Special stain in HIstopathology

Presented by Dr. Mahesh

Guide: Dr. S. P. Hiryur

- Associate Professor

• Special stains are used to identify certain normal and abnormal substance present in the cells and tissue, which can not be identified on routine Haematoxylene & Eosin staining or are better appreciated on special stain.

Periodic Acid-Schiff (PAS) stain • Principle:

Substance containing vicinal glycol groups or their amino or alkylamino derivatives are oxidized by periodic acid into dialdehydes which on reaction with schiff’s reagent give insoluble purple-magenta compound.

• Dyes : 1%periodic acidSchiff’s reagent

• Control : Appendix• Result :

PAS +ve substance : Purple –magenta colorNuclei : Blue

PAS positive substances :

• Glycogen

• Neutral mucoprotein

• Glycoprotein

• Glycolipid

• Basement membrane

• All fungi

• Phosphorylated sugar

• Cerebrosides

• PAS Diastase reaction : Glycogen is diastase sensitive, hence section containing glycogen when pretreated with diastase, the enzyme will digest the glycogen and will give negative PAS reaction.

• The purpose of using PAS with diastase staining procedure is to differentiate glycogen from other PAS positive elements such as mucin that may be present in the tissue sample.

Pocedure : •Dewax the section and bring to water level

•Dip the slide in copplin jar containing Per iodicacid for 10 min

•Wash well in tap water for 15-20 min

• Dip the slide in coplin jar containing Schiffreagent for 20 min

•Rinse in distilled water

•Counter stain with hematoxylene for 5-10seconds

•Wash in tap water

• Clear in xylene and mount in DPX.

• Uses of PAS:• PAS is used to demonstrate glycogen and neutral

mucoprotein.

• In diagnosis of poorly differentiated adenocarcinoma ofvarious tissue like stomach, pancreas, lung.

• In diagnosis of hepatocellular carcinoma.

• Seminoma, Dysgerminoma

• RCC- Renal cell carcinoma: Clear cell type

• To demostrate – intracytoplasmic crystal in alveolar softpart sarcoma

• To demostrate basement membrane e.g. adenoid cysticca.

• To demostrate neutral mucin in gastrointestinal tract

• To demostrate fungi in tissue sample which are positive due to high carbohydrate content in their cell wall

like. CandidiasisActinomycosisHistoplasmosisBlastomycosis

• It can be used to confirm the metastatic malignancy. Finding mucin positive tumor cells in an area that does not contain mucin producing cells would indicate the tumor did not arise from that area.

• In diagnosis of ALL- block positivity and AML- diffuse cytoplasmic positivity in M5, M3 and cytoplasmicgranular/block positivity in M6 and cytoplasmic granular and blebs positivity of blast in M7.

Gastric signet ring cell carcinoma on PAS stain

Candida seen on PAS stain

• Presence of glycogen will be evidenced by loss of staining after enzyme treatment when compared to the untreated sections.

PAS StainPAS with Diastase

Mucin• Mucins are high moleqular weight substance containing

acidic groups and divided into two major categories-epithelial and stromal.

• Epithelial mucin (membrane bound or secreted) are composed of central protein core and sialic acid containing chains of carbohydrate(polysaccharides)-that may be sulfated or nonsufated.

• Stromal mucin known as glycosaminoglycans contain hyaluronic acid may be sulfated or non sulfated. They are better known as myxoid substance.

• Their functions are lubrication, chemical barrier and cell signaling.

The types of mucin are as follows:

Neutral - Found in glands of stomach and in prostate. They stain with PAS but not with Alcian blue, colloidal iron and mucicarmine.

Acid (simple, or non-sulfated) - Are the typical mucins of epithelial cells containing sialic acid. They stain with PAS, Alcian blue at pH 2.5, colloidal iron. They resist hyaluronidase digestion. E.g. salivary glands.

Acid (simple, sulfated -mesenchymal) - These contain hyaluronic acid and are found in tissue stroma. They do not stain with PAS, but do stain with Alcian blue at pH 2.5, colloidal iron, and metachromatic dyes. They digest with hyaluronic acid. They can be found in sarcomas.

