Fixation PowerPoint Fall 2023.pptx

Fixation HLAB 1260 & CAPSTONE  Definition  Fixation is the stabilization of protein.  Fixative is a chemical that alters tissue by stabilizing protein in such a way that the tissue is resistant to further changes. In the fields of histology, pathology, and cell biology, fixation is a ch...

Fixation HLAB 1260 & CAPSTONE  Definition  Fixation is the stabilization of protein.  Fixative is a chemical that alters tissue by stabilizing protein in such a way that the tissue is resistant to further changes. In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay, either through autolysis or putrefaction. Fixation terminates any ongoing biochemical reactions, and may also increases the mechanical strength or stability of the treated tissues. Fixation Function of Fixatives:  To kill the tissue so that the postmortem activities of decay or putrefaction (bacterial attack) and autolysis (enzyme attack) are prevented. 1. Autolysis is the destruction or digestion of tissues and cells by the enzymes normally present in the cell. 2. Putrefaction is the further tissue breakdown by bacteria. To help maintain the proper relationship between cells and extracellular substances, e.g. collagen, reticulin, elastin  To bring out differences in refractive indexes and to increase the visibility of. Fixation  The purpose of fixation is to preserve a sample of biological material (tissue or cells) as close to its natural state as possible in the process of preparing tissue for examination. To achieve this several conditions must usually be met.  The aims of fixation are: 1. Prevent postmortem (PM) degeneration Prevent autolysis. 2. It is effective against hydrolytic enzymes. 3. Stop the bacterial effect. 4. Harden the tissues, as fixation causes coagulation of proteins 5. Fixation has a mordanting effect, facilitating subsequent staining of tissues. Actions of Fixatives  Fixatives make tissue more receptive to dyes and in many cases act as mordants, which link the dye to the tissue (creates a bridge).  Each fixative produces its own morphologic pattern and set of artifacts visible in the nuclear chromatin patterns, cell membranes, or staining intensities of various cellular elements.  Physical Methods 1. Heat - the method of heat is an added energy that breaks the internal bonds of the tissue. Not very used in Histology, except when using the microwave. 2. Desiccation is a physical method of fixing protein, but rarely used. Air drying of a slide is the closest to this method. Action of Fixatives  The primary method of stabilizing protein in tissues involves the use of 1 or more chemical reagents. These reagents are classified as : 1. Additive or nonadditive 2. Coagulant or Noncoagulant  Additive fixatives – chemically link, or add themselves, to the tissue and change it with this action. Common additive reagents are mercuric chloride, chromium trioxide, picric acid, formaldehyde, glutaraldehyde, glyoxal, osmium tetroxide, and zinc sulfate or chloride.  Nonadditive fixatives – mostly organic compounds, such as acetone and the alcohols act tissue without chemically combining with it. These work is to dissociate bound water molecules from tissue protein groups. (It can result in shrinkage and hardening of tissue)  Action of Fixatives  Coagulant – it establishes a network in tissue that allows solutions to readily penetrate or gain entry into the interior of the tissue. 1. Coagulant fixatives are zinc salt, mercuric chloride, cupric sulfate, ethyl alcohol, methyl alcohol, acetone, and picric acid. Acetic acid can also be considered a coagulant of nucleic acids, but a noncoagulant of cell cytoplasm.  Noncoagulant – these fixatives act by creating a gel that makes penetration by subsequent solutions difficult. Not great especially in tissue processing. 1. Noncoagulant fixatives are formaldehyde, glutaraldehyde, glyoxal, osmium tetroxide, potassium dichromate, and acetic acid. Factors affecting Fixation  Temperature- the temp. at which fixation is carried out may affect tissue morphology.  Size – the thickness of the tissue is important because of its effect on reagent penetration.  No more than 3mm thick  Volume ratio – the ratio of tissue volume to fixative is very important -  15 to 20 times greater than the tissue  Time – important for 2 reasons – 1. Quick placement of tissue in fixative - must but fixative on tissue as soon as possible after removal 2. Duration of fixation is also critical, decreasing the time in fixation can result in many problems. Adequate fixation is needed for morphology preservation and tissue will not be distorted by subsequent processing steps. Formalin should have at least 6-8 hours before dehydration is started. This is dependent on tissue size. This Photo by Unknown Author is licensed under CC BY Choice of Fixative There are many choices of fixatives so each lab will have a preferred choice. The universally used fixative is 10% formalin. If there is a special study request, then a special fixative or no fixative is needed ( Immunofluorescence or enzyme profile, even some IHC’s can require special fixation). Choosing a fixative is dependent on:  Penetration – for proper fixation  Tissue storage – for further studies of wet tissue  pH – influences the reactivity of the fixative  Buffered formalin is to a pH 7.2 to pH 7.4  Osmolality  Refers to number of particles in solution  Reactions of the Cell with Fixatives  Nucleus – how does the fixative affect the DNA, RNA, chromatin patterns and all internal cell structures.  