9. MUST to KNOW in Histopathology

Germ layers Categories of tissues

Covering Epithelia Simple squamous Simple cuboidal Simple columnar Stratified squamous Stratified columnar Pseudostratified columnar Glandular Epithelia Exocrine glands

Endocrine glands Pancreas Merocrine Apocrine Holocrine Connective Tissues Connective tissue Collagen

MUST TO KNOW IN HISTOPATHOLOGIC TECHNIQUES 1. Ectoderm 2. Mesoderm 3. Endoderm 1. Epithelial = 3 germ layers 2. Nervous = endoderm 3. Muscular = mesoderm 4. Connective = mesoderm Bowman’s, endothelium, loop of Henle, alveoli Ducts of glands, walls of thyroid follicles Gallbladder (nonciliated) Uterine tube (ciliated) Skin (keratinized) Vagina, esophagus, cervix (nonkeratinized) Male urethra Female reproductive tract (nonciliated) Trachea (ciliated), Epididymis w/ ducts Tubular = stomach, uterus Acinar/alveolar = pancreas, salivary glands Tubuloacinar = prostate Ductless Exocrine = enzymes Endocrine = hormones No loss of cytoplasm Goblet cells, sweat glands w/ cytoplasmic loss Distal portion is pinched off Mammary glands Disintegrating cell and its constituents released Complete breakdown of cell Sebaceous gland Support Framework Cells are widely separated Major ingredient of connective tissues Stains: “VgMMAK” Van Gieson Mallory’s aniline blue Masson’s trichrome Alcian blue Krajian’s aniline blue

General Connective Tissues Loose CT Wharton’s jelly (acid MPS) BM (reticular) Lymph node (reticular) Embryo (mesenchyme) Hypodermis lec.mt 04 |Page | 255

Dense CT

Dermis Capsules Tendons Stroma of cornea

Special Connective Tissues Cartilage Hyaline = trachea Fibrous = intervertebral discs Elastic = ear, epiglottis Bone Cancellous/spongy/trabecular = epiphysis/ends of long bones Compact/cortical = diaphysis/shaft Others Blood Lymph Hematopoietic tissues Acid mucopolysaccharides Fixative: Lead fixatives Stain: Alcian blue Osteogenesis imperfect Brittle bone disease Defective production of collagen Deposits found in Connective Tissues (Eosinophilic) Fibrin Early: yellow Old: blue Stains: Mallory’s PTAH Lendrum’s MSB Fibrinoid Necrotizing vasculitis Staining reactions identical to fibrin Mixture of exudates & altered cytoplasmic constituents Hyaline Degenerated collagen Hypertension, atheroma, diabetic kidney Stain: PAS Amyloid TB, leprosy, osteomyelitis Stains: “CoMT” Congo Red Metachromatic stain Thioflavine Muscle Tissues Smooth Involuntary Intestines, blood vessels Skeletal Striated, voluntary Skeletal muscles Cardiac Striated, involuntary Heart Nervous Tissues CNS Brain, spinal cord PNS Peripheral nerves Special receptors Ear, eye, nose Inflammation Inflammation Latin word: Inflammare (to set afire) 5 Cardinal Signs of Inflammation 1. Rubor Redness Blood flow  Injury 2. Calor Heat  lec.mt 04 |Page | 256

3. Tumor

Swelling

4. Dolor

Pain

 Fluid extravasation  Sensory nerves Loss of function Destruction of functional units Acute inflammation Vascular & exudative ---(Tissue)---> Microphages Subchronic inflammation Intergrade between acute & chronic Chronic inflammation Vascular & fibroblastic ---(Tissue)---> Macrophages Inflammation according to Characterisics of Exudate Serous inflammation Serum/secretions from serosal mesothelial cells (3P’s) Pulmonary TB Fibrinous inflammation Diphtheria, rheumatoid pericarditis Early stage of pneumonia Catarrhal inflammation Hypersecretion of mucosa Hemorrhagic inflammation Blood + exudates Bacterial infections & other infections Suppurative/purulent inflammation 5. Functio laesa

