Prussian Blue- A Histology Stain For Iron

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 its original formula was described by German pathologist, Max Perls – hence why is often known as ‘Perls’ Prussian blue’.

The PB stain is not a true special staining technique but rather, a histochemical reaction.

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 or safranin O.

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.

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.

Similarly, researchers investigating any of these types of diseases may also routinely examine PB-stained tissue sections to detect the presence of iron in tissues of interest. And others may use the stain simply because they come across a brown granular pigment in tissue sections that needs to be identified.

How do you use Prussian blue in your lab?

Want to know more about histology? Visit the Bitesize Bio Histology Hub for tips and trick for all your histology experiments.