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 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.


  • 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


  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.


Revealing Iron Stores in Bone Marrow: Positive Perls' Stain Highlights Blue-Colored Iron Granules (x400 Magnification)
The presence of blue-colored iron granules in a bone marrow smear, revealed by Perls’ staining, provides valuable insights into the body’s iron status. These granules, composed of ferric iron bound to proteins, represent the body’s storage form of iron and serve as a reservoir for essential cellular processes.

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