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Ch.4 Fixation
Overview of the chapter
| Autolysis | Destruction or digestion of tissue and cells by the enzymes |
| Fixation | The stabilization of protein |
| Artifact | A structure or substance not normally present but produced by some external force or action |
| Pigment | a heterogeneous group of substances that contain enough natural color to be visible without any further staining |
| Denaturation | To change the nature of |
| Non aqueous fixative | nonadditive, coagulating fixative. primarily acetone and methyl or ethyl alcohol. very;flammable only used when desired tissue components are destroyed or dissolved by the aqueous fixative |
| Coagulating fixative | establish a network in tissue that allows solutions to readily penetrate or gain entry into the interior of the tissue |
| Additive fixative | a chemical or substance that adds onto; combines with another substance usually improving,strengthening, or altering it |
| Hypertonic | causes the water to leaves the cell and the cell shrinks |
| Isotonic | fluids into which normal animals cells can be placed without causing either swelling or shrinking of the cells |
| Hypotonic | water will be drawn into the cell causing swelling a possible rupture of the cell membrane |
| Putrefaction | Tissue breakdown by bacteria |
| What does fixation prevent? | Autolysis(enzyme attack) and putrefication(bacterial attack) |
| What does fixation do? | Transforms soluble cellular components into insoluble substances so that they are not lost in subsequent processing steps. |
| What is denaturation? | The change in the 3-D structure of macromolecules. |
| What is the older definition of fixative action? | A fixative kills, penetrates, and hardens tissue. |
| Why is penetration important? | Penetration is important because it ensures fixation of the interior of the tissues as well as the few exterior cell layers. |
| _____________ has long been recognized as a preservative? | Wine, or alcohol |
| Major functions of the fixatives are __________________________. | Autolysis, putrefaction, and insolubilize cellular components |
| List 2 functions of a fixative. | 1. To kill the tissue by denaturing proteins so the postmortem activities of decay, or putrefaction(bacterial attack and autolysis(enzyme attack) are prevented. 2. To help maintain the proper relationship between cells and substances |
| List 2 more functions of a fixative. | 3. to bring out differences in RI and increase visibility of different tissue elements 4. help aid in rendering cell constituents insoluble, with proteins serving as the primary target for stabilization |
| Which one cannot be prevented? Autolysis or putrefication | Autolysis( enzyme attack) |
| Why does autolysis occur? | Autolysis occurs because enzymes continue their metabolic processes, after interruption of blood supply, until something stops the enzymes in action. |
| How can bacterial attack(putrefaction) be prevented in most fresh tissue? | By observing very strict antiseptic techniques. |
| How does fixation prevent autolysis? | the shape of the enzymes are altered and biological activity is destroyed |
| What happens to severely autolysed tissue? | The tissue will fail to stain |
| Why is stabilization important? | Distortion in tissue elements could occur |
| Refractive Index | the ratio of the velocity of light in air to the velocity of light in a liquid or solid medium |
| Enhancing differences in the refractive indexes of various tissue structures will increase the contrast between those structures. True or False | True |
| How is fixation enhanced and what happens to tissue if not fixed correctly? | Fixation is enhanced by staining and tissue not fixed correctly will stain poorly. |
| Some fixatives help stabilize or retain _________ and _________ initially, but, most of the time are lost | Lipids, carbohydrates |
| Does fixation make tissue firmer or softer? Why? | Firmer, so that gross dissection and collection of the thin sections required for processing becomes much easier. |
| Actions of fixatives | Make tissues more receptive to dyes and acts as mordants, which serve to link the dye to the tissue |
| Where are the differences of each fixatives morphologic pattern and set of artifacts seen in light microscopy? | These differences may be visible in the nuclear chromatin patterns, cell membranes, or staining intensities of various cellular elements. |
| Name physical methods of fixation. | Heat fixation(microwave oven) and Dessiccation |
