Romanowsky staining is a prototypical

staining Staining is a technique used to enhance contrast in samples, generally at the Microscope, microscopic level. Stains and dyes are frequently used in histology (microscopic study of biological tissue (biology), tissues), in cytology (microscopic ...

technique that was the forerunner of several distinct but similar stains widely used in

hematology Hematology (American and British English spelling differences#ae and oe, spelled haematology in British English) is the branch of medicine concerned with the study of the cause, prognosis, treatment, and prevention of diseases related to bloo ...

(the study of blood) and

cytopathology Cytopathology (from Greek , ''kytos'', "a hollow"; , ''pathos'', "fate, harm"; and , ''-logia'') is a branch of pathology that studies and diagnoses diseases on the cellular level. The discipline was founded by George Nicolas Papanicolaou in ...

(the study of diseased cells). Romanowsky-type stains are used to differentiate

cell Cell most often refers to: * Cell (biology), the functional basic unit of life * Cellphone, a phone connected to a cellular network * Clandestine cell, a penetration-resistant form of a secret or outlawed organization * Electrochemical cell, a de ...

s for microscopic examination in

pathological Pathology is the study of disease. The word ''pathology'' also refers to the study of disease in general, incorporating a wide range of biology research fields and medical practices. However, when used in the context of modern medical treatme ...

specimens, especially

blood Blood is a body fluid in the circulatory system of humans and other vertebrates that delivers necessary substances such as nutrients and oxygen to the cells, and transports metabolic waste products away from those same cells. Blood is com ...

and

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 ...

films, and to detect parasites such as

malaria Malaria is a Mosquito-borne disease, mosquito-borne infectious disease that affects vertebrates and ''Anopheles'' mosquitoes. Human malaria causes Signs and symptoms, symptoms that typically include fever, Fatigue (medical), fatigue, vomitin ...

within the blood. The staining technique is named after the Russian physician Dmitri Leonidovich Romanowsky (1861–1921), who was one of the first to recognize its potential for use as a blood stain. Stains that are related to or derived from the Romanowsky-type stains include

Giemsa Giemsa stain (), named after German chemist and bacteriologist Gustav Giemsa, is a nucleic acid stain used in cytogenetics and for the histopathological diagnosis of malaria and other parasites. Uses It is specific for the phosphate groups of ...

, Jenner,

Wright Wright is an occupational surname originating in England and Scotland. The term 'Wright' comes from the circa 700 AD Old English word 'wryhta' or 'wyrhta', meaning worker or shaper of wood. Later it became any occupational worker (for example, a ...

Field Field may refer to: Expanses of open ground * Field (agriculture), an area of land used for agricultural purposes * Airfield, an aerodrome that lacks the infrastructure of an airport * Battlefield * Lawn, an area of mowed grass * Meadow, a grass ...

, May–Grünwald, Pappenheim and Leishman stains. They differ in protocols and additives and their names are often confused with one another in practice.

Mechanism

The value of Romanowsky staining lies in its ability to produce a wide range of hues, allowing cellular components to be easily differentiated. This phenomenon is referred to as the ''Romanowsky effect'', or more generally as

metachromasia {{inline citations needed, date=November 2024 Metachromasia (var. metachromasy) is a characteristic change in the color of staining carried out in biological tissues, exhibited by certain dyes when they bind to particular substances present in th ...

. Eosin part of the stain is responsible for pink-orange hue of

erythrocytes Red blood cells (RBCs), referred to as erythrocytes (, with -''cyte'' translated as 'cell' in modern usage) in academia and medical publishing, also known as red cells, erythroid cells, and rarely haematids, are the most common type of blood cel ...

and granules inside cytoplasms of eosinophilic leukocytes.

Romanowsky effect

In 1891 Romanowsky developed a stain using a mixture of

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 ...

(typically

eosin Y Eosin Y, also called C.I. 45380 or C.I. Acid Red 87, is a member of the triarylmethane dyes. It is produced from fluorescein by bromination. Use Eosin Y is commonly used as the red dye in red inks. It is commonly used in histology, most nota ...

) and aged solutions of

methylene blue Methylthioninium chloride, commonly called methylene blue, is a salt used as a dye and as a medication. As a medication, it is mainly used to treat methemoglobinemia. It has previously been used for treating cyanide poisoning and urinary trac ...

that formed hues unattributable to the staining components alone: distinctive shades of purple in the

chromatin Chromatin is a complex of DNA and protein found in eukaryote, eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important r ...

of the cell nucleus and within granules in the

cytoplasm The cytoplasm describes all the material within a eukaryotic or prokaryotic cell, enclosed by the cell membrane, including the organelles and excluding the nucleus in eukaryotic cells. The material inside the nucleus of a eukaryotic cell a ...

of some

leukocytes White blood cells (scientific name leukocytes), also called immune cells or immunocytes, are cells of the immune system that are involved in protecting the body against both infectious disease and foreign entities. White blood cells are genera ...

