Ebook Histopathology of the skin: Part 1

(BQ) Part 1 book Histopathology of the skin presents the following contents: Histology of normal skin, techniques of skin biopsy, dermoepidermal junction, the cells of the skin and their identification, common terminologies used in dermatopathology, staining techniques in dermatopathology,... Invite you to consult.

of the Skin

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Skin Institute and School of Dermatology

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Late Prof Pran Nath Behl

Indeed it is intriguing to have a title on Histopathology of the Skin in the Indian context It is unique for it embraces the

various relevant facets which need to be appraised to the native students The endeavor to write a book on the title hasbeen made by Dr Ashok Aggarwal who had done his postgraduate degree in Dermatology and Venereology in the year

1978 from Maulana Azad Medical College, New Delhi He had the privilege of working under Dr Pran Nath Behl in SkinInstitute and School of Dermatology Over the years his interest in histopathology of skin was recognized and he wentfor short training to Edinbur gh under Prof Hunter and his group After his return he had a privileged opportunity ofstreamlining the services in dermatopathology in the Institute He has now utilized his acumen in preparing a dissertation

in the form of a booklet hugging various dermatoses attempting to bring about a correlation for the purposes of ultimatediagnosis of the disease It’s a worthwhile endeavor and should help all those who are interested in clinical histopathologicaland treatment aspect of various dermatoses The illustrations contain therein are of good quality , and may serve as aready reckoner or accomplishing for the diagnosis of dermatoses in question The sincere attempt therefore is worthapplauding and augurs well for the future of the specialty

Virendra N Sehgal MD, FNASc, FAMS, FRAS (Lond.) Former Professor/Head Department of Dermatology and

Venereology, Goa Medical College, Panji

• Professor/Head Department of Dermatology and Venereology,

UCMS, Safdarjung Hospital

• Professor/Head (Acting Dean), Director-Professor, Department of Dermatology and Venereology, Maulana Azad Medical College/LNJP Hospital, New Delhi

• Principal/Medical Superintendent/Director-Professor Dermatology and

Venereology, Lady Hardings Medical College, New Delhi

• Director-Professor Dermatology/Venereology/Medical Superintendent,

UCMS-GTB Hospital, Delhi Sehgal Nursing Home Dermato-Venereology (Skin/VD) Centre A-6 Panchwati, Delhi-110033 (India)

The book “Histopathology of the Skin” is a much awaited book in the Indian context on dermatopathology designed for

postgraduate students of dermatology It is also meant for general pathologist dealing with the cutaneous morbid conditions.The chapters on Normal Histology of Skin and Recognition of Inflammatory Cells will enable beginners to identifycommon skin structures and inflammatory cells in tissue sections The chapter on Special Stains has been included togive insight into the role of these stain in diagnoses of different skin conditions The histopathological findings ofdifferent skin diseases are described in detail but in a simple and straightforward fashion A lar ge number ofmicrophotographs included in this book will further help the readers to assess their histopathological tissue sections

Ashok Aggarwal

1 Histology of Normal Skin 1

2 Techniques of Skin Biopsy 14

3 Dermoepidermal Junction 18

4 The Cells of the Skin and their Identification 20

5 Common Terminologies Used in Dermatopathology 26

6 Staining Techniques in Dermatopathology 33

7 Dermatitis and Eczema (Spongiotic Dermatitis) 46

8 Lichen Planus 53

9 Vesiculobullous Disorders 63

10 Dermatophytosis (Superficial Fungal Infections) 74

11 Yeast Infections 80

12 Viral Infections of Skin 83

13 Tuberculosis of the Skin 92

14 Hansen’s Disease (Leprosy) 98

15 Leishmaniasis 106

16 Disorders of Hypopigmentation 110

17 Erythema Multiforme 114

18 Lichen Sclerosus et Atrophicus (LSA) 116

19 Scleroderma 118

20 Lichen Simplex Chronicus (Circumscribed Neurodermatitis) 123

21 Pigmented Purpuric Dermatosis (Schamber g’s Disease) 126

22 Dermatitis Herpetiformis 129

23 Sarcoidosis 135

24 Lupus Erythematosus (LE) 138

25 Pyoderma Gangrenosum 146

26 Urticaria Pigmentosa (UP) 148

27 Gyrate and Annular Erythemas 150

28 Seborrheic Keratosis (Senile Warts) 152

29 Basal Cell Carcinoma (BCC) 156

30 Verrucous Epidermal Nevus 160

31 Epidermal or Infundibular Cyst 163

32 Keratoacanthoma 167

33 Squamous Cell Carcinoma (SCC) 169

34 Actinic Keratosis (Solar Keratosis) 172

35 Bowen’s Disease 174

Contents

xii Histopathology of the Skin

36 Oral Leukoplakia 176

37 Paget’s Disease 178

38 Alopecias 180

39 Psoriasis 188

40 Lymphocytoma Cutis (Cutaneous Lymphoid Hyperplasia) 198

41 Disorders of Hyperpigmentation 200

42 Syringoma/Trichoepithelioma/Cylindroma 208

43 Acne Vulgaris 211

44 Polymorphous Light Eruption 216

45 Dermatofibroma (Benign Fibrous Histiocytoma, Sclerosing Hemangioma) 220

46 Mycosis Fungoides (MF) 226

47 Ichthyosis Vulgaris 229

48 Porokeratosis 231

49 Xeroderma Pigmentosum 233

50 Erythema Nodosum (EN) 235

51 Urticaria 238

52 Erythema Elevatum Diutinum 241

53 Pityriasis Lichenoides 243

54 Acanthosis Nigricans 245

55 Fixed Drug Eruption 247

56 Granuloma Annulare 249

57 Hemangiomas Lymphangiomas 251

58 Syringocystadenoma Papilliferum 258

59 Leiomyoma 261

60 Sweet’s Syndrome 263

61 Secondary Syphilis 264

62 Arthropod Bite Reactions (Papular Urticaria) 266

63 Keratosis Pilaris 268

64 Lichen Amyloidosis and Macular Amyloidosis 269

65 Malignant Melanoma 271

66 Cutaneous Neurofibroma 274

Index 277

• Epidermis

• Dermis and

• Hypodermis or subcutaneous fat which actually is a soft tissue and not truly a part of the skin, but because of itsexceedingly close anatomic relationship to the skin and its tendency to respond together with the skin in manypathological processes, it is considered to be a third compartment of the skin

