Special stains in histopathology

… Dr. Imran Shaikh M.D. Pathology KEM Hospital Mumbai

What is a special stain ?

• H&E stain is routine stain. It is the preliminary or the first stain applied to the tissue sections Give diagnostic information in most cases.

• A special stain is a staining technique to highlight various individual tissue components once you have got preliminary information from the H&E stain.

• special stain is applied to diagnose non-neoplastic as well as neoplastic lesions.

• the term usual special stains generally does not include immunoperoxidase methods.

• A control is necessary

Classification

Special stains can be broadly classified according to the tissue which they stain:-

1. Stains for glycogen and mucins

2. Stains for amyloid

3. Connective tissue stains

4. Lipid stains

5. Stains for neuropathology

6. Stains for microorganisms

7. Stains for pigments and minerals

8. Special stains in hematology

Stains for glycogen and mucin

Periodic acid Schiff(PAS) method

Principle: Periodic acid(HIO2) bring about oxidative cleavage of C-C bond in glycol ortheir amino/alkylamic derivatives to form dialdehydes. These aldehydes react with Schiff’s reagent to produce insoluble magenta coloured compound

Solutions:1) Periodic acid- oxidizing agent2) Schiff’s reagent- 1) Basic fuschin 2) Potassium metabisulphite 3) HCL 4) Activated charcoal3) Hematoxylin- nuclear counterstain

Schiff’s reagent should be- Clear or pale yellow in color- Discard when pink color develops- Stored at 40 C in dark container.

Procedure:1. Control is must.2. Deparaffinize and hydrate to water.3. Oxidize with 0.5% periodic acid for 3 min. (time is imp)4. Wash in water for 5 min.5. Cover slide with Schiff’s reagent for 10 min till tissue becomes pink.6. Wash in running tap water for 10 min.7. Do acid alcohol and then wash the slide for 5-6 min.8. Counterstain with Ham’s hematoxylin for 1 min.9. Wash in water for for 1min.10. Dehydrate in alcohol, clear in xylene, mount in DPX.

Results:Glycogen and other PAS positive substances- Magenta coloured(purple red)Nuclei-blue

Control: Liver or Muscle

PAS positive substances

1. Polysaccharides - glycogen ( starches and celluloses in plant) - many blood leucocytes contain glycogen - capsule of fungi and certain bacteria contain neutral polysaccharides( chitin)

2. Glycoproteins ( largest group) - mucins - gonadotrophic hormone, TSH, thyroglobulin - serum mucoproteins - some plasma cells(Rusell bodies) - cytoplasm of megakaryocytes - basement membranes, reticulin, collagen

3. Glycolipids - gangliosides - cerebrosides - whipples disease - Tay Sacs disease - Krabbe’s leucodystrophy

4. Sphingomyelin- in Niemann pick’s disease

5. Certain pigments - ceroid - lipofuschin pigment - pigment in melanosis coli - Dubin Johnson’s pigment

6. Mast cells

7. Amyloid

** Acid mucopolysaccharides i.e. hyaluronic acid, chondrotin sulphate are PAS negative

Enzyme digestion( PAS with diastase)

Principle: pretreatment with some enzymes(malt diastase) will remove glycogen and PAS reaction becomes negative.Use: It is used to differentiate glycogen from other PAS positive substances.

Normal Kidney: PAS Stain

Membranous GN : with PAS stain the thickening of capillary walls is easily seen

KW lesions of diabetic nephropathy

PAS stain showing pseudo hyphae of candida

Rhinosporidiasis: PAS stain showing trophozoites and cysts of the organism

α1-Antitrypsin (AAT) deficiency. A: Characteristic hyaline globules are barely discernible by hematoxylin and eosin. B: The inclusions are periodic acid-Schiff (PAS) positive and diastase resistant.

