Bhavya Swarnkar, Neetu Bhari
It is important to know what normal skin looks like in order to identify the abnormalities in a skin biopsy. The skin is composed of several different types of cells and connective tissue components. The three anatomically separable but functionally dependent structures forming the skin are: (1) epidermis, (2) dermis, and (3) subcutaneous tissue.
In adults, the skin weighs around 5 kg and covers a surface area of approximately 1.73 m2. The epidermis ranges from 0.05 to 0.1 mm in thickness in different areas. The dermis is also of variable thickness, ranging from 0.5 mm on the eyelid or scrotum to >5 mm on the back. The thickness of subcutis also varies across our body, thinnest over eyelids and external genitalia (<1 mm) and thickest over abdomen and buttocks (>3 cm). The localization and thickness of subcutaneous tissue also differs by gender. The accumulation of fat is more over abdomen and shoulders in males while it is more around hips, thighs and buttocks in females.
EPIDERMIS
The epidermis is composed largely of keratinocytes with small populations of melanocytes, Langerhans cells, neuroendocrine cells known as Merkel cells and unmyelinated axons. The epidermis also contains specialized cuboidal epithelium of the terminal portion of eccrine sweat ducts. An ultrastructurally complex basement membrane zone separates epidermis from dermis but appears as a barely perceptible eosinophilic line in normal biopsies.
Two dimensionally, the epidermal layer has an undulant undersurface, with downward invaginations termed rete ridges that interdigitate with projections of the dermis termed dermal papillae. In three-dimensions, dermal papillae are rounded, conical elevations surmounted by epidermis that forms a honeycomb of interconnected rete ridges.
From the bottom up, the epidermis consists of the basal cell layer (stratum basale), prickle cell layer (stratum spinosum), granular cell layer (stratum granulosum), and horny layer (stratum corneum) (Fig. 1). On the palms and soles, an additional thin, clear layer (stratum lucidum) is also noted between the granular layer and the horny layer.
Keratinocytes
Keratinocytes are large cells with a vesicular nucleus typically containing a conspicuous nucleolus and abundant cytoplasm. They show intercellular bridges connecting them with adjacent keratinocytes. In the basal layer, keratinocytes are cuboidal or columnar in shape, contain basophilic cytoplasm, and are smaller and closer together than in upper layers. Usually, small numbers of mitoses may be evident. Basal keratinocytes form a single layer and lie with their long axes perpendicular to the underlying basement membrane. There is a variable quantity of melanin in the cytoplasm of keratinocytes, chiefly in basal cells, corresponding to the color of the skin.
Fig. 1: Layers of the epidermis: Stratum corneum, stratum granulosum, stratum spinosum, and stratum basale (from above to below). Occasional clear cells seen in the basal layer are melanocytes (arrows) (H&E, ×200).
Prickle layer cells are arranged in 5–10 layers, are polygonal in outline, have abundant eosinophilic cytoplasm and oval vesicular nuclei, often with conspicuous nucleoli. This layer becomes progressively flattened as it moves toward the outer skin surface.
Granular cells are flattened tetrakaidecahedron shaped, and their cytoplasm is filled with keratohyaline granules that are deeply basophilic and irregular in size and shape. This layer is 1–3 cell layers thick in areas in which the horny layer is thin can be up to 10 layers in areas with a thick, horny layer, such as the palms and soles.
The stratum corneum is acellular, lacks nuclei and consists of eosinophilic keratin plates that are arranged in a basket weave pattern.
Melanocytes, Langerhans cells, and Merkel cells
Melanocytes are melanin-synthesizing dendritic cells located within the basal layer of the epidermis (Fig. 1), hair bulb, and outer root sheath of hair follicles. In the surface epidermis, melanocytes are seen in the basal layer as vacuolated cells, one for every 10 basal keratinocytes. They appear more frequent at the sides and bases of rete pegs. Melanocytes contain a round to oval, dark-stained nucleus surrounded by a small amount of irregularly shaped pink cytoplasm. The cytoplasm is retracted from adjoining keratinocytes because of shrinkage during tissue processing giving it a pseudovacuolated appearance. This helps to distinguish them from glycogenated keratinocytes which have true spaces in their cytoplasm, surrounding a vesicular nucleus with a conspicuous nucleolus. The density of melanocytes varies: the highest concentration of about 2,000/mm2 is found on the face and the male genitals and the lowest of about 800/mm2 on the trunk.
