2. SPECIAL STAINS
• "Special stain" - alternative staining techniques.
• Used when the traditional H&E does not provide all
the information the pathologist needs from a slide.
• "Special stains" are processes that employ a dye or
chemical that has an affinity for particular tissue
component that is to be demonstrated.
• They allow the presence/ absence of certain cell
types, structures and microorganisms to be viewed
microscopically.
3. RETICULIN STAIN
• It demonstrates both reticular fibres and basement
membrane material.
• Reticular fibres consists of very thin fiber of type III
collagen - widespread in connective tissue throughout
the body.
• It stains the reticulinic fibers of the stroma and makes it
possible to specify the architecture of the tumors.
• Reticulin fibers cannot be visualized in a hematoxylin &
eosin (H&E) slide.
4. • It is based on the silver impregnation of reticulin
fibers that can not be detected with H&E.
• It uses the argyrophilic properties of the reticulin
fibers.
• Argyrophilic cells can adsorb silver but cannot
reduce it.
• Argyrophilic cells need a reducer/developer to
convert the invisible silver salts to a visible
metallic silver.
5. • This silver staining process is known as silver
impregnation.
• With this staining, the reticulin fibers are stained
black.
• Examples of reticulin stain using different
methodologies:
• Gordon & Sweets, Wilder, Gridley, Snook,
Laidlaw, and Gomori.
7. USES
• It is a key staining in the study of lesions affecting
the organs whose frame is rich in reticulin such as
the liver, bone marrow.
• Characteristic reticulin patterns can help diagnose
cirrhosis of the liver, early fibrosis in bone marrow,
hemangiosarcomas ,fibroblastic tumors, and
rhabdomyosarcomas.
• They can also help diagnose epithelial vs non-
epithelial tumors.
8. • In carcinoma reticulin are seen around tumor nests
but not in between tumor cells.
• In most sarcoma and large cell lymphoma reticulin
seperates individual tumor cells.
• To differentiate in situ and invasive carcinoma.
• Fibrosarcoma or Fibrothecoma (stains each cell).
• Endothelial cell tumors (stains outside of all cells).
USES
9. USES
• Fibrosis or the excess formation of fibrous tissue
is commonly demonstrated in bone marrow
biopsies of myeloproliferative disorders such as
polycythemia vera, primary or idiopathic
myelofibrosis, essential thrombocytosis, or
chronic myeloid leukemia.
• Additionally, fibrosis can be noted on bone
marrow specimens that have significant tumor
metastasis.
11. • The fibers are first oxidized, by potassium
permanganate.
• Followed by bleaching with oxalic acid, potassium
metabisulphite or periodic acid.
• The sensitization is a pre-impregnation step.
• Done by the formation of organometallic complexes
between the reticulin fibers and metal salts.
• The preferred sensitizers are ferric salts.
12. • For the impregnation stage, an ammoniacal silver
nitrate solution is most often used.
• The silver will deposit on the reticulin fibers in the
form of a brown oxide which will become metallic
silver during the reduction.
• Ionic silver is reduced to metallic silver in the
presence of a developing agent, formalin.
• We obtain a black deposit visible to the naked eye.
13. • Turning is a treatment of gold chloride.
• In which gold is substituted for silver for better
resolution and more stable metal deposit.
• A further reduction is required, sodium thiosulfate
which removes non-specific silver or ionic gold
residues.
• Counterstaining: most often, neutral red is used.
14.
15. • 1. Bring slides to water after dewaxination and
dehydration.
• 2. Oxidize in potassium permanganate - 10 min
• 3. Rinse well in running water - 3 mins
• 4. Oxalic acid - 1 mins ( removes excess KMnO4 )
• 5. Rinse well in running water - 2 mins
• 6. Sensitize in ferric ammonium sulfate - 15 mins
( provides basic ph )
• 7. Rinse in distilled water - 2 mins
• 8. Impregnate in ammoniacal silver - 2 mins
• 9. Rinse in distilled water - 1 min
STEPS
16. STEPS
• 10. Add 20% unbuffered formalin - 1 min
• 11. Rinse well in running water - 2 mins
• 12. Tone with 0.1% gold chloride - 3-5 mins
• 13. Rinse in water - 1 min
• 14. 5% sodium thiosulfate - 1 min
• 15. Rinse in water - 1 min
• 16. Counterstain in nuclear fast red/ light green - 3-5
mins
• 17. Rinse briefly in water - 15 sec
• 18. Dehydrate, clear and mount with DPX.
19. TROUBLESHOOTING THE GOMORI’S
RETICULIN
• Silver solution should be made fresh with distilled
water and clean glassware.
• Using old, ammonia depleted ammonium hydroxide
can cause the solution to fail.
• Do not use excessive ammonium hydroxide to clear
the silver/sodium hydroxide solution.
•
• Excess ammonium hydroxide will cause weak
staining.
20. CAUTION
• Ammoniacal silver salts in the dry state are
potentially dangerous due to their explosive
properties and they should be stored in solution
in black plastic rather than glass bottles.
