Prepared by
ROY ELLIS
IMVS Division of Pathology
The Queen Elizabeth Hospital
Woodville Road, Woodville, South Australia 5011
Principle
Dilute mineral acid hydrolysis releases ferric ions from protein bound tissue deposits, which, in the presence of ferrocyanide ions, is precipitated as the highly coloured and highly water-insoluble complex, potassium ferric ferrocyanide, Prussian blue.
FeCl3 + K4Fe(CN)6 = KFeFe(CN)6¯ + 3KCl
Ferrous ions do not produce a coloured reaction product, thus are excluded from the visualisation.
Tissue deposits containing ferric ions are invariably haemosiderin.
The original method of Perls applied the ferrocyanide and acid as separate reagents. The “mixed method” (as written here) is well suited for a routine laboratory, but it must be kept in mind that heavy deposits of haemosiderin in tissue sections may lead to leaching of the coloured end product, with subsequent artefactual background staining of collagen.
Asbestos is the name given to a special form of silica which exists in the form of long, thin, crystalline fibres. The fibres become coated with protein which contains haemosiderin and therefore appears brown on unstained and H&E sections and blue by the perl's prussian blue reaction. The asbestos fibres with their protein are known as "asbestos bodies" and the characteristic birefringence is lost.
Technical Points
1. A known positive control section must be used to ensure correct differentiation has been achieved.
2. Neutral buffered formalin gives good results. Other fixatives may be used, but acidic fixatives, dichromate fixatives, and acidic decalcification fluids should be avoided. These reagents will cause progressive hydrolytic loss of ferric ions from tissues, and a negative result must be viewed with suspicion.
3. (step 5) - Red background staining results if the neutral red stain is applied directly from tap water.
4. (step 6) - Differentiation of neutral red staining is achieved during dehydration.
Method
1. Bring sections to distilled water.
2. On a rack, flood with equal parts mixture of ferrocyanide and hydrochloric acid for 10 min (asbestos bodies for 30 mins)
3. Wash well in distilled water, several changes 5 min
4. Counterstain with filtered neutral red stain 1 min
5. Rinse in distilled water
6. Rapidly dehydrate in absolute alcohol, clear and mount.
Results
- ferric salts..................................................... deep blue
- nuclei........................................................... red
- erythrocytes................................................... yellow
- asbestos bodies............................................... blue/black
Reagent Formulae
1. aq hydrochloric acid (Analytical Reagent grade)
2. aq potassium ferrocyanide (Analytical Reagent grade)
CARE - toxic hazard
3. neutral red stain
neutral red (CI 50040)........................................ 1.0 g
distilled water................................................. 100.0 ml
glacial acetic acid............................................. 1.0 ml
Dissolve the dye in the distilled water. Add the acid. Mix well. Filter into the reagent bottle. Keeps well. The reagent is supplied from MedVet already made up.
References
Perls M,(1867),Arch.Pathol V39, p42.