Langerhans cells are bone marrow-derived, dendritic, antigen presenting cells. They may be observed in sections stained with hematoxylin and eosin as clear cells in the suprabasal epidermis. It may sometimes be difficult to distinguish them from intraepidermal T lymphocytes, melanocytes and macrophages. Their dendritic cytoplasmic processes may not be discernible in routine histopathology. Enzyme immunohistochemistry using ATPase and aminopeptidase may be used to highlight these cells. These cells are also positive for prothymocyte differentiation cell surface glycoprotein CD1a, HLA-DR antigen (that also stains acrosyringium), and S100 protein (that also stains melanocytes). The concentration of these cells varies between 460 and 1,000 cells/mm2 over the skin. Apart from epidermis of skin, they are also found in oral mucosa, vagina, dermis, lymph node and thymus. There is a considerable site variation in the number of these cells and their number might decrease with time due to ultraviolet light exposure. Their number changes significantly during pathological processes like infection.
Merkel cells are located in the basal layer but are difficult to see in normal sections. They may represent a rudimentary touch receptor, are quite scarce, irregularly distributed and are occasionally arranged in groups.
Mucosal epithelium
With the exception of the dorsum of the tongue and the hard palate, the mucous membrane of the mouth possesses neither a granular nor a horny layer. The epithelial cells may appear pale and vacuolated, largely as a result of their high glycogen content (Fig. 2).
BASEMENT MEMBRANE
Basement membrane separating epidermis and dermis is mostly inconspicuous on hematoxylin and eosin staining but can be made out easily with periodic acid–Schiff (PAS) staining (Fig. 3), especially if it is thickened as seen in lupus erythematosus.
Fig. 2: Mucosal epithelium: Note the absence of granular layer and pale keratinocytes (arrow) (H&E, ×40).
DERMIS
The normal dermis consists of collagen, elastin and mucin, blood vessels, nerve twigs, hair follicles and eccrine glands, and a mild infiltrate of lymphocytes that traffic through the skin. Conventionally, the dermis is divided into the papillary dermis representing a thin zone immediately beneath the epidermis and between the rete ridges (Fig. 4). The collagen in the papillary dermis is thin and fine and is vertically oriented. The dermis immediately around cutaneous appendages has the same character as papillary dermis and is termed the adventitial dermis. The remainder of the dermis extending downward to the junction with the subcutis is the reticular dermis. The reticular collagen is more thick and eosinophilic and is composed of coarse large collagen fibers.
The junction of the dermis with the subcutis is irregular with extensions of subcutis into the dermis, around eccrine glands. The collagen of the septa in the subcutis is continuous with collagen of the deep reticular dermis.
Elastic fibers are found with collagen but are not visible in standard hematoxylin and eosin stained sections. In the dermis, the arrangement and size of elastic fibers varies from very large fibers in perianal skin to almost no fibers in the scrotum. Bluish, thin strands of dermal mucin may be seen in small quantities around appendages and between collagen bundles (Fig. 5).
Small vessels are seen in both the superficial and deep dermis. They are lined by a single layer of endothelial cells and may occasionally contain red blood cells. Medium-sized vessels are seen in the deep dermis and subcutis where they appear as rounded, moderately large, luminated structures with an endothelial lining (Fig. 6). Characteristically, they contain concentric bundles of muscle in the wall. Marked variation in the cutaneous blood supply is found between areas of distensible skin such as the eyelid and more rigid areas such as the fingertips. Lymphatic vessels are not visible in normal skin but in the presence of dermal edema, they are seen as dilated, thin-walled spaces lined with endothelial cells.
