Haematoxylin and its types

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1. HAEMATOXYLIN AND ITS TYPES

2. INTRODUCTION The word hematoxlin is drived from old Greek word Haimato (blood) and Xylon (wood), reffering to its dark red color in natural state and to its origin(wood). A natural dye extracted from the log wood of tree Haematoxylon Campechianum . It is the most important and most used dye in the histopathology, cytology and immunohistochemistry . Basic in nature and stains acidic component of the tissue, nucleus, mitochondria etc. It is one of the best nuclear stain.

3. Also use to stain metal ions e.g iron, lead etc. Stains tissue components in various shades of blue, pink, red etc, depending on the nature of the mordant used and the second stain. It is itself not an active dye, it is its oxidative product Haematein (natural dye). Haematein is a weak dye have poor affinity for tissue and requires a mordant for adequate staining. Mordant is a chemical substance that increases the affinity of dye for tissue and its staining efficiency by forming link between the stain and the tissue. Mordants are the metallic salts e.g salts of aluminum and iron etc.

4. Extraction: It is extracted by boiling the heart wood of Hematoxylon Campechianum in hot water. It is then precipitated out of the aqueous solution using urea or ether. Oxidation: Two methods of oxidation: 1. Natural oxidation: Exposed to air and sunlight. Takes 3-4 months to complete Resultant solutions have longer useful life.

5. Examples are Ehrlich’s and Delafield’s haematoxylin . 2. Chemical oxidation: Chemical oxidizing agents are used, e.g sodium iodate , mercuric oxide, hydrogen peroxide, potassium permanganate etc. Instant conversion of haematoxylin to haematein , immediately ready for use. Resultant solutions have a shorter useful life. Examples are Mayer’s and Harris’s haematoxylins .

6. TYPES OF HAEMATOXYLIN

7. ALUM HAEMATOXYLIN Mordant used are alumminum salts, either 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. All types are commonly reffered as haemalums . Haemalums are used when counter stain does not contain an acid. Can be used progressively or regressively.

8. Types of Alum hematoxylin

9. Stain Mordant Oxidation Applications Staining time Life span Ehrlich’s hematoxylin Potash alum Natural Nuclear stain used with eosin P= 20-45 min More than a year Mayer’s hematoxylin Ammonium or potash alum Chemical (Sodium iodate ) Nuclear stain used with eosin P=5-10 min R=10-20 min 3-4 months Hariss’s hematoxylin Ammonium or sodium alum Chemical (Mercuric oxide) Nuclear stain used with eosin P= 4-30 s R=5-15 min About 3 months Gill’s hematoxylin Aluminium sulphate Chemical (Sodium iodate ) Nuclear stain used with eosin R=5-15 min About 3 months Cole’s hematoxylin Potash alum Chemical (Alcoholic iodine solution) Nuclear stain used with eosin 20-45 min 3 months Delafield’s hematoxylin Ammonium alum Natural Nuclear stain used with eosin 15-20 min More than a year Carazzi’s hematoxylin Potash alum Chemical (Potassium iodate ) Nuclear stain used with eosin P= 1-2 min R= 10 min 6 months

10. Compositions of haemalums Ehrlich’s hematoxylin Hematoxylin 2g Absolute alcohol 100ml Distilled water 100ml Glycerin 100ml Glacial acetic acid 10ml Potash alum 15g (approx) Mayer’s hematoxylin Hematoxylin 1g Distilled water 1000ml Potash alum 50g Sodium iodate 0.2g Citric acid 1g Chloral hydrate 50g

11. Hariss’s hematoxylin Hematoxylin 2.5g Absolute alcohol 25ml Distilled water 500ml Mercuric oxide 1.5g Glacial acetic acid 20ml Potash alum 50g Gill’s hematoxylin Hematoxylin 2g Sodium iodate 0.2g Aluminium sulfate 17.6g Distilled water 750ml Ethylene glycol 250ml Glacial acetic acid 20ml

12. Cole’s hematoxylin Hematoxylin 1.5g Saturated aqueous potash alum 700ml 1% iodine in 95% alcohol 50ml Distilled water 250ml Delafield’s hematoxylin Hematoxylin 4g 95% alcohol 125ml Saturated aqueous potash alum 400ml Glycerin 100ml Carazzi’s hematoxylin Hematoxylin 5g Potash alum 25g Potassium iodate 0.1g Distilled water 400ml glycerol 100ml

13. IRON HEMATOXYLIN Iron salts are used as mordant, these are also oxidizing agents. Commonly used iron salts are ferric chloride and ferric ammonium sulphate . Overoxidation of the prepared and stored iron hematoxylins is the problem. To avoid this problem hematoxylin and mordant solutions are either mixed just before use (as in weigert’s hemtoxylin ) or used consecutively (as in heidenhain’s hematoxylin) These are used when the counter stain is acidic ( e.g Van Gieson and other Trichome stains). Demonstrate a much wider range of tissue structure but is time consuming due to differentiation stage.

14. Types of Iron hematoxylins

15. . Stain Mordant Oxidation Applications/ Results Staining time Weigert’s hematoxylin Ferric chloride Natural Nuclear stain with acid dye Stains the nucleus brown to black 15-30 min Heidenhain’s hematoxylin Ferric ammonium sulphate Natural Intracellular details (mitochondria, chromatin, nucleus, centrioles etc) and muscle fiber striations Stains cellular components black or dark gray-black 30-45 min at 60ºC 12-24 Hours at room temp. Verhoeff’s hematoxylin Ferric chloride Natural Stains elastic fibers black 25-60 min Loyez hematoxylin Ferric ammonium sulphate Natural Myelin

16. Compositions of Iron hematoxylins Weigert’s hematoxylin a) Hematoxylin solution b) Iron solution Hematoxylin 1g 30% aqueous ferric chloride 4ml Absolute alcohol 100ml Hydrochloric acid 1ml Distilled water 95ml Heidenhain’s hematoxylin a) Hematoxylin solution b) Iron solution Hematoxylin 0.5g Ferric ammonium sulphate 5g Absolute alcohol 10ml Distilled water 100ml Distilled water 90ml Verhoeff’s hematoxylin Stock solutions a) 5% alcoholic h ematoxylin 10ml b)10% ferric chloride 4ml c) Lugol’s iodine 4ml

17. Tungstun hematoxylin Mallory phosphotungstic acid hematoxylin (PTAH) is an example. Phosphotungstic acid is used as mordant. Hematoxylin is oxidized naturally, can also be oxidized using potassium permanganate. Can be prepared using hematein , no oxidation required. Routine stain for nervous tissue, also used to stain muscle striations and fibrin. Stains in shades of blue and red. Regressive stain with staining time of 1-16 hours at room temperature and 1-2 hours at 60ºC.

18. Molybdenum hematoxylin Molybdic acid is used as mordant. Very rare, most accepted molybdenum hematoxylin is Thomas hematoxylin . Hydrogen per oxide is used for oxidation. Used to stain chollagen and granules in endocrine cells. Stains: Chollagen - violet to black Argentaffin cells - black Nuclei - pale black

19. Solcia hematoxylin Lead salts are used as mordant No oxidation Used to demonstrate granules in endocrine cells. Mallory Hematoxylin No mordant required No oxidation Used to demonstrate various minerals in the tissue e.g iron, copper, lead.