Wikipedia

Romanowsky stain

Romanowsky stainingis a prototypicalstainingtechnique that was the forerunner of several distinct but similar stains widely used inhematology(the study of blood) andcytopathology(the study of diseased cells). Romanowsky-type stains are used to differentiatecellsfor microscopic examination inpathologicalspecimens, especiallybloodandbone marrowfilms,[1]and to detect parasites such asmalariawithin the blood.[2][3][4][5]

Blood filmwithGiemsa stain.Monocytessurrounded byerythrocytes.

The staining technique is named after the Russian physicianDmitri Leonidovich Romanowsky(1861–1921), who was one of the first to recognize its potential for use as a blood stain.[6]

Stains that are related to or derived from the Romanowsky-type stains includeGiemsa,Jenner,Wright,Field,May–Grünwald,PappenheimandLeishmanstains. They differ in protocols and additives and their names are often confused with one another in practice.

Mechanism

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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 theRomanowsky effect,or more generally asmetachromasia.[7]

Eosin part of the stain is responsible for pink-orange hue oferythrocytesand granules insidecytoplasmsofeosinophilic leukocytes.

Romanowsky effect

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Blood filmstained withGiemsashowingPlasmodium(center of image), the parasite that causesmalariainfections.

In 1891 Romanowsky[8][9][10]developed a stain using a mixture ofeosin(typicallyeosin Y) and aged solutions ofmethylene bluethat formed hues unattributable to the staining components alone: distinctive shades of purple in thechromatinof the cell nucleus and within granules in thecytoplasmof someleukocytes.This became known as the Romanowsky effect.[11][12][6][13][4]Eosin and pure methylene blue alone (or in combination) do not produce the Romanowsky effect,[13][4]and the active stains which produce the effect are now considered to beazure Band eosin.[14][3][13]

Polychromed methylene blue

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Romanowsky-type stains can be made from either a combination of pure dyes, or from methylene blue that has been subject tooxidativedemethylation,which results in the breakdown of methylene blue into multiple other stains, some of which are necessary to produce the Romanowsky effect.[15][4]Methylene blue that has undergone this oxidative process is known as "polychromed methylene blue".[15][4]Polychromed methylene blue may contain up to 11 dyes, includingmethylene blue,azure A,azure B, azure C,thionine,methylene violet Bernthesen, methyl thionoline and thionoline.[4]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.[15]

Common method of rapid oxidation uses increasing pH of the solution withpotassium carbonateand boiling it, which introduces atmosphericoxygen.[16]Other methods have been employed, as well, such as oxidation in acidic medium withdichromate anion.

Although azure B and eosin have been shown to be the required components to produce the Romanowsky effect,[14][3][13]these stains in their pure forms have not always been used in the formulation of the staining solutions.[4]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.[3][13]Ernst Malachowskyin 1891 was the first to purposely polychrome methylene blue for use in a Romanowsky-type stain.[15][17]

Types

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Wright stain

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Main article:Wright's stain

Wright's stain can be used alone or in combination with the Giemsa stain, which is known as the Wright-Giemsa stain.[1]Wright's stain is named afterJames Homer Wrightwho in 1902[18]published a method using heat to produce polychromed methylene blue, which is combined with eosin Y.[19][20][21][1]The polychromed methylene blue is combined with eosin and allowed to precipitate, forming an eosinate which is redissolved inmethanol.[4]The addition of Giemsa to Wright's stain increases the brightness of the "reddish-purple" color of the cytoplasmic granules.[1][21]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.[21][1]

Jenner stain

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Main article:Jenner's stain

Jenner's stain is used inmicroscopyforstainingblood smears.The stain is dark blue and results in very observable clearly stained nucleus.

Giemsa stain

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Main article:Giemsa stain

Giemsa stain is composed of "Azure II" and eosin Y with methanol and glycerol as the solvent.[15]"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.[4][15]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.[5]

May-Grünwald stain

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Main article:May–Grünwald stain

The May-Grünwald-Giemsa is used for thestainingof slides obtained byfine-needle aspirationin a histopathology lab for the diagnosis of tumorous cells.