Staining methods for glycogen

1. Iodine: - oldest method, now absolute - iodine stains glycogen mahogany brown. starch- dark blue - not specific for glycogen as amyloid, some proteins substances, lecithin also stain positive

2. Best’s Carmine method: - though empirical, it is highly specific for glycogen. - also stains mast cell granules, mucin and fibrin but in a lighter shade - Results: glycogen: brilliant red Nuclei: blue

3. PAS with diastase: now method of choice to detect glycogen

Stains for Mucin

Mucin: it is a polysaccharideIt is classified asA) Acidic Mucin 1) Strongly sulphated: Connnective tissue mucins a. Chondrointin sulphate A- hyaline cartilage b. Chondrointin sulphate B- dermis, aorta, heart valves c. Chondrointin sulphate C- umblical cord, cartilage, dermis d. Heparin/heparan sulphate- mast cell/ aorta e. Keratan sulphate- cornea, nucleus pulposus 2) Weakly sulphated epithelial mucin- colonic goblet cells 3) Carboxylated sialomucin- Bronchial submucus glands, submandibular salivary glands, goblet cells of small intestine 4) Sulphated sialomucins – prostatic carcinomas 5) Hyaluronic acid – umblical cord, dermis, cardiac conn tissue

B) Neutral mucin Brunner’s gland, gastric lining cells, prostatic glands

Alcian blue stain

Use: for demonstration of acid mucins. Neutral mucins are not stained by alcian blue.

Principle: Alcian blue is a positively charged( cationic) dye and it forms electrostatic bonds with tissue polyanions bearing either carboxyl or sulphate groupof acid mucin.

Solution:1. 1% alcian blue in 3% acetic acid at PH 2.52. Hematoxylin/neutral red- nuclear counter stain

Procedure:1. Control slide is preferred2. Bring sections to the water3. Stain with alcian blue soln for 10min at RT4. Wash in running water for 5 min5. Counterstain with hematoxylin for 5 min6. Wash in running water for 5 min

Results:Acid mucin- blueNuclei- dark blue- hematoxylin red- neutral red

Alcian blue soln is used at various PH to separate & identify different mucins• Strongly sulphated mucin: pH<1.0• Weakly sulphated epithelial mucin: pH 2.5-1• Hyaluronic acid and Carboxylated sialomucin: pH 3.2-1.7• Sulphated sialomucins: pH 1.5

Alcian blue positive substances1. Acid mucins and Acid MPS2. Cartilage ground substance3. Cell bodies of fungi4. Mucoid capsules of organisms e.g. pneumococci5. Mast cell granules

Alcian blue with PAS

This combined technique is used to demonstrate neutral mucins

Principle: First, acid mucin is stained blue with Alcian blue and is unable to react with PAS stain and then neutral mucins and glycogen are stained magenta with subsequent PAS staining.

Results:Acidic mucin: bluePAS and Neutral mucin: magenta

Barrett esophagus: Alcian blue/PAS stain in showing incomplete intestinal metaplasia

Undiffrentiated gastric signet ring adenocarcinoma: Alcian blue/PAS

Mucicarmine stain

Use:1. It is used for staining of epithelial mucin and mucin secreting adenocarcinomas.2. Also used to demonstrate capsule of cryptococcus.

Results:Mucin and capsule of cryptococcus – deep rose to redOther tissue elements- yellow

Cryptococcosis of lung – mucicarmine stain

Stains for amyloid

1. Lugol’s iodine - done on gross slices - Lugol’s iodine contains iodine crystals – 1gm potassium iodide – 2 gms distilled water – 100 ml - apply Lugol’s iodine soln to gross slices of liver, spleen amyloid if present is seen as pin head sized mahogony areas. With application of dilute H2SO4, color changes to blue.

