Stains and dyes have played a vital part in most all laboratory disciplines. They have been instrumental in "rendering color" to cells, cell constituents, entities and artifacts for clear visibility through microscopy.
Antoni van Leeuwenhoek used one of the first true stains, a solution of saffron in alcohol to color muscle fibers. Reichel used plant extracts to color plant tissues in 1758 and Hill introduced cochineal a few years later. What a difference a dye makes in rendering color to almost all types of cells and tissue structures in the diagnosis of normal and diseased tissues.
Fixation of tissue sections and cytology preparations also plays a vital role in the proper staining of tissue entities, microorganisms and structures. Fixation is the key that unlocks the door to quality stains. Ten percent neutral formalin is the most universal fixative that allows numerous stains to be performed and demonstrate specific tissue entities and components. Stain procedures and protocols usually state the recommended fixative for the proper demonstration of a structure. Many entities and tissue structures can not be demonstrated unless proper fixation is performed.
The Biological Stain Commission (BSC) serves the dye industry through the certification of stain and dye powders by chemical analysis and assessment of their use for biological and medical purposes. By certification and assay testing, quality dyes are brought to market that are guaranteed to perform well in specific staining applications and to ensure lot-to-lot consistency. Upon certification the BSC seal is placed on the bottle of dye powder (Fig. 1).
Special stains that incorporate silver nitrate solutions are really "impregnations." The silver ions deposit silver onto the tissue entities such as fungi, reticulum fibers and membranes.
Reticulum or reticulin are usually referred to as "young collagen." The reticular fibers form delicate networks and are smaller than collagen. The reticulum stain is useful in demonstrating reticulin in the diagnosis of hepatocellular carcinoma. A positive reticulin network can be preserved within the tumor; however, the absence or decreased reticulin stain or even an abnormal reticulin pattern with widened trabeculas aides in the diagnosis of a well-differentiated hepatocellular carcinoma. Reticulum stains can highlight the growth patterns of neoplasm or new growths (Fig. 2).
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The Gomori's Methenamine stain or Grocott's Methenamine Silver stain is used for many fungi and the protozoa Pneumocystis jiroveci. The cell walls are stained demonstrating a black color (Fig. 3) and even a kinetoplast can be demonstrated in the Pneumocystis jiroveci organism. Spirochetes are filamentous microorganisms that can be demonstrated by either the Warthin Starry silver stain or the Steiner silver stain. These stains are used to demonstrate spirochetes, usually in the tertiary stage of syphilis. The Jones Methenamine Silver stain is useful in many kidney diseases and is used particularly to demonstrate the basement membrane around the glomerus and other components of the kidney. Melanin pigment can often be demonstrated by the Fontana Masson silver stain.
Several mucin stains can be used to demonstrate types of mucin and mucosubstances in tissue sections. The Mayer's or Southgate's Mucicarmine stain is very specific for epithelial mucins (Fig. 4). Periodic Acid Schiff techniques will demonstrate glycogen, mucin, neutral mucins and some simple acid mucins. Alcian Blue techniques will stain some acid mucins at pH 2.5 and others at pH 1.0. In Alcian Blue techniques the pH is critical to the proper demonstration of certain types of mucins and mucosubstances. Colloidal Iron (AMP) techniques will also demonstrate mucin but require formalin fixation. The pH is critical and can be adjusted to give more specificity. The blue color is demonstrated by a Prussian Blue reaction.
Melanin is a "coloring" pigment found in the skin, eye and the substantia nigra. Melanocytes are cells that produce the dark pigment. A melanoma is a malignant tumor of the melanocytes and is usually found in the skin, but may also appear in other parts of the body, such as the small bowel or rectum.
The Schmorl's method demonstrates melanin by reducing the properties of melanin to stain the granules a blue-green color. Lillie's Ferrous Ion Uptake method will also demonstrate melanin. Iron stains can be used in a melanoma panel to rule out iron pigment since both melanin and iron can be brown in color in tissue sections.
Immunohistochemistry is often used to demonstrate melanin pigment in conjunction with special stains, such as the S-100 or Melanin A antibodies.
Hemosiderin or storage of iron granules can be seen in old hemorrhage or in the disease hemochromatosis. Bone marrow cores are often stained with an iron stain to demonstrate iron stores. Hemochromatosis is a condition of excess iron or iron overload that is associated with organ failure. Tissues affected have a high iron content and stain very positive, even "bleeding" into the connective tissue and stroma (Fig. 5). There are several staining techniques for iron but the Perl's method is the classic for demonstrating iron in tissue sections.