Epidermis

The epidermis is the thinnest portion of skin, varying in thickness from 0.04 mm on the eyelids to 1.6 mm on the palms,the average thickness being 0.1 mm It is a metabolically active, stratified squamous, cornifying epithelium The lowerportion of the epidermis has an undulating surface with downward invagination termed rete and interdigitating mesenchymalcones called dermal papillae (Fig 1.1)

Two main types of cells constitute the epidermis:

• Keratinocytes and

• Dendritic cells

2 Histopathology of the Skin

The keratinocytes constitute more than 90 percent of cell population of epidermis and differ from the dendritic cells

or clear cells by possessing intercellular bridges and ample amount of stainable cytoplasm The main dendritic cells of theepidermis are melanocytes, Langerhans’ cells and indeterminate cells Other cells that are present in epidermis are Merkelcells (neuroendocrine) as well as unmyelinated axons

The epidermis shows four distinct but related cellular layers (Fig 1.2) which from below upwards are:

• Basal cell layer (Stratum basale)

• Squamous cell layer (Stratum spinosum)

• Granular layer (Stratum granulosum)

• Horny layer (Stratum corneum)

The term stratum malpighii is often applied to the three lower layers which includes the basal, squamous and granularcells and comprises the viable nucleated portion of the epidermis

Stratum lucidum is the lowest portion of stratum corneum seen in those areas of skin where the horny layer normally

is very thick, i.e on palms and soles It is seen as a thin homogenous eosinophilic zone and is rich in protein-bound lipids

It is also called stratum conjunctum in contrast to the overlying basket weave stratum corneum which is also calledstratum dysjunctum

Basal Cell Layer

The basal cells form a single layer, are columnar in shape and lie with their long axis perpendicular to the dividing linebetween the epidermis and the dermis They have a deeply basophilic cytoplasm and a dark-staining oval or elongatednucleus They are connected with each other and with the overlying squamous cells by intercellular bridges or desmosomes.These desmosomes are less distinct than those in the squamous cell layer At their base the basal cells are attached to thesubepidermal basement membrane zone The amount of melanin present in the basal cells parallels the skin color

Squamous Cell Layer

The cells of the squamous cell layer are polygonal and are named “spinous cells” because of the presence of distinct butdelicate spine like intercellular bridges or desmosomes that with conventional microscopy are seen to cross intercellularspaces and form contact between adjacent cells (Fig 1.3) Squamous cell layer is five to ten layers thick The cells

become flattened towards the surface with their long axis arranged parallel to the skin surface

Fig 1.2: Epidermis showing horny layer , granular

layer and squamous cell layer Fig 1.3: Squamous cell layer showing polygonal cells

and delicate spine like intercellular bridges or desmosomes

Granular Cell Layer

The cells of the granular layer are diamond-shaped or flattened Their cytoplasm is filled with keratohyaline granules thatare deeply basophilic and irregular in size and shape (Fig 1.4) The thickness of the granular layer in normal skin isproportional to the thickness of the horny layer; it is only one to three-cell layers thick in areas in which the horny layer

is thin but measures up to ten layers in thickness in areas with a thick horny layer , such as palms and soles

Horny Layer

Horny layer or stratum corneum is the outermost anucleated layer of the skin, which stains eosinophilic in contrast to theunderlying stratum malpighii It consists of layers of flattened anucleated cells with dense cell wall and apparently emptyinteriors (Fig 1.5) In formalin fixed histologic sections it is difficult to ascertain the exact thickness of stratum corneumbecause some of the outer cell layers get detached during fixation and processing

Melanocytes

Under light microscopy in hematoxylin and eosin stained skin sections melanocytes appear as clear cells in and immediatelybeneath the row of epidermal basal cells The clear space characteristic of melanocytes is actually an artifact of fixationduring which cytoplasm shrinks and becomes concen-trated around the nucleus, thereby creating a space Melanocytes

in fact are not clear cells but possess abundant translucent cytoplasm The nucleus of a melanocyte is smaller and moredeeply basophilic than that of a basal keratinocyte and the cytoplasm of it is dendritic, unlike that of a keratinocyte.Dendrites of melanocytes are revealed more effectively with silver salts that stain melanin black; they then are seen toarborize in all directions among neighboring keratinocytes and even into the upper most part of the dermis It has beenestimated that, a single melanocyte is connected to about 36 keratinocytes, constituting “the epidermal melanin unit.” Insections stained by hematoxylin and eosin, the average ratio of melanocytes to basal keratinocytes is about 1 to 10

Langerhans’ Cells

Langerhans’ cells are dendritic cells that are present just above the middle of the spinous zone of the epidermis Langerhans’cells represent about 4 percent of the entire population of cells in the epidermis Fine unmyelinated nerve fibers ascendinto the epidermis where they touch Langerhans’ cells, thereby creating a link between the nervous and immune systems

Fig 1.4: Granular layer showing diamond- shaped

or flattened cells filled with kerato-hyaline granules

that are deeply basophilic and irregular in size and

shape

Fig 1.5: Basket weave horny layer comprising of flattened anucleated cells with dense cell wall and apparently empty interiors

4 Histopathology of the Skin

In sections stained by hematoxylin and eosin Langerhans’ cells like melanocytes appear as clear cells As identification

of Langerhans’ cell is difficult in conventional sections, special stains are generally required for their detection Enzymehistochemical stains employed for such purpose are adenosine triphosphatase and aminopeptidase These stains demonstrateLangerhans’ cells in the mid epidermis from each of which up to 12 dendritic cytoplasmic processes extend betweenkeratinocytes to reach the granular zone above and the dermoepidermal junction below Electron microscopy is the onlyreliable method of identifying Langerhans’ cells

Merkel Cells

Merkel cells are present within the basal cell layer of the epidermis, oral mucosa and in the bulge region of hair follicles.They cannot be recognized in light microscopic sections

Dermoepidermal Junction (Basement Membrane Zone)

With conventional light microscopy the basement membrane zone is best visualized with Periodic acid-Schiff (PAS) stainwhich makes the basement membrane zone appear as magenta colored linear band situated immediately beneath the basalkeratinocytes (Fig 1.6)