Retrogressive Changes = Organ/Tissue smaller than normal Developmental defects: AAHA Aplasia Incomplete/defective development of a tissue/organ Ex. amastia (breast aplasia) Atresia Failure to form an opening Hypoplasia Failure of an organ to reach its matured size Agenesia Complete non-appearance of an organ Atrophy Physiologic atrophy Natural Thymus, brain, sex organs Pathologic atrophy Vascular atrophy Pressure atrophy Atrophy of disuse Exhaustion atrophy Endocrine atrophy Brown atrophy Lipofuscin Progressive Changes = Organ/Tissue larger than normal Hypertrophy Increased tissue size due to increased cell size Physiologic: ásize of uterus Pathologic: Systemic hypertension Hyperplasia Increased tissue size due to increased cell number Physiologic: Glandular proliferation of the female breast, ásize of uterus (preg.) Pathologic: Skin warts due to HPV Compensatory hyperplasia Ex. Enlargement of one kidney Pathologic hyperplasia Ex. Endometrial hyperplasia Congenital hypertrophy Phenytoin-induced Degenerative Changes = Tissues have abnormalities Metaplasia Reversible One adult cell type ↔ Another adult cell type lec.mt 04 |Page | 257

Dysplasia Anaplasia/ Dedifferentiation Neoplasia/tumor Oncology Parts of a tumor Types of tumor

Benign Malignant Leukemia Lymphoma Squamous cell papilloma Squamous cell carcinoma Hepatoma/ hepatocarcinoma Melanoma/ melanocarcinoma Ectopic pregnancy Grading

Grade I II III IV Staging UICC AJCS TNM system T

Reversible One type of adult cell ↔ Changes in structural components Irreversible Criterion toward malignancy Adult cell  More primitive cells (release tumor markers) Continuous abnormal proliferation of cells w/o control (no purpose/function) Ex. Leukemia Study of neoplasm Tumors 1. Parenchyma = active elements (tumor cells) 2. Stroma = CT framework 1. Capacity to produce death: - Benign (Ex. mole) - Malignant 2. Histologic characteristics: - Medullary = cells (parenchyma) > supporting tissues (stroma) - Scirrhous = supporting tissues (stroma) > cells (parenchyma) “-oma” “SaMe CarE” “-sarcoma” = mesenchymal/CT “-carcinoma” = epithelial tissues Malignant Benign Malignant Malignant Malignant Fallopian tube pregnancy Grading Aggressiveness/level of malignancy Differentiated cells = resemble normal cells Undifferentiated cells = younger cells Broder’s Classification (Grading) Differentiated Cells Undifferentiated Cells 75-100% 0-25% 50-75% 25-50% 25-50% 50-75% 0-25% 75-100% Staging Size, extent of spread to lymph nodes, +/- metastases TNM classification Grading + staging TNM System Applicable to all forms of neoplasia 1’ tumor #: denotes the size of tumor and its local extent Tis = carcinoma in situ Ta = non-invasive Tx = cannot be evaluated T0 = free of tumor

Treatment Surgery ↓ ↓ Radiation

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N

M

Teratomas

Apoptosis Necrobiosis Necrosis Types of Necrosis Coagulation necrosis

Liquefaction/colliquative necrosis Caseous/caseation necrosis Gangrenous necrosis Fat necrosis Fatty degeneration 1’ changes 2’ changes Algor mortis (1st)

T1 = lesion Pig (3’) > Sheep (4’) > Horse (5’) > Guinea pig (6’) Mice

“LUBO” = paired Lung Uterus Breast Ovary (+) CK 20 Stomach (-) CK 7 Colon (+) CK 7 Transitional cell carcinoma of the bladder (+) CK 20 Mucinous ovarian tumor (-) CK 7 HCC (-) CK 20 RCC SCC Thyroid carcinoma Prostatic adenocarcinoma EMA (Epithelial membrane (+) carcinoma “BuLK” = paired antigen) Breast Lungs Kidney CEA Oncofetal antigen GI carcinoma Differentiates adenocarcinoma (+) & mesothelioma (-) TTF-1 (Thyroid Differentiates lung adenocarcinoma & mesothelioma Transcription Factor) (+): Thyroid, lung, neuroendocrine tumors PSA Prostate cancer Intermediate Filament Markers Actin Smooth muscle Skeletal muscle lec.mt 04 |Page | 278

