Perls’ / Prussian Blue Staining
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.
The granules (containing ferric iron) react with pottassium ferrocyanide [K4Fe(CN)6] to form a blue compound ferriferrocynanide), Prussian blue reaction.
- 2% Potassium Ferrocyanide ((K4Fe(CN)6.3H2O)
- 2 N HCl
- 1% aqueous safranin (counterstain)
- Air dried peripheral blood or bone marrow smear
- 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.
- Fix the slides in absolute methanol for 10-20 minutes. Leave it to dry.
- 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).
- Submerge the fixed and dried slides into the Coplin jar containing the working solution.
- Leave it at room temperature or incubate in a water bath at 50°C for 20 minutes.
- Wash the slides in running tap water for 3-5 minutes.
- Rinse thoroughly in distilled water, and then counterstain with safranin similar to steps 4 – 6.
- Dry and cover the slides with cover slips with depex.