| The physical method of heat fixation | is not generally been used in histopathology, but is finding more use because of the microwave. The added energy breaks internal bonds of tissue, molecule unwinds, new formed bonds create a stabilized molecule. |
| Oscillate | swing back and forth |
| Microwaves are a form of _____________ radiation. | Nonionizing |
| What 2 fixatives are are mainly used before microwaving? | Glyoxal and formaldehyde |
| What technique is desiccation used for? | Air drying of touch preparations for Wright staining |
| What is the primary method used for stabilizing protein in tissues? | Chemical method using 1 or more reagents |
| Classification of Chemical Reagents | Additive v Nonadditive, Coagulant v Noncoagulant |
| Additive | Chemically link or add themselves to the tissue and change it with this action |
| What is 2 step fixation? | 1st step immerse large specimens in saline to make tiss firm for gross dissection 2nd step fixation of 2mm thick blocks immered in saline and heated to 50-68C or 45-55C |
| If a tissue shows pyknotic overstained nuclei, what was the cause? | Temperatures exceeded 68 C |
| Denaturation of proteins from overheating causes.... | a loss of enzyme activity, antigenicity, false localization of nucleic acids and lysis of red cells. |
| Common Additive Reagents | Mercuric chloride, Chromium trioxide, picric acid, formaldehyde, glutaraldehyde, glyoxal, osmium tetroxide, zinc sulfate, and zinc chloride. |
| Nonadditive fixatives | Alcohols, Acetic Acid, and Acetone |
| Nonadditive | Act on tissue without chemically combining with it |
| Excessive removal of bound water causes | shrinkage and hardening |
| Coagulation | Establishes a network in tissue that allows solutions to readily penetrate or gain entry into the interior of the tissue |
| Coagulant fixatives | Zinc salts, mercuric chloride, cupric sulfate, ethyl alcohol, methyl alcohol, actetone, and picric acid |
| Action of noncoagulant fixatives | act by creating a gel that makes penetration difficult |
| What two processes are inferior for noncoagulant fixatives? | paraffin embedding and infiltration |
| A fixative that is classified as a coagulant of nucleic acids, but a noncoagulant of cell cytoplasm | Acetic Acid |
| Noncoagulant fixatives | formaldehyde, glutaraldehyde, glyoxal, osmium tetroxide, potassium dichromate, and acetic acid |
| Factors that affect the quality of fixation | temperature, size, volume ratio, and time |
| What does temperature affect? | Tissue morphology |
| If the temperature increases what happens? | an increase in temperature increases the rate of autolysis and diffusion of cellular elements |
| Ideal temperature range for fixation of specimens for electron microscopy | 0-4 C, however some labs have moved away from cold fixation |
| What temperature is ideal for formaldehyde fixation and ultrastructural preservation? | room temperature |
| Higher temperatures are are being used in which two instruments | microwave oven and tissue processors |
| Why is the size(thickness) of a tissue important? | Its effect on reagent penetration |
| What happens if large specimens(small intestine) are held for extended periods without being surgically opened to expose all layers?What is the corrective action? | The fixative will have difficulty penetrating through the entire wall to the inner epithelial surface. Results in autolysis. Ca=open before they are placed in fixative solution. |
| How should solid organs such as spleen or kidney be placed? | They should be bread-loafed as soon as possible and covered i fixative. |
| How thick should sections be cut? | < or equal to 3 mm thick |
| True or false: Tissues should be so thick they touch both the top and bottom of the tissue processing cassette. | False, They should not touch the top and bottom of the tissue processing cassette |
| How much greater should the fixative volume be than the tissue volume? | 15-20X |
| What happens if the tissue volume is greater than the fixative volume? | If the tissue volume is greater than the solution, the fixation composition is compromised. |
| What two problems are really the result of poor fixation because of the inadequate volume of fixation? | Staining and/or processing |
| Time is important in what 2 respects? | Ischemic time and duration |
| Ischemic time | The interval between interruption of blood supply and placement of the tissue in fixative |
| What happens when more time elapses between interruption of the blood supply and fixation ? | The more postmortem changes that can be demonstrated microscopically |