Vimentin Desmin GFAP (Glial Fibrillary Acidic Protein) NF (Neurofilament)

S100 protein Neuroendocrine Markers NSE (Neuron-specific enolase) Others

Cardiac muscle Melanomas Schwannomas Leiomyoma (smooth muscle) Rhabdomyosarcoma (skeletal muscle) Astrocytoma Neuroblastoma Ganglioneuromas Neuroma Chemodectoma Pheochromocytoma Low MW Ca2+-binding protein CNS glial cells, Schwann cells Strong evidence of neural/neuroendocrine differentiation Chromogranin Synaptophysin

Germ Cell tumor markers HCG

Synthesized by syncytiotrophoblasts Choriocarcinoma AFP Endodermal sinus tumors showing yolk sac differentiation PLAP (Placenta-like ALP) Germinomas Mesenchymal Tumor Markers Myogenic tumors Myo-D1 Myoglobin Myogenin Fibrohistiocytic tumors -Vascular tumors Factor VII-related antigen CD31 UEA: Ulex europaeus I Melanomas -Lymphomas LCA: Leukocyte common antigen (CD45) Cell Proliferation Markers Ki67 MIB-1: reference monoclonal antibody for Ki67 demonstration PCNA Proliferating cell nuclear antigen Controls Positive control Known Contains antigen in question Negative control Done using a parallel section from the tissue Internal tissue control A.k.a. “built-in control” Contains the target antigen Other Topics Faults During Tissue Processing Brittle/hard tissue

Clearing agent  Milky

Incomplete dehydration lec.mt 04 |Page | 279

On trimming, tissue smells of clearing agent Tissue is opaque Tissue shrinks away from wax Tissue is soft when block is trimmed Air holes on tissue Wax appears crystalline Paraffin block is moist & crumbles Sections fail to form ribbons

Sections roll up on cutting… adhere & get broken against the knife edge Ribbon is curved, crooked, or uneven Sections are compressed, wrinkled or jammed

Sections are squashed Hole in section Sections of unequal thickness Sections adhere to knife or other parts of the microtome

Insufficient impregnation Insufficient clearing Insufficient dehydration Incomplete clearing & impregnation Incomplete fixation Incomplete impregnation Contaminated wax Block not cooled rapidly enough Insufficient paraffin impregnation Surface & edges of block not parallel Wax too hard Knife tilted too much Thick sections Dull knife Blunt knife Dirty knife edge Irregular knife edge Edges of block are not parallel Knife not parallel to the block Impure paraffin Blunt/dull knife Block is warm & soft Knife edge coated w/ paraffin Thin sections Microtome screw is loose Tilt: vertical Bevel of knife is lost Incorrect sharpening Bubble/dirt Hard spot in the tissue (Ca2+) Screw/holder is loose Large & hard blocks Static electricity Dirty knife edge Dull knife edge

Ribbon is split

Nicks/damage on knife Dirty embedding Dirty knife

Chatters are seen

Knife vibrates (hard tissue)

Section: sometimes thin & thick

Blunt knife

Frozen tissue crumbles & comes off when the block

Knife/block holder is loose Inadequate freezing lec.mt 04 |Page | 280

holder when cut Frozen tissue chips into fragments

Tissue is frozen too hard

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PAS w/ diastase ctrl

Stains Substance Stained (+) Color/Result CHO, Glycogen, Mucins, PAS (+):Magenta red Bacteria & Fungi, basement membrane Glycogen Red