• Acid (complex, epithelial) - These are found in adenocarcinomas of colon, breast,ovary, mucoepidermoidcarcinoma . PAS is usually positive. Alcian blue is positive at pH 1, and colloidal iron, mucicarmine, and metachromaticstains are also positive. They resist digestion with hyaluronidase.

• Acid (complex, connective tissue) - Found in tissue stroma, cartilage, and bone and include substances such as chondroitin sulfate or keratan sulfate. They are PAS negative but do stain selectively with Alcian blue at pH 0.5.

Stains for mucin

• Alcian blue

• PAS

• Mayer mucicarmine

• Hale colloidal iron stain

Alcian blue

• PRINCIPLE

• Alcian blue is a copper phthalcyanin dye and contains positively charged groups capable of salt linkage with certain polyanions. These polyanions consist of the sulphate and carboxyl radicals of the acid mucinsand the phosphate radicals of the nucleic acids thatdo not react. Consequently, only the acid mucins are stained. By varying the pH of the solution of Alcianblue more information can be gained concerning the types of acid mucin present.

Alcian blue • At pH 2.5 it stains – Acidic mucin that include acid-simple or

non-sulfated and acid –simple mesenchymal mucin.

• At pH 1 it stains acid-complex or sulfated mucin and at pH 0.5 it stains acid-complex connective tissue mucin.

• It does not stain neutral mucin.

• Dyes : 1%Alcian blue in 3%acetic acid

Results : Acid MPS – deep blue

Nuclei – faint blue

• PAS-Alcian blue is best ‘pan-mucin’ stain combination, since it demostrates mucin both neutral, slightly acidic and highly acidic.

REAGENTS: 3% Glacial Acetic Acid Alcian Blue Solution:

3% glacial acetic acid-100.0mlAlcian blue -1.0 gm

Mix, adjust pH to 2.5, using acetic acid. Filter, add a crystal of thymol, label with initial and date.

Nuclear Fast Red (Kernechtrot):

Aluminum sulfate 25.0 gmDistilled water 500.0 mlNuclear fast red 0.5 gmDissolve the aluminumsulfate in the water. Add

the nuclear fast red, dissolve

with aid of heat. Filter,add a crystal of thymol

SPECIMENStandard paraffin section fixed in neutral buffered formalin

PROCEDURE: ( Alcian Blue)

1. Hydrate slides to distilled water.2. Stain with3% acetic acid, 3 minutes.3. Stain with alcian blue solution, for 30 seconds.4. Wash in running water for 2 minutes, rinse in

distilled.5. Nuclear-fast red, 5 minutes, wash in tap water.6. Dehydrate, clear, and mount.

USES

• It is useful to differentiate gastric metaplasia into complete and incomplete. Complete metaplasia is small intestinal type, shows well defined brush border and well formed goblet cells. Whereas incomletemetaplasia is colonic type and shows multiple irregular mucin droplets of varying size in the cytoplam and absence of brush border.

• It is useful in diagnosis of salivary gland pleomorphicadenoma, mucoepidermoid carcinoma, in stroma of chordoma at sacrococcygeal region.

• Excessive amounts of non-sulfated acidic mucosubstances are seen in mesotheliomas, certain amounts occur normally in blood vessel walls but increase in early lesions of atherosclerosis.

The goblet cells of the gastrointestinal tract are filled with abundant acid mucin and stain pale blue with this Alcian blue stain.

Colonic mucosa showing sialomucin that have acid and neutral mucopolysaccharide stain purple on alcian blue-PAS.

Mixed salivary gland. Acid mucopolysaccharides in mucous cells are stained blue (Alcian blue at pH 2.5).

Chordoma on alcian blue stain at ph 2.5

MUCICARMINE STAIN –SOUTHGATE’S – MUCIN

PRINCIPLE :-

Positively charged carmine-mucicarmine complexbonds with the negatively charged acid mucin. Thecarmine solution is highly complex. Stronglysulphated mucins are variable in their reaction,neutral mucin do not stain at all, while other acidicmucin stain strongly.

