9.2.3 Basics in Dermatopathology

The most efficient method in the diagnosis of skin diseases is the combined validation of clinical and histopathological features, i.e., clinico-pathologic correlation. The diagnostic impact of histopathology may be decisive or just confirmative of one or more clinical differential diagnoses. The majority of of skin biopsies can have a specific diagnosis and the remainder can be classified into a particular group of disorders. However, there are some dermatoses in which a biopsy is not indicated because typical histopathologic features are lacking.

The process of interpretation of skin biopsies starts with the differentiation of an inflammatory disorder from a neoplastic condition. As for inflammatory conditions, the interpretation consists into the integration of 2 morphological features: the tissue reaction pattern, and the composition of the infiltrate. There are at least 6 major tissue reaction patterns: psoriasiform, spongiotic, lichenoid, vesiculo-bullous, granulomatous, and vasculopathic/vasculitic.

1. Taking a biopsy

1.1. When

When considering clinicopathologic considerations in approaching a diagnosis there basically are four scenarios, in which the diagnostic impact of histopathology may be high, moderate, low or none.

1.2. Where

In inflammatory processes, infectious or non-infectious, the biopsy should be taken from the border of the lesion, where the initial changes without regenerative, necrotic, fibrous or secondary granulomatous reactions are found.

In tumors, however, the biopsy should be taken from the center of the lesion or in some cases from the border, including both normal and neoplastic tissue. In very large lesions one from the center and one from the margin is recommended.

1.3. How

It is fundamentally important, that the dermatopathologist, to whom the specimen is referred for microscopic evaluation, is able to describe his findings in the context of the clinical features, which the clinician has written down on the application form when sending in. The specimen must include the critical tissue levels and must show the pathologic changes. One of the most frequent mistakes is a superficial biopsy, missing the pathologic changes in the deep dermis or subcutaneous tissue.

In addition, the clinician should provide the pathologist with clinical differential diagnoses, too.

Depending on the size and the depth of the lesion, it can be either completely excised-especially small tumors- (by intention excisional biopsy) or a spindle- or wedge-shaped biopsy is taken from the border (incisional biopsy).

In tumorous lesions reaching to the surface of the skin, a flat slice can be removed horizontally with a scalpel (shave biopsy).

Punch biopsies can be applied for homogeneous tumor lesions (size of diameter between 4 to 8mm).

Fragmented material from superficial curettage may be sufficient in special situations for confirming infectious (warts, mollusca contagiosa) or neoplastic origin (actinic keratosis), when the morphology of a small cluster of tumor cells is sufficient for making a diagnosis. Swaps from the nostril area or from the superficially scratched earlobe may provide material for microbiologic investigations under the microscope or in appropriate culture media.

Tape strips for diagnosing mycotic infections from the body sites (M.furfur) with a mythelene stain is easy to perform and confirms a fast a diagnosis within minutes.

Electrocaustic retrieval of a specimen should be avoided, since important tissue structures may be boiled.

1.4. 3-D dermatopathology (Mohs-technique)

The basic idea of the micrographic¹ controlled surgery (MCS), as invented originally by Frederic Mohs in (Madison/Wi, USA), was to confirm by 3-dimensional sectioning that the borders of the excised specimens were tumor free towards the periphery and towards the depth and by serial surgical procedures to completely remove the tumor and saving uninvolved tissue. This technique, originally as pre-fixed, later on as fresh tissue and finally today mostly as post-fixed procedure is indicated for solid tumors with superficially undetermined, “subclinical” borders;, especially in basal cell carcinomas; not for inflammatory dermatoses. Today, in addition, immunostaining can be done immediately on a Moh’s section to identify specifically tumor cells to be more precise when evaluating the section border.

¹This hybrid expression refers to the procedure, in which the microscopic investigation of a histologic specimen is guided by colored margins depicted also in a corresponding macroscopic mapping of the specimens.

2. Sample processing

The biopsy is trimmed to an optimal size of 1x2x2 cm and fixed in buffered formalin (4% aqueous solution) for several hours before embedded into a paraffin block. These procedures today can run automatically.

Sections of 2-4 microns perpendicularly from the epidermis to the subcutis are cut with a microtome on to a glass slide, which is transferred to an automatic staining device.

