Special Stains in Dermatopathology

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1 Application Special Stains in Dermatopathology Jameel Ahmad Brown, MD Bruce R. Smoller, MD Fellow: Division of Dermatopathology University of Arkansas for Medical Sciences 4301 W. Markham St. Little Rock, AR, USA Chair: Department of Pathology University of Arkansas for Medical Sciences 4301 W. Markham St. Little Rock, AR, USA Histochemical Stains Dual staining with hematoxylin, which stains nuclei blue, and eosin, a pink cytoplasmic stain, is the mainstay in routine examination of cutaneous tissue under light microscopy. The ability to delineate differences in tissue type and cellular components is greatly enhanced by exploiting the inherent uptake properties of various tissues with these stains. When particular characteristics are not easily identified with hematoxylin and eosin, histochemical staining becomes of great value. Its merit is recognized diagnostically and economically, as histochemical staining is much less expensive than immunohistochemical staining and is readily available in virtually all dermatopathology practice settings. Commonly Used Special Stains Periodic Acid-Schiff (PAS) Periodic Acid-Schiff (PAS) is one of the most frequently employed special stains in the dermatopathology laboratory. A positive stain is perceived as purplish-red or magenta. PAS is used to demonstrate the presence of neutral mucopolysaccharides, especially glycogen. The process by which tissues are stained with PAS involves the oxidation of hydroxyl groups in 1,2 glycols to aldehyde and subsequent staining of the aldehydes with fuschin-sulfuric acid. PAS also has utility when predigestion with diastase (PASD) has been performed. The diastase removes glycogen from tissue sections but leaves other neutral mucopolysaccharides behind. PAS stain allows for the recognition and highlighting of basement membrane material and thickness, such as in lupus erythematosus. In addition, PASD accentuates fungal cell walls, helps identify the presence of glycogen in tumor cells versus lipid, and illuminates many PAS positive inclusion bodies, secretions, and granules. Non-cutaneous pathology finds PAS equally useful (Fig. 1). Alcian Blue Alcian blue facilitates visualization of acid mucopolysaccharides or acidic mucins. The staining procedure is performed at ph of 2.5 and 0.5 respectively. Positive staining is perceived as blue. The mucopolysaccharide differential staining pattern is created by and based on the principle that sulfated mucopolysaccharides, like chondroitin sulfate and heparan sulfate, will stain at both ph values while non-sulfated ones stain at a ph of 2.5 only. The most common non-sulfated mucopolysaccharide of the skin is hyaluronic acid, found normally in scant amounts in the papillary dermis, surrounding cutaneous appendages, and encircling the vascular plexuses. Alcian blue is also an excellent special stain when attempting to detect acidic mucin in neoplasms, inflammatory conditions, and dermatoses including granuloma annulare, lupus erythematosus, dermatomyositis, scleromyxedema, pre-tibial myxedema, scleredema, and follicular mucinosis. Additionally, sialomucin produced in extramammary Paget s disease demonstrates positive alcian blue staining. Of note, colloidal iron demonstrates a staining profile which is essentially indistinguishable from alcian blue, and the use of one over the other will be laboratorydependent; based on the familiarity of the operator with the nuances of each stain and the quality of results obtained. 156 Connection 2010

2 Figure 1. PAS-D showing fungal organisms in a case of onychomycosis. Figure 2. Fite stain demonstrating acid fast bacilli in a case of lepromatous leprosy. Connection

