Hematoxylin and eosin stain (orhaematoxylin and eosin stain or hematoxylin-eosin stain; often abbreviated as H&E stain or HE stain) is one of the principal tissue stains used in histology.[1][2][3] It is the most widely used stain in medical diagnosis[1] and is often the gold standard.[4] For example, when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E.
Main types of staining seen on H&E stain.
Retina (part of the eye) stained with hematoxylin and eosin, cell nuclei stained blue-purple and extracellular material stained pink.
H&E is the combination of two histological stains: hematoxylin and eosin. The hematoxylin stains cell nuclei a purplish blue, and eosin stains the extracellular matrix and cytoplasm pink, with other structures taking on different shades, hues, and combinations of these colors.[5][6] Hence a pathologist can easily differentiate between the nuclear and cytoplasmic parts of a cell, and additionally, the overall patterns of coloration from the stain show the general layout and distribution of cells and provides a general overview of a tissue sample's structure.[7] Thus, pattern recognition, both by expert humans themselves and by software that aids those experts (in digital pathology), provides histologic information.
This stain combination was first introduced in 1876 by A. Wissowzky.[8][7]
The H&E staining procedure is the principal stain in histology[3][7][2][5] in part because it can be done quickly,[7] is not expensive, and stains tissues in such a way that a considerable amount of microscopic anatomy[9][10] is revealed,[7][5][4] and can be used to diagnose a wide range of histopathologic conditions.[8] The results from H&E staining are not overly dependent on the chemical used to fix the tissue or slight inconsistencies in laboratory protocol,[11] and these factors contribute to its routine use in histology.[7]
H&E staining does not always provide enough contrast to differentiate all tissues, cellular structures, or the distribution of chemical substances,[9] and in these cases more specific stains and methods are used.[10][7]
Rack of slides being removed from a bath of hematoxylin stain.
There are many ways to prepare the hematoxylin solutions (formulation) used in the H&E procedure,[11][12][6] in addition, there are many laboratory protocols for producing H&E stained slides,[9] some of which may be specific to a certain laboratory.[7] Although there is no standard procedure,[11][9] the results by convention are reasonably consistent in that cell nuclei are stained blue and the cytoplasm and extracellular matrix are stained pink.[7] Histology laboratories may also adjust the amount or type of staining for a particular pathologist.[7]
After tissues have been collected (often as biopsies) and fixed, they are typically dehydrated and embedded in melted paraffin wax, the resulting block is mounted on a microtome and cut into thin slices.[6] The slices are affixed to microscope slides at which point the wax is removed with a solvent and the tissue slices attached to the slides are rehydrated and are ready for staining.[6] Alternatively, H&E stain is the most used stain in Mohs surgery in which tissues are typically frozen, cut on a cryostat (a microtome that cuts frozen tissue), fixed in alcohol, and then stained.[9]
The H&E staining method involves application of haematoxylin mixed with a metallic salt, or mordant, often followed by a rinse in a weak acid solution to remove excess staining (differentiation), followed by bluing in mildly alkaline water.[13][8][14] After the application of haematoxylin, the tissue is counterstained with eosin (most commonly eosin Y).[6][8][7]
Although hematein, an oxidized form of hematoxylin,[5][16][14] is the active colorant (when combined with a mordant), the stain is still referred to as hematoxylin.[8][13] Hematoxylin, when combined with a mordant (most commonly aluminum alum) is often considered to "resemble"[10] a basic, positively charged, or cationic stain.[5] Eosin is an anionic (negatively charged) and acidic stain.[5][10] The staining of nuclei by hemalum (a combination of aluminum ions and hematein)[14] is ordinarily due to binding of the dye-metal complex to DNA, but nuclear staining can be obtained after extraction of DNA[14] from tissue sections. The mechanism is different from that of nuclear staining by basic (cationic) dyes such as thionine or toluidine blue.[10] Staining by basic dyes occurs only from solutions that are less acidic than hemalum, and it is prevented by prior chemical or enzymatic extraction of nucleic acids. There is evidence to indicate that co-ordinate bonds, similar to those that hold aluminium and hematein together, bind the hemalum complex to DNA and to carboxy groups of proteins in the nuclear chromatin.