Proteins  Lipids  Carbohydrates This Photo by Unknown Author is licensed under CC BY-NC  Simple Aqueous Fixatives or Fixative Ingredients Individual Ingredients that make up compound fixatives, can sometime be use alone to fix a tissue or sample. The following are water based (aqueous) fixatives. 1. Acetic acid (glacial acetic is concentrated form) A. The precipitation and preservation of nucleoproteins; ability to fix nuclei B. Precipitates DNA C. Added to counteract the shrinking effect of another reagent D. Does not fix or destroy carbohydrates and does not fix lipids. E. Penetrates very rapidly and leave tissue soft. F. Increases protein swelling (disadvantage) G. RBC’s are lysed  Simple Aqueous Fixatives or Fixative Ingredients 2. Formaldehyde – is a colorless gas and is sold as a 37% to 40% solution in water Stock solutions are 37% to 40% formaldehyde 10% formalin, most commonly used in the labs, it is prepared by diluting 1 part of the stock formaldehyde with 9 parts of water. (10% formalin is equal to 3.7% - 4.0% formaldehyde.  Paraformaldehyde, a highly polymeric form of formaldehyde, may be deposited as a white powder in concentrated solution.  Formaldehyde  Is both noncoagulant and an additive fixative.  It penetrates very quickly, but it fixes very slowly because it takes longer to cross-link the tissue proteins.  Hardens tissue and It causes less shrinkage than any other fixatives.  Inexpensive and stable and allows for many special stains techniques.  Formic acid (pigment) forms when formaldehyde becomes acidic and reacts with oxygen and when the pH drops below 6.0. It can be treated with alcoholic picric acid or alkaline alcohol. Cont.  Types of Formalin  10% Aqueous Formalin (formaldehyde, 37% to 40% + water)  10% Formalin saline (formaldehyde, 37% to 40% + sodium chloride + distilled water)  Calcium formalin (formaldehyde, 37% to 40% + calcium chloride + distilled water)  Preservation of phospholipids in tissues  Acetate formalin (formaldehyde, 37% to 40% + sodium acetate + distilled water)  Formalin Ammonium Bromide (formaldehyde, 37% to 40% + Ammonium bromide + distilled water)  For tissues of the central nervous system  10% Neutralized formalin (formaldehyde, 37% to 40% + distilled water + Calcium or magnesium carbonate)  Not recommended because solution becomes acidic  10% Neutral buffered formalin (formaldehyde, 37% to 40% + distilled water + sodium phosphate, monobasic & sodium phosphate dibasic  Most widely used for routine fixation and pH is approximately 6.8 – 7.2 Cont.  Modified Millonig formalin (formaldehyde, 37% to 40% + distilled water + sodium phosphate, monobasic & sodium hydroxide.  Is dual-purpose fixative, allowing electron microscopy on stored tissue  Phosphate- buffered paraformaldehyde  Alcoholic formalin (formaldehyde, 37% to 40% + ethyl alcohol + distilled water  Is a compound fixative and a useful fixative on the tissue processors, because it speeds the dehydration process. Use of Formaldehyde has to be  monitored in a 8-hour period for Exposure of employees  Permissible exposure limit (PEL) is set at 0.75ppm Short term exposure limit (STEL) for 2ppm over a 15 minute period Simple Aqueous Fixatives or Fixative Ingredients 3. Glutaraldehyde  Is also an aldehyde but a dialdehyde  Fixes at rate which it penetrates, but penetrates slowly and poorly  Gives a false positive when used with Schiff reagent  Most frequently used for the fixation of specimens for electron microscopy; preserves ultrastructure  It tends to overharden tissue if prolonged (usually 2 hours or less)  It is unstable substance that breaks down on exposure to oxygen and should be stored in the refrigerator. Simple Aqueous Fixatives or Fixative Ingredients 4. Glyoxal  Is the smallest dialdehyde and is supplied as a 40% aqueous solution  Has replaced formaldehyde in some labs because it is less toxic and are extremely rapid  Penetration rate is equivalent if not faster than NBF  Specimens are fixed in 4 to 6 hour exposures and biopsy specimens can be processed in 45 minutes  Artifacts seen in formaldehyde are not seen in glyoxal  No problems with PAS reaction but staining of Helicobacter pylori is unsatisfactory.  