Leishman stain

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A Leishman stain showing the schizont stage of Plasmodium vivax malaria parasite Plasmodium vivax malaria.jpg
A Leishman stain showing the schizont stage of Plasmodium vivax malaria parasite

Leishman stain, also known as Leishman's stain, is used in microscopy for staining blood smears. It is generally used to differentiate between and identify white blood cells, malaria parasites, and trypanosomas. It is based on a methanolic mixture of "polychromed" methylene blue (i.e. demethylated into various azures) and eosin. The methanolic stock solution is stable and also serves the purpose of directly fixing the smear eliminating a prefixing step. If a working solution is made by dilution with an aqueous buffer, the resulting mixture is very unstable and cannot be used for long. Leishman stain is named after its inventor, the Scottish pathologist William Boog Leishman. It is a version of the Romanowsky stain, and is thus similar to and partially replaceable by Giemsa stain, Jenner's stain, and Wright's stain.

Contents

Preparation and storage

Many companies sell Leishman Stain in the form of a dry powder, which is later reconstituted with methanol. [1] Heat and bright light oxidize the stain, and cause the precipitation of insoluble precipitates, such as methylene violet (Bernthsen) or Azure-Eosinate salts. [2]

Advantages and disadvantages

Advantages

According to Sir John Vivian Dacie's textbook, Practical Haematology, "Amongst the Romanowsky stains now in use, Jenner's is the simplest and Giemsa's the most complex. Leishman's stain, which occupies an intermediate position, is still widely used in the routine staining."

The WHO protocol [3] mentions:

There are a number of different combinations of these dyes which vary in their staining characteristics. May-Grunwald-Giemsa is a good method for routine work. Wright's stain is a simpler method, whilst Leishman's is also a simple method which is especially suitable when a stained blood film is required urgently or the routine stain is not available (e.g. at night). Field's stain is a rapid stain used primarily on thin films for malarial parasites. Whichever method is used, it is important to select dyes that are not contaminated with other dyes or metallic salts.

Sir William Boog Leishman of London and Karl Reuter of Germany independently discovered in 1901 what is considered "Perhaps the most practical modifications of Malachowski’s stain" [4] They adopted the best aspects of the stains developed by Malachowski and Jenner, i.e., they used both polychromed methylene blue (accidentally done by Romanowsky who got the name, systematically done by Ernst Malachowski even before Romanowsky, and rediscovered by Bernhard Nocht, but unknown to Jenner, May, Grunwald and many others who used simple methylene blue) and filtering the Azure Eosinate precipitate from the aqueous mixture and redissolving in an alcoholic solvent (Jenner's wisdom). Differences between Leishman's and Reuter's methods were: Leishman used methanol (like Jenner) and substituted Eosin B for Eosin Y, whereas Reuter used ethyl alcohol and rightly stressed the importance of using an absolutely pure solvent. Both methods produced a stable stain and the desired purple color.

Good contrast

Leishman stain generally shows the brilliant violet color of the nucleus and the neutrophil granules for which differential count becomes convenient and makes the quality of staining better than the stains that are simple methylene blue and Eosin based which do not produce enough contrast between the cytoplasm and the nucleus. Since Leishman, like other Romanowsky stains, stain cytoplasmic details and granules better, hematologists generally prefer them (however see below about cytologists). Compared to the costly and toxic pure synthetic AZure B and Eosin Y based reagents used by the ICSH reference methods which are also not free from the disadvantage of getting oxidized and eventually giving grey tone instead of the optimal blue colors for cytoplasm, Leishman is a cheap easily available and easy to do technique which gives a fairly acceptable contrast.[ citation needed ]

Good sensitivity for malaria parasite

It has been documented that Leishman staining is more sensitive than Field's stain and as good as fluorescent stains for detection of malaria parasite. [5]

Disadvantages

Difficulty controlling molar ratio

Composition of polychromed methylene blue mixed with Eosin is never as good as the directly weighed and mixed proportions in Giemsa type stains. Albert Plehn in 1890 [6] had figured out that the molar ratio of basic to acidic dyes had to be increased from 2:1 to 3:1, however from Jenners time (1899) the use of the Azure Eosinate crystals brought the ratio back to the unsatisfactory 2:1 and the depth of color became less. It was only the work by Gustav Giemsa and the likes who again manually controlled the proportion of these two components that brought the depth of staining back. However Giemsa and others who artificially controlled the proportion sometimes went to the other extreme (a large molar excess of Azure up to a ratio of 16.1) which was probably unnecessary. per ICSH the optimal ratio is 6.5 to 7.3. [7]