Hair Follicle

Three types of hairs are seen on a human body:

“Lanugo” hairs (L Lana “wool”) which are soft, fine, lightly pigmented hairs that cover the body of a fetus “Vellus” hair(L.Vellus “fleece”) are fine hairs that cover most of the body of children and adults “T erminal hairs” are long coarsepigmented hairs with larger diameter present on the scalp, beard, eye-brows, eyelashes, axillae and pubes Terminal hairsare the only type of hair that consistently possesses a medulla

The hair follicle as seen in longitudinal sections of skin is divided into two segments: upper segment and lowersegment Upper segment includes infundibulum and isthmus Infundibulum is the uppermost part of hair follicle thatextends from the ostium or surface opening of the follicle to the entrance of the sebaceous duct below Isthmus is theshorter portion of the hair follicle that extends from entry of sebaceous duct above to attachment site of arrectorespilorum muscle below Lower segment also consists of two parts, i.e stem and a bulb S tem is that portion of the hairfollicle, which stretches from the base of the isthmus to the end of the keratogenous zone at Adamson’s fringe Bulb is thelowest portion of the follicle below the Adamson’s fringe

Fig 1.6: Periodic acid-Schiff stain showing basement membrane zone which appears as magenta coloured linear band immediately beneath the basal keratinocytes



Adamson’s fringe represents the zone where the process of keratinization starts and forms the boundary between thenucleated cells in the bulb of a follicle and anucleated cells of stem of a follicle The bulk of a follicular bulb consists ofmatrical epithelial cells among which melanocytes are interspersed The bulb in its lower most part is expanded to enclose

a follicular papilla formed of connective tissue which is continuous with the connective tissue sheath (perifollicular) thatenvelops the rest of the follicle up to the infundibulum The infundibulum is surrounded by a connective tissue which issimilar to connective tissue in the papillary dermis

The perifollicular sheath has two components, an outer one with bundles of collagen arranged longitudinally and aninner one with bundles of collagen that encircle the follicle Sheath has fibrocytes that produce it and capillaries that lieparallel to collagen bundles of the outer compartment

A “glassy” periodic acid-Schiff positive basement membrane separates the follicular papillae and perifollicular sheathfrom the follicle itself Abutting the basement membrane are pale or clear epithelial cells of the outer sheath that representsthe periphery of the follicular bulb These cells are columnar in shape and arrange in a palisade Melanocytes in considerablenumber are interspersed among matrical cells in the bulb, but only few melanocytes are found along the course of thestem and the isthmus; in the infundibulum melanocytes are present in a manner similar to that in the normal epidermis.The matrical cells of follicular bulb differentiate along six different pathways, giving rise to six different layers of hairfollicles which from outside inwards are (Fig 1.7):

• Henle’s layer of the inner sheath—one cell thick with prominent, brightly eosinophilic trichohyaline granules and thefirst to cornify

• Huxley’s layer of the inner sheath—two cells thick and characterized by numerous trichohyaline granules

• Cuticle of the inner sheath—one cell thick

• Cuticle of the hair—single layer of imbricated squames that wrap around the hair shaft and interdigitate with cornifiedcells of the cuticle of the inner sheath

• Cortex of the hair

• Medulla of the hair—present only in terminal hair

Matrical epithelium of the follicular bulb consists of a pool of undifferentiated cells that have large round pale stainingfinely stippled monomorphous nuclei with a prominent nucleolus They differ from germinative cells who have smallerdarker nuclei devoid of discernible nucleoli

Hair grows in a cyclical fashion comprising of three phases:

• Anagen (growing phase)

• Catagen (involuting phase) and

• Telogen (resting phase)

Fig 1.7: Cross sections through different levels of the bulb and stem of hair follicles in the subcutaneous tissue



6 Histopathology of the Skin

In histological sections identification of hair in different phases is important

Fully developed anagen hair is identified by the presence of hair bulb consisting of matrical cells and enveloping afollicular papilla (Fig 1.8)

Early catagen (Fig 1.9) is characterized by follicular bulb that looses its bulbous shape, becomes flattened formingthe base of the inferior segment of the involuting follicle Follicular papilla lies adjacent to the flattened base A markedlythickened basement membrane surrounds the atrophic lower segment of the follicle and separates it from the perifollicularsheath

In well advanced catagen the lower segment of the involuting follicle consists of an effete column of epithelial cellssurrounded by thickened, corrugated basement membrane At the base of the column is present an ill formed follicularpapilla

Far-advanced catagen shows a shrunken column of epithelial cells, the residuum of the lowest segment of a follicle,forming a pincer around a well defined follicular papilla and the lower segment is surrounded by a markedly thickened,corrugated basement membrane Tracking behind the involuting follicle is a fibrous tract within which the events of thefollicular cycle takes place (Fig 1.10)

Late catagen is characterized by remnants of follicular epithelium resembling that of normal isthmus The follicularpapilla is seen only as scattered fibrocytes at the base of a follicle now entering into resting stage

Telogen or resting phase consists only of infundibulum and an isthmus (Fig 1.1 1) Few scattered fibrocytes whichare remanants of a follicular papilla are seen at the base of the isthmus Periphery of the isthmus shows columnar cellsaligned in a palisade The bowed cords of undifferentiated epithelial cells that emanate from the junction of infundibulumand isthmus are called mantles

Sebaceous Glands

The sebaceous gland is a lipid producing gland that is present all over the body with the exception of the palms, soles anddorsa of feet Most of the sebaceous glands are connected to a hair follicle In areas like buccal mucosa and vermilion ofthe lip (Fordyce’ s spots), areolae of women (Montgomery’ s tubercles) internal fold of the prepuce (T yson’s glands),labia minora and eyelids (Meibomian glands), sebaceous glands are joined to hair follicles that consist of infundibula only;

no bulb or stem is present

Fig 1.8: Fully developed anagen hair follicle is

identified by the presence of hair bulb and a follicular

papilla

Fig 1.9: Early catagen showing flattened base of the inferior segment of the involuting hair follicle