REAGENTS:

• Southgate's Mucicarmine Solution:Carmine, alum lake -1.0 gmAluminum hydroxide -1.0 gm50% alcohol -100.0 mlAluminum chloride, anhydrous- 0.5 gm

• Mayer's Hematoxylin:

PROCEDURE: ( Mucicarmine stain)

1. Deparaffinize and hydrate to distilled water.2. stain with mayer's hematoxylin for 2 minutes.3. Wash in running tap water.4. Differentiate in acid alcohol. 5. Rinse in tap water.6. Blue in scott’s tap water substitute.7. Wash in running tap water.8. Stain with mucicarmine solution for 20

minutes.9. Wash in running tap water.

10. Dehydrate, clear and mount.

Uses:• Mucicarmine is useful to identify acidic mucin.

• It is used to identify adenocarcinoma particularlyof gastrointestinal tract.

• It also stains the capsule of fungus- cryptococcusneoformans found in lung and nervous tissue ofimmuno-compromised patient.

Colonic Mucosa showing sialomsucin --- magenta

RESULTS:

Mucin - magenta

Nuclei - black

Other tissue elements- yellow

Cryptococcus neoformans in lung of AIDS patientcapsule stains red

HALE’S COLLOIDAL IRON STAIN

• Positive staining with hale’s colloidal iron stain is considered diagnostic feature of chromophobe renal cell carcionoma and has been used as discriminatory feature to differentiate it from other renal tumour.

Result

• Acid mucopolysaccharides: blue

• Nuclei: red

Reagents

• Acetic acid 12%

• Potassium ferrocyanide 2% aqueous

• Hydrochloric acid 2%

• Colloidal iron suspension

• Working colloidal solution

Colloidal iron suspension 1 vol

Acetic acid 1 vol

• Perls’ solution

2% potassium ferrocyanide 1 vol

2% hydrochloric acid 1 vol

Procedure: (Hale’s colloidal iron stain)

• Bring the section to water level.

• Place into working colloidal iron for 15-20 minutes.

• Wash with distilled water.

• Wash with running tap water for 5 minutes to remove all traces of colloidal iron.

• Wash with distilled water.

• Place into freshly made perls’ solution for 10 minutes.

• Wash with distilled water.

• Counter stain with neutral red for 1 minute.

• Dehydrate, clear ad mount.

a.Classic chromophobe RCCb. classic chromophobe RCC on hale’s colloidal iron stain

c. Eosinophilic chromophobed. Eosinophilic chromophobeon hale’s colloidal iron stain

e.oncocytomaf. oncocytoma diffusely negative cytoplasmic

reaction with hale‘s colloidal iron stain

RETICULIN STAIN-GOMORI’S METHOD

• Reticulin stain demonstrate both reticular fibersand basement membrane material.

• Reticular fibers consist of very thin fiber of type III collagen which are widespread in connective tissue throughout the body.

• Basement membrane are largely composed of type IV collagen and laminin.

Principle:

Reticulin fibers have little natural affinity for silver solutions. On treatment with potassium permangenate it produce sensitised sites on fiberswhere silver deposition can be initiated. The silver is in the form readily able to precipitate as metallic silver. The optimal ph for maximum uptake of silver ions is 9.0. A reducing agent formalin causes deposition of silver in the form of metal.

• Dyes used: Silver nitrate 10%NaOH 10%

KMnO4 1% aqu.

oxalic acid 5% aquIron alum 2.5%

Formalin 10%

• Control: Cirrhosis of liver

• Result: Reticular fiber – Black

Nuclei- Gray

Other elements- According to counter

stain used

Pocedure : •Dewax the section and bring to water

•Treat with 1% potassium permanganate solution for 3 min

•Rinse in tap water

•Bleach in 5% oxalic acid solution for 2 minute

•Wash well in tap water

•Treat with 2.5% iron alum for 1 min

•Rinse in distilled water

•Impregnation with ammonical silver solution for 3 min

•Distilled water wash

•Reduce in 10% aqueous formalin with agitation for 3 minute

•Wash in tap water

•Dehydrate ,clean and mount.

Uses of reticulin stain1. Diagnosis of liver cirrhosis.

