Hematoxylin and Eosin Staining (H& E Staining)

Hematoxylin and Eosin Staining

1. HEM A T OXYLIN AND EOSIN STAINS Sunil Kumar. P St.John’s Medical College Bangalore 10/13/2018 1 SUNIL KUMAR. P

2. CON T E N T Hematoxylin Principles of hematoxylin Oxidation Mordant Classification of hematoxylin Alum hematoxylin Method of use of alum hematoxylin Bluing Differentiation Deterioration of the hematoxylin Types of alum hematoxylin Staining time with alum hematoxylin Disadvantages of alum hematoxylin 10/13/2018 2 SUNIL KUMAR. P

3. Iron hematoxylin Tungsten hematoxylin Lead hematoxylin Tungsten hematoxylin Molybedenum hematoxylin Hematoxylin without mordant Weigert Pal technique – of mordanting blocks Eosin Introduction Types of eosin commerrcially available Substitutes for eosin Differentiation Difficulties encountered 10/13/2018 3 SUNIL KUMAR. P

4. Staining: Is the process of coloring cells, cellular constituent & tissue fibers to facilitate optical differentiation by microscopic examination. Is the union between a colored dye & a tissue substrate which resists simple washing. . 10/13/2018 4 SUNIL KUMAR. P

5. It involves visual labeling of some entity by attaching, or depositing in its vicinity a marker of characteristic color or shape. Stain is the marker or reagent used to generate the marker. 10/13/2018 5 SUNIL KUMAR. P

6. 6 Chromophore Chromogen Principles of dye chemistry: All the dyes have an aromatic hydrocarbon benzene as a central component. HC CH HC CH HC CH 10/13/2018 6 SUNIL KUMAR. P

7. CHROMO G EN AUXOCHROME S U B S T R A TE A dye/stain is a coloured compound that binds to a substrate. It consists of a chromogen (colour) and auxochrome ( substrate binding component). Chromogen = benzene derivative + chromophore (colouring agent ). 10/13/2018 7 SUNIL KUMAR. P

8. CHROMO G EN AUXOCHROME S U B S T R A TE Auxochrome : give +ve or –ve charge to the chromogen. The ionized stain is capable of binding to cell structures with opposite charges. 10/13/2018 8 SUNIL KUMAR. P

9. Acidic dyes Acid dyes usually stain basic components such as cytoplasm, acidophil granules etc. e.g. Eosin, Acid fuchsin Basic dyes Usually stain acidic components such as nucleus, basophil granules etc. e.g. Hematoxylin , Basic Fuchsin, Methylene blue. 10/13/2018 9 SUNIL KUMAR. P

10. Electrostatic bonding Hydrogen bonding Van der Waal’s forces Covalent bonding Hydrophobic bonding Dye aggregation Tissue permeability STAINING MECHANISMS 9 10/13/2018 10 SUNIL KUMAR. P

11. ELECTROSTATIC BONDING ‘ Salt linkage’ & ‘Ionic bonding’ 1. Elecrostatic bonding: The affinity between opposite ionic groups of dye & tissue. Forces involved – Coulombic attraction Dyes are classified as Acidic dyes – Have a negative charge Basic dyes – Have a positive charge 10 10/13/2018 11 SUNIL KUMAR. P

12. 11 Acid dyes Anionic Chromogen Cationic Auxochrome Basic Dyes Cationic Chromogen Anionic Auxochrome Dyes thus carry an organic, charged moeity & an inorganic salt with an opposite charge 10/13/2018 12 SUNIL KUMAR. P

13. Binding of dye to tissue: When dye goes into tissue, they ionize or dissociate. Acid dyes provide available anions (chromogen) & cations that represent the auxochrome or salt. Basic dyes provide cationic chromogen & anionic auxochrome. The degree of ionization is pH dependent. 10/13/2018 13 SUNIL KUMAR. P

