Normal Histopath Anatomy

Objectives

• Present a introduction to histology of the skin Normal Skin Histology • Discuss the different cell types in the skin • Cover the reginal variation and artifacts that are seen in the skin Jason Stratton

I have no conflicts of interest Duplicate Function Layers

1. Epidermis • Protectionor / mechanical barrier – UV, chemical, thermal insult – Dehydration 2. Dermis • Thermoregulation & electrolyte balance – Prevents & facilitates heat loss • Sensation – Receptors for pressure & pain • Immunologic organ • Sensuality & psychological well-being • Metabolic 3. Subcutaneous Adipose Tissue – Subcutaneous fat for energy – Vitamin D synthesis

Distribute Epidermal Cell Function Dermal Cell Function

• Immune organ • Supportive – Langerhan cells – antigen presenting cells – Fibroblasts – produce fibers & ground substance – Keratinocytes – produce interleukins, interferons, & growth • Collagen fibers – tensile strength factors • Elastic fibers – retractile properties • Protection UV • Immunologic function – Melanocytes – Dendritic cells, lymphocytes, mast cells, macrophages Not• Produce melanin from tyrosine via tyrosinase & store within • Provides nutrients melanosomes – Vascular networks • Transfer melanosomes to adjacent keratinocytes • Mechanical barrier • Thermoregulation – Keratinocytes – structural integrity via keratin filaments, – Vascular networks Do desmosomes, hemidesmosomes – Nerve supply

1 Epidermis Keratinocytes

• Stratified keratinizing squamous epithelium – Continuously renewing itself • Stratified 4/5 layers – Maintains nl thickness via desquamation – Stratum corneum (cornified / horny layer) • Cells (4) • Cornified/keratin – Keratinocytes (80-90%) – Stratum lucidum • Keratin = intermediate filament (tonofilament) • Only palms & soles – Dendritic cells • Melanocytes – Stratum granulosum (granular layer) • Langerhan cells (3-5%) • Thickest on palms & soles – Stratum spinosum (squamous / prickle cell – Merkel cells (neuroendocrine cells) Stratum layer) Malpighi • Also contains – Stratum basalis / germinativum (basal – Opening eccrine ducts (acrosyringium) layer) – Opening hair follicles (acrotrichium) Duplicate Keratinocytes Basal layer (Stratum basalis, germinative layer) • Interface b/w epidermis • Single layeror of mitotically & dermis is irregular active cells above the – Dermal papillae basement membrane that • Upward projections of papillary dermis into give rise to other epidermis keratinocytes – Rete ridges • Basal cells contain melanin • Portion epidermis covering apical surface of separating the dermal papillae nucleus • Attach basement membrane via hemidesmosomes

SquamousDistribute Layer Granular Layer (Stratum spinosum) (Stratum granulosum) • Flattened cells •5-10layers keratinocytes •2 to 3cell layers thick – Generally proportional to thickness horny • Maturation layer – Suprabasal cells are polyhedral • Filled with basophilic keratohyalin granules – Form 2 structures with abundant cytoplasm and a • Interfibrillary matrix / filaggrin vesicular nucleus w/ a prominent – Cements keratin filaments together • Marginal band nucleolus – Inner lining of horny cells – ↑ sulfur (cysteine disulfide bonds) → – SuperficialNot cells larger, flattened, strength & stability eosinophilic, oriented parallel • Tonofilaments ↓ sulfhydryl groups → soft & flexible → “soft” keratin • In contrast, hair & nails, which lack • Keratinocytes connected via keratohyaline granules, incorporate disulfide desmosomes (intercellular bonds themselves → “hard” keratin Dobridges)

2 Cornified Layer Basement Membrane Zone (Stratum corneum) • Specialized region at the interface between the • Flattened eosinophillic, anucleated epidermis & dermis • Dead • Homogeneous, • Basket-weave keratin eosinophilic band below pattern (orthokeratotic) Basement membrane (PAS) – Portion horny cytoplasm w/ “Basket-weave” the epidermis disulfide cystine bonds Stratum Corneum shrinks formalin fixation – 1.0 microns thick • Thinnest in eyelid and – Highlighted by periodic genitals acid-Schiff (PAS) stain • Thickest on palms and soles • Continuous around epidermal appendages Duplicate Melanocytes