Instability

The Leishman stain if reconstituted with buffer becomes very unstable (in contrast with Giemsa which is relatively more stable due to Glycerol, or the ICSH reference stains which use methanol+DMSO in 6:4 v/v ratio) and starts precipitating and needs repeated filtering. If not carefully supervised, water absorption, methanol evaporation from an opened container and the repeated filtering changes the composition and required frequent changing and makes this method wasteful. If not filtered, the precipitate deposits on the smear may be confused with platelets. It is important not to shake the bottle of stain before use, else settled precipitates would be resuspended and will deposit on films during staining and cause numerous artifacts and make microscopy very difficult.[ citation needed ]

Cytoplasmic contrast good but nuclear contrast not as good as H&E

Like all other Malachowski-Romanowsky-Giemsa methods, it fades with time and cannot be stably archived for long. Also its above counterparts it stains the nuclei dark purple and the nuclear feature details are not as clear as Hematoxylene and Eosin, which are thus preferred by some cytopathologists. But that way it is a complementary method (since it stains cytoplasmic details granules etc. better than H&E stain).[ citation needed ]

Related Research Articles

Romanowsky stain Family of related stains for examination of blood including the detection of parasites

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 and cytopathology. Romanowsky-type stains are used to differentiate cells for microscopic examination in pathological 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 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.

Methylthioninium chloride, commonly called methylene blue, is a salt used as a dye and as a medication. Methylene blue is a thiazine dye. As a medication, it is mainly used to treat methemoglobinemia by converting the ferric iron in hemoglobin to ferrous iron. Specifically, it is used to treat methemoglobin levels that are greater than 30% or in which there are symptoms despite oxygen therapy. It has previously been used for cyanide poisoning and urinary tract infections, but this use is no longer recommended.

William Boog Leishman British Army general

Lieutenant-General Sir William Boog Leishman, was a Scottish pathologist and British Army medical officer. He was Director-General of Army Medical Services from 1923 to 1926.

Staining Technique used to enhance visual contrast of specimens observed under a microscope

Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses of disease at a microscopic level. Stains may be used to define biological tissues, cell populations, or organelles within individual cells.

Blood smear Stained blood on microscope slide

A blood smear, peripheral blood smear or blood film is a thin layer of blood smeared on a glass microscope slide and then stained in such a way as to allow the various blood cells to be examined microscopically. Blood smears are examined in the investigation of hematological (blood) disorders and are routinely employed to look for blood parasites, such as those of malaria and filariasis.

Wrights stain Hematologic stain that facilitates the differentiation of blood cell types

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 marrow aspirates, which are examined under a light microscope. In cytogenetics, it is used to stain chromosomes to facilitate diagnosis of syndromes and diseases.

Giemsa stain

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.

Basophilic

Basophilic is a technical term used by pathologists. It describes the appearance of cells, tissues and cellular structures as seen through the microscope after a histological section has been stained with a basic dye. The most common such dye is haematoxylin.

Eosin methylene blue

Eosin methylene blue is a selective stain for Gram-negative bacteria. EMB contains dyes that are toxic to Gram-positive bacteria. EMB is the selective and differential medium for coliforms. It is a blend of two stains, eosin and methylene blue in the ratio of 6:1. EMB is a differential microbiological medium, which slightly inhibits the growth of Gram-positive bacteria and provides a color indicator distinguishing between organisms that ferment lactose and those that do not. Organisms that ferment lactose display "nucleated colonies"—colonies with dark centers.

Periodic acid–Schiff stain Histological staining method

Periodic acid–Schiff (PAS) is a staining method used to detect polysaccharides such as glycogen, and mucosubstances such as glycoproteins, glycolipids and mucins in tissues. The reaction of periodic acid oxidizes the vicinal diols in these sugars, usually breaking up the bond between two adjacent carbons not involved in the glycosidic linkage or ring closure in the ring of the monosaccharide units that are parts of the long polysaccharides, and creating a pair of aldehydes at the two free tips of each broken monosaccharide ring. The oxidation condition has to be sufficiently regulated so as to not oxidize the aldehydes further. These aldehydes then react with the Schiff reagent to give a purple-magenta color. A suitable basic stain is often used as a counterstain.

Papanicolaou stain Histological staining method

Papanicolaou stain is a multichromatic (multicolored) cytological staining technique developed by George Papanicolaou in 1942. The Papanicolaou stain is one of the most widely used stains in cytology, where it is used to aid pathologists in making a diagnosis. Although most notable for its use in the detection of cervical cancer in the Pap test or Pap smear, it is also used to stain non-gynecological specimen preparations from a variety of bodily secretions and from small needle biopsies of organs and tissues. Papanicolaou published three formulations of this stain in 1942, 1954, and 1960.