14. Reactive tissue groupings consist of Bound moiety of one charge Mobile moiety of opposite charge Staining occurs when a chromogen of one charge attracts to bound tissue moiety of opposite charge 13 10/13/2018 14 SUNIL KUMAR. P

15. STAINING OF BASIC CYTOPLASMIC PROTEIN BY ACID DYE EOSIN Tissue Eosin Stained tissue Free salt NH2 Cl + Chromogen Na = NH2 Chromogen + Na Cl 14 An acid dye has a coloured acid radical which attaches to a basic tissue compo nent 10/13/2018 15 SUNIL KUMAR. P

16. IN T ROD U C T ION Hematoxylin & eosin stain Is the most widely used histologic stain. Hematoxylin component stains  cell nuclei blue/black with good intranuclear detail. Eosin stains  cell cytoplasm & most connective tissue fibers in varying shades & intensities of pink, orange & red. 10/13/2018 16 SUNIL KUMAR. P

17. HEM A T OXY L IN It is extracted from the core of the tree HAEMATOXYLON CAMPECHIANUM. Hematoxlin - Greek word Haimato(blood) and Xylon(wood) , reffering to its dark red color in natural state and to its origin(wood). The hematoxylin is extracted from logwood with hot water and then precipitated out from the aqueous solution using urea. 10/13/2018 17 SUNIL KUMAR. P

18. For years it was used in textile industry until WALDEYER established its use in histology in 1862. Two years later Bohmer combined haematoxylin with alum as a mordant and obtained more specific staining. 10/13/2018 18 SUNIL KUMAR. P

19. Historical aspect of hematoxylin In 1891 Heidenhain introduced his classical Iron alum-haematoxylin method which today is still the standard technique of the cytologist. 10/13/2018 19 SUNIL KUMAR. P

20. Ehrlich (1886) who overcame the instability of hematoxylin and alum by the additions of glacial acetic acid and at the same time produced his formula for haematoxylin as it is used today . Historical aspect of hematoxylin 10/13/2018 20 SUNIL KUMAR. P

21. Hematoxylin is a misnomer!!!! natural extract obtained from the logs, hematoxylin is not an active dye. Hematoxylin is extracted and it is oxidised to haematein. Haematin is responsible for staining properties. In this process of oxidative conversion to haematin, hematoxylin loses 2 hydrogen atoms & assumes a quinoid arrangement in one of its rings. 10/13/2018 21 SUNIL KUMAR. P

22. Chemical Structures: Hematoxylin & Hematein He m ato x ylin He m atein Oxidation (loss of electron) is demonstrated by the loss of hydrogen and its electron from the Hematoxylin structure 10/13/2018 22 SUNIL KUMAR. P

23. NATURALLY RIPENED HEMATOXYLINS CHEMICALLY RIPENED HEMATOXYLINS Ripening by exposure to light & air Ripening by exposure to chemical oxidizing agents. Slow process (3-4 months) Ripening instantaneous, ready to use immediately after preparation Long shelf life, retain stability for a long time Shorter shelf life (because of continuing oxidation process in air & light eventually destroys much of the hematein converting it into a colourless compound) Example Ehrlich’s hematoxylin Delafield’s hematoxylin. Example Sodium iodate in Mayer’s hematoxylin (SIM) Mercuric chloride in Harris’s hematoxylin (MCh) 10/13/2018 23 SUNIL KUMAR. P

24. MARSHALL AND HOROBIN 1972 –said about overoxidation Over ripening leads to production of a large number of compounds which are colorless and useless. So correct amount of oxidant should be used. GLYCEROL - added to prevent over oxidation and reduce evaporation STABILISER Improve staining properties 10/13/2018 24 SUNIL KUMAR. P

25. MORD A NTS “ To bite” Biological staining – substance intermediate between dye and tissue + acid , base or neutral Bas i c 24 Mordant Dye LAKE 10/13/2018 25 SUNIL KUMAR. P

