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Untitled Flashcards
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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
BIOCHEMICAL FIXATION
Preservation of certain biochemical molecules for demonstration - Also known as histochemical fixation
LIPID FIXATION
For preserving lipids - Must be fixed with a solution that mustn't affect lipid composition - Examples of fixatives: mercuric chloride, potassium dichromate - Frozen tissue sections and lipid stains may be used to preserve and stain lipids
PHOSPHOLIPIDS
Found in cell membrane - Fixed with aldehydes - For cholesterol demonstration: Bakers formol citrate, digitonin
CARBOHYDRATE FIXATION
For preserving carbohydrates - Do not use fixatives with high water content - Examples: alcoholic fixatives, Rossmans fluid
PROTEIN FIXATION
For preserving proteins - For amino acid histochemistry: NB Formol-Saline or formaldehyde vapor
FIXATION FOR EM, ENZYME, HC, IF, AND IHC ELECTRON MICROSCOPY
Specialized fixation for electron microscopy, enzyme histochemistry, immunofluorescence, and immunohistochemistry
Fixed with aldehydes
Used for preserving tissue structure and proteins in a fixed state.
Baker's formol citrate
A fixative used specifically for preserving carbohydrates.
Digitonin for cholesterol demonstration
A fixative used to demonstrate cholesterol content in tissues.
Carbohydrate Fixation
Fixation method for preserving carbohydrates in tissue samples.
Hydrophilic
Tendency to attract and hold water; hydrophilic fixatives do not have a high water content.
Alcoholic Fixatives
Fixatives containing alcohol, used for preserving tissue structure and proteins.
Rossmann's fluid
A fixative recommended for preserving glycogen in tissue samples.
Protein Fixation
Technique for preserving protein structure and activity in tissue samples.
Amino Acid Histochemistry
Method for studying the distribution of amino acids in tissues using fixation techniques.
NB Formal Saline or Formaldehyde vapor
Recommended fixatives for preserving proteins for histochemistry.
ENZYME HC, IF, and IHC
Techniques involving enzyme histochemistry, immunofluorescence, and immunohistochemistry for tissue analysis.
Electron Microscopy
Method for studying tissue ultrastructure at a microscopic level, requiring specific fixation techniques.
Glutaraldehyde
Recommended fixative for tissue samples intended for electron microscopy.
Osmium Tetroxide
Secondary fixative used after glutaraldehyde for electron microscopy.
Maximal Preservation of Enzyme Activity
Goal of enzyme histochemistry fixation techniques to maintain enzyme activity and location in tissues.
Overnight Fixation
Extended fixation duration using 4% formaldehyde or formal saline for tissue preservation.
Fixed with acetone or formaldehyde for frozen sections
Fixation methods specifically tailored for preserving tissues for frozen section analysis.
Immunofluorescence and Immunohistochemistry
Methods involving antigen-antibody interactions for detecting specific antigens in tissue samples.
Epitopes of Antigens
Specific binding sites on antigens that must be preserved for antibody detection.
Antigen Retrieval
Technique used to restore antigenicity after prolonged or harsh fixation, often involving heat-induced epitope retrieval (HIER).
Microwave Fixation
Use of microwave heat to aid in tissue fixation or assist in the permeation of chemical fixatives.
Advantages of Microwave Fixation
Rapid fixation, preservation of neurochemical substances, reduced processing time for immunohistochemistry, and preparation of tissues for electron microscopy.
B COMPOUND FIXATIVES
Combination of two or more fixatives that combines effects of individual action of fixatives.
II ACCORDING TO ACTION
A. MICROANATOMICAL FIXATIVES: Permits microscopic study, doesn't distort the structural patterns of the cells, and preserves the structure of the entire cell. B. CYTOLOGICAL FIXATIVES: Preserves specific parts such as nuclear contents, cytoplasmic contents, and histochemical molecules inside the tissue for demonstration. SECONDARY FIXATION: Fixing an already preserved tissue in a different fixative for special staining. MORDANT: Ensures further hardening and preservation of tissues before staining. WASHING OUT: Process of removing excess fixative by rinsing with tap water or 50-70% alcohol. BIOCHEMICAL FIXATION: Preservation of specific biochemical molecules for demonstration.