. This became known as the Romanowsky effect. Eosin and pure methylene blue alone (or in combination) do not produce the Romanowsky effect, and the active stains which produce the effect are now considered to be azure B and eosin.

Polychromed methylene blue

Romanowsky-type stains can be made from either a combination of pure dyes, or from methylene blue that has been subject to

oxidative Redox ( , , reduction–oxidation or oxidation–reduction) is a type of chemical reaction in which the oxidation states of the reactants change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is ...

demethylation Demethylation is the chemical process resulting in the removal of a methyl group (CH3) from a molecule. A common way of demethylation is the replacement of a methyl group by a hydrogen atom, resulting in a net loss of one carbon and two hydrogen at ...

, which results in the breakdown of methylene blue into multiple other stains, some of which are necessary to produce the Romanowsky effect. Methylene blue that has undergone this oxidative process is known as "polychromed methylene blue". Polychromed methylene blue may contain up to 11 dyes, including

azure A Azure A is an organic compound with the chemical formula A chemical formula is a way of presenting information about the chemical proportions of atoms that constitute a particular chemical compound or molecule, using chemical element symbo ...

, azure B, azure C,

thionine Thionine, also known as Lauth's violet, is the salt of a heterocyclic compound. It was firstly synthesised by Charles Lauth. A variety of salts are known including the chloride and acetate, called respectively thionine chloride and thionine ac ...

, methylene violet Bernthesen, methyl thionoline and thionoline. The exact composition of polychromed methylene blue depends on the method used, and even batches of the stain from the same manufacturer may vary in composition. Common method of rapid oxidation uses increasing pH of the solution with

potassium carbonate Potassium carbonate is the inorganic compound with the formula . It is a white salt, which is soluble in water and forms a strongly alkaline solution. It is deliquescent, often appearing as a damp or wet solid. Potassium carbonate is mainly used ...

and boiling it, which introduces atmospheric

oxygen Oxygen is a chemical element; it has chemical symbol, symbol O and atomic number 8. It is a member of the chalcogen group (periodic table), group in the periodic table, a highly reactivity (chemistry), reactive nonmetal (chemistry), non ...

. Other methods have been employed, as well, such as oxidation in acidic medium with dichromate anion. Although azure B and eosin have been shown to be the required components to produce the Romanowsky effect, these stains in their pure forms have not always been used in the formulation of the staining solutions. The original sources of azure B (one of the oxidation products of methylene blue) were from polychromed methylene blue solutions, which were treated with oxidizing agents or allowed to naturally age in the case of Romanowsky. Ernst Malachowsky in 1891 was the first to purposely polychrome methylene blue for use in a Romanowsky-type stain.

Types

Wright stain

Wright's stain can be used alone or in combination with the Giemsa stain, which is known as the Wright-Giemsa stain. Wright's stain is named after

James Homer Wright James Homer Wright (April 8, 1869 – January 3, 1928) was an early and influential American pathologist, who was chief of pathology at Massachusetts General Hospital from 1896 to 1926. Wright was born in Pittsburgh, Pennsylvania. In 1915, he jo ...

who in 1902 published a method using heat to produce polychromed methylene blue, which is combined with eosin Y. The polychromed methylene blue is combined with eosin and allowed to precipitate, forming an eosinate which is redissolved in

methanol Methanol (also called methyl alcohol and wood spirit, amongst other names) is an organic chemical compound and the simplest aliphatic Alcohol (chemistry), alcohol, with the chemical formula (a methyl group linked to a hydroxyl group, often ab ...

. The addition of Giemsa to Wright's stain increases the brightness of the "reddish-purple" color of the cytoplasmic granules. The Wright's and Wright-Giemsa stains are two of the Romanowsky-type stains in common use in the United States and are mainly used for the staining of blood and bone marrow films.

Jenner stain

Jenner's stain is used in

microscopy Microscopy is the technical field of using microscopes to view subjects too small to be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of microscopy: optical mic ...

for

blood smears. The stain is dark blue and results in very observable clearly stained nucleus.

Giemsa stain

Giemsa stain is composed of "Azure II" and eosin Y with methanol and glycerol as the solvent. "Azure II" is thought to be a mixture of azure B (which Giemsa called "azure I") and methylene blue, although the exact composition of "azure I" is considered a trade secret. Comparable formulations using known dyes have been published and are commercially available. Giemsa stain is considered to be the standard stain for detection and identification of the malaria parasite.