26. PRINCIPLE OF MORDANT Hematin is anionic . Tissue  is also anionic. Therefore  hematin has poor affinity for tissue Making hematin inadequate as a nuclear stain without the presence of a 3 rd element (mordant). Mordant forms a link between the “tissue and the stain” Dye mordant tissue complex 10/13/2018 26 SUNIL KUMAR. P

27. MOLECULAR PROPERTY OF MORDANTS Mordants are always di-valent and tri-valent salts or hydroxides of metals. They combine as hydroxides with the dye by displacing a hydrogen atom from the dye. The remaining valences of the mordant serves to attach/bind the dye-mordant complex to the tissue components (phosphate groups of the nucleic acid) Although simple salts such as sulfates and chlorides will do, generally double sulfates or alums are used. The double sulfates have An active usually trivalent metal such as iron, aluminium or chromium, Together with potassium or ammonium as a second cation. 10/13/2018 27 SUNIL KUMAR. P

28. DYE – TISSUE INTERACTIONS Covalent bonds between the metal ions and mordant dyes – are thought to facilitate dye-tissue binding (mordanting) INCORPORATION OF MORDANT It can be incorporated into the hematoxylin staining solution (most common way). The tissue section can be pretreated with mordant before staining Heidenhain’s iron hematoxylin . ADVANTAGES OF MORDANT The dye-mordant complex is virtually insoluble in most fluids. 10/13/2018 28 SUNIL KUMAR. P

29. MORDANTS USED WITH HEMATIN Most Commonly Used Salts of aluminium  in the form of potash alum or ammonium alum . Salts of iron Salts of Tungsten Less commonly used Salts of Lead Salts of molybdenum . 10/13/2018 29 SUNIL KUMAR. P

30. CLASSIFICATION OF HEMATOXYLIN Based on the Oxidation Procedure Natural oxidation – Ehrlich’s and Delafield’s Chemical Oxidation - Mayer’s and Harris II. Based on the Mordant Used Alum hematoxylin Iron hematoxylin Tungsten hematoxylin Lead hematoxylin Molybedenum hematoxylin Hematoxylin without mordant 10/13/2018 30 SUNIL KUMAR. P

31. Types of Alum hematoxylin Ehri l ch ’ s haem a t o xyl i n . Mayer’s haematoxylin . Harris’s haematoxylin . Gill’s haematoxy lin. Cole’s haematoxylin . Delafield’s haematoxylin . Carazzi’s haematoxylin . 10/13/2018 31 SUNIL KUMAR. P

32. 32 ALUM HEMATOXYLIN Routinely used Mordant - “ potash alum ” (aluminium potassium sulfate) or “ ammonium alum ” (aluminium ammonium sulfate) METHOD OF USE OF ALUM HEMATOXYLIN Alum hematoxylin can be used in 2 ways Regressively - the section is over stained & then differentiated in acid alcohol, followed by “bluing”. Progressively – stained for a pre determined time so as to adequately stain the nuclei but leave the background tissue relatively unstained. 10/13/2018 32 SUNIL KUMAR. P

33. DIF F E R E N TI A TION  provides a more controllable method in removing excess stain from tissue component and glass slide . Traditional HCl/alcohol acts quickly and indiscriminately, is more difficult to control, and can result in light nuclear stain. 1ml of 5 – 10% solution of acetic acid in 99ml of 70 – 95% alcohol detaches dye molecules from the cytoplasm/nucleoplasm while keeping nucleic acid complexes intact. 10/13/2018 33 SUNIL KUMAR. P

34. BLUING Int r oduction After differentiating the hematoxylin with acid alcohol, the nuclei in tissue are red colour This red colour is converted to blue black when section is washed in weak alkali solution  “ BLUING ”. Principle of bluing ALUM ( POTTASIUM ALUMINIUM SULPAHTE ) (ACIDIC SOLUTION) 10/13/2018 34 SUNIL KUMAR. P