•Origin – Neural crest or • Function – Produce melanin, which protects against non-ionizing ultraviolet irradiation • Histology – Clear peripheral halo surrounding small deep purple ovoid nucleus along basal layer • Ratio of melanocytes to keratinocytes – Normally varies from 1:4 to 1:10 – Does not depend on race (type of melanosome does) • Immunohistochemistry – S-100 protein, Sox-10, Melan-A / MART-1 (Melanoma Antigens Recognized by T cells-1), HMB-45 Melanocytes Melanocytes •EM – Abundant pale cytoplasm – Scattered solitary melanosomes – No tonofibrils / desmosomes

Distribute Melanosomes

• Stages (4) – Round w/o melanin • I: seen in balloon cell melanoma – Ellipsoidal w/ numerous longitudinal filaments • II: melanin deposits start Not • III: melanin deposits are prominent • IV: fully-packed w/ melanin obscuring internal structures Melanocyte – Transferred to keratinocytes » Epidermal melanin unit = 1 melanocyte : 36 keratinocytes DoMelanocyte

3 Melanosomes Langerhan Cells Lightly Darkly •Origin Pigmented Pigmented – Dendritic cells derived from bone marrow Skin Skin • Function – Antigen presenting cells Melanization Stages II, III Stage IV>III • Important role in cutaneous immunity – Allergic contact dermatitis, allograft rejection & immune tolerance and immune surveillance • Depleted by chronic UVL, steroid application, HIV/AIDS Size 0.3-0.5 0.5-0.8 • Histology microns microns – Clear cells (appear to lie w/i lacunae) in the suprabasal epidermis w/ reniform nucleus Number per <20 >200 • Immunohistochemistry cell – POS: HLA-DR antigen, CD4, S-100 protein, CD1a, langerin – Also positive: Fc & C3 receptors, CD1c (M241), LCA, actin-like & Distribution Groups of 2- Singly vimentin filaments, ATPase, & aminopeptidase – PNA (peanut agglutinin) – stains w/ characteristic halo and/or dot 10 dispersed pattern •EM Degradation Fast Slow – Cerebriform nucleus with Birbeck granules = tennis racquet shaped membrane bound vesicle w/ a dilated end and laminated elongated handle Duplicate or

Langerhans Cells Langerhans Cells Langerhan Cells (CD1a) Langerhan Cells (CD1a)

Distribute Merkel Cells (Neuroendocrine cells)

• Location – Scattered irregularly throughout basal layer – May group together above enlarged terminal sensory nerve fibers • Function – Mediate tactile sensation Not • Histology – Not recognized • Immunohistochemistry – Express a synaptophysin, neuron specific enolase, cytokeratin 20 (and other non-specific low molecular weight cytokeratins such as CK 8, CK 18, & CK 19) Langerhans Cell •EM – Possess membrane-bound electron-dense (neurosecretory) Do granules that vary in size between 80 and 200 nm

4 Merkel Cell Merkel Cells (CK20) Merkel Cells (CK20) Duplicate

Dermis • Composition • Layers or – Glycoprotein matrix – Papillary • Ground substance largely • Thin, superficial •c/o finely woven collagen nonsulfated acid fibers mucopolysaccharides such • Periadnexal dermis of as hyaluronic acid similar quality (adventitial – Stains w/ colloidal iron & dermis) digests w/ hyaluronase • Predominantly type III – Type I collagen (90%) collagen • Fine elastic fibers • Eosinophilic fibers in orientated perpendicular birefringent bundles – Reticular – Elastic fibers • Wider, deeper • Wavy, eosinophilic fibers • Coarse thick collagen • Verhoeff Van Gieson bundles elastic staining • Predominantly type I • Orcein / resorcin-fuchsin collagen Dermis stains • Elastic fibers thicker & parallel to the surface epithelium Dermis Distribute Oxytalan

