3.0-Histopathology-Fixation.pdf
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Fixation PATRICK JUMAR S. BUENAFLOR,RMT, MSMT(C) Histotechnology Isthe art and science performed by the histotechnologist to produce a tissue section of good quality that will enable the pathologist to diagnose the presence or absence of disease. Tissue Processing Fresh tissues are...
Fixation PATRICK JUMAR S. BUENAFLOR,RMT, MSMT(C) Histotechnology Isthe art and science performed by the histotechnologist to produce a tissue section of good quality that will enable the pathologist to diagnose the presence or absence of disease. Tissue Processing Fresh tissues are usually examined when there is an immediate need for evaluation Reasons Permanently preserved Stained for demonstration of specific structure Permanent keeping Tissue Processing 1. Fixation 2. Dehydration 3. Clearing 4. Infiltration (Impregnation) 5. Embedding 6. Trimming 7. Section-cutting 8. Staining 9. Mounting 10. Labeling Fixation First and Critical step in histotechnology “Quality of the section on the slide is only as good as the quality of the fixed tissue” Preserved the morphological and chemical integrity of the cell in as life-like manner as possible Prevents degeneration, putrefaction, decomposition, and distortion after removing from the body Fixation Itharden and protect tissue from the trauma of further handling Certain special study requires specific fixation procedure Most important reactions in fixation is stabilizing proteins Fixation To preserve the tissue To prevent breakdown of Cellular elements To coagulate or precipitate protoplasmic substances Mechanisms in Fixation Additive fixation Chemical constituent of the fixative is taken in and becomes part of the tissue by forming cross-links or molecular complexes and giving stability to the proteins Formalin, Mercury and Osmium tetroxide Non-additive fixation Fixing agent is not incorporated into the tissue, but to alters the tissue composition and stabilizes the tissue by removing the bound water attached to the H-bond of certain groups of protein molecule Alcohol Factors Involved Fixation Hydrogen Ion Concentration Temperature Thickness of section Osmolality Concentration Duration of fixation Practical Consideration Speed Penetration Volume Duration of fixation Effects of Fixatives They harden soft and friable tissues and make the handling and cutting of sections easier They make the cells resistant to damage and distortion caused by hypotonic and hypertonic solutions They inhibit bacterial decomposition They increase the optical differentiation of cells and tissue components Act as mordants to promote and hasten staining Reduce risk of infections during handling Characteristic of a Good Fixatives It must be cheap It must be stable It must be safe to handle It must kill the cell quickly It must inhibit bacterial decomposition It must produce minimum shrinkage of tissue It must permit rapid and even penetration of tissues Characteristic of a Good Fixatives It must harden tissues It must be isotonic It must make cellular components insoluble Itmust permit the subsequent application of many staining procedure Types of Fixation Physical Fixation Heating Microwave Freezing Chemical Fixation According to COMPOSITION According to ACTION According to COMPOSITION Formaldehyde Aldehyd es Glutaraldehyd e Simple Fixative Potassium Mercuric Dichromat Chloride Metallic e Compositio Chromic n Fixative Acid Compou Chromate nd Fixative Picric Fixative Acid Lead Acetic Fixatives Acid Acetone Alcohol Osmium tetroxide According to ACTION Micro-anatomical Fixatives General microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues Cytological Fixatives Preservespecific parts and particular microscopic elements of the cell itself 10% Formol Saline 10% Neutral Buffered Formalin Heidenhain’s Susa Formol sublimate Zenker’s solution Zenker-formol (Kelly’s Solution) Bouin’s solution Flemming’s Brasil’s solution