Perls’ Prussian Blue

Perls’ Prussian Blue – IRON

Perls’ Prussian blue stains are used to detect and identify ferric (Fe3+) iron in tissue preparations, blood smears, or bone marrow smears. Minute amounts of ferric acid are commonly found in bone marrow and in the spleen. Abnormal amounts of iron can indicate haemochromatosis and hemosiderosis. Lack of iron identification can indicate deficient anemia.

Haemochromatosis is caused by excessive iron uptake. In the liver, the deposits may lead to cirrhosis following hepatocyte death.

What is Prussian Blue?

Prussian blue (PB) was actually the first synthetic color to be discovered during the Industrial Revolution. It was developed accidentally in 1704 by a chemist who was trying to produce another color. It wasn’t used as a histochemical stain until 1867 when German pathologist, Max Perls – hence described its original formula why is often known as ‘Perls’ Prussian blue’.

What does it Stain?

PB is used to detect the presence of IRON in tissues. It is an extremely sensitive test, and can even detect single granules of iron in cells.

Although iron is essential for life, it is also toxic due to its ability to form free radicals that can damage cells. So the body must protect itself from this element, and it does so by using iron-storage proteins. Haemosiderin is one type of iron-storage complex that is found inside cells. It is mostly found within phagocytic macrophages, and is especially prominent in these cells following haemorrhage, when haemoglobin breaks down.

Microscopically, the presence of iron is therefore identified by demonstrating iron-containing haemosiderin, which shows up as a granular brown pigment in cells when examined with haematoxylin and eosin (H&E) stain. Although visible with H&E, other pigments can stain a similar color, so haemosiderin needs to be differentiated from other brown pigments using the PB histochemical staining technique.

General Principles of the Stain

Various staining protocols have emerged since Perls’ original description, but all serve to achieve the same result – demonstration of iron in tissues.

The procedure involves 3 basic steps:

1.            Tissue sections are treated with hydrochloric acid to denature the binding proteins of the haemosiderin molecule, and thereby release ferric (3+) ions.

2.            Potassium ferrocyanide is then introduced. The ferric ions combine with this solution, resulting in the formation of ferric ferrocyanide, an insoluble bright blue pigment – otherwise known as Prussian blue. Although the hydrochloric acid and potassium ferrocyanide can be introduced as separate solutions, most formulations now typically use them in combination.

3.            If required, the PB reaction can then be followed by a red counterstain, such as neutral red.

Consequently, the end product of the reaction is such that iron in tissue sections is seen as blue or purple deposits, while other tissue components, such as nuclei and cytoplasm, are highlighted red by the counterstain (1% neutral red).

Who Uses Prussian Blue Stain?

This stain is used widely for both diagnostic and research purposes. In diagnostic labs, PB is widely used by pathologists to detect the presence of iron in biopsy specimens, especially in tissues such as bone marrow and spleen.

This procedure is particularly helpful when they need to evaluate patients with pathological conditions that involve haemosiderin deposits. In addition to haemorrhage, this can occur in patients with haemolytic anaemia, as well in conditions such as haemochromatosis (where excessive amounts of iron may form in organs due to iron overload), some liver diseases, and in the lungs of patients with congestive heart failure.

RESULTS:

Ferric iron (haemosiderin) – Blue

Nuclei – Red

Background (cytoplasm) – Pink