Pappenheim stain

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Main article:Pappenheim stain

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

Leishman stain

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Main article:Leishman stain

In 1901William Leishman[22]developed a stain that was similar to Louis Jenner's but with the replacement of pure methylene blue with polychromed methylene blue.[19][15][4]Leishman's stain is prepared from the eosinate of polychromed methylene blue and eosin Y using methanol as the solvent.[4]

Field's stain

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Main article:Field stain

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

Clinical importances

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Blood and bone marrow pathology

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Bronchoalveolar lavagespecimen stained withDiff-Quik,a commercial Romanowsky stain variant widely used in cytopathology

Romanowsky-type stains are widely used in the examination of blood, in the form ofblood films,and in the microscopicexamination of bone marrowbiopsiesand aspirate smears.[1][23]Examination of both blood and bone marrow can be of importance in the diagnosis of a variety of blood diseases.[1][23]In the United States the Wright and Wright-Giemsa variants of the Romanowsky-type stains are widely used,[1]while in Europe Giemsa stain is commonly employed.[1]

Detection of malaria and other parasites

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Of the Romanowsky-type stains, the Giemsa stain is especially important inthe detection and identification of malaria parasites in blood samples.[5][15]Malaria antigen detection testsare an alternative to the staining and microscopic examination of blood films for the detection of malaria.[5]

Use in cytopathology

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Romanowsky-type stains are also used for the staining ofcytopathologicspecimens such as those produced fromfine-needle aspiratesandcerebrospinal fluidfromlumbar punctures.[24]

History

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Dmitri Leonidovich Romanowsky (1861-1921)

Although debate exists as to who deserves credit for this general staining method, popular usage has attributed it toDmitri Leonidovich Romanowsky.[14][17][19]

In the 1870sPaul Ehrlichused a mixture of acidic and basic dyes includingacid fuchsin(acid dye) andmethylene blue(basic dye) to examine blood films.[25][26][27][28][17]In 1888 Cheslav Ivanovich Chenzinsky used methylene blue, but substituted the acid fuchsin used by Ehrlich with eosin.[14][27][28]Chenzinsky's stain combination was able to stain themalariaparasite (a member of thegenusPlasmodium).[28][19]Neither Ehrlich's or Chenzinsky's stains produced the Romanowsky effect as the methylene blue they used was not polychromed.[17]

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.[6]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.[17]Romanowsky's 1890 publication did not include a description of how he modified his methylene blue solution,[6][17]but in his 1891 doctoral thesis he described methylene blue best as used after mold began forming on the surface.[6][17]Other than the use of an aged methylene blue solution, Romanowsky's stain was based on Chenzinsky's stain technique.[17]Romanowsky's use of his method to study the malaria parasite has been attributed to the continued interest in his staining method.[26]

Ernst Malachowsky

Ernst Malachowskyhas been credited with independently observing the same stain combination as Dmitri Romanowsky in 1891,[6][13]although he has also been credited with being the first to do so.[17][19]Malachowsky was the first to use a deliberately polychromed methylene blue solution,[15]which Malachowsky accomplished by the addition ofboraxto the staining mixture.[17]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".[19]Both the Romanowsky and Malachowsky methods were able to stain thenucleusandcytoplasmof themalariaparasite,when until this point the stains used had only colored the cytoplasm.[19]

Gustav Giemsa

In 1899,Louis Leopold Jennerdeveloped a more stable version of the methylene blue and eosin stain by collecting theprecipitatethat forms in water-based mixtures and redissolving it inmethanol.[28][15][4]Romanowsky-type stains prepared from the collected precipitates are sometimes known aseosinates.[4]Besides increasing the stability of the stain, the use of methanol inJenner's stainhad the effect offi xingthe blood samples,[4]although Jenner's version of the stain does not produce the Romanowsky effect.[28][19][15]

Richard May andLudwig Grünwaldin 1892 published a version of the stain (now known as theMay–Grünwald stain) which is similar to the version proposed by Jenner in 1899, and likewise does not produce the Romanowsky effect.[28][19][15]

In 1901, bothKarl ReuterandWilliam Leishman[22]developed stains that combined Louis Jenner's use of alcohol as the solvent and Malachowsky's use of polychromed methylene blue.[19][15][4]Reuter's stain differed from Jenner's in usingethyl alcoholinstead of methanol, and Leishman's differed from Jenner's by usingeosin Binstead ofeosin Y.[19][4]