Microanatomi Fluid cal Fixative Carnoy’s Fluid Bouin’s Fluid ACTION Nuclear Newcomer’s Fixative Fluid Heidenhain’s Flemming’s w/o Acetic Cytological AcidFluid Fixative Kelly’s Fluid Cytoplasmic Fixative Formalin with “post- chroming” Regaud’s (Muller’s Fluid) Orth’s FormolFluid Saline 10% Histochemical Absolute Ethyl Alcohol Fixative Acetone Newcomer’s Fluid Cytological Fixative Nuclear Fixative Preservesthe nuclear structure (E.G. Chromosomes) Contains glacial acetic acid Cytoplasmic Fixative Preserves the cytoplasmic structures Do not have acetic acid Histochemical Fixative Preserves the chemical constituents of cells and tissues Aldehyde Fixatives Formaldehyde (Formalin) Commercially available formalin contains 35- 40% gas by weight Pure stock solution of 40% is unsatisfactory for routine examination The commonly used formalin uses as 4% solution, giving 10% formalin for tissue fixation It is usually buffered to pH 7 with phosphate buffer Usual fixation time is 24 hours Aldehyde Fixatives Formaldehyde (Formalin) Preserves fats, mucin and glycogen Does not over-harden tissues It preserves but does not precipitate It is a “tolerant fixative” It is irritating It may produce considerable shrinkage of tissues It is a soft fixative Aldehyde Fixatives 10% Formol-saline Saturated formaldehyde(40%) diluted with 10% sodium chloride Fixation of CN tissue and general post-mortem tissues for histochemical examination Preserves enzymes and nucleoproteins Ideal for most staining techniques (Silver Impregnation) Slow fixative Tissues tends to shrink during alcohol dehydration Acid dye stains less brightly Aldehyde Fixatives 10% Neutral Buffered Formalin (Phosphate Buffered Formalin) Storage and preservation of surgical, post- mortem and research specimens Best fixative for tissues containing iron pigments Requires no post-treatment after fixation Produce a gradual loss in basophilic staining of stain Positivity of mucin to PAS is reduced Reactivity of myelin to Weigert’s iron hematoxylin stain is reduced Aldehyde Fixatives Formal-Corrosive (Formal-Sublimate) Recommended for routine post-mortem tissues It penetrates small pieces of tissues Excellent for many staining procedures (Silver Reticulum Method) Cytological structure and Blood cells are well preserved No need for “washing out” Penetration is slow ( tissue sections should not be more than 1 cm. thick) Forms mercuric chloride deposits Aldehyde Fixatives Alcohol Formalin (Gendre’s) Post-fixationwith phenol-formalin for 6 hours or more can enhance immunoperoxidase studies on the tissues Used for rapid diagnosis Fixation is faster It is used to fix sputum Causes partial lysis of RBC Preservation of iron-containing pigment is poor Aldehyde Fixatives Glutaraldehyde Made up of two formaldehyde residues linked by 3 carbon chains Acts in a manner similar to formaldehyde Utilized in routine light microscopic work Buffered glutaraldehyde → osmium tetroxide Satisfactory for electron microscopy 2.5% solution (small tissues and needle biopsy) 4% solution (larger tissue less than 4 mm thick) Fixation time nay varies Aldehyde Fixatives Glutaraldehyde More stable effect on tissues Preserves plasma proteins Recommended for enzyme histochemistry and electron microscopy More pleasant and not irritating smell More expensive Penetrates tissue slowly Tends to make tissue more brittle Reduces PAS positivity Metallic Fixatives Mercuric Chloride Common metallic fixative and most widely used secondary fixative It penetrates poorly and causes shrinkage of tissues Tissues fixed with solution containing mercuric chloride contains black precipitates Penetrates and harden tissues rapidly Nuclear components are shown in detail Precipitates all protein Greater affinity to acid dyes Metallic Fixatives Mercuric Chloride Trichrome staining is excellent Routine fixative of choice for preservation of cell detail in tissue photography Permits brilliant metachromatic staining of cells