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Fixation

The first and most critical step in histopathologic and cytologic techniques that prevents autolysis, putrefaction, and decomposition of tissue, and preserves its morphology.

Autolysis

The process of self-digestion of tissue caused by the release of innate enzymes when tissue is not fixed properly, leading to distortion of tissue morphology.

Putrefaction

The decomposition of tissue caused by the growth of bacteria and fungi, which can distort the overall morphology of the tissue.

Fixative

A substance or chemical used to harden and protect tissue from trauma, and to preserve its morphology in a life-like manner.

Enzymatic destruction

The breakdown of cellular and extracellular molecules by enzymes, which fixation aims to minimize.

Macromolecular structures

The large-scale structures of molecules, such as proteins, nucleic acids, carbohydrates, and lipids, which fixation aims to maintain.

Microorganisms

Bacteria and fungi that can cause putrefaction and decomposition of tissue, which fixation aims to protect against.

Physical fixation

A method of fixation that involves direct heating, microwaving, or freeze-drying of tissue to preserve its morphology.

Chemical fixation

The most commonly used method of fixation in the histopathology lab, which involves exposing tissue to different chemicals to fix it.

Immersion fixation

A type of chemical fixation where tissue is completely submerged in a fixative agent.

Injection fixation

A type of chemical fixation where the fixative agent is directly injected into the tissue, which may introduce tissue distortion due to the insertion of a syringe.1. Coagulant:A substance that disrupts tertiary protein structures, causing proteins to coagulate or clump together. Examples include alcohol, acetone, picric acid, and trichloroacetic acid.

Cross-linking

The formation of molecular bridges within tissue, which masks epitopes and prevents antibodies from binding to target antigens. Aldehydes, such as formalin, are commonly used for cross-linking.

Compound

A combination of fixatives, often mixed together to take advantage of the benefits of different fixative groups. Alcoholic formalin is an example of a compound fixative.

Aldehydes

A type of fixative that is commonly used for cross-linking. Formalin is an example of an aldehyde fixative.

Oxidizing agents

A group of fixatives that contain metallic ions and have oxidizing properties.

Alcohol-based

A group of fixatives that are alcohol-based, often used for dehydrating and coagulating tissues.

Metallic

A group of fixatives that contain metal ions, still in liquid form.

Proteins

Fixatives disrupt the tertiary structure of proteins, either by coagulating or cross proteins.

Nucleic acid

Cross-linkers can entrap nucleic acids, while alcohol can denature and precipitate them. Coagulants make nucleic acid samples insoluble.

Carbohydrates

Generally, pure forms of carbohydrates are washed away during the fixation process. Glycoproteins can be fixed by formalin, and carbohydrates can be oxidized by osmium tetroxide.1. Fixation:The process of preserving tissue samples for histological slide preparation.

Rossman's fluid

A fixative solution used for preserving glycogen in tissue samples.

Osmium tetroxide

A fixative that can cross-link lipids and make them insoluble.

Lipids

Organic compounds that are typically yellow in color and can be stained black.

Adipose tissue

Tissue that stores fat and appears as white areas.

Formalin

A fixative solution that has no action on lipids.

Cross-links

Bonds formed between molecules, in this case, between lipids and double bonds.

Factors affecting fixation

Various variables that can influence the effectiveness of the fixation process.

Temperature

The degree of heat, which can affect the penetration rate and risk of autolysis.

Penetration rate

The speed at which the fixative solution diffuses into the tissue.

Volume ratio

The ratio of fixative solution volume to tissue volume, which should be at least 10:1.

Hydrogen Ion concentration

The measure of acidity or alkalinity, which can affect fixation.

Osmolality

The concentration of solutes in a solution, which can affect fixation.

Autolysis

The self-destruction of cells or tissues due to the activation of enzymes.

Tissue processing

The steps involved in preparing tissue samples for examination.

Medical Technologist

The professional responsible for processing tissue samples.

Room temperature

The ambient temperature at which fixation is ideally done.

Electron microscopy

A technique used to study the ultrastructures of cells.