May-Grünwald stain

The May-Grünwald-Giemsa is used for the

of slides obtained by

fine-needle aspiration Fine-needle aspiration (FNA) is a diagnostic procedure used to investigate lumps or masses. In this technique, a thin (23–25 gauge (0.52 to 0.64 mm outer diameter)), hollow needle is inserted into the mass for sampling of cells that, a ...

in a histopathology lab for the diagnosis of tumorous cells.

Pappenheim stain

This method is a combination of May-Grünwald and Giemsa staining.

Leishman stain

In 1901

William Leishman Lieutenant-general, Lieutenant-General Sir William Boog Leishman, ( ; 6 November 1865 – 2 June 1926) was a Scottish pathologist and British Army medical officer. He was Director General Army Medical Services, Director-General of Army Medical S ...

developed a stain that was similar to Louis Jenner's but with the replacement of pure methylene blue with polychromed methylene blue. Leishman's stain is prepared from the eosinate of polychromed methylene blue and eosin Y using methanol as the solvent.

Field's stain

Field stain is used for staining thick blood films in order to discover

malarial Malaria is a Mosquito-borne disease, mosquito-borne infectious disease that affects vertebrates and ''Anopheles'' mosquitoes. Human malaria causes Signs and symptoms, symptoms that typically include fever, Fatigue (medical), fatigue, vomitin ...

parasites.

Clinical importances

Blood and bone marrow pathology

Macrophages_in_bronchial_wash_specimen_--_high_mag

Romanowsky-type stains are widely used in the examination of blood, in the form of blood films, and in the microscopic examination of bone marrow

biopsies A biopsy is a medical test commonly performed by a surgeon, an interventional radiologist, or an interventional cardiologist. The process involves the extraction of sample cells or tissues for examination to determine the presence or extent of ...

and aspirate smears. Examination of both blood and bone marrow can be of importance in the diagnosis of a variety of blood diseases. In the United States the Wright and Wright-Giemsa variants of the Romanowsky-type stains are widely used, while in Europe Giemsa stain is commonly employed.

Detection of malaria and other parasites

Of the Romanowsky-type stains, the Giemsa stain is especially important in the detection and identification of malaria parasites in blood samples.

Malaria antigen detection tests Malaria antigen detection tests are a group of commercially available rapid diagnostic tests of the rapid antigen test type that allow quick Medical diagnosis, diagnosis of malaria by people who are not otherwise skilled in traditional laboratory t ...

are an alternative to the staining and microscopic examination of blood films for the detection of malaria.

Use in cytopathology

Romanowsky-type stains are also used for the staining of cytopathologic specimens such as those produced from fine-needle aspirates and

cerebrospinal fluid Cerebrospinal fluid (CSF) is a clear, colorless Extracellular fluid#Transcellular fluid, transcellular body fluid found within the meninges, meningeal tissue that surrounds the vertebrate brain and spinal cord, and in the ventricular system, ven ...

from lumbar punctures.

History

Although debate exists as to who deserves credit for this general staining method, popular usage has attributed it to Dmitri Leonidovich Romanowsky. In the 1870s

Paul Ehrlich Paul Ehrlich (; 14 March 1854 – 20 August 1915) was a Nobel Prize-winning German physician and scientist who worked in the fields of hematology, immunology and antimicrobial chemotherapy. Among his foremost achievements were finding a cure fo ...

used a mixture of acidic and basic dyes including

acid fuchsin Acid fuchsin or fuchsine acid, (also called Acid Violet 19 and C.I. 42685) is an acidic magenta dye with the chemical formula C20H17N3Na2O9S3. It is a sodium sulfonate derivative of fuchsine. Acid fuchsin has wide use in histology, and is one of ...

(acid dye) and

(basic dye) to examine blood films. In 1888 Cheslav Ivanovich Chenzinsky used methylene blue, but substituted the acid fuchsin used by Ehrlich with eosin. Chenzinsky's stain combination was able to stain the

parasite (a member of the

genus Genus (; : genera ) is a taxonomic rank above species and below family (taxonomy), family as used in the biological classification of extant taxon, living and fossil organisms as well as Virus classification#ICTV classification, viruses. In bino ...

Plasmodium ''Plasmodium'' is a genus of unicellular eukaryotes that are obligate parasites of vertebrates and insects. The life cycles of ''Plasmodium'' species involve development in a Hematophagy, blood-feeding insect host (biology), host which then inj ...

