Title | MUST-KNOW Histopathologic Techniques |
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Course | Medical Laboratory Science |
Institution | University of San Agustin |
Pages | 35 |
File Size | 1.1 MB |
File Type | |
Total Downloads | 46 |
Total Views | 383 |
MUST TO KNOW IN HISTOPATHOLOGIC TECHNIQUESGerm layers 1. Ectoderm 2. Mesoderm 3. Endoderm Categories of tissues 1. Epithelial = 3 germ layers 2. Nervous = endoderm 3. Muscular = mesoderm 4. Connective = mesoderm Covering Epithelia Simple squamous Bowman’s, endothelium, loop of Henle, alveoli Simple ...
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
General Connective Tissues Loose CT
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 Wharton’s jelly (acid MPS) BM (reticular) Lymph node (reticular) Embryo (mesenchyme) lec.mt 04 |Page | 1
Hypodermis 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 lec.mt 04 |Page | 2
1. Rubor
Redness
2. Calor
Heat
3. Tumor
Swelling
4. Dolor
Pain
5. Functio laesa
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 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 lec.mt 04 |Page | 3
Compensatory hyperplasia Pathologic hyperplasia Congenital hypertrophy Metaplasia 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
Ex. Enlargement of one kidney Ex. Endometrial hyperplasia Phenytoin-induced Degenerative Changes = Tissues have abnormalities Reversible One adult cell type ↔ Another adult cell type 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
Treatment Surgery ↓ ↓ Radiation
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TNM system T
N
M
Teratomas
Apoptosis Necrobiosis Necrosis Types of Necrosis Coagulation necrosis
Liquefaction/colliquative necrosis Caseous/caseation necrosis Gangrenous necrosis
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 T1 = lesion Pig (3’) > Sheep (4’) > Horse (5’) > Guinea pig (6’) Mice “LUBO” = paired Lung Uterus Breast Ovary Stomach Colon Transitional cell carcinoma of the bladder Mucinous ovarian tumor HCC RCC SCC Thyroid carcinoma Prostatic adenocarcinoma (+) carcinoma “BuLK” = paired Breast Lungs Kidney Oncofetal antigen GI carcinoma Differentiates adenocarcinoma (+) & mesothelioma (-) Differentiates lung adenocarcinoma & mesothelioma (+): Thyroid, lung, neuroendocrine tumors Prostate cancer
TTF-1 (Thyroid Transcription Factor) PSA Intermediate Filament Markers Actin Smooth muscle Skeletal muscle Cardiac muscle Vimentin Melanomas Schwannomas Desmin Leiomyoma (smooth muscle) Rhabdomyosarcoma (skeletal muscle) GFAP (Glial Fibrillary Acidic Astrocytoma Protein) NF (Neurofilament) Neuroblastoma
lec.mt 04 |Page | 25
S100 protein Neuroendocrine Markers NSE (Neuron-specific enolase) Others Germ Cell tumor markers HCG
Ganglioneuromas Neuroma Chemodectoma Pheochromocytoma Low MW Ca2+-binding protein CNS glial cells, Schwann cells Strong evidence of neural/neuroendocrine differentiation Chromogranin Synaptophysin Synthesized by syncytiotrophoblasts Choriocarcinoma Endodermal sinus tumors showing yolk sac differentiation Germinomas
AFP PLAP (Placenta-like ALP) 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 On trimming, tissue smells of clearing agent Tissue is opaque Tissue shrinks away from wax Tissue is soft when block is
Incomplete dehydration Insufficient impregnation Insufficient clearing Insufficient dehydration Incomplete clearing & impregnation Incomplete fixation lec.mt 04 |Page | 26
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
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+)
Sections of unequal thickness
Sections adhere to knife or other parts of the microtome
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 holder when cut Frozen tissue chips into fragments
Knife/block holder is loose Inadequate freezing
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)
Gomori’s aldehyde fuchsin stain
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
Stain PAS
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-Periodic-Schiff method Alkaline fast-green method
Gangliosides
Metachromatic redbrown or yellow Red
Sakaguchi’s test Gomori calcium method Gomori lead method Lead method Metal precipitation
