Principle
The test is based on the principle of a positive Prussian blue reaction by iron particles. Non-haem iron reacts with potassium ferrocyanide to form the blue colored compound ferriferrocyanide.
Equipment and reagents
1) 2% hydrochloric acid
2) 2% potassium ferrocyanide
3) Slides and spreader
Procedure
1) Take an air-dried slide of a bone marrow smear.
2) Fix it in methanol for 20 minutes.
3) Prepare solution by mixing 2% hydrochloric acid & 2% potassium ferrocyanide, 2 ml each.
4) Pour this solution onto methanol-fixed slide.
5) Keep it for 20 minutes.
6) Wash well in running water for 20 minutes.
7) Counterstain with eosin & keep for 10–15 seconds.
8) Then view the slide under microscope.
Results
Grade 0: No iron granules are seen.
Grade 1: Small granules in reticulum cells are seen only under oil-immersion.
Grade 2: Few small granules visible with low-power lens.
Grade 3: Numerous small granules in all marrow particles.
Grade 4: Large granules in small clumps.
Grade 5: Dense large clumps of granules.
Grade 6: Very large deposits obscuring the marrow cells.
Interpretation
Grade 0: Iron deficiency (a minimum of 7 particles must be available before concluding that hemosiderin is absent).
Grade 1 and 2: Normal iron stores.
Grade 3 to 6: Increased iron stores.
Image source: ASH Image Bank