H&E stain Histological stain method

Hematoxylin and eosin stain is one of the principal tissue stains used in histology. It is the most widely used stain in medical diagnosis and is often the gold standard. For example, when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E.

Field stain

Field stain is a histological method for staining of blood smears. It is used for staining thick blood films in order to discover malarial parasites. Field's stain is a version of a Romanowsky stain, used for rapid processing of the specimens.

Thionine Chemical compound and histologic stain C12H10N3S

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 acetate. The dye is structurally related to methylene blue, which also features a phenothiazine core. The dye's name is frequently misspelled with omission of the e, and is not to be confused with the plant protein thionin. The -ine ending indicates that the compound is an amine.

Gustav Giemsa German scientist

Gustav Giemsa was a German chemist and bacteriologist who was a native of Medar-Blechhammer. He is remembered for creating a dye solution commonly known as "Giemsa stain". This dye is used for the histopathological diagnosis of malaria and parasites such as Plasmodium, Trypanosoma, and Chlamydia.

Diff-Quik

Diff-Quik is a commercial Romanowsky stain variant used to rapidly stain and differentiate a variety of pathology specimens. It is most frequently used for blood films and cytopathological smears, including fine needle aspirates. The Diff-Quik procedure is based on a modification of the Wright-Giemsa stain pioneered by Harleco in the 1970s, and has advantages over the routine Wright-Giemsa staining technique in that it reduces the 4-minute process into a much shorter operation and allows for selective increased eosinophilic or basophilic staining depending upon the time the smear is left in the staining solutions.

Pappenheimer bodies

Pappenheimer bodies are abnormal basophilic granules of iron found inside red blood cells on routine blood stain. They are a type of inclusion body composed of ferritin aggregates, or mitochondria or phagosomes containing aggregated ferritin. They appear as dense, blue-purple granules within the red blood cell and there are usually only one or two, located in the cell periphery. They stain on a Romanowsky stain because clumps of ribosomes are co‐precipitated with the iron‐containing organelles.

Jaswant Singh–Bhattacharji stain, commonly referred to as JSB stain, is a rapid staining method for detection of malaria. It is useful for the diagnosis of malaria in thick smear samples of blood. The JSB stain is commonly used throughout India, but rarely used in other countries.

Liu's stain (劉氏染色法) is a staining technique used to stain animal cells. It is an improved staining based on Romanowsky stain, and was introduced by professor Chen-Hui Liu(劉禎輝), faculty of National Taiwan University, in 1953. The method sees a wide variety of usage in Taiwan. Comparing to other staining methods, Liu's stain is relatively fast, taking no more than 3 minutes to complete the process. In pathology, Liu's stain is primarily used to distinguish blood cells, but it can also apply on vaginal discharge, sputum, and pus as a simple stain.

White blood cell differential Blood test

A white blood cell differential is a medical laboratory test that provides information about the types and amounts of white blood cells in a person's blood. The test, which is usually ordered as part of a complete blood count (CBC), measures the amounts of the five normal white blood cell types – neutrophils, lymphocytes, monocytes, eosinophils and basophils – as well as abnormal cell types if they are present. These results are reported as percentages and absolute values, and compared against reference ranges to determine whether the values are normal, low, or high. Changes in the amounts of white blood cells can aid in the diagnosis of many health conditions, including viral, bacterial, and parasitic infections and blood disorders such as leukemia.

References

  1. District Laboratory Practice in Tropical Countries, Part 2 By Monica Cheesbrough, Appendix I, Preparation of Reagents and Culture Media, p396
  2. District Laboratory Practice in Tropical Countries, Part 2 By Monica Cheesbrough, Section 8 Hematological Tests, subsection 8.7 Blood Films, p322
  3. Blood Safety and Clinical Technology Guidelines on Standard Operating Procedures for HAEMATOLOGY Chapter 11 - Preparation and Staining of Blood Films, (Last update: 27 April 2006) Archived 1 April 2012 at the Wayback Machine
  4. Biotech Histochem. 2011 Feb;86(1):7-35. The Color Purple: from Royalty to Laboratory, with Apologies to Malachowski. Krafts KP, Hempelmann E, Oleksyn BJ.
  5. Indian Journal of Medical Microbiology, (2006) 24 (1):49-51
  6. Plehn F (1890b) Zur Aetiologie der Malaria. Berl. Klin. Wochenschr. 27: 292–294.
  7. ICSH reference method for staining of blood and bone marrow films by AZure B and Eosin Y (Romanowsky stain) INTERNATIONAL COMMITTEE FOR STANDARDIZATION IN HAEMATOLOGY Article first published online: 12 MAR 2008 DOI: 10.1111/j.1365-2141.1984.tb02949.x