Histopathology Techniques - Fixation PDF

HISTOPATHOLOGY TECHNIQUES FIXATION M.Sc : Eman mahjoub Dr; Eman mahjoub Cross-linking fixative:  Gluteraldehyde:  Ideal for ultra structural studies due to detailed preservation.  Rapid Fixation, crosslinking achieved quickly, ensuring fine cellular detail. ...

HISTOPATHOLOGY TECHNIQUES FIXATION M.Sc : Eman mahjoub Dr; Eman mahjoub Cross-linking fixative:  Gluteraldehyde:  Ideal for ultra structural studies due to detailed preservation.  Rapid Fixation, crosslinking achieved quickly, ensuring fine cellular detail.  High Fixation Strength: Preserves subcellular structures like organelles and membranes which make it optimal for electron microscopy Dr; Eman mahjoub electron microscopy Light microscopy image image Dr; Eman mahjoub  Osmium Tetroxide Fixatives:  Properties: 1. Lipid Fixation:  Binds to unsaturated lipids, preserving and contrasting cell membranes. 2. Secondary Fixation:  Often used after aldehyde-based fixation for better ultrastructure preservation. Dr; Eman mahjoub 3. Staining Properties:  Give strong electron density and dark-staining cellular structures for visibility  Mercuric Chloride :  Properties : 1. Excellent for preserving protein structures and maintaining tissue morphology. 2. Adds density, enhancing tissue staining for light microscopy. 3. Often combined with other fixatives like formalin in Zenker's and Helly's solutions Dr; Eman mahjoub 3- Compound fixative:  Pathologists use formaldehyde-based fixatives to preserve histomorphometric patterns.  Other agents may be added to formaldehyde to produce specific effects which are not possible with formaldehyde alone  As ethanol added to formaldehyde produce alcoholic formalin to preserves molecules such as glycogen.  Eg: Compound fixatives( Carnoy fixative, Susa, Zenker ) Dr; Eman mahjoub Dr; Eman mahjoub Dr; Eman mahjoub  Disadvantage of fixative: 1- Molecular loss. 2- Swelling or shrinkage 3-Variation in the quality of biochemical and immunohistochenmical stain. 4- Loss of antigen immunorecognition. Dr; Eman mahjoub  Factors effect quality of fixation: 1- PH :  As in formalin when used in acid ph with cause brown – black insoluble crystalline pigment so use of neutral buffer formalin NBF to avoid this artifact. 2- Duration of fixative and size of specimens:  The depth reached by fixatives is directly proportional to square root of duration of fixative. d=k √ t Dr; Eman mahjoub 3- Temperature:  The diffusion of molecules increase with rising temperature. 4- Concentration of fixative:  Effectiveness and solubility determine the appropriate concentration of fixatives.  Concentration of formalin above 10% tend to cause increase hardening and shrinking.  Ethanol concentration below 70 % do not remove free water from tissue efficiently. So appropriate concentration for appropriate fixation. Dr; Eman mahjoub 5- Osmolality and ionic composition:  Hypertonic fixative lead to shrinking , hypotonic fixative lead to swallowing of the fixed tissue.  The best morphological result obtained with slightly hypertonic solution ( 400 -450 ) mosm. 6- Additives:  The addition of electrolytes and non electrolytes to fixatives improves the morphology of fixed tissue. Dr; Eman mahjoub  This electrolytes include calcium chloride, potassium thiocyanate , ammonium sulfate, ….  Electrolytes may react with proteins causing denaturation  Non electrolytes substances such as sucrose, dextran has also reported to improve fixation.  Fixative artifact: 1- formalin pigments:  Produce from fixation of non buffer formalin  Removed by saturated alcoholic picric acid. Dr; Eman mahjoub formalin pigments Dr; Eman mahjoub 2- Mercuric pigments:  Produce from fixation in mercuric chloride.  Removed by alcohol. 3- Chrome oxide pigment:  Produce from fixation in chrome acid.  Removed by 1% acid alcohol. Dr; Eman mahjoub Mercuric pigments Dr; Eman mahjoub Good luck Dr; Eman mahjoub