2. To distinguish epithelial neoplasms from non-epithelial neoplasms. Foci of carcinoma have reticulin around tumour nest but not in between tumour cell, whereas in most sarcomas and large cell lymphoma reticulinseparates single cells.

3. To differentiate between in-situ and invasive carcinoma

Reticulin fiber- Black Nuclei -Black

Trichrome stain

• This stain is mainly used to evaluate the type and amount of extracellular material like-collagen, fibrin, muscle and elastic fiber. Various technique includes:

• Masson trichrome stain

• Van gieson stain

• Mallory, Phosphotungstic or phosphomolybdicacid stain

• Verhoeff-Van Gieson(VVG) stain

The general rule in trichrome staining is thatthe less porous tissues are colored by thesmallest dye molecule; whenever a dye of largemolecular size is able to penetrate, it willalways do so at the expense of the smallermolecule.

Masson trichome stain• This method is used for detection of collagen

fibers in the tissues such as skin, stomach, intestine and lung.

• The collagen fibers will be stained blue and their nuclei will be stained black.

Masson trichrome stain

Principle:

As per the name 3 dyes are used which selectively stain muscle, collagen fibers, fibrin and erythrocytes. As general rule in trichromestain less porous tissues are stained by small dye molecule. Acid fuchsine stain all the connective tissue, PMA( phosphomolybdicacid) competes with fuchsine and gain access to collagen displacing fuchsine. If reaction stopped at appropriate time, collagen will be free to be stained by Methyl Blue.

• Reaent used – Weigerts iron hematoxylin solution

- Phosphomolybdic- phosphotungstic

acid solution

- Biebrich scarlet acid fucshin solution

- Aniline blue solution

- 1% acetic acid solution

• Result - Nuclei : Black

Collagen : Blue

Cytoplasm, Muscle, RBC : Red

• Positive control : skin, lung, stomach, intestine

Pocedure: Masson trichrome stain

•Dewax the section and bring to water

•Wash in tap water

•Stain with weigerts iron hematoxylene for 10 minute

•Rinse well in running tap water for 10 minute

•Wash in distilled water

•Stain in biebrich scarlet acid fuchsin for 10-15 minute

•Wash in distilled water.

•Differentiate in Phosphomolybdic-phosphotungstic acidsolution for 10-15 minute

•Transfer section directly(without rinse) to aniline bluesolution and stain for 5-10 minute

•Rinse briefly in distilled water and differentiate in1%acetic acid solution for 2-5 minute•Wash in distilled water•Dehydrate through alcohol, clear in xylene and mount inDPX.

ResultCollagen- Blue

Cytoplasm, RBCs- Red

• Membranoproliferative glomerulonephritis on massontrichome stain

Uses of Masson trichrome stain• It is used to differentiate between collagen and smooth

muscle in tumour.

• To identify increased collagen deposition in condition like cirrhosis, keloid, benign prostatic hyperplasia, membranoproliferative glomerulonephritis etc.

VAN GIESON STAINPrinciple

When using combined solution of picric acid andacid fuchsin, the small molecules of picric acid penetrate allthe tissue rapidly, but are only firmly retained in the closetextured red blood cells and muscle. The larger moleculesof ponceau S displaces picric acid molecule from collagenfibres, which has larger pores and allow larger molecules toenter.

It is used for detection of collagen.

Result -- Nuclei : Blue / Black

Collagen : Red

Cytoplasm, muscle, fibrin, RBCs : Yellow

• Results:

• Collagen – deep red

• Muscle, – yellowcornified epithelium

• Nuclei – blue to black

Phosphotungstic acid- Hematoxylenetest (PTAH) test:

• Principle

There is much more phosphotungstic acid in the solution then hematein. The phosphotungstic acid binds all the available hematein to form blue lake pigment. This lake stains muscle cross striations, fibrin, nuclei, and other tissue elements blue. The rest of phosphotungstic acid stains the red-brown components, such as collagen.

USES

• Traditionally used for demonstration of muscle striations, glial cells and fibrin.