MICROANATOMICAL FIXATIVES
Permits microscopic study, doesn't distort the structural patterns of the cells, and preserves the structure of the entire cell.
CYTOLOGICAL FIXATIVES
Preserves specific parts such as nuclear contents (pH 4-6), cytoplasmic contents (pH 4-6), and histochemical molecules inside the tissue for demonstration.
SECONDARY FIXATION
Fixing an already preserved tissue in a different fixative for special staining. Allows the demonstration of particular substances (e.g., lipids) and enhances staining quality.
MORDANT
Ensures further hardening and complete preservation of tissues before staining. Applied before dehydration and on deparaffinized tissues before staining.
WASHING OUT
Process of removing excess fixative by rinsing with tap water or 50-70% alcohol. Improves staining and removes artifacts.
BIOCHEMICAL FIXATION
Also known as histochemical fixation. Involves the preservation of specific biochemical molecules inside the tissue for demonstration.
LIPID FIXATION
Preservation of lipids in tissues. Requires fixatives containing HgCl2 or potassium dichromate. Frozen tissue sections may also be used to preserve lipids, and a general lipid stain is used for staining.
CARBOHYDRATE FIXATION
Preservation of carbohydrates in tissues. Rossmann's fluid can be used for glycogen fixation.
PROTEIN FIXATION
Preservation of proteins in tissues. Formol-Saline or formaldehyde vapor can be used for amino acid histochemistry.
FIXATION FOR EM, ENZYME HC, IF, AND IHC
Fixatives used for electron microscopy, enzyme histochemistry, immunofluorescence, and immunohistochemistry.
Aldehydes
Used for protein fixation, enzyme histochemistry, and immunofluorescence.
Formol
Used for carbohydrate fixation, protein fixation, and overnight fixation.
Citrate
Used in Bakers' fixative for tissue demonstration.
Digitonin
Used for cholesterol demonstration in fixatives.
CARBOHYDRATE FIXATION
Requires hydrophilic fixatives and should not be high in water content.
Alcoholic fixatives
Used for preservation of tissues and reduction of fixation time.
Rossmans fluid
Used for preservation of glycogen in tissues.
PROTEIN FIXATION
Involves the use of Formol-Saline or formaldehyde vapor for preserving enzyme activity.
ENZYME HISTOCHEMISTRY
Aims to preserve enzyme activity and structural integrity during fixation.
ELECTRON MICROSCOPY
Requires glutaraldehyde as the recommended fixative, followed by a secondary fixation using Osmium tetroxide.
ENZYME HISTOCHEMISTRY
Maximally preserves enzyme activity and structural integrity in tissues.
4C TEMPERATURE
Recommended for electron microscopy and fixation to preserve tissue structure at around freezing temperatures.
Acetone
Used for fixing frozen tissue sections.
IMMUNOFLUORESCENCE AND IMMUNOHISTOCHEMISTRY
Relies on the preservation of epitopes of antigens for antibodies to bind and detect antigens.
Antigen retrieval
Can be done to restore antigenicity using heat-induced epitope retrieval.
MICROWAVE FIXATION
Employing heat to fix tissue, either by allowing heat to permeate and fix the tissue itself, or using microwave-assisted fixation.
Advantages of MICROWAVE FIXATION
Allows examination of rapid cellular processes, preservation of neurochemical substances, rapid fixation of tissues, reduction of processing time for immunohistochemistry, and production of tissues for electron microscopy.
Disadvantages of Microwave Tissue Fixation
- May only penetrate tissues with 10-15 mm thickness - No significant crosslinking of proteins, resulting in soft tissues - Spores and other pathogens may remain in the tissues
Chemical Fixation
- Common ways to fix tissues - Use of chemical reagents to preserve tissues - Chemical fixatives have different actions: crosslinks with proteins, denatures proteins out of the tissue, precipitates proteins in the process
Formaldehyde and Formalin
- Used as a standard fixative - Sold as 35-40% weight in volume solution - Stabilized by methanol at 15% - Must be diluted to a 10% formalin solution in phosphate-buffered saline - Advantages: cheap, easy to prepare, penetrates tissues well, can be stored for a long time - Disadvantages: very irritating, chronic use is carcinogenic
Paraformaldehyde
- White powder formaldehyde polymers - Used for paraffin embedding, sectioning, and IHC - Effects are reversible by excess water - Avoids pigmentation produced by formalin - Long-term shortage - Similar disadvantages to formaldehyde
Glutaraldehyde
- Standard fixative for electron microscopy (EM) - Less dangerous compared to paraformaldehyde
Formalin
An organic solution of formaldehyde in water, commonly used as a tissue fixative in histology and pathology.
