HCT-FIXATIVES-TABLE.pdf PDF

1. ALDEHYDE FIXATIVES • • Commonly used fixative; satisfactory for routine paraffin sections. Electron microscopy, histochemical and enzyme studies. FIXATIVE CHARACTERISTICS FORMALDEHYDE AND FORMALIN • • • • Gas produced by oxidation of methanol (methyl alcohol). 10% recommended, buffered to 7.0...

1. ALDEHYDE FIXATIVES • • Commonly used fixative; satisfactory for routine paraffin sections. Electron microscopy, histochemical and enzyme studies. FIXATIVE CHARACTERISTICS FORMALDEHYDE AND FORMALIN • • • • Gas produced by oxidation of methanol (methyl alcohol). 10% recommended, buffered to 7.0. Formalin has a different formaldehyde concentration. Concentrations: o 4% Formaldehyde – pure concentration/stock solution; not recommended to use in a routine fixation. ▪ Can overbrittle/overharden the tissue. o 10% Formaldehyde ▪ Paraformaldehyde → white crystalline precipitation. ▪ Formic acid → reacts with hemoglobin, which forms acid formaldehyde hematin. • • • • • • 10% NEUTRAL BUFFERED FORMALIN • • • • 10% FORMOL SALINE • • • Best general tissue fixative. Recommended for the preservation and storage of surgical, post-mortem, and research specimen. Most widely used fixative for routine histology. Fixative of choice for: o Paraffin embedding; o Immunohistochemistry; o Fluorescent in-situ hybridization (FSH). pH: 7 (because of phosphate buffer). Microanatomical fixative. Made up of formaldehyde diluted to 10% NaCl. PRECAUTIONS Paraformaldehyde formation. Prolonged storage may induce precipitation – filter or add 10% METHANOL. Well-ventilated room. o Can cause irritation to the eyes and nose otherwise. Not neutralized if concentrated – can cause an explosion. o 40% formaldehyde is too concentrated. Buffered or neutralized by adding magnesium carbonate or CaCO3 to remove acid formaldehyde hematin. o Should be put in a wide mouth bottle to prevent explosion; can explode due to insufficient space for the CO2 release. Bleaching prevented by changing formalin. o If the formalin/formaldehyde used is old, change it every 3 mos. N/A ADVANTAGES • • • • • • • • • • • • • • • DISADVANTAGES Cheap • Readily • available Easy to prepare ✓ Stable ✓ Compatible w/ stains ✓ Penetrates ✓ tissues well Preserves fat ✓ Mucin ✓ Glycogen ✓ For tissue photography. ✓ y Penetrates and fixes tissues well. Minimum shrinkage and distortion. Does not overharden tissues. Best fixative for iron pigments and elastic fibers. Penetrates and fixes tissues well. Irritating fumes ✓ Prolonged fixation may bleach tissues/precipitation. FIXATION TIME 24 HOURS ✓ • • • Longer to prepare Time-consuming Inert towards lipids. • Slow (>24 h). 4 – 24 HOURS 12 – 24 HOURS liyah ♡ • • FORMOLCORROSIVE (FORMOL SUBLIMATE) • • UNBUFFERED ZINC FORMALIN • • • GLUTARALDEHYDE • • • • KARNOVSKY’S FIXATIVE • • • • • • • Recommended for CNS and postmortem tissues for histochemical exam. Preservation of lipids (phospholipids). Has mercuric chloride. Recommended for routine mortem tissues. BLANK • • • • • post• Components: o Formaldehyde (HCHO); o Unbuffered zinc. Alternatives to mercuric chloride formulations Improved results with immunohistochemistry (IHC). Light microscopy, enzyme, histochemistry, and electron microscopy. Larger molecule than formaldehyde. Made up of 2 formaldehyde residues linked by 3 carbon chains. Concentrations: o 2.5% Glutaraldehyde → small tissues, fine needle biopsies; fixed for 2 – 4 hours at RT. o 4% Glutaraldehyde → larger tissues; 6 – 24 hours at RT. Mixture of 4% paraformaldehyde and 1% glutaraldehyde in a 0.1 M phosphate buffer. Suitable for use when preparing samples for light microscopy in resin embedding and sectioning, and for electron microscopy. Should be freshly prepared. KARDASEWITCH’S METHOD Bring down to water. Place in a mixture of 70% ETOH and 28% Ammonia water (5 mins – 3hrs). Wash with water. • Minimum shrinkage and distortion. Does not overharden tissues. Minimum shrinkage and hardening. No need for washing out from fixative to alcohol. • • Slow Forms mercuric deposit. 