The structures do not have to be acidic or basic to be called basophilic and eosinophilic; the terminology is based on the affinity of cellular components for the dyes. Other colors, e.g. yellow and brown, can be present in the sample; they are caused by intrinsic pigments such as melanin. Basal laminae need to be stained by PAS stain or some silver stains, if they have to be well visible. Reticular fibers also require silver stain. Hydrophobic structures also tend to remain clear; these are usually rich in fats, e.g. adipocytes, myelin around neuron axons, and Golgi apparatus membranes.
Examples of H&E stained tissues
Bone, cell nuclei (blue-purple), bone matrix (pink).
Ductal carcinoma in situ (DCIS) in breast tissue, cell nuclei (blue-purple), extracellular material (pink).
Lung tissue taken from an emphysema patient. Cell nuclei (blue-purple), red blood cells (bright red), other cell bodies and extracellular material (pink), and air spaces (white).
Muscle tissue, cell nuclei (blue-purple), extracellular material (pink).
^ abTitford, M. (2005). "The long history of hematoxylin". Biotechnic & Histochemistry. 80 (2): 73–80. doi:10.1080/10520290500138372. PMID 16195172. S2CID 20338201.
^ abSmith C (2006). "Our debt to the logwood tree: the history of hematoxylin". MLO Med Lab Obs. 38 (5): 18, 20–2. PMID 16761865.
^ abDapson RW, Horobin RW (2009). "Dyes from a twenty-first century perspective". Biotech Histochem. 84 (4): 135–7. doi:10.1080/10520290902908802. PMID 19384743. S2CID 28563610.
^ abRosai J (2007). "Why microscopy will remain a cornerstone of surgical pathology". Lab Invest. 87 (5): 403–8. doi:10.1038/labinvest.3700551. PMID 17401434.
^ abcdefghChan JK (2014). "The wonderful colors of the hematoxylin-eosin stain in diagnostic surgical pathology". Int J Surg Pathol. 22 (1): 12–32. doi:10.1177/1066896913517939. PMID 24406626. S2CID 26847314.
^ abcdefStevens, Alan (1982). "The Haematoxylins". In Bancroft, John; Stevens, Alan (eds.). The Theory and Practice of Histological Techniques (2nd ed.). Longman Group Limited. p. 109.
^ abcdefghijklWittekind D (2003). "Traditional staining for routine diagnostic pathology including the role of tannic acid. 1. Value and limitations of the hematoxylin-eosin stain". Biotech Histochem. 78 (5): 261–70. doi:10.1080/10520290310001633725. PMID 14989644. S2CID 10563849.
^ abcdefTitford, Michael (2009). "Progress in the Development of Microscopical Techniques for Diagnostic Pathology". Journal of Histotechnology. 32 (1): 9–19. doi:10.1179/his.2009.32.1.9. ISSN 0147-8885. S2CID 26801839.
^ abcdeLarson K, Ho HH, Anumolu PL, Chen TM (2011). "Hematoxylin and eosin tissue stain in Mohs micrographic surgery: a review". Dermatol Surg. 37 (8): 1089–99. doi:10.1111/j.1524-4725.2011.02051.x. PMID 21635628. S2CID 2538853.{{cite journal}}: CS1 maint: multiple names: authors list (link)
^ abcdefRoss, Michael H.; Pawlina, Wojciech (2016). Histology : a text and atlas : with correlated cell and molecular biology (7th ed.). Wolters Kluwer. pp. 984p. ISBN978-1451187427.
^ abcSchulte EK (1991). "Standardization of biological dyes and stains: pitfalls and possibilities". Histochemistry. 95 (4): 319–28. doi:10.1007/BF00266958. PMID 1708749. S2CID 29628388.
^ abOrtiz-Hidalgo C, Pina-Oviedo S (2019). "Hematoxylin: Mesoamerica's Gift to Histopathology. Palo de Campeche (Logwood Tree), Pirates' Most Desired Treasure, and Irreplaceable Tissue Stain". Int J Surg Pathol. 27 (1): 4–14. doi:10.1177/1066896918787652. PMID 30001639.