Heme Pigment
The pigment responsible for the brown color observed in tissues with high blood content when exposed to formalin fixation.
Tissue Penetration
The ability of a fixative to penetrate and preserve tissue structures, often a desirable quality for effective tissue fixation.
Long-term Shortage
A condition where there is a prolonged scarcity of a particular fixative, which may require alternative fixative options.
Disadvantages of Formalin
Include potential health hazards, irritating effects, and properties similar to formaldehyde. It may also cause tissue distortion and shrinkage.
Glutaraldehyde
A standard fixative for electron microscopy (EM) with less danger compared to paraformaldehyde or formaldehyde, and a more pleasant smell. It is often used in EM at concentrations of 2%, 2.5%, or 4%.
Electron Microscopy (EM)
A technique for obtaining high-resolution images of biological specimens, commonly used in research and diagnostic laboratories.
Karnovsky's Fixative
Also known as Karnovsky's paraformaldehyde-glutaraldehyde solution, a fixative composed of 4% paraformaldehyde and 1% glutaraldehyde mixed together in 0.1 M Phosphate Buffer. It is suitable for embedding tissues in resin and is commonly used in electron microscopy.
Alcoholic Fixatives
Fixatives like methanol, ethanol, and chloroform solutions used to preserve tissue samples and cytologic smears. They are known to denature proteins and are often used in slide samples or for specific applications.
Methanol
An example of an alcoholic fixative that denatures proteins, not routinely used for tissues due to potential tissue distortion. It is used in cytologic smear fixation and for preserving peripheral blood smears and bone marrow aspiration smears.
Isopropanol
A fixative used for the preparation of touch or imprint slides, often used as a 95% solution for this purpose.
Ethanol
Used as a fixative and dehydrating agent at concentrations of 70% to 100%. It is also used in the preparation of various fixative solutions and techniques.
Carnoy's Fluid
A fixative composed of absolute alcohol and chloroform, often used to preserve Nissl bodies, cytoplasmic granules, nucleoproteins, and nucleic acids in brain tissues for rabies diagnosis.
Alcoholic Formalin
A fixative that involves the use of formalin mixed with absolute alcohol, often used for fixing large fatty specimens and in certain diagnostic techniques.
Clark's Solution
A fixative used on frozen tissue sections, typically containing formaldehyde and other components to preserve the tissue structure during the freezing process.
Formol-Acetic Alcohol
A fixative used for fixing tissues for diagnostic cryostat sections, typically containing formalin and acetic acid mixed with alcohol.
Gendre's Fluid
A fixative used to enhance immunoperoxidase studies for glycogen and microincineration, often containing specific components for these purposes.
Newcomer's Fluid
A fixative used for fixing mucopolysaccharides and nuclear proteins, often composed of specific components for these purposes.
Metallic Fixatives
Fixatives made from metallic compounds, the mechanisms of action for tissue preservation are still not fully understood. They can produce good to excellent staining quality but often have poor tissue penetration due to their large molecules.
Tissue Shrinkage
A reduction in tissue size or volume, often observed as a result of certain fixatives and fixation techniques.
Ability to penetrate tissue
Poor penetration produces shrinkage.
Molecule size
Large molecules of fixatives result in poor penetration compared to alcoholic and aldehyde fixatives.
Staining quality
Good to excellent staining quality.
