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Untitled Flashcards
Study
FIXATION
Process of preserving tissues to retain the state of the tissue as it was removed from the body, prevent decay, inactivate enzymes, and stabilize cellular structures.
GOALS OF FIXATION
1. Preserve the chemical and morphological integrity of the cell, making it appear lifelike. 2. Prevent decay and putrefaction. 3. Stabilize cellular structures and provide rigidity to the tissue.
Facts about tissue processing
1. Fixation makes cells slightly swell and tissues lose 20-30% of their total volume. 2. Some fixatives contain a high amount of water, which can affect the degree of staining. 3. It stops the activity of enzymes, preventing the breakdown of cellular components.
What is autolysis in the process of fixation?
Autolysis refers to the premature release of enzymes inside the cell that leads to the breakdown of different cellular components. In the process of fixation, enzymes are inactivated by fixatives, preventing them from acting on the cellular components.
Why is it important to use an appropriate volume of fixative in tissue fixation?
The volume of fixative must be 20 times greater than the volume of the tissue, or a range of around 10-25 times the volume of the fixative compared to the tissue. This ensures proper penetration and preservation of the tissue components.
What is the role of hydrogen ion concentration (pH) in fixation?
Fixatives are known to have a somewhat neutral or almost neutral pH (around 6-8). The pH level affects the preservation of tissue components and the activity of fixatives.
How does temperature affect tissue fixation?
Cold temperatures retard the process of fixation, while warm temperatures enhance the diffusion of fixative inside the tissues. High temperatures above 45 degrees Celsius can lead to tissue maceration, affecting the fixation process. Some procedures may require specific temperature conditions, such as refrigeration for electron microscopy or freezing for frozen sections.
What is the relationship between tissue thickness and fixative penetration?
Thicker tissues allow less fixative penetration, while thinner tissues allow better fixative penetration. The thickness of the tissue affects the effectiveness of fixation.
What temperature should tissues be kept at for optimal cutting?
5 to 10 degrees Celsius.
What does OCT stand for?
Optimal Cutting Temperature.
How does the thickness of tissue sections affect fixative penetration?
Thicker tissue results in less fixative penetration, while thinner tissue allows for better fixative penetration.
What is the significance of osmolality in fixatives?
Most fixatives are kept at a slightly hypertonic osmolality to maintain tissue integrity.
Why is it important to dilute fixatives?
Diluting fixatives eliminates brittleness while providing enough water to contribute to tissue volume without extensive cell swelling.
What is the general concentration of formalin used as a fixative?
10% concentration.
DURATION OF FIXATION
The duration of fixing a tissue depends on the tissue thickness and the type of fixative used. Different fixatives have different durations to produce their expected effects. For example, 10% formalin requires an overnight or 24-hour fixation, while metallic fixatives may only require a few hours.
TIME INTERVAL
The time interval from tissue excision to fixation. Tissues should be submerged in fixative within 1 hour of removal from the body to avoid the effects of cell death on the tissue.
CONSIDERATIONS ON FIXING TISSUES
Tissues must be fixed within 1 hour of excision to prevent the effects of cell death. The tissuetofixative ratio must be at least 1:10 or in general 1:20. Anatomical barriers should be incised or removed to enhance fixative penetration. Large specimens may need to be sectioned or inflated for deeper penetration of fixatives. Tissues can be pinned on a corkboard or a wick inserted into tubular structures to maintain shape.
DURATION AND PENETRATION RATE
Not all fixatives have the same penetration rate, so the duration of fixation must be considered based on the tissue type and fixative used. Prolonged fixation can lead to the loss of IHC antigenicity, affecting the process of Immunohistochemistry (IHC).
IMMUNOHISTOCHEMISTRY (IHC)
IHC is the process of detecting antigens in tissues using antibodies, essential for diagnosis. Prolonged fixation can lead to the loss of antigenicity, impacting the antigen-antibody interaction necessary for IHC examination.
Antigen-Antibody Reaction
In Immunohistochemistry (IHC), this interaction is necessary for the examination process. Prolonged tissue fixation can lead to loss of epitopes and antigenicity properties.
Methods of Fixation
1. Physical: Includes heating (microwaving, cryopreservation) 2. Chemical: Includes immersion, perfusion, vaporfix.
Fixation Mechanisms
1. Additive Fixation: Incorporates fixative inside cells, forms crosslinks and provides rigidity. 2. Nonadditive Fixation: Alters tissue composition, removes water by competing with H-bonds.
Types of Fixation
1. Aldehydes: Crosslinks proteins, deposits on cellular cytoskeleton for rigidity. 2. Oxidizing Agents: Crosslinks proteins, deposits on cellular cytoskeleton for rigidity. 3. Alcohol-based Fixatives: Protein-denaturing agents, removed or precipitated outside the cell. 4. Metallic Fixatives: Forms insoluble metallic precipitates to enhance staining qualities.
According to Composition
Fixatives do not synergize with each other and produce individual effects on tissues.
Simple Fixatives
Contains one component: a. Aldehydes b. Metallic Fixatives c. Picric Acid d. Acetic Acid e. Acetone f. Alcohol g. Osmium Tetroxide
B COMPOUND FIXATIVES
Combination of two or more fixatives Combines effects of the individual action of fixatives
II ACCORDING TO ACTION
A MICROANATOMICAL FIXATIVES - Permits microscopic study - Doesn't distort the structural patterns of the cells - Preserves the structure of the entire cell B CYTOLOGICAL FIXATIVES - Preserves specific parts - Nuclear (pH 4-6) - preserves contents of the nucleus only, acidic - Cytoplasmic (pH 4-6) - preserves only the cytoplasmic contents inside the cell, somewhat basic components of cytoplasm - Histochemical - preserves the biochemical molecules inside the tissue for demonstration (e.g., lipid, fats, proteins, nucleic acids)
SECONDARY FIXATION
Fixing an already preserved tissue in a different fixative - Allows for demonstration of particular substances (e.g., lipids) - Makes special staining possible
MORDANT
Ensures further and complete hardening and preservation of tissues - Done before dehydration and on deparaffinized tissues before staining - Done before dehydration and after microtomy after removing the excess paraffin wax or resin - Postchromatization using potassium dichromate enhances the staining quality of tissues
WASHING OUT
Process of removing excess fixative - Rinsing out the excess fixatives present on the tissue - Improves staining and removes artifacts - Solutions used: tap water, 50-70% alcohol, alcoholic iodine
