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:
There are a variety of stains for mucin:
- 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.
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.
- 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.
Gastrointestinal tract goblet cells stained with Alcian blue.
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):
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.
- Argyrophil (pre-reduction step necessary)
Types of stains for argentaffin include:
Types of stains for chromaffin include:
- Diazo (diazonium salts)
Types of stains for argyrophil include:
- Modified Giemsa
Gastrointestinal tract enteroendocrine cells stained with Fontana-Masson argentaffin stain.
- Grimelius (Bouin's fixative preferred)
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.
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.
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.
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.
The rare autosomal recessive disorder known as Wilson's disease results from decreased serum ceruloplasmin, the blood protein that transports serum copper. This leads to excessive copper accumulation in brain, eye, and liver. Hepatic copper accumulation results in fatty change, acute hepatitis, chronic hepatitis, and eventual cirrhosis. Urinary copper excretion is increased.
The rubeanic acid and rhodanine stains are utilized to detect the cytoplasmic accumulation of copper in the liver.
Copper accumulation in liver, medium power microscopic.
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