Mercuric Chloride
- Most common metallic fixative - Used as a 5-7% aqueous solution - Works by additive and coagulative means - Incorporates itself in cells and precipitates protein, denaturing them - Gives excellent nuclear detail - Allows excellent trichrome staining
Zenker's Solution
- Recommended for congested specimens - Used for trichrome staining and PTAH (Phosphotungstic Acid Hematoxylin) staining - Excellent fixative for bone marrow, liver, spleen, pituitary glands, and cardiac muscle
Lillie's B5 Fixative
- Used for fixing hematopoietic BM biopsies and lymphoid tissues
Heidenhain's Susa
- Used for tumor biopsies of the skin - Permits easier sectioning of large blocks of fibrous connective tissue
Dezenkerization
- Removal of mercury deposits from tissues fixed with mercury or mercury-based fixatives - Involves using Lugol's Iodine or alcoholic iodine solution, followed by rinsing with sodium thiosulfate 5% solution
Oxidizing Agents
- Include Permanganates, Dichromates, Chromic Acid, Osmium Tetroxide, and Picric acid or Picrate - React with various side chains of proteins and other biomolecules, forming crosslinks to stabilize tissue structure
Osmium Tetroxide
- Used as a secondary fixative in electron microscopy - Causes complete denaturation of proteins - Preserves cytoplasmic structures - Penetrates at a slower rate compared to metallic fixatives - Gives off a black stain
Traditionally used in EM fixative
Osium-based fixatives
Good fixative and stain for lipids
Osium-based fixatives
Preserves cytoplasmic structures, Penetrates at a slower rate vs metallic fixatives
Osium-based fixatives
May give off a black stain, Extremely volatile, prolonged exposure to vapors may cause conjunctivitis, Must be done under a fume hood
Osium-based fixatives
Flemmings Fluid
Osium-based fixatives, most common permanent fixative for fat
Flemmings without acetic acid
Osium-based fixatives, recommended for demonstrating mitochondria
Chromic Acid 12
Chromate fixatives, good in preserving mitochondria at pH 4-5, most notable
Regauds Fluid
Chromate fixatives, recommended for demonstrating mitochondria, mitotic figures, Golgi bodies, RBC, and colloid-containing tissues
Orth's Fluid
Chromate fixatives, recommended for studying early degenerative processes and tissue necrosis, advisable for demonstrating rickettsiae and other bacteria
Fixatives created using picric acid or salts of picric acid
Picrate fixatives
Bouin's Fluid
Picrate fixatives, recommended for fixing embryos, pituitary biopsies, and glycogen
Hollande's Solution
Picrate fixatives, recommended for gastrointestinal tract specimens, endocrine tissues, and has some decalcifying properties
Gendre's Fluid
Picrate fixatives, highly recommended for glycogen and carbohydrate preservation, produces minimal distortion of microanatomical structures
Brasil's Alcoholic Picroformol Fixative
Picrate fixatives, better and less messy than Bouin's, excellent for preserving glycogen
Glacial Acetic Acid
Anhydrous, often used in conjunction with other fixatives, enables cell swelling, precipitates and fixes nucleoproteins
Considered as a metallic fixative because of the presence of lead
Lead fixatives
Recommended for fixing acid mucopolysaccharides and mucin
Lead fixatives
What is the substance that needs to be removed?
Lead carbonate deposits
What methods can be used to remove lead carbonate deposits?
Several methods can be used to remove lead carbonate deposits, including chemical cleaning, mechanical scraping, and electrolytic treatment.
TRICHLOROACETIC ACID (TCA)
Fixative and decalcifying agent that precipitates proteins and nucleic acids. It is helpful for the demonstration of nucleic acids. Used only on small pieces and penetrates poorly.
ACETONE
Nonalcoholic compound not recommended as a morphological fixative. Used in fixing cryostat sections and for tissues requiring enzyme preservation. Used to fix tissues at cold temperatures (0-4°C). Recommended for fixing brain tissues to diagnose rabies. Preserves glycogen poorly but dissolves fats.
MICHELS SOLUTION
Used for transporting specimens in a reference laboratory. Not a fixative. Can keep tissues for 5 days in transit. Stable medium for transporting fresh unfixed tissues that will undergo frozen sectioning or IF (immunofluorescence) studies. Not suitable for keeping tissues for FISH (Fluorescent in situ hybridization). Specimens can be kept in the solution at an ambient temperature of 4-22°C.
Decalcification
The process of removing calcified structures inside tissue samples to facilitate an easier cutting process.
Removal of Calcium in tissues
The process of removing calcium or magnesium ions that contribute to the hardening of structures in tissues, such as bones and other calcified tissues.
Softening of Tissues
The process of making tissue as soft as the embedding medium to facilitate easier cutting for histotechnologists during microtomy.
Calcified Tissues vs. Decalcified Tissues
Calcified tissues are hard and dense, while decalcified tissues are softened and easier to cut.
