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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,[1] and to detect parasites such as malaria within the
blood.[2][3][4][4][5] 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 Blood film with Giemsa stain. White blood cells
(center) surrounded by red blood cells.
potential for use as a blood stain.[6]

Contents

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.[7]

Romanowsky effect

In 1891 Romanowsky[8][9][10] 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.[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 be azure B and eosin.[14][3][13]
Blood film stained with Giemsa showing
Plasmodium (center of image), the parasite that
causes malaria infections.
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 demethylation, 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 that has undergone this oxidative process is
known as "polychromed methylene blue".[15][4] 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.[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]

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 Malachowsky in 1891 was the
first to purposely polychrome methylene blue for use in a Romanowsky type stain.[15][16]

Types

May-Grünwald-Giemsa

Main article: May–Grünwald 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.[17]

Wright's and Wright-Giemsa stains

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 after James Homer Wright who 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 in methanol.[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]

Leishman stain

Main article: Leishman stain

In 1901 William 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]

Giemsa stain

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]

Clinical importance

Blood and bone marrow pathology

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 and 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]
Bronchoalveolar lavage specimen
stained with Diff-Quik, a commercial
Romanowsky stain variant widely
Detection of malaria and other parasites used in cytopathology

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

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 from lumbar punctures.[24]

History

Although debate exists as to who deserves credit for this general staining method, popular usage has
attributed it to Dmitri Leonidovich Romanowsky.[14][16][19]

In the 1870s Paul Ehrlich used a mixture of acidic and basic dyes including acid fuchsin (acid dye) and
methylene blue (basic dye) to examine blood films.[25][26][27][28][16] 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 the malaria parasite (a member of the genus
Plasmodium).[28][19] Neither Ehrlich's or Chenzinsky's stains produced the Romanowsky effect as the
methylene blue they used was not polychromed.[16]
Dmitri Leonidovich
Dmitri Romanowsky in 1890 published preliminary findings of his blood stain (a combination of aged Romanowsky (1861-
methylene blue and eosin), including the results when applied to malaria infected blood.[6] This use of 1921).

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.[16] Romanowsky's
1890 publication did not include a description of how he modified his methylene blue solution,[6][16] but in his 1891 doctoral
thesis he described methylene blue best as used after mold began forming on the surface.[6][16] Other than the use of an aged
methylene blue solution, Romanowsky's stain was based on Chenzinsky's stain technique.[16] Romanowsky's use of his method
to study the malaria patasite has been attributed to the continued interest in his staining method.[26]

Ernst Malachowsky
Ernst Malachowsky has 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.[16][19] Malachowsky was the first to use a deliberately polychromed methylene blue solution,[15]
which Malachowsky accomplished by the addition of borax to the staining mixture.[16] 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 the nucleus and cytoplasm of the malaria parasite, when until this point the stains
used had only colored the cytoplasm.[19]

In 1899, Louis Leopold Jenner developed a more stable version of the methylene blue and eosin stain
by collecting the precipitate that forms in water-based mixtures and redissolving it in
methanol.[28][15][4] Romanowsky type stains prepared from the collected precipitates are sometimes
known as eosinates.[4] Besides increasing the stability of the stain, the use of methanol in Jenner's
stain had the effect of fixing the blood samples,[4] although Jenner's version of the stain does not
produce the Romanowsky effect.[28][19][15]

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
Gustav Giemsa.
produce the Romanowsky effect.[28][19][15]

In 1901, both Karl Reuter and William 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 using ethyl alcohol
instead of methanol, and Leishman's differed from Jenner's by using eosin B instead of eosin Y.[19][4]

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

Gustav Giemsa's name 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 during
World War I, which caused increased utilization of James Homer Wright's method for polychroming methylene blue.[19][1]

See also

Liu's stain

Papanicolaou stain

Malaria antigen detection tests

Staining (biology)

References

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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. ISBN 978-0-7020-
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3. ^ a b c d Horobin, 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):
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4. ^ a b c d e f g h i j k l m n o p q Marshall, P. N. (1978). "Romanowsky-type stains in haemotology". The Histochemical Journal.

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8. ^ Романовсĸiй Д.Л. (1890). "Къ вопросу о строенiи чужеядныхъ малярiи". Врачъ. 52: 1171–1173.

9. ^ Романовсĸiй Д.Л. Къ вопросу о паразитологіи и терапiи болотной лихорадĸи. Диссертацiя на степень доĸтора
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13. ^ a b c d e f g Wittekind, D. H. (1983). "On the nature of Romanowsky-Giemasa staining and its significance for
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14. ^ a b c d Cooksey, CJ (2017). "Quirks of dye nomenclature. 8. Methylene blue, azure and violet". Biotechnic &
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15. ^ a b c d e f g h i j k l m n o Lillie, Ralph Dougall (1977). H. J. Conn's Biological stains (9th ed.). Baltimore: Williams & Wilkins.
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16. ^ a b c d e f g h i j Lillie, 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.
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17. ^ Piaton, E.; Fabre, M.; Goubin-Versini, I.; Bretz-Grenier, M-F.; Courtade-Saïdi, M.; Vincent, S.; Belleannée, G.; Thivolet, F.;
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20. ^ a b c Gatenby, J. B.; Beams, H. W. (1950). The Microtomist's Vade-Mecum (11th ed.). Philadelphia: The Blackstone
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21. ^ a b c Woronzoff-Dashkoff KK (2002). "The wright-giemsa stain. Secrets revealed" . Clin Lab Med. 22 (1): 15–23.
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22. ^ a b Leishman 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 . PMC 2507168 . PMID 20759810 .

23. ^ a b c Peterson, Powers; McNeill, Sheila; Gulati, Gene (2012). "Cellular Morphologic Analysis of Peripheral Blood".
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24. ^ Krafts, KP; Pambuccian, SE (2011). "Romanowsky staining in cytopathology: history, advantages and limitations".
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25. ^ Ehrlich P (1880). "Methodologische Beiträge zur Physiologie und Pathologie der verschiedenen Formen der
Leukocyten" (PDF). Z Klin Med. 1: 553–560. Archived from the original (PDF) on 2011-07-19.

26. ^ a b c Bracegirdle, Brain (1986). A history of microtechnique : the evolution of the microtome and the development of
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27. ^ a b Krafts, Kristine; Hempelmann, Ernst; Oleksyn, Barbara J. (2011). "In search of the malarial parasite". Parasitology
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