Perls’ / Prussian Blue Staining

Red Blood Cell Resources
Associated Diseases

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.

Principle

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 N HCl
  • 1% aqueous safranin (counterstain)
  • Air dried peripheral blood or bone marrow smear

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. Wash the slides in running tap water for 3-5 minutes.
  7. Rinse thoroughly in distilled water, and then counterstain with safranin similar to steps 4 – 6.
  8. Dry and cover the slides with cover slips with depex.

Positive Perls’ stain with normal iron stores (blue colored) x400 magnification