INTRO_TO_TISSUE_PROCESSING

Overview

Title: The Gross Room / Surgical Cut-Up

Tissue Processing for Diagnosis

Tissues are collected from patients for diagnosing various disease processes. These tissues must undergo processing in a specialized histopathology laboratory to prepare them for examination by pathologists. The primary objective of this process is to produce high-quality microscopic slides that can be analyzed for diagnostic purposes, enabling the identification of diseases such as cancer, infections, and other pathological conditions.

Role of Histotechnologists

Histotechnologists play a crucial role in the processing of tissue samples. Their responsibilities include:

• Precisely processing tissues to ensure that they maintain their structural integrity during preparation.

• Creating high-quality glass microscopic slides that are essential for accurate examination by pathologists.

• Implementing standardized procedures to obtain consistent results across various tissue samples.

Sample Stains and Specimens

Different types of tissue specimens are examined using specific staining techniques to enhance visibility of certain features:

• Colon Polyp: H&E stain, useful for evaluating cellular morphology.

• Heart & Valve: Trichrome stain, aids in distinguishing between collagen and muscle fibers.

• Sigmoid Colon: H&E stain, critical for identifying abnormalities in tissues.

• Adrenal Pheochromocytoma: H&E stain highlights chromaffin cells.

• Leprosy: Bacit Acid Fast Stain, specifically targets mycobacteria.

• Appendix (Inflamed): Trichrome stain to assess the degree of inflammation.

• Lung (Asbestosis): Gomon's Stain, detects asbestos fibers.

• Basal Cell Carcinoma: H&E stain is standard for identifying skin cancers.

• AIDS Pneumonia: Silver Stain helps visualize specific pathogens.

• Spleen: Putt's Stain, useful for examining hematological tissues.

Skin Punch Biopsy

A 5 mm skin punch biopsy is illustrated from multiple angles to show:

• Above view: Provides information about the surface characteristics of the tissue.

• Side view: Shows depth and relation of layers within the skin tissues.

• Bisected view: Displays internal structure, allowing for a thorough examination of the tissue architecture.

Fixation Considerations

• Speed: Specimens should be placed in fixative as soon as they are removed from the body to ensure effective preservation.

• Penetration: Fixative penetration occurs at approximately 1 mm per hour, slowing down as it reaches deeper tissues.

• Volume: It is recommended to use 10-25 times the volume of the tissue being fixed to ensure adequate exposure and effectiveness of the fixative.

• Duration of Fixation: The duration required for fixation varies depending on the structure of the tissue; duration can be reduced through methods such as heat application, vacuum techniques, agitation, or microwave use.

Agitation in Tissue Processing

• The rate of exchange between the tissue and processing reagent is dependent on the exposed surface of the tissue that is in contact with the reagent.

• Automated processors utilize vertical or rotary oscillation or pressurized removal and replacement of fluids at timed intervals.

• Efficient agitation can reduce the overall processing time by up to 30%.

Agitation in tissue processing refers to how we mix and move tissue samples when they're being prepared for examination. The more of the tissue's surface that is exposed to the chemical solutions used in the process, the better the results will be.

Heat in Tissue Processing

• Heat increases the rate of penetration and fluid exchange during tissue processing.

• It must be used sparingly to reduce the possibility of shrinkage, hardening, and brittleness of the tissue.

• Effective temperatures are limited to 45˚C; higher temperatures may negatively impact subsequent immunohistochemical staining.

Viscosity in Tissue Processing

• Viscosity is the property of resistance to the flow of a fluid.

• The smaller the size of the molecules in the solution, the faster the rate of fluid penetration (low viscosity).

• If the molecular size is larger, the rate of exchange is slower (high viscosity).

Vacuum in Tissue Processing

• Using reduced pressure to increase the rate of infiltration.

• If used on the automated processor, should not exceed 15 inches of Hg (mercury) to prevent damage and deterioration to the tissue.

Simple Fixatives
- Made up of only one component substance.

Examples of Simple Fixatives

• Aldehydes: formaldehyde, glutaraldehyde

• Metallic fixatives: mercuric chloride, chromate fixatives

• Other examples include: picric acid, acetic acid, acetone, alcohol, osmium tetroxide.