In histologic sections sebaceous gland is seen to be formed of several lobules Each lobule has an outer row of

undifferentiated somewhat flattened germinative cells with large nuclei and a homogenous basophilic cytoplasm Thecells in the central portion of the lobule are larger in size with a characteristic foamy pale staining cytoplasm and nucleithat become scalloped owing to their compression by lipid vacuoles (Fig 1.12)

The sebaceous duct is lined by cornifying squamous epithelium and contains a thin granular zone The sebaceousgland is surrounded by a PAS- positive basement membrane zone and a thin highly vascular zone of periadnexal connectivetissue

Apocrine Glands

Apocrine glands are found only in certain areas of the body like axillae, areola, periumbilical region, perineal and circumanalareas, prepuce, scrotum, mon spubis, labia minora, external auditory canals (ceruminous glands) and on the eyelids(Moll’s glands)

Fig 1.10: Advanced stage of cat agen showing

involuting hair follicle and a tracking fibrous tract Fig 1.11: Telogen hair follicle consist s of

infundibulum and isthmus

Fig 1.12: Sebaceous gland showing several lobules Nuclei of cells nearest to the peripheral germinative layer of a sebaceous lobule are round but become scalloped as they differentiate, owing to compression by droplets of lipid



8 Histopathology of the Skin

In histologic sections apocrine gland consists of secretary part and excretory duct Secretory part of the gland is acoiled structure that is situated in the lower part of the dermis or in the subcutaneous fat In cross section, the apocrinesecretary coil has a diameter almost ten times greater than that of the eccrine coil (Fig 1.13) The lumen of the secretorycoil is lined by a single layer of cells that are either cuboidal or columnar in shape, and have abundant pale stainingcytoplasm and round nuclei situated near the base of the cells The convex apical borders of the secretory cells projectwithin the lumen to a variable extent The secretory cells are surrounded by a layer of contractile myoepithelial cells, aPAS-positive basement membrane zone and fibers of the periadnexal dermis

Excretory duct of the gland is a straight structure that empties into the infundibulum of a hair follicle, slightly abovethe entrance of sebaceous duct Apocrine duct may also open directly on to the skin surface It is composed of two layers

of cuboidal cells with an inner periluminal cuticle and an outer myoepithelial lining

By conventional microscopy it is difficult to distinguish between apocrine and eccrine ducts

Eccrine Glands (Sweat Glands)

Eccrine gland is the sweat producing gland They are present all over the body including the glans penis and prepuce butexcluding the oral lips, clitoris, labia minora and external auditary canal Palms, soles, axillae and forehead are areas thatshow maximum number of sweat glands The sweat glands are located around the junction of the dermis and thesubcutis

The eccrine unit consists of secretory part, an irregularly coiled structure which leads to coiled dermal excretory ductwhich leads to straight duct that traverse the dermis The spiraled intraepidermal portion of the duct is called acrosyringium(Fig 1.14)

In histologic sections coiled glandular portion of the eccrine unit is found to be formed of two rows of cells (Fig.1.15):

• An outer row of spindle shaped, contractile myoepithelial cells which are located in a discontinuous manner

• A single inner row of secretory epithelial cells which are pyramidal in shape and line the lumen of the gland The row

of secretory epithelial cells also consists of two types of cells Large pale or clear cells (containing glycogen) andsmaller dark cells (containing mucopolysaccharide) The dark cells mostly line the luminal surface, whereas the palecells are situated peripheral to the dark cells and for the most part do not abut the lumen itself Peripheral to the outerrow of myoepithelial cells is a basement membrane that separates glandular epithelium from the richely vascularconnective tissue of the periadnexal dermis

Fig 1.13: Cross section of an apocrine gland which is identified

by its diameter which is ten times greater than that of the

eccrine gland

Fig 1.14: Acrosyringium which is the intraepidermal portion of the eccrine duct It spirals through the epidermis in a cork screw pattern

The dermal portion of the eccrine duct is lined by a double row of small, darkely basophilic, cuboidal epithelialcells A homogenous eosinophilic cutical lines the luminal margin of the entire eccrine duct.

The cells that contribute the intraepidermal portion of the eccrine duct are called as acrosyringeal cells.Theacrosyringeal cells that line the lumen are designated “cuticular” and peripheral cells are named “poroid” Theacrosyringium spirals through the epidermis in a corkscrew pattern

DERMIS

The dermis consists mostly of relatively noncellular connective tissue composed of collagen bundles, elastic fibers andground substance A variety of cells are scattered in variable numbers throughout the mature dermis: fibrocytes, dermaldendrocytes, histiocytes, Langerhan’s cells, mast cells and rarely lymphocytes Within the dermis are lodged nerves,blood vessels, lymph vessels, smooth muscles and epithelial structures of adnexa, namely the folliculo-sebaceous apocrineunits and the eccrine units The dermis is 15 to 40 times thicker than the epidermis, depending on the anatomic site

A fully formed dermis is divisible into two distinct compartments

A thin zone immediately beneath the epidermis (papillary dermis) and around adnexa (periadnexal dermis) Papillaryand periadnexal dermis together constitute the adventitial dermis Adventitial dermis is formed of thin haphazardly arrangedcollagen bundles, delicate branching elastic fibers, plentiful fibrocytes, abundant ground substance and many capillaries

A thick zone that extends from the base of the papillary dermis to the surface of the subcutaneous fat (reticulardermis) The reticular dermis is formed predominantly of thick bundles of collagen arranged in an orthogonal pattern.Elastic fibers course among these bundles Proportionately fewer fibrocytes and blood vessels and less ground substanceare present in the thick reticular dermis than in the thin adventitial dermis Adipocytes of the subcutaneous fat commonlyextend in broad columns into the reticular dermis, where they envelop eccrine units and terminate at bases of hairfollicles



10 Histopathology of the Skin

zone between rete ridges and subpapillary blood vessels The pilosebaceous units and the eccrine and apocrine glands arealso encircled by a thin meshwork of collagen fibers similar to that present in the papillary dermis Thus the papillary andthe periadnexal dermis forms an anatomical unit, the adventitial dermis The blood vessels of the dermis are also surrounded

by a thin layer of fine collagen fibers

In the reticular dermis which is the major portion of the dermis the collagen fibers occur as thick bundles (Fig 1.16).These collagen bundles are oriented horizontally and extend into various directions