3 Elastic Tissue Stain (Verhoff von Gieson) The elastic tissue stain (ETS), a silver stain, is used to demonstrate and evaluate the quantity and quality of tissue elastic fibers. Positive staining is perceived as black. ETS serves as a dependable adjunct when evaluating tissue sections for entities under the umbrella of elastolytic granulomata; these diseases include actinic granuloma, atypical facial necrobiosis lipoidica, and granuloma multiforme. Similarly, when assessing a patient for anetoderma (focal dermal elastolysis) and cutis laxa, the elastic tissue stain aids in identifying complete elastolysis, fragmented elastic fibers in the papillary and mid-reticular dermis, and the morphology of existing elastic fibers. Anomalous distribution and organization of elastic-rich tissue, as in collagenoma and other variants of connective tissue nevi, can also be detected. Most recently, the elastic tissue stain has received a new place in the spotlight of the dermatopathology literature, primarily because of its utility in differentiating the invasive component of melanoma from a benign underlying or adjacent melanocytic nevus when the two are associated. Brown and Brenn Stain The Brown and Brenn stain is a tissue Gram stain that is a practical method of identifying and differentially staining bacteria and resolving them into two primary groups Gram-negative and Gram-positive. The baceteria s staining characteristics are based on the physical properties of their cell walls. Gram-positive bacteria have thick cell walls composed of high proportions of peptidoglycan which retain the purple color during the staining process. Peptidoglycan comprises only a small fraction of the thin cell walls of Gram-negative bacteria which also have a lipid outer membrane; both characteristics contribute to their pink color as the purple dye (crystal violet) is washed away during the decolorization step. In addition to the color contrast created by the stain, morphology can also be elucidated and one can appreciate bacilli, cocci, or coccobacilli. The tissue Gram stain is not ideal for all bacterial species, as some have ambiguous staining patterns or are simply not highlighted. The Brown and Brenn stain is very difficult to interpret in skin sections, thus its diagnostic value is limited in dermatopathology practice. Additionally, cultures of suspected cutaneous infections are superior in sensitivity and reproducibility to the Brown and Brenn stain. Acid Fast Stain (AFB) The classic acid-fast bacilli stain used most commonly in dermatopathology settings is the Ziehl-Neelsen stain. Acid-fast organisms are those whose cell walls contain high lipid in the form of mycolic acids and long-chain fatty acids. These characteristics permit Special stains are economical tools and have rapid turn around times, giving them significant advantages to many more sophisticated ancillary diagnostic techniques. strong binding and retention of carbol fuchsin dye after decolorization with acid-alcohol. An AFB positive organism stains brightly red after completion of the procedure. Frequently, a counterstain of methylene blue is used to serve as a contrasting background. AFB stain is employed most often when there is a high clinical suspicion of a mycobacterial infection and when granulomatous inflammation is seen in the absence of foreign body material on histopathologic examination. The stain is an excellent adjunct to light microscopy when seeking the presence of mycobacterium tuberculosis; however, not all mycobacterial species stain positively. Modified versions of the classic stain include modified bleach Ziehl-Neelsen, Kinyoun, Ellis and Zabrowarny, auramine-rhodamine (most sensitive of all), and Fite. The Fite stain highlights mycobacteria in general but is specifically used to identify mycobacterium leprosum. The stronger acid used in the classic method is deemed too harsh for M. leprae and the lipid in the cell s membrane is washed away, making visualization of the organism difficult. Fite uses a weaker acid in the decolorization phase of the procedure, preserving the more delicate cell walls of the organism. The Fite and the Ziehl-Neelsen methods share their positive acid-fast profiles; red is read as a positive stain (Fig. 2). Giemsa Giemsa is a metachromatic stain in that many tissues/organisms stain differently than the dye color itself. The dye mixture methylene blue, azure, and eosin compounds is blue, but positive staining is designated by a purplish hue. Giemsa is frequently used to identify mast cells; their granules stain positively. Of note, the immunohistochemical stains mast cell tryptase and CD 117 are also used to detect mast cells. Urticaria and urticaria pigmentosa are among the disease entities marked by a significant increase in mast cell volume, thus Giemsa finds its utility here. Further contributing to the value of Giemsa is the positive staining of several infectious organisms; spirochetes, protozoans, and cutaneous Leishmania in particular. 158 Connection 2010

4 Figure 3. Mast cell tryptase highlighting secretory granules of mast cells. Figure 4. Fontana-Masson stain highlighting melanin in epidermal keratinocytes, melanocytes, and dermal melanophages. Connection