Recommended for renal tissues, fibrin, connective tissues and muscles It causes marked shrinkage of cells Metallic Fixatives (Mercuric chloride) Zenker’s Fluid Made up of mercuric chloride solution and glacial acetic acid Itis a good general fixative for adequate preservation of all kinds of tissue Recommended for fixing small pieces of liver, spleen, connective tissue fibers and nuclei Metallic Fixatives Helly’s Solution (Zenker-formol) Excellent microanatomic fixative for pituitary gland, bone marrow and blood containing organs It preserves cytoplasmic granules well Metallic Fixatives Heidenhain’s Susa Solution Recommended mainly for tumor biopsies especially of the skin Excellent cytologic fixative Fixation time: 3-12 hours B-5 Fixative Commonly used for bone marrow biopsies Metallic Fixatives (Chromate) Chromic Acid Used in 1-2% aqueous solution It precipitates all proteins and adequately preserves carbohydrates It is a strong oxidizing agent Potassium Dichromate Used in 3% aqueous solution Preserves lipid Preserves mitochondria(pH 4.5-5.2) Metallic Fixatives (Chromate) Regard’s (Muller’s) Fluid FixationTime: 12-48 hours Recommended for demonstration of chromatin, mitochondria, mitotic figures, golgi bodies, RBC and Colloid-containing tissues Deteriorate and darkens on standing Nuclear staining, Glycogen penetration is poor Metallic Fixatives (Chromate) Orth’s Fluid Recommended for study of early degenerative proceces and tissue necrosis Demonstrate rickettsiae and other bacteria Preserves myelin better Fixation time: 36-72 hours Metallic Fixatives (LEAD) Lead Fixatives Used in 4% aqueous solution of basic lead acetate Recommended for acid mucopolysaccharides Fixes connective tissue mucin Takes up CO2 to form insoluble lead carbonate on prolong standing Metallic Fixatives (Picric Acid) Picric Acid Fixatives Normally used in strong saturated aqueous solution It can also dyes the tissues Excellent fixative for glycogen demonstration Suitable for Aniline Stain It is not suitable for frozen section Picric must never be washed with water It must be kept moist Interferes with Azure eosin method Metallic Fixatives (Picric Acid) Bouin’s Solution Recommended for fixation of embryos and pituitary biopsies It is an excellent fixative for preserving soft and delicate structures It is the preferred fixative for tissues to be stained by Masson’s trichrome and Does not need washing out Destroys cytoplasmic structures Picrates are soluble in water Not suitable for fixing kidney structures Metallic Fixatives (Picric Acid) Brasil’s alcoholic Picroformol Fixative Better and less messy than bouin’s Excellent fixative of glycogen Metallic Fixatives (Glacial Acetic Acid Acid) Glacial Acetic Acid Normally used in conjunction with other fixatives Fixes and precipitates nucleoprotein Precipitates chromosomes and chromatin material Useful in the study of nuclear components of cell It causes tissue to swell Contraindicated for cytoplasmic fixation Metallic Fixatives (Alcohol) Alcohol Rapidly denatures and precipitates proteins by destroying hydrogen and other bonds It must be used in concentration ranging 70-100% Absolute alcohol can be used to fix and preserve glycogen, pigments, blood, tissue films and smears Preserved color of the specimen Used both as fixative and dehydrating agent Preserves nuclear stain Dissolves lipid and fats It causes polarization of glycogen granules Metallic Fixatives (Alcohol) Methyl Alcohol Excellentfor fixing dry and wet smears, blood smears and bone marrow Fix and dehydrates at the same time Penetration is slow Isopropyl alcohol 95% Used for fixing touch preparations For special staining procedures (Wright-Giemsa Stain) Metallic Fixatives (Alcohol) Ethyl Alcohol Used at 70-100% concentrations Simple fixative Fixes blood, tissue films and smears Fixing Time: 18-24 hours Can lyse RBC Carnoy’s Fluid Recommended for fixing chromosomes, lymph glands and urgent biopsies Rapid in action (5 hours) Used