Reticulum fibers are special type of very thin collagen fibers, unrecognized with routine stains In normal skin they areonly present in certain areas like basement membrane zone, the region of the adventitial dermis that lies closest to theepidermis and its appendages, around blood vessels and as a basket- like capsule around each fat cell Special stains arerequired for identifying reticulum fibers

Elastic Fibers

Elastic fibers are inconspicuous in routine hematoxylin-eosin stained sections seen under light microscopy Special stainssuch as orcein or resorcin-fuschin are required for their visualization The elastic fibers are thinner than collagen fibers,wavy and are found entwined among the collagen bundles As elastic fibers are wavy , in histopathologic sections theyappear fragmented as only a small part of the fiber is included in any one section In the lower portion of the dermis,elastic fibers are thicker and towards the epidermis they become thinner

an empty look The lobules are separated by thin fibrous septae through which small vessels course

Fig 1.16: Collagen occurs as finely woven

network of fibers in the subepidermal papilla.

The reticular dermis collagen fibers occur as

thick bundles

Fig 1.17: Subcutaneous fat showing adipocytes

of loosely wound branching axons that form irregular masses.

“Unspecialized” receptors are devoid of distinctive structural features and include most of the sensory nerves thatsupply the skin, including those linked to Merkel cells (Figs 1.18 and 1.19)

Blood Vessels

The dermal vasculature consists of a superficial and deep plexuses of arterioles and venules (Fig 1.20) Communicatingvessels connect superficial and deep plexuses The deep plexus is situated in the lower part of the reticular dermis at thedermal-subcutis interface and the superficial plexus is positioned at the junction of the papillary dermis with the reticulardermis The arterioles have a homogenous basement membrane, discontinuous subendothelial elastic lamina and 4-5layers of smooth muscle cells In contrast, the venules have a multilayer basement membrane along with 5-6 layers ofpericytes In larger venules the pericytes take on the characteristic of smooth muscle cells and are separated by layers ofelastic fibers

Capillary networks also surround the sweat glands and hair bulbs The capillary walls lack elastic fibers and contain

a basement membrane which changes from homogenous to multilayered as the capillary changes from an arteriolarcapillary to a venous capillary

Lymphatic Vessels

Lymph vessels are difficult to recognize in histologic sections because of their resemblance to blood vessels In areas oflymph stasis they are seen as spaces lined only by a few endothelial cells In contrast to blood capillaries they lack

Fig 1.18: Peripheral nerve A thin fibrous

capsule, the perineurium surrounds the bundle

of myelinated nerve axons

Fig 1.19: Nerve bundle showing wavy nerve fibers and spindle shaped nuclei

12 Histopathology of the Skin

pericytes and do not possess a basement membrane The lumina are surrounded by loosely arranged collagen fibers andelastic fibers

Mucosal Epithelium

Granular and horny layer is absent in epithelium of the normal oral mucosa with the exception of the dorsum of thetongue and hard palate (Fig 1.21) In the absence of granular and horny layer, the epithelial cells migrate from the basallayer to the surface first appearing vacuolated because of the presence of glycogen, then these cells shrink and finallydesquamate

Fig 1.20: Blood vessels in the dermis Fig 1.21: Oral mucosal epithelium devoid

of granular and horny layer

Fig 1.22: Arrector pilorum muscle showing cigar shaped nuclei with rounded ends The nucleus is present in the center of muscle cells



DERMAL MUSCLE CELLS

Smooth Muscle(Involuntary Muscle)

Smooth muscle shows absence of striations and the nucleus is present in the center of the muscle cell The nuclei are

“cigar-shaped” with rounded ends (Fig 1.22) In skin smooth muscle is present as arrectoris pilorum which arises in theconnective tissue of the upper dermis and is attached to the hair follicle below the attachment of the sebaceous gland.Other smooth muscles of the skin include tunica dartos of the external genitals and in the areola of the nipple

Striated Muscle(Voluntary Muscle)

Striated muscle fibers show characteristic cytoplasmic cross-striations and their nuclei are located at the periphery of thefibers (Fig 1.23) They occur in the skin of neck as platysma and in the skin of the face as muscles of expression

Fig 1.23: Striated muscle bundles



Techniques of Skin Biopsy

C H A P T E R

A biopsy is defined as a process by which a diseased living tissue is removed from the body and then utilized for

microscopic and/or other investigations to establish a precise diagnosis

Skin biopsy is a common investigation performed by a dermatologist, and a thorough understanding of its indications,the various techniques and its limitations is essential This helps a dermatologist to provide maximum useful informationfrom the study of biopsy

Skin biopsy can also be subjected to:

• Immunofluorescence study

• Frozen section

• Tissue crust

• Electron microscopy

• Bacteriological and mycological cultures

A proper selection of site, type of biopsy and type of lesion is very important for a good histopathological preparationand precise diagnosis Following points are to be considered before performing a skin biopsy

• The lesion biopsied should be an early and untreated lesion, representative of the skin disorder as a whole

• If lesions are present at different stages of evolution, it is appropriate to biopsy more than one type of lesion

• Normal skin should be included with a diagnostic biopsy Inclusion of perilesional skin is important when submittingbiopsies for direct immunofluoresence studies

• It is important to ensure that the biopsy is deep enough

• It is always sensible to avoid areas that are liable to heal badly, e.g areas over bony prominences and the lower limbs

• It is also very important to avoid cosmetically important areas while taking a biopsy

• It is equally important that the specimen be removed with the minimum of trauma and that it is properly orientedbefore being placed in a fixative

Any sort of trauma or damage to biopsy specimen should be avoided while performing the skin biopsy This can beachieved by:

• The use of skin hooks in manipulating the specimen during the procedure

• Division of small specimens into multiple smaller portions of tissue for different diagnostic purposes should beavoided to prevent trauma artifact

• Biopsies taken for ultrastructural studies should be small to allow for adequate fixation

• Biopsy specimen once taken, should be placed epidermal side uppermost on a small portion of filter paper to preventcurling and transferred promptly to appropriate transport medium

Skin biopsy is usually done:

• To confirm the diagnosis

• For special pathological investigative techniques that aid in confirming the diagnosis, e.g Immunopathological tests,Enzyme histochemistry, Resin embedding techniques, Autoradiography, Electron microscopy

• For microbiological investigations, e.g mycetoma, deep fungal infections, cutaneous tuberculosis, leprosy

• For assessing the prognosis of certain diseases, e.g LE.

