Romanowsky stain

Romanowsky staining, also known as Romanowsky–Giemsa staining, is a prototypical staining technique that was the forerunner of several distinct but similar stains widely used in hematology (the study of blood) and cytopathology (the study of diseased cells). Romanowsky-type stains are used to differentiate cells for microscopic examination in pathologic specimens, especially blood and bone marrow films, and to detect parasites such as malaria within the blood. Stains that are related to or derived from the Romanowsky-type stains include Giemsa, Jenner, Wright, Field, May–Grünwald stain and Leishman stains. 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. Romanowsky staining, also known as Romanowsky–Giemsa staining, is a prototypical staining technique that was the forerunner of several distinct but similar stains widely used in hematology (the study of blood) and cytopathology (the study of diseased cells). Romanowsky-type stains are used to differentiate cells for microscopic examination in pathologic specimens, especially blood and bone marrow films, and to detect parasites such as malaria within the blood. Stains that are related to or derived from the Romanowsky-type stains include Giemsa, Jenner, Wright, Field, May–Grünwald stain and Leishman stains. 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. 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. In 1891 Romanowsky developed a stain using a mixture of eosin (typically eosin Y) and aged solutions of methylene blue that formed hues unattributable to the staining components alone: distinctive shades of purple in the chromatin of the cell nucleus and within granules in the cytoplasm of some white blood cells. This became known as the Romanowsky or Romanowsky-Giemsa 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. Romanowsky type stains can be made from either a combination of pure dyes, or from methylene blue that has been subject to oxidative demethylation, 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 that has undergone this oxidative process is known as 'polychromed methylene blue'. Polychromed methylene blue may contain up to 11 dyes, including methylene blue, azure A, azure B, azure C, thionine, 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. 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. The May-Grünwald-Giemsa stain is a two step procedure that includes first staining with May-Grünwald stain, which does not produce the Romanowsky effect, followed by staining the Giemsa stain which does produce the Romanowsky effect. 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 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 with is redissolved in methanol. 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. In 1901 William Leishman 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. 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.