1. Dr huzlinda hussin Dept of pathology 13 march 2018
SPECIAL STAINS
Dr huzlinda hussin
Dept of pathology
13 march 2018

2. 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

3. CLASSIFICATION:
Stains for carbohydrates
Stains for amyloid
Nucleic acid stains
Lipid stains
Stains for microorganisms
Connective tissue stains
Stains for pigments and minerals

4. CARBOHYDRATES SIMPLE CARBOHYDRATES GLYCOCONJUGATES
(molecules composed purely of
carbohydrates)
GLYCOCONJUGATES
(molecules composed of carbohydrates and other molecules such as protein and lipid)
Monosaccharides (glucose,mannose,galactose)
Oligosaccharides (sucrose,maltose)
Polysaccharides
(glycogen,starch)
Proteoglycans
Mucins
Others glycoproteins

5. 1. STAINS FOR CARBOHYDRATE
Periodic acid schiff(PAS) technique
Alcian blue method
Combined alcian blue- PAS techhnique
Mucicarmine technique
Colloidal iron technique
Metachromatic staining
Iodine staining for glycogen
Enzymatic digestion technique Diastase digestion, Sialidase digestion, Hyaluronidase digestion

6. Possible uses & comments
CARBOHYDRATE
STAINING
Components stained
Possible uses & comments
1. Periodic acid Schiff (PAS)
Glycogen
Starch
Mucin (neutral mucin; GI tract, prostate and acid-simple non-sulfated and acid- complex sulfated mucins)
Basement Membrane
Alpha Anti Trypsin
Reticulin
Fungi(capsules)
Pancreatic Zymogen Granules
Thyroid Colloid
Corpora Amylacea
Russell Bodies
**stains MAGENTA/ RED
Nuclei: BLUE
Glycogen storage disorder.
Basement membrane in glomerular disease
Staining macrophages in Whipple's disease
Mucins in adenocarcinoma of large intestine
Demonstration of fungi
Classification of tumours with glycogen(e.g.Seminoma,
rhabdomyosarcoma,ewing’s/ PNET, renal cell carcinoma).
Spironolactone bodies.
tissue can be pre-digested with diastase to remove glycogen (PAS-diastase).
Some mucins are PASD (PAS with predigestion with diastase) positive (i.e. stain is present after diastase predigestion; also called diastase resistant); glycogen is PASD negative (also called diastase sensitive because diastase removes PAS staining)

7. Candida in lung, PAS stain.
Glycogen in Ewing's sarcoma, PAS stain
This is nodular glomerulosclerosis involving a glomerulus in the kidney, as seen with the PAS stain.

8. Note PAS +ve cytoplasmic granules partly resistant to diastase

9. Possible uses & comments
CARBOHYDRATE
STAINING
Components stained
Possible uses & comments
2. Alcian blue
Acid mucins – BLUE
Acid-simple non-sulfated:
Contain sialic acid
Found in epithelium (gallbladder [benign, adenocarcinoma], intestinal metaplasia in stomach)
Positive for PAS, Alcian blue at pH 2.5, colloidal iron, and metachromatic dyes
They resist hyaluronidase digestion
Acid-simple mesenchymal:
Contain hyaluronic acid and digest with hyaluronic acid
Found in tissue stroma and sarcomas
Positive for Alcian blue at pH 2.5, colloidal iron, and metachromatic dyes
Negative for PAS
Acid-complex sulfated:
Found in adenocarcinomas
Usually positive for PAS, Alcian blue at pH 1, colloidal iron, mucicarmine, and metachromatic stains
Acid-complex connective tissue:
Found in tissue stroma, cartilage, and bone
Includes chondroitin sulfate, keratan sulfate
Positive for Alcian blue at pH 0.5
Nuclei- RED
Cytoplasm- PINK
The pH of this stain can be adjusted to give more specificity.
3. Alcian blue/ PAS
This is a combined method utilising the properties of both the PAS and Alcian blue methods to demonstrate the full complement of tissue proteoglycans.
The rationale of the technique is that by:
First staining all the acidic mucins with Alcian blue, those remaining acidic mucins which are also PAS positive will be chemically blocked and will not react further during the technique. 
Those neutral mucins which are solely PAS positive will subsequently be demonstrated in a contrasting manner.  Where mixtures occur, the resultant colour will depend upon the dominant moiety.
Results
acidic mucins blue
neutral mucins magenta
mixtures of above blue/purple
nuclei deep blue
demonstrates both acidic- neutral- and mixtures of acidic and neutral mucins.

10. Alcian blue/ PAS stain
Acid mucins - blue. Neutral mucins - magenta. Mixtures - purple. Nuclei - grey/blue.
The goblet cells of the gastrointestinal tract are filled with abundant acid mucin and stain pale blue with this Alcian blue stain.

11. Possible uses & comments
CARBOHYDRATE
STAINING
Components stained
Possible uses & comments
4. Mucicarmine
Mucin/ capsule of cryptococcus – deep rose to red
Nuclei – black
Other tissue elements – light yellow
Mucicarmine is the most specific stain for mucin (very specific for epithelial mucin), but it is very insensitive.
Useful for identification of adenocarcinoma
( especially of GIT).
Capsule of fungus – cryptococcus neoformans is also detected
CRYPTOCOCCUS STAINED BY MUCICARMINE
The cytoplasm of the cells lining this neoplastic gland in a colonic adenocarcinoma are pink with the mucicarmine stain, indicative of mucin production typical for an adenocarcinoma at this site

12. 2. STAIN FOR AMYLOID I. Congo red -Also called amyloid stain -Must examine stained tissue with standard and polarized light -Amyloid under polarized light has apple green birefringence, based on the molecule being in an antiparallel beta-pleated sheet. - amyloid-red; nuclei- blue

13. 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

14. Amyloid displays apple-green birefringence under polarized light.
AMYLOID BY CONGO RED STAIN UNDER
LIGHT MICROSCOPE

15. 3. Stains for lipid
Oil Red O
Sudan black B

16. LIPIDS SIMPLE LIPIDS COMPOUND LIPIDS DERIVED LIPIDS FATS OILS WAXES
Derived from simple or compound lipids by hydrolysis
cholesterol
Bile acids
- c/o fatty acids, alcohol and one more group such as phosphorus or nitrogen

17. Simple lipid is best demonstrated with fresh frozen sections
Best fixative – Formal calcium (2% calcium acetate + 10% formalin)

18. I. Oil Red O
Stain identifies neutral lipids and fatty acids
Fresh smears / cryostat sections of tissue are necessary because alcohols used in tissue processing remove lipids
Rapid and simple routine stain
Uses:
Differentiate fibroma from thecoma (not that important a distinction)
Diagnose renal cell carcinoma, sebaceous gland tumors of skin, lipid-rich carcinomas
Identify fat emboli in lung tissue or clot sections of peripheral blood

19. Oil red O stain of fat emboli in lung.