James Homer Wrightin 1902 published[18]a method using heat to polychrome the methylene blue, which he combined with eosin Y. This technique is known asWright's stain.[19][20]

Gustav Giemsa'sname has also become associated with the stain as he is credited with publishing a useful formulation and protocol in 1902.[13][6][26]Giemsa attempted to use combinations of pure dyes rather than polychromed methylene blue solutions which are highly variable in composition.[20][19][15]Giemsa sold the rights to produce his stain, but never fully published details on how he produced it,[19]although it is thought that he used a combination of azure B and methylene blue.[15]Giemsa published a number of modifications of his stains between 1902 and 1934. In 1904[29]he suggested adding glycerin to his stain, along with the methanol, to increase its stability.[23][19]

Giemsa stain powders produced in Germany were widely used in the United States until the interruption of the supply duringWorld War I,which caused increased utilization of James Homer Wright's method for polychroming methylene blue.[19][1]

See also

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References

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  1. ^abcdefghijTheil, Karl S. (2012). "Bone Marrow Processing and Normal Morphology".Laboratory Hematology Practice.pp.279–299.doi:10.1002/9781444398595.ch22.ISBN9781444398595.
  2. ^Bain, Barbara J.; Bates, Imelda; Laffan, Mike A. (11 August 2016). "Chapter 4: Preparation and staining methods for blood and bone marrow films".Dacie and Lewis Practical Haematology(12 ed.). Elsevier Health Sciences.ISBN978-0-7020-6925-3.
  3. ^abcdHorobin, RW (2011). "How Romanowsky stains work and why they remain valuable — including a proposed universal Romanowsky staining mechanism and a rational troubleshooting scheme".Biotechnic & Histochemistry.86(1):36–51.doi:10.3109/10520295.2010.515491.ISSN1052-0295.PMID21235292.S2CID207513741.
  4. ^abcdefghijklmnopMarshall, P. N. (1978). "Romanowsky-type stains in haemotology".The Histochemical Journal.10(1):1–29.doi:10.1007/BF01003411.PMID74370.S2CID37939306.
  5. ^abcdLi, Qigui; Weina, Peter J.; Miller, R. Scott (2012). "Malaria Analysis".Laboratory Hematology Practice.pp.626–637.doi:10.1002/9781444398595.ch48.ISBN9781444398595.
  6. ^abcdefgBezrukov, AV (2017). "Romanowsky staining, the Romanowsky effect and thoughts on the question of scientific priority".Biotechnic & Histochemistry.92(1):29–35.doi:10.1080/10520295.2016.1250285.ISSN1052-0295.PMID28098484.S2CID37401579.
  7. ^Ribatti, Domenico (2018)."The Staining of Mast Cells: A Historical Overview".International Archives of Allergy and Immunology.176(1):55–60.doi:10.1159/000487538.hdl:11586/227987.ISSN1018-2438.PMID29597213.
  8. ^Романовскiй Д.Л. (1890). "Къ вопросу о строенiи чужеядныхъ малярiи".Врачъ.52:1171–1173.
  9. ^Романовскiй Д.Л. Къ вопросу о паразитологіи и терапiи болотной лихорадки. Диссертацiя на степень доктора медицины. Спб. 1891 г., 118 с.
  10. ^Romanowsky D (1891). "Zur Frage der Parasitologie und Therapie der Malaria".St Petersburg Med Wochenschr.16:297–302,307–315.
  11. ^Horobin RW, Walter KJ (1987)."Understanding Romanowsky staining. I: The Romanowsky-Giemsa effect in blood smears".Histochemistry.86(3):331–336.doi:10.1007/bf00490267.PMID2437082.S2CID25723230.
  12. ^Woronzoff-Dashkoff KK. (2002)."The wright-giemsa stain. Secrets revealed".Clin Lab Med.22(1):15–23.doi:10.1016/S0272-2712(03)00065-9.PMID11933573.