Elaunin Not

DoElastic Fibers Elastic Fibers Collagen & Fibroblasts

5 Dermal Vessels

•Two plexi – Superficial plexus • Located at the papillary & reticular dermal junction – Deep plexus • Located at the dermal & subcutaneous junction • Connected by vertically oriented communicating vessels • Endothelial cells –POS: CD34, CD31, Ulex europaeus lectin, factor VIII-related antigen (vWF) – Contain HLA class I (HLA-A, B, C) & HLA II (HLA-DR) antigens Duplicate Dermal Lymphatics Glomus Body

• Specializedor arteriovenous (AV) shunts (Sucquet-Hoyer canal-arterial segment) • Location • Location – Collapsed in normal dermis – Pads & nail beds of fingers & toes, central • May be difficult to distinguish from blood face and ears vessels, unless valve represented • Function – Form a complex network and follow the distribution of the blood vessels – Modified smooth muscle cells that regulate • Histology blood flow and temperature – Lined by a single layer of flattened cells • Histology – Have a thin wall without pericytes – Large cells with uniform, round to oval nuclei – Surrounded by a discontinuous basement and abundant cytoplasm membrane – Surround endothelial lined lumen • Immunohistochemistry • Immunohistochemistry – POS: CD31, D2-40 – NEG: FVIII-related antigen, CD34 – POS: Smooth muscle actin (SMA)

Nerves ofDistribute Dermis Pacinian Corpuscle (Vater-Pacini Corpuscle) • Sensory & autonomic nerves • Location – Permeate entire dermis – Travel along course along superficial – Weight-bearing surfaces of & deep plexi deep dermis & subcutis • Sensory (fingers & toes) – Mediate pain, touch, itch, • Function temperature,Not pressure & vibration – Mediates sense of pressure & • Autonomic vibration – Derived from the sympathetic nervous • Histology system – Innervate blood vessels, arrector pili, – Concentrically arranged eccrine and apocrine glands Schwann cells with an axon Do (Onion skin)

6 Meissner Corpsuscle Subcutaneous Tissue • Location (Subcutis, hypodermis) – Tips of dermal papillae • Function (approx. q4), volar skin – Thermal regulation, insulation, provision energy, (fingertips) protection mechanical injuries • Function • Histology – Mediates sense of touch – Consists of lobules of mature adipocytes separated by thin fibrous septa • Histology – Adipocytes are round cells w/ cytoplasm filled w/ – Ovoid structures composed lipids which compresses the nucleus to one side – Abundant blood vessels, nerves & lymphatics are of transversely arranged present laminar Schwann cells • Immunohistochemistry containing an axon – S-100 protein, vimentin Duplicate orAdnexal Structures • Pilosebaceous Unit – Hair and Hair Follicles – Sebaceous Glands – Sensory End Organ – Arrector Pili • Apocrine Glands • Eccrine Glands

Subcutis Adipocytes

Infundibulum PilosebaceousDistribute Unit (Pilar Unit) Isthmus • Pilar Unit = hair follicle, sebaceous gland, arrector pili muscle, & (when present) apocrine glands • Division

–Not Entry of sebaceous duct (upper arrow) Inferior Segment divides infundibulum above from isthmus below – Insertion of pilar arrector muscle (lower arrow) divides isthmus above from inferior follicle below Do Pilosebaceous Unit

7 Hair Follicle Hair Follicle

• Everywhere on the body • Growth phase – Except palms, soles, & mucocutaneous junctions – Anagen (84%) • Types • Active growth phase – Lanugo hairs • Length: years (scalp longest) • Fine hair on the fetal body; shed in utero or during 1st wk of life • Amount related to length of hair – Vellous hairs – Catagen (2%) • Fine, very short, non-pigmented, nearly imperceptible, & cover most of the body • Involuting phase (apoptosis-driven regression) • Similar to terminal hair follicles but proportionately smaller, • Length: 2-3 wks usually non-medullated, & without an assoc. arrector pili muscle – Telogen (14%) – Terminal hairs • Resting phase • Larger, scalp and eyebrows • Length: few months Duplicate Hair Follicle

