Dermatopathology Histologic stains in dermatopathology Mark E. Logan, MD, a and M. Tarif Zaim, MDa.b Cleveland, Ohio This article reviews the theory and results of commonly used stains in dermatopathology and summarizes their specific applications. (J AM ACAD DERMATOL 1990;22:820-30.)

The most commonly used routine stain in dermatopathology laboratories is hematoxylin and eosin. Although it is sufficient for the diagnosis of most skin diseases, some diseases require further elucidation of specific tissue components by other stains. Pinkus 1 and Mehrcgan et aP· 3 advocate the use of a second routine stain, the acid orcein-Giemsa method. It stains elastic fibers and amyloid and differentiates melanin from hemosiderin (Fig. 1). We will briefly review the more commonly used histologic stains in dermatopathology. Hematoxylin and eosin. Hematoxylin is a naturally occurring, basic (cationic) dye that comes from the tree Haematoxylon campechianum. 4 Hematoxylin requires an aluminum mordant (a mordant links the dye to the tissue more strongly) to stain tissue adequately. The active coloring agent is hematein, which stains negatively charged, acidic elements (e.g., DNA). Eosin is a synthetic, acidic (anionic) xanthene dye that stains cytoplasm and connective tissue fibers various shades of pink. With the hematoxylin-and-eosin stain, nuclei and calcium are blue-black; cytoplasm, collagen, muscle, nerve, and fibrin are pink to red; and elastic fibers are unstained (Fig. 2). COLLAGEN STAINS (trichrome stains) (Table I)

Different tissue structures have varying permeabilities that restrict dye penetration into them. Relatively impermeable structures such as red blood cells are colored only by small-molecule dyes (e.g., From the Departments of Dermatology" and Pathology," University Hospitals of Cleveland, and Case Western Reserve University School of Medicine. Reprint requests: M. Tarif Zairn, MD, Department of Dermatology, University Hospitals of Cleveland, 2074 Abington Rd., Cleveland,

OH 44106.

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acid fuchsin). Whenever a large-molecule dye (e.g., methylene blue) can penetrate, it will do so at the expense of the smaller-molecule dye. For example, collagen is permeable to both small- and large-molecule dyes and is colored by either dye when used separately. Collagen is colored by the large-molecule dye when it is used in combination with a smallmolecule dye. van Gieson stain. The van Gieson stain, a mixture of hematoxylin, acid fuchsin, and trinitrophenol, stains collagen red." Acid fuchsin is a small-molecule dye that penetrates collagen. It is not displaced because no larger-molecule dye is present. Nuclei stain blue-black with hematoxylin (Fig. 3). Masson trichrome. Masson trichrome is a combination of hematoxylin, acid fuchsin, and methylene blue. Methylene blue dyes collagen blue because it is a larger-molecule dye that overrides the effect of acid fuchsin. Methylene blue also stains reticulum fibers, basement membranes, and osteoid. Cytoplasm, muscle, and fibrin are stained red by acid fuchsin, which is not displaced by methylene blue in these tissues because of their impermeability to the large-molecule methylene blue stain (Figs. 4 and 5). ELASTIC FIBER STAINS (Table I)

Elastic fiber stains establish hydrogen bonds between the stain and the elastic fibers. Acid orcein. Orcein is a dye that occurs naturally in lichens." When combined with hydrochloric acid, this dye stains elastic fibers dark brown (Fig. 1). This stain also differentiates melanin from hemosiderin, which stain dark green and greenish yellow, respectively. Amyloid deposits are stained sky blue. Verhoeff stain. Verhoeff stain, made of hematoxylin and iodine combined with ferric chloride, stains elastic fibers black. Other tissues stain variably, depending on the counterstain. This stain is occasionally used in combination with the van Gieson stain

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Fig. 1. Acid orcein-Giemsa stain: Brown elastic fibers and purple mast cell granules in scarring alopecia. Note anelastotic fibrotic tract. Fig. 2. Hematoxylin-and-eosin stain: Pink fibrotic tract in scarring alopecia (same case shown in Fig. 1). Fig. 3. van Gieson stain: Red collagen extruding through the epidermis in reactive perforating collagenosis. Fig. 4. Masson trichrome stain: Red muscle fibersof leiomyoma with interspersed blue collagen.

to highlight both collagen and elastic tissue." The Verhoeff-van Gieson stain results in black elastic fibers and red collagen (Fig. 6). Gomori's aldehyde fuchsin. Gomori's aldehyde

fuchsin, a mixture of potassium permanganate, aldehyde fuchsin, and eosin, stains elastic fibers blue-purple.' Mast cell granules, mucins, cartilage, and some fungi are also stained blue-purple.

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Fig. 5. Higher magnification of field in Fig. 4. Fig. 6. Verhoeff-van Gieson stain: Black elastic fibers in pseudoxanthoma elasticum.

agent (such as KOH), however, sensitized sites are produced on the fibers and bind silver. Reticulum fibers and nerve fibers are stained black, and nuclei appear gray. Other tissue components stain variably, depending on the counterstain.

