Special stains in histopathology
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SPECIAL STAINS
DR. EKTA JAJODIA
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H&E stain is routine stain. - It is the preliminary or the first stain applied to the tissue sections - Gives diagnostic information in most cases.
A special stain is a staining technique to highlight various individual tissue component once we have preliminary information from the H&E stain
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Classification
1. Stains for carbohydrates
2. Stains for amyloid
3. Nucleic acid stains
4. Lipid stains
5. Stains for microorganisms
6. Connective tissue stains
7. Stains for pigments and minerals
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CARBOHYDRATES
SIMPLE CARBOHYDRATES(molecules composed purely of carbohydrates)
• Monosaccharides(glucose,mannose,galactose)
• Oligosaccharides(sucrose,maltose)
• Polysaccharides(glycogen,starch)
GLYCOCONJUGATES(molecules composed of carbohydrates and other molecules such as protein and lipid)
• Proteoglycans
• Mucins
•Others glycoproteins
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90-95% of their molecular weight is due to the carbohydrate component
The carbohydrate component is known as glycosaminoglycans(GAG)
A GAG is composed of repeating disaccharide units , each made up of 2 different monosaccharides
Each disaccharide is composed of a carboxylated uronic acid (glucuronic or iduronic acid) and a hexosamine such as N-acetylglucosamine or N-acetylgalactosamine
TYPES OF GYCOSAMINOGLYCANS
Chondroitin sulfate Dermatan sulfate Keratan sulfate Heparin sulfate Heparin Hyaluronic acid
PROTEOGLYCANS
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MUCINS
1. Neutral mucins : surface epithelia of gastric mucosa brunner’s glands prostatic epithelia
2. Sialomucins : bronchial submucous glands goblet cells salivary glands
3. Sulfomucins : bronchial mucous glands
Sialomucins and sulfomucins are acidic mucins
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FIXATIVES
Tissue placed in fixative promptly after removal – capable of autolytic reaction
If immediate fixation not possible, tissue refrigerated until adequate fixation possible
Recommended fixatives : Rossman’s fluid Alcoholic formalin with picric acid
Glutaraldehyde fixatives avoidedfree aldehyde groups are capable of undergoing schiff reaction – so increased background staining
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HEPATOCYTES SHOWING STREAMING ARTIFACT
Fixation should be carried out at 4 G C to minimize streaming artifact
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1. Periodic acid schiff (PAS) technique2. Alcian blue method3. Combined alcian blue- PAS techhnique4. Mucicarmine technique5. Colloidal iron technique6. Metachromatic staining7. Iodine staining for glycogen8. Enzymatic digestion technique Diastase digestion, Sialidase digestion, Hyaluronidase digestion
CARBOHYDRATE STAINING
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PERIODIC ACID SCHIFF METHOD
1st histochemical use was by McManus for demonstration of mucin
Reagents – 1. periodic acid 2. schiff reagent
0.5-1% solution of periodic acid (oxidant) used for 5-10 minutes oxidation of hydroxyl group within
the formation of two free aldehyde groups
free aldehydes react with schiff reagent
carbohydrate
bright
red magenta end productRESULT – glycogen and mucins : magenta nuclei : blue
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CANDIDA IN PAS STAIN
ALPHA ANTITRYPSIN IN PAS STAIN
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Other oxidants like potassium permanganate/ chromic
acid not used – further oxidise aldehyde groups to
carboxylic groups – not reactive to schiff reagent
MILD PAS TECHNIQUE –
0.01% periodic acid used for shorter period for N-acetyl sialic acid containing mucins as the hydroxyl groups are
highly susceptible to periodic acid oxidation
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PAS REACTIVE CELLS AND TISSUE COMPONENTS
1.GLYCOGEN2. STARCH3. MUCIN4. BASEMENT MEMBRANE5. ALPHA ANTI TRYPSIN6. RETICULIN7. FUNGI(CAPSULES)8. PANCREATIC ZYMOGEN GRANULES9. THYROID COLLOID10. CORPORA AMYLACEA11. RUSSELL BODIES
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Diagnosis of several medical conditions:
Glycogen storage disorder
Staining macrophages in Whipple's disease
Mucins in adenocarcinoma of large intestine
Demonstration of fungi
Seminoma,rhabdomyosarcoma,ewing’s sarcoma contain glycogen
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ALCIAN BLUE