• This technique has been largely replaced by immunohistochemistry techniques

RESULTS

• Muscle, cross striations : Blue- black

• Fibrin and neuroglia – Deep blue

• Connective tissue: Pale orange-pink to brownish red

• Bone and cartilage- yellowish to brownish red

Skeletal muscle striation on mallory’s PTAH stain

Gliosis on PTAH stain

Verhoeff-Van Gieson(VVG) stain :

• This method is used for identifying elastic fiber in tissue such as skin, aorta etc.

• Result – Elastic fiber: Blue-black to black

- Nuclei: Blue to black

- Collagen: Red

- other tissue elements: Yellow

Vessel wall on Verhoeff van gieson stain

This section shows elastic cartilage and elastic fibers (arrow),which are dark-stained linear structures embeddedin the cartilage matrix.

Fibrin

• Fibrin is formed by polymerization of smaller soluble fibrinogen.

• Found in tissue damage and acute inflammation, fibrinoid necrosis in vessel wall.

• Stained by : Mallory PTAH

MSB Stain

MSB (Maritus, Scarlet, Blue)Stain for fibrin:

• This is trichrome method for selective demostration of fibrin.

• Dyes Used : Maritus YellowCrystal ScarletMethyl Blue, PTA.

• Results : Fibrin : RedRBC’S : YellowMuscle : RedCollagen : Blue

Fibrin stains red, collagen stains blue, muscle stains red, nuclei stains blue/black, red cells stain yellow

Fibrin stains red, collagen stains blue, muscle stains red, nuclei stains blue/black, red cells stain yellow

Stain for Amyloid

Amyloids are insoluble fibrous protein. They arise from inappropriately folded protein and polypeptides present naturally in the body. These misfolded structure alter their configuration and interact with each other or other cell components forming insoluble fibrils. Abnormal accumulation of amyloid fibrils leads to amyloidosis and play role in various pathological disease.

Stains used to demostrate amyloid:

• Congo red

• Crystal/methyl violet

Congo red stain

Principle :

Amyloids are homogeneous and eosinophillic, proteinaceous deposits, that are extracellular and may become sufficiently large enough to cause damage to surrounding tissues. When stained with the congo red stain the amyloid will show birefringence an apple green color, under the polarizing microscope.

Reagent:

• Solution a - 0.5% Congo red in 50% alcohol

• Solution b - 0.2% potassium hydroxide in 80% alcohol

• RESULTS:

Amyloid, elastic fibers, --- red to pink

eosinophilic granules,

Nuclei – blue

PROCEDURE: (Congo red stain)

1. Deparaffinize and hydrate to water.

2. Stain in Congo red solution for5 minutes.

3. Differentiate with alcoholic potassium hydroxide

solution for 3-10 seconds.

4. Wash in water, stain nuclei in alum hematoxyllin, differentiate and blue.

7. Dehydrate, clear in xylene & mount.

It is used to demostrate amyloid deposits in

• renal amyloidosis- in patients on dialysis for long time

• Medulary carcinoma of thyroid,

• Vessel wall in case of Alzheimer’s disease

• Cardiac arrhythmias, etc.

Positive red staining is present around the large central artery and

a smaller vessel to its upper right. The right panel shows the green

birefringence that is diagnostic of amyloid when the Congo red

stain is viewed with polarized light.

All amyloids have a fibrillar ultrastructure that gives this reaction.

Crystal/Methyl violet for amyloid

• Crystal violet or methyl violet stain are used for metachromatic amyloid staining.

• They stain amyloid as purple-red in blue background.

Reagents used

• Crystal/methyl violet

• 95% alcohol

• 1% aqueous ammonium oxalate

• 0.2% acetic acid

Preparation of solutions

• Dissolve 2 gm crystal/methyl violet in 20ml 95% alcohol and 80ml of 1% aqueous ammonium oxalate dissolve using minimum of heat. Cool and filter

Procedure• Bring the section to water level.

• Stain in crystal/methyl violet solution for 5 minute.

• Wash and differentiate in 0.2% acetic acid controlling differentiation microscopically arresting differentiation in water and repeating untill good contrast is obtained between amyloid and background.

• Wash and mount in DPX.