Immunohistochemistry

A method that uses antibodies to detect specific antigens in tissues.

Volume of tissue

The size or amount of tissue being fixed, which affects penetration.

Fixative-tissue volume ratio

The correct ratio of fixative solution volume to tissue volume.

Staining artifacts

Abnormalities or distortions in the staining of tissue samples.

Coefficient of diffusibility

A measure of how easily a substance can diffuse through a medium.

Underfixing

Insufficient fixation of the center of tissue, leading to autolysis.

Optimum time

The ideal duration of fixation, which varies depending on specimen size.

Over/underfixed

Tissue that has been fixed for too long or too short a time, respectively.

pH

The measure of acidity or alkalinity in a solution.1. Fixation:The process of preserving tissue samples by immersing them in a fixative solution, typically buffered formalin, to prevent decay and maintain cellular structure.

pH

A measure of the acidity or alkalinity of a solution. In fixation, a pH of 6-8 (near neutral) is ideal to prevent the formation of pigments that can interfere with tissue visualization.

Hypoxia

A condition characterized by a lack of oxygen supply to tissues. Submerging tissue directly into fixative can promote hypoxia, leading to an acidic environment and chemical reactions that can obscure or interfere with tissue visualization.

Formalin-heme pigment

A pigment formed when formalin reacts with heme, which can obscure or interfere with the visualization of tissue.

Buffered fixative

A fixative solution that has been adjusted to maintain a stable pH. Not all fixatives are buffered, so washing tissue with plain water or alcohol may be necessary after fixation.

Osmolality

A measure of the concentration of solutes in a solution. In fixation, it is important to use isotonic solutions to prevent damage to phospholipids and maintain the osmolality of the tissue.

Hypertonic

A solution with a higher solute concentration than the surrounding tissue. If a fixative is hypertonic, it can cause cells to swell.

Hypotonic

A solution with a lower solute concentration than the surrounding tissue. If a fixative is hypotonic, it can cause cells to burst or swell.

Factors affecting fixation

Various factors, including temperature, specimen size, volume-tissue ratio, time, pH, osmolality, and ionic compositions, can influence the effectiveness of fixation.

Loss of Nuclear chromatin

Poor fixation can result in the loss of nuclear chromatin, which is important for pathologists to view cells.

Disappearance of some cells (Epithelial cells)

Poor fixation can cause the disappearance of epithelial cells, which are important for orienting oneself with the tissue and are naturally found in open sources or cavities.

Cell shrinkage

Poor fixation can lead to cell shrinkage, which can affect the identification of tissue.

Complete Fixation

Proper fixation results in well-defined cells, detailed and prominent epithelial cells, and no shrinkage or washing off of cells.

Incomplete Fixation

Poor fixation results in washed-off and shrunk cells, distorted epithelial cells, and overall compromised tissue visualization.

Difficulties caused by improper fixation

Improper fixation can lead to problems such as failure to arrest autolysis, removal of soluble substances and fixing agents, presence of artifact pigments on tissue sections, soft and feather-like tissues, loss/inactivation of enzymes needed for study, shrinkage and swelling of cells, brittle and hard tissue blocks, and separation of tissue components during sectioning.1. Fixation:The process of preserving biological tissues by treating them with a fixative to prevent decay and maintain their structure.

Fixative

A chemical solution used to preserve biological tissues during the process of fixation.

Simple fixatives

Fixatives that consist of a single type of chemical compound.

Compound fixatives

Fixatives that are a mixture of different types of chemical compounds.

Formaldehyde

The most common and ideal fixative for histopathology, used to preserve tissues. It targets proteins and stabilizes them by forming a methylene bridge.

10% NBF

A recommended tissue fixative that is similar to formaldehyde but with a buffer added.

Glutaraldehyde

A fixative used for electron microscopy that preserves ultrastructure.

Paraformaldehyde

A polymerized form of formaldehyde that is suitable for paraffin embedding and sectioning.

Alcohol

Ethanol, a fixative that denatures proteins and is good for cytologic smears.

Acetic acid

A fixative that preserves nucleic acids by precipitation and swells proteins.