• For assessing the progress of certain diseases e.g leprosy on treatment

• For diagnosis of recurrence of condition after adequate therapy , e.g leprosy

• As a therapeutic remedy in conditions like acquired melanocytic nevi, small basal cell carcinoma, etc

Skin biopsy can be avoided in circumstances like:

• History of bleeding diathesis

• Active infection at the site of biopsy

• Shave biopsy (Epidermal)

• High speed rotatory punch biopsy

Epinephring should not be used in biopsies taken from the fingers or toes or glans penis as occasional intensevasospasm can result in tissue necrosis

Topical anesthetic (PRIMLA) gels are being used and are helpful in biopsies performed in children They are mixtures

of prilocaine and lignocaine These can be used 1-½ to 2 hours before performing biopsy The prepration can be applied

at the site generously and under occlusion

• Punch biopsy:

It is the most common, simple, fast and usually a satisfactory method of skin biopsy The biopsy punch is a metalcylinder of variable diameters with a sharp cutting edge at one end, usually attached to a plastic handle A 4 mm punchprovides an adequate tissue sample for histopathological examination

After giving the proper local anesthesia, the sharp cutting edge of the punch is pressed into the skin with adownward twisting movement as it descends vertically through the dermis into the subcutis As it enters the subcutis,there is a feeling of giving away The punch is then withdrawn slowly The tissue specimen is lifted with the help of

a skin hook and cut at its base and put in a fixative

The wound may be left to heal without suturing, after ensuring adequate hemostasis For cosmetic reasons it may

be closed with simple interrupted sutures and dressed

Punch biopsies are very useful in busy outpatient departments and office practices, particularly for diagnosis andmanagement of small cutaneous lesions

• Incisional biopsy:

This is a type of biopsy done from active edge of lesion that also includes the surrounding normal skin This is usuallyindicated when an adequate tissue specimen is required for histopathological examination

An elliptical incision, perpendicular to the surface and down to the subcutaneous fat, is made The tissue is lifted

at one end with a hook (Kilner/Gillie’ s) or with a needle and the other end is cut with a sharp pointed scissor(preferably iris scissor) Once the specimen is removed, it is placed with epidermis upward on a small blotting paper

16 Histopathology of the Skin

It is then placed in a jar of fixative (10% neutral buffered formalin) This fixative is a protein coagulant and helps thespecimen to stick to the blotting paper without getting wrinkled No toothed forceps to be used during biopsy as it willinjure the tissue

In very cold areas, hills, etc Lillie's fixative may be used:

is important when suturing the wound to ensure that the sutures include the corners of the wound thus avoiding anydead space and subsequent hematoma formation

• Shave biopsy (Epidermal biopsy):

It is a type of biopsy which consists of simply shaving through the superficial part of the lesion (epidermis and upperdermis) It is a tissue sparing procedure that leaves the lower level of dermis intact for better cosmetic result Shavebiopsy can be superficial (for flat and curved surfaces) or deep (saucerization)

With the help of left thumb and index finger , the lesion is stretched and stabilized (two-point finger traction).Outline the perimeter of the lesion to be removed with the very superficial pass of the blade Now hold the blade (no

15 or 22 or razor blade) parallel to the skin surface and insert the belly of the blade through the perforated skin Thelesion is carefully separated by to and fro sweeps through the entire lesion In a vesiculobullous lesion forceps may beused to hold and stabilize the edge of biopsy site to prevent rupture of the vesicle At the same time tissue can begrasped at its center gently with the help of the forcep to provide upward traction

• High speed rotatory punch biopsy:

This instrument allows a biopsy to be performed without anesthesia This is useful if the tissue is used for research asthere is no chance for chemical change due to anesthesia

• Skin surface biopsy:

This technique is used for inspecting horny layer by removing strips of stratum corneum This method is useful in

detection of scabies mite, dermatophytic fungae, Candida species, P.ovale, microorganisms causing erythrasma and

studying the functions of epidermis and hyperkeratotic skin conditions This is not a routinely done method

A drop or two of cyanoacrylate adhesive is placed on the skin for rapid bonding A clean glass slide is then appliedover it and rolled off the skin surface, taking with it a strip of stratum corneum The slide is fixed and then stainedwith various chemicals as indicated

• Curette biopsy:

The curette can also be used to provide tissue for biopsy It is done by scooping the friable part of the lesion andrendering it for histological examination

• Mucosal biopsy:

Sometimes biopsy from a mucosal area is needed and this can be obtained by any of the techniques mentioned earlier,except from lesions occurring deep within the nose or in the eye, which should be referred to their respectivespecialists

Common sites for mucosal biopsy:

a Oral cavity—lips, buccal mucosa, anterior aspect of tongue

b Genitals—penis, including glans penis and scrotum in males, vulva in females

c Rarely eyes, nose, etc

Types of mucosal biopsies:

a Punch biopsy

b Shave biopsy for superficial growths or vesicles

c Excisional biopsy for small, benign growths

d Incisional biopsy for leukoplakia, suspected malignancies, etc

Complications of skin and mucosal biopsies:

a Hemorrhage—primary and secondary

b Secondary infection

c Scarring

d Anaphylaxis to local anesthesia

e Injury to underlying structures

f Change of contours of lip or eye

Dermoepidermal junction can be divided into four major zones:

1 Epidermal portion of the dermoepidermal junction which includes: intermediate filaments, hemidesmosomal plaquesand plasma membrane of the basal keratinocytes Keratin intermediate filaments (keratin 5 and 14) attach tohemidesmosomes (HD) located on the basal plasma membrane Hemidesmosomes have two components:

– Intracellular component which forms the major portion of hemi-desmosome and

– Transmembrane component

Intracellular component of HD includes 230kD bullous pemphigoid antigen (BPAg1) and plectin

The transmembrane component of HD includes α6 β4 integrin and the 180kD bullous pemphigoid antigen (BPAg 2)

• BPAg1 is targeted by autoantibodies from patients with bullous pemphigoid

• BPAg2 is targeted by autoantibodies from patients with bullous pemphigoid, pemphigoid gestationis (PG), cicatricialpemphigoid (CP) and a subgroup of linear IgA bullous dermatosis (LABC)

Fig 3.1: Diagrammatic representation of dermoepidermal junction

• Congenital deficiency of BP Ag2 is observed in some cases of generalized atrophic benign epidermolysis bullosa(GABEB).