Fig. 4: Dermis: Papillary dermis enclosed by rete pegs (above line) with reticular dermis beneath it (H&E, ×200).
The entire dermis is permeated by sensory and autonomic nerve fibers. Over palms, soles, and mucocutaneous junction, some of the sensory nerves end in special end-organs namely mucocutaneous end organs, Vater–Pacini end organs, and Meissner corpuscles.
In routine histopathological examination, only large myelinated nerve fibers, Vater–Pacini end organs, and Meissner corpuscles are appreciated. Nerve twigs are seen in the vicinity of blood vessels and consist of a small collection of spindled cells surrounded by a thin, slightly eccentric perineurium. They are larger in the deep dermis and subcutis (Fig. 7). Finer nerves require special staining for demonstration.
Meissner corpuscles are situated in the papillary dermis and mediate fine touch. They resemble a pine cone and possess a capsule composed of multiple layers of flattened Schwann cells arranged transverse to the long axis of the corpuscle, forming the characteristic concentric pattern (Fig. 8). Vater–Pacini end organs are larger, located in the subcutis and mediate pressure sensation. They have variable shapes varying from ovoid, flattened sphere to irregular and are composed of a body proper with a core and thick capsule and a stalk. The core shows a granular substance surrounding the ascending nerve while the thick capsule is composed of multiple concentric, loosely arranged lamellae.
Fig. 6: Dermal vessels: Thin-walled small vessels (black arrow) and a thick walled, rounded medium vessel (red arrow) showing concentric bundles of smooth muscles in the wall, both with red blood cells (RBCs) in the lumen (H&E, ×200).
Fig. 7: Nerve twigs: Fascicle of spindle cells with wavy nuclei invested by thin perineurium (arrow) (H&E, ×200).
A small number of lymphocytes are seen around vessels in the superficial and deep dermis. They constitute a normal finding but may be incorrectly interpreted as representing an inflammatory infiltrate, particularly in biopsies that show no other change.
Adnexal structures
Adnexal structures extend from the epidermis into the dermis and consist of the hair follicle and associated sebaceous glands and apocrine glands, and eccrine sweat glands. The number, size and type of adnexal structures vary from one body site to another; e.g., glabrous skin of the palms and soles lacks hair follicles and associated structures, but has numerous eccrine sweat glands. The scalp has large hair follicles that extend deep into subcutaneous tissue whereas the face has small vellus hair follicles that are located in the dermis and are accompanied by relatively large sebaceous glands. Apocrine glands are found in the axilla, areolae, pubic and perianal area. Eccrine glands are seen over the entire skin except the vermilion border of lip, external ear canal, nail beds, glans penis, clitoris and labia minora. Sebaceous glands are active in the newborn and from puberty onward. They accompany hair follicles all over the skin but are functionally most prominent on the scalp, face, chest and back. Detailed descriptions of the structure of normal hair follicles and nails are provided in Chapters 21 and 22, respectively.
Fig. 8: Meissner corpuscle: In the dermal papilla, with multiple layers of transversely arranged Schwann cells (H&E, ×400).
Fig. 9: Eccrine glands and ducts: Glands show a single layer of pale cells around a lumen (red arrow); ducts show two layers of cuboidal cells with a eosinophilic cuticle on the luminal aspect (black arrow) (H&E, ×200).
Eccrine glands and ducts
Eccrine glands are the secretory portion of the eccrine apparatus and lie in the lower dermis or at the junction of the dermis and the subcutaneous fat. They are often surrounded by adipocytes that form a layer of perieccrine fat. Eccrine glands show large, pale cells and small, dark cells attached to the basement membrane (Fig. 9). However, individual sections may at times suggest a double layer of cells. Outside the basement membrane, there are longitudinally arranged myoepithelial cells.