Picric acid

A fixative and stain that coagulates nucleoproteins and is ideal at low pH.

Osmium tetroxide

A secondary fixative commonly used for lipid fixation and ultrastructures in electron microscopy.

Mercuric chloride

A fixative that reacts with amine groups and coagulates proteins, commonly used as an alternative metal fixative.

Carnoy's solution

A compound fixative that is rapid acting and commonly used for fixation, with good nuclear preservation and retention of glycogen.

Zenker's solution

A compound fixative that is commonly used for fixation and preserves nuclear morphology.

Helly's solution

A compound fixative that is commonly used for fixation and preserves nuclear morphology.

Bouin's solution

A compound fixative that is commonly used for fixation and preserves nuclear morphology.

Precipitates

The formation of solid particles from a solution, in this case, nucleic acids being precipitated by ethanol.

Cross-linking

The formation of chemical bonds between molecules, in this case, proteins being cross-linked by glutaraldehyde and formaldehyde.

Denaturant

A substance that disrupts the structure of proteins, in this case, alcohol denaturing proteins.1. Carnoy's solution:A fixative solution composed of absolute ethanol, chloroform, and acetic acid, used to preserve and prepare tissue samples for microscopic examination.

Bouin's solution

A fixative solution that stains tissue bright yellow due to picric acid and preserves glycogen, composed of formaldehyde, picric acid, and acetic acid.

Zenker's solution

A fixative solution suitable for bloody specimens and trichrome staining, composed of distilled water, mercuric chloride, potassium dichromate, sodium sulfate, and glacial acetic acid.

Helly's solution

A fixative solution used for bone marrow and intercalated discs of cardiac muscles, composed of distilled water, potassium chloride, sodium sulfate, mercuric chloride, and 40% formaldehyde.

Karnovsky's fixative

A type of fixative solution used in electron microscopy.

Zinc formalin

A fixative solution containing zinc, used for preserving tissue samples.

B-5 fixative

A fixative solution used for preserving tissue samples, particularly for lymph nodes.

Clarke's solution

A fixative solution used for preserving tissue samples, particularly for nerve tissue.

Formol acetic alcohol

A fixative solution composed of formaldehyde, acetic acid, and alcohol, used for preserving tissue samples.

Alcoholic Formalin

A fixative solution composed of formaldehyde and alcohol, used for preserving tissue samples.

Rossman's fluid

A fixative solution used for preserving tissue samples, particularly for bone marrow.

Metallic fixative supplement

Additional substances such as zinc, cobalt, copper, mercury, cadmium, and lead that can be added to fixative solutions to enhance their preservation properties.

Characteristics of a Good Fixative

Cheap, inhibits bacterial decomposition, stable, produces minimum shrinkage of tissue, safe to handle, rapid and even penetration, kills cells quickly, makes components insoluble, hardens tissue, permits subsequent application of reagents, and has an ionic composition that is isotonic to the tissue.

Autolysis

The self-digestion of cells or tissues by their own enzymes after death.

Hypotonic solution

A solution with a lower concentration of solutes compared to the surrounding cells or tissues.

Hypertonic solution

A solution with a higher concentration of solutes compared to the surrounding cells or tissues.

Mordants

Substances that enhance the binding of dyes to tissues during staining.

Optical differentiation

The ability to distinguish and visualize cells and tissue components more clearly during microscopic examination.

Precautions in Handling and Fixation of Specimens

Proper labeling and identification of tissue specimens, immediate fixation or refrigeration of surgical specimens to prevent drying and tissue distortion, avoidance of freezing and repeated freezing and thawing, consideration of fresh or incompletely-fixed tissue as potentially infectious, and prevention of drying to avoid shrinkage and distortion of tissue.1. Fixation:The process of preserving tissue samples for examination by preventing decay or degradation.

Autolysis

The self-digestion of cells or tissues by their own enzymes.

Purulent material

Pus-like fluid containing dead white blood cells, bacteria, and tissue debris.

Fixative

A chemical solution used to preserve tissue samples during fixation.