SPECIAL STAINS

DR. EKTA JAJODIA

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

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

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

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

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

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

HEPATOCYTES SHOWING STREAMING ARTIFACT

Fixation should be carried out at 4 G C to minimize streaming artifact

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

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

CANDIDA IN PAS STAIN

ALPHA ANTITRYPSIN IN PAS STAIN

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

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

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

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

REAGENTS :

1. Alcian blue

2. Aluminium sulfate 3. Nuclear fast red

RESULTS

sulfomucin,sialomucin

Proteoglycans

Hyaluronic acid

Nucleus red

Blue

GOBLET CELLS BY ALCIAN BLUE

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)

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

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

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

COCCUS CACTI

CRYPTOCOCCUS STAINED BY MUCICARMINE

GOBLET CELLS BY MUCICARMINE

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

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

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

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

DNA BY FEULGEN STAIN

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

NUCLEIC ACIDS BY METHYL GREEN PYRONIN

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

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

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)

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

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

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

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

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

Fixation – Not critical

Solution- 0.5 % Congo red in 50% alcohol

0.2% Potassium Hydroxide in 80% alcohol Results- Amyloid - red Nuclei - Blue

TECHNIQUE

AMYLOID BY CONGO RED

ALKALINE CONGO-RED TECHNIQUE

High concentration of NaCl is used

Background electrochemical staining is reduced

hydrogen bonding of congo-red to amyloid is enhanced

POLARIZING MICROSCOPY AND CONGO-RED

Under polarized light, congo red stained amyloid exhibits apple-green birefringence

Most reliable diagnostic test for amyloid currently

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

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

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

STAINS FOR MICROORGANISMS

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

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

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

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

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

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

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

Warthin Starry Method for Spirochetes

REAGENTS :

1. Acetate buffer pH-3.6

2. 1% silver nitrate

RESULTS :

SPIROCHETES – black

BACKGROUND – golden -yellow

SPIROCHETES BY WARTHIN STARRY

FUNGAL STAINS

Fungal cell walls are rich in polysaccharides which can be converted by oxidation to dialdehydes

Dialdehydes are then detected by silver solution

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

Results

Fungi , Pneumocystis, melanin - Black

Mucin & Glycogen - dark grey

Background - Pale green

Hyphae & yeast form - sharply delineated in black against green background

CRYPTOCOCCUS BY GMS STAIN

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

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

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

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

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

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

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

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

Trichrome stain showing slight mesangial prominance

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

PIGMENTS AND MINERALS

ENDOGENOUS PIGMENTS

1. hematogenous2. non-

hematogenous

EXOGENOUS PIGMENTS

1. asbestos2. silica3. lead4. carbon

ARTIFACT PIGMENTS

1. formalin2. malaria3. mercury4. schistosome

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

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

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

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

BILE PIGMENTS BY MODIFIED FOUCHET’S TECHNIQUE

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

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

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

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

Special stains enhance detection & localization of individual tissue component

But should not be substituted for routine H&E

CONCLUSION