35. Principle . Alum acts as mordant and hematoxylin containing alum stains the nucleus light blue . ... Bluing step converts the initial soluble red color within the nucleus to an insoluble blue color. The counterstaining is done by using eosin which imparts pink color to the cytoplasm. 10/13/2018 35 SUNIL KUMAR. P

36. Principle of bluing ALUM ( POTTASIUM ALUMINIUM SULPAHTE ) INSOLUBLE ALUMINIUM HYDROXIDE BLUE IN COLOUR OH FROM WATER SULPHURIC ACID (ACIDIC SOLUTION) FREE H ATOMS FROM WATER COMBINES WITH SULPHATE LACK OF OH GROUPS LACK OF INSOLUBLE ALUMINIUM HYDROXIDE RED COLOUR SO TO NEUTRALIZE A A LKALINE S OLUTION IS ADDED- BLUE INK 10/13/2018 36 SUNIL KUMAR. P

37. Alkaline solutions used for bluing Tap water is alkaline enough to produce this colour change. Substitute for alkaline solutions Scott’s tap water substitute Saturated Lithium carbonate (disadvantage – lithium has a tendency to form crystalline deposits unless the slides are agitated in it and well washed afterwards). Ammonia in distilled water (disadvantage – ammonia is “hard” on delicate tissues and will loosen sections from the slide). 10/13/2018 37 SUNIL KUMAR. P

38. DETERIORATION OF ALUM HEMATOXYLIN Deterioration is marked by the formation of a precipitate in the stored stain. At this stage the stain should be filtered before use and the staining time need to be increased. It is advised to prepare fresh batch of stain every month . Since it would be uneconomical it is prepared in small batches. TYPES OF ALUM HEMATOXYLIN Ehrlich’s hematoxylin (Ehrlich 1886) Delafield’s hematoxylin (Delafield 1885) Mayer’s hematoxylin (Mayer 1903) Harris hematoxylin (Harris 1900) Cole’s hematoxylin (Cole 1943) Carazzi’s hematoxylin (Carazzi 1991) Gill’s hematoxylin (Gill et al 1974) 10/13/2018 38 SUNIL KUMAR. P

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43. STAINING TIMES WITH ALUM HEMATOXYLIN Time varies according to the factors such as 1.Type of hematoxylin used Erhlcih’s  20 – 45 mins Mayer’s  10 – 20 mins 2. Age of stain As the stain ages  staining time has to be increased. 3. Intensity of use of stain Heavily used hematoxylin will lose its staining power more rapidly and longer staining times will be necessary. 4. Method of use of stain When used progressively  Mayer’s hematoxylin  5 – 10mins When used regressively  Mayer’s hematoxylin  10 – 20mins. 10/13/2018 43 SUNIL KUMAR. P

44. 5. Pre treatment of tissues or sections - Length of time In fixative In acid decalcifying solution or Whether paraffin or frozen sections Post treatment of sections – subsequent acid stains such as van Geison. Personal preference . General rule – Time Shortened  for frozen sections Increased  for decalcified tissues Increased  for those that have been stored for a long time in non buffered formalin. 10/13/2018 44 SUNIL KUMAR. P

45. DISADVANTAGES OF ALUM HEMATOXYLIN The sensitivity of these stains to any subsequently applied acidic staining solutions . Van Geison and other trichrome stains. Application of picric acid fucshin mixture in van Geison stain removes most of the hematoxylin so that the nuclei are barely discernable. Rectification Using iron mordanted hematoxylin such as Weigert’s hematoxylin , which is resistant to the effects of picric acid. Using a combination of Celestian blue staining solution with an alum hematoxylin . Commonly used Effect of celestian blue solution Celestian blue is resistant to the effects of acid Ferric salt in the prepared Celestine blue solution strengthens the bond between the nucleus and the alum hematoxylin to provide a 10/13/2018 45 SUNIL KUMAR. P