• Histology (anagen hair) Hair Follicle or – Layers (beginning from innermost) • 1. Hair medulla 2. hair cortex 3. hair cuticle 4. three concentric layers inner root sheath 5. Huxley’s layer 6. Henle’s layer 7. outer root sheath 8. basement membrane • At level isthmus – inner root sheath disappears, outer root sheath keratinizes (trichilemmal keratinization) – trichohyaline granules red in H&E- stained sections • Normal flora of follicular infundibulum – Staphylococcus epidermis – Yeasts of Pityrosporum – Demodex folliculorum mite

Distribute Not Do

8 Demodex Folliculorum

Demodex Folliculorum

Pityrosporum Duplicate

Yeasts of Pityrosporum Sebaceous Glands • Malassezia spp. – 11 or species • Malassezia spp. – • All cutaneous surfaces 11 species – Except palms & soles –Forms – M. globosa • Yeast (Pityrosporum ovale / orbiculare) - – M. slooffiae Commensals of normal human skin; ?role seborrhoeic dermatitis, atopic dermatitis, – M. obtusa • Free sebaceous glands unassociated with folliculitis – M. sympodialis hair follicles – M. sympodialis, M. globosa, M. – M. furfur – Areola & nipple = Montgomery's tubercles restricta – M. restricta • Fungal – pityriasis (tinea) versicolor – M. pachydermatis – Labia minora and glans = Tyson's gland – M. globosa & M. sympodialis – M. dermatis – Eyelids = meibomian glands of the tarsal plate – Physiology & biology – M. japonica – Eyelashes = Zeis glands • Lipophilic (all except M. pachydermatis) = – M. nana – Buccal mucosa & vermilion border of lips = require source (olive oil) long-chain fatty – M. yamotoensis Fordyce's spot acids • 2-4 micron, broad-base budding yeast w/ collarette

Sebaceous GlandsDistributeSebaceous Glands • Histology Apocrine Glands – Lobules lead to common • Always connected to pilosebaceous follicle excretory duct lined by stratified squamous • Located – Axillae, anogenital region, external auditory canal epithelium (ceruminous glands), eyelids (glands of Moll), – Cells of lobule areola, periumbilically, & uncommonly on face and scalp • PeripheralNotrow of basophilic undifferentiated • Consist of cells (germinative cells) – Secretory portion • Central cells with • Situated in the lower dermis or subcutaneous fat, vacuolated cytoplasm-lipid – Straight ductal (excretory) portion accumulation (sebocytes) • Opens into follicular infundibulum above opening of sebaceous duct or to epidermal surface – Will undergo holocrine-type Do excretion (sebum)

9 Apocrine Glands Eccrine Glands • Arise independently of pilosebaceous • Histology unit • Location – Single layer of columnar cells – Present on almost all skin surfaces, esp. abundant on palms, soles & in with abundant eosinophilic axillae • Function cytoplasm with round, basal – Involved in temperature regulation nuclei • Consist of –Secretoryportion Acrosyringium – Outer layer of myoepithelial • Coiled secretory portion present at the lower dermis or at the dermal- cells subcutaneous junction – Excretory portion – luminal cells – Decapitation secretion produce PAS-D eosinophilic cuticle • Coiled duct leads to secretory coils • Apical portions of cytoplasm • Straight intradermal duct • Spiraled intra-epidermal duct forms dome shaped projections (acrosyringium) into lumen which are "pinched off" Eccrine Glands Duplicate Cutaneous Muscles or • Types – Smooth muscle • Location – Arrector pili most common » Attaches to the hair follicle below the sebaceous gland – Scrotum (tunica dartos) & areola – Blood vessel walls • Histology – Centrally placed nuclei – No cross-striations – Skeletal muscle • Location – Facial muscles, eyelid, lip & neck (platysma) • Histology – Peripheral nuclei – Cross-striations Arrector Pili Areola

Distribute Inflammatory Cells

• Small number may be found in normal dermis – Perivascular areas Not – Immune surveillance • Inflammatory dermatoses – Larger number of various types of inflammatory cells found in dermis & occasionally epidermis Do Lip

10 Neutrophils Eosinophils • Histology • Histology – Deeply basophillic, – Refractile pink-red segmented nuclei granular cytoplasm – Pale pink granular – Bilobed nuclei cytoplasm