Alcian blue 8 GX – recommended
Comprised of copper containing pthalocyanine ring linked to 4 isothiouronium groups – strong bases - account for cationic nature of the dye
Sulfate and carboxylate groups of proteoglycans ionised at pH 2.5 and carry a negative charge
Sialo- and sulfo mucins also reactive at pH 2.5
So, they stain with alcian blue at pH 2.5
Neutral mucins are not reactive with alcian blue
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REAGENTS :
1. Alcian blue
2. Aluminium sulfate 3. Nuclear fast red
RESULTS
sulfomucin,sialomucin
Proteoglycans
Hyaluronic acid
Nucleus red
Blue
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GOBLET CELLS BY ALCIAN BLUE
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COMBINED ALCIAN BLUE- PAS TECHNIQUE
PRINCIPLE
Demonstrate presence of mucin
Differentiate acid mucin from neutral mucin
1st stain all acid mucin with alcian blue (blue)
Those acid mucin which are PAS +ve will not be stained on PAS reaction only neutral mucin will be stained(magenta)
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ALCIAN BLUE WITH VARYING ELECTROLYTE CONCENTRATIONS
This technique is based upon phenomenon known as CRITICAL ELECTROLYTE CONCENTRATION (CEC)
CEC is defined as point at which amount of electrolyte such as MgCl2 is sufficient to prevent staining from AB
Competition between cations of salt and dye occurs for polyanionic sites within tissue
Different acidic carbohydrates have different CEC value
So can differentiate acidic mucins and proteoglycans
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MUCICARMINE Active dye molecule is aluminium – carminic acid complex known as CARMINE
carminic acid produced from dried bodies of female Coccus Cacti insects
Carmine complex has a positive charge and so attracts polyanions such as sialomucins and sulfomucins
Useful for identification of adenocarcinoma ( especially of GIT)
Capsule of fungus – cryptococcus neoformans is also detected
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REAGENTS :
1.Southgate’s mucicarmine solution2.Alcoholic hematoxylin3.Acidified ferric chloride solution4.Weigert’s iron hematoxylin solution5.Metanil yellow solution
RESULTS :
Acidic mucins – deep rose to red Nuclei – black Other tissue elements – light yellow
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COCCUS CACTI
CRYPTOCOCCUS STAINED BY MUCICARMINE
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GOBLET CELLS BY MUCICARMINE
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NUCLEIC ACIDS 2 nucleic acids are :
1. DNA ( In the nucleus)
2. RNA (In the cytoplasm)
They consist of : Sugar (Deoxyribose / Ribose), Phosphate and Nitrogenous base
Demonstration of Nucleic acids depends upon either :
1. Reaction of the dyes with the phosphate groups , or ,
2. Production of aldehydes from the sugar (deoxyribose)
No histochemical methods are available to demonstrate the nitrogenous base
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1. Feulgen technique ( demonstrate sugar)
2. Methyl green pyronin technique (demonstrate phosphate)
3. Acridine orange (by fluorescent method)
4. Gallocyanin-chrome alum method
Demonstrates both DNA and RNA
The last staining method do not separate the 2 nucleic acids (stains both DNA and RNA blue) and suitable extraction technique must be used
DNA IS DEMONSTRATED BY
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EXTRACTION TECHNIQUES
1. DIGESTION METHODS : Pure deoxyribonuclease will digest DNA and pure
ribonuclease will digest RNA
2. CHEMICAL METHODS :
a) By perchloric acid : To remove RNA – 10% perchloric acid at 4G C overnight
b) Trichloroacetic acid
c) Hydrochloric acid
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FEULGEN STAIN SOLUTIONS USED ARE : A)1M HCL acid - used for acid hydrolysis to break the purine-
deoxyribose bond and yield an aldehyde. - Done at 60G C (HCL should be preheated to
60 G C ) - Time (minutes) depends upon the fixative
used - For carnoy’s and formalin – 8 minutes used
B) Schiff reagent - The aldehydes are then demonstrated by schiff’s reagent
C) Bisulfite solution
RESULT DNA : red-purple CYTOPLASM : green
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DNA BY FEULGEN STAIN
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METHYL GREEN PYRONIN METHODReagents :
1.Methyl green - impure dye contains methyl violet –
removed by washing with chloroform - pure methyl green specific for DNA - NH2 of dye reacts with phosphate of DNA
2.Pyronin - binds to any negatively charged tissue
constituent - apart from RNA, binds to acid mucins and