46. STEPS IN STAINING PROCEDURE FOR ALUM HEMATOXYLIN Dewaxing the sections (hot plate and then into xylene) Hydrating the sections (through graded alcohols 100%, 90%, 80%) Bring the sections to water Nuclear stain (Hematoxylin – harris – 5 – 10 mins) Differentiation (1% acid alcohol = 1% HCl in 99ml 70% alcohol) – 5-10s Wash well in tap water until sections are ‘blue’(10-15 minutes) Bluing - Blue by dipping in an alkaline solution (eg.ammonia water), followed by 5 min tap water wash. Stain in 1% Eosin Y for 10 min Dehydration Clearing Mounting 10/13/2018 46 SUNIL KUMAR. P

47. EOSIN IN T ROD U C T ION Most suitable stain to combine with alum hematoxylin. It has the ability for proper differentiation  to distinguish Between the cytoplasm of different types of cells & Between the different types of connective tissue fibers and matrices, by staining them different shades of red and pink. Eosins are xanthine dyes ( tetrabromofluorescein ) TYPES OF EOSIN - commercially available Eosin Y Ethyl eosin Eosin B 10/13/2018 47 SUNIL KUMAR. P

48. Eosin Y Eosin yellowish Most widely used It is water & alcohol soluble. Used as a cytoplasmic stain - 0.5-1% solution in distilled water with a Crystal of thymol - prevent fungal growth. Addition of Acetic acid (0.5 ml to 1000 ml) - sharpens the staining Ethyl eosin (eosin alcohol-soluble) Eosin B (eosin bluish, erythrosine B) 10/13/2018 48 SUNIL KUMAR. P

49. SUBSTITUTE FOR EOSIN Phloxine Bierbrich scarlet – gives a more intense red color to the tissues. They are rarely as amenable to subtle differentiation as eosin and are generally less valuable DIFFERENTIATION OF EOSIN Occurs in the subsequent tap water wash Further differentiation occurs during the dehydration through the alcohols DIFFICULTIES ENCOUNTERED  Under circumstances Eosin staining is intense and difficulty may be experienced in obtaining adequate differentiation (this may occur after mercuric fixation) Over differentiation of the eosin may be continues until only red blood cells and granules of eosniphil polymorph are stained red. This is occasionally used to facilitate the location and identification of eosinophils. 10/13/2018 49 SUNIL KUMAR. P

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52. IRON HEMATOXYLINS INTRODUCTION Here iron salts are used both as oxidizing agent and as mordant Most commonly used iron salts are ferric chloride and ferric ammonium sulfate TYPES OF IRON HEMATOXYLIN Weigert hematoxylin Heidenhan hematoxylin  muscle striation, mitochondria & Verhoeff hematoxylin for elastin fibers Loyez hematoxylin for myelin 10/13/2018 52 SUNIL KUMAR. P

53. DISADVANTAGE OF IRON HEMATOXYLIN 1. It has strong oxidizing ability - resulting in over oxidation of the hematoxylin. To overcome this Separate mordant /oxidant and hematoxylin solutions are prepared 2 solutions are Mixed immediately before use (example – Weigert hematoxylin) Used consecutively (example – Heidenhan’s and Loyez hematoxylin). Time consuming Since the staining technique incorporates a differentiation stage it needs microscopic control for accuracy. 10/13/2018 53 SUNIL KUMAR. P

54. DIFFERENTIATION OF HEMATOXYLIN Because of the strong oxidizing ability of the solution containing iron salts  the same iron solutions are used as a Mordanting fluid  before hematoxylin staining Differentiating fluid after hematoxylin staining. Method Iron solution is used first  acts as a mordant . The section is then treated with the hematoxylin solution until it is over-stained It is then differentiated with same iron solution under microscopic control – acts as a differentiating agent . ADVANTAGE OF IRON HEMATOXYLIN Capable of demonstrating a much wider range of tissue structures than alum hematoxylin 10/13/2018 54 SUNIL KUMAR. P

