot sections of peripheral blood.

Oil red O stain of fat emboli in lung.

Connective tissue stains

The trichrome stain helps to highlight the supporting collagenous stroma in sections from a variety of organs. This helps to determine the pattern of tissue injury. Trichrome will also aid in identifying normal structures, such as connective tissue capsules of organs, the lamina propria of gastrointestinal tract, and the bronchovascular structures in lung.

Chronic active hepatitis with collapse in liver, trichrome stain.

Cerebral abscess in brain, trichrome stain.

Scleroderma with fibrosis of submucosa in stomach, trichrome stain.

The reticulin stain is useful in parenchymal organs such as liver and spleen to outline the architecture. Delicate reticular fibers, which are argyrophilic, can be seen. A reticulin stain occasionally helps to highlight the growth pattern of neoplasms.

Normal liver at medium magnification, reticulin stain.

Normal spleen at high magnification, reticulin stain.

An elastic tissue stain helps to outline arteries, because the elastic lamina of muscular arteries, and the media of the aorta, contain elastic fibers. The van Gieson method for elastic fibers provides good contrast.

Normal aorta, media with parallel elastic fibers, van Gieson stain.

Giemsa stain

There are a variety of "Romanowsky-type" stains with mixtures of methylene blue, azure, and eosin compounds. Among these are the giemsa stain and the Wright's stain (or Wright-Giemsa stain). The latter is utilized to stain peripheral blood smears. The giemsa stain can be helpful for identifying components in a variety of tissues.

One property of methylene blue and toluidine blue dyes is metachromasia. This means that a tissue component stains a different color than the dye itself. For example, mast cell graules, cartilage, mucin, and amyloid will stain purple and not blue, which is helpful in identifying these components.

Skin with mast cells in dermis, giemsa stain.

Esophagus with eosinophils, giemsa stain.

Peripheral blood smear, Wright's stain.

Microorganisms

Bacteria appear on H and E as blue rods or cocci regardless of gram reaction. Colonies appear as fuzzy blue clusters. Tissue gram stains are all basically the same as that used in the microbiology lab except that neutral red is used instead of safranin. Gram positive organisms usually stain well, but gram negatives do not (because the lipid of the bacterial walls is removed in tissue processing). Brown and Brenn (or the Brown and Hopps modification) is the method most commonly used.

Fungi stain blue with H and E and red with PAS. The most sensitive method for demonstrating them is Methenamine silver.

Spirochetes are very difficult to stain. The best method is the Warthin-Starry. A Giemsa stain may help demonstrate donovan bodies and leishmania.

Spirochetes with Warthin-Starry silver stain.

AFB (acid fast bacilli) stain

This stain uses carbol-fuchsin to stain the lipid walls of acid fast organisms such as M. tuberculosis . The most commonly used method is the Ziehl-Neelsen method, though there is also a Kinyoun's method. A modification of this stain is known as the Fite stain and has a weaker acid for supposedly more delicate M. leprae bacilli. However, much of the lipid in mycobacteria is removed by tissue processing, so this stain can, at times, be very frustrating and cause you to search extensively for organsisms you are sure are in a big granuloma. The most sensitive stain for mycobacteria is the auramine stain which requires a fluorescence microscope for viewing.

There are things other than mycobacteria that are acid fast. Included are cryptosporidium, isospora, and the hooklets of cysticerci.

Mycobacterium tuberculosis in lung, Ziehl-Neelsen acid fast stain.

Mycobacterium avium-complex, Ziehl-Neelsen acid fast stain.

Cryptosporidium in stool specimen, Ziehl-Neelsen acid fast stain.

Mycobacterium tuberculosis in lung, Auramine stain, fluorescence.

Nocardia asteroides in lung, acid fast stain.

Gomori methenamine silver stain

This stain, often abbreviated as "GMS", is used to stain for fungi and for Pneumocystis carinii . The cell walls of these organisms are stained, so the organisms are outlined by the brown to black stain. There is a tendency for this stain to produce a lot of artefact from background staining, so it is essential to be sure of the morphology of the organism being sought.

GMS stain for Pneumocystis carinii.

GMS stain for Cryptococcus neoformans.

GMS stain for Coccidioides immitis.

PAS (periodic acid-Schiff)

This an all-around useful stain for many things. It stains glycogen, mucin, mucoprotein, glycoprotein, as well as fungi. A predigestion step with amylase will remove staining for glycogen. PAS is useful for outlining tissue structures--basement membranes, capsules, blood vessels, etc. It does stain a lot of things and, therefore, can have a high background. It is very sensitive, but specificity depends upon interpretation.

Candida in lung, PAS stain.

Glycogen in Ewing's sarcoma, PAS stain.

Nodular glomerulosclerosis in kidney, PAS stain.