Principle of Decalcification
Sections are immersed or exposed to methods that facilitate the exchange of ions to form calcium salts, often through the use of chelating agents or acrylic/epoxy resins.
Chelating Agents
Substances applied to calcified structures that bind calcium and other metallic ions to soften tissues for decalcification.
Use of Resins
Resins such as acrylic or epoxy can be used to eliminate the need for decalcification by replacing calcium ions or other metallic ions contributing to tissue hardness.
Factors Influencing Decalcification
Volume, fluid access, size and consistency, agitation, and temperature are factors that influence the decalcification process.
Volume Ratio
The ratio of decalcifying agent to tissue, with a lower ratio retarding the decalcification process.
Fluid Access
All sides of the tissue must be exposed to the decalcifying agent to ensure even decalcification.
Suspended Tissue
Tissue can be suspended using gauze and thread, with gauze holding the tissue and thread suspending it above the base of the container.
Size and Consistency
Dense tissues, which have a lower penetration rate, must be soaked for around 14 days and changed daily for proper penetration during decalcification.
Agitation
Swirling the decalcifying agent to facilitate circulation around the container and prevent uneven decalcification.
Temperature
Increased temperature enhances the penetration of the decalcifying agent, but excessive temperature can have damaging effects.
Temperature and Decalcification
Increased temperature excites molecules, facilitating easier penetration of decalcifying agent. Low temperature retards the process.
Acid Decalcification
Use of various acids to form soluble salts. Most common method.
Chelation in Decalcification
Binds with calcium to soften tissue. Chelation means binding.
Ion Exchange Resin
Removes calcium from decalcifying agents. May contain formic acid.
Electrophoresis
Use of electrodes to remove calcium ions. Based on their charges.
Microwave Decalcification
Application of microwaves to hasten the process.
Tissue Softening
Use of decalcifying agent to soften unexpected foci of calcium during microtomy. Applied during microtomy process.
What is the purpose of tissue softening in the process of decalcification?
Tissue softening is important to facilitate an easier cut during the decalcification process.
What are the basic compositions of decalcifying agents?
Decalcifying agents are composed of strong acids, weak acids, or chelating agents.
How can dilute mineral acids be used as decalcifying agents?
Dilute mineral acids can be used only if the endpoint is monitored carefully to avoid tissue maceration.
What is the potential effect of prolonged decalcification with strong acids?
Prolonged decalcification with strong acids can macerate the tissues and compromise cellular details.
What is the potential consequence of tissue maceration during decalcification?
Excessive softening of tissue can lead to a pop-like consistency that facilitates poor tissue cutting and compression during microtomy process.
What is the potential consequence of using strong acids as decalcifying agents on nuclear staining?
Strong acids can affect the staining of nuclear contents but not acidophilic cell components, requiring post-decalcification removal and adjustments on the staining process.
How can post-decalcification be used to correct the effects of strong acids on staining?
Post-decalcification can remove the excess strong acids that may have remained inside the cell, facilitating a greater staining process during routine staining.
What is the common property of nitric acid-based decalcifiers and why does it impart a yellow color?
Nitric acid-based decalcifiers are common and impart a yellow color due to the presence of nitrous acid resulting from the reduction of nitric acid.
What is the difference between hydrochloric acid and nitric acid as decalcifying agents?
Hydrochloric acid is inferior to nitric acid in terms of its action and produces greater distortion during the decalcifying process.
How can hydrochloric acid be used as a decalcifying agent?
Hydrochloric acid can act as an enhancer of nuclear stain and may be used as a surface decalcifier or as 1 HCl in 70 alcohol or tissue softener.
HYDROCHLORIC ACID-BASED DECALCIFIERS
Produces a greater form of distortion during decalcifying process, slow action, and produces greater distortion. It may act as an enhancer of nuclear stain and may be used as a surface decalcifier.
Von Ebner's fluid
Weak acids, mostly organic acids but some are inorganic like formic acid and trichloroacetic acid. Better suited for urgent biopsy samples, moderate acting, and recommended for post-mortem research tissue studies.
Formic Acid (HCOOH)
Moderate acting formic acid, recommended for post mortem research tissue studies. Used as a 10% aqueous solution or combined with formalin or a buffer. Only weak acid extensively used as a primary decalcifier because it causes less distortion to the cells compared to strong acids.