RESULTAmyloid - Purple

Backgrond - Blue

Amylod – purple Background - blue

Part II

Stains for Lipid

- Oil Red O

- Sudan Black B

Oil red o stain

PRINCIPLE :

Staining with oil-soluble dyes is based onthe greater solubility of the dye in the lipidsubstances than in the usual hydroalcoholicdye solvents.

Result

Lipid – RedNuclei- Blue

• REAGENTS : • 60% isopropanol• Alum hematoxylin:• Glycerin Jelly mounting medium• Oil Red O working solution:

o To make oil red O stock solution• Oil red O - 0.5gm• Isopropanol 100 ml

Dissolve the dye in isopropanol, using the very gentle heat of water bath. This is the stock solution.

o To make working oil red O solution• Dilute 30ml of stock stain with 20ml of distilled water

and allow it to stand for 10 minutes, filter into coplin jar and cover immedeiately.

• Working solution should be made fresh each time.

PROCEDURE : (Oil red O stain)

• Fix slides in 10% formalin if fresh.

• Wash well in tap water for 1-10 minutes to drain off excess water.

• Rinse with 60% isopropanol.

• Stain with freshly prepared oil red O working solution for 15 minutes.

• Rinse with 60% isopropanol.

• Lightly stain nuclei with alum hematoxylin 5 dips.

• Rinse with distilled water.

• Mount with aqueous mounting media, Glycerin Jelly.

Fat emboli seen as red dot within capillaries of lung on Oil red O stain

Fatty liver -stain Oil red O

Uses• Oil red O stain is used for staining neutral

triglycerides, lipids and lipoprotein.

• Tumors arising from fat cells (liposarcomas) can be differentiated from other types of tumors.

• Fat occurring in an abnormal place, such as fatty emboli that may develop after either a bone fracture or an injury that crushes a fatty body area.

• Lipid storage disorder like nieman-pick disease

• To demonstrate fat or lipids in conidition like fatty liver.

• In hematological condition like burkitt’s lymphoma and sea-blue histiocytic syndrome

FAT - SUDAN BLACK B - PROPYLENE GLYCOL

PRINCIPLE :Sudan Black B is a dye that is insoluble in water but dissolves in fat. Therefore this dye will accumulate in fat globules within cells.

• It is slightly basic dye and will combine with acidic groups in compound lipids, thus staining phospholipids also.

ResultFat- Blue/ BlackNuclei- Red

REAGENTS :

• 85% Propylene Glycol:

Propylene glycol -85.0 ml

Distilled water -15.0 ml

• Hematoxylin:

• Sudan Black B/Propylene:

Sudan Black B -0.7 gm

Propylene glycol -100.0 ml

PROCEDURE : ( Sudan black stain)

1. Fix slides in 10% formalin if fresh.

2. Wash well in tap water, rinse in distilled water, drain off excess water.

3. Propylene glycol, two changes, 5 minutes each.

4. Stain with Sudan Black, 7 minutes, agitate.

5. Dip in 85% Propylene glycol, 3 minutes.

6 Rinse in distilled water.

7. Stain with Nuclear Fast Red, 3 minutes.

8. Wash in water.

9. Wash in tap water, rinse in distilled.

10. Mount with aqueous mounting media, Glycerin Jelly.

Uses• Sudan black is used for staining neutral

triglycerides, lipids, lipoprotein and phospholipid.

• In hematological disorder it can be used to differentiate blast as it stains the myeloblast, but does not stain lymphoblast.

Myeloblast on Sudan black stain

MASSON FONTANA METHOD- FOR MELANIN

Melanin is a nonlipid, non hematogenouspigment. It is a brown-black pigment present normally in the hair, skin, retina, iris and certain parts of the CNS.

PRINCIPLE:

Melanin is insoluble in organic solvents but soluble in 1M sodium hydrooxide.

It is slowly bleached by strong oxidising agents.

The solutions of ammoniacal silver nitrate are reduced by melanin to black metallic silver this is the basis of Masson Fontana method for demostrating melanin.

USES:

• To identify melanin and argentaffingranules.

• In diagnosis of malignant melanoma

• Argentaffin granules are found in carcinoid tumors.