CARBOHYDRATE STAINS (Table II)

Fig. 7. Alcian blue stain: Blue AMPS in metastatic adenocarcinoma.

RETICULUM FIBER AND NERVE STAIN (Table I) Reticulum fibers are type III collagen. They are argyrophilic and therefore need an external reducing agent to reduce a silver nitrate solution to metallic silver. Bodian (Gomori's silver nitrate). Reticulum fibers and nerves have little natural affinity for silver solutions. When mixed with a suitable reducing

Acid mucopolysaccharides (AMPS) are carbohydrates that can be produced by fibroblasts, endothelial cells, osteocytes, chondrocytes, and mast cells. Sulfated AMPS include chondroitin sulfate A, B, and C; heparin; and heparan sulfate. Carboxylated AMPS include hyaluronic acid and sialomucin. Alcian blue. Alcian blue is a cationic dye that forms electrostatic bonds with certain tissue polyanions that contain either sulfate or carboxyl groups. It does not react with the phosphate groups of nucleic acids because the high density of DNA and RNA prevents entry of this large-molecule dye into the nucleus. The dye is specific to AMPS, but calcium salts may also stain. The tissue component will stain most intensely if the dye is used at a pH at which the reacting groups are fully ionized. Sulfated AMPS react at a lower pH than do carboxylated AMPS. Therefore chondroitin sulfate, heparin, and heparan sulfate stain at pH 2.5 and 0.5, whereas hyaluronic acid and sialomucin stain at pH 2.5 only.80 10 The pH must be strictly controlled to avoid false-negative results. With Alcian blue, AMPS stain blue and nuclei appear red (Fig. 7). A combination of the Alcian blue and the periodic acid-

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Fig. 8. PAS reaction: Magenta neutral mucopolysaccharides at basementmembrane and in vessel walls in lupus erythematosus. Fig. 9. PAS reaction: Diastase-resistant, magentaspores and hyphaein superficial dermatomycosis. Fig. 10. Crystal violet stain: Red-purple dermal amyloid deposits in macular amyloidosis. Schiff reaction is occasionally used to demonstrate both acidic and neutral mucopolysaccharides.'! Colloidal iron. At low pH, colloidal iron is adsorbed onto tissue polyanions (sulfate and carboxyl groups). The adsorbed iron is then visualized by conversion to ferric ferrocyanide (blue). This stain is more sensitive and intense than Alcian blue but is also more time consuming and complex." The results of the colloidal iron stain are similar to those of Aldan blue; AMPS stain blue and nuclei red. Toluidine blue. Toluidine blue is a basic, cationic dye of the thiazine series. It is a metachromatic stain. This means that certain tissue structures (chromotropes). react with it to produce a color that is different from that of the dye. AMPS are chromotropes that react with metachromatic dyes. Sulfated

AMPS stain at a pH of less than 1.5, whereas carboxylated AMPS stain at a pH of 3.0 to 6.0.8 , 12 With this dye AMPS, nucleic acids, and mast cell granules stain metachromatically purple. Periodic acid-8chiff reaction. The periodic acidSchiff (PAS) reaction involves carbohydrate oxidation by periodic acid to produce a dialdehyde, The dialdehyde reacts with basic fuchsin and sulfurous acid to produce an alkyl sulfonate, which appears magenta (Figs. 8 and 9). The PAS reaction takes place with neutral mucopolysaccharides and glycogen. Digestion with diastase before the PAS reaction makes the stain specific to neutral mucopolysaccharides. 13Therefore diastase-resistant PAS positivity is indicative of neutral mucopolysaccharides and is normally seen in the basement membrane

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Fig. 11. Thioflavine T stain: Yellow fluorescenceof dermal amyloid depositsin lichen amyloidosis. Fig. 12. Oil red 0 stain: Red lipid droplets in xanthoma. Fig. 13. Fontana-Masson stain: Black melanin pigment in amelanotic spindle cell melanoma.

zone (Fig. 8), the dark cells of eccrine glands, the lumen of eccrine ducts, the apocrine secretion, and fungi (Fig. 9). On the other hand, a PAS-positive, diastase-labile result is indicative of glycogen and is normally seen in cutaneous and mucosal stratified squamous cells, pilar outer root sheath cells, and clear cells of eccrine glands. AMYLOID STAINS (Table III)

Fig. 14. Perls' Prussian blue stain: Blue hemosiderin pigment in stasis dermatitis.

Amyloid is an amorphous, eosinophilic, faintly refractile protein. It is variably PAS-positive, and stains green or blue with Masson trichrome and khaki with van Gieson stain. Amyloid staining has no absolute standard, although the new cotton dyes have many advantages overolder methods. They are simple, long lasting, specific, and stain most intensely. Thioflavine T has maximal sensitivity. Cotton dyes (Congo red, Pagoda red, RIT Scarlet No.5, RIT Cardinal red No.9). The tertiary structure of cotton dye molecules is linear whereas

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Histologic stains in dermatopathology

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Histologic stains in dermatopathology.

Dermatopathology Histologic stains in dermatopathology Mark E. Logan, MD, a and M. Tarif Zaim, MDa.b Cleveland, Ohio This article reviews the theory a...
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