SODIUM CITRATE TRICHLOROACETIC ACID (TCA)
Slow-acting not recommended for urgent examinations. Does not require washing out. Excess may be washed out using several changes of 90% alcohol. May be used for minute bone pieces.
CHROMIC ACID
Flemming's fixative and decalcifier. May form insoluble pigments. Highly corrosive, carcinogenic, and an environmental hazard.
CITRIC ACID / CITRATE BUFFER SOLUTION
Very slow tissue decalcification, seldomly used for routine decalcification. May be used as a chelating agent.
CHELATING AGENTS (EDTA)
Binds calcium and magnesium ions to soften bone and other calcified tissues. EDTA, most common chelating agent used, will not act like other acids, as it is adjusted to a pH of 7 or 8. More preferred if studying DNA or histochemistry. May take 1-3 weeks to decalcify small bone tissues and 6-8 weeks for dense cortical bone.
ENHANCEMENT PROCEDURES
Sonication with EDTA: use of sound waves to enhance the movements of EDTA. Microwave: act the same as sonication. Electrolytic decalcification.
EDTA
Ethylene diamine tetraacetic acid (EDTA) is a chelating agent used to bind and sequester metal ions.
Sound Waves
Used to enhance the movements of EDTA by agitating the solution and improving chelation.
Microwave
Acts similarly to sonication in enhancing the movements of EDTA and improving chelation process.
Electrolytic Decalcification
A process that uses an electric current to aid in the removal of calcium from tissues, similar in some ways to the mechanisms of EDTA chelation.
Physical Method for End Point
Using a probe to poke the tissue, but can distort the tissue due to the physical force.
Chemical Method for End Point
Utilizes several agents for decalcification. Titrating diluting agent using a base solution to check if decalcification process is completed. Look for cloudiness or turbidity in the solution to indicate incomplete decalcification.
Titration Process
Neutralizes acids with the addition of a strong ammonia solution. Presence of cloudiness indicates incomplete decalcification.
X-ray Method for End Point
Most ideal, sensitive, and reliable method. However, it is the most expensive.
Post-Decalcification Steps
Remove excessive decalcifying agent, neutralize with saturated lithium carbonate or 5-10% aqueous sodium bicarbonate, and rinse with tap water. Store tissues accordingly based on decalcification method.
Storage of Acid-Decalcified Tissues (Frozen Sections)
Wash with water or store in formol-saline with 15% sucrose or PBS (phosphate buffer saline) with 15-20% sucrose.
Storage of EDTA-Decalcified Tissues
Store in formol-saline/PBS before dehydration. Tissues that have undergone EDTA decalcification should be stored in formol-saline/PBS before dehydration.
Surface Decalcification and Tissue Softeners
Used when tissues are unexpectedly hard during microtomy. 1% HCl in 70% alcohol is a common type of tissue softener and surface decalcifier. Additional softeners like Perenyi's fluid and Molliflex may be used.
Dehydration
Complete substitution of water from tissues being processed via conventional tissue processing.
Dehydration Process
Occurs before clearing in tissue processing to remove water from tissues, allowing the entry of clearing agents.
Clearing Agents
Essentially water immiscible, used after dehydration to penetrate tissues for impregnation.
Impregnating Agents
Enter tissues via substitution of water using organic solvents that act as dehydrating agents.
Gradual Dehydration
Involves slowly substituting water with a strong organic solvent in gradual increasing series to minimize shrinkage and extraction of cellular components.
Effects of Dehydration
May cause distortion of different cellular components inside the tissues.
Considerations for Dehydrating Agent
Includes water miscibility, cost, and common dehydrating agents such as alcohols, acetone, dioxane, cellosolve, triethyl phosphate, and tetrahydrofuran.
Complete Dehydration Test
Involves placing anhydrous copper sulfate crystals in a filter paper at the bottom of the container; the crystals will turn blue when water is completely removed.
Dehydrating Agent's Duration
Varies in acting time to produce the expected dehydration result; can be checked using anhydrous copper sulfate to detect the presence of water.
Alcohols for Dehydration
Ethanol is recommended for routine dehydration in histopathology, using gradual increasing concentrations to minimize tissue distortion.
