Medical Laboratory Science - FIXATIVES PDF

BROKENSHIRE COLLEGE, INC HISTOPATH FIXATIVE MEDICAL LABORATORY SCIENCE  Remember: To beat the su...

BROKENSHIRE COLLEGE, INC HISTOPATH FIXATIVE MEDICAL LABORATORY SCIENCE  Remember: To beat the summer Heat, Alden Richards  It fixes lipids, especially neutral fats and goes to Mall Of AsiAA! (HA-MOAAA) phospholipids.  Fixation time: 3-24 hours  H → heat  A → aldehyde 5. ALCOHOLIC FORMOLIN  M → metallic  AKA: Gendre’s fixative  O → osmic  It contains ethyl alcohol saturated with picric acid.  A → acids  It enhances immunoperoxidase studies for EM if  A → acetone post-fixed with phenol-formalin for 6 hours or  A → alcohol more.  It fixes and dehydrates at the same time and fixes sputum since it coagulates mucus. I. ROUTINE FORMALIN FIXATIVES (FCNFACB) SPECIAL FORMALIN FIXATIVES: A. FORMALDEHYDE 1. Cajol’s Formol-ammonium bromide (CFAB) 1. 10% Formol-Saline  Good fixative for nervous tissue (astrocytes).  Most commonly used fixative in pathology.  Best fixative for Central Nervous tissues and 2. Fixatives for acid mucopolysaccharides. General post-mortem tissues for histochemical examination. 3. Baker’s Formol-calcium  FIXATION TIME:  Used for the preservation of lipids since most  24 hours at 35ºC formalin fixatives are inert to lipids.  48 hours at 20-25ºC  ADVANTAGES: REMOVAL OF FORMALIN PIGMENTS:  Should be changed every 3 months so large specimens can be fixed. 1. Lillies Method  Ideal for silver impregnation.  Involves placing formaldehyde fixed specimens in  DISADVANTAGES: acetone, 28% ammonia water and hydrogen  Similar to formaldehyde with following peroxide. It uses 70% alcohol as a rinsing agent. addition:  It shrinks during alcohol dehydration. 2. Kardasewitch’s  Amyloid metachromatic action is reduced.  A method of formaldehyde clearance involving 70% ethanol and 28% ammonia water. 2. Calcium Acetate Formalin (Lillie’s)  It is used to preserve phospholipids 3. Saturated Alcoholic Picric Acid  Replaced formol-saline as the most commonly  To remove formalin pigments. used fixative in pathology because:  It is simple to prepare B. GLUTARALDEHYDE  Buffered to pH 7 by acetate  It is made up of 2 formaldehyde residues, linked by 3. 10% Neutral Buffered Formalin/Phosphate Buffered three carbon chains. It is utilized for Light microscopy. Formalin (pH7)  Buffered Formaldehyde with secondary fixation in  Best General tissue fixative OSMIUM TETROXIDE is satisfactory for Electron  Best fixative for frozen sections. Microscopy.  Prevents precipitation of acid formalin pigments.  2.5% for small tissue fragments and needle  Best fixative for iron containing pigments and biopsies for 2-4 hours at room temperature elastic fibers.  4% large tissue fragments less than 4 mm thick for  FIXATION TIME: 4-24 hours. 6-8 hours up to 24 hours.  Disadvantages:  Fixation time: ½-2 hours.  PAS positivity to mucin is reduced.  ADVANTAGES OVER FORMALIN:  Myelin reactivity to Weigert’s Iron  Recommended for HISTOCHEMISTRY and hematoxylin is reduced. ELECTRON MICROSCOPY.  Inert to neutral fats and phospholipids.  DISADVANTAGES:  Reduces PAS + of reactive MUCIN. 4. FORMOL CORROSIVE  AKA: Formol-sublimate, Formol-mercuric chloride II. METALLIC FIXATIVES  Recommended for routine post-mortem tissues.  It is excellent for silver reticulum methods. A. Mercuric chloride BAUTISTA 3C  The most common metallic fixative.  Frequently used in aqueous saturated solutions of 5-7%. 6. Carnoy-Lebrun  The routine fixative of choice for preservation of cell  These are rapid fixatives giving excellent NUCLEAR detail in Tissue photography. preservation.  Tissues fixed with mercury chloride containing compounds produce black precipitates except 7. Ohlmacher HEIDENHAIN’S SUSA.  