HAEMATOXYLIN AND EOSIN STAIN.pdf

HAEMATOXYLIN AND EOSIN STAIN BY B. A. OWUSU  WORKFLOW 02/03/2018 OBRIGHTA 2 MANUAL HEMATOXYLIN AND EOSIN 02/03/2018 OBRIGHTA 3 INTRODUCTION § The H&E stain is the most widely used histological stain. § It demonstrates clearly many different...

HAEMATOXYLIN AND EOSIN STAIN BY B. A. OWUSU  WORKFLOW 02/03/2018 OBRIGHTA 2 MANUAL HEMATOXYLIN AND EOSIN 02/03/2018 OBRIGHTA 3 INTRODUCTION § The H&E stain is the most widely used histological stain. § It demonstrates clearly many different tissues prepared in many different ways § It is simple to perform. § The hematoxylin stains the nuclei blue black with good intranuclear detail § Eosin stains the cytoplasm and connective tissue fibers different shades from pink –orange-red. 02/03/2018 OBRIGHTA 4  HAEMATOXYLIN § Hematoxylin is extracted from heartwood or bark of a tropical tree Haematoxylon campechianum § Originally from Mexico, Jamaica etc. § Extracted with hot water, precipitated with urea from aqueous solution. § The active stain is not the raw hematoxylin but the oxidation product called haematein. Hematoxylin is therefore not a dye to some degree. § Although 02/03/2018 a natural dye most modern haematoxylins OBRIGHTA are synthesized. 5 OXIDATION OF HAEMATOXYLIN TO HAEMATEIN (NATURAL OXIDATION) a. Exposure to light and air. b. Process slow. c. Takes 3-4 months. d. Dye solution retains ability to stain for long time. 02/03/2018 OBRIGHTA 6  OXIDATION OF HAEMATOXYLIN TO HAEMATEIN (CHEMICAL OXIDATION: by oxidizing agents) § Almost instantaneous ripening by chemicals. § The stain is ready for use immediately after preparation. § The dye solution retains its ability to stain for only a short period because further oxidation of the hematein occurs in air and light till it becomes a colorless compounds. 02/03/2018 OBRIGHTA 7 RIPENING AGENTS § Sodium iodate § Mercuric oxide § Iodine § Potassium iodate § KMnO4 § H2O2 02/03/2018 OBRIGHTA 8  MORDANTS § Hematein is weakly anionic and on its own has poor affinity for tissue it is therefore inadequate as a nuclear stain without a mordant. § Mordants are the salts or hydroxides of divalent or trivalent metals that promote binding between stain and tissue by acting as an intermediate binding agent. § They do this by conferring a net positive charge on the dye by virtue of their negative metallic ions. § The compound formed between a dye radical and the mordant is called a dye lake. § When attached to the tissue the dye lake is relatively permanent. § It is insoluble in neutral solutions and does not decolorize during dehydration and subsequent staining. 02/03/2018 OBRIGHTA 9 APPLICATION OF MORDANTS § Mordants can be used before applying the dye, at the same time as the dye or after the dye. § The type of mordant used influences very strongly the type of tissue components stained and their final color. § Mordants are either incorporated into the stain or the tissue is soaked in it before the stain is applied. 02/03/2018 OBRIGHTA 10 HAEMATOXYLINS ARE CLASSIFIED ACCORDING TO THE TYPES OF MORDANTS § ALUMMINIUM (ALUM) § IRON § TUNGSTEN § MOLYBDENUM § LEAD § CHROMIUM § DICHROMATE § WITHOUT MORDANTS 02/03/2018 OBRIGHTA 11 ALUM HAEMATOXYLINS § Mayer’s § Harris § Erhlichs § Delafield's § Cole § Carrazzi 02/03/2018 OBRIGHTA 12 ALUM HAEMATOXYLINS (2) (GENERALLY) § Mordants: (Usually) aluminium potassium sulphate (potash alum) aluminium ammonium sulphate (ammonium alum) § Initially stains the nuclei red but during bluing in weak alkaline solutions they become blue black § They are used as regressive stains as well as progressive stain. 02/03/2018 OBRIGHTA 13 ALUM HAEMATOXYLIN (ERHLICHS) § Mordant is alum. § Oxidant is natural air and light for about 2 months. § Lasts for years on bench in bottle and months in coplin jar. § Excellent nuclear stain. Intense and crisp and fades very slowly as compare to other alum hematoxylins. § Also stains mucins, cartilage and bones. § Use: very good for tissues exposed to acid – decalcified TISSUE; stored for long time in formalin (becomes acidic); Bouin’s fixative (acidic). § Not good for frozen sections § Delafield: similar to Erhlichs in longevity. Naturally ripening. 3-4 months. 