histology Histology, also known as microscopic anatomy or microanatomy, is the branch of biology that studies the microscopic anatomy of biological tissue (biology), tissues. Histology is the microscopic counterpart to gross anatomy, which looks at large ...

histopathology Histopathology (compound of three Greek words: 'tissue', 'suffering', and '' -logia'' 'study of') is the microscopic examination of tissue in order to study the manifestations of disease. Specifically, in clinical medicine, histopatholog ...

, and

clinical pathology Clinical pathology is a medical specialty that is concerned with the diagnosis of disease based on the laboratory analysis of bodily fluids, such as blood, urine, and tissue homogenates or extracts using the tools of chemistry, microbiology, ...

, Perls Prussian blue is a commonly used method to detect the presence of

iron Iron is a chemical element; it has symbol Fe () and atomic number 26. It is a metal that belongs to the first transition series and group 8 of the periodic table. It is, by mass, the most common element on Earth, forming much of Earth's o ...

in tissue or cell samples. Perls Prussian Blue derives its name from the German pathologist Max Perls (1843–1881), who described the technique in 1867. The method does not involve the application of a dye but rather causes the pigment

Prussian blue Prussian blue (also known as Berlin blue, Brandenburg blue, Parisian and Paris blue) is a dark blue pigment produced by oxidation of ferrous ferrocyanide salts. It has the chemical formula . It consists of cations, where iron is in the oxidat ...

to form directly within the tissue. The method stains mostly iron in the

ferric In chemistry, iron(III) or ''ferric'' refers to the chemical element, element iron in its +3 oxidation number, oxidation state. ''Ferric chloride'' is an alternative name for iron(III) chloride (). The adjective ''ferrous'' is used instead for i ...

state which includes

ferritin Ferritin is a universal intracellular and extracellular protein that stores iron and releases it in a controlled fashion. The protein is produced by almost all living organisms, including archaea, bacteria, algae, higher plants, and animals. ...

and

hemosiderin Hemosiderin image of a kidney viewed under a microscope. The brown areas represent hemosiderin Hemosiderin or haemosiderin is an iron-storage complex that is composed of partially digested ferritin and lysosomes. The breakdown of heme gives ri ...

, rather than iron in the

ferrous In chemistry, iron(II) refers to the chemical element, element iron in its +2 oxidation number, oxidation state. The adjective ''ferrous'' or the prefix ''ferro-'' is often used to specify such compounds, as in ''ferrous chloride'' for iron(II ...

state.

Uses

Hemochromatosis liver iron prussian blue

Perls's method is used to indicate "non-

heme Heme (American English), or haem (Commonwealth English, both pronounced /Help:IPA/English, hi:m/ ), is a ring-shaped iron-containing molecule that commonly serves as a Ligand (biochemistry), ligand of various proteins, more notably as a Prostheti ...

" iron in tissues such as ferritin and hemosiderin, the procedure does not stain iron that is bound to

porphyrin Porphyrins ( ) are heterocyclic, macrocyclic, organic compounds, composed of four modified pyrrole subunits interconnected at their α carbon atoms via methine bridges (). In vertebrates, an essential member of the porphyrin group is heme, w ...

forming heme such as

hemoglobin Hemoglobin (haemoglobin, Hb or Hgb) is a protein containing iron that facilitates the transportation of oxygen in red blood cells. Almost all vertebrates contain hemoglobin, with the sole exception of the fish family Channichthyidae. Hemoglobin ...

and

myoglobin Myoglobin (symbol Mb or MB) is an iron- and oxygen-binding protein found in the cardiac and skeletal muscle, skeletal Muscle, muscle tissue of vertebrates in general and in almost all mammals. Myoglobin is distantly related to hemoglobin. Compar ...