3. Staining

3.1. Routine

Ha(e)matoxylin and Eosin (HE): blue and red

3.2. Special stains

• Alcian blue: blue. Acid glycosamines

• Berlin Blue: blue. Hemosiderin (iron)

• Elastica: black. Elastic fibres

• Fite-Faraco: red. Mycobacterium leprae

• Giemsa: violet blue. Mast cells, amastigotes in leishmaniasis

• Gram: blue (Gram positive bacteriae) or red (Gram negative bacteriae)

• Grocott: black. Fungi

• Congo red: pink or green in polarized light. Amyloid

• Kossa: black. Calcium

• Masson-Fontana: black. Melanin

• Naphthol-AS-D Chloroacetate esterase: (Leder’s stain) red. Mast cells

• Mast cells Periodic Acid Schiff (PAS): violet, purple. Stains glycogen, fungi, basal lamina

• Sudan (cryosection): orange or yellow. Lipids

• Toluidine Blue: bluish granules. Mast cells

• Van Gieson-Elastica: red (collagen), black (elastic fibres), muscle fibres (yellow)

• Warthin-Starry: black. Spirochetes, fungi

• Ziehl-Neelsen: red. Acid fast bacteria (tubercle bacilli, leishmania)

3.3. Molecular Morphology²

• Immune histochemistry and immune phenotyping: Labelling of antigens (epithelial, mesenchymal, vascular, melanocytic, nerval, lymphocytic, dendritic, histiocytic, bacterial, fungal and proliferation markers), membrane markers or cell-to-cell contacts with specific mono- or polyclonal antibodies, which often stain specific or several epitopes of different cells or structures.

• Direct Immunofluorescence (DIF): Use of antibodies labelled with a fluorochrome. The most important applications include autoimmune-bullous dermatoses, vasculitis and lupus erythematosus. Double labelling or labelling with up to 50 markers is possible today (multi epitop ligand cartography).

• In-situ-Hybridization (ISH): Detection of microorganisms and confirmation of clonality of i.e. in lymphoproliferative disorders (lymphomas).

• Fluorescence-in-Situ Hybridization (FISH): Detection of chromosomal aberrations, i.e. in neoplasia.

• Polymerase-Chain-Reaction (PCR): extremely sensitive for detection of i.e. microorganisms, clonality (receptor rearrangement), mutations, oncogenes.

²Cryocuts from unfixed tissue or from samples fixed in special Michel’s solution needed for many antibodies. The results always have to be interpreted in conjunction with clinical and histological findings.

4. Structure of normal skin: https://www.cyberderm.net/en/learn/structure-of-the-skin

There are striking regional differences with respect to thickness of the skin-layers, sensory receptors, vessels, connective tissue, subcutaneous fat and adnexae.

4.1. Epidermis

Multilayer squamous epithelium of 0.3-0.8 mm thick. From the outside to the inside, the following layers are distinguished:

4.1.1. Stratum corneum consists of flat, plate-like, interlocking coreless keratinocytes, which are held together by intercellular “barrier lipids” (e.g. ceramides). Since it is the outermost protective layer of the body, it exerts a mechanical barrier function, prevents excessive water loss and protects from penetration of microorganisms, chemicals or allergens.

4.1.2. Stratum granulosum exhibits moisturizing and UV-protective (pyrrolidone carboxylic acid) functions. The granules contain lipids and enzymes, which are released into the intercellular space and lead to the formation of a water-resistant putty substance in the stratum corneum.

4.1.3. Stratum spinosum together with the statum basale forms the stratum germinativum (stratum germinativum). The histologic processing of skin samples results in shrinking of the sections, which accentuates their intercellular desmosomal contacts (spiny cell layer). The keratinization process begins in this layer. During their migration to the outermost layer of the epidermis, the cells become flattened and cease to divide, keeping a delegate balance between gain and loss of cells by peeling off the skin surface.

4.1.4. Stratum basale (stratum germinativum). Cells of this layer exhibit qualities of determined stem cells. Following division one cell migrates to the skin surface within the next 4 weeks, whereas the other remains and divides again.