Copper pigment can be demonstrated in tissue sections and is usually seen in the autosomal recessive disorder Wilson's disease. The accumulation of copper is most often seen in the liver but also can be seen in the brain and the eye. Hepatic copper abundance can result in fatty changes, acute hepatitis chronic hepatitis and cirrhosis. The rubeanic acid and rhodanine stains are used to demonstrate the cytoplasmic accumulation of copper pigment in the liver, which stains a "penny red" color.
Connective Tissue Stains
Connective tissue consists of fibers such as collagen, elastic and reticulum; cells such as fibroblasts, adipose, mast cells, plasma cells, blood cells and macrophages and amorphous ground substance.
The Masson Trichrome stain can demonstrate collagen and connective tissue and can also aide in the demonstration of tissue injury. In forensics, the trichrome stain can demonstrate the entrance or exit of a gunshot wound. Diseases such as liver cirrhosis, cerebral abscess in the brain and acute myocardial infarction in the heart can be demonstrated with the trichrome stain. Normal structures such as bronchovascular structures, connective tissue capsules or increases in collagen can also be seen with the trichrome stain (Fig. 6).
The Movat Pentachrome is a combination of five stains that will demonstrate collagen, connective tissue, elastic fibers, muscle and mucin. Elastic stains demonstrate elastic fibers and outlines arteries. In some lung tumors, an elastic stain may be implemented to demonstrate the invasion of a tumor or in anastomosis case where new blood vessels may be forming. Periodic Acid Schiff can be used to demonstrate glycogen in Ewing's sarcoma or nodular glomerulosclerosis in kidney diseases.
Fungi, bacteria and viral inclusions are demonstrated in tissue sections, smears and cytologic preparations. The Periodic Acid Schiff stain is helpful for the demonstration of many fungi. The Grocott's Methenamine Silver is the most sensitive method for most fungi.
Bacteria are best demonstrated with the Brown and Brenn gram stain, demonstrating the gram-positive bacteria a blue-purple color and the gram-negative bacteria a red color. The Gram Twort's Gram stain is also helpful in demonstrating bacteria. The Stainer stain can be used as a "screening" stain for many types of bacteria. The Giemsa techniques can demonstrate Donovan Bodies, Leishmania and malaria. The Acid Fast technique Ziehl-Neelsen or Kinyoun stain is valuable to demonstrate the lipid cell walls of M. tuberculosis, cryptosporidium or isospora (Fig. 7). Fite-Faraco will demonstrate M. leprae in dermatology cases and Nocardia asteroids in lung and brain.
Urate or uric acid crystals can be seen in urine and tissue sections. They are soluble in aqueous solutions and must be fixed in 100 percent alcohol. The crystals can be seen with the H and E stain and should be polarized, and the Gomori's Methenamine Silver stain will demonstrate the urate crystals a black color.
Hematoxylin and Eosin
Hematoxylin and Eosin (H and E) stains can demonstrate tissue entities, structures or artifacts in tissue sections. The H and E is a special stain for the nucleus and cytoplasm and can provide accurate microscopic evaluation of the architecture of tissues and cells. Microorganisms such as some fungi and Helicobacter can be seen with the H and E, but often times are documented with a special stain. Some fungal organisms can be initially seen with the H and E stain, and it can accurately diagnose bone disorders and tumors.
Antibodies are often used to demonstrate normal and abnormal structures in tissue sections. Although immunohistochemistry is referred to as stains, it really refers to expressions. IHC is comprised of proteins that are stacked on top of proteins and visualized by a coloring component known as a chromagen, which is a dye substance such as DAB, AEC or alkaline phosphatase (Fig. 8). IHC is often used to determine the origin, prognosis and treated of a tumor. The knowledge required is critical to the performance of IHC, all to include fixation, processing, microtomy, quality control, technique and an understanding of antibodies.
Patient diagnosis for tissue and cytology specimens may change with present and future technology and automation, yet the H and E, special stains and immunohistochemistry will remain as diagnostic tools for patient care.
Lamar Jones is with the Pathology Department, Emory University Hospital, Atlanta, and HT program director, School of Histotechnology, Davidson County Community College, Lexington, NC.
1. Carson, FL, & Hladik, C. Histotechnology A Self Instructional Text, 3rd edition. ASCP Press, Chicago, 2009.
2. Jones, ML. Interpretation of Special Stains, Scientific Workshop, 2010.
3. Jones, ML. How Formalin Effects the Outcome of the H and E and Special Stains, Biological Stain Commission lecture, 2007.
4. Penny, DP, Frank, M., Willis, C. Stain and Dye Stability Over a 30-year Period: A Comparsion of Certified Dye Powders by the Biological Stain Commission. Biotechnic and Histochemistry, October 2009, 84(1): 11-15.