cartilage
RESULTS – DNA : green-blue RNA : red
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NUCLEIC ACIDS BY METHYL GREEN PYRONIN
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1. Most suitable technique for identifying DNA is In-situ hybridization
2. Bouin’s fixative is not suitable as it causes over hydrolysis of the nucleic acid during fixation
3. RNA cannot be demonstrated by feulgen stain because ribose-purine bond is unaffected by hydrolysis/ 1 M HCL
4. Control method for the standard feulgen technique is – Naphthoic acid hydrazide (NAH) method – DNA is acid hydrolysed by 1M HCL. Aldehydes are coupled with naphthoic acid and then again coupled with diazonium salt, fast blue B. results are identical to true feulgen reaction
5. Blue thionin-feulgen reaction – used for studying cancer cell nuclear morphology and ploidy. Here DNA is stained blue and cytoplasm remains unstained
POINTS TO REMEMBER
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LIPIDS
SIMPLE LIPIDS
- FATS- OILS- WAXES
COMPOUND LIPIDS
- c/o fatty acids, alcohol and one more group such as phosphorus or nitrogen
DERIVED LIPIDS
- Derived from simple or compound lipids by hydrolysis- cholesterol- Bile acids
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Lipids with melting point below staining temperature can be stained with fat stains
So only lipids which are liquid at staining temp. are stained. Those in solid or crystalline state remains unaffected
Melting point of a lipid is inversely related to its fatty acid chain length
Simple lipid is best demonstrated with fresh frozen sections
Best fixative – Formal calcium (2% calcium acetate + 10% formalin)
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1st Sudan dye was Sudan 3
Most sensitive of all fat dyes is – Sudan black B
Sudans must be dissolved in organic solvents to penetrate fats
Some organic solvents used are –
1. 70% ethanol2. Isopropanol3. Propylene glycol4. Triethyl phosphate
SUDAN BLACK B
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Sudan black b has 2 fractions – 1st stains neutral fats blue-black
2nd stains phospholipids gray
This gray reaction can be enhanced as a bronze dichroism if section is viewed in polarised light
It fails to stain crystalline cholesterol, lecithin and free fatty acids
Bromine pre treatment converts crystalline cholesterol to oily derivatives and hence permeable to Sudan dye
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AMYLOID
Extracellular , amorphous , eosinophilic material
Composed of protein in an antiparallel -pleated sheet configuration
In H&E stain , can be confused with hyaline and fibrinoid substances
Earliest special stain used for amyloid was Iodine by Virchow
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CONGO RED STAIN
Acidic dye and composed of 2 identical halves
Each half has a phenyl ring bound to a naphthalene moiety by a diazo group
2 phenyl groups bound by a diphenyl bond - gives a linear dye molecule
It stains amyloid by hydrogen bonding and other tissue components by electrochemical bonds
Electrochemical staining of other tissues can be suppressed by – using alkaline-alcoholic solvents using competitive inhibition by salt solution
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2 factors are important to the congo red-amyloid reaction
1.Linearity of the dye molecule
2. -pleated sheet configuration of the amyloid
If the spatial configuration of either is altered, the reaction fails
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Fixation – Not critical
Solution- 0.5 % Congo red in 50% alcohol
0.2% Potassium Hydroxide in 80% alcohol Results- Amyloid - red Nuclei - Blue
TECHNIQUE
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AMYLOID BY CONGO RED
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ALKALINE CONGO-RED TECHNIQUE
High concentration of NaCl is used
Background electrochemical staining is reduced
hydrogen bonding of congo-red to amyloid is enhanced
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POLARIZING MICROSCOPY AND CONGO-RED
Under polarized light, congo red stained amyloid exhibits apple-green birefringence
Most reliable diagnostic test for amyloid currently
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POINTS TO REMEMBER
Thickness of section is critical – 8-10 micro meter is ideal
Too thin section – show faint red color
Too thick section – show yellow birefringent
Other structures giving apple-green birefringence : 1. neurofibrillary tangles of alzheimer’s 2. intracellular inclusions seen in adrenal cortical cells 3. cellulose and chitin 4. dense collagen Thioflavin T – flourescent method