| Tissues rich in enzymes such as liver,brain,and pancreas are more subject to ______________ than those with a predominance of connective tissue fibers. | Autolysis |
| Duration | current trend of decreasing the time allowed for fixation. This is resulting in many problems. |
| _____________ preserves the morphology of the tissue and keeps tissue from being distorted. | Adequate Fixation |
| Artifact that is attributed to fixation with formalin alone, however, Dapson attributes it to the specimen not being completely fixed before dehydration is begun. | Nuclear bubbling |
| How long should formalin have to act before the remainder of the processing schedule is begun? | 6-8 hrs |
| Where does processing occur and why? | in the dehydrating alcohols for fixation to occur in fixative solution |
| What is Her2 and what are the guidelines? | Human epidermal growth factor receptor 2 in invasive breast cancer. Current guiddelines recommend that the incisional and excisional biopsy specimens used are fixed in 10% neutral buffered formalin for a min. of 6 hrs and a max. of 72 hrs |
| If tissue is allowed to remain in glutaraldehyde, Helly solution, Zenker solution, and Bouin solution too long, the tissue becomes ____________ and ___________ may be impaired. | Overhardened and staining |
| How should the specimen be prepared for immunofluorescence study or if an enzyme profile is needed? | Must be frozen without fixation |
| What solutions should be used when muscle cross striations are to be stained with phosphotungstic acid- hematoxylin? | Zenker and Bouin solution |
| When tissues are to be stained with a trichrome technique what solution should be used? | Bouin Solution |
| Postfixation(mordanting) | tissue section that has been fixed with 1 reagent can be treated with another fixative |
| When is postfixation is used? How is it fixed? | Masson trichrome technique is fixed in formalin then mordanted with Bouin solution before staining. |
| ________________cannot be demonstrated after formalin fixation. | Pheochromocytomas |
| For the demonstration of chromaffin granules, tissue must be fixed in a primary dichromate fixative such as ______________. | Orth Solution |
| ____________________are water soluble and requires a nonaqueous fixative such as _________________________. | Urate Crystals, Absolute alcohol |
| A physical process and fixatives do this at different rates. | Penetration |
| Law of diffusion | d= k/t d=depth k= coefficient t= root of time/hours k increases as penetration increases |
| k=0.78 for 10% formalin | Rounding to 1 this means 10% formalin would penetrate 1mm per hour. |
| Factors that influence the penetration rate | increase in pressure, distance increases with time, type of tissue, presence of muscle fibers and capillaries as well as an increased surface area with crypts. |
| What does the increase in pressure do? | improves formalin penetration |
| The type of tissue influences the ____________________ of penetration. | Rate |
| The factors that determine the minimum length of time that a fixative should act are the _____________________ and _____________. | rate of penetration and the mode of action |
| Which fixative penetrates faster than any of the common fixative ingredients? | Formaldehyde |
| Fixative ingredients, in order of decreasing speed of penetration are (6 of them). | formaldehyde, acetic acid, mercuric chloride, methyl alcohol, osmium tetroxide, and picric acid |
| What affects the rate of penetration? | heat |
| A noncoagulant that penetrates fast, but continues to crosslink proteins for a long time after penetration is complete | Formaldehyde |
| Why is tissue storage important? | Wet tissue is often needed for additional studies |
| Tissue is typically stored in what fixative? if its immunohistochemical stains are required in the future should be stored in __________________. | NBF, and must be transferred from formalin to 70% alcohol to stop crosslinking. |
| How does pH affect fixation? | It influences the reactivity of the fixative, and in electron microscopy influences ultrastructural preservation. |
| Osmolality | the # of particules in a solution |
| What is the osmolality of body fluids? | 340 mOsm or 0.3 Osm. |
| 1- Osm can be defined as | 1 formula weight of a nondissociating compound(sucrose) per 1000 g of solution |
| 1 formula weight of a dissociating compound(sodium chloride) per 1000 g of solution is defined as | 2- Osm or 2000 mOsm |
| The most rapidly penetrating component of an aqueous fixative | Water |
| Used as a holding solution for tissue | Normal isotonic saline solution |
| Hypotonic | The cells swells because water was drawn from the surrounding solution in the cell |