02/03/2018 OBRIGHTA 14 ALUM HAEMATOXYLIN (Mayer’s) § Chemical ripening with sodium iodate. § Used progressively and regressively. § Progressively as a counterstain in special stains+ where the differentiation stage will damage the cytoplasmic stain. E.g. glycogen demonstration. 02/03/2018 OBRIGHTA 15 ALUM HAEMATOXYLIN (HARRIS) Ripened chemically by mercuric oxide. A General purpose hematoxylin. Gives clear nuclear staining. Used in cytology with eosin in a progressive method. In routine H&E used as a regressive stain. Addition of glacial acetic acid gives more precise and selective staining of nuclei. The quality reduces in months with formation of crystals in stored stain. Filter before use and stain for longer time. Prepare new stain each month. 02/03/2018 OBRIGHTA 16 OTHER ALUM HAEMATOXYLINS Cole- ripened by alcoholic iodine. Staining result and use are like Harris. Carrazzi – ripened by K Iodate. Used as a progressive nuclear counterstain like Mayer’s. Largely used in frozen section H&E modified rapid method. Good nuclear stain. Gills ripened with sodium iodate, May be used by a single (normal), double or triple dips in the stain. 02/03/2018 OBRIGHTA 17 IRON HAEMATOXYLINS § Weigert’s § Heidenhain’s § Verhoef § Loyez § Weil’s 02/03/2018 OBRIGHTA 18 CONTINUATION § Mordants are mainly iron salts: FeCl3, FeNH4SO4 § Iron Hematoxylins form stronger nucleic acid – dye bonds. § Particularly useful when vigorous counterstains like Van Gieson is to be used. § They have a problem with over-oxidation so, the hematoxylin and mordants/oxidant solutions are prepared separately. 02/03/2018 OBRIGHTA 19 CONTINUATION (2) § These oxidant/mordant solutions are strong oxidizers and are used before the hematoxylin for mordanting and also after the hematoxylin as differentiating fluid. ADVANTAGE § Stain wide range of tissues DISADVANTAGES § Time consuming, need differentiating steps controlled under the microscope for accuracy. § Staining times may vary with different fixatives. § If over-differentiated beyond desired end point, re-stain before attempting to re- differentiate. 02/03/2018 OBRIGHTA 20 IRON HAEMATOXYLINS (Weigert’s) § Mordant is FeCl3 (Ferric chloride). § Oxidant/mordant and hematoxylin are mixed just before use § Used with Van Gieson stain, § CNS tissues in H&E 02/03/2018 OBRIGHTA 21 IRON HAEMATOXYLINS (Heidenhain’s) § Mordant is ferric ammonium sulphate § Ferric ammonium sulphate is also used as differentiating fluid. § Mordant /oxidant are used sequentially with Heidenhain’s and Loyez. § Used for mitochondria, muscle striations, nuclear chromatin, myelin. RBCs and keratin § Staining times may vary with different fixatives. § If over-differentiated beyond desired end point, re-stain before attempting to re- differentiate. 02/03/2018 OBRIGHTA 22 TUNGSTEN HAEMATOXYLINS § The only widely used one is Mallory’s Phosphotungstic acid hematoxylin -Mallory’s PTAH. § Mordant is 1% aqueous Phosphotungstic acid. § Pretreatment of tissue in acid dichromate solution or Mallory bleach procedure is necessary for good result. § Demonstrates- muscle striations, neuroglial fibers, fibrin, amoebae- dark blue § Nuclei, cilia, rbc – blue § Myelin – light blue, 02/03/2018 OBRIGHTA 23 OTHER HAEMATOXYLINS § MOLYBDENUM HAEMATOXYLIN Rare mordant is molybdic acid. Used for collagen –stains violet and black. Reticulin fibers, argentaffin cell granules- black. § LEAD HAEMATOXYLIN Lead is mordant. Demonstrate endocrine cells in tumors of doubtful origin. Localization of gastrin cells in stomach. Solcia stain: used in the investigation of diffuse neuroendocrine system § CHROMIUM-COPPER HAEMATOXYLIN Weigert-PAL stain for myelin has mordant as chromium-copper 02/03/2018 OBRIGHTA 24 HAEMATOXYLIN WITHOUT MORDANTS § Freshly prepared hematoxylin with no mordant is used to demonstrate metals and minerals in tissue. lead, iron copper. § Principle: Unmordanted hematoxylin forms blue-black lakes with metals * Now superseded by other methods. 