Haematoxylin

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Haematoxylin
Haematoxylin powder
Skeletal formula of haematoxylin
Ball-and-stick model of the haematoxylin molecule
Names
Preferred IUPAC name
(6aS,11bR)-7,11b-Dihydroindolo[2,1-c] [1]benzopyran-3,4,6a,9,10(6H)-pentol
Other names
Hematoxylin; Natural Black 1; Hematoxyline; Hydroxybrazilin; Hydroxybrasilin; C.I. 75290
Identifiers
3D model (
JSmol
)
ChEMBL
ChemSpider
ECHA InfoCard
 100.007.490 Edit this at Wikidata
MeSH Hematoxylin
UNII
  • InChI=1S/C16H14O6/c17-10-2-1-8-13-9-4-12(19)11(18)3-7(9)5-16(13,21)6-22-15(8)14(10)20/h1-4,13,17-21H,5-6H2/t13?,16-/m0/s1 checkY
    Key: WZUVPPKBWHMQCE-VYIIXAMBSA-N checkY
  • InChI=1/C16H14O6/c17-10-2-1-8-13-9-4-12(19)11(18)3-7(9)5-16(13,21)6-22-15(8)14(10)20/h1-4,13,17-21H,5-6H2/t13?,16-/m0/s1
    Key: WZUVPPKBWHMQCE-VYIIXAMBBJ
  • Oc2cc3C[C@]4(O)COc1c(O)c(O)ccc1C4c3cc2O
Properties
C16H14O6
Molar mass 302.282 g·mol−1
Melting point 100-120 °C
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
☒N verify (what is checkY☒N ?)

Haematoxylin

cytology specimens.[1][14]

Although the stain is commonly called haematoxylin, the active colourant is the

haematein, which forms strongly coloured complexes with certain metal ions (commonly Fe(III) and Al(III) salts).[1][7][8][15][16] In its pure form, haematoxylin is a colourless and crystalline solid,[7][17] although commercial samples are typically light to dark brown based on the level of impurities present.[2][18]

Extraction and purification

Logwood (Haematoxylum campechianum) chips

Haematoxylin has been synthesized,[19][20] although never in commercially viable amounts.[14][21] Historically the logwood was exported and the haematoxylin extracted in Europe. More recently extraction takes place closer to where the logwood is harvested.[18] 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.[9][22] Once extracted, the dye can be sold as a liquid concentrate or dried and sold in a crystalline form.[9] 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.[18]

The commercial product may vary from batch to batch and between manufacturers[18] in both the level of impurities and in the ratio of haematoxylin to haematein.[23][2][24] For histologic use, this variability can affect the stains interaction with biological tissue samples, and is therefore of concern to histologists and pathologists.[23][2][18] 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.[23]

Use as a histologic stain

Hematoxylin staining shown as "basophilic" at top, seen with dual staining with hematoxylin and eosin (H&E).

Haematoxylin stain is commonly followed (or counterstained) with another histologic stain,

cytology specimens, notably in the PAP test used to detect cervical cancer.[14][1]

Principally used as a

cell nuclei dark blue to black.[1][7][25][10] 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.[1][10][15]

Stain formulations

Hematoxylin solution for staining microscopy slides.

Haematoxylin stain formulations can be broadly classified based on how the haematoxylin is oxidized (or ripened) and by choice of the mordant used.[1] Haematoxylin stain formulations may either be natural oxidized by exposure to air and sunlight, or more commonly, especially in commercially prepared solutions,[7] chemically oxidized using sodium iodate.[1][26][11] 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.[13][27][11] Of the metallic salts used as mordants, aluminium is the most common,[11] other mordants include salts of iron, tungsten, molybdenum and lead.[1]

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.[11][25][1] Removal of unwanted staining, or differentiation, typically involves a solution of diluted ethanol and hydrochloric acid.[11][1][20]

Table of significant formulations

Formula name Reference Mordant Oxidation method Typical use
Ehrlich's Haematoxylin[26] Ehrlich, 1886 Potassium alum Natural Nuclear stain in
H&E
Delafield's Haematoxylin[26] Prudden, 1855 Ammonium alum Natural Nuclear stain in H&E
Mayer's Haematoxylin[26] Mayer, 1903 Potassium or Ammonium alum Sodium iodate Nuclear stain in H&E
Harris's Haematoxylin[26] Harris, 1900[28] Potassium alum
Mercuric oxide
 Nuclear stain in H&E, also used in the classical versions of the Papanicolaou stain[29]
Cole's Haematoxylin[1] Cole, 1943[30] Potassium alum Iodine Nuclear stain in H&E
Carazzi's Haematoxylin[1] Carazzi, 1911 Potassium alum Potassium iodate Nuclear stain in H&E, urgent biopsy sections
Weigert's Haematoxylin[26] Weigert, 1904
Ferric chloride
 Natural Nuclear stain in H&E, resistant to acids
Verhoeff's Haematoxylin[1] Verhoeff, 1908 Ferric chloride Iodine
elastic fibers, myelin[20]
phosphotungstic acid Haematoxylin[1]
 Mallory, 1897 Phosphotungstic acid Natural or chemical Fibrin, muscle striations
Gill's Haematoxylin (I, II, and III) Culling et al. 1985 [11][27] Aluminium sulfate Sodium iodate Nuclear stain in H&E

