Hematoxylin and eosin staining

basics of Hematoxylin and eosin staining

1. HEMATOXYLIN AND EOSIN STAINING Dr Aarthiprabha K R DNB Resident Dept of Pathology

2. HISTORY OF HEMATOXYLIN It was independently introduced in 1865 and 1875, by Böhmer and Fischer respectively . Primary diagnostic technique in the histo-pathology laboratory. Waldeyer established the use of hematoxylin in histology in 1862.

3. Bohmer combines hematoxylin with alum as a mordant & obtained more specific staining in 1864. Heindenhan introduced classical Iron - Alum hematoxylin method Ehrlich overcame the instability of alum he m a t ox y lin by a d ding g l a c i a l a c e tic a c i d .

4. HE M A T OXY L IN The word hematoxylin is derived from old Greek word Haimato (blood) & Xylon (wood) , referring to its dark red colour in natural state. A natural dye extracted from the log wood of the tree Haematoxylon campechianum , mainly seen in Campeche state of Mexico & also available in West Indies

5. Hematoxylin itself is not a stain On oxidation it produces hematein (poor dye). Commonly used dye in histopathology, cytology, immuno histochemistry. Best nuclear stain. Basic in nature, stains acidic component of the tissue, nucleus, mitochondria etc.

6. OXIDATION Oxidation of hematoxylin is called RIPENING. Hematein can be produced in 2 ways Natural ripening Chemical ripening

7. 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 ) Cole’s hematoxylin Carazzi’s hematoxylin Gill’s hematoxylin

8. DIFFRENTIATION P rovides 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.

9. BLUEING Blueing is the process of shifting the color from reddish to purple blue by the application of a weak alkaline solution Blueing is utilized in both progressive and regressive techniques

10. Alkaline solutions used for bluing Tap water is alkaline enough to produce this colour change 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 )

11. MORDANT Hematin is anionic . Tissue is also anionic. Therefore hematin has poor affinity for tissue Making hematin adequate as a nuclear stain with the presence of a 3rd element (mordant) Mordant forms a link between the “tissue and the stain”

12. The chelate formed from a mordant dye and a metal is called a lake

13. MORDANT-DYE APPLICATION Mordant is applied first, followed by the dye. e.g Heidenhain’s iron hematoxylin Mordant and dye are mixed together and then applied. Commonly done in histotechnology e.g Alum hematoxylin solutions Dye applied first, followed by the mordant. Hardly done in histotechnology

14. TYPES OF HEMATOXYLIN According to the mordant used Alum hematoxylin Iron hematoxylin Tungsten hematoxylin Molybdenum hematoxylin Lead hematoxylin Hematoxylin with out mordant

15. ALUM HEMATOXYLIN Mordant used are aluminum salts aluminum potassium sulphate (potash alum) or aluminum ammonium sulphate (ammonium alum). Mainly used in routine H and E staining. Has different types but all of them stain the nuclei blue- black. These are used when counter stain does not contain an acid. Can be used progressively or regressively.

16. PROGRESSIVE STAINING Tissue is left in the stain just enough to reach the endpoint Frequent monitoring of stain quality may be needed to determine when staining is complete The staining intensity is controlled by the time it is immersed in the solution Eg : progressive staining using Gill’s Hematoxylin

17. REGRESSIVE STAINING Involves over-staining where the dye completely saturates all tissue elements The tissue is then selectively de-stained using a process called differentiation Eg :Harris Hematoxylin is popularly used regressively in many histology labs for routine H & E staining Sharper degree of staining is obtained

18. TYPES OF ALUM HAEMATOXYLIN Ehri l ch ’ s haem a t o xyl i n Mayer’s haematoxylin Harris’s haematoxylin Gill’s haematox ylin Cole’s haematoxylin Delafield’s haematoxylin Carazzi’s haematoxylin

19. 1. EHRLICH’S HEMATOXYLIN (1886) Strong stain for nuclei, stains intensely & crisply Oxidized naturally Stained sections fade slowly. Not ideal for frozen sections e

20. 2. HARRI’S HEMATOXYLIN (1885) Widely used in exfoliative cytology as a nuclear stain Chemical oxidation – mercuric oxide Life span is short : 2-3 months

21. 3. MAYER’S HEMATOXYLIN (1903) Used as a nuclear counterstain where the cytoplasmic material needed to demonstrate. Used as a progressive stain Chemical oxidation – sodium iodate Gives little or no staining of mucopolysaccharide material.

22. 4. DELAFIELD’S HEMATOXYLIN (1900) Naturally ripened Similar properies of Ehrlich’s hematoxylin

23. 5. COLE’S HEMATOXYLIN (1943) Chemically ripened with alcoholic iodine solution

24. 6. CARAZZI’S HEMATOXYLIN (1991) Chemically ripened with potassium iodate Used as a progressive nuclear stain. Largely confined to use with frozen sections

25. 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.

26. 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.