• Encountered • Encountered Eosinophils – In many inflammatory, – In bullous pemphigoid, neoplastic & infectious Well’s syndrome, diseases allergic dermatoses • i.e. LCV, Sweet’s, bacterial infections Duplicate Lymphocytes Plasma Cells • Histology • Histology – Large oval-shaped cells – Small mononuclear – Abundantor purple cytoplasm cells with scant – Paranuclear Hof cytoplasm • Perinuclear clearing due to Golgi apparatus • Encoutered – “Clock-face” chromatin pattern w/i eccentric placed – Predominate cell type Lymphocytes nuclei in subacute & chronic • Immunohistochemistry inflammatory – CD79a, CD138, kappa, dermatoses lambda • Encountered • i.e. Lupus, lichen – Syphilis, rhinoscleroma, planus, GVHD granuloma inguinale

HistiocytesDistribute Mast Cells • Histology • Histology – Large pink cells (low N:C ratio) – Normally found in small numbers in • Abundant pale eosinophilic the skin and mucous membrane in a perivascular distribution cytoplasm – Medium sized mononuclear oval to • Eccentric to centrally placed spindle-shaped cells with a vesicular, lightly staining elongated to moderate amount of finely granular footprint-shaped nucleus w/ a clearly amphophilic cytoplasm and a centrally placed round to oval visibleNot nuclear membrane nucleus • Encountered • Histochemistry –Any metachromatic stain – In large numbers in many • Geimsa, toluidine blue, alcian blue granulomatous processes • Immunohistochemistry • i.e. tuberculoid, sarcoidal, fungal, – CD117 (ckit), tryptase parasitic and foreign body • Encountered granulomas Histiocytes – Mastocytosis, urticaria, & variety Do benign soft tissue tumors

11 Histologic Differences of Skin Acral Skin With Age (Intrinsic) • Location • Newborns & children – Sole of foot & palm of hand – Epidermis: same thickness, ↑melanocytes & Langerhan cells • Histology – Dermis: ↑fibroblasts & eccrine glands; – Thick, compact stratum apocrine glands not dev. until after puberty corneum – Subcutis: ↑brown fat (thermogenesis) • Loss characteristic basket-weave pattern • Elderly: atrophy & ↓most elements – Stratum lucidum – Epidermis: ↓melanocytes, melanosomes, – Thick stratum pigmentation, Langerhan cells granulosum – Dermis: thinned, ↓cellularity, ↓vascularity, – Abundant eccrine glands ↓collagen, ↓elastin, ↓ground substance, ↓glands (but eccrine glands ↑size – – Absence of sebaceous hyperplasia), ↓hair follicles pilosebaceous units (vellus→terminal) Solar Elastosis – Prominent epidermal – Subcutis: thinned face, shins, hands, feet; ridges thickened abdomen (men) & thighs (women) – Dense dermis *Hallmark extrinsic aging is solar elastosis Acral Skin Duplicate Other Regional Variations Conjunctiva • Mucosa – Location: oral cavity, vulva – Histology: stratum corneum & 1. Cornea granular layer absent; clear cytoplasm due to glycogen 2. lens or • Eyelid – Thin stratum corneum 3. fornix – Many vellus hairs in mid dermis 4. marginal conjunctiva – Modified apocrine glands (Moll’s glands) 5. palpebral portion of the lacrimal gland •Nose 6. tarsal conjunctiva – Numerous, well-developed large sebaceous glands Face •Ear Scalp • 2-5 layers of columnar or – Abundant vellus hairs cuboidal cells •Back • Epithelial cells may have – Thickened reticular dermis eosinophilic cytoplasm and •Scalp apical snouts – Hair follicles extending into Back subcutis • goblet cells that secrete •Face mucin – Prominent pilosebaceous units • melanocytes • Axilla – Pilosebaceous units & apocrine • Langerhans cells glands Mucosa • intraepithelial lymphocytes Eyelid

Artifacts • Fixation Distribute – Poor / inadequate fixation The end of Normal Histology • Slide preparation • Produced at time excision – Cautery – vertical elongation of keratinocytes – Excessive squeezing Cautery • Storage – Low temp – freezing artifact • CharacteristicNot of certain pathology – Basal cell carcinoma – holes – Toxic epidermolytic necrolysis – lack of epidermis Do Freezing

12