Histopathology

Primary aim: preserve the morphological and chemical integrity of the cell in as life-like manner. – Shape, structure, intercellular relationship and chemical constituents of tissues are preserved. – Prevents degeneration, decomposition, putrefaction, and distortion of tissues after removal from the body. Secondary goal: harden and protect the tissue from the trauma of further handling MAIN FACTORS INVOLVED IN FIXATION: Hydrogen Ion Concentration – pH 6 and 8 . Temperature – Formalin heated at 60C Thickness of section – 2cm queb for light microscopy Osmolality – slightly hypertonic Concentration – low conc. of glutaraldehyde Duration of fixation – 2-6 h in buffered formalin EFFECT OF FIXATIVES harden soft and friable tissues make the cells resistant to damage and distortion inhibit bacterial decomposition increase optical differentiation of cells and tissues act as mordants or accentuators reduce the risk of infection CHARACTERISTICS OF A GOOD FIXATIVE Cheap Stable Safe to handle Kills the cell quickly producing minimum distortion of cell constituents. Inhibit bacterial decomposition Produce minimum shrinkage of tissues Harden tissues making cutting sections easier Isotonic, causing minimal physical and chemical alteration of the cells and their constituents. Make cellular components insoluble to hypotonic solutions TYPES OF FIXATIVES :- 1. According to composition : A. Simple Fixative – made up of only one component substance such as- Formaldehyde ( Most used fixative), Glutaraldehyde, Mercuric Chloride, Potassium dichromate, Chromic acid , Picric Acid, Acetic Acid, Acetone ,Alcohol, Osmium tetra oxide etc. B. Compound Fixative – made up of two or more fixatives such as Zenker’s solution, Bouins Fluid etc. 2. According to Action A. Microanatomical Fixatives – permits the general microscopic study of tissue structures such as 10% Formol Saline 10% Neutral Bufered Formalin Heidenhain’s Susa Formol sublimate Zenker’s solution Zenker formol Ouin’s solution Brasil’s solution B. Nuclear Fixative – Preserve nuclear structures such as, Flemming’s fluid Carnoy’s fluid Bouin’s fluid Newcomer’s fluid Heidenhain’s Susa C. Cytological Fixatives – preserves cytoplasmic structures such as, Flemming’s fluid without acetic acid Kelly’s fluid Formalin with “post-chroming” Regaud’s fluid (Muller’s fluid) Orth’s fluid Histochemical Fixatives – preserve chemical contents of cells and tissues such as   D. LIPID FIXATIVE – Mercuric chloride and Potassium dichromate PHOSPHOLIPIDS FIXATIVE – Baker’s formal calcium CARBOHYDRATE FIXATIVE – Alcoholic formaldehyde PROTEIN FIXATIVE – Neutral buffered formal saline or formaldehyde GLYCOGEN FIXATIVE – Rossman’s fluid or absolute alcohol Composition, Advantage, Disadvantage & Use of Fixative Formaldehyde – A. 10% formaline widely used (10% formalin) Disadvantage – fumes are irritating to the nose and eyes prolonged storage may induce precipitation of white paraformaldehyde Notes –   Removal of precipitate is addition of 10% methano B. 10% formol – Saline – – 40% Formaldehyde + NaCl + Distilled water fixation of CNS Tissues and General post-mortem tissues preserves enzymes and proteins C. 10% Neutral Buffered Formalin/Phosphate-Buffered Formalin – Sodium dihydrogen phosphate + Disodium hydrogen phosphate + 40%Formaldehyde + Distilled water Preservation of surgical, post-mortem and research specimens Best fixative for iron-containing tissues D. Formol-Corrosive (Formol Sublimate) Aq. Mercuric Chloride + 40% Formaldehyde Routine post-mortem tissues Excellent in silver reticulum methods Fixes lipids, especially neutral fats and phospholipids E. Alcoholic Formalin (Gendre’s Fixative) 95% Ethyl Alcohol saturated with picric acid + Strong formaldehyde solution + glacial acetic acid. Immunoperoxidase studies on tissues Used for rapid diagnosis Good for preservation of glycogen and for micro-incineration Used to fix sputum, since it coagulate mucus F. Glutaraldehyde two formaldehyde residues linked by 3C chains used for enzyme histochemistry and electron microscopy preserves plasma proteins 2. METALLIC FIXATIVES   A. MERCURIC CHLORIDE Mercuric Chloride + Potassium Dichromate + Sodium Sulfate + Distilled Water most common metallic fixative Tissues fixed with mixtures containing mercuric chloride (except Susa) contain black precipitates of mercury. Routine fixative of choice for preservation of cell detail in tissue photography. Renal tissues, Fibrin, Connective tissues and muscles Black deposits may be removed by adding saturated iodine solution in 96% alcohol, the iodine being decolorized with absolute alcohol in the subsequent stages of dehydration. B. Zenker’s Fluid Mercuric Chloride + Glacial Acetic Acid fixing small pieces of liver, spleen, connective tissue and nuclei may act as mordant Mercuric deposits may be removed by immersing tissues in an alcoholic iodine solution. “de-zenkerization” C. Zenker-formol (Helly’s solution)   Mercuric chloride + Potassium dichromate + Sodium sulphate + Distilled water + Strong formaldehyde (40%) Fixative for pituitary gland, bone marrow and blood-containing organs such as spleen and liver. Preserves cytoplasmic granules Brown pigments are produced if tissues are allowed to stay for more than 24 hours. Pigments can be removed by immersing the tissue in saturated alcoholic picric acid or sodium hydroxide   D. Heidenhain’s Susa Solution   Mercuric chloride + Sodium chloride + Trichloroacetic acid + Glacial Acetic Acid + Formaldehyde (40%) + Distilled water tumor biopsies especially of the skin Excellent cytological fixative Mercuric chloride deposits may be removed by immersion on alcoholic iodine solution the tissue should be transferred directly to a high-grade alcohol, to avoid undue swelling of tissues caused by treatment with low-grade alcohol or water. E. B-5 Fixative Distilled water + Mercuric Chloride + Sodium acetate Commonly used for bone marrow biopsies