• Mutation of the β4 subunit of α6β4 integrin is responsible for junctional epidermolysis bullosa (EB) associated withpyloric atresia

2 The lamina lucida is an electron-lucent zone between basal keratinocytes and lamina densa

Anchoring filaments traverse the lamina lucida extending from hemi-desmosome in basal keratinocytes to the underlyinglamina densa

The antigens associated with these anchoring filaments include:

– Laminin 5 and the surrounding lamina lucida is composed of laminin1 and the nidogen

Autoantibodies to epiligrin, the α3 subunit of laminin 5, result in one form of cicatricial pemphigoid Nonsensemutations of the laminin 5 gene are responsible for Herlitz subtype of junctional epidermolysis bullosa

3 The lamina densa is an electron-dense layer that lies parallel to and contiguous to the lamina lucida It is composedprimarily of type IV collagen Other antigenic components are:

– Laminin 1

– Nidogen and

– Heparan sulphate proteoglycans

4 Sublamina densa region that is the zone below lamina densa shows the presence of short curved structures calledanchoring fibrils which fan out at either end, the distal part inserting into the lamina densa and the proximal partterminating either in the papillary dermis or looping around and merging in the lamina densa The major component ofanchoring fibrils is type VII collagen

Autoantibodies against type VII collagen have been described in:

– Epidermolysis bullosa acquisita (EBA)

– Bullous systemic lupus erythematosus and

– Some variants of linear IgA disease (IgA mediated EBA)

Nonsense mutations of type VII collagen gene are associated with dystrophic epidermolysis bullosa (EB)

Epidermis: Inflammatory cells seen in the epidermis due to exocytosis are usually mononuclears or polymorphs.

Balloon cells In viral infections large swollen up epidermal cells known as balloon cells are seen These cells have

homogenous and intensely eosinophilic cytoplasm

Dyskeratotic cells are abnormal prematurely keratinized epidermal cells taking strongly eosinophilic stain Corps ronds are

dyskeratotic cells with a central pyknotic nucleus, surrounded by a halo and homogenous cytoplasm Grains are ellipsoiddyskeratotic cells seen in stratum corneum with eosinophilic cytoplasm and are larger than parakeratotic cells (Fig 4.1)

Civatte body is an apoptotic keratinocyte that presents as round to ovoid, homogenous eosinophilic structure They

measure approximately 10 µ and are seen in the lower epidermis and papillary dermis (Fig 4.2)

Fig 4.1: Dyskeratotic cells—corps

Malignant dyskeratosis also referred to as individual cell keratinization manifests itself as homogenous, eosinophilic

bodies about 10 µ in diameter that occasionally still show remnants of their nuclei

Paget’s cells are large pale cells with clear cytoplasm and centrally placed nuclei containing a variable amount of densely

stained chromatin (Fig 4.3)

Tzanck cells (acantholytic cells) are epidermal cells which are rounded and not polygonal in shape with a central dark

staining large nuclei and peripherally condensed cytoplasm They are found as a single cell not attached to other epidermalcells (Fig 4.4)

Dermis: The cells seen in the dermis are neutrophils, eosinophils, histiocytes, lymphocytes (B and T), plasma cells, mast

cells, basophils, epithelioid cells, foamy cells, melanoma cells, basiloma cells, all types of giant cells, nevus cells, glomuscells, atypical fibroblasts, melanophages, smooth muscle cells, various benign and malignant tumors and secondarymetastasis from tumors of other organs

Neutrophils or polymorphonuclear leukocytes measures 10 to 15 µ in diameter and are characterized by segmented nuclei

displaying usually 2 to 5 lobes and by cytoplasm containing pink or violet pink granules (Fig 4.5)

Eosinophils can be identified with the light microscope by the presence of a bilobed nucleus in a round cell whose

cytoplasm contains numerous coarse granules that take up eosin and other acid dye stains (Fig 4.6)

Histiocytes are mononuclear cells that range in size from 15 to 25 microns Their nuclei are generally larger and paler than

those of lymphocytes and are gray-blue in sections stained with hematoxylin and eosin Histiocyte nuclei may be round,reniform, deeply indented or multilobed The cytoplasm of histiocyte is more abundant than that of the lymphocyte (Fig.4.7) In tissues the histiocytes often exist for months without undergoing a single cell division The number of histiocytes

in normal skin is small In acute inflammatory reactions there is a prompt influx of monocytes from the blood If thereaction persists for more than a few days, the histiocytes at the site begin to divide

Lymphocytes: T and B lymphocytes are indistinguishable by light microscopy Both can be of various sizes (small, 5 to 8

microns; medium, 8 to 12 microns; and large, 12 to 30 microns) with darkly staining or slightly indented round nucleisurrounded by a narrow rim of light-blue cytoplasm (Fig 4.8) These small, medium and large lymphocytes are actuallystages in a progression of size Just as it is difficult to distinguish some circulating blood monocytes from circulating

Fig 4.3: Paget’s cells Fig 4.4: Civatte body

22 Histopathology of the Skin

lymphocytes, so, too, it may be impossible to differentiate some histiocytes from lymphocytes in inflammatory skindiseases

Plasma cells are rarely found in normal skin, but they are relatively plentiful in the lamina propria of normal mucous

membranes The distinctive features of plasma cells are seen in sections stained with hematoxylin and eosin They areovoid cells with eccentric round or oval nuclei and deeply purple cytoplasm (Fig 4.9) The nuclear chromatin is scattered

in coarse clumps at the periphery of the nucleus, giving it a “clock-face” appearance A pale staining perinuclear area, orperinuclear halo can usually be detected Older plasma cells often contain homogenous eosinophilic globules of varyingsize within their cytoplasm These accumulations of glycoproteins are known as Russell bodies