^ abcdefKiernan JA (2018). "Does progressive nuclear staining with hemalum (alum hematoxylin) involve DNA, and what is the nature of the dye-chromatin complex?". Biotech Histochem. 93 (2): 133–148. doi:10.1080/10520295.2017.1399466. PMID 29320873. S2CID 13481905.
^ abLeeson, Thomas S.; Leeson, C. Roland (1981). Histology (Fourth ed.). W. B. Saunders Company. p. 600. ISBN978-0721657042.
^Kahr, Bart; Lovell, Scott; Subramony, Anand (1998). "The progress of logwood extract". Chirality. 10 (1–2): 66–77. doi:10.1002/chir.12.
Further reading
Kiernan JA (2008) Histological and Histochemical Methods: Theory and Practice. 4th ed. Bloxham, UK: Scion.
Lillie RD, Pizzolato P, Donaldson PT (1976) Nuclear stains with soluble metachrome mordant lake dyes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues. Histochemistry 49: 23–35.
Puchtler H, Meloan SN, Waldrop FS (1986) Application of current chemical concepts to metal-haematein and -brazilein stains. Histochemistry 85: 353–364.
External links
Wikimedia Commons has media related to H&E stain.
Protocol
Routine Mayer's Hematoxylin and Eosin Stain (H&E)
Hematoxylin & Eosin (H&E) Staining Protocol
February 21, 2023
stain, redirects, here, other, uses, disambiguation, hematoxylin, eosin, stain, haematoxylin, eosin, stain, hematoxylin, eosin, stain, often, abbreviated, stain, principal, tissue, stains, used, histology, most, widely, used, stain, medical, diagnosis, often, . H amp E redirects here For other uses see H amp E disambiguation Hematoxylin and eosin stain or haematoxylin and eosin stain or hematoxylin eosin stain often abbreviated as H amp E stain or HE stain is one of the principal tissue stains used in histology 1 2 3 It is the most widely used stain in medical diagnosis 1 and is often the gold standard 4 For example when a pathologist looks at a biopsy of a suspected cancer the histological section is likely to be stained with H amp E Main types of staining seen on H amp E stain Retina part of the eye stained with hematoxylin and eosin cell nuclei stained blue purple and extracellular material stained pink H amp E is the combination of two histological stains hematoxylin and eosin The hematoxylin stains cell nuclei a purplish blue and eosin stains the extracellular matrix and cytoplasm pink with other structures taking on different shades hues and combinations of these colors 5 6 Hence a pathologist can easily differentiate between the nuclear and cytoplasmic parts of a cell and additionally the overall patterns of coloration from the stain show the general layout and distribution of cells and provides a general overview of a tissue sample s structure 7 Thus pattern recognition both by expert humans themselves and by software that aids those experts in digital pathology provides histologic information This stain combination was first introduced in 1876 by A Wissowzky 8 7 Contents 1 Uses 2 Method of application 3 Results 4 Mode of action 5 Examples of H amp E stained tissues 6 References 7 Further reading 8 External links 8 1 ProtocolUses EditThe H amp E staining procedure is the principal stain in histology 3 7 2 5 in part because it can be done quickly 7 is not expensive and stains tissues in such a way that a considerable amount of microscopic anatomy 9 10 is revealed 7 5 4 and can be used to diagnose a wide range of histopathologic conditions 8 The results from H amp E staining are not overly dependent on the chemical used to fix the tissue or slight inconsistencies in laboratory protocol 11 and these factors contribute to its routine use in histology 7 H amp E staining does not always provide enough contrast to differentiate all tissues cellular structures or the distribution of chemical substances 9 and in these cases more specific stains and methods are used 10 7 Method of application EditMain articles Histology Sample preparation and Haematoxylin Rack of slides being removed from a bath of hematoxylin stain There are many ways to prepare the hematoxylin solutions formulation used in the H amp E procedure 11 