Haematoxylin
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Haematoxylin or hematoxylin (Template:IPAc-en), also called natural black 1 or C.I. 75290, is a compound extracted from heartwood of the logwood tree (Haematoxylum campechianum)<ref name="Bancroft and Stevens, 1982"/><ref name="Lillie, 1977" /> with a chemical formula of Template:Chem. This naturally derived dye has been used as a histologic stain, as an ink<ref name="Mitchell, 1908" /><ref name="Barrow, 1948" /><ref name="Centeno 2016" /><ref name="Neevel, 2003" /> and as a dye in the textile and leather industry.<ref name="Titford, 2005" /><ref name="Ortiz-Hidalgo and Pina-Oviedo, 2019"/> As a dye, haematoxylin has been called palo de Campeche,<ref name="Ortiz-Hidalgo and Pina-Oviedo, 2019" /> logwood extract,<ref name="Titford, 2005" /> bluewood<ref name="Ponting, 1973" /> and blackwood.<ref name="Ponting, 1973" /> In histology, haematoxylin staining is commonly followed by counterstaining with eosin.<ref name="Bancroft and Stevens, 1982" /><ref name="Chan, 2014" /><ref name="Llewellyn, 2009" /> When paired, this staining procedure is known as H&E staining and is one of the most commonly used combinations in histology.<ref name="Bancroft and Stevens, 1982" /><ref name="Titford, 2005" /><ref name="Smith, 2006" /><ref name="Kiernan, 2006" /><ref name="Dapson and Horobin, 2009" /> In addition to its use in the H&E stain, haematoxylin is also a component of the Papanicolaou stain (or Pap stain) which is widely used in the study of cytology specimens.<ref name="Bancroft and Stevens, 1982" /><ref name="Dapson and Horobin, 2009" />
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).<ref name="Bancroft and Stevens, 1982" /><ref name="Titford, 2005" /><ref name="Ortiz-Hidalgo and Pina-Oviedo, 2019" /><ref name="Titford, 2009" /><ref name="Kahr et al., 1998" /> In its pure form, haematoxylin is a colourless and crystalline solid,<ref name="Titford, 2005" /><ref name="Bettinger and Zimmermann, 1991" /> although commercial samples are typically light to dark brown based on the level of impurities present.<ref name="Lillie, 1977" /><ref name="Dapson et al., 2010" />
Extraction and purificationEdit
Haematoxylin has been synthesized,<ref name="Morsingh and Robinson, 1970" /><ref name="Puchtler et al., 1986" /> although never in commercially viable amounts.<ref name="Dapson and Horobin, 2009" /><ref name="Cooksey, 2010" /> Historically the logwood was exported and the haematoxylin extracted in Europe. More recently extraction takes place closer to where the logwood is harvested.<ref name="Dapson et al., 2010" /> 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.<ref name="Ponting, 1973" /><ref name="Lillie, 1974" /> Once extracted, the dye can be sold as a liquid concentrate or dried and sold in a crystalline form.<ref name="Ponting, 1973" /> Modern production methods use water, ether or alcohol as a solvent, at which point the extracts may be further refined to the level of purity needed.<ref name="Dapson et al., 2010" />
The commercial product may vary from batch to batch and between manufacturers<ref name="Dapson et al., 2010" /> in both the level of impurities and in the ratio of haematoxylin to haematein.<ref name="Schulte, 1991" /><ref name="Lillie, 1977" /><ref name="Marshall and horobin, 1974" /> For histologic use, this variability can affect the stain's interaction with biological tissue samples, and is therefore of concern to histologists and pathologists.<ref name="Schulte, 1991" /><ref name="Lillie, 1977" /><ref name="Dapson et al., 2010" /> Haematoxylin, like other biological stains, may be certified by the Biological Stain Commission, signifying that a particular batch of stain works in a standardized test, although this does not specify the dye's actual purity.<ref name="Schulte, 1991" />
Use as a histologic stainEdit
Haematoxylin stain is commonly followed (or counterstained) with another histologic stain, eosin.<ref name="Chan, 2014" /><ref name="Llewellyn, 2009" /><ref name="Bancroft and Stevens, 1982" /> When paired, this staining procedure is known as H&E staining, and is one of the most commonly used combinations in histology.<ref name="Bancroft and Stevens, 1982" /><ref name="Smith, 2006" /><ref name="Titford, 2005" /><ref name="Dapson and Horobin, 2009" /> 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.<ref name="Dapson and Horobin, 2009" /><ref name="Bancroft and Stevens, 1982" />