It does not give off fumes but it has serious health hazard rating Simple Aqueous Fixatives or Fixative Ingredients 5. Mercuric Chloride  Few labs use it because of its extreme toxicity  Very corrosive chemical and all contact with metallic object must be avoided  It is used as a compound fixative because it is a very powerful protein coagulant and enhances staining by leaving the tissue receptive to dyes  Produces a mercury pigment that cannot be prevented but can be removed. (Iodine plus sodium thiosulfate)  Mercury is very toxic compound capable of affecting the central nervous system Simple Aqueous Fixatives or Fixative Ingredients 6. Osmium Tetroxide  Not frequently used for fixation  Primary use in the fixation of specimens for electron microscopy  Preserves lipids  May be used to fix small amounts of fat during paraffin processing  Is a very expensive reagent that is very hazardous because it vaporizes readily Simple Aqueous Fixatives or Fixative Ingredients 7. Picric Acid  It is unique that it is both a fixative and a stain  It is a coagulant fixative  It stains for DNA and RNA  It will decalcify tissue containing small deposits of calcium  It must be washed out of the tissue before processing and can be washed out with 50% alcohol  Major safety concern is its hazard as an explosive compound Simple Aqueous Fixatives or Fixative Ingredients 8. Potassium Dichromate  Rarely used alone in fixation  Chromium will attach to some lipids rendering them insoluble  Can yield fixation pigments that can be removed with 1% hydrochloric acid in 70% alcohol  Chromium is highly toxic by both inhalation and ingestion Simple Aqueous Fixatives or Fixative Ingredients 9. Zinc Salt  Zinc sulfate has found acceptance as a replacement for mecury.  Only used today in combination with formaldehyde for routine fixation  It only carries moderate health risks  Compound or Combined Fixatives Most the fixatives solutions are combined in such a way that the disadvantage of 1 will be counterbalanced by an advantage (or disadvantage) of another. Example: the swelling caused by acetic acid is a disadvantage that can be countered by the shrinkage effect of picric acid. Most commonly used fixatives are: 1. B-5 – Mercury-based solutions  For hematopoietic and lymphoreticular tissue because of its beautiful nuclear detail  Tissue must be treated for mercury removal  Tissue can not remain in this solution indefinitely  Excellent results with many special stains and tissue antigens  B-5 fixative stock solution (Mercuric chloride, sodium acetate, distilled water)  Working solution: (B-5 stock + formaldehyde) Compound or Combined Fixatives 2. Gendre Solution  A alcoholic Bouin solution excellent in preserving some carbohydrates, especially glycogen  Fixative must be washed out before processing with 80% alcohol  Gendre contains formaldehyde so all regulations governed the use is applicable  Alcohol with saturated picric acid, formaldehyde, 37% to 40% and glacial acetic acid  Compound or Combined Fixatives 3. Bouin Solution: (Picric Acid, Formaldehyde, Acetic Acid)  Lyses RBC’s  Excellent for tissue for trichrome stain  Preserves structures with soft and delicate textures  Swelling effect of acetic acid is balance by shrinking effect of picric acid  Hardening effect of formaldehyde is counteracted by the soft fixation of picric acid  Brilliant nuclear cytoplasmic staining  Yellow color must be removed  Can not leave tissue in Bouin fixative  Great on Gastrointestinal biopsies- gives great nuclear staining Compound or Combined Fixatives 4. Hollande solution  Widely used as a fixative for gastrointestinal tract biopsies  Will decalcify small specimens of bone  The cupric acetate in the solution stabilizes RBC membrane  Specimens must be washed or the salts present in the solution will form a precipitate.  It is moderately toxic if ingested and may cause dermatitis  Copper acetate, Picric acid, Formaldehyde, acetic acid and water Compound or Combined Fixatives 5. Zenker and Helly Solutions  It is stable and can be prepared in large quantities  Tissues must be treated for mercury pigment removal  Zenker solution will lyse erythrocytes because of the acetic acid and Helly will preserve the erythrocytes  Most staining is satisfactory after fixation  Specimens can not remain in solution indefinitely  It is still very toxic  Mercuric chloride, Potassium dichromate, Sodium sulfate, and water  Working Zenker=acetic acid Working Helly=Formaldhyde Compound or Combined Fixatives 6. Orth Solution (potassium dichromate, sodium sulfate, water, and formaldehyde)  No mercuric chloride  Formaldehyde added just before use 7. Zamboni Solution  Only has Paraformaldehyde and Picric acid  Used in electron microscopy Compound or Combined Fixatives 8. Zinc Formalin Solutions  Gained prominence in immunohistochemistry  Antigenicity is not lost in long term storage  Cross linking is prevented  Aqueous Zinc Formalin  Orignal formula ( zinc sulfate, formaldehyde, water)  Unbuffered Aqueous Zinc Formalin (zinc sulfate, water, formaldehyde)  Produces formalin pigment, minimum 4-6 hr fixation and 6-8 hr for other tissue  Alcoholic Zinc Chloride Formalin (Zinc chloride, water, Isopropyl alcohol, formaldehyde)  Recommended as a post fixative Nonaqueous Fixatives Nonaqueous fixative ingredients are primarily acetone and ethyl or methyl alcohol. They are nonadditive, coagulating fixatives. Very flammable, tend to overharden. 