# Hematoksilinsko-eozinska boja

Mrežnjača (dio oka), obojena hematoksilinom i eozinom: čelijska jedra obojene su plavo-ljubičastom, a vančelijski materijal ružičasto.
**Boja od hematoksilina i eozina** (često skraćeno kao "'boja H&E ili **HE boja**) jedno je od glavnih tkivnih bojila koja se koristi u histologiji. To je najčešće korištena boja u medicinskoj dijagnostici i često je zlatni standard. Naprimjer, kada patolog pogleda biopsiju sumnjivu na karcinom, histološki preparat će vjerovatno biti obojen H&E.

H&E je kombinacija dvije tkivne boje: hematoksilinom i eozinom. Hematoksilin boji ćelijsko jedro u plavo, a eozin vanćelijski matriks i citoplazmu u ružičastu boju, a druge strukture poprimaju različite nijanse, prelibe i kombinacije ovih boja. Otuda patolog može lahko razlikovati jedarni i citoplazmatski dio ćelije, a osim toga, ukupne obrasce obojenja od emisije boja, opći raspored i distribuciju ćelija i pruža općeniti pregled strukture uzorka tkiva. Dakle, prepoznavanje uzoraka, stručnjačko prepoznavanje uzorka i pomoću softvera koji pomaže stručnjacima, pruža histološke informacije.
Ovu kombinaciju boja je prvi put predstavio u A. Wissowzky, 1876.

## Upotreba

Postupak bojenja pomoću H&E glavno je bojenje u histologiji ref name="Dapson and Horobin, 2009"/> dijelom i zato što se to može učiniti brzo, nije skupo i bojenje tkiva na takav način omogućava da se otkriva značajna količina mikroskopske anatomije i može se koristiti za dijagnozu širokog spektra histopatoloških stanja. Rezultati bojanja pomoću H&E ne ovise pretjerano o hemikalijama koja se koristi za fiksiranje tkiva ili male nedosljednosti u laboratorijskom protokolu, i ovi faktori doprinose njegovoj rutinskoj upotrebi u histologiji.

H&E bojenje ne pruža uvijek dovoljan kontrast za razlikovanje svih tkiva, ćelijskih struktura ili distribuciju hemijskih supstanci,, a u tim slučajevima se koriste specifičnije boje i metodi.

## Metod primjene

 Stalak preparata koji se uklanjaju iz kupke s bojom od hematoksilina.
Postoji mnogo načina za pripremu rastvora hematoksilina (formulacija) koje se koriste u postupku H&E, pored toga, postoji mnogo laboratorijski protokola za proizvodnju H&E obojenih preparata, od kojih neki mogu biti specifični za određenu laboratoriju. Iako ne postoji standardni postupak, rezultati po dogovoru su razumno dosljedni u tome što su ćelijska jedra obojene plavo, a citoplazma i vanćelijski matriks ružičasto. Histološke laboratorije mogu također prilagoditi količinu ili tip bojenja za određene patološka stanja.

Nakon što se tkiva prikupe (često kao biopsijama) i fiksiraju, obično se dehidriraju i ugrade u rastopljeni parafinski vosak, a rezultirajući blok se postavi na mikrotom i iseče na tanke kriške. Odsječci su pričvršćene na mikroskopsko stakalce, a zatim se vosak uklanja rastvaračem, a kriške tkiva pričvršćene za preparate su rehidrirane i spremne za bojenje. Alternativno, H&Eboja je najčešće korištena u Mohsovoj hirurgiji u kojoj se tkiva obično zamrzavaju, režu na kriostatu (mikrotom koji reže smrznuto tkivo), fiksirana u alkoholu, a zatim obojena.

Metod H&E bojenja uključuje primjenu hematoksilina pomiješanog sa metalnom solju ili mordantom, nakon čega slijedi ispiranje u rastvoru slabe kiseline, kako bi se uklonila suvišna boja (*diferencijacija*), nakon čega slijedi *modrenje* u blago alkalnoj vodi. Nakon nanošenja hematoksilina, tkivo se kontrastira dolijevanjem eozina (najčešće eozin Y).

## Rezultati

Hematoksilin uglavnom boji ćelijska jedra plavo ili tamnoljubičasto, zajedno s nekoliko drugih tkiva, poput keratohijalinskih granula i kalcificiranog materijala. Eozin boji citoplazmu i neke druge strukture uključujući vanćelijski matriks kao što je kolagen u do pet nijansi ružičaste boje. eozinofili (supstance koje eozin boji) strukture se uglavnom sastoje od unutarćelijskih ili vanćelijskih proteina. Lewyjeva tijela i Malloryjeva tijela su primjeri eozinofilnih struktura. Većina citoplazme je eozinofilna i postaje ružičasta. Crvena krvna zrnca su obojena intenzivno crvenom bojom.