The intradermal sweat duct consists of two layers of uniform cuboidal cells that are lined on the luminal aspect by a deeply eosinophilic, homogeneous cuticle that is PAS positive and diastase resistant (Fig. 9).7
The intraepidermal portion of the sweat duct is called the acrosyringium and may be seen winding its way through the epidermis. This is best appreciated on the palms and soles. The cells lining the acrosyringium may be difficult to distinguish from the surrounding epidermal keratinocytes but are arranged around a lumen unlike the orderly stacking of the epidermis.
Apocrine glands and ducts
Apocrine glands are a part of the pilosebaceous follicular unit and drain into the infundibular portion of the hair follicle, just above the sebaceous duct. The ductal portion of the apocrine unit is identical to the eccrine duct. The secretory glands are usually much larger than eccrine glands and show a single layer of secretory cells with an outer layer of myoepithelial cells. Apocrine glands show decapitation secretion with pinching off of the tips of the secretory cells into the lumen (Fig. 10). Apoeccrine glands show features of both apocrine and eccrine glands.
Fig. 10: Apocrine glands: Large glands lined by cuboidal cells showing decapitation secretion (arrow) (H&E, ×200).
Fig. 11: Sebaceous gland: Mature sebocytes showing multiple vacuoles around a rounded or scalloped nucleus opening into the sebaceous duct lined with a eosinophilic cuticle (H&E, ×200).
Sebaceous glands
A sebaceous gland consists of one/several sebaceous lobules leading into a common excretory duct which opens into infundibulum of the hair follicle (Fig. 11). The follicle may be a terminal or vellus hair follicle but the size of the sebaceous gland is not related to the size of the hair shaft. The short excretory duct is lined by stratified squamous epithelium with an eosinophilic cuticle on the luminal aspect.
Each sebaceous lobule has a peripheral, germinative layer of cuboidal, deeply basophilic cells that usually contain no lipid droplets. From this arises the inner zone of lipid-laden vacuolated cells with a central crenellated nucleus. The lipid in sebocytes can be detected if lipid stains are used on frozen sections but routine processing methods extract the lipid and hence the cytoplasm appears as a delicate network of fine cytoplasmic vacuoles. Sebaceous cells disintegrate in the portion of the lobule lying closest to the duct to form sebum.
SUBCUTANEOUS FAT
Groups of adipocytes are arranged in lobules, with most adipocytes of about the same size and most lobules measuring approximately 1 cm in diameter. Normal adipocytes appear vacuolated with an inconspicuous nucleus pushed to the side (Fig. 12). In neonates and infants, the nucleus of adipocytes and adipoblasts are more prominent and occupy a larger proportion of the cell. Lobules are separated from each other by interlobular septa composed of collagen and reticulin fibers. These interlobular septa appear as thin bands of collagen that can be traced upward to the reticular dermis. Small and medium-sized vessels and nerve twigs are seen in both the septa and the lobules.
Fig. 12: Subcutis: Lobules of adipocytes separated by thin fibrous septa. Note the upward extension of fat around the eccrine glands (arrow) and continuity of septa with dermal collagen (H&E, ×40).
SOME VARIATIONS AND NORMAL FINDINGS
- Skeletal muscle is seen at the base of the biopsy in lesions from the head and neck including oral mucosa. Biopsies from the tongue show an impressive amount of skeletal muscle (Fig. 13).
- Smooth muscle is seen scattered through the dermis in biopsies from the scrotum and labia majora and the nipple-areola complex (Fig. 14).
- The smooth muscle of arrectores pilorum may be difficult to recognize when a small part is cut in tangential or cross section.
- The dermis in biopsies from the back is thick and may appear slightly sclerotic. Be careful not to overdiagnose this as morphea.
Fig. 14: Genital skin: Dilated vessels and smooth muscle bundles (arrows) are seen in the dermis (H&E, ×100).
SUGGESTED READING
- Yousef H, Alhajj M, Sharma S. Anatomy, Skin (Integument), Epidermis. [Updated 2021 Nov 19]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan. Available from: https://www.ncbi.nlm.nih.gov/books/NBK470464/