Perl’s Prussian Blue for Ferric Iron
Purpose
The detection of ferric (Fe3+) iron in tissues. Ferric iron is normally found in small amounts in the bone marrow and spleen. Abnormally large deposits may be seen in hemochromatosis and hemosiderosis.
Principle
Major deposits of iron are in the red cells as hemoglobin and in phagocytic cells as ferritin and hemosiderin. In these compounds the metal is present in the “ferric” (oxidation state 3+) form but being complexed to protein it is not available as ions to participate in simple chemical reactions. The iron of ferritin and hemosiderin is readily released as Fe3+ ions by treatment with a dilute mineral acid. In the presence of ferrocyanide ions (potassium ferrocyanide), Prussian blue is immediately precipitated.
Solutions
2% aqueous Potassium ferrocyanide (use acid washed glassware)
- Potassium ferrocyanide 2 g
- Distilled water 100 mL
Expiry: 1 month
2% aqueous Hydrochloric acid (use acid washed glassware)
- Concentrated HCl 2 mL
- Distilled water 98 mL
Expiry: 1 year
Working Solution of Potassium Ferrocyanide
- 2% aqueous Potassium ferrocyanide 25 mL
- 2% aqueous Hydrochloric acid 25 mL
Prepare working solution fresh in acid cleaned graduated cylinder and filter into acid cleaned coplin jar before use
Nuclear Fast Red (Kernechtrot)
- Aluminum sulphate Octadecahydrate 25 g
- Distilled water 500 ml
- Nuclear fast red (CI 60760) 0.5 g
- Dissolve aluminum sulfate in DH2O.
- Add nuclear fast red and dissolve with aid of heat.
- Cool, Filter and add a crystal/grain of thymol.
Stable for 2 months.
Caution: Irritant avoid contact and inhalation
METHOD
- Take sections to water
- Place in freshly prepared Working solution for 30 minutes
- Wash well in water (see notes)
- Counterstain in 0.1% nuclear fast red for 8 minutes
- Wash in tap water for at least 1 minute
- Dehydrate, clear and mount
Results
Hemosiderin and ferric salts – blue
Nuclei – red
Other pigments – retain their natural color
Notes
- Distilled water must be iron free.
- Iron containing forceps must not be used. Use plastic forceps (non metallic) or paraffin-dipped metal forceps
- All traces of acid ferrocyanide must be washed out of section before counter-staining with nuclear fast red or a dark red fine precipitate may form.
- Acid fixative and/or acid decalcification must not be used for iron to be demonstrated, and the acidic conditions will cause the ferric iron to be leached from the tissue.
Control
Spleen from hemosiderosis
Liver and spleen from hematochromatosis
References
IWK. (2007). IWK Laboratory Histology Staining Manual, Special Stains.
Carson, F. L., & Cappellano, C. H. (2015). Histotechnology: A Self-Instructional Text (4th ed.). American Society of Clinical Pathologists Press.