  Types Of Fixatives

• Compound Fixatives
  - Made up of two or more fixatives which have been added together to obtain the optimal combined effect of their individual actions upon the cells and tissue constituents.

  A. Microanatomical Fixatives

  • Designed to preserve the overall architecture of tissues for detailed microscopic examination.

  B. Cytological Fixatives

  1. Nuclear Fixatives

     • Target and preserve the morphology of the cell nucleus.

  2. Cytoplasmic Fixatives

     • Focus on preserving the cytoplasm and its components.

  C. Histochemical Fixatives

  • Used to maintain the chemical composition of tissues for specific staining techniques that identify cellular components.

• A. Microanatomical Fixatives

  • Permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question.

  • Examples include:

    • 10% Formal saline

    • 10% Neutral buffered formalin

    • Heidenhain’s Susa

    • Formal sublimate (formol corrosive)

    • Zenker’s solution

    • Zenker-formal (Kelly’s solution)

    • Bouin’s solution

    • Brasil’s solution

  B. Cytological Fixatives

  • Preserve specific parts and particular microscopic elements of the cell itself.

  1. Nuclear Fixatives

  • Preserve the nuclear structures (e.g., chromosomes).

  • Usually contain glacial acetic acid.

  • Examples include:

    • Flemming’s fluid

    • Carnoy’s fluid

    • Bouin’s fluid

    • Newcomer’s fluid

    • Heidenhain’s Susa

  Cytoplasmic Fixatives

  • Preserve cytoplasmic structures.

  • Examples include:

    • Flemming’s fluid without acetic acid

    • Kelly’s fluid

    • Formalin with “post-chroming”

    • Regaud’s fluid (Muller’s fluid)

    • Orth’s fluid

Histochemical Fixatives

• Preserve the chemical constituents of cells and tissues.

• Examples include:

  • 10% Formal saline

  • Absolute ethyl alcohol

  • Acetone

  • Newcomer’s fluid

• Types of Fixations:

  Lipid Fixation

  Lipid fixation is a fundamental technique in histology focused on preserving lipid structures within cells and tissues, ensuring accurate analysis for subsequent studies. The following methods and agents are commonly utilized for effective lipid fixation:

  • Cryostat and Frozen Section Techniques rapidly freeze tissue samples to prevent degradation and maintain lipid and protein integrity, enabling the preparation of thin sections for microscope slides. Specific lipid staining techniques, like Sudan Black or Oil Red O, are used post-sectioning to visualize lipids in the preparations.

  • Mercuric Chloride is a common fixative for lipid fixation, effective in preserving cellular morphology. It is generally used in a 5-7% aqueous solution and can interact with proteins and nucleic acids, making it useful for analyzing both lipid and non-lipid components. Due to its toxicity, it requires careful handling and strict waste disposal protocols.

  • Potassium Dichromate is an effective fixative known for its oxidizing properties that stabilize lipid molecules for enhanced visualization in staining. However, it is a carcinogen and should be handled with care to prevent oxidative damage to tissue samples.

  Techniques Involved in Lipid Fixation

  1. Cryostat Sectioning:

     • This technique involves quick freezing of tissue samples to maintain lipid and protein integrity, allowing for thin section preparation. The rapid processing enables immediate staining for diagnostic evaluations in surgical pathology.

  2. Frozen Sections:

     • Utilizing frozen sections permits real-time processing, crucial when urgent results are needed during surgical interventions. This method facilitates rapid histological assessments that inform treatment decisions on-site.

  3. Microscopy Applications:

     • The visualization of preserved lipid structures can be performed using either light microscopy or electron microscopy, depending on the resolution needed for analysis. Electron microscopy is particularly advantageous for examining the ultrastructure of lipid-rich organelles, such as adipocytes (fat cells).

  The Significance of Lipid Fixation

  • Lipid fixation plays an essential role in studying metabolic disorders, obesity, and conditions affecting lipid metabolism (e.g., diabetes and atherosclerosis). By enabling precise examination of lipid changes at a cellular level, this technique aids in the development of targeted therapeutic strategies and enhances our understanding of associated pathophysiological mechanisms.

