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PAP Stain) for Cytology Principle of the H&E Stain

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PAP Stain) for Cytology Principle of the H&E Stain Tissue Stains (H&E) (PAP) Prepared by M Hlasek March 2017 Reviewed February 2020 Aims of staining Commonly used medical process in the medical diagnosis of tumors Technique used to enhance contrast in samples Make the cell structure visible Show variation in structure Indicate the chemical nature of tissue entities Staining methods Haematoxylin o Three main types: Alum Iron Tungsten o Other: Lead Molebdenum Haematoxylin without mordant These haematoxylins are named as such because of the mordant that is used. Alum Haematoxylin The mordant contains aluminium eg Potassium aluminium sulphate in Mayer’s Haematoxylin. Disadvantage: very sensitive to acid solutions Iron Haematoxylin Here the mordant is an iron salt eg ferric chloride or ferric ammonium. These salts acts also as the oxidising agent. Disadvantage: Over oxidise and “ripen” very quickly Tungsten Haematoxylin The haematoxylin can be ripened chemically with potassium permanganate or left to ripen in sunlight. Disadvantage: only Mallory’s phosphotungstic acid haematoxylin is the only one widely used Mordant Is a substance, typically an inorganic oxide, that combines with a dye or stain and thereby fixes it in a material/tissue section. Ionic bonding / Coulombic attractions Acid and basic dyes, and other ionic reagents, including inorganic salts Hydrogen bonding Is a dye-tissue attraction arising when a hydrogen atom lies between two electronegative atoms (e.g. oxygen or nitrogen) Van der Waals forces Intermolecular attractions as dipole-dipole, dipole-induced dipole and dispersion forces. These occur between all reagents and tissue substrates. Van der Waals Forces cont. Covalent bonds Between tissue and stain also occurs, which bonds may be regarded merely as another source of stain-tissue affinity Covalent bonds cont. Hydrophobic interaction Staining systems using aqueous solutions of dyes or other organic reagents; enzyme substrates for example Hydrophobic interaction Histology classification of dyes Basic dyes – these are cationic dyes and will stain anionic or acidic materials (e.g. the phosphates in nucleic acid) in the tissue e.g. Methylene Blue, Safranin, Basic fuchsin. Acidic substances that stain with basic dyes are termed basophilic Acidic dyes - these are anionic dyes and will stain cationic or basic groups in tissue such as amino groups e.g. Picric acid, Eosin, Acid fuchsin. Substances that stain with acid dyes are called acidophilic Histology classification of dyes continue Neutral Dyes- simply compounds of basic and acidic dyes. Such dye complexes will stain both nucleus and cytoplasm e.g. Romanowsky stains, EosinY or Eosin B Amphoteric dyes - have both anionic and cationic groups, but on the same ion. Such dyes stain either the nucleus or the cytoplasm if conditions are appropriate e.g. Celestine blue B, Acid fuchsin Natural dyes – dye substances extracted from natural sources e.g. Haematoxylin from the logwood of a tree. Important dyes in histology Nuclear stains Haematoxylin Carmine and Carminic acid Methylene blue Neutral Red and Safranine O Methyl green Toluidine blue Cytoplasmic stains Eosin Methyl blue and aniline blue Fast green FCF and Light green SF Orange G Routine stains Haematoxylin and Eosin (H &E Stain) for histology Papanicolaou stain (PAP Stain) for cytology Principle of the H&E Stain The staining method is based on the chemical attraction between tissue and dye. Charges on the dye and tissue are opposite and therefore attract (Van der Waals forces or Ionic bonding) The Nuclei is acidic (-ve charges) and reacts with haematoxylin a basic dye The cytoplasm or connective tissue are basic (+ve charges) components reach with Eosin an Acid dye. Method H&E Stain a. Scott’s Tap Water Sodium hydrogen carbonate 3.5gm/17.5gm Magnesium sulphate 20gm/100 gm Tap water 1000ml/ 5000mlL b. 1% Acid alcohol Ethanol 70 ml Distilled water 30 ml Use 99ml of above, add HCL 1ml OR Ethanol 3500ml Distilled water 1500ml Use 4950ml of above, add HCL 50ml H&E stain continue c. Eosin/Phloxine solution 1% Eosin Stock Solution Eosin Y 1gm Distilled water 100ml 1% Phloxine