Best Carmine

Glycogen

Bright red

Langhan’s iodine method

Glycogen

Mahogany brown

Alcian blue

Acid mucins

Blue

Alcian Blue-PAS

Any mucins (acid/neutral) Acid MPS Sulfated mucins Carboxylated mucins Cryptococcus neoformans Mucins Acid mucins

Acid mucin: blue Neutral mucin: magenta Sulfated mucins: purple Carboxylated mucins: blue Mucin: red

Acid mucins/MPS Fungi

Stain PAS

Gomori’s aldehyde fuchsin stain Mucicarmine stain Colloidal (Dialyzed) iron technique Acridine orange Sudan black Sudan IV (Scharlach R)

Lipids Lipids (TAG)

Oil red O Osmium tetroxide Nile blue sulfate method

Lipids Lipids Neutral fat Cholesterin esters Cholesterin fatty acids Fatty acids & soap Cerebrosides

Brilliant red Black = Pinkish red = Light red = Light red = Deep blue to violet = Light blue

Toluidine blue-acetone mtd

Sulfatide

Borohydride-PeriodicSchiff method Alkaline fast-green method

Gangliosides

Metachromatic redbrown or yellow Red

Peracetic acid-Alcian blue Sakaguchi’s test Gomori calcium method Gomori lead method Lead method Metal precipitation Calcium cobalt method α-naphthyl acetate method

Avoid Ehrlich’s hematoxylin

Dark blue Acid MPS: black Fungi: greenish red fluorescence Blue black red

Histones Protamines Cystine Cysteine Arginine Alkaline phosphatase Acid phosphatase 5’-nucleotidase ATPase ATPase Nonspecific esterase

Comments Basic fuchsin: essential component of Schiff reagent Method of choice for glycogen staining Selective & highly specific for glycogen Obsolete Not specific for glycogen Avoid celloidinization of slides Avoid Ehrlich’s hematoxylin

Green

Lasts for only 2 hrs

Most commonly used stain Nile blue: preliminary indicator of the type of lipid present -Red oxazone (dissol. neutral lipids) -Blue oxazone (reacts w/ PL and FFA)

Fast green stains basic groups in tissues

Blue-green Orange-red Brownish-black Black Blackish brown deposits Dark brownish-black ppt Cobalt phosphate ppt Reddish brown

Uses Milton reagent Substrate: β-glyceroPO4 For skel. muscle biopsies For skel. muscle biopsies lec.mt 04 |Page | 282

Stain Indoxyl acetate method Tetrazolium method Feulgen technique

Substance Stained Nonspecific esterase Monoamine oxidase DNA

(+) Color/Result Blue Bluish black Red-purple

Methyl green-pyronin

RNA DNA Reticulin fibers

RNA (nucleoli): red DNA (chromatin): green black

Collagen Muscle, cytoplasm, RBC, fibrin Collagen & mucus Muscle, RBC & keratin Collagen fibers, cytoplasm, fibroglia fibrils, axon cylinders, neuroglia Elastic fibers RBCs, myelin sheets CT Glomerular basement membrane Amyloid & mucous colloid Elastic fibers Elastic fibers Elastic fibers Elastic fibers Fibrin & CT RBC RBCs Muscle Collagen Fibrin Fibrin, muscle striations, neuroglia, amoeba RBCs Myelin Collagen, osteoid, cartilage, elastic fibers Amyloid Amyloid

= Pink/deep red = Yellow

Amyloid

Yellow fluorescence

Muscle fibers Collagen Muscles, RBC Collagen

= Red = Green = Red = Yellow

Gomori’s silver impregnation stain Van Gieson’s stain Masson’s trichrome stain Mallory’s aniline blue

Azocarmine

Weigert’s Verhoeff’s Taenzer-Unna-Orcein mtd Krajian’s technique Martius-Scarlet-Blue

Mallory’s PTAH

Congo red Methyl violet-crystal violet method Thioflavin-T fluorescent staining Modified Gomori’s Trichrome stain Lissamine fast red