12 6 in addition there are many laboratory protocols for producing H amp E stained slides 9 some of which may be specific to a certain laboratory 7 Although there is no standard procedure 11 9 the results by convention are reasonably consistent in that cell nuclei are stained blue and the cytoplasm and extracellular matrix are stained pink 7 Histology laboratories may also adjust the amount or type of staining for a particular pathologist 7 After tissues have been collected often as biopsies and fixed they are typically dehydrated and embedded in melted paraffin wax the resulting block is mounted on a microtome and cut into thin slices 6 The slices are affixed to microscope slides at which point the wax is removed with a solvent and the tissue slices attached to the slides are rehydrated and are ready for staining 6 Alternatively H amp E stain is the most used stain in Mohs surgery in which tissues are typically frozen cut on a cryostat a microtome that cuts frozen tissue fixed in alcohol and then stained 9 The H amp E staining method involves application of haematoxylin mixed with a metallic salt or mordant often followed by a rinse in a weak acid solution to remove excess staining differentiation followed by bluing in mildly alkaline water 13 8 14 After the application of haematoxylin the tissue is counterstained with eosin most commonly eosin Y 6 8 7 Results EditHematoxylin principally colors the nuclei of cells blue or dark purple 6 15 14 along with a few other tissues such as keratohyalin granules and calcified material Eosin stains the cytoplasm and some other structures including extracellular matrix such as collagen 5 7 14 in up to five shades of pink 8 The eosinophilic substances that are stained by eosin 5 structures are generally composed of intracellular or extracellular proteins The Lewy bodies and Mallory bodies are examples of eosinophilic structures Most of the cytoplasm is eosinophilic and is rendered pink 10 15 Red blood cells are stained intensely red Mode of action EditAlthough hematein an oxidized form of hematoxylin 5 16 14 is the active colorant when combined with a mordant the stain is still referred to as hematoxylin 8 13 Hematoxylin when combined with a mordant most commonly aluminum alum is often considered to resemble 10 a basic positively charged or cationic stain 5 Eosin is an anionic negatively charged and acidic stain 5 10 The staining of nuclei by hemalum a combination of aluminum ions and hematein 14 is ordinarily due to binding of the dye metal complex to DNA but nuclear staining can be obtained after extraction of DNA 14 from tissue sections The mechanism is different from that of nuclear staining by basic cationic dyes such as thionine or toluidine blue 10 Staining by basic dyes occurs only from solutions that are less acidic than hemalum and it is prevented by prior chemical or enzymatic extraction of nucleic acids There is evidence to indicate that co ordinate bonds similar to those that hold aluminium and hematein together bind the hemalum complex to DNA and to carboxy groups of proteins in the nuclear chromatin The structures do not have to be acidic or basic to be called basophilic and eosinophilic the terminology is based on the affinity of cellular components for the dyes Other colors e g yellow and brown can be present in the sample they are caused by intrinsic pigments such as melanin Basal laminae need to be stained by PAS stain or some silver stains if they have to be well visible Reticular fibers also require silver stain Hydrophobic structures also tend to remain clear these are usually rich in fats e g adipocytes myelin around neuron axons and Golgi apparatus membranes Examples of H amp E stained tissues Edit Bone cell nuclei blue purple bone matrix pink Ductal carcinoma in situ DCIS in breast tissue cell nuclei blue purple extracellular material pink Lung tissue taken from an emphysema patient Cell nuclei blue purple red blood cells bright red other cell bodies and extracellular material pink and air spaces white