1. Acetone  Nonadditive protein coagulant  Used to demonstrate enzymes  Used for fixative for brain tissue  Causes extreme shrinkage, distortion and overhardening  Highly flammable Nonaqueous Fixatives 2. Alcohol  Both ethyl and methyl alcohols are used for fixation  Methyl used frequently for touch preps and blood smears  Ethyl preserves most pigments, dissolves fat and overhardens and shrinks the tissue Nonaqueous Fixatives  Alcohol – containing solutions A. Carnoy Solution (Absolute ethyl alcohol, chloroform, acetic acid)  Used in cytology and is rapid acting, preserves glycogen and exhibits nuclear preservation  Erythrocytes are lysed  Causes excessive shrinkage and hardening  Fixation should not exceed beyond 4 hours  Repeated or prolonged exposure can damage central nervous system  Chloroform is a suspected carcinogen  Absolute ethyl alcohol, Chloroform and Acetic acid Nonaqueous Fixatives  Alcohol containing solutions B. Clarke Fluid  It is mixed before use  Oldest fixative and is excellent of subsequent paraffin embedding  Great for microanatomical preservation  Absolute alcohol and Glacial acetic acid  Transport Solutions  If unfixed tissue is held only for a brief period or transported only for a short distance, it is best to be placed in a saline-dampened gauze, enclosed in a tightly sealed container and then placed in ice.  If un fixed tissue is held for several days or transported over a long distance, then Michel transport medium is recommended.  It is important the pH is at 7.0 to 7.2 because lower pH can cause variable results  Michel Transport Medium  Ingredients: (Anhydrous citric acid, Ammonium sulfate, N-ethylmaleimide, magnesium sulfate, water)  Not recommended for muscle biopsies but used routinely for kidney biopsies that are to be mailed.  The solution is commercially available Fixatives for Electron Microscopy The primary fixatives for ultrastructural studies are:  Osmium tetroxide – can not be used for light microscopy 1. Provides excellent preservation of cytologic detail, renders lipids insoluble, giving excellent membrane preservation, can only leave in fixative 2-4 hours or less, penetration is poor, histochemical studies cannot be done.  Aldehydes (formaldehyde and glutaraldehyde)- allows better penetration of fixative, histochemical studies can be done, if formaldehyde is used EM can be done, serve dual purpose ( can be for both light and EM microscopy), lipids are not preserved.  Buffered PAF (Zamboni) Solution – can leave specimen in fixative at room temp. indefinitely, penetrates rapidly and stabilizes cellular proteins, can fix tissues for both light and electron microscopy, lipids are not well preserved, some cytoplasmic granules and lysosomes may not be preserved, some background substances may not be well preserved Removal of Fixation Pigments  Removal of Fixation Pigments- Formalin pigment resists extraction by most strong acids, water, alcohol, or acetone. Can remove by treating deparaffinized and hydrated slides (sections) with 1 of the following: 1. Absolute alcohol saturated with picric acid 10 min. to 3 hours, then wash with water 2. 70% Alcohol with ammonium hydroxide for 30 minutes to 3 hours, then wash in 1% acetic acid, wash again then stain.  Mercury pigments: Removed by: 1. Treat with depariffized and hydrated slide with gram or Lugol iodine for 10 minutes, then wash in running water, then place slides in 5% sodium thiosulfate for 3 minutes, then wash slides for 10 minutes.  Troubleshooting Fixation Problems The major problems encountered with fixation are because of delayed or incomplete fixation. 1. Autolysis – caused by delayed fixation. To prevent: a. Place specimen in fixative solution ASAP, ensuring the volume is 15 – 20 times the tissue b. Open specimens that are whole ( uterus, colon, etc.) c. Slicing organ resections d. Bisecting lymph nodes 2. Incomplete Fixation – a. Increase time allowed in fixative solution b. Change to another fixative c. Place formalin alcohol in the first 3 stages of processing cycle d. Ensure that the grossing sections are thin enough for good reagent penetration e. Ensure formalin solution is clean in the processor f. Do not pack cassettes in the processor holder (baskets) g. Use agitation of cassettes in fixation solution Egg in Fixatives