4.1.5. Up to 8% of the cells of the basal layer of the epidermis are melanocytes which produce melanin, a dark pigment blocking UV rays. Ethnic different skin colors are due to the packaging and distribution pattern of the pigment grains (loose fine granules in black people or packed in melanosomes in Caucasians).

4.1.6. Langerhans cells are another type of dendritic cells in the germinative layers of the epidermis and also present as dendritic reticulum cells (DRC) in the dermis. They derive from the bone marrow and play a pivotal role in the immunological defense and in the recognition of antigens.

4.1.7. Merkel cells are a third type of dendritic cells in the epidermis. Localized in the basal layer they fulfill sensory receptor functions (pressure).

4.2. The Dermo-epidermal junction (grenz-zone) comprises a complex PAS positive basal membrane, which is anchored to the dermis by hemidesmosomes. Metabolic products, dendritic immune cells and lymphocytes are exchanged between the two compartments. Under the electron microscope the complex structure of the junction zone is revealed, comprising of (1) the cytoskeleton of the basal keratinocytes with intermediate filaments composed of keratin 5 and 14; (2) the anchorfilaments of the hemidesmosomes (Lamina lucida), consisting of various molecular structures; (3) lamina densa with collagen IV as the main component besides other components; (4) the sublamina densa, which is directly adjacent to the lamina densa and consists of collagen type I, III, IV and type VII, anchoring to the papillary dermis. These structural proteins and their antigenic determinants play an important role in the pathophysiology of some autoimmuno- and geno- dermatoses.

4.3. Dermis

4.3.1. The Papillary dermis is the upper part of the dermis, protruding as rete ridges between the epidermal papillae. The capillary loops provide optimal nutrition for the epidermis and fine nerve endings give sensory protection.

4.3.2. The Reticular dermis shows a texture of collagen fibres, which is more compact than in the papillary dermis. Moreover, it harbors vascular, nervous and adnexal elements between fibroblasts, histiocytes, macrophages, dendritic and other cells, permanently migrating through the skin.

4.4. The Subcutis consists of fat lobuli separated by sepal fibres, connecting it to both the dermis and the deeper tissue. It serves as energy and heat storage, and as mechanical protective pad.

4.5. Adnexal structures

4.5.1. Hairs are found on almost all body surfaces except for soles and palms. They consist of dead, fish-scale-like horn cells produced in the hair follicles of the dermis. The hair follicle consists of an epithelial layer that surrounds the hair root. The lower part of the hair follicle, the hair bulb, is embedded in a net of nerve endings and capillaries. Hair growth occurs in a cycle: (1) anagen (growth) phase (2-6 years; 0.3 mm per day) in which 85% of our head hairs are; (2) 2-week catagen (transition) phase; and (3) telogen (resting) phase (3-4 months) in which 50-100 hairs per day are lost.

Sebaceous gland follicles are endocrine adnexal organs per se and smaller glands are linked to the terminal and vellus hair follicles. They are under control of androgens, growth factors and other mediators and produce a high number of different lipids (sebum), which protect skin and hair from drying out, water loss and growth of pathogenic bacteria.

4.5.2. Sweat glands.

4.5.2.1. The eccrine sweat glands are spread over almost the entire body surface. The twisted part in the deep dermis with a secretory and an excretory part which leads via a straight dermal duct directly to the epidermis where it is coiling and reaching the skin surface and produces an aqueous secretion, which is rich in urea, uric acid, ammonia, salts and other substances. They are used for temperature regulation and the excretion of metabolic products.

4.5.2.2. The apocrine sweat glands are found in the axilla, the groin, the anogenital area and the beard hair in men. The secretory portion lies mainly in the subcutis with the excretory duct flowing into the upper hair follicle canal.

5. Pathologic changes

5.1. Epidermis.

5.1.1. Orthokeratosis: Basket weave stratum corneum in the fixed tissue section.

Example: Normal skin.

5.1.2. Hyperkeratosis: Thickening of the stratum corneum due to increased epidermal proliferation or retention of horny cells by reduced scaling.

Example: Ichthyosis.

5.1.3. Parakeratosis: Remnants of nuclei remain in the stratum corneum due to disturbance of the keratinization process (dyskeratosis).

Example: Eczema.