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To differentiate AA and AL amyloid :
Section pretreatment with trypsin or potassium permanganate done
AA amyloid lose their affinity for congo-red but AL amyloid is resistant
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METHYL /CRYSTAL VIOLET METHOD
Methyl violet contains a mixture of tetra- , penta- , and hexa- methyl pararosaniline
Amyloid stained due to selective affinity for one of the colored fractions
Hence, polychromasia seen
Ammonium oxalate accentuates polychromatic effect
RESULT : AMYLOID, MUCIN , HYALINE – red-purpleBACKGROUND - blue
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STAINS FOR MICROORGANISMS
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Gram staining of Bacteria( MODIFIED BROWN-BRENN METHOD)
Reagents :(1) Crystal violet stain(2) Gram’s iodine solution(3) Ethyl alcohol – acetone solution(decolorizer)(4) Acetone-xylene solution(5) Basic Fuchsin(6) Picric acid, 0.1% in acetone
RESULTS : GRAM POSITIVE BACTERIA – blue GRAM NEGATIVE BACTERIA – red NUCLEI – red OTHER TISSUE ELEMENTS - yellow
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Dry picric acid is explosive – recommended to purchase picric acid – acetone solution pre made
Do not allow sections to dry at any point in the staining process – decolorization will be difficult and uneven
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Acid Fast Staining for Bacteria
Mycobacteria cannot be demonstrated by gram’s stain – possess a capsule containing long chain fatty acid (mycolic acid) that makes them hydrophobic
Can be stained by a strong stain like carbol fuchsin
Fatty capsule resist the removal of stain by acid-alcohol solution (acid and alcohol fastness)
Mycobacteria are PAS positive due to carbohydrate content of their cell wall
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Ziehl Neelson (ZN) stain
Reagents
(1) Carbol fuchsin solution
(2) 1% acid alcohol
(3) 0.1%Methylene blue solution
RESULTS
Acid fast bacilli bright red Other tissue Pale blue Caseous material very pale grayish
blue
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Blue counterstain may be patchy if extensive caseation is present
Avoid over counterstaining – scant organism can easily be obscured
Decalcification using strong acids may destroy acid-fastness - so formic acid recommended
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MODIFIED FITE TECHNIQUE
REAGENTS :
1. Carbol fuchsin solution2. 5% sulphuric acid in 25% alcohol3. Methylene blue solution
RESULTS: M.leprae – bright red nuclei and other tissue elements – pale
blue
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Fite stain Modified ZN
Uses mixture of xylol & liquid paraffin prior to stain
Does not use
Incubation in ZN carbolfuchsin at 37ºc for 45 min
Incubation In preheated ZN carbol fuchsin at 56ºc for 60min
Decolorize with 5% H2SO4 1% acid alcohol. Acid :20% H2SO4
Demonstrates M leprae M tuberculosis
AFB : Fite vs modified ZN
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Warthin Starry Method for Spirochetes
REAGENTS :
1. Acetate buffer pH-3.6
2. 1% silver nitrate
RESULTS :
SPIROCHETES – black
BACKGROUND – golden -yellow
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SPIROCHETES BY WARTHIN STARRY
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FUNGAL STAINS
Fungal cell walls are rich in polysaccharides which can be converted by oxidation to dialdehydes
Dialdehydes are then detected by silver solution
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Gomori methenamine silver nitrate(GMS) technique
Reagents
(1) 4% chromic acid (2) 1% sodium bisulfite (3) 5% sodium Thiosulfate(4) 0.21% Silver nitrate(stock)(5) Gold chloride 0.1% aqueous solution(6) Light green solution
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Results
Fungi , Pneumocystis, melanin - Black
Mucin & Glycogen - dark grey
Background - Pale green
Hyphae & yeast form - sharply delineated in black against green background
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CRYPTOCOCCUS BY GMS STAIN
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MISCELLANEOUS STAINS
Cresyl violet acetate method for helicobacter pylori
Macchiavello’s stain for rickettsia and viral inclusions
Lendrum’s phloxine – tartrazine stain for viral inclusions
Giemsa stain for parasites
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CONNECTIVE TISSUES
Provide a matrix that connects and binds the cells and organs and ultimately gives support to the body.