to fix brain tissue Metallic Fixatives (Alcohol) Newcomer’s Fluid Recommended for fixing mucopolysaccharides and nuclear proteins Produces better reaction in Feulgen stain Acts both as nuclear and histochemical fixative Metallic Fixatives (Osmic Acid) Osmium Tetroxide Yellow powder which is dissolved in water to form strong oxidizing solutions (6% at 20⁰C) Causes complete denaturation of protein Fixes conjugated-fats and lipids permanently Used extensively for neurological tissues Produces brilliant nuclear staining with safranin Adequately fixes material for ultrathin sectioning It inhibit hematoxylin Readily reduced (organic material and sunlight) Metallic Fixatives (Osmic Acid) Flemming’s Solution Most common chrome-osmium acetic acid fixative Recommended for nuclear preparation Poor penetrating agent Solution deteriorates rapidly Has a tendency to form an artifact pigment Flemming’s Solution w/o acetic acid Made up of only chromic and osmic acid Recommended for cytoplasmic structures Metallic Fixatives (Trichloroacetic Acid) Trichloroacetic Acid Sometimes incorporated into compound fixatives Marked swelling effect on tissue Used as weak decalcifying agent Softening effect on dense fibrous tissues Poor penetrating Metallic Fixatives (Acetone) Acetone Used at ice cold temperature Recommended for the study of water diffusible enzymes Fixing brain tissues Use as solvent for certain metallic salt Produces inevitable shrinkage and distortion Evaporates rapidly Dissolves fat and poor preservation of glycogen Metallic Fixatives (Heat Fixation) Heat fixation Involves thermal coagulation of tissue proteins for rapid diagnosis Frozen tissue and bacterial smear Fixation is better Produces considerable shrinkage and distortion Destroys RBCs Dissolves starch and glycogen Other Procedures Secondary Fixation To facilitate and improve the demonstration of particular substance Make special staining techniques possible Ensure further and complete hardening and preservation of tissue Post-Chromatization It is a form of secondary fixation whereby the fixed tissue is place in aqueous sol’n of 2.5-3% potassium dichromate for 24 hours Washing-Out Process of removing excess fixative from the tissue after fixation Washing-Out Tap water – used to remove Excess chromates (Kelly’s,Zenker’s and Flemming’s) Excess formalin Excess osmic acid 50-70% alcohol Wash excess amount of picric acid (Bouin’s) Alcohol Iodine Used to remove excessive mercuric fixatives Factors that Affect Fixation of Tissues Retarded by Enhanced Size and thick of the tissue by specimen Size and Presence of mucus thickness of Presence of fat tissues Presence of Agitation blood Cold temperatures Fixation Artifacts Well-known artifacts that may be produced under acid conditions Buffered formalin and phenol- formalin stops the formation of artifacts Crush Artifacts may be found in surgical specimens particularly in liver biopsies Microwave Technique Works as a physical agent similar to vacuum, oven(heat) and agitation to increase the movement of molecules and accelerate fixation Also used to accelerate staining, decalcification, immunohistochemistry and electron microscopy Microwave Technique Advantage Tissue is heated right through the block in very short time Non-chemical technique that is useful in preserving neurochemical substances Used for rapid fixation Significantly reduces the time Microwave Technique Disadvantage Commercial oven penetrates tissue to a thickness of 10-15 mm Thereis no significant cross-linking of protein molecules Viable spores and other pathogens may remain in tissues Microwave Technique Fixation 10% formalin buffered to pH 7.3 Procedure 1. Fix tissue (no more than 5mm) in formalin – 4 hours 2. Soak blocks in water at room temp – 1 hour 3. Place blocks in 100mL of Formalin 4. Place in microwave, 450 watts, 55C, 1.5-4 hours 5. Remove blocks and slice tissue to 2mm thick 6. Place directly in alcohol THANK YOU QUIZ NEXT MEETING