Stock Solution Phloxine B 1gm Distilled water 100ml Eosin/Phloxine Working Solution 1% Eosin stock solution 80ml/380ml 1% Phloxine solution 40ml/160ml Distilled water 120ml/480ml H&E continue Method Dewax and hydrate sections to distilled water Stain nuclei with the alum haematoxylin for 5-6 minutes Rinse in running tap water Differentiate with 1.0% acid alcohol for ±2 seconds (if staining regressively) Rinse in tap water Blue in Scott's tap water for 1minute OR blue in running tap water for 5 minutes Rinse well in tap water Counterstain with Eosin for 1- 2 minutes Rinse well in tap water Dehydrate, clear and mount. H&E results Collagen Pale pink Muscle Deep pink Acidophilic cytoplasm Red Basophilic cytoplasm Purple Nuclei Blue Erythrocytes Cherry red Calcium deposits Blue Bacteria Blue Mucin Pale blue/Grey Cartilage Pale blue/grey H&E results continue H&E Troubleshoot Special stains vs H&E Papanicolaou Stain Principle of the PAP stain The pap stain is a special stain for keratin that is capable of detecting minimal or focal evidence of squamous cell differentiation. As cells keratinized, the glassy cytoplasm progress from blue-green (abundant free ribosomes and prekeratin) to intensely orange (true keratin). Principle of the PAP stain Papanicolaou stain (also Pap stain) is a multi- chromatic staining histological technique histological technique. Pap stain involves five dyes in three solutions: A nuclear stain, haematoxylin, is used to stain cell nuclei. OG-6 counterstain. The Orange G is used to stain keratin. Its original role was to stain the small cells of keratinizing squamous cell carcinoma present in sputum. EA (Eosin Azure) counterstain, comprising of three dyes; the number denotes the proportion of the dye e.g. Eosin Y stains the superficial epithelial squamous cells, nucleoli, cilia, and red blood cells. Light Green SF yellowish stains the cytoplasm of all other cells. Bismarck brown Y stains nothing and in contemporary formulations it is often omitted. Method of PAP stain a. Scott’s tap water Sodium Hydrogen Carbonate 3.5gm OR 17.5gm Magnesium Sulphate 20gm OR 100gm Tap Water 1000ml OR 5000ml b. 95% Ethanol Ethanol 50ml OR 5ml DDW 950ml OR 95ml c. Solutions OG-6 and EA-50 (commercial) PAP stain continue Procedure 2 (Modified Pap Procedure): Fix in 95% Ethanol 15 minutes Rinse in 2 changes DDW - 10 dips each Stain in Gill’s Haematoxylin for 2 minutes Rinse in DDW - 10 dips Blue in Scott's tap water for 1 minute Rinse in 2 changes 95% Ethanol - 10 dips Stain in OG-6 stain for 5-7 minutes Rinse in 3 changes 95% Ethanol - 10 dips Stain in EA-50 (or EA-65 stain) for 6-10 minutes Rinse in 3 changes 95% Ethanol - 20-30 dips Rinse in Absolute ethanol - 10 dips Clear in xylene, mount Results Cell nuclei Crisp blue to black Cells (high content of keratin) Yellow/orange Glycogen stains Yellow Superficial cells Pink to orange Intermediate & para-basal cells Turquoise green to blue Metaplastic cells Blue/green and pink at once. Results continue Results continue A. REGRESSIVE - Differentiator Haematoxylin stain is applied Tissue is overstained with haematoxylin Differentiator is used to aggressively remove excess haematoxylin Stain procedure continues on with counterstain B. PR0GRESSIVE - No differentiator Haematoxylin stain is applied Tissue is stained with haematoxylin only to a point Traditionally no differentiator is used to remove excess haematoxylin Stain procedure continues on with counterstain PAP Stain Troubleshoot Causes of inconsistent staining . Varying thickness of material on slide . Type of fixative used . Inadequate filtering of stain solutions . Age of staining solution Degree of usage of staining solutions . Use of chlorinated tap water . pH of water can effect nuclear staining . Temperature of water and reagents . Insufficient rinsing after acid . Speed of dipping slides in reagents - agitation . NB Improper draining of slides during staining. PAP Stain Troubleshoot PAP Stain Troubleshoot References John D. Bancroft, Christopher Layton and S.Kim Suvarna, (2013), Bancroft’s Theory and Practice of Histological Techniques, 7thEdition, Elsevier, China 3.J.A.Kiernan,(2015)Histological and Histochemical Methods, 5th Edition, Scion, UK Pathcare Academy Notes.
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