Muscle tissue cell nuclei blue purple extracellular material pink Basal cell carcinoma of the skin cell nuclei blue purple extracellular material pink References Edit a b Titford M 2005 The long history of hematoxylin Biotechnic amp Histochemistry 80 2 73 80 doi 10 1080 10520290500138372 PMID 16195172 S2CID 20338201 a b Smith C 2006 Our debt to the logwood tree the history of hematoxylin MLO Med Lab Obs 38 5 18 20 2 PMID 16761865 a b Dapson RW Horobin RW 2009 Dyes from a twenty first century perspective Biotech Histochem 84 4 135 7 doi 10 1080 10520290902908802 PMID 19384743 S2CID 28563610 a b Rosai J 2007 Why microscopy will remain a cornerstone of surgical pathology Lab Invest 87 5 403 8 doi 10 1038 labinvest 3700551 PMID 17401434 a b c d e f g h Chan JK 2014 The wonderful colors of the hematoxylin eosin stain in diagnostic surgical pathology Int J Surg Pathol 22 1 12 32 doi 10 1177 1066896913517939 PMID 24406626 S2CID 26847314 a b c d e f 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 a b c d e f g h i j k l Wittekind D 2003 Traditional staining for routine diagnostic pathology including the role of tannic acid 1 Value and limitations of the hematoxylin eosin stain Biotech Histochem 78 5 261 70 doi 10 1080 10520290310001633725 PMID 14989644 S2CID 10563849 a b c d e f Titford Michael 2009 Progress in the Development of Microscopical Techniques for Diagnostic Pathology Journal of Histotechnology 32 1 9 19 doi 10 1179 his 2009 32 1 9 ISSN 0147 8885 S2CID 26801839 a b c d e Larson K Ho HH Anumolu PL Chen TM 2011 Hematoxylin and eosin tissue stain in Mohs micrographic surgery a review Dermatol Surg 37 8 1089 99 doi 10 1111 j 1524 4725 2011 02051 x PMID 21635628 S2CID 2538853 a href Template Cite journal html title Template Cite journal cite journal a CS1 maint multiple names authors list link a b c d e f Ross Michael H Pawlina Wojciech 2016 Histology a text and atlas with correlated cell and molecular biology 7th ed Wolters Kluwer pp 984p ISBN 978 1451187427 a b c Schulte EK 1991 Standardization of biological dyes and stains pitfalls and possibilities Histochemistry 95 4 319 28 doi 10 1007 BF00266958 PMID 1708749 S2CID 29628388 Llewellyn BD 2009 Nuclear staining with alum hematoxylin Biotech Histochem 84 4 159 77 doi 10 1080 10520290903052899 PMID 19579146 S2CID 205713596 a b Ortiz Hidalgo C Pina Oviedo S 2019 Hematoxylin Mesoamerica s Gift to Histopathology Palo de Campeche Logwood Tree Pirates Most Desired Treasure and Irreplaceable Tissue Stain Int J Surg Pathol 27 1 4 14 doi 10 1177 1066896918787652 PMID 30001639 a b c d e f Kiernan JA 2018 Does progressive nuclear staining with hemalum alum hematoxylin involve DNA and what is the nature of the dye chromatin complex Biotech Histochem 93 2 133 148 doi 10 1080 10520295 2017 1399466 PMID 29320873 S2CID 13481905 a b Leeson Thomas S Leeson C Roland 1981 Histology Fourth ed W B Saunders Company p 600 ISBN 978 0721657042 Kahr Bart Lovell Scott Subramony Anand 1998 The progress of logwood extract Chirality 10 1 2 66 77 doi 10 1002 chir 12 Further reading EditKiernan JA 2008 Histological and Histochemical Methods Theory and Practice 4th ed Bloxham UK Scion Lillie RD Pizzolato P Donaldson PT 1976 Nuclear stains with soluble metachrome mordant lake dyes The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues Histochemistry 49 23 35 Llewellyn BD 2009 Nuclear staining with alum hematoxylin Biotech Histochem 84 159 177 Puchtler H Meloan SN Waldrop FS 1986 Application of current chemical concepts to metal haematein and brazilein stains Histochemistry 85 353 364 External links Edit Wikimedia Commons has media related to H amp E stain SIGMA ALDRICH H amp E Informational PrimerProtocol Edit Routine Mayer s Hematoxylin and Eosin Stain H amp E Hematoxylin amp Eosin H amp E Staining Protocol Rosen Lab Department of Molecular and Cellular Biology Baylor College of Medicine Step by step protocol Retrieved from https en wikipedia org w index php title H 26E stain amp oldid 1136269273, wikipedia, wiki, book, books, library,