These are rapid fixatives giving excellent NUCLEAR  Permits brilliant METACHROMATIC staining; Trichrome preservation. staining is excellent.  Alcoholic Iodine and Sodium thiosulfate. B. CHROMATE FIXATIVES (CROP)  DISADVANTAGES: 1. It causes marked shrinkage of cells.  A class of fixatives which are strong oxidizing agents  RESOLUTION: Addition of an acid (Gl. Hac) or used for precipitating proteins and preserving Secondary Fixative. carbohydrates. Recommended for Chromaffin tissue, 2. Has Black Granular deposits and is extremely Adrenal medulla and Mitochondria. corrosive to metals. 1. CHROMIC ACID 1. ZENKER’S FLUID  Used in 1-2 % used as a constituent of a compound  Mercuric chloride + glacial acetic acid just before fixative. its use.  It precipitates all proteins and adequately  Good general fixative for all kinds of tissue. preserves carbohydrates.  Recommended for fixing small pieces of liver,  Formaldehyde must be added to spleen, connective tissue fibers and nuclei. chrome-containing tissues before use to prevent  Fixative Time: 12-24 hours. counteracting effects and consequent  Contains Gl. Acetic Acid which makes the solution decomposition of solution upon prolonged unstable. standing. 2. Zenker-formol 2. REGAUD’S FLUID  AKA: Helly’s fixative  AKA: Moeller’s/Muller’s  Fixative time: 12-24 hours  Recommended for demonstration of Chromatin,  Mercuric chloride + 40% Formaldehyde just before Mitochondria, Mitotic figures, Golgi bodies, RBC’s its use. and colloid-containing tissues.  It is an excellent microanatomic fixative for pituitary gland, bone marrow and blood-ours 3. ORTH’S FLUID containing organs such as the liver and due spleen.  Recommended for study of early degenerative  Disadvantage: Brown pigments are produced if processes and tissue necrosis. tissues are allowed to stay in the fixative for more  Demonstrates RICKETTSIA and other bacteria. than 24 to RBC lysis.  Preserves Myelin better than buffered formalin.  Fixation time: 36-72 hours. 3. Heidenhain’s Susa Solution  Recommended for TUMOR BIOPSIES especially of 4. POTASSIUM DICHROMATE the skin.  Preserves mitochondria (pH 4.5-5.2)  It is an excellent Cytologic fixative.  Used in 3 % aqueous solution.  Produces minimum shrinkage and hardening of  It fixes but does not precipitate cytoplasmic tissues due to the counter-balance of the swelling structures. effects of acids and the shrinkage effect of mercury. C. Lead Fixatives  SUSA: Su = Sublimat Sa = Saure  Fixation time: 3-12 hours 1. Lillie’s alcoholic lead nitrate formalin 2. Lead subacetate 4. Schaudinn’s Solution/Sublimated alcohol  A solution of mercuris chloride, sodium chloride, ADVANTAGES: alcohol, and glacial acetic acid.  It is recommended for Acid mucopolysaccharides.  Used on wet smears for cytologic examinations.  It fixes connective tissue Mucin. 5. B-5 Fixative DISADVANTAGES:  Composed of mercuric chloride and sodium  It takes up CO2, to form insoluble Lead carbonate acetate especially on prolonged standing. This may be removed  Commonly used for Bone Marrow Biopsies by filtration or by adding Acetic Acid drop by drop to  Just prior to use, add 1mL of 40% formaldehyde to lower the pH and dissolve the residue. 10 mL of B5  III. PICRIC ACID FIXATIVES (PBB) 1. Methyl alcohol 100%  Used as in Wright’s stain a diluent.  Excellent for glycogen demonstration.  Excellent for fixing touch preparations, although  Normally used in strong saturated aqueous solution. some are air dried and not fixed, for certain  It dyes the tissues YELLOW, thus preventing the tissue produces such as WRIGHT-GIEMSA staining. fragments from being overlooked. On the other hand, this hinders proper staining. 2. ISOPROPYL ALCOHOL 95%  Suitable for aniline stains and trichrome method.  It is used for fixing touch preparations, although some are air dried and not fixed, for certain  The chemical name for the general picric acid fixatives: procedures such as WRIGHT-GIEMSA staining. 