Melanin pigment of skin showing

black color

RESULTS:

Melanin, argentaffingranules - Black

Nuclei -red

Melanin pigment in cells of malignantmelanoma, Fontana-Masson stain.

VON KOSSA METHOD FOR CALCIUM

PRINCIPLE:

Tissue sections are treated with silver nitrate solution, silver is deposited by replacing the thecalcium and then it is reduced by the strong light and visualized as metallic silver.

USE:

Abnormal deposits of calcium may be found in any area of the body. With the H&E stain, calcium appear deep blue-purple. On von kossa method it appear black.

Coronary artery showing calcified atheromatous plaque

RESULTS :

Calcium salts -black

Nuclei -red

Cytoplasm -pink

PERL’S- PRUSSIAN BLUE REACTION FOR IRON

PRINCIPLE :

Dilute mineral acid hydrolysis releases ferric iron from protein bound tissue deposits, which in the presence of ferrocyanide ions, is precipitated as highly coloured and highly water soluble complex, potassium ferric ferrocyanide- prussian blue.

• Ferrous ion do not produce a coloured reaction.

• Tissue deposits containing ferric iron are invariably hemosiderin

USE:

• To demonstrate ferric iron in tissue sections.

• Small amounts of iron are found normally in spleen and bone marrow.

• Excessive amount are present in hemochromatosis- with deposits found in the liver and pancreas and hemosiderosis- with deposits in the liver, spleen, and lymph nodes.

• To access the bone marrow iron content

REAGENTS:

• 2% Potassium Ferrocyanide:Potassium ferrocyanide - 2.0 gm

Distilled water - 100.0 ml• Neutral-fast Red:

Neutral red - 1gmDistilled water - 100mlGlacial acetic acid - 1ml

• 2% Hydrochloric Acid:Conc. Hydrochloric acid, - 2.0 ml

Distilled water - 100.0 ml

CONTROL: Postmortem lung – high number of heart failure cells that contain hemosiderin

PROCEDURE: ( Perl’s prussian blue)

1. Deparaffinize and hydrate to distilled water.

2. Stain with mixture of equal volume of aqueous 2% potassium ferrocyanide and 2% HCl solution for 30 minutes.

3. Thorough wash with distilled water.

4. Counter stain with 0.5% Neutral red lightly.

5. Wash in tap water.

6. Dehydrate, clear, and mount.

Hemosiderin in liver

RESULTS:

Iron (hemosiderin) -blue

Nuclei -red

Background - pink

Staining for Microorganism

- Ziehl- Neelsen (ZN) stain

- Wade fite faraco stain

- Gomori Methenamine (hexamine) silver stain

- Warthin-Starry stain

- Giemsa stain

- Gram staining

Ziehl-Neelsen (ZN) Stain for Mycobacterium Bacillus

Mycobacteria are difficult to demonstrate by the Gram technique because they possess a capsule containing a long chain fatty acids that make them hydrophobic.

Phenolic acid or heat may be used to reduce the surface tension and increase the porosity.

• PRINCIPLEMycobacterias (tubercle bacilli) have a lipid-rich cell wall which is capable of taking up strong phenol dye solutions (eg. carbol fuchsin solution) such that the dye is retained upon subsequent differentiation in acid or alcohol. They are said to be acid and alcohol fast (AAFB = acid and alcohol fast bacilli).

Results

• Mycobacteria - Red

• Background - pale Blue

Method

• Bring the section to water level.

• Flood sections with carbol-fuchsin and heat to steaming by passing the flame of spirit swab underneath the slides on metal rack till, vapour just being formed.

• Wash the slides in distilled water. Shake off excess liquid.

• Decolourise the slides with 20% H2SO4

• Wash well in tap water, till no more red colour runs off the surface when the slides is dipped in water

• Wash thoroughly with water.

• Counter stain in methylene blue solution, 30 seconds.

• Blot and differentiate by alternate dehydration and rehydration until the background is a delicate pale blue.

• Examine microscopically screening at high power and confirming all suspicious organism with an oil immersion lens.