3 – 24 HOURS black chloride 4 – 8 HOURS • • More stable effect Less tissue shrinkage, less irritating. REMOVAL OF FORMALIN PIGMENT LILLIE’S METHOD Bring down to water. Place in mixture of acetone, H2O2, 28% Ammonia water (1 – 5 mins). WASH WITH 70% ETOH. Wash with water. • • • • • More expensive Slow penetration. PICRIC ACID METHOD Bring down to water. Place in saturated alcoholic picric acid (5 mins – 2 hrs). Wash in running water for 10 – 15 mins. liyah ♡ 2. ALCOHOLIC FIXATIVES Acts as both a fixative and dehydrating agent. Rapidly denatures and precipitates proteins. Not normally used on tissues because it can cause too much brittleness and hardness. Polarization – movement of glycogen granules towards the ends of the cells. FIXATIVE COMPONENTS ADVANTAGES 100% METHYL ALCOHOL Excellent for fixing dry and wet smear. • Used for blood smears and bone marrow tissue. 95% ISOPROPYL ALCOHOL Used for fixing touch preparations and preparation of • ☆ Most commonly used air-dried specimens. alcoholic fixative. • • • • ETHYL ALCOHOL (70 – 100%) • Not usually used (esp. in blood smears) because it causes hemolysis and does not preserve leukocytes. CARNOY’S FIXATIVE GENDRE’S FIXATIVE NEWCOMER’S FLUID CLARKE’S SOLUTION ALCOHOLIC FORMALIN FORMOL-ACETIC ALCOHOL • Used when staining with WrightGiemsa; used for cytologic smears. Fixes blood, tissue films, and smears. • • • Absolute alcohol (100%) Chloroform Glacial acetic acid. • • • • • • • • • • • Ethyl alcohol Formaldehyde Glacial acetic acid Isopropyl alcohol Proprionic acid Petroleum Ether Acetone Dioxone Ethyl alcohol Glacial acetic acid DISADVANTAGES Slow penetration. FIXATION TIME 6 – 24 HOURS Strong reducing agent. 18 – 24 HOURS Strong reducing agent ☆ Most rapid fixative – fixation of chromosomes and brain tissues, which is used for the diagnosis of rabies. Coagulates mucus; can be used for sputum. 1 – 3 HOURS 4 – 18 HOURS Recommended for fixing mucopolysaccharides and nuclear proteins; both a nuclear and histochemical fixative. 12 – 18 HOURS AT 30°C Used on frozen section and smear. 3 – 4 HOURS Used for fixation and post-fixation of large fatty specimens (breast). Faster acting agent than alcoholic formalin. ☆ Sometimes used to fix diagnostic cryostat sections. 12 – 24 HOURS 1 – 6 HOURS 3. METALLIC FIXATIVES Used for the fixation of hematopoietic and reticuloendothelial tissues. o Increases the staining brightness, which is helpful for the staining procedure; gives excellent nuclear detail. FIXATIVE CHARACTERISTICS PRECAUTIONS ADVANTAGES DISADVANTAGES liyah ♡ • MERCURIC CHLORIDE FIXATIVE ZENKER’S FLUID (HgCl2 + Glacial Hac) • • ZENKER-FORMOL (HELLY’S) SOLUTION HEIDENHAIN’S SUSA LILLIE’S B-5 FIXATIVE • • • • • • • Most common metallic • Produces black precipitates of • For tissue photography. • fixative. mercury – remove by 0.5% • Permits brilliant • iodine solution in 70% • Recommended for renal metachromatic staining of ethanol then decolorize iodine tissue, fibrin, connective cells. • using 5% sodium thiosulfate. tissues, and muscles. EXAMPLES OF MERCURIC CHLORIDE COMPONENTS CHARACTERISTICS ADVANTAGES DISADVANTAGES Mercuric chloride • Recommended for • Poor penetration. liver, spleen, CT fibers, Glacial acetic acid. nuclei. • Recommended for trichrome staining (for connective tissue). • Excellent • Brown pigment microanatomic fixative produced if fixed more for pituitary, BM, than 24 hours due to spleen, liver. lysis of RBC – remove by saturated picric acid or NaOH. Mercuric chloride • Recommended for • Black precipitate – tumor biopsies (skin). immerse in alcoholic Sodium chloride iodine. Trichloroacetic acid • ☆ Excellent cytological fixative. Formaldehyde 4% formaldehyde with 