## Način djelovanja

Iako hematein, oksidirani oblik hematoksilina,<ref name ="Kiernan, 2018">je aktivno sredstvo za bojenje (kada se kombinira s lijekom), boja se i dalje naziva "hematoksilin".
 Hematoksilin, u kombinaciji sa metalom (najčešće aluminijumom, često se smatra da "nalikuje" osnovnoj, pozitivno nabijenoj ili kationskoj boji. Eozin je anionska (negativno nabijena) i kisela boja. Bojenje jedara **hemalumom** (kombinacija iona aluminija i hemateina) obično je složen zbog vezanja metala za bojenje DNK, ali jedarno bojenje može se dobiti nakon ekstrakcije DNK iz odsječka tkiva. Mehanizam se razlikuje od mehanizma nujedarnog bojenja osnovnim (kationskim) bojama, kao što je tionin ili toluidinsko plavo. Bojenje osnovnim bojama dolazi samo od rastvora koji su manje kiseli od hemaluma, a to se sprečava prethodnom hemijskom ili enzimskom ekstrakcijom nukleinskih kiselina. Postoje dokazi koji ukazuju na to da koordinatne veze, slične onima koje drže aluminij i hematein zajedno, vežu kompleks hemaluma za DNK i karboksi grupe proteina u jedarnom hromatinu. Strukture ne moraju biti kisele ili bazne da bi se nazivale bazofilne i eozinofilne; terminologija se zasniva na afinitetu ćelijskih komponenata prema bojama. Ostale boje, npr. žuta i smeđa, mogu biti prisutne u uzorku; uzrokuju ih unutrašnji pigmenti kao što je melanin. Baznu lamelu treba bojiti PAS ili nekib srebrenim bojama, ako moraju biti dobro vidljive. Mrežasta vlakna zahtijevaju i srebrnu boji. Hidrofobne strukture također ostaju jasne; to su obično bogate mastima, npr. adipociti, mijelin oko neuronskih aksona i Golgijev aparat membrana.

## Primjeri tkiva obojenih H&E

Hrskavica, ćelijska jedra (plavo-ljubičasta), vanćelijski materijal (ružičasti)
Duktusni karcinom *in situ* (DCIS) u tkivu dojke, ćelijskim jedrima (plavo-ljubičasta), vanćelijskom materijalu
Plućno tkivo puzeto iz emfizema pacijenta. Ćelijska jedra (plavo-ljubičasta), crvene krvne ćelije (jarko crvene), druga ćelijska tijela i vanćelijski materijal (ružičast) i zračni prostori (bijeli)
Mišićno tkivo, ćelijska jedra (plavo-ljubičasta), vanćelijski materijal (ružičast)
Karcinom baznih ćelija kože, ćelijska jedra (plavo-ljubičasta), vanćelijski materijal (ružičast)

## Reference

## Dodatna literatura

* 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.
* 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.

## Vanjski linkovi

* SIGMA-ALDRICH H&E Informational Primer

### Protokol

* Routine Mayer's Hematoxylin and Eosin Stain (H&E) Arhivirano 2. 6. 2023. na Wayback Machine
* Hematoxylin & Eosin (H&E) Staining Protocol Arhivirano 5. 3. 2022. na Wayback Machine
* Rosen Lab, Department of Molecular and Cellular Biology, Baylor College of Medicine) Step by step protocol