Principally used as a nuclear stain (to stain the cell nucleus), haematoxylin will also stain rough endoplasmic reticulum, ribosomes, collagen, myelin, elastic fibers, and acid mucins.<ref name="Chan, 2014" /> Haematoxylin alone is not an effective stain, but when oxidized to hematein, and combined with a mordant, stains chromatin in cell nuclei dark blue to black.<ref name="Bancroft and Stevens, 1982" /><ref name="Titford, 2005" /><ref name="Kiernan, 2018" /><ref name="Chan, 2014" /> The colour and specificity of haematoxylin stains are controlled by the chemical nature, and amount, of the mordant used, and the pH of the staining solution, thus, a variety of haematoxylin formulations have been developed.<ref name="Bancroft and Stevens, 1982" /><ref name="Chan, 2014" /><ref name="Titford, 2009" />
Stain formulationsEdit
Haematoxylin stain formulations can be broadly classified based on how the haematoxylin is oxidized (or ripened) and by choice of the mordant used.<ref name="Bancroft and Stevens, 1982" /> Haematoxylin stain formulations may either be natural oxidized by exposure to air and sunlight, or more commonly, especially in commercially prepared solutions,<ref name="Titford, 2005" /> chemically oxidized using sodium iodate.<ref name="Bancroft and Stevens, 1982" /><ref name="Gatenby and Beams, 1950" /><ref name="Llewellyn, 2009" /> 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.<ref name="Kiernan, 2006" /><ref name="Gill, 2010" /><ref name="Llewellyn, 2009" /> Of the metallic salts used as mordants, aluminium is the most common,<ref name="Llewellyn, 2009" /> other mordants include salts of iron, tungsten, molybdenum and lead.<ref name="Bancroft and Stevens, 1982" />
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.<ref name="Llewellyn, 2009" /><ref name="Kiernan, 2018" /><ref name="Bancroft and Stevens, 1982" /> Removal of unwanted staining, or differentiation, typically involves a solution of diluted ethanol and hydrochloric acid.<ref name="Llewellyn, 2009" /><ref name="Bancroft and Stevens, 1982" /><ref name="Puchtler et al., 1986" />
Table of significant formulationsEdit
| Formula name | Reference | Mordant | Oxidation method | Typical use |
|---|---|---|---|---|
| Ehrlich's Haematoxylin<ref name="Gatenby and Beams, 1950" /> | Ehrlich, 1886 | Potassium alum | Natural | Nuclear stain in H&E |
| Delafield's Haematoxylin<ref name="Gatenby and Beams, 1950" /> | Prudden, 1855 | Ammonium alum | Natural | Nuclear stain in H&E |
| Mayer's Haematoxylin<ref name="Gatenby and Beams, 1950" /> | Mayer, 1903 | Potassium or Ammonium alum | Sodium iodate | Nuclear stain in H&E |
| Harris's Haematoxylin<ref name="Gatenby and Beams, 1950" /> | Harris, 1900<ref name="Harris, 1900" /> | Potassium alum | Mercuric oxide | Nuclear stain in H&E, also used in the classical versions of the Papanicolaou stain<ref name="Gill, 2013" /> |
| Cole's Haematoxylin<ref name="Bancroft and Stevens, 1982" /> | Cole, 1943<ref name="Cole, 1943" /> | Potassium alum | Iodine | Nuclear stain in H&E |
| Carazzi's Haematoxylin<ref name="Bancroft and Stevens, 1982" /> | Carazzi, 1911 | Potassium alum | Potassium iodate | Nuclear stain in H&E, urgent biopsy sections |
| Weigert's Haematoxylin<ref name="Gatenby and Beams, 1950" /> | Weigert, 1904 | Ferric chloride | Natural | Nuclear stain in H&E, resistant to acids |
| Verhoeff's Haematoxylin<ref name="Bancroft and Stevens, 1982" /> | Verhoeff, 1908 | Ferric chloride | Iodine | elastic fibers, myelin<ref name="Puchtler et al., 1986" /> |
| Mallory's phosphotungstic acid Haematoxylin<ref name="Bancroft and Stevens, 1982" /> | Mallory, 1897 | Phosphotungstic acid | Natural or chemical | Fibrin, muscle striations |
| Gill's Haematoxylin (I, II, and III) | Culling et al. 1985 <ref name="Llewellyn, 2009" /><ref name="Gill, 2010" /> | Aluminium sulfate | Sodium iodate | Nuclear stain in H&E |
Early use as a histologic stainEdit
In 1758, Georg Christian Reichel used haemotoxylin, without a mordant, to stain plant tissues.<ref name="Bracegirdle 1986" /><ref name="Smith, 2006" /><ref name="Allison, 1999" /> John Thomas Quekett in an 1852 book,<ref name="Quekett, 1852" /> suggests using "logwood" (haematoxylin) to dye translucent material for examination under the microscope.<ref name="Allison, 1999" /><ref name="Bracegirdle 1986" /> In 1863, Wilhelm von Waldeyer-Hartz used haematoxylin on animal tissue without a mordant (with limited success),<ref name="Mann, 1902" /> and is sometimes credited as being the first to do so,<ref name="Ortiz-Hidalgo and Pina-Oviedo, 2019" /><ref name="Smith, 2006" /><ref name="Cook, 1997" /><ref name="Mann, 1902" /> although this is not universally accepted.