Perls' Prussian Blue
for Hemosiderin

Hemosiderin forms the body's iron stores. The iron is in the ferric state, and may be demonstrated by releasing it from hemosiderin with hydrochloric acid, forming ferric chloride. The iron reacts with potassium ferrocyanide to form ferric ferrocyanide. This is an insoluble, blue compound known as Prussian blue or Berlin blue. The intensity of the colour gives some indication as to amount, but it is qualitative only. Other sources of ferric iron will also be demonstrated.

Solutions

Neutral red

Stock solution A
Potassium ferrocyanide	2	g
Distilled water	100	mL

Stock solution B
Hydrochloric acid, conc.	2	mL
Distilled water	100	mL

Working solution
Stock solution A	1	volume
Stock solution B	1	volume

Tissue sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Avoid iron containing materials and jars while fixing as these may contaminate the tissue. Acid containing fixatives may remove some of the iron deposits, but apart from that most are satisfactory.

Method

1. Bring sections to distilled water with xylene and ethanol.
2. Place into the working solution for 15 minutes.
3. Rinse with distilled water, then tap water.
4. Stain with neutral red for one minute.
5. Rinse well with tap water.
6. Dehydrate with ethanol.
7. Clear with xylene.

Expected results

• Ferric iron  –  blue
• Nuclei  –  red

Notes

1. The working solution should be made immediately before use.
2. Avoid washing with tap water before placing into the working solution, as rust in the water or tap fixtures could cause false positive staining.
3. Wash well at step 3, as traces of iron will form a granular red deposit with neutral red.
4. Iron ores can be demonstrated, but the acid concentration in solution A may need to be increased to 10% or more.

 

Reference
Culling, C.F.A., Allison, R.T. and Barr, W.T.
Cellular Pathology Technique, Ed.4.
Butterworth, London, UK.

Susan Budavari, Editor, (1996)
The Merck Index, Ed. 12
Merck & Co., Inc., Whitehouse Station, NJ, USA