Perls' Prussian blue stain (iron)

Principle

• Acid releases ferric iron (Fe3+) from haemosiderin, which reacts with potassium ferrocyanide to form insoluble blue ferric ferrocyanide (Prussian blue).
• Ferritin iron in solution and iron within haemoglobin are not stained.

Specimen

• Bone marrow aspirate films containing particles, trephine sections (after decalcification caution), blood films, urine sediment (haemosiderinuria).

Reagents and equipment

• Fixative (methanol), freshly mixed equal volumes of about 2% potassium ferrocyanide and about 2% hydrochloric acid, neutral red or eosin counterstain, iron-free glassware.

Procedure

• Fix air-dried films in methanol (about 10–20 min).
• Cover with the fresh ferrocyanide–HCl mixture for the time stated in the local method (commonly 10–30 min).
• Rinse well in water, counterstain briefly, rinse, dry.
• Include a known positive control slide.

Results

• Iron appears as blue-green granules; nuclei red (neutral red) and cytoplasm pale.
• Storage iron in macrophages within particles is graded (e.g. 0 to 4+ or 0 to 6).
• Sideroblasts: erythroblasts containing blue granules. Ring sideroblasts: at least 5 granules covering at least one-third of the nuclear circumference (WHO definition).

Interpretation

• Absent storage iron: iron deficiency.
• Increased storage iron with few sideroblasts: anaemia of chronic disease.
• Increased iron: haemochromatosis, transfusion overload.
• Ring sideroblasts: sideroblastic anaemias (congenital, alcohol, lead, drugs, copper deficiency) and MDS with ring sideroblasts (at least 15%, or at least 5% with SF3B1 mutation).

Quality control and pitfalls

• Assessment requires marrow particles; aspirates without particles cannot be graded.
• Iron contamination from glassware or reagents gives false positives; stain precipitate can mimic granules.
• Decalcified trephines may lose iron.

Clinical use

• Gold standard for marrow iron stores; identifies sideroblastic anaemia and siderocytes (Pappenheimer bodies) in blood.