Perls’ / Prussian Blue Staining

Introduction

Prussian blue (Perls’) reaction is a method for staining non-haem iron in normoblasts (siderocytes), macrophages (haemosiderin), and other cells containing particulate iron. The granules are formed of a water-insoluble complex of ferric iron, lipid, protein and carbohydrate. The method allows assessment of both the amount of iron in reticulo-endothelial stores and availability of iron to developing erythroblasts for example in iron deficiency anemia.

Principle of Perl’s stain

The granules (containing ferric iron) react with pottassium ferrocyanide [K₄Fe(CN)₆] to form a blue compound (ferriferrocynanide), Prussian blue reaction.

Materials

• 2% Potassium Ferrocyanide ((K₄Fe(CN)₆.3H2O)
• 2% HCl
• 1% aqueous safranin (counterstain)
• Air dried peripheral blood or bone marrow smear
• Distilled water
• Methanol
• Coplin jars

Method

1. Choose a suitable sample as a positive control and the stain together with a test sample. Label the slides as control and patient name / registration number (R/N) accordingly.
2. Fix the slides in absolute methanol for 10-20 minutes. Leave it to dry.
3. Prepare the working solution by adding 30 mL potassium ferrocyanide and 30 mL HCl in a Coplin jar (v/v potassium ferrocyanide:HCl = 1:1).
4. Submerge the fixed and dried slides into the Coplin jar containing the working solution.
5. Leave it at room temperature or incubate in a water bath at 50°C for 20 minutes.
6. Rinse the slide with slow running running tap water for 3-5 minutes.
7. Rinse thoroughly in distilled water, and then counterstain with safranin similar to steps 4 – 6.
8. Wipe the back of the slide and edges with Kim wipes. Be careful not to touch the smear. 
9. Dry the slide using a hair dryer on the lowest speed (not more than 10 seconds at a time) or air dry in a tilted position.
10. Mount the slide with Depex and cover the zone of morphology with a cover slip. 
11. This slide is now ready for viewing.

Interpretation