What is the recommended alcohol for routine dehydration in histopathology?
Ethanol
What is the most common type of dehydrating agent used in routine histopathology?
Ethanol
What changes of gradual increasing concentrations of alcohols must be used in routine dehydration?
To minimize the effect of tissue distortion
For routine dehydration process, what concentration of ethanol should be started with and what should it be increased to?
Start with 70% ethanol until 95% solution of pure ethanol
What concentration of alcohol should be used to start dehydration of delicate tissues such as embryos?
50% solution instead of 70% ethanol
What may happen if tissues are soaked in 70% alcohol for a prolonged time?
Maceration may occur, enhancing softening of the tissue and making it prone to compressing effects of microtome during cutting process
What are the potential effects of using alcohols as storage solutions for tissues?
Quality of staining may be affected due to different types of alcohol and concentration in tissues
Why should high water content tissues, like embryos, start with an alcohol solution of 50% instead of 70%?
They require shorter dehydration time and will gradually be exposed to increasing concentration of alcohols
What is a reliable but tedious alcohol dehydrating agent, considered as the best dehydrating agent?
Ethanol
What are the characteristics of butanol as a dehydrating agent?
Excellent for slow processing, odorous, slow-acting, and not recommended for routine processing
What are the characteristics of tert-butanol as a dehydrating agent?
Can be used as a clearing and dehydrating agent, mixes well with water, ethanol, xylene, and paraffin, more expensive compared to butanol, and may tend to solidify at 25°C
25C
25 degrees Celsius
ISOPROPANOL
Isopropyl Alcohol: Best all-round substitute for ethanol. No government restrictions. Compared to ethanol because of its possibility for abuse. Cannot be used for celloidin embedding. Cannot be used for preparing staining solutions.
PENTANOL
Miscible with 90 alcohol, toluene, and xylene. Toxic, must not be used in poorly-ventilated rooms.
ACETONE
Cheap, rapid-acting. Rapid action compared to alcohols. Cheap alternative for dehydrating agent. Often used for urgent biopsies. Not recommended for routine processing, considerable shrinkage is observed. Extremely volatile and flammable. Can dissolve fats (lipids). Consider acetone in demonstrating lipids or fats.
DIOXANE
Excellent dehydrating and clearing agent. RI 1.42. Less shrinkage, tissues can be left in solution for long periods, even months or weeks. Produces tissues that ribbon poorly. Expensive. Extremely dangerous. Highly toxic cumulative effect. Peroxides formed are explosive.
CELLOSOLVE
Ethylene glycol monoethyl ether. Tissues can be stored for months without injury. Toxic to the different systems of the body: Reproductive, Urinary, Circulatory. If possible, must be substituted by propylene-based glycol ethers.
TRIETHYLPHOSPHATE
Readily removes water. Compared to alcohols. Miscible in alcohols, benzene, xylene, toluene, ether, and chloroform. May be used as a dehydrating agent in staining.
TETRAHYDROFURAN (THF)
Dissolves many substances, fats. Causes less shrinkage, facilitates easier cutting since it will try to somewhat soften the tissues. Staining procedures are improved. Toxic, irritant to nose and eyes, and has an offensive odor. Similar to formalin solutions.
DEHYDRATION FOR ELECTRON MICROSCOPY
Dehydration is carried out using ethanol and propylene oxide. Ethanol: main dehydrating solvent. Propylene Oxide: transitional fluid that is used after the dehydration process. In the process of Electron Microscopy, a transitional fluid is necessary to perform the procedure.
PROPYLENE OXIDE
Completely miscible with resins. Infiltrates tissues readily. Highly flammable, volatile, can evaporate easily, and potentially carcinogenic. It can completely dissolve resins that are deposited in tissues.
ACETONITRILE
Non-carcinogenic, less toxic, not as flammable. Miscible to alcohols, water, acetone, and epoxy resins. Preserves phospholipids well.
NOTES TO REMEMBER
In the process of dehydration, you are not completely removing water from the tissues; rather, you are substituting the water molecules with a dehydrating agent that will allow for the permeation of clearing agents.
Dehydrating agent
A substance used to remove water from a material or environment.
Permeation
The process of a substance passing through a medium or membrane.
Clearing agents
Substances used to make tissues or cells more transparent for microscopic examination.
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