. The stain is an important

histochemical Histology, also known as microscopic anatomy or microanatomy, is the branch of biology that studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures visi ...

stain used to demonstrate the distribution and amount of iron deposits in liver tissue, often in the form of a biopsy. Perls's procedure may be used to identify excess iron deposits such as

deposits (

hemosiderosis Hemosiderosis is a form of iron overload disorder resulting in the accumulation of hemosiderin. Types include: * Transfusion hemosiderosis * Idiopathic pulmonary hemosiderosis * Transfusional diabetes Organs affected: * Hemosiderin deposi ...

) and in conditions such as

hereditary hemochromatosis Hereditary haemochromatosis type 1 (HFE-related haemochromatosis) is a genetic disorder characterized by excessive intestinal absorption of Human iron metabolism, dietary iron, resulting in a pathological increase in total body iron stores. Huma ...

. Perls Prussian blue is commonly used on

bone marrow Bone marrow is a semi-solid biological tissue, tissue found within the Spongy bone, spongy (also known as cancellous) portions of bones. In birds and mammals, bone marrow is the primary site of new blood cell production (or haematopoiesis). It i ...

aspirates to indicate levels of iron storage and may provide reliable evidence of iron deficiency.

Method of application

Perls did not publish a detailed procedure other than indicating a dilute

potassium ferrocyanide Potassium hexacyanidoferrate(II) is the inorganic compound with formula K4 e(CN)6�3H2O. It is the potassium salt of the coordination complex e(CN)6sup>4−. This salt forms lemon-yellow monoclinic crystals. Synthesis In 1752, the French chemi ...

solution was applied to the tissue followed by

hydrochloric acid Hydrochloric acid, also known as muriatic acid or spirits of salt, is an aqueous solution of hydrogen chloride (HCl). It is a colorless solution with a distinctive pungency, pungent smell. It is classified as a acid strength, strong acid. It is ...

Ferric In chemistry, iron(III) or ''ferric'' refers to the chemical element, element iron in its +3 oxidation number, oxidation state. ''Ferric chloride'' is an alternative name for iron(III) chloride (). The adjective ''ferrous'' is used instead for i ...

iron deposits in tissue (present mostly as ferric iron within the storage protein

) then react with the soluble ferrocyanide in the stain to form the insoluble

pigment (a complex hydrated ferric ferrocyanide substance). These deposits are then visualizable microscopically as blue or purple deposits. Many methods of performing Perls Prussian blue stain for iron have been published, Drury and Wallington (1980) give a protocol that uses a mixture of 1 part 2% hydrochloric acid and 1 part 2% potassium ferrocyanide that is applied to the section for 20–30 minutes followed by a rinse in distilled water and application of a

counterstain A counterstain is a stain with colour contrasting to the principal stain, making the stained structure easily visible using a microscope. Examples include the malachite green counterstain to the fuchsine stain in the Gimenez staining techniqu ...

such as

eosin Eosin is the name of several fluorescent acidic compounds which bind to and from salts with basic, or eosinophilic, compounds like proteins containing basic amino acid residues such as histidine, arginine and lysine, and stains them dark red ...

safranin Safranin (Safranin O or basic red 2) is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring cell nuclei red. This is the classic counterstain in both Gram stains and endo ...

neutral red Neutral red (toluylene red, Basic Red 5, or C.I. 50040) is a eurhodin dye used for staining in histology. It stains lysosomes red. It is used as a general stain in histology, as a counterstain in combination with other dyes, and for many stain ...

Mode of action

Potassium ferrocyanide in the staining solution combines with the ferric iron forming the Prussian blue pigment. The addition of hydrochloric acid increases the availability of iron within the tissue for reaction with the potassium ferrocyanide. The chemical reaction for the conversion of iron to Prussian blue is provided as follows in Drury and Wallington (1980):
4FeCl₃ + 3K₄Fe(CN)₆ → Fe₄ e(CN)₆sub>3 + 12KCl (ferric iron) + (potassium ferrocyanide) → (ferric ferrocyanide or Prussian blue)

References

{{Stains Anatomical pathology Perls Prussian blue Hematopathology Staining Blue symbols