0.22 • Mercuric chloride BM biopsy. M mercuric chloride and Sodium acetate 0.22 acetic acid. Formaldehyde FIXATIVE 1 – 2% CHROMIC ACID POTASSIUM DICHROMATE REGARD’S/MULLER’S FLUID ORTH’S FLUID 4. CHROMATE FIXATIVES CHARACTERISTICS Preserves carbohydrates. Preserves lipids and mitochondria. Used for chromatin, mitochondria, Golgi, and RBCs. Used for rickettsia, bacteria, and myelin. Hardens outer layers only. Black granular deposits formed. Corrosive to metals. FIXATION TIME 12 – 24 HOURS 4 – 24 HOURS 3 - 12 HOURS 4 – 8 HOURS FIXATION TIME 24 – 48 HOURS 12 – 48 HOURS 36 – 72 HOURS 5. LEAD FIXATIVES • ADVANTAGE For acid mucopolysaccharides and fixes connective tissue mucin. o Color (when stained): Alcian blue. • • • • 6. PICRIC ACID FIXATIVES Highly explosive when dry. Normally used in strong aqueous solution (1%). Excessive yellow in color – remove color by dipping 70% ETOH followed by 5% sodium thiosulfate and running water. ☆ Excellent for glycogen demonstration. FIXATIVE CHARACTERISTICS ADVANTAGES DISADVANTAGES • DISADVANTAGE Forms insoluble lead carbonate – remove by filtering or adding acetic acid. FIXATION TIME liyah ♡ BOUIN’S BRASIL’S ALCOHOLIC PICROFORMOL (W/ TCA) HOLLANDE’S FIXATIVE Components: • Picric acid; • Formaldehyde; • Glacial acetic acid. Commonly used picric acid. Excellent for glycogen preservation. • • • • • • Recommended for fixation of embryos and pituitary biopsies. Excellent for glycogen preservation. Better and less messy than Bouin’s. Recommended for GIT specimens, endocrine tissues. Produces less lysis than Bouin’s. Has decalcifying properties. • Not good for kidneys, mitochondria, hemolyzes RBC. 4 – 18 HOURS AT RT 24 HOURS 4 – 18 HOURS GLACIAL ACETIC ACID • • • • • • ADVANTAGES Fixes nucleoproteins, chromosomes, and chromatin materials. Causes tissues to swell; should have a counterpart. CHARACTERISTICS Color: Pale yellow. Pale yellow powder that dissolves in water. Upon prolonged exposure, it causes a black precipitate (mercuric chloride). Used in electron microscopy (fixative and heavy metal stain). FIXATIVE FLEMMING’S SOLUTION FLEMMING SOLUTION W/OUT ACETIC ACID TRICHLOROACETIC ACID ACETONE • • • • DISADVANTAGES Destroys mitochondria and Golgi bodies. 7. OSMIUM TETROXIDE FIXATIVES PRECAUTIONS ADVANTAGES Should be kept in a dark colored, • Fixed conjugated fats and lipids. chemically clean bottle to prevent • Preserves cytoplasmic structures. evaporation and reduction by sunlight or organic matter. Prevention: Add saturated aqueous mercuric chloride. Remedy: Black osmic oxide crystals may be dissolved on cold water. CHARACTERISTICS Components: Aqueous chromic acid Aqueous osmium tetroxide Glacial acetic acid (nuclear) • Most common chromeosmium acetic acid fixative used. Components: Chromic and osmic acid. • Decalcifier and fixative. • Used for the precipitation of proteins and nucleic acids. • Incorporated into compound fixatives. • Not recommended as morphological fixative for ADVANTAGES Recommended for nuclear preparation (chromosomes). Recommended for cytoplasmic structures (mitochondria). • Precipitates proteins. • Weak decalcifying agent. • For enzyme studies (lipase and phosphatase). DISADVANTAGES • • • • • DISADVANTAGES Expensive Poor penetration Reduced w/ sunlight → black precipitate. Acid vapor → irritation of eyes, conjunctivitis, osmic oxide in cornea → blindness. Inhibits hematoxylin and extremely volatile. FIXATION TIME 24 – 48 HOURS • • Poor penetration. Suitable for only small pieces of tissues or bones. • • Dissolves fat. Preserves glycogen poorly. SEVERAL MINUTES: Cell smears, cryostat sections. liyah ♡ • • tissue blocks; can cause the shrinkage and has poor preservation effects. Fixation of cryostat sections. Use at ice cold temp (0°C to 4°C). • Fixes brain tissue. • Evaporates rapidly. SEVERAL HOURS: 1 – 24 hours for small tissue blocks. liyah ♡