''). Neither Ehrlich's or Chenzinsky's stains produced the Romanowsky effect as the methylene blue they used was not polychromed. Dmitri Romanowsky in 1890 published preliminary findings of his blood stain (a combination of aged methylene blue and eosin), including the results when applied to malaria infected blood. This use of polychromed methylene blue differentiated Romanowsky's stain (and the subsequent formulations) from those of Ehrlich and Chenzinsky, which lacked the purple hue associated with the Romanowsky effect. Romanowsky's 1890 publication did not include a description of how he modified his methylene blue solution, but in his 1891 doctoral thesis he described methylene blue best as used after mold began forming on the surface. Other than the use of an aged methylene blue solution, Romanowsky's stain was based on Chenzinsky's stain technique. Romanowsky's use of his method to study the malaria parasite has been attributed to the continued interest in his staining method. Ernst Malachowski

Ernst Malachowsky has been credited with independently observing the same stain combination as Dmitri Romanowsky in 1891, although he has also been credited with being the first to do so. Malachowsky was the first to use a deliberately polychromed methylene blue solution, which Malachowsky accomplished by the addition of

borax The BORAX Experiments were a series of safety experiments on boiling water nuclear reactors conducted by Argonne National Laboratory in the 1950s and 1960s at the National Reactor Testing Station in eastern Idaho.

to the staining mixture. Malachowsky is reported to have demonstrated the stain on June 15, 1890, and in the same year to have published a paper "describing his public demonstration". Both the Romanowsky and Malachowsky methods were able to stain the

nucleus Nucleus (: nuclei) is a Latin word for the seed inside a fruit. It most often refers to: *Atomic nucleus, the very dense central region of an atom *Cell nucleus, a central organelle of a eukaryotic cell, containing most of the cell's DNA Nucleu ...

and

of the

parasite Parasitism is a Symbiosis, close relationship between species, where one organism, the parasite, lives (at least some of the time) on or inside another organism, the Host (biology), host, causing it some harm, and is Adaptation, adapted str ...

, when until this point the stains used had only colored the cytoplasm. In 1899, Louis Leopold Jenner developed a more stable version of the methylene blue and eosin stain by collecting the

precipitate In an aqueous solution, precipitation is the "sedimentation of a solid material (a precipitate) from a liquid solution". The solid formed is called the precipitate. In case of an inorganic chemical reaction leading to precipitation, the chemic ...

that forms in water-based mixtures and redissolving it in

. Romanowsky-type stains prepared from the collected precipitates are sometimes known as ''eosinates''. Besides increasing the stability of the stain, the use of methanol in Jenner's stain had the effect of fixing the blood samples, although Jenner's version of the stain does not produce the Romanowsky effect. Richard May and Ludwig Grünwald in 1892 published a version of the stain (now known as the May–Grünwald stain) which is similar to the version proposed by Jenner in 1899, and likewise does not produce the Romanowsky effect. In 1901, both Karl Reuter and

developed stains that combined Louis Jenner's use of alcohol as the solvent and Malachowsky's use of polychromed methylene blue. Reuter's stain differed from Jenner's in using

ethyl alcohol Ethanol (also called ethyl alcohol, grain alcohol, drinking alcohol, or simply alcohol) is an organic compound with the chemical formula . It is an alcohol, with its formula also written as , or EtOH, where Et is the pseudoelement symbol ...

instead of methanol, and Leishman's differed from Jenner's by using

eosin B Eosin B is a form of 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 lys ...

instead of

in 1902 published a method using heat to polychrome the methylene blue, which he combined with eosin Y. This technique is known as

Wright's stain Wright's stain is a hematologic stain that facilitates the differentiation of blood cell types. It is classically a mixture of eosin (red) and methylene blue dyes. It is used primarily to stain peripheral blood smears, urine samples, and bone marr ...

. Gustav Giemsa's name has also become associated with the stain as he is credited with publishing a useful formulation and protocol in 1902. Giemsa attempted to use combinations of pure dyes rather than polychromed methylene blue solutions which are highly variable in composition. Giemsa sold the rights to produce his stain, but never fully published details on how he produced it, although it is thought that he used a combination of azure B and methylene blue. Giemsa published a number of modifications of his stains between 1902 and 1934. In 1904 he suggested adding glycerin to his stain, along with the methanol, to increase its stability. Giemsa stain powders produced in Germany were widely used in the United States until the interruption of the supply during

World War I World War I or the First World War (28 July 1914 – 11 November 1918), also known as the Great War, was a World war, global conflict between two coalitions: the Allies of World War I, Allies (or Entente) and the Central Powers. Fighting to ...

, which caused increased utilization of James Homer Wright's method for polychroming methylene blue.

References

{{Stains Anatomical pathology Cytopathology Hematology Hematopathology Histology Romanowsky stains Staining