<ref name="Cook, 1997" /><ref name="Ortiz-Hidalgo and Pina-Oviedo, 2019" /> Franz Böhmer in 1865 published a haematoxylin formula using alum as a mordant,<ref name="Mann, 1902" /><ref name="Cooksey, 2010" /><ref name="Smith, 2006" /><ref name="Ortiz-Hidalgo and Pina-Oviedo, 2019" /><ref name="Cook, 1997" /><ref name="Bracegirdle 1986" /> and in 1891, Paul Mayer published a formulation using a chemical oxidizer to convert haematoxylin into haematein.<ref name="Gatenby and Beams, 1950" /><ref name="Bracegirdle 1986" /><ref name="Smith, 2006" /> 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.<ref name="Titford, 2009" /><ref name="Bracegirdle 1986" /> By the early 1900s, haematoxylin had become widely accepted as a histologic stain.<ref name="Smith, 2006" />
Shortages and possible alternativesEdit
During World War I, the late 1920s, World War II, the early 1970s (summer 1973<ref name="Lillie, 1974" />) and in 2008, there were shortages of haematoxylin due to interruptions in its extraction from logwood.<ref name="Dapson et al., 2010" /> These shortages prompted a search for alternative nuclear stains.<ref name="Lillie, 1974" /><ref name="Dapson et al., 2010" /> Several synthetic dyes have been recommended as replacements, notably celestine blue (CI 51050),<ref name="Dapson et al., 2010" /> gallocyanine<ref name="Titford, 2005" /><ref name="Llewellyn, 2009" /> (CI 51030), gallein<ref name="Dapson et al., 2010" /> (CI 45445) and eriochrome cyanine R<ref name="Dapson et al., 2010" /><ref name="Llewellyn, 2009" /> 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,<ref name="Dapson and Horobin, 2009" /> who have spent years of training with H&E stained slides, to examine the slides and make medical diagnoses.<ref name="Titford, 2005" /> None of proposed replacement stains have been widely adopted.<ref name="Dapson and Horobin, 2009" /><ref name="Titford, 2005" />
Use as a textile dyeEdit
Haematoxylin was first used as a dye by the Mayans and Aztecs in Central America where logwood trees grow natively.<ref name="Ortiz-Hidalgo and Pina-Oviedo, 2019" /><ref name="Ponting, 1973" /> The dye was first introduced to Europe by the Spanish, and soon after was widely adopted.<ref name="Ponting, 1973" /><ref name="Ortiz-Hidalgo and Pina-Oviedo, 2019" /> 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.<ref name="Ponting, 1973" /> As a blue dye (with alum as a mordant), the initial results were not as lightfast as those produced using indigo.<ref name="Titford, 2005" /><ref name="Ponting, 1973" /> 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.<ref name="Ortiz-Hidalgo and Pina-Oviedo, 2019" /><ref name="Ponting, 1973" /> After the introduction of synthetic black dyes in the late 19th century, haematoxylin was first replaced as a dye for cotton.<ref name="Ponting, 1973" /> 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".<ref name="Georgievics, 1902" /> 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.<ref name="Ponting, 1973" /> Contemporary usage of haematoxylin includes the dyeing of silk, leather, and sutures.<ref name="Titford, 2005" />
Use as a writing and drawing inkEdit
Haematoxylin has been used as the primary component of writing and drawing inks, although the timing of first use as an ink is unclear.<ref name="Centeno 2009" /> Haematoxylin was also added to some iron gall inks, which take time to fully darken when applied to paper.<ref name="Barrow, 1948" /><ref name="Centeno 2009" /> In this case the Haematoxylin provided some initial colour before the iron gall reached its full depth of colour.<ref name="Barrow, 1948" /><ref name="Centeno 2009" /> William Lewis in 1763 is credited with being the first to use haematoxylin as an additive in iron gall inks.<ref name="Neevel, 2003" /> 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.<ref name="Neevel, 2003" /> Van Gogh is known to have used haematoxylin ink with a chrome mordant in a number of his drawings and letters.<ref name="Neevel, 2003" /><ref name="Centeno 2016" /><ref name="Centeno 2009" />
See alsoEdit
- Staining (biology)
- Histology
- Eosin
- H&E stain
- Haematein
- Verhoeff's stain
- Papanicolaou stain
- Natural dye
- Haematoxylum campechianum
Further readingEdit
- 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.
- Puchtler, H., Meloan, S.N. & Waldrop, F.S. 1986. Application of current chemical concepts to metal-haematein and -brazilein stains. Histochemistry 85: 353–364.
- Stainsfile