Histones Protamines Cystine Cysteine Arginine Alkaline phosphatase Acid phosphatase 5’-nucleotidase ATPase
Avoid Ehrlich’s hematoxylin
Dark blue Acid MPS: black Fungi: greenish red fluorescence Blue black red
Peracetic acid-Alcian blue
Acid mucins/MPS Fungi
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
Uses Milton reagent Substrate: β-glyceroPO4 For skel. muscle biopsies lec.mt 04 |Page | 29
Calcium cobalt method α-naphthyl acetate method Stain Indoxyl acetate method Tetrazolium method Feulgen technique
ATPase Nonspecific esterase Substance Stained Nonspecific esterase Monoamine oxidase DNA
Cobalt phosphate ppt Reddish brown (+) 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
= Pink/deep 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
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
= Blue = Red = Red
For skel. muscle biopsies 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 Heidenhain’s modification of Mallory’s aniline blue stain = Deep blue Dark-blue/blue-black Black Dark-brown = Bright red = Dark blue = Orange-yellow = Yellow = Red = Blue = Red = Dark blue
Rapid method
Early fibrin = yellow Old fibrin = blue
= Blue = Lighter blue = Deep brownish-red Red Purplish red
Amyloid
Yellow fluorescence
Muscle fibers
= Red lec.mt 04 |Page | 30
Trichrome stain Lissamine fast red Stain Schmorl’s Picro-Thionin method Bielschowsky’s technique
Bodian’s stain Sevier-Munger technique
Toluidine blue Polychrome methylene blue Thionine Cresyl fast violet Weigert-Pal technique Luxol fast blue Weil’s method Cajal’s gold sublimate method Perl’s Prussian blue Gomori’s Prussian blue Turnbull’s blue Benzidine-nitroprusside stain Mod. Fouchet’s technique Gmelin technique Stein’s iodine test
Schmorl’s ferric ferricyanide method
Gomori’s aldehyde fuchsin Mallory’s fuchsin stain Masson Fontana technique
Von Kossa’s silver nitrate
Collagen Muscles, RBC Collagen Substance Stained Lacunae & canaliculi Bone matrix Neurofibril, axons & dendrites Neuroglia & collagen Nerve fibers & nerve endings Peripheral neuritis Axons Myelin sheath Neuritic plaques & tangles Argentaffin granules Nissl granules & nucleoli Nissl granules & nucleoli Nissl granuls & nucleoli Nissl substance Neurons Myelin sheath Myelin Myelin Astrocytes Hemosiderin Iron pigments Ferrous iron (Hemosiderin) Hemoglobin & oxidase granules Bile pigments Bile & hematoidin Bile pigments
= Green = Red = Yellow (+) Color/Result = Dark brown-black = Yellow/brownish-yellow = 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
Purple Red = Black = Black
Argentaffin reaction: melanin reduces ammoniacal silver solutions w/o use of a reducer lec.mt 04 |Page | 31
method Lindquist modified rhodanine technique
Stain Gram-Twort stain
Brown & Brenn method Wade-Fite technique Toluidine blue Cresyl violet acetate mtd Dieterle method Levaditi’s method Modified Steiner & Steiner technique Warthin-Starry method Grocott Methenamine Silver
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 H2O2 Helly’s Formalin ammonium bromide Alcohol as 1’ fixative
Copper
Red to orange-red
Substance Stained Gram (+) organisms Gram (-) organisms RBCs Elastic fibers Gram (+) bacteria Gram (-) bacteria M. leprae H. pylori H. pylori L. pneumophila & spirochetes Spirochetes Spirochetes, Donovan bodies, fungi, bacteria Spirochetes Fungi
(+) Color/Result = Blue-black = Pink-red = Green = Black = Blue = Red Golden yellow Dark blue against blue BG Blue-violet Dark brown to black
Mucin & glycogen Mycelia & hyphae RBCs Viral inclusions
Comments
Black on a yellowish BG Black Black = Sharply outlined I black = Gray-black = Old rose = Yellow Bright red
HBsAg Brown-black Bacteria = Blue Mast cell granules = Deep blue Eosinophilic granules = Red Nuclei = Blue Cytoplasm = Pink 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
Recommended for blood and BM parasites, inclusion conjunctivitis, Toxoplasma, spirochetes & other bacteria
Contains formalin, K2CrO4 and HAc Fixative for CNS (gold/silver stain) Increased tissue shrinkage lec.mt 04 |Page | 32
Glutaraldehyde Carnoy’s Orth’s Zenker’s Formaldehyde Ethanol 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
Not satisfactory for PAS Nonaqueous fixative Pheochromocytoma PTAH for cross-striations Wash tissue in water a...