Iron Staining

Normally ferritin is present in all cells in the body and haemosiderin is mainly found in macrophages in the bone marrow, liver and spleen.

In iron deficiency anaemia the iron stores are completely depleted while in sideroblastic anaemia prominent increase in the number of iron granules is observed in the bone marrow smear. By means of a cytochemical stain, the amount of stainable iron in the bone marrow can be visualized and quantitatively assessed . This is of particular importance in the diagnosis of iron deficiency anaemia and sideroblastic anaemia .

• Principle

The iron containing compounds, haemosiderin or ferritin  ( ironic iron ) reacts with potassium ferrocyanide in acid medium to produce a Prussian blue colour compound . This reaction is called as Prussian-blue (Perls’) reaction .

• Specimen



Air dried bone marrow smear

Air dried bone marrow smear

• Requirements

0.2 mol / l  HCl

20 g/l  Potassium ferrocyanide

Absolute methanol

1 g/ l Eosin solution

Distilled water

Staining jar


Positive & Negative control slides

• Procedure

Air dried bone marrow smear is fixed  in absolute methanol for 10 – 20 minutes . Equal volumes of 0.2 mol / l  HCl and 20 g/l  potassium ferrocyanide are mixed in Staining jar . Because this solution is unstable , should prepare immediately before use .

Iron staining - Procedure

When the slide is dry , it places in the freshly prepared solution and leaves for 10 minutes  at about 20 0 C . After that , the slide washes in running tap water for 20 minutes and it rinses thoroughly in distilled water .

Iron staining - Procedure

Finally the slide counter stains with eosin for 10 – 15 seconds . The air dried slide screens under high power objective and examine under oil- immersion lens .

Iron staining - Procedure
Iron staining - Procedure

• Results

Iron granules appear as bright blue or blue – green aggregates contrasting sharply with the pink -stained background .

• Limitation

  • Iron that may have present in slide and glass wares , can be contaminated with test and it may give false positive results . So glass wares prepare by soaking in 3 mol/l HCl overnight before washing to avoid iron contamination.

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