Histopathology Techniques - Fixation PDF

HISTOPATHOLOGY TECHNIQUES FIXATION M.Sc : Eman mahjoub  Fixation  Fixation is preserve tissue by harden and prevent loss of tissue molecules.  Fixation is important to maintain clear and consistent morphological features.  The appropriate fixation...

HISTOPATHOLOGY TECHNIQUES FIXATION M.Sc : Eman mahjoub  Fixation  Fixation is preserve tissue by harden and prevent loss of tissue molecules.  Fixation is important to maintain clear and consistent morphological features.  The appropriate fixation of tissues for histological examination is central to all histology tests, as without this process all tissues would degrade and analysis would  Function of fixative: 1- Prevent autolysis( break down by enzyme) 2- Prevent putrefaction ( break down by microorganism) 3- Preserve cell as living state. 4- Preserve tissue from harmful effect of tissue processing. 5- Facilitate staining process. 6- Minimize the diffusion of soluble molecules from their original location. Good fixed tissue autolysis  Type of fixative: 1. Physical fixative. 2. Chemical fixative.  Physical fixative:  Use heat, microwave and freeze dry.  Not commonly used in routine practical.  Chemical fixative:  Most commonly used in histopathology lab  Use liquid fixative.   Use Organic or non-organic solutions to maintain adequate morphological preservation.  Chemical fixatives can be considered as members of three major categories: 1. Coagulation fixative. 2. Cross-linking fixative 3. Compound fixative  Coagulant fixatives:  Both organic and non-organic solutions may coagulate proteins making them insoluble.  Cellular architecture is maintained primarily by lipoproteins and fibrous proteins such as collagen.  Coagulating these proteins maintains tissue histomorphology at the light microscope level. 1- Coagulation fixative - Dehydrating coagulation fixative:  Remove of water from tissue by break down of hydrophobic bond, which responsible of treachery structure of protein  Disruption of the tertiary structure of proteins (i.e. denaturation) changes their physical properties, potentially causing insolubility Eg: ( ethanol, methanol)  Acid coagulation fixative:  using acid as picric acid and acetic acid disrupt the electrostatic and hydrogen bond  may also insert a lipophilic anion into a hydrophilic region  All this disrupt the tertiary structures of proteins  Acetic acid Acetic acid coagulates nucleic acids but does not fix or precipitate proteins. It is therefore added to other fixatives to prevent the loss of nucleic acids.  Picric acid dissolves slightly in water to form an acid solution (pH 2.0). Its react an forms salts with basic groups of proteins causing the proteins to coagulate. 2- Cross-linking fixative:  Several chemicals were selected as fixatives to their potential actions of forming cross- links between proteins, individual protein, within nucleic acid and nucleic acids and proteins.  Formaldehyde:  10% neutral buffered formalin (NBF) is the most common fixative used in diagnostic pathology.  Pure formaldehyde is a vapor which, when completely dissolved in water forms a solution containing 37–40% formaldehyde and this aqueous solution is ‘formalin  The usual ‘10% formalin’ used in the fixation of tissues contains approximately 4% weight to volume of formaldehyde.  Mostly used fixatives in histopathology lab due to their effectiveness in preserve tissue morphology and cellular details.  Stabilizes tissue, prevents degradation and maintains cellular structure by forms bonds between amino groups in proteins and nucleic acids.  Suitable for stains like H&E, IHC, and ISH.  Types of formalin: 1. Acidic formalin 2. Neutral buffered formalin ( NBF):  Acidic formalin :  PH: below 7 , usually around 4-5  Less commonly used for general fixation  Prolong fixation in acidic formalin may lead to formalin pigment especially in blood rich tissue.  Also may effect staining quality in some protocol.  Neutral buffered formalin ( NBF):  PH: neutral typically at 7.0  Is most commonly used in histopathology lab because its minimize pigment formation and better tissue preservation and its compatible with various histological and molecular techniques.  All types of formalin are carcinogenic, respiratory irritant, and skin sensitizer.  Use ventilation, wear PPE (gloves, goggles, lab coat), and store in sealed containers Good luck