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58. APPLICATION OF HEIDENHAIN’S HEMATOXYLIN It can be used to demonstrate many structures according to the degree of differentiation . After staining  all components are black or dark grey black . The hematoxylin staining is removed progressively from different tissue structures at different rates using the iron alum solution . The black color disappears first from mitochondria  then from muscle striations  then from nuclear chromatin  more prolonged differentiation will remove the stain from almost all structures, although red blood cells and keratin retain the stain the longest. Therefore  Mitochondria, muscle striations, nuclear chromatin and myelin can all be demonstrated. 10/13/2018 58 SUNIL KUMAR. P

59. TUNGSTEN HEMATOXYLIN Only one wide l y used T ungsten he m atoxy l in  M a l l or y ’ s PTAH. MALLORY PTAH COMPOSITION Hematoxylin 15% aqueous phosphotungstic acid  mordant 10/13/2018 59 SUNIL KUMAR. P

60. OXIDATION Hematoxylin oxidation can be achieved by 3 ways No ripening Hematein can be used directly instead of hematoxylin Oxidation process is unnecessary Staining solution can be used immediately. But its activity is short comparatively. Chemical ripening Hematoxylin can be oxidized chemically by using potassium permanganate solution. Solution can be used within 24 hours. Natural ripening - Of tungstein hematoxylin solution in light and air. Most satisfactory method of preparation But time consuming because it may take months to ripen. But will remain usable for many years. Applications – demonstrate CNS material and general tissue structure. 10/13/2018 60 SUNIL KUMAR. P

61. Modifications of Mallory PTAH  based on the oxidation process PTAH technique using hematein  no oxidation PTAH solution  chemically oxidized with Potassium permanganate PTAH solution  naturally oxidized. 10/13/2018 61 SUNIL KUMAR. P

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64. WEIGERT-PAL technique - For demonstration of myelin Is a hematoxylin method in which the tissue block is mordanted in a chromate solution before embedding and sectioning Further mordanting of the section in a copper acetate solution is often performed before the hematoxylin is applied. The major mordant is chromium compound. 10/13/2018 64 SUNIL KUMAR. P

65. The H & E stain is popular It can be applied to tissues differing widely in nature and pretreatment (e.g., fixation) technically simple to use. 10/13/2018 65 SUNIL KUMAR. P

66. How does H & E staining relate to an ‘ ideal routine histological stain’’? 10/13/2018 66 SUNIL KUMAR. P

67. Theory and practice of histological techniques – Bancroft 5 th edition Cellular pathology technique – CFA Culling 4 th edition Histological staining methods – Disbray and Rack Koss Diagnostic Cytology and its Histopathologic basis - Leopold G Koss : volume one : 5 th edition Manual & Atlas of Fine Needle Aspiration Cytology : Svante R Orell REFER E NC E S 67 10/13/2018 67 SUNIL KUMAR. P

68. Thank you…… 10/13/2018 68 SUNIL KUMAR. P

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70. Qtn s : Which is the hematoxylin used in ihc ? - mayers. Which is the eosin used in ihc? – Eosin y What do u mean by1% acid alcohol?- 1% acid alcohol = 1% HCl in 99ml 70% alcohol) What h stain used in our lab? – Harris Harris is regreesively or progressively stained ? – both done, regressively for histology & progresively for cytology. Which is better for bluing ? Tap water or scotts tap water ?- tap water is not stable always…scott tap water more alkaline…. Composition of scott tap water? 10/13/2018 70 SUNIL KUMAR. P

71. Scott’s Tap Water/Bluing Magnesium sulfate (MgSO4)30.0 gm Sodium bicarbonate 2.0 gm Tap water 3000.0 ml Ammonia Water Ammonium hydroxide 5.0 ml Distilled water 1000.0 ml 0.05% Lithium Carbonate Lithium carbonate 0.5 gm Distilled water 1000.0 ml 10/13/2018 71 SUNIL KUMAR. P