Early use as a histologic stain

In 1758,

Wilhelm von Waldeyer-Hartz used haematoxylin on animal tissue without a mordant (with limited success),[34] and is sometimes credited as being the first to do so,[8][12][35][34] although this is not universally accepted.[35][8] Franz Böhmer in 1865 published a haematoxylin formula using alum as a mordant,[34][21][12][8][35][31] and in 1891, Paul Mayer published a formulation using a chemical oxidizer to convert haematoxylin into haematein.[26][31][12] 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.[15][31] By the early 1900s, haematoxylin had become widely accepted as a histologic stain.[12]

Shortages and possible alternatives

During

medical diagnoses.[7] None of proposed replacement stains have been widely adopted.[14][7]

Use as a textile dye

Haematoxylin was first used as a dye by the

copperas or chrome as a mordant) for wool until the 1920s when a black synthetic dye compatible with wool became available.[9] Contemporary usage of haematoxylin includes the dyeing of silk, leather, and sutures.[7]

Use as a writing and drawing ink

Haematoxylin has been used as the primary component of writing and drawing

Van Gogh is known to have used haematoxylin ink with a chrome mordant in a number of his drawings and letters.[6][5][37]

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.
  • 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

References

  1. ^ a b c d e f g h i j k l m n o p q r s Stevens, Alan (1982). "The Haematoxylins". In Bancroft, John; Stevens, Alan (eds.). The Theory and Practice of Histological Techniques (2nd ed.). Longman Group Limited. p. 109.
  2. ^ a b c d Lillie, Ralph Dougall (1977). H. J. Conn's Biological stains (9th ed.). Baltimore: Williams & Wilkins. pp. 692p.
  3. doi:10.1039/AN9083300080
    .
  4. ^
    ISSN 0360-9081
    .
  5. ^
    ISSN 0377-0486
    .
  6. ^
    ISBN 9780300191875
    .
  7. ^
    S2CID 20338201
    .
  8. ^
    PMID 30001639
    .
  9. ^
    ISSN 2499-8281
    .
  10. ^
    S2CID 26847314
    .
  11. ^
    S2CID 205713596
    .
  12. ^
    PMID 16761865
    .
  13. ^
    ISSN 1472-3581
    .
  14. ^
    S2CID 28563610
    .
  15. ^
    S2CID 26801839
    .
  16. doi:10.1002/chir.12
    .
  17. S2CID 23504301
    .
  18. ^
    S2CID 7698557
    .
  19. doi:10.1016/0040-4020(70)85029-3
    .
  20. ^
    S2CID 7384777
    .
  21. ^
    S2CID 5297820
    .
  22. ^
    PMID 4139897
    .
  23. ^
    S2CID 29628388
    .
  24. PMID 4135791
    .
  25. ^
    S2CID 13481905
    .
  26. ^ a b c d e f g Gatenby, J. B.; Beams, H. W. (1950). The Microtomist's Vade-Mecum (11th ed.). Philadelphia: The Blackstone Company.
  27. ^
    S2CID 207513639
    .
  28. ^ Harris, H. F. (1900). "On the rapid conversion of haematoxylin into haematein in staining reactions". Journal of Applied Microscopic Laboratory Methods. 3 (3): 777.
  29. ISSN 1574-9053
    .
  30. ISSN 0038-9153
    .
  31. ^
    ISBN 978-0940095007
    .
  32. ^
    PMID 10605407
    .
  33. ^ Quekett, John Thomas (1848). A Practical treatise on the use of the microscope. Library of illustrated standard scientific works. Vol. VI. Paris: Hippolyte Bailliere.
  34. ^ a b c Mann, Gustav (1902). Physiological Histology, Methods and Theory. Clarendon Press. p. 488.
  35. ^
    PMID 9389971
    .
  36. ^ Georg von Georgievics (1902). The Chemical Technology of Textile Fibres: Their Origin, Structure, Preparation, Washing, Bleaching, Dyeing, Printing and Dressing. Scott, Greenwood & Co. p. 180.
  37. ^
    ISSN 0377-0486
    .
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