Haematoxylin or hematoxylin (), also called natural black 1 or C.I. 75290, is a compound extracted from heartwood of the logwood tree ( Haematoxylum campechianum)
Although the stain is commonly called haematoxylin, the active colourant is the oxidized form haematein, which forms strongly coloured complexes with certain metal ions (commonly Fe(III) and Al(III) salts).
Extraction and purification
Haematoxylin has been synthesized,
although never in commercially viable amounts.
Historically the logwood was exported and the haematoxylin extracted in Europe. More recently extraction takes place closer to where the logwood is harvested.
Extraction of haematoxylin from logwood on industrial scales has been accomplished in the 'French process' by boiling the wood chips or in the 'American process' with
steam and pressure.
Once extracted, the dye can be sold as a liquid concentrate or dried and sold in a crystalline form.
Modern production methods use water,
diethyl ether or
ethanol as a
solvent, at which point the extracts may be further refined to the level of purity needed.
The commercial product may vary from batch to batch and between manufacturers
Use as a histologic stain
Haematoxylin stain is commonly followed (or counterstained) with another histologic stain,
eosin.
When paired, this staining procedure is known as H&E staining, and is one of the most commonly used combinations in
histology.
Haematoxylin is also a component of the Papanicolaou stain (or PAP stain) which is widely used in the study of
cytology specimens, notably in the
Pap test used to detect
cervical cancer.
Principally used as a cell nuclei stain (to stain the cell nucleus), haematoxylin will also stain rough endoplasmic reticulum, ribosomes, collagen, myelin, elastic fibers, and acid mucins.
Stain formulations
Haematoxylin stain formulations can be broadly classified based on how the haematoxylin is oxidized (or
ripened) and by choice of the mordant used.
Haematoxylin stain formulations may either be natural oxidized by exposure to air and sunlight, or more commonly, especially in commercially prepared solutions,
chemically oxidized using
sodium iodate.
Commonly only enough oxidizer is added to convert one half of the haematoxylin to haematein, allowing the remainder to naturally oxidize during use, this extends the staining solution's useful life as more haematein is produced, while some haematein is further oxidized to oxyhaematein.
Of the metallic salts used as mordants,
aluminium is the most common,
other mordants include salts of
iron,
tungsten,
molybdenum and
lead.
Depending on the formulation or staining technique, haematoxylin stains may be used in what is called a progressive manner, in which the length of time the tissue remains in contact with the staining solution is used to control the amount of colouration, or in a regressive manner, in which the tissue is over-stained, and excess stain is removed in a secondary step of the procedure.
Table of significant formulations
|
|
| Nuclear stain in H&E |
| Nuclear stain in H&E |
| Nuclear stain in H&E |
Nuclear stain in H&E, also used in the classical versions of the Papanicolaou stain |
| Nuclear stain in H&E |
| Nuclear stain in H&E, urgent biopsy sections |
| Nuclear stain in H&E, resistant to acids |
elastic fibers, myelin |
| Fibrin, muscle striations |
| Nuclear stain in H&E |
Early use as a histologic stain
In 1758, Georg Christian Reichel used haemotoxylin, without a mordant, to stain plant tissues.
John Thomas Quekett in an 1852 book,
suggests using "logwood" (haematoxylin) to dye translucent material for examination under the
microscope.
In 1863, Wilhelm von Waldeyer-Hartz used haematoxylin on animal tissue without a mordant (with limited success),
and is sometimes credited as being the first to do so,
although this is not universally accepted.
Franz Böhmer in 1865 published a haematoxylin formula using alum as a mordant,
and in 1891, Paul Mayer published a formulation using a chemical oxidizer to convert haematoxylin into haematein.
The first use of haematoxylin with eosin as a counterstain, which is currently the most used stain combination in histology, was first suggested by A. Wissowzky in 1876.
By the early 1900s, haematoxylin had become widely accepted as a histologic stain.
Shortages and possible alternatives
During World War I, the late 1920s, World War II, the early 1970s (summer 1973
) and in 2008, there were shortages of haematoxylin due to interruptions in its extraction from logwood.
These shortages prompted a search for alternative nuclear stains.
Several synthetic dyes have been recommended as replacements, notably celestine blue (CI 51050),
gallocyanine
(CI 51030), gallein
(CI 45445) and eriochrome cyanine R
also called chromoxane cyanine R and solochrome cyanine (CI 43820). All four have Fe(III) as the
mordant. An alternative is the aluminium complex of oxidized
brazilin, which differs from haematoxylin by only one hydroxyl group. A replacement stain for haematoxylin in H&E staining must also not disrupt the ability of histologists and pathologists,
who have spent years of training with H&E stained slides, to examine the slides and make
diagnoses.
None of proposed replacement stains have been widely adopted.
Use as a textile dye
Haematoxylin was first used as a dye by the
Mayans and
Aztecs in
Central America where logwood trees grow natively.
The dye was first introduced to
Europe by the
Spain, and soon after was widely adopted.
Haematoxylin was used to produce blacks, blues and purples on various textiles, and remained an important industrial dye until the introduction of suitable replacements in the form of
synthetic dyes.
As a blue dye (with alum as a mordant), the initial results were not as
lightfast as those produced using
indigo dye.
In reaction to this perceived inferiority of the quality of the blue colour produced with haematoxylin, its use to dye fabric was barred in England from 1581 to 1662.
After the introduction of synthetic black dyes in the late 19th century, haematoxylin was first replaced as a dye for cotton.
A 1902 German treatise on the dyeing textiles notes "...logwood in the black dyeing of cotton has suffered considerably from the competition of aniline black".
Haematoxylin remained important as a black dye (using
copperas or chrome as a mordant) for wool until the 1920s when a black synthetic dye compatible with wool became available.
Contemporary usage of haematoxylin includes the dyeing of silk, leather, and
Surgical suture.
Use as a writing and drawing ink
Haematoxylin has been used as the primary component of writing and drawing
inks, although the timing of first use as an ink is unclear.
Haematoxylin was also added to some iron gall inks, which take time to fully darken when applied to paper.
In this case the Haematoxylin provided some initial colour before the iron gall reached its full depth of colour.
William Lewis in 1763 is credited with being the first to use haematoxylin as an additive in iron gall inks.
In 1848, Friedlieb Ferdinand Runge produced a heamatoxylin ink that was non-acidic, using a potassium chromate as the mordant, which had the advantage of not corroding steel pens.
is known to have used haematoxylin ink with a chrome mordant in a number of his drawings and letters.
See also
Further reading
-
Jocelyn H. Bruce-Gregorios, M.D.: Histopathologic Techniques, JMC Press Inc., Quezon City, Philippines, 1974.
-
Meloan, S. M. & Puchtler, H. 1987. "Harris hematoxylin," what Harris really wrote and the mechanism of hemalum stains. Journal of Histotechnology 10: 257–261.
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Puchtler, H., Meloan, S.N. & Waldrop, F.S. 1986. Application of current chemical concepts to metal-haematein and -brazilein stains. Histochemistry 85: 353–364.
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