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« 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.

° 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.

° control is necessary
vlassification

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

1. Stains for glycogen and mucins

2. Stains for amyloid

3. vonnective tissue stains

4. Lipid stains

5. Stains for neuropathology

6. Stains for microorganisms

7. Stains for pigments and minerals

8. Special stains in hematology

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!eriodic acid Schiff(! S) method

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

Solutions:
1) Periodic acid- oxidizing agent
2) Schiff¶s reagent-
1) Basic fuschin
2) Potassium metabisulphite
3) HCL
4) ctivated charcoal
3) 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.
!rocedure:
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 P S positive substances- Magenta coloured(purple red)
Nuclei-blue

vontrol: Liver or Muscle

 ! S positive substances

1. !olysaccharides 4. Sphingomyelin- in Niemann pick¶s disease

- glycogen ( starches and celluloses in plant)
- many blood leucocytes contain glycogen 5. vertain pigments
- capsule of fungi and certain bacteria - ceroid
contain neutral polysaccharides( chitin) - lipofuschin pigment
- pigment in melanosis coli
2. Glycoproteins ( largest group) - Dubin Johnson¶s pigment
- mucins
- gonadotrophic hormone, TSH, thyroglobulin 6. Mast cells
- serum mucoproteins
- some plasma cells(Rusell bodies) 7. myloid
- cytoplasm of megakaryocytes
- basement membranes, reticulin, collagen

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

** cid mucopolysaccharides i.e. hyaluronic acid, chondrotin sulphate are

P S negative
Enzyme digestion( ! S with diastase)

!rinciple: pretreatment with some enzymes(malt diastase) will remove

glycogen and P S reaction becomes negative.
Use: It is used to differentiate glycogen from other P S positive substances.
Normal Kidney: P S Stain KW lesions of diabetic nephropathy

Membranous GN : with P S stain the

thickening of capillary walls is easily seen
 P S stain showing pseudo hyphae of
candida

Rhinosporidiasis: P S stain showing

trophozoites and cysts of the organism
31- ntitrypsin (
) deficiency.
: Characteristic hyaline globules are barely discernible by hematoxylin and eosin.
B: The inclusions are periodic acid-Schiff (P S) 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 varmine 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. ! S with diastase: now method of choice to detect glycogen

Stains for Mucin

Mucin: it is a polysaccharide
It is classified as
) cidic Mucin
1) Strongly sulphated: Connnective tissue mucins
a. Chondrointin sulphate - 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
 lcian blue stain

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

!rinciple: lcian blue is a positively charged( cationic) dye and it forms

electrostatic bonds with tissue polyanions bearing either carboxyl or sulphate group
of acid mucin.

Solution:
1. 1% alcian blue in 3% acetic acid at !H 2.5
2. Hematoxylin/neutral red- nuclear counter stain

!rocedure:
1. Control slide is preferred
2. Bring sections to the water
3. Stain with alcian blue soln for 10min at RT
4. Wash in running water for 5 min
5. Counterstain with hematoxylin for 5 min
6. Wash in running water for 5 min
Results:
cid mucin- blue
Nuclei- dark blue- hematoxylin
red- neutral red
 lcian blue soln is used at various !H 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

lcian blue positive substances

1. cid mucins and cid MPS
2. Cartilage ground substance
3. Cell bodies of fungi
4. Mucoid capsules of organisms e.g. pneumococci
5. Mast cell granules
 lcian blue with ! S

This combined technique is used to demonstrate neutral mucins

!rinciple: First, acid mucin is stained blue with lcian blue and is unable
to react with P S stain and then neutral mucins and glycogen are stained
magenta with subsequent P S staining.

Results:
cidic mucin: blue
P S and Neutral mucin: magenta
 Undiffrentiated gastric signet ring adenocarcinoma
: lcian blue/P S

Barrett esophagus: lcian blue/P S

stain in showing incomplete intestinal
metaplasia
Mucicarmine stain

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

Results:
Mucin and capsule of cryptococcus ± deep rose to red
Other tissue elements- yellow

vryptococcosis 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. vongo red (Benhold method)

- sections should be 10-12 um thick.
- congo red is a diazo dye which attaches itself parallel to fibrils of amyloid
- lcohol 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: L amyloid (secondary) looses it affinity for
congo red
Results: amyloid- deep pink to red
nuclei- blue
Procedure:
1. Control slide is must
2. Bring sections to the water
3. Stain with filtered 1% congo red soln for 45 min
4. Do not wash
5. 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 ± 1min
8. 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
 L amyloidosis
: salmon pink staining of amyloid with Congo
red.
B: pple green birefringence is elicited on
polarization of sample shown in
v: Thioflavin-T stain depicts mesangial amyloid
deposits.
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ypes of collagen

It is the most abundant protein in the human body

There are 4 major and several minor variants

ype1: 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.

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

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

young fibroblasts before the tougher type I collagen is synthesized.
lso called Reticulin fiber.

ype 4: Found in basement membrane.

vollagen Vs reticulin fibers

vollagen
° 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 niline 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. P S
Masson¶s trichrome
Use:
1. Routine stain for liver and kidney biopsies.
2. Used to differentiate between collagen and smooth muscle in
tumors, and
3. the increase of collagen in diseases such as cirrhosis.