2,4,6 – Trinitrophenol. 3. ETHYL ALCOHOL A. BOUIN’S SOLUTION  It is used in 70-100% concentrations. Lower concentrations (70-80%) will cause RBC lyses,  Fixation of embryos and pituitary biopsies. inadequate WBC, preservation.  It is an excellent fixative for preserving, soft and delicate  FIXATION TIME: 18-24 hours structures.  It fixes blood, tissue films and smears  Produces minimal distortion.  Used for HISTOCHEMISTRY especially for ENZYME  Yellow stain is used in FRAGMENTARY biopsies. studies.  Preferred fixative for MASSON’S TRICHROME staining  Can be both used as a simple and compound for collagen, elastic or connective tissue. fixative.  FIXATION TIME: 6-24 hours. 4. CARNOY’S FLUID B. BRASIL’S ALCOHOLIC PICROFORMOL FIXATIVE  The most rapid tissue fixative.  Recommended for fixing chromosome, Lymph  Best routine fixative for glycogen glands and Urgent biopsies.  It is BETTER and less MESSY than Bouin’s solution.  It is used to fix brain tissue for rabies diagnosis.  Overnight tissue fixation by automatic processing  FIXATION TIME: 1-3 hours technique may utilize 3-4 changes of BRASIL’S FIXATIVE  It preserves Nissl granules and very suitable for at ½ - 2 hours each, succeeded directly by absolute curetting’s. alcohol. 5. ALCOHOLIC FORMALIN IV. GLACIAL ACETIC ACID  Better preserve’s glycogen.  Also a Formalin fixative.  It solidifies at 17 °C (Black gregoriou: 16°C).  Capable of coagulating MUCOS and is used as a  Fixes and precipitates nucleoproteins. fixative for sputum.  It precipitates chromosomes and chromatin materials  Causes tissues to swell specially those containing 6. NEWCOMER’S FLUID collagen  For fixing mucopolysaccharides and nuclear  Disadvantages: It is contraindicated for cytoplasmic proteins. fixation because it destroys mitochondria and Golgi  It produces better reaction in Feulgen stain than elements. Carnoy’s fluid  It is both a nuclear and histochemical fixative. V. ALCOHOLIC FIXATIVES  FIXATION TIME: 12-18 hours @ 3 °C  FORMULA: Isopropyl alcohol, Propionic acid,  Denatures and precipitates proteins Petroleum ether, Acetone and Dioxane.  70-100% concentrations are used; Less concentrations results to RBC lysis (hypotonicity) VI. TRICHLOROACETIC ACID  Advantages:  May be used both as a Fixative and dehydrating  Sometimes incorporated into compound fixatives. agent  It precipitates proteins & nucleic acid.  Excellent for glycogen preservation  Causes marked swelling effect on tissues.  Disadvantages:  Can be used as a weak decalcifying agent.  Causes RBC lysis and dissolves fats and lipids  Has softening effect on dense fibrous tissue.  Tissue shrinks on long usage  Suitable only for small pieces of tissues or bone because  Polarization (major disadvantage) of its poor penetration. General Rule: VII. OSMIUM TETROXIDE (OSMIC ACID) (OFF)  Alcohol-containing fixatives are contraindicated when lipids are to be studied.  It is a pale yellow powder which dissolves in water (up to 6% at 20°C) to form strong oxidizing solution.  Adequately fixes materials for ultrathin sectioning in  alcoholic fixatives are generally recommended for electron microscopy, since it rapidly fixes small pieces glycogen fixation. of tissues and aids in their staining  Alcoholic formaldehyde is a better fixative in  Disadvantage: Inhibits Hematoxylin and makes human skin compared with NBF counterstaining difficult. 3. Protein Fixation / Amino acid Histochemistry 1. Flemming's Solution  Neutral buffered formalin or formaldehyde vapor  Fixation time: 24-48 hours are the most commonly used.  The most commonly used Chrome-Osmium acetic acid, recommended for nuclear preparation of 4. Glycogen Fixation such sections.  