5.1.4. Atrophy: Reduced spinal layer and flattening of the basal undulation.

Examples: Aged skin; lichen sclerosus et atrophicus.

5.1.5. Acanthosis: Thickening of the epidermis to due broadening of the spinal layer following increased proliferation and turnover of keratinocytes.

Example: Chronic eczema; psoriasis.

5.1.6. Papillomatosis: Hyperplasia of epidermis with resulting dermal papillae forming an undulating wave like basal lamina.

Example: Psoriasis.

5.1.7. Hypergranulosis: Broadening of the stratum granulosum due to disturbances in the keratinization process or hyperproliferation.

Example: Lichen planus.

Hypogranulosis: reduced or absent stratum granulosum.

Example: psoriasis, ichthyosis.

5.1.8. Spongiosis: Dehiscence of keratinocytes with intercellular edema up to intraepidermal blister formation; desmosome bridges may be preserved.

Example: Acute eczema.

5.1.9. Acantholysis: Loss of intercellular connections (desmosomes, desmogleins, desmoplakins) between keratinocytes, resulting in blister formation.

Example: Pemphigus vulgaris.

5.1.10. Dyskeratosis: Premature or altered (abnormal) keratinocyte differentiation with preservation of nuclei in the stratum corneum.

Example: Dyskeratosis follicularis Darier.

5.1.11. Ballooning: Intracellular edema, blowing up the cell size.

Examples: Herpes virus infections.

5.1.12. Necrolysis: Complete damage and lysis of the celluar structure and substance.

Example: Toxic epidermal necrolysis (TEN).

5.1.13. Apoptosis: programmed cell death by regulating different events to the epidermal cells (physiologic or in response to premalignant or malignant processes).

Examples: UV light damage, immuno surveillance, Herpes virus infections.

5.2. Dermo-epidermal junction (grenz-zone)

5.2.1. Interface dermatitis: Obscuring of the border between epidermis and papillary dermis due to edema (vacuolation), epidermotropism of inflammatory cells or to cellular infiltrate in the papillary zone (lichenoid type).

Examples: Lupus erythematosus; lichen planus.

5.2.2. Subepidermal blistering: Supra- or infrabasal blistering separation of epidermis and the papillary dermis.

Example: Bullous pemphigoid.

5.2.3. Subepidermal edema: Edema in the papillary dermis without blister formation.

Example: Erythema exsudativum multiforme.

5.3. Dermis

5.3.1. Fibrosis: Increased numbers and activity of fibroblasts.

Examples: Fibroma; histiocytoma.

5.3.2. Sclerosis: Compact densely packed collagen fibres usually paucicellular (reduced number of connective tissue cells).

Example: Scleroderma

5.3.3. Elastosis: Degenerative changes of collagen and elastic tissue with development of coarse fibres.

Example: Actinic elastosis.

5.4. Vessels

5.4.1. Vasculitis: Swelling of endothelia, extravasation of fibrin and erythrocytes, leuco- or lymphocytic perivascular inflammation,leucocytoclasia.

Example: Vasculitis allergica.

Thrombotic vasculopathy: occlusion of vessels by thrombi in the absence of inflammatory infiltrate.

5.4.2. Calcification: Calcium deposits in and around (elastica) vascular endothelial cells.

Example: Calciphylaxis

5.4.3. Proliferation: Endothelia cells and vascular spaces.

Examples: benign (hemangioma), “semimalignant” (Kaposi) or malignant processes (hemangiosarcoma).

5.5. Deposits and Storage

5.5.1. Foreign bodies: Inorganic material (pigment) inserted from outside intentionally (tattoo), or accidentally, into the dermis is ingested by macrophages or -if the particles are too big- leads to enclosure by a granulomatous inflammatory infiltrate with various types of multinucleated giant cells.

Examples: Tattoo; foreign body granuloma.

5.5.2. Other deposits: Melanin, lipids, urate, mucin, iron (hemosiderin), porphyrins, calcium, amyloid and other organic or inorganic substances, produced during pathologic disease processes may be deposited intracellularly (macrophages) or extracellularly between or attached to collagen bundles in the dermis.

Examples: Nevi; xanthoma; gout; mucinosis; pilomatricoma (Malherbe).