= Blue = Red = Red

Comments Most reliable & specific histochemical staining technique for DNA Contains Schiff’s reagent Reticulin = Argyrophilic (silver stain) Contains acid fuchsin & picric acid

(-) Fuchsin: Excellent & colorful method of demonstrating CT fibers

= Pale pink/yellow = Yellow

= Deep blue Dark-blue/blue-black Black Dark-brown = Bright red = Dark blue = Orange-yellow = Yellow = Red = Blue = Red = Dark blue

Heidenhain’s modification of Mallory’s aniline blue stain

Rapid method Early fibrin = yellow Old fibrin = blue

= Blue = Lighter blue = Deep brownish-red Red Purplish red

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Stain Schmorl’s Picro-Thionin method

Substance Stained Lacunae & canaliculi Bone matrix

Bielschowsky’s technique

Neurofibril, axons & dendrites Neuroglia & collagen Bodian’s stain Nerve fibers & nerve endings Sevier-Munger technique Peripheral neuritis Axons Myelin sheath Neuritic plaques & tangles Argentaffin granules Toluidine blue Nissl granules & nucleoli Polychrome methylene blue Nissl granules & nucleoli Thionine Nissl granuls & nucleoli Cresyl fast violet Nissl substance Neurons Weigert-Pal technique Myelin sheath Luxol fast blue Myelin Weil’s method Myelin Cajal’s gold sublimate Astrocytes method Perl’s Prussian blue Hemosiderin Gomori’s Prussian blue Iron pigments Turnbull’s blue Ferrous iron (Hemosiderin) Benzidine-nitroprusside Hemoglobin & oxidase stain granules Mod. Fouchet’s technique Bile pigments Gmelin technique Bile & hematoidin Stein’s iodine test Bile pigments Schmorl’s ferric ferricyanide method Gomori’s aldehyde fuchsin Mallory’s fuchsin stain Masson Fontana technique

Von Kossa’s silver nitrate method Lindquist modified rhodanine technique

(+) Color/Result = Dark brown-black = Yellow/brownishyellow = Black on a grayish BG

Comments

= Lightly stained Diagnosis of Alzheimer’s disease = Black = Black = Light brown = Black = Black Deep blue Deep blue Purple = Purple-dark blue = Pale purple blue Blue black Blue-green Black Black on a light brownish BG Deep blue Bright blue Blue

Nissl granules: a.k.a. Tigroid substances

Dark blue Emerald to blue green Blue-purple then green Depend on the oxidation of the pigment to green biliverdin by iodine

Bile, lipofuscins, melanin, argentaffin cells, chromaffin, thyroid colloid Lipofuscin Hemofuscin Melanin Argentaffin cell granules

Dark blue

Calcium

Black

Copper

Red to orange-red

Purple Red = Black = Black

Argentaffin reaction: melanin reduces ammoniacal silver solutions w/o use of a reducer

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Stain Gram-Twort stain

Brown & Brenn method Wade-Fite technique Toluidine blue

Substance Stained Gram (+) organisms Gram (-) organisms RBCs Elastic fibers Gram (+) bacteria Gram (-) bacteria M. leprae H. pylori

Cresyl violet acetate mtd Dieterle method

H. pylori L. pneumophila & spirochetes Levaditi’s method Spirochetes Modified Steiner & Steiner Spirochetes, Donovan technique bodies, fungi, bacteria Warthin-Starry method Spirochetes Grocott Methenamine Silver Fungi

Lendrum’s phloxinetartrazine method Orcein method Giemsa stain

In situ hybridization PCR Chondrocalcinosis Kardasewitsch method 0.1% urea + 5% NaSO4 Metastasis Degree of localization Dunn-Thompson K2MnO4 H2 O 2 Helly’s Formalin ammonium bromide Alcohol as 1’ fixative Glutaraldehyde Carnoy’s Orth’s Zenker’s Formaldehyde Ethanol