27. DISADVANTAGE OF ALUM HEMATOXYLIN Alum hematoxylin nuclear stain is sensitive to the subsequently applied acidic solutions . Common examples are Van Gieson & trichrome stain Satisfactory staining can be achieved by using Iron mordanted hematoxylin, which resist effect of picric acid . Combination of celestine blue with an alum

28. CELESTINE BLUE Oxazine Dye Has little useful coloring property of its own It forms an additional strong mordant with certain hematoxylins. Used as a preliminary to alum hematoxylin staining. Resistant to the effects of acid. Ferric salt in the celestine blue solution strengthens the bond between the nucleus and the alum hematoxylin to provide a strong nuclear stain which is reasonably resistant to acid.

29. Celestine blue B Ferric ammonium suphate Glycerine Distilled water : 2.5g : 25g : 70ml : 500ml Procedure Ferric ammonium sulphate is dissolved in cold distilled water with stirring. The celestine blue B is added to this solution and the mixture is boiled for few minutes. Filtered Glycerine is added, filter

30. IRON HEMATOXYLIN Iron salts used both as mordant & oxidizing agent Commonly used salts ferric chloride ferric ammonium sulphate. Over oxidation is the problem to avoid this prepare mordant / oxidant & hematoxylin seperately & mix them immediately before use.

31. Stain Mordant oxidation A p p li c a t i on / results Staining time Weigert’s Ferric chloride Natural Nuclear stain 15-30 min hematoxylin with acid dye, Stains nucleus brown to black Heidenhains’s Ferric Natural mitochondria, 30-45 min at hematoxylin ammonium chromatin & 60º C sulphate muscle fiber 12- 24 hrs at striation stains RT black or dark grey- black Verhoeff’s Ferric chloride Natural Stains elastic 25- 60 min hematoxylin fibers as black Loyez Ferric Natural Myelin hematoxylin ammonium sulphate

32. TUNGSTEN HEMATOXYLIN Mallory phosphotungstic acid hematoxylin (PTAH) is an example Phosphotungstic acid is used as mordant . Possible to prepare a staining solution using hematein ; instead of hematoxylin. Oxidation not required, can be used immediately , but short lived.

33. Can be naturally oxidized, takes months to ripen U sable for many years Can be oxidized chemically using potassium permanganate U sed to stain N ervous tissue M uscle striations F ibers

34. MOLYBDENUM HEMATOXYLIN Molybdic acid as the mordant. For the demonstration of collagen, coarse reticulin& granules in endocrine cells. Hydrogen per oxide is used for oxidation.

35. LEAD HEMATOXYLIN Lead salts act as the mordant. Identification of endocrine cells in some in tumors.

36. ALTERNATIVES TO HEMATOXYLIN Celestine blue Gallo cyanine Solochrome cyanine

37. EOSIN Most suitable stain to combine with alum hematoxylin . Eosins are xanthine dyes (tetrabromofluorescein) TYPES OF EOSIN - commercially available Ethyl eosin Eosin B Eosin Y

38. 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 sharpens the staining ETHYL EOSIN ( eosin alcohol-soluble ) EOSIN B (eosin bluish, erythrosine B)

39. ALTERNATIVES FOR EOSIN Phloxine Bierbrich scarlet saffranine

40. DIFFERENTIATION OF EOSIN Occurs in the subsequent tap water wash Further differentiation occurs during the dehydration through the alcohols

41. BASIC STEPS OF STAINING

42. R E S U L T

43. 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.

44. How does H & E staining relate to an ‘ ideal routine histological stain’’?

45. TROUBLESHOOTING IN H&E STAINING

46. P r o b l e m s Possible causes Too much differentiation Too less time in haematoxylin Due to excessive decalcification Haematoxylin is over oxidized Remedies 1. Stain in haematoxylin again Keep in haematoxylin for longer duration Not possible to correct Change the haematoxylin solution Darkly stained nuclei Too short differentiation Too much time in haematoxylin Thick section 1. Decolorize and do optimum differentiation Decolorize and give appropriate time in haematoxylin Recut thin section Nuclei looks reddish brown Insufficient bluing Haematoxylin is degenerating Restain by giving more time in bluing step Check the oxidation status of haematoxylin Pale stained nuclei

47. P r ob l e m s Pale coloured cytoplasm by eosin Too thin section The eosin solution has pH more than 5 Too much dehydration o f the section in alcohol 1. Recut the section properly May be due to dilution of eosin by the carryover bluing solution.Check pH of eosin solution Do not keep the slide in alcohol for a long time Incomplete dehydration Remove the mounting medium and coverslip. Keep the section in absolute alcohol Water bubbles in the section Possible causes Remedies

48. Bluish-black precipitate Staining is irregular and spotty May be due to precipitation of haematoxylin Improper deparaffinization Due to heating artefact for using electrocautery Filter the haematoxylin staining solution Keep the slide in xylene for longer time to remove the paraffin No solution Dark-blue stain at the edge of the tissue sections P r ob l e m s Possible causes Remedies

49. Pale stained nuclei Too dark nuclear stain

50. Incomplete deparaffinization Cytoplasmic staining is dark

51. Weak staining of eosin Uneven staining