2. Congo red (Benhold method) - sections should be 10-12 um thick. - congo red is a diazo dye which attaches itself parallel to fibrils of amyloid - Alcohol is the best fixative. - It also stains dense connective tissue and elastica. - +ve congo red staining and apple green birefrengence when seen under polarized light is the most useful test to diagnose amyloid. - Prior treatment with KMnO4: AL amyloid (secondary) looses it affinity for congo red

Results: amyloid- deep pink to red nuclei- blueProcedure:1. Control slide is must2. Bring sections to the water3. Stain with filtered 1% congo red soln for 45 min4. Do not wash5. Differentiate with alcoholic NaOH soln till light pink color appears.6. Wash in running tap water for 5 min.7. Counterstain with Meyer’s hematoxylin for 45sec – 1min8. Wash in water for 5 min.9. Dehydrate, clear & mount in DPX.

2. Metachromatic stains a. methyl violet – pinkish red b. crystal violet – purplish red

4. Fluoroscent stain for amyloid - fixation not critical, can be performed on frozen section - 1% aq thioflavin T is used

5. Silver impregnation techniques Van Gieson – amyloid appears khaki coloured

AL amyloidosis A: salmon pink staining of amyloid with Congo red. B: Apple green birefringence is elicited on polarization of sample shown in AC: Thioflavin-T stain depicts mesangial amyloid deposits.

Connective tissue stains

Types of collagen

It is the most abundant protein in the human body There are 4 major and several minor variants

Type1: This is the most abundant collagen in the human body. It is present in scar tissue. It is found in tendons, skin, arterial walls, fibrocartilage, and the organic part of bones and teeth.

Type 2: found in Hyaline cartilage, vitreous humour of the eye.

Type 3: This is the collagen of granulation tissue, and is produced quickly by

young fibroblasts before the tougher type I collagen is synthesized. Also called Reticulin fiber. Type 4: Found in basement membrane.

Collagen Vs reticulin fibers

Collagen• Thick & stronger• Type 1 collagen is most common Tough bundles of collagen called collagen fibers are a major component of the extracellular matrix that supports most tissues and gives cells structure from the outside.• Can be appreciated with H&E.• stains: 1. Massons trichrome 2. Mallory’s Aniline blue 3. Van Gieson’s Picrofuschin

Reticulin fibres• refers to type 3 collagen• forms supporting framework of solid parenchymatous organs like liver, spleen• arranged in 3D configuration around parenchymal cells.• not seen on H&E• stains: 1. Silver impregnation method 2. gold staining 3. PAS

Masson’s trichromeUse:1. Routine stain for liver and kidney biopsies.2. Used to differentiate between collagen and smooth muscle in tumors, and3. the increase of collagen in diseases such as cirrhosis.

Principle: As the name implies, three dyes are employed selectivelystaining muscle, collagen fibers, fibrin, and erythrocytes. The general rulein trichrome staining is that the less porous tissues are colored by thesmallest dye molecule. Whenever a dye of large molecular size is able topenetrate, it will always do so at the expense of the smaller molecule.

Fixative: Bouin's is preferred, 10% formalin.

Solutions:- The trichrome is applied by immersion of the fixated sample into Weigert's iron hematoxylin, and then three different solutions, labeled A, B, and C are applied.- Weigert's hematoxylin is a sequence of three solutions: ferric chloride in diluted hydrochloric acid, hematoxylin in 95% ethanol, and potassium ferricyanide solution alkalized by sodium borate. It is used to stain the nuclei. - Solution A, also called plasma stain, contains acid fuchsin, Xylidine Ponceau, glacial acetic acid, and distilled water. - Solution B contains phosphomolybdic acid in distilled water. - Solution C, also called fibre stain, contains Light Green SF yellowish. It is used to stain collagen..

Results:Nuclei-blackCytoplasm, muscle, erythrocytes-redCollagen-blue

Liver cirrhosis: Portal fibrosis with hepatocellular regenerative nodules (Masson’s Trichrome stain)

Kidney (Masson's Trichrome): Interstitial fibrosis,tubular loss and mononuclear interstitial infiltration

EVG: Elastic Van Geison (Verhoeff’s method)

Use: This stain is useful in demonstrating atrophy of elastic tissue in cases of emphysema, and the thinning and loss of elastic fibers in arteriosclerosis, and

other vascular diseases and connective tissue diseases

Principle: The tissue is stained with a regressive hematoxylin, consisting of ferric chloride and iodine. The differentiation is accomplished by using excess mordant (ferric chloride) to break the tissue-mordant dye complex. The dye will be attracted to the larger amount of mordant in the differentiating solution and will be removed from the tissue. The elastic tissue has the strongest affinity of the iron hematoxylin Soln complex and will retain the dye longer than the other tissue elements.

