Special Stains: Important & Application
By: Dr. Rajaa Ali
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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.
Though molecular techniques have become increasingly important in this sophisticated approach to diagnosis, pathologists and histotechnologists still require an extensive arsenal of diagnostic tools so that they can provide a complete picture of a patient’s disease. Certainly, immunohistochemistry and flow cytometry will continue to play a key role, as will special staining techniques.
Special stains (histochemical methods) are used to help visualize and/or identify structures and substances in sections.
Special stains have two broad areas of application: research and diagnostic. In research, special stains are used as probes to identify certain chemical constituents in normal and abnormal cells. The information so obtained is used as a basis for further study and also as a baseline against which the results of special staining can be compared in diagnostic applications.Special stains belong to a diverse family of slide-based stains that rely on basic chemical reactions for microscopic visualization and general identification of various tissues, structures, cells, organelles, carbohydrates, minerals and microorganisms. Introduced to all college biology students through the simple bacterial test known as the Gram stain, special stains use both science and art to provide valuable and cost-effective information for the pathology laboratory.
The story of special stains begins several centuries ago in the early laboratories and workshops of physicians, chemists, botanists and businessmen who were just beginning to explore the world of cells and microorganisms. These histology pioneers dabbled with household goods, such as soap for embedding and wine for staining, to study diverse specimens under crude microscopes.
These early efforts enabled today’s widespread use of basic staining mechanisms for morphologic evaluation. Initial protocols were limited to coloring the cell’s nucleus and cytoplasm. However, as morphologic evaluation became the most important tool used by pathologists to guide patient treatment, physicians and scientists developed numerous staining procedures that now include a vast array of tinctorial stains.
Today, these stains augment tissue morphology, demonstrate important cellular components, aid in the evaluation of disease states, and identify pathogenic organisms.
In relation to their clinical relevance, special stains remain important tools for many pathologists, providing a powerful complement to immunohistochemistry, flow cytometry, in situ hybridization and other diagnostic technologies that ultimately define a patient’s medical profile. Many special stains are key to detecting and identifying pathogens, while others play an important role in diagnosing and monitoring cancer. Mucicarmine stains, for instance, demonstrate a poorly differentiated adenocarcinoma, Giemsa stains are useful in leukemia classification, and elastic stains gauge a tumor’s degree of vascular invasion. Other clinical applications for special stains cover a wide range of diseases. Iron stains can indicate hemochromatosis or iron deficiency, the Masson’s Trichrome stain demonstrates changes in collagen and muscle cells associated with cirrhosis, and the Periodic Acid-Schiff stain provides information about glycogen storage disease and lupus-associated renal disease.
Mucicarmine stain is used for visualization of neutral epithelial mucins in small intestine. The mucins are stained rose to red, nuclei are blue/black, and other tissue elements are yellow; Verhoeff’s elastic stain colors elastin fibers in skin black, nuclei appear blue/black, and collagen is red .Iron stain is used for visualization of ferric iron in liver sections, ferric depositis are stained blue, nuclei are red, and background tissue elements are pink to red; Masson’s trichrome stain differentiates between collagen and smooth muscle in the liver, with muscle staining red, collagen blue and fibrin pink; PAS stain is used for the visualization of mucopolysaccharides and glycogen in renal tissue. Mayer’s hematoxylin counterstains the nuclei blue. Listed below are a few examples of special stains :
Acid fast stains (various methods) for mycobacteria [Mycobacterium avium, lung]
Congo red for amyloid [Islet amyloid (polaroscopy)]
Gram stains (various methods) for bacteria [Corynebacterium bovis, skin]
Grocott’s methenamine silver (GMS) for fungi [Pneumocystis carinii, lung]
Jones methenamine silver for basement membrane and mesangium [Glomerulonephritis]
Luxol fast blue-PAS for demyelination [Demyelinating myelitis]
Masson’s trichrome for collagen & muscle [Renal arteritis, mouse]
Periodic acid-Schiff (PAS) [Hypersensitivity bronchitis]
Phosphotungstic acid-hematoxylin (PTAH) for fibrin [Glomerular thrombosis]
Prussian blue for ferric iron [Hemosiderin, spleen]
Sudan black for lipids & lipochrome pigments [Lipochrome pigment, heart]
Verhoeff s method for elastin [Renal arteritis]
Warthin-Starry method for bacteria [Helicobacter hepaticus, liver]
As the medical community demands greater standardization, special stain protocols have become increasingly automated. For today’s histology and anatomic pathology laboratories, the result is new levels of productivity, flexibility and environmental safety. With special staining techniques remaining an integral part of any pathologic diagnosis, this technology will continue to evolve.
References:
1. Anderson J. (2019): An Introduction to Routine and Special Staining.
2. Carson FL. Histotechnology: A self-instructional text. 2nd ed. Chicago: ASCP Press; 1997.
3. Disease information. 2003; Available at: http://www.cdc.gov/ncidod/dbmd/diseaseinfo.
4. Mitra A. P. (2010): Education Guide Special Stains and H & E ,Second Edition
5. Wulff Sonja Hafer Laurie, Cheles Mary, Couture Rick , Holliday Jamie M. , Smith Susie and Stanforth David A. ( 2004): Guide to Special Stains. DakoCytomation, Carpinteria, California, USA.