Parent cell is embryonic mesenchyme
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COLLAGEN FIBRES
1. Masson ‘s trichrome technique2. Van Gieson’s stain3. Mallory’s Phosphotungstic Acid
Hematoxylin4. MSB Technique5. PAS6. Heidenhain’s Azan stain7. lillie’s allochrome method8. Luxol fast blue G
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FACTORS AFFECTING TRICHROME STAINING
1. TISSUE PERMEABILITY AND DYE MOLECULAR SIZE
When protein component of a tissue exposed to a fixative – insoluble protein network formed
Structure of the protein network directly related to the staining reactions
Erythrocyte protein – dense network with small pores
Muscle cells – larger pores
Collagen – least dense network and quite porous
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2. Heat : Increases rate of staining and penetration
3. pH : Low pH ( 1.5 – 3)
4. Nuclear stain for trichrome Iron hematoxylin preferred More resistant to acidity of dye solutions Alum hematoxylins are decolorized Can use Celestin blue- alum hematoxylin sequence
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EFFECT OF FIXATION
10% NBF will not yield optimal results
Treatment of formaldehyde fixed tissue with picric acid /mercuric chloride solution enhances intensity and radiance of trichrome
Recommended fixatives are: Bouin’s Zenker’s, Formal-mercury
Zinc formalin
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Demonstrate collagen and muscle in normal tissue
Differentiate collagen and Muscle in tumors Identify an increase in collagenous tissue
Indicate fibrotic change in cirrhosis of liver
Indicate fibrotic change in pyelonephritis
Distinguish tumors that have arisen from muscle cells and fibroblasts
Masson ‘s trichrome technique
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REAGENTS
1. Weigert’s iron hematoxylin2. Acid fuchsin3. Glacial acetic acid4. Phosphomolybdic acid5. Methyl blue
RESULT
Nuclei – Blue/ Black Cytoplasm, muscle , RBC → Red Collagen → Blue
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Trichrome stain showing slight mesangial prominance
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Van Gieson Technique
REAGENT :
Weigert’s iron hematoxylin Saturated Picric acid solution Acid fuchsin
RESULTS :
Collagen – bright red Nuclei – Blue/Black Cytoplasm, muscle, RBC , elastin , reticulin -yellow
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PIGMENTS AND MINERALS
ENDOGENOUS PIGMENTS
1. hematogenous2. non-
hematogenous
EXOGENOUS PIGMENTS
1. asbestos2. silica3. lead4. carbon
ARTIFACT PIGMENTS
1. formalin2. malaria3. mercury4. schistosome
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Hemosiderin
Breakdown product of haemoglobin composed of ferric iron and protein
Seen as yellow-brown granules
3 methods for demonstration :
1.Perl’s prussian blue reaction – for ferric ion
2. Lillie’s method – for ferrous iron
3. Hukill and putt’s method – for both ferric and ferrous iron
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PERL’S STAINPrinciple : unmasking of ferric iron in hydroxide form by dilute HCl
PRUSSIAN BLUE REACTION – Ferric Hydroxide + potassium ferrocyanide = Ferric ferrocyanide (insoluble blue compound)
Results Ferric iron –Blue Nuclei – Red
Reagents 2% aq. Potassium ferrocyanide 2% HCl Counterstain with 1% neutral red or saffranin
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Best positive control – postmortem lung tissue that contains a reasonable amount of iron positive macrophages (heart failure cells)
In Hb and myoglobin , iron bound tightly within protein complex – cannot be demonstrated by traditional technique
Treatment with hydrogen peroxide releases iron - stained with perl’s stain
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Modified Fouchet’s technique: bile pigment
Demonstrates liver bile pigment Most common routine method
Reagents a)Fouchet ‘s reagent : 25% aq trichloracetic acid 10% aq ferric chlorideb)Van Gieson stain :acid Fuchsin + saturated aq picric acid
RESULTBile pigment : emerald to blue greenMuscle :yellowCollagen :red
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BILE PIGMENTS BY MODIFIED FOUCHET’S TECHNIQUE
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MELANIN
Normally occur as light brown to black granules in substantia nigra,hair , skin and eye
Found Pathologically throughout the body :benign nevus,malignant melanoma
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MELANIN DEMONSTRATED BY :
1. Reducing methods : a) Masson fontana silver technique
b) Schmorl’s ferric-ferricyanide reduction test
2. Enzyme methods – DOPA reaction
3. Solubility and bleaching characteristics
4. Fluorescent method
5. Immunohistochemistry
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MASSON FONTANA STAIN
ARGENTAFFIN REACTION – reduction of ammoniacal silver solution to form metallic silver without the use of extraneous reducer.
Masson’s method( using fontana’s silver solution) rely on melanin’s argentaffin property
Melanins are blackened by acid silver nitrate solution
RESULT :
Melanin – black
Nuclei - red
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Schmorl’s ferric-ferricyanide reduction test
Schmorl reaction – Melanin reduce ferricyanide to ferrocyanide with production of prussian blue in the presence of ferric salts
RESULT :
Melanin – dark blue
Nuclei - red
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Special stains enhance detection & localization of individual tissue component
But should not be substituted for routine H&E
CONCLUSION
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