Mucin & glycogen Mycelia & hyphae RBCs Viral inclusions

(+) Color/Result = Blue-black = Pink-red = Green = Black = Blue = Red Golden yellow Dark blue against blue BG Blue-violet Dark brown to black

Comments

Black on a yellowish BG Black Black = Sharply outlined I black = Gray-black = Old rose = Yellow Bright red

HBsAg Brown-black Bacteria = Blue Recommended for blood Mast cell granules = Deep blue and BM parasites, Eosinophilic granules = Red inclusion conjunctivitis, Nuclei = Blue Toxoplasma, spirochetes Cytoplasm = Pink & other bacteria Most sensitive technique for identifying DNA DNA amplification Pseudogout Pigment removal 70% ETOH + 28% NH3 water Remove yellow color of HNO3 Most definitive of malignancy Most reliable indicator of prognosis of malignant tumors Hgb = emerald green Removes excess melanin Contains formalin, K2CrO4 and HAc Fixative for CNS (gold/silver stain) Increased tissue shrinkage Not satisfactory for PAS Nonaqueous fixative Pheochromocytoma PTAH for cross-striations Wash tissue in water after fixation in Zenker’s Combines w/ amino group Nonadditive fixative lec.mt 04 |Page | 285

pH >6.0 (formalin) Universal solvents Soft paraffin Weigert’s hematoxylin Natural resins (mounting) Formalin-alcohol Churukian-Schenk technique Masson-Fontana Muscle biopsies Paraffin sections Zamboni’s PAF Glutaraldehyde 10% NBF Paraformaldehyde Warthin-Starry stain Iris diaphragm Substage condenser Pathology Cornelius Celsus Littoral cells Hoffbauer cells Cancer Biohazards Mercuric chloride According to the presence of a mordant According to the presence of differentiator According to the resultant color Vital staining

Neurons

Prevent brown microcrystalline deposits in H & E stain Both dehydrates & clears For thick sections Not easily decolorized w/ acidic solutions Inherently acidic Microincineration Substance that can bind silver but need a chemical reducer Substance that can both bind & reduce silver Isopentane at -150’C If isopentane is low, dust muscle w/ talc then freeze in liquid nitrogen Naphthol AS-D chloroacetate esterase Specimens may remain indefinitely Specimens may be removed after 2-4 hrs pH 7.2-7.4 Pure polymer of formaldehyde Calibrate pH meter to 7.0 Increase amount of light Adjust to focus the image of the substage diaphragm Greek: Pathos = suffering 1st to describe the 4 signs of inflammation Splenic macrophages Placental macrophages Latin: Cancrum = crab Infectious agents Contaminated solutions Contaminated specimens Corrode all metals except for the nickel alloy Monel Methods of Staining a. Direct staining = w/o mordant b. Indirect staining = w/ mordant: serves as a link/bridge between the tissue & the dye a. Progressive = w/o differentiator/decolorizer b. Regressive = w/ differentiator/decolorizer *1’ stain = acidic (decolorizer: basic) *2’ stain = basic (decolorizer: acidic) a. Orthochromatic = “ortho”: correct/same | same color = dye & tissue b. Metachromatic = “meta”: after/change | different color = dye & tissue Selective staining of living cells a. Intravital stain = injection of dye  animal body - Ex. Lithium, Carmine, India ink b. Supravital stain = staining of cells immediately after removal from the animal body. Examples are: - Neutral red = Best vital dye - Janus green = mitochondria - Trypan blue - Nile blue - Thionine - Toluidine blue Functional cells of the CNS Nerve fibers: a. Dendrites (Greek: “Tree”) = conduct impulses to the cell body lec.mt 04 |Page | 286

b. Axons = conduct impulses away from the cell body Criteria for the diagnosis of Marked progesterone effect normal pregnancy At least 50% of intermediate cells in clusters At least some typical pregnancy cells present 2mm  embed in a single paraffin block Shave biopsy Curettage specimens Wedge biopsy Specimen is subdivided w/ a razor blade Marginal excision Shell-out end

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