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| Boje sortirane od tamnih prema svjetlim |
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| Navy | #000080 |
| Purple | #800080 |
| Indigo | #4B0082 |
| MidnightBlue | #191970 |
| DarkRed | #8B0000 |
| DarkCyan | #008B8B |
| DarkBlue | #00008B |
| DarkMagenta | #8B008B |
| DarkOliveGreen | #556B2F |
| SaddleBrown | #8B4513 |
| ForestGreen | #228B22 |
| OliveDrab | #6B8E23 |
| SeaGreen | #2E8B57 |
| DarkGoldenrod | #B8860B |
| DarkSlateBlue | #483D8B |
| Sienna | #A0522D |
| MediumBlue | #0000CD |
| Brown | #A52A2A |
| DarkTurquoise | #00CED1 |
| DimGray | #696969 |
| LightSeaGreen | #20B2AA |
| DarkViolet | #9400D3 |
| FireBrick | #B22222 |
| MediumVioletRed | #C71585 |
| MediumSeaGreen | #3CB371 |
| Chocolate | #D2691E |
| Crimson | #DC143C |
| Goldenrod | #DAA520 |
| SteelBlue | #4682B4 |
| MediumSpringGreen | #00FA9A |
| LawnGreen | #7CFC00 |
| DarkOrchid | #9932CC |
| Red | #FF0000 |
| OrangeRed | #FF4500 |
| DarkOrange | #FF8C00 |
| Orange | #FFA500 |
| Gold | #FFD700 |
| Yellow | #FFFF00 |
| YellowGreen | #9ACD32 |
| Chartreuse | #7FFF00 |
| LimeGreen | #32CD32 |
| Lime | #00FF00 |
| SpringGreen | #00FF7F |
| Aqua | #00FFFF |
| Cyan | #00FFFF |
| CadetBlue | #5F9EA0 |
| DeepSkyBlue | #00BFFF |
| Blue | #0000FF |
| Fuchsia | #FF00FF |
| Magenta | #FF00FF |
| Gray | #808080 |
| SlateGray | #708090 |
| Peru | #CD853F |
| BlueViolet | #8A2BE2 |
| LightSlateGray | #778899 |
| DeepPink | #FF1493 |
| MediumTurquoise | #48D1CC |
| DodgerBlue | #1E90FF |
| Turquoise | #40E0D0 |
| RoyalBlue | #4169E1 |
| SlateBlue | #6A5ACD |
| DarkKhaki | #BDB76B |
| MediumOrchid | #BA55D3 |
| IndianRed | #CD5C5C |
| GreenYellow | #ADFF2F |
| MediumAquamarine | #66CDAA |
| Tomato | #FF6347 |
| Orchid | #DA70D6 |
| RosyBrown | #BC8F8F |
| DarkSeaGreen | #8FBC8F |
| MediumPurple | #9370DB |
| PaleVioletRed | #DB7093 |
| Coral | #FF7F50 |
| CornflowerBlue | #6495ED |
| DarkGray | #A9A9A9 |
| SandyBrown | #F4A460 |
| MediumSlateBlue | #7B68EE |
| Tan | #D2B48C |
| DarkSalmon | #E9967A |
| BurlyWood | #DEB887 |
| HotPink | #FF69B4 |
| Salmon | #FA8072 |
| LightCoral | #F08080 |
| Violet | #EE82EE |
| SkyBlue | #87CEEB |
| LightSalmon | #FFA07A |
| Khaki | #F0E68C |
| Plum | #DDA0DD |
| LightGreen | #90EE90 |
| Aquamarine | #7FFFD4 |
| Silver | #C0C0C0 |
| LightSkyBlue | #87CEFA |
| LightSteelBlue | #B0C4DE |
| PaleGreen | #98FB98 |
| LightBlue | #ADD8E6 |
| PowderBlue | #B0E0E6 |
| Thistle | #D8BFD8 |
| PaleGoldenrod | #EEE8AA |
| PaleTurquoise | #AFEEEE |
| LightGrey | #D3D3D3 |
| Wheat | #F5DEB3 |
| NavajoWhite | #FFDEAD |
| Moccasin | #FFE4B5 |
| LightPink | #FFB6C1 |
| Gainsboro | #DCDCDC |
| PeachPuff | #FFDAB9 |
| Pink | #FFC0CB |
| Bisque | #FFE4C4 |
| BlanchedAlmond | #FFEBCD |
| LemonChiffon | #FFFACD |
| LightGoldenrodYellow | #FAFAD2 |
| AntiqueWhite | #FAEBD7 |
| Beige | #F5F5DC |
| PapayaWhip | #FFEFD5 |
| Cornsilk | #FFF8DC |
| LightYellow | #FFFFE0 |
| LightCyan | #E0FFFF |
| MistyRose | #FFE4E1 |
| Linen | #FAF0E6 |
| Lavender | #E6E6FA |
| OldLace | #FDF5E6 |
| WhiteSmoke | #F5F5F5 |
| Seashell | #FFF5EE |
| FloralWhite | #FFFAF0 |
| Ivory | #FFFFF0 |
| Honeydew | #F0FFF0 |
| Azure | #F0FFFF |
| AliceBlue | #F0F8FF |
| LavenderBlush | #FFF0F5 |
| MintCream | #F5FFFA |
| GhostWhite | #F8F8FF |
| Snow | #FFFAFA |
| White | #FFFFFF |