Mycobacteria seen as red rods

Acid fast bacteria seen on intestinal biopsy

Wade fite faraco stain• This stain is used for staining leprocy bacilli in

tissue sample.

• Leprocy bacilli are much less acid and alcohol fast than tuberculous bacilli. Therefore alcohol is removed from hydrating and dehydrating steps and 10% sulphuric acid is used as decolouriser in place of acid-alcohol solution.

• The section are also deparaffinised using ground nut oil and xylene mixture to protect more delicate waxy coat of lepra bacilli.

PROCEDURE• Warm section and deparaffinised using mixture of two part

xylene and one part of ground nut oil for 10 minutes.

• Blot dry and wash in water untill section is uniformly wetted.

• Stain with carbol fuschin for 20-30 minutes.

• Wash in tap water and blot dry.

• Decolorize in 10% sulphuric acid till no more red colour appear while dipping in water.

• Wash in tap water and counter stain with 0.2% methyleneblue for 5-10 seconds.

• Blot dry and do not mount.

• Smears should be seen in oil immersion lens.

Leprrocy and other mycobacteria- redBackground- blue

Warthin-Starry method for spirochetes (Warthin & Starry 1920)

PRINCIPLEOrganisms are demonstrated by silver impregnation technique.

SolutionsAcetate buffer. pH 3.6

Sodium acetate 4.1 gAcetic acid 6.25 mlDistilled water 500 ml

1 % silver nitrate in pH 3.6 acetate buffer.

• It can also be used to demostrate

- Helicobacter pylori in gastric mucosa

- Leptospira organism in renal biosy

- Cat scratch disease associated bacilli on

lymph node biopsy

H. Pylori on gastric mucosa on Warthin starry stain

Leptospira organism Renal biopsy

Result:

Organisms - black

Background - golden

yellow

Cat scratch disease bacilli in lymph node biopsy

Gomori methenamine silver stain for fungi

• GMS staining is a silver staining technique fordemonstrating fungi in tissue sections.

• It is primarily based on staining the polysaccharides in fungal cell walls, and can be used to demostratethe basement membrane.

PRINCIPLEThis method depends upon the reduction of the silver by the aldehyde groups produced after oxidation of fungal wall components with chromic acid.

Pneumocystis carinii

Result:

Fungi, pneumocystis carinii, melanin - black

Mucin and glycogen-- grey-black

RBCs - yellow

Background - pale green

GMS stain for Cryptococcus neoformans

Basement membrane on GMS stain

Giemsa stain

• PRINCIPLEThis method is a modified version of the original Giemsa technique used for haematological smears and gives good results for sections. Giemsa is a Romanowsky stain which contains azure B and eosin Y and is capable of making subtle distinction in shades of staining. The acidic groupings of the nucleic acids and proteins of the cell nuclei determine their uptake of the basic dye azure B and the presence of basic groupings result in an affinity for acidic dyes and their staining by eosin.

• It can be used for histopathological diagnosis of malaria and some other spirochete protozoan, and blood parasites.

Results

• Micro-organism, fungi - purplish-blue

parasites

• Nuclei- blue to violet

• Erythrocytes- salmon pink

• cytoplasm - light blue

• Collagen, muscle and bone - pale pink

Aspergillus fumigatus on giemsa stain

Toxoplasma gondii

Gram method for Bacteria • Gram staining differentiate bacteria into 2 classes

depending on their cell wall structure and composition

• Gram positive and Gram negative.

• PRINCIPLECrystal Violet stains the nucleic acids of the bacteria (and background tissue) and after treatment with iodine, the sections are differentiated in acetone and counterstained with basic fuchsin. The tissue background and Gram-negative bacteria lose their blue staining and are subsequently stained with counter stain basic fuchsin. Gram-positive bacteria resist the decolourisation and retain the crystal violet/iodine blue staining.

Result:Gram-positive organisms - blueGram – negative organisms - red.

Gram Positive

Gram Negative

Cutaneous anthrax – Gram positive Bacilli stained blue on skin biopsy

References

• Rosai and ackerman’s surgical pathology- 10th edition

• Textbook of microbiology-Ananthanarayan and paniker’s

• Internet

Thank you