Verhoefff’s Soln(a+b+c)Soln A – alcoholic hematoxylin … 2.0 mlSoln B – Ferric chloride … 8 mlSoln C – Lugol’s iodine … 8 ml

Procedure:1. Bring the sections to water.2. Stain with working Verhoeff’s soln for 25 min. wash in water.3. Diferentiate with 2% aqeous ferric chloride soln till tissue appears light grey.4. Rinse with water.5. Keep in 95% ethanol for 10 min.6. Counterstain with Van dieson soln for 3 min.7. Don not wash. Blot the slide on filter paper to remove extra stain.8. Dehydrate, clean and mount inDPX.

Control : artery or skin

Results:Elastic tissue fibres - black

EVG stain:A: normal aortaB: extensive fragmentation of IEC of aorta in a case of Marfan’s syndrome

Temporal arteritis(EVG) stain:

A B

Reticulin stain Gomori’s method for reticulin fibres

Principle: The tissue is oxidized, then sensitized with the iron alum, which is replaced with silver. The silver is reduced with formalin to its visible metallic state.Soln:1. 0.5% KMnO42. 2% oxalic acid3. 2% Iron alum4. 0.2% gold chloride5. 2% Na thiosulphate6. Ammonical Silver Soln

Procedure:1. Deparaffinize and hydrate to water2. Oxidize with KMnO4 for 3 min. Rinse in water for 5 min3. Bleach with oxalic acid for 2 min. Rinse in water for 5 min4. Treat with Iron alum for 20 min. Wash in running tap water.5. Treat with freshly prepared ammonical silver soln till slide is filled with silver scum all over ( 5-7 min)6. Rinse in water for 2 min7. Treat with 10% formalin till tissue becomes blackish brown( 1 min)8. Do not wash. Pour off the soln9. Add 0.5% gold chloride for 10 min10. Oxalic acid for 2 min11. Treat with Na thiosulphate for 3 min till soapy appearance is seen12. Wash in water. Dehydrate, clear and mount in DPX

Diagnostic uses of silver reticulin stains1. Kidney biopsy: helpful in differentiating certain kidney lesions2. In liver biopsies, reticulin stains are used to define early cirrhosis.3. Also used to define early fibrosis of bone marrow.4. Certain tumors produce abundant reticulin and often assumes characteristic patterns. eg- rhabdomyosarcomas, hemangiosarcomas, angiomatous tumors, fibroblastic tumors.5. Differentiation of epithelial from non-epithelial neoplasms.6. Differentiating in situ from invasive carcinoma.

Results:Reticulin – blackNuclei – greyCollagen – grey purple

Reticulin stains demonstrates both reticulin fibers and basement membrane.

Normal liver: reticulin stain

Cirrhotic liver: reticulin stain highlights the Nodular architecture

Silver methanamine stain

Use: It in mainly used in kidney biopsies to highlight glomerular capillary basement membrane.

Procedure: 1. Deparaffinise, hydrate to water.2. Oxidize with 1% periodic acid soln for 10 min.3. Wash in running tap water for 5 min.4. Stain with freshly prepared Hexamine complex working soln. Dip the slide in above soln

for 40 min at 600 C in water bath. Check microscopically for the blue color.5. Wash in water.6. Dip in gold chloride for 10 min.7. Wash in water.8. Na thiosulphate for 10 min.9. Wash in water.10. Counterstain with light green for 2 min.11. Dehydrate, clear and mount in DPX.