02/03/2018 OBRIGHTA 25 EOSINS § Eosins are the most suitable counterstain for alum hematoxylins. § Stains connective tissue fibers and can distinguish between cytoplasm of different cells when well differentiated. § Can also differentiate between different types of connective tissue fibers by staining different shades of pink –orange-red. § Eosins are xanthene dyes. Industry- for dying wool and silk. § Three types: Eosin Y, ethyl eosin S, and Eosin B 02/03/2018 OBRIGHTA 26 EOSIN Y §Eosin Y is the sodium salt of tetra bromo-fluorescence. The most widely used. §Usually prepared as a 0.5 to 1% solution in distilled water and alcohol soluble with a crystal of thymol added to inhibit the growth of fungi. §Acid, usually acetic acid is added to increase hydrogen ion concentration which helps improve the staining intensity “sharpens the dye”. 02/03/2018 OBRIGHTA 27 CONTINUATION § A crystal of thymol is added to inhibit fungi. § The eosin is differentiated in 70% alcohol and continues in the tap water and dehydration stages in alcohol during H&E stain. § It stains more intensely at high pH. § Eosin S and B is rarely used except for Negri bodies demonstration by Harris method 02/03/2018 OBRIGHTA 28 CONTINUATION § Under certain circumstances eosin staining is intense and difficulty may be experienced in obtaining adequate differentiation; this may occur after mercuric fixation. § Over-differentiation of eosin may be continued until only the red blood cells and granules of eosinophil polymorph are stained red. NB: Combining eosin Y and Phloxine B (10ml 1% Phloxine B, 100 ml 1% eosin Y, 780 ml 95% alcohol, 4 ml glacial acetic acid) produces a cytoplasmic stain, which more dramatically demonstrates various tissue components. 02/03/2018 OBRIGHTA 29  MOUNTING MEDIA 02/03/2018 OBRIGHTA 30 MOUNTANTS § Low viscosity media designed for mounting of tissue sections and cell smears. § In order to provide a maximum degree of transparency to stained tissue sections the refractive index should around 1.53 and 1.54. 02/03/2018 OBRIGHTA 31 Cover-Slipping 02/03/2018 OBRIGHTA 32 COVER GLASSES USED IN HISTOPATHOLOGY Care has to be exercised in selecting cover glasses for mounting, these are available in variable sizes and thickness and are supplied usually in 10 gm packings. Following sizes are commonly available 22 x 22 mm 25 x 50mm 22 x 30 mm Circular 22 x 40 mm Cover glass should preferably be the No. 1 thickness (0.13 - 0.16 mm), but never more than No. 1 1⁄2 thickness (0.16 - 0.19 mm). 02/03/2018 OBRIGHTA 33  AUTOMATED COVERSLIPPER 02/03/2018 OBRIGHTA 34 TYPES OF MOUNTANTS § Aqueous mountants. § Resinous mountants § Fluorescent-free mountant 02/03/2018 OBRIGHTA 35 AQUEOUS MOUNTANTS § Water based mounting media are designed for the permanent mounting of hydrated tissues, which may be damaged with organic solvents § Such samples include cell smears, with peroxidase and alkaline phosphatase chromogens. § It also preserves Fast Red, Aminoethyl carbazole (AEC), Tetrazolium salt (NBT) etc 02/03/2018 OBRIGHTA 36 ORGANIC BASED MOUNTANTS § These are xylene or other organic based mountants which can be used to permanently preserve chromogen that allow routine dehydration and clearing. § Since the procedures are the most common, the mountants are the most widely used 02/03/2018 OBRIGHTA 37 ORGANIC BASED MOUNTANTS Example § DPX mountant is a mixture of distrene, plasticizer (dibutyl phthlate) and xylene used as a synthetic resin mounting media. § DPX mountant dries quickly and preserves stain. § DPX mountant is suitable for HE- (Hematoxylin - Eosin) and Many other stains. § It is therefore the most widely used. 02/03/2018 OBRIGHTA 38 ORGANIC BASED MOUNTANTS Example § Limonene-Mount is made with limonene, a natural product from orange peels. It preserves tissues and cell smears that can be dehydrated with organic solvents in immunohistochemistry, e.g. DAB § Limonene-Mount also works well with alkaline phosphatase chromogens, and organic solvent resistant Supper Fast Red. § It is a good choice for mounting H & E stained slides 02/03/2018 OBRIGHTA 39 FLUORESCENT GEL MOUNTANT § Fluoro-Gels are water based mounting media designed for the permanent mounting of fluorescent stained tissues. § This prevents rapid photo-bleaching of fluorochromes FITC, Texas Red, Tetramethyl rhodamine, Phycoerythrin (RP-E), 02/03/2018 OBRIGHTA 40 02/03/2018 OBRIGHTA 41 THANK YOU, ANY QUESTION?? 02/03/2018 OBRIGHTA 42