5 Warthin-Starry Warthin-Starry stain is a silver nitrate stain. Tissue sections are incubated in a 1% silver nitrate solution followed by a developer such as hydroquinol. Warthin-Starry is used chiefly in the identification of spirochetes in diseases such as syphilis, Lyme disease, and acrodermatitis chronica atrophicans. A positive stain will reveal black spirochetes. Like the Verhoff von Gieson stain, the Warthin-Starry stain has the disadvantage of non-specific elastic tissue fiber staining, making it difficult to interpret in skin sections. Further complicating interpretation is positive staining of melanocytes. The use of this stain is often supplanted by antibody stains and/or serologies. Masson s Trichrome Masson s trichrome is a special stain which is typically used to characterize and discriminate between various connective and soft tissue components. Smooth muscle and keratin stain pink-red, collagen stains blue-green, and elastic fibers appear black. Either phosphotungstic or phosphomolybdic acid is used along with anionic dyes to create a balanced staining solution. Masson s trichrome stain is frequently employed when the histopathologic differential includes leiomyomatous and neural tumors. The finer characteristics of collagen in the dermis can be better appreciated, as evidenced by the highlighted and welldelineated pattern demonstrated in collagenomas. Perivascular fibrosis, scar formation, and sclerotic lesions are also better appreciated with the use of Masson s trichrome. Congo Red Detection of amyloid in tissue sections is greatly enhanced and confirmed by positive staining with congo red. The stain itself is red-pink. Examination of tissue sections suspected of involvement by amyloidosis must be performed under both light microscopy and polarized light. When polarized, amyloid has a characteristic apple green birefringence. The color and tinctorial subtleties observed with congo red staining are attributed to amyloid s physical structural arrangement and antiparallel beta-pleated sheets. The elastolytic material in colloid milium and the degenerating keratinocytes in lichen amyloidosis also stain with congo red. Other stains such as crystal violet, thioflavin T, and sirius red are also capable of staining amyloid but they are far less convenient and are less widely available. Fontana-Masson Silver is often a component of special stains in dermatopathology. Not only is it used in the Verhoff von Gieson elastic tissue stain and the Warthin-Starry spirochete stain described above, silver is a constituent of the Fontana-Masson melanin stain as well. In the latter case, the observer relies on the reduction of silver to form a black precipitate. In the context of traumatized melanocytic lesions or melanocytic entities superimposed upon hemorrhagic processes, the Fontana-Masson can aid in the distinction between melanin and hemosiderin pigments. Similarly, secondary dermal pigment deposits produced by specific drug exposures can be distinguished from those with melanocytic origins. Occasionally, Fontana-Masson is used in the evaluation of vitiligo and post-inflammatory hyperpigmentation. Some observers report that the Fontana-Masson stain is difficult to interpret when only rare granular staining is present (Fig. 4). Chloroacetate Esterase The lineage-specific cytoplasmic granules of myeloid cells are readily identified with the use of chloroacetate esterase. A positive stain is observed as an intense bright red hue. The stain finds its relevance in the investigation of malignant hematopoietic infiltrates in which the lineage of the suspicious cells is unclear. It can help distinguish acute lymphocytic leukemia from acute myeloid leukemia, although up to approximately one-quarter of myeloid cases stain negatively, with false negative findings attributed to excessive immaturity of the granulocytes and significant monocytic expression. Lastly, mast cells also stain positively with chloroacetate esterase. In summary, special stains are an effective adjunct to routine staining with hematoxylin and eosin in the practice of dermatopathology. While some are easier to interpret than others, most find diagnostic utility in everyday practice. Special stains are economical tools and have rapid turn around times, giving them significant advantages to many more sophisticated ancillary diagnostic techniques. Selected Bibliography 1. Aroni K, Tsagroni E, Kavantzas N, Patsouris E, Ioannidis E. A study of the pathogenesis of rosacea: how angiogenesis and mast cells may participate in a complex multifactorial process. Archives of Dermatological Research 2008; 300: Bancroft JD, Gamble M. Theory and practice of histologic techniques. 6th Edition. Philadelphia: Churchill Livingstone Elsevier, Bergey DH, Holt JH. Bergey s Manual of Determinative Bacteriology. 9th Edition. Philadelphia: Lippincott Williams & Wilkins, Kamino H, Tam S, Tapia B, Toussaint S. The use of elastin immunostain improves the evaluation of melanomas associated with nevi. Journal of Cutaneous Pathology 2009; 36(8): Connection 2010