Histopathology Fixation Techniques

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