!rinciple: s the name implies, three dyes are employed selectively

staining muscle, collagen fibers, fibrin, and erythrocytes. The general rule
in trichrome staining is that the less porous tissues are colored by the
smallest dye molecule. Whenever a dye of large molecular size is able to
penetrate, 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 , 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 , also called plasma stain, contains acid fuchsin, Xylidine Ponceau,
glacial acetic acid, and distilled water.
- Solution B contains phosphomolybdic acid in distilled water.
- Solution v, also called fibre stain, contains Light Green SF yellowish. It is
used to stain collagen..

Results:
Nuclei-black
Cytoplasm, muscle, erythrocytes-red
Collagen-blue
 Kidney (Masson's
richrome): Interstitial fibrosis,
tubular loss and mononuclear interstitial infiltration

Liver cirrhosis: Portal fibrosis with hepatocellular

regenerative nodules (Masson¶s Trichrome stain)
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

!rinciple: 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 ± alcoholic hematoxylin « 2.0 ml
Soln B ± Ferric chloride « 8 ml
Soln C ± Lugol¶s iodine « 8 ml
!rocedure:
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.

vontrol : artery or skin

Results:
Elastic tissue fibres - black
 EVG stain:
: normal aorta
B: extensive fragmentation of IEC of aorta in a case of
Marfan¶s syndrome

emporal arteritis(EVG) stain:

Reticulin stain
Gomori¶s method for reticulin fibres

!rinciple: 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% KMnO4
2. 2% oxalic acid
3. 2% Iron alum
4. 0.2% gold chloride
5. 2% Na thiosulphate
6. mmonical Silver Soln

!rocedure:
1. Deparaffinize and hydrate to water
2. Oxidize with KMnO4 for 3 min. Rinse in water for 5 min
3. Bleach with oxalic acid for 2 min. Rinse in water for 5 min
4. 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 min
7. Treat with 10% formalin till tissue becomes blackish brown( 1 min)
8. Do not wash. Pour off the soln
9. dd 0.5% gold chloride for 10 min
10. Oxalic acid for 2 min
11. Treat with Na thiosulphate for 3 min till soapy appearance is seen
12. Wash in water. Dehydrate, clear and mount in DPX
Results:
Reticulin ± black
Nuclei ± grey
Collagen ± grey purple

Reticulin stains demonstrates both reticulin fibers and basement membrane.

`iagnostic uses of silver reticulin stains

1. Kidney biopsy: helpful in differentiating certain kidney lesions
2. In liver biopsies, reticulin stains are used to define early cirrhosis.
3. lso 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.
 Normal liver: reticulin stain

virrhotic liver: reticulin stain highlights the

Nodular architecture
Silver methanamine stain
Use: It in mainly used in kidney biopsies to highlight glomerular
capillary basement membrane.

!rocedure:
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: black
Background: light green
 SM stain: normal glomerulus SM with P S: MGN with spikes

MPGN showing tram tracking or reduplication of BM

Fibrin stain
Use: mainly used in kidney biopsies to evaluate certain lesions in
various forms of glomerulonephritis.

vontrol slide is a must.

Stains for fibrin

1. H&E- homogeneous eosinophilic(pink) substance
2. Mallory¶s PT H- blue to purple
3. P S +ve
4. Gram- Weigert stain- app Gm+ve
5. Picro-Mallory method
6. MSB method
±  
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

`emonstration of fat:
1. Sudan dyes
2. Secondary Fluorescence
3. Nile blue staining
4. Osmic acid staining
5. Extraction techniques: supportive evidence

Sudan dyes
Sudan III- first Sudan dye to be introduced
Sudan IV- darker staining than Sudan III
Oil red O- even more darker staining
Sudan black O ± most sensitive
Sudan III stain
Done on frozen sections

!rocedure:
1. Cut frozen sections. Use albuminized slides
2. Cover Sudan III soln over tissue for 10 min.
3. Wash in water.
4. Counterstain with hematoxylin for 45sec- 1 min
5. Wash in water.
6. Mount in glycerine

Results:
Fat- red globules
Nucleus-blue

dipocytes stained with sudan III

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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 ML and LL. LL blasts are MPO ±ve
ii. in ML 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. vhloroacetate (v E):
- 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 ML
- also focally positive in M6, M7 and T- LL
5. ! S reaction:
- positive in L1, L2 subtypes of LL(B cell LL)
- negative in T-cell LL and L3
- focally positive in M5, M6

6. cid phosphatase ( !):

- !rincipe: 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 LL
- focal activity is also seen in ML M6 and M7.
- tartrate resistant acid phoshatase activity is seen in hairy cell leukemia.

7. Leukocyte alkaline phosphatase (L !)

- The L P stain is used to determine if an increase of cells is due to CML or a
noncancerous reaction to an infection.
- !rinciple: The L P 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.
4 Interpretation:
Normal Range: 11-95
Increased L P score : Leukemoid reaction, pregnancy, polycythemia vera
Decreased L P score: Chronic Myeloid Leukemia, PNH
ML M0: MPO positivity
 NSE positivity in ML M5

Block like positivity of P S in LL