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

histopathology Histopathology (compound of three Greek words: ''histos'' "tissue", πάθος ''pathos'' "suffering", and -λογία '' -logia'' "study of") refers to the microscopic examination of tissue in order to study the manifestations of disease. Spe ...

, 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 with symbol Fe (from la, ferrum) 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, right in f ...

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 or, in painting, Parisian or Paris blue) is a dark blue pigment produced by oxidation of ferrous ferrocyanide salts. It has the chemical formula Fe CN)">Cyanide.html" ;"title="e(Cyani ...

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

ferric In chemistry, iron(III) refers to the element iron in its +3 oxidation state. In ionic compounds (salts), such an atom may occur as a separate cation (positive ion) denoted by Fe3+. The adjective ferric or the prefix ferri- is often used to spe ...

state which includes

ferritin Ferritin is a universal intracellular 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. It is the primary ' ...

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 rise ...

, rather than iron in the

ferrous In chemistry, the adjective Ferrous indicates a compound that contains iron(II), meaning iron in its +2 oxidation state, possibly as the divalent cation Fe2+. It is opposed to "ferric" or iron(III), meaning iron in its +3 oxidation state, such a ...

state.

Uses

Hemochromatosis liver iron prussian blue

Perls's method is used to indicate "non-

heme Heme, or haem (pronounced / hi:m/ ), is a precursor to hemoglobin, which is necessary to bind oxygen in the bloodstream. Heme is biosynthesized in both the bone marrow and the liver. In biochemical terms, heme is a coordination complex "consisti ...

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

porphyrin Porphyrins ( ) are a group of heterocyclic macrocycle organic compounds, composed of four modified pyrrole subunits interconnected at their α carbon atoms via methine bridges (=CH−). The parent of porphyrin is porphine, a rare chemical com ...

forming heme such as

hemoglobin Hemoglobin (haemoglobin BrE) (from the Greek word αἷμα, ''haîma'' 'blood' + Latin ''globus'' 'ball, sphere' + ''-in'') (), abbreviated Hb or Hgb, is the iron-containing oxygen-transport metalloprotein present in red blood cells (erythrocyte ...

and

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

. The stain is an important

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

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 depositi ...

) and in conditions such as

hereditary hemochromatosis Hereditary haemochromatosis type 1 (HFE-related Hemochromatosis) is a genetic disorder characterized by excessive intestinal absorption of dietary iron, resulting in a pathological increase in total body iron stores. Humans, like most animals, h ...

. Perls Prussian blue is commonly used on

bone marrow Bone marrow is a semi-solid tissue found within the 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 is composed of hematopoietic ce ...

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 ferrocyanide 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 chemist Pierr ...

solution was applied to the tissue followed by

hydrochloric acid Hydrochloric acid, also known as muriatic acid, is an aqueous solution of hydrogen chloride. It is a colorless solution with a distinctive pungent smell. It is classified as a strong acid Acid strength is the tendency of an acid, symbol ...

Ferric In chemistry, iron(III) refers to the element iron in its +3 oxidation state. In ionic compounds (salts), such an atom may occur as a separate cation (positive ion) denoted by Fe3+. The adjective ferric or the prefix ferri- is often used to spe ...

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 technique a ...

such as

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

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 endospo ...

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 staini ...

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