Fig 4.5: Neutrophils Fig 4.6: Eosinophils

Fig 4.7: Histiocytes Fig 4.8: Lymphocytes

Mast cells arise from undifferentiated mesenchymal cells unlike neutrophils, eosinophils, histiocytes, and lymphocytes,

all of which originate in the bone marrow Mast cells have a characteristic appearance when stained with hematoxylin andeosin and viewed through the light microscope They vary in size from 8 to 15 microns and have a central dark staininground to ovoid nucleus Mast cell cytoplasm is replete with tiny (0.6 to 0.7 microns) amphophilic granules Mast cellgranules demonstrate metachromasia, by which it means a reaction in which a dye selectively stains certain tissuesubstances in a color that differs from the color of the dye itself (Fig 4.10) The production of metachromasia resultsfrom the presence of free electronegative charges of a certain minimal density Acid mucopolysaccharides, nucleic acids,and other acidic groups, if present in suf ficient quantity, will promote metachromasia Mast cell granules aremetachromatically stained purple with basic dyes, such as toluidine blue and methylene blue The metachromasia of mastcell granules results from their contents of the strongly acidic sulfated mucopolysaccharide heparin

Basophils: In human skin connective tissue, mast cells and blood basophils appear to be independent cell types, despite

the similar staining properties of their granules Morphologically , these two cells dif fer in the shape of their nuclei, the

Fig 4.9: Plasma cells



Fig 4.10: Mast cells



24 Histopathology of the Skin

basophil nucleus being multilobed and the mast cell being single-lobed, and in the size of their granules, the basophilgranules being slightly larger than those of the mast cell Because basophils cannot be identified in sections stained withhematoxylin and eosin, but with special stains only, they are not crucial to the histologic diagnosis of inflammatory skindiseases

Epithelioid cells arise from macrophages, after they have completed phagocytosis of a digestible foreign product Epithelioid

cells can develop in granulomas mostly due to delayed hypersensitivity reaction Epithelioid cells usually have a large ovalpale vesicular nucleus and have abundant ill defined slightly eosinophilic cytoplasm (Fig 4.11)

Giant cells are macrophages which show numerous nuclei fused together and large cytoplasm The following types of

giant cells are described:

• Foreign body giant cell—many nuclei, irregularly arranged in the cytoplasm

• Langhans giant cell—multiple nuclei in horse-shoe fashion arranged peripherally and leaving large cytoplasm (Fig.4.12)

Fig 4.11: Epithelioid cells Fig 4.12: Langhans type giant cells

Fig 4.13: Melanophages



• Wreath giant cell—multiple nuclei arranged in a ring fashion like a wreath of flowers seen in xanthomas.

• Rosette giant cell—nuclei arranged in ovoid shaped ring pattern seen in compound nevi

• Tuoton giant cell—foamy cytoplasm and a central ring of multiple nuclei seen in lipidoses

• Ground glass giant cell—random multiple nuclei and homogenous cytoplasm seen in reticulohistiocytoma

• Tumor giant cells—Reidsternburg cell also called a tumor giant cell

In the dermis one has to identify sometimes wavy Schwann cells in a nerve fiber and these cells are usually mistakenfor smooth muscle cells in arrectores pilorum muscle If elastic fiber stain is done, arrectores pilorum muscle will showpresence of elastic fibers whereas a nerve fiber will not Difference between foamy cell and adult fat cell can also be madeout The cytoplasm of foamy cells will be hazy or have ground glass appearance whereas in H and E stain adult fat cellwill appear empty with nucleus pushed to one side

Melanophages are dermal macrophages containing melanin granules (Fig 4.13).

Nevus cells basically are modified melanocytes originating from the junctional zone of epidermis and as they migrate into

dermis they leave their melanocytic activity and loose their dendritic processes and are usually arranged in clusters ornests Varying cellular forms are seen: small dark cells, epithelioid type cell, giant cell and spindle shaped cells Nevus cells

in the upper dermis commonly resemble epithelioid cells, usually oval or cuboidal in shape with large round or ovalnucleus and may contain melanin pigment (Fig 4.14) In lower dermis they resemble fibroblasts and are elongated inshape and show a spindle shaped nucleus

Glomus cells: These cells are found clustered around the vascular channels These are large cells with a clear cytoplasm

resembling epithelioid cell and a large pale, uniform oval or round nuclei (Fig 4.15) The glomus cells irregularly extendfrom the wall of blood vessels deeper into the stroma of the collagenous tissue

Malignant cells usually pose little difficulty in identification They are irregular in shape with dark hyperchromatic nuclei,

and show abnormal mitotic figures

Fig 4.14: Nevus cells Fig 4.15: Glomus cells

Common Terminologies Used in Dermatopathology

Argentaffin

Ability to reduce silver salts to metallic silver Melanin possesses phenolic groups capable of reducing the silver salts thatare present in ammoniated silver nitrate to free black silver (The Fontana - Masson stain contain ammoniated silvernitrate)

Ballooning Degeneration

A type of degeneration of epidermal cells in which the affected cells swell and loose their attachment to adjacent cells,thus separating from them (secondary acantholysis) The cytoplasm of these cells becomes homogenous and intenselyeosinophilic and some are multinucleate

Corps and Ronds

These are solitary or small groups of dyskeratotic cells in the upper malpighian and horny layer They have a centralhomogenous, basophilic pyknotic nucleus and a clear perinuclear halo Peripheral to the halo lies basophilic, dyskeratoticmaterial as a shell The non-staining halo in some cases is partially replaced by homogenous, eosinophilic dyskeratoticmaterial Because of their size and prominent halo corps ronds stand out clearly

of outline of bundles) and bluish staining

Desmoplasia

Desmoplasia or desmoplastic reaction means the formation and development of fibrous tissue in increased amountsresulting in fibrosis

Dyskeratosis

Faulty and premature keratinization of individual keratinocytes Two types of dyskeratosis are recognised:

Acantholytic dyskeratosis occurs as corps ronds, which consists of a central, homogenous, basophilic, pyknoticnucleus surrounded by a clear halo; peripheral to the halo lies a shell of basophilic, dyskeratotic material Neoplasticdyskeratosis referred also as individual cell keratinization manifests itself as homogenous, eosinophilic bodies about 10

μm in diameter that occasionally still show remnants of their nucleus