  13. ^abcdefgWittekind, D. H. (1983). "On the nature of Romanowsky-Giemasa staining and its significance for cytochemistry and histochemistry: an overall view".The Histochemical Journal.15(10):1029–1047.doi:10.1007/BF01002498.ISSN0018-2214.PMID6196323.S2CID23896062.
  14. ^abcdCooksey, CJ (2017). "Quirks of dye nomenclature. 8. Methylene blue, azure and violet".Biotechnic & Histochemistry.92(5):347–356.doi:10.1080/10520295.2017.1315775.ISSN1052-0295.PMID28598697.S2CID46746062.
  15. ^abcdefghijklmnoLillie, Ralph Dougall (1977).H. J. Conn's Biological stains(9th ed.). Baltimore: Williams & Wilkins. pp. 692p.
  16. ^Goodpasture, E. W. (1917)."An Acid Polychrome-Methylene Blue Solution for Routine and Special Staining".Journal of the American Medical Association.LXIX(12): 998.doi:10.1001/jama.1917.25910390002016c.Retrieved2024-01-16.
  17. ^abcdefghijLillie, R. D. (1978). "Romanowsky–Malachowski Stains the So-Called Romanowsky Stain: Malachowski's 1891 Use of Alkali Polychromed Methylene Blue for Malaria Plasmodia".Stain Technology.53(1):23–28.doi:10.3109/10520297809111439.PMID78544.
  18. ^abWright JH (1902)."A Rapid Method for the Differential Staining of Blood Films and Malarial Parasites".J Med Res.7(1):138–144.PMC2105822.PMID19971449.
  19. ^abcdefghijklmnopKrafts, KP; Hempelmann, E; Oleksyn, BJ (2011). "The color purple: from royalty to laboratory, with apologies to Malachowski".Biotechnic & Histochemistry.86(1):7–35.doi:10.3109/10520295.2010.515490.ISSN1052-0295.PMID21235291.S2CID19829220.
  20. ^abcGatenby, J. B.; Beams, H. W. (1950).The Microtomist's Vade-Mecum(11th ed.). Philadelphia: The Blackstone Company.
  21. ^abcWoronzoff-Dashkoff KK (2002)."The wright-giemsa stain. Secrets revealed".Clin Lab Med.22(1):15–23.doi:10.1016/S0272-2712(03)00065-9.PMID11933573.
  22. ^abLeishman W (1901)."Note on a Simple and Rapid Method of Producing Romanowsky Staining in Malarial and other Blood Films".Br Med J.2(2125):757–758.doi:10.1136/bmj.2.2125.757.PMC2507168.PMID20759810.
  23. ^abcPeterson, Powers; McNeill, Sheila; Gulati, Gene (2012). "Cellular Morphologic Analysis of Peripheral Blood".Laboratory Hematology Practice.pp.10–25.doi:10.1002/9781444398595.ch2.ISBN9781444398595.
  24. ^Krafts, KP; Pambuccian, SE (2011). "Romanowsky staining in cytopathology: history, advantages and limitations".Biotechnic & Histochemistry.86(2):82–93.doi:10.3109/10520295.2010.515492.ISSN1052-0295.PMID21395493.S2CID5168332.
  25. ^Ehrlich P (1880)."Methodologische Beiträge zur Physiologie und Pathologie der verschiedenen Formen der Leukocyten"(PDF).Z Klin Med.1:553–560. Archived fromthe original(PDF)on 2011-07-19.
  26. ^abcBracegirdle, Brian (1986).A history of microtechnique: the evolution of the microtome and the development of tissue preparation(2nd ed.). Lincolnwood, IL: Science Heritage Ltd.ISBN978-0940095007.
  27. ^abKrafts, Kristine; Hempelmann, Ernst; Oleksyn, Barbara J. (2011). "In search of the malarial parasite".Parasitology Research.109(3):521–529.doi:10.1007/s00436-011-2475-4.ISSN0932-0113.PMID21660627.S2CID1823696.
  28. ^abcdefWoronzoff-Dashkoff, Kristine Pauline Krafts (1993). "The Ehrlich-Chenzinsky-Plehn-Malachowski-Romanowsky-Nocht-Jenner-May-Grünwald-Leishman-Reuter-Wright-Giemsa-Lillie-Roe-Wilcox Stain: The Mystery Unfolds".Clinics in Laboratory Medicine.13(4):759–771.doi:10.1016/S0272-2712(18)30406-2.ISSN0272-2712.PMID7508837.
  29. ^Giemsa G (1904). "Eine Vereinfachung und Vervollkommnung meiner Methylenazur-Methylenblau-Eosin-Färbemethode zur Erzielung der Romanowsky-Nochtschen Chromatinfärbung".Centralbl F Bakt Etc.37:308–311.
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