| Hypertonic | The cell shows shrinkage because water was drawn from the cell into the surrounding solution |
| Isotonic | The cell neither swells or shrinks |
| What are added to prevent the damage caused by a hypotonic solution? | Unreactive salts with small rapid diffusing ions (sodium sulfate and sodium chloride) |
| What is the protocol for biopsy specimens that cannot be placed in a fixative immediately? | dampen a piece of gauze with saline, squeeze out the excess, and place tissue on dampened gauze. Tissue treated this way can be sealed in plastic container and placed on ice for short term holding. |
| What should kidney biopsy specimens be placed in for immunofluorescence? | Michel transport solution |
| What are found in the nucleus? | DNA, RNA, and attached protein histones |
| Preferred fixatives for nucleic acids | Acetic Acid and Carnoy Solution |
| To make formaldehyde react with DNA and RNA the temperature must be___________for RNA and ________ for DNA. | 45 C or 65 C |
| What is nuclear fixation? | The entrapment of RNA and DNA molecules by the fixed or stabilized nuclear proteins |
| According to Banks, what type of fixatives render tissue more resilient to the disruptive effects of sectioning , deparaffination, and staining. What are the results? | coagulating or precipitating results in sharper, more intact appearing nuclei. |
| What type of artifact is caused in the deparaffinization step on formalin fixed tissue or the specimen being imcompletely fixed in NBF before dehydration. | Nuclear Bubbling |
| List protein structures | primary, secondary, tertiary, and quanternary |
| Primary protein structure | amino acid sequence |
| Secondary protein structure | shape of the major polypeptide chain(backbone), common shapes are alpha helix or the beta pleated sheet and are held by hydrogen bonds |
| Tertiary Protein structure | Complete 3D shape of the protein and is determined by the interactions of the side chain( R groups) of amino acids |
| Quaternary Protein structure | applies to proteins that have multiple polypeptide chains that are joined together |
| What does it mean if an additive fixative changes the electrical charge? | That charge was a force helping to maintain the conformation(shape) of the protein, then the tertiary structure may be changed. |
| What does the protein structure determine? | The function of the protein |
| ONLY 2 fixatives will insolubilize lipids so that they are not lost in processing. What are they? | Osmium tetroxide and chromic acid |
| An additive and crosslinking fixative. It reacts with double bonds of unsaturated lipids. It produces dark-brown color and can now work as a stain. | Osmium Tetroxide |
| Correct the statement Most carbohydrates are lost during fixation in nonaqueous solutions. | Corrected statement Some carbohydrates are lost during fixation in aqueous solutions. |
| Hallmarks of good fixation | Nuclei with various crisp chrmatin patterns and a crisp blue nuclear membrane, not cell shrinkage or artifactual spaces, cell cytoplasm well preserved and stain well with eosin |
| What does formalin pigment resist? | extraction by most strong acids, water, alcohol, or acetone |
| How is formalin and malarial pigments removed? | absolute alcohol saturated with picric acid(10 min-3hr), wash with water or 70% alcohol(100mL) containing 3 mL ammonium hydroxide(30 min-3 hrs) and rinse in 1% acetic acid |
| Removal procedure for mercury pigment | Gram or Lugol iodine for 10 min., wash in running water, place sections in 5% sodium thiosulfate(hypo) for 3 min, wash scetion for 10 min and stain as desired. |
| Major problems encountered with fixation are caused by ____ or__________. | Delayed or incomplete fixation |
| Autolysis is caused by ____________ fixation. | delayed |
| Autolysis is prevented by | placing specimen in fixative asap(15-20X greater), opening uterus specimens upon receipt so fixative can come into contact with the endometrium, opening andpinning GI tract open so fixative gets mucosal surface, cutting thin sections, bisecting lymph node |
| What causes incomplete fixation | Rapid turnaround time, specimens not allowed enough time in the fixative solution to fix completely before beginning dehydration |
| Corrective action(s) of incomplete fixation | Increase time allowed in fixative, change to another fixative, place formalin alcohol in 1st 3 stages, ensure sections are thin enough for penetration, do not pack cassette tightly, use agitation, solution not depleted from overuse |
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