Most useful fixatives are alcohol based such as  Excellent for nuclear structures such as Rossman's fluid or cold absolute alcohol chromosomes  Section is coated with Celloidin for better retention  Permanently fixes FATS. of glycogen.  Disadvantages:  Very expensive (less amount i is required I for 5. Mixture of Fixatives (EM) fixation).  karnovsky's paraformaldehyde-glutaraldehyde  Formation of artefact pigments/black solution (electron histochemistry and electron precipitate immunocytochemistry)  Prolonged exposure to acid vapors causes eye  acrolein - mixture with glutaraldehyde or irritation (conjunctivitis) or black osmic oxide formaldehyde. deposition in the cornea (blindness) 2. Flemming's Solution without Acetic Acid  Fixation time: 24-48 hours  Recommended for Cytoplasmic structures such as MITOCHONDRIA.  The removal of acetic acid from the formula serves to improve the cytoplasmic detail of the cell. VIII. ACETONE  It is used at ice cold temperature from -5°C to 4 °C  Recommended for the study of water diffusible enzymes especially Lipases and phosphatases.  Used in fixing brain tissues for diagnosis of rabies (Negri Compatibility & Incompatibility bodies)  Few fixatives permit the use of all stains. Roup Some  Used in Freeze-Substitution techniques as a solvent for fixatives may act as a mordant for one of dyes and an certain metallic salts inhibitor for another set of stains.  Evaporates rapidly & also flammable. Secondary Fixation IX. HEAT FIXATION  The process of placing an already fixed tissue in a second fixative in order to:  This procedure involves Thermal congelation of tissue A. To facilitate and improve the demonstration of proteins for rapid diagnosis, usually employed for frozen particular substances. Lokdichromate; zenker's tissue sections and preparations of bacteriologic Potassium avastoncus smears. B. To make special staining techniques possible (secondary fixative acting as mordant) 1. Lipid Fixation C. To ensure further and complete hardening and  Cryostat or Frozen sections should be used for preservation of tissues demonstrating lipids in tissues mercuric chloride  Not recommended when primary fixation for specific and K dichromate are used in cryostat sections. observations is possible in the first instance.  Aldehydes  This is done before dehydration and on deparaffinized  Baker's formol-calcium sections before  Imidazole osmium tetroxide-improved  10% Formalin/10% formol-saline -commonly used ultrastructural demonstration of lipids primary fixative.  Cholesterol may be fixed with digitonin for ultrastructural demonstration Post-chromatization  AKA: Post-chroming/Post-mordanting 2. Carbohydrate Fixation  A secondary fixation whereby a primarily fixed tissue is placed in aqueous solution of 2.5 -3% Potassium dichromate for 24 hours, to act as mordant for better staining effects and to aid in cytologic preservation of tissues Washing Out  The process of removing excess fixative from the tissue after fixation in order to improve staining and remove artefacts from the tissues. Solutions that may be used: 1. Tap water - used to remove: a. Excess Chromates b. Excess Formalin  10% formalin is extracted more rapidly in 70% alcohol than in water c. Excess Osmium tetroxide or Osmic acid 2. 50-70 % alcohol - used to wash out excess amount of Picric acid 3. Alcoholic iodine - used to remove excessive mercury. SPECIAL FACTORS AFFECTING FIXATION A. RETARDED BY: 1. Size and thickness 2. Presence of Mucus 3. Presence of fats 4. Presence of Blood 5. Cold temperature 6. Hot temperature B. ENHANCED BY: 1. Size and thickness 2. Agitation 3. Moderate Heat IMP. FIX 1. Failure to arrest early cellular autolysis 2. Too brittle and too hard blocks 3. Soft and feather-like tissues 4. wrong choice of fixative 5. Incomplete washing of fixative 6. Shrinkage and swelling of cells and tissue structure.