Results:Basement membrane of glomerulus: blackBackground: light green

SM stain: normal glomerulus SM with PAS: MGN with spikes

MPGN showing tram tracking or reduplication of BM

Fibrin stainUse: mainly used in kidney biopsies to evaluate certain lesions in

various forms of glomerulonephritis.

Control slide is a must.

Stains for fibrin1. H&E- homogeneous eosinophilic(pink) substance2. Mallory’s PTAH- blue to purple3. PAS +ve4. Gram- Weigert stain- app Gm+ve5. Picro-Mallory method6. MSB method

Lipid stains

Introduction

- Fat is best demonstarted on frozen sections.- there is no ideal fixative for fats- fixation if desired can be done by - Formol calcium - Chromate fixatives - Osmium tetraoxide

Demonstration of fat:1. Sudan dyes2. Secondary Fluorescence3. Nile blue staining4. Osmic acid staining5. Extraction techniques: supportive evidence Sudan dyesSudan III- first Sudan dye to be introducedSudan IV- darker staining than Sudan IIIOil red O- even more darker stainingSudan black O – most sensitive

Sudan III stain Done on frozen sections

Procedure:1. Cut frozen sections. Use albuminized slides2. Cover Sudan III soln over tissue for 10 min.3. Wash in water.4. Counterstain with hematoxylin for 45sec- 1 min5. Wash in water.6. Mount in glycerine

Results: Fat- red globules Nucleus-blue

Adipocytes stained with sudan III

Special stains in Hematology

1. Myeloperoxidase stain: - Myelopeoxidase an enzyme located in the azurophil(primary) granules of the myeloid cells. - MPO positivity appears as coloured granules in the cytoplasm mainly at the site of enzyme activity (golgi zone). - use: i. to differentiate between AML and ALL. ALL blasts are MPO –ve ii. in AML it is positive in M0, M1, M2, M3.

2. Sudan Black B (SBB) - Phospholipids in the membrane of neutrophilic granules are stained by SBB - It’s activity and use is same as that of MPO.

3. Chloroacetate (CAE): - present in all cells of neutrophilic series( MO, M1, M2, M3). - it is more specific than MPO but less sensitive. - not present in monocytic series.

4. Non- specific esterase(NSE) reaction: - it is an enzyme present in cells of monocytic series - it is positive in M4, M5 AML - also focally positive in M6, M7 and T-ALL

5. PAS reaction: - positive in L1, L2 subtypes of ALL(B cell ALL) - negative in T-cell ALL and L3 - focally positive in M5, M6

6. Acid phosphatase (AP): - Principe: The acid phosphatase within the cell hydrolyzes the substrate naphthol phosphoric acid. The hydrolyzed substrate then couples with the dye (hexagotized pararosaniline) and because the colored complex is insoluble, it precipitates out at the site of enzyme activity. - Strong focal positivity is seen in T cell ALL - focal activity is also seen in AML M6 and M7. - tartrate resistant acid phoshatase activity is seen in hairy cell leukemia.

7. Leukocyte alkaline phosphatase (LAP) - The LAP stain is used to determine if an increase of cells is due to CML or a noncancerous reaction to an infection. - Principle: The LAP within the cell hydrolyzes the substrate naphthol phosphate. The hydrolyzed substrate then couples with the dye (fast red‑violet salt L.B.) and precipitates out at the site of enzyme activity.

- Method of Scoring Count 100 segmented and band neutrophils, rating the cells as below O = unstained cells 1+ = cells stained faintly with a few small granules 2+ = moderate number of small granules 3+ = medium to large granules fill the cell 4+ = cells deeply stained with granules obscuring the nucleus of the cell. - Interpretation: Normal Range: 11‑95 Increased LAP score : Leukemoid reaction, pregnancy, polycythemia vera Decreased LAP score: Chronic Myeloid Leukemia, PNH

AML M0: MPO positivity

NSE positivity in AML M5

Block like positivity of PAS in ALL