 |

| Boje sortirane po abecedi |
| --- |
| AliceBlue | #F0F8FF |
| AntiqueWhite | #FAEBD7 |
| Aqua | #00FFFF |
| Aquamarine | #7FFFD4 |
| Azure | #F0FFFF |
| Beige | #F5F5DC |
| Bisque | #FFE4C4 |
| Black | #000000 |
| BlanchedAlmond | #FFEBCD |
| Blue | #0000FF |
| BlueViolet | #8A2BE2 |
| Brown | #A52A2A |
| BurlyWood | #DEB887 |
| CadetBlue | #5F9EA0 |
| Chartreuse | #7FFF00 |
| Chocolate | #D2691E |
| Coral | #FF7F50 |
| CornflowerBlue | #6495ED |
| Cornsilk | #FFF8DC |
| Crimson | #DC143C |
| Cyan | #00FFFF |
| DarkBlue | #00008B |
| DarkCyan | #008B8B |
| DarkGoldenrod | #B8860B |
| DarkGray | #A9A9A9 |
| DarkGreen | #006400 |
| DarkKhaki | #BDB76B |
| DarkMagenta | #8B008B |
| DarkOliveGreen | #556B2F |
| DarkOrange | #FF8C00 |
| DarkOrchid | #9932CC |
| DarkRed | #8B0000 |
| DarkSalmon | #E9967A |
| DarkSeaGreen | #8FBC8F |
| DarkSlateBlue | #483D8B |
| DarkSlateGray | #2F4F4F |
| DarkTurquoise | #00CED1 |
| DarkViolet | #9400D3 |
| DeepPink | #FF1493 |
| DeepSkyBlue | #00BFFF |
| DimGray | #696969 |
| DodgerBlue | #1E90FF |
| FireBrick | #B22222 |
| FloralWhite | #FFFAF0 |
| ForestGreen | #228B22 |
| Fuchsia | #FF00FF |
| Gainsboro | #DCDCDC |
| GhostWhite | #F8F8FF |
| Gold | #FFD700 |
| Goldenrod | #DAA520 |
| Gray | #808080 |
| Green | #008000 |
| GreenYellow | #ADFF2F |
| Honeydew | #F0FFF0 |
| HotPink | #FF69B4 |
| IndianRed | #CD5C5C |
| Indigo | #4B0082 |
| Ivory | #FFFFF0 |
| Khaki | #F0E68C |
| Lavender | #E6E6FA |
| LavenderBlush | #FFF0F5 |
| LawnGreen | #7CFC00 |
| LemonChiffon | #FFFACD |
| LightBlue | #ADD8E6 |
| LightCoral | #F08080 |
| LightCyan | #E0FFFF |
| LightGoldenrodYellow | #FAFAD2 |
| LightGreen | #90EE90 |
| LightGrey | #D3D3D3 |
| LightPink | #FFB6C1 |
| LightSalmon | #FFA07A |
| LightSeaGreen | #20B2AA |
| LightSkyBlue | #87CEFA |
| LightSlateGray | #778899 |
| LightSteelBlue | #B0C4DE |
| LightYellow | #FFFFE0 |
| Lime | #00FF00 |
| LimeGreen | #32CD32 |
| Linen | #FAF0E6 |
| Magenta | #FF00FF |
| Maroon | #800000 |
| MediumAquamarine | #66CDAA |
| MediumBlue | #0000CD |
| MediumOrchid | #BA55D3 |
| MediumPurple | #9370DB |
| MediumSeaGreen | #3CB371 |
| MediumSlateBlue | #7B68EE |
| MediumSpringGreen | #00FA9A |
| MediumTurquoise | #48D1CC |
| MediumVioletRed | #C71585 |
| MidnightBlue | #191970 |
| MintCream | #F5FFFA |
| MistyRose | #FFE4E1 |
| Moccasin | #FFE4B5 |
| NavajoWhite | #FFDEAD |
| Navy | #000080 |
| OldLace | #FDF5E6 |
| Olive | #808000 |
| OliveDrab | #6B8E23 |
| Orange | #FFA500 |
| OrangeRed | #FF4500 |
| Orchid | #DA70D6 |
| PaleGoldenrod | #EEE8AA |
| PaleGreen | #98FB98 |
| PaleTurquoise | #AFEEEE |
| PaleVioletRed | #DB7093 |
| PapayaWhip | #FFEFD5 |
| PeachPuff | #FFDAB9 |
| Peru | #CD853F |
| Pink | #FFC0CB |
| Plum | #DDA0DD |
| PowderBlue | #B0E0E6 |
| Purple | #800080 |
| Red | #FF0000 |
| RosyBrown | #BC8F8F |
| RoyalBlue | #4169E1 |
| SaddleBrown | #8B4513 |
| Salmon | #FA8072 |
| SandyBrown | #F4A460 |
| SeaGreen | #2E8B57 |
| Seashell | #FFF5EE |
| Sienna | #A0522D |
| Silver | #C0C0C0 |
| SkyBlue | #87CEEB |
| SlateBlue | #6A5ACD |
| SlateGray | #708090 |
| Snow | #FFFAFA |
| SpringGreen | #00FF7F |
| SteelBlue | #4682B4 |
| Tan | #D2B48C |
| Teal | #008080 |
| Thistle | #D8BFD8 |
| Tomato | #FF6347 |
| Turquoise | #40E0D0 |
| Violet | #EE82EE |
| Wheat | #F5DEB3 |
| White | #FFFFFF |
| WhiteSmoke | #F5F5F5 |
| Yellow | #FFFF00 |
| YellowGreen | #9ACD32 |

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