  Carbohydrate Fixation

  • Function: Alcohol fixatives are primarily used for the fixation of carbohydrates.

  • Action: They denature proteins and precipitate carbohydrates, which aids in the preservation of glycogen and other sugar-related structures in tissue samples.

  Protein Fixation

  • Definition: A method used to preserve the structural integrity of proteins in tissue samples during histological processing.

  • Common Fixatives:

    • Neutral Buffered Formalin: One of the most widely used fixatives that helps maintain pH stability and prevents pigment formation.

    • Formol Saline: A combination of formaldehyde and saline, beneficial for preserving cellular details while preventing shrinkage.

    • Formaldehyde: An effective fixative that penetrates tissues well, but should be used with caution due to its potential to form pigment granules.

  Glycogen Fixation:

  • Composition: Alcohol-based fixatives such as Rossman’s fluid or cold absolute alcohol are utilized for effective glycogen preservation.

  • Function: These fixatives denature proteins and precipitate carbohydrates, aiding in the preservation of glycogen and similar sugar-related structures within tissue samples.

Mixtures of Fixatives

• Karnovsky’s Paraformaldehyde-Glutaraldehyde Solution

  • Two aldehyde fixative mixtures have been particularly useful for electron cytochemistry.

  Acrolein

  • Another aldehyde which has been introduced as a mixture with glutaraldehyde or formaldehyde.

Aldehyde Fixatives

Formaldehyde (Formalin)

• 10% Formalin

• Usual fixation time: 24 hours

• One of the most widely used fixatives; compatible with many stains and penetrates tissues well.

Disadvantages:

• Irritating fumes and may cause skin irritation upon contact.

• Forms pigment granules; precipitates may be removed by filtration or addition of 10% methanol.

• Unsuitable for electron microscopy.

10% Formal-Saline

• Saturated formaldehyde diluted to 10% with NaCl.

• Recommended for fixation of CNS (central nervous system) and general post-mortem tissues for histochemical examination.

• Slow fixative; recommended duration is over 24 hours or more.

10% Neutral Buffered Formalin (Phosphate Buffered Formalin)

• Recommended for preservation and storage of surgical, post-mortem, and research specimens.

• Prevents precipitation of pigments.

• Best fixative for tissues containing iron pigments and elastic fibers.

Formol-Corrosive (Formol-Sublimate)

• Recommended for routine post-mortem tissues.

• Excellent for many staining procedures including silver reticulum methods.

Alcoholic Formalin (Gendre’s) Fixative

• Can be used for rapid diagnosis, fixes and dehydrates at the same time.

• Good for preservation of glycogen and for microincineration technique.

• Can fix sputum.

Glutaraldehyde

• Buffered glutaraldehyde, followed by secondary fixation in osmium tetroxide is satisfactory for electron microscopy.

• 2-5% concentration is used for small tissue fragments.

• 4% concentration is recommended for larger tissues.

• It is also recommended for enzyme histochemistry and electron microscopy.

Metallic Fixatives

Mercuric Chloride

• Most common metallic fixative (5-7% aqueous solution).

• Mercury deposits are removed from deparaffinized sections before staining by treating with 0.5% iodine solution in 70% ethanol for 5-10 minutes.

• Routine fixative of choice for preservation of cell detail in tissue photography.

Examples:

Zenker’s Fluid

• Contains glacial acetic acid to prevent turbidity and formation of dark precipitate.

• Recommended for fixing small pieces of liver, spleen, connective tissue fibers, and nuclei.

• Recommended for trichrome staining.

• De-Zenkerization is done using alcoholic iodine.

  Zenker-Formol (Helly’s Solution)

  • Microanatomic fixative for pituitary gland, bone marrow, and blood-containing organs.

  • Pigment removal can be done using saturated alcoholic picric acid or NaOH.

  Heidenhain’s Susa Solution

  • Recommended mainly for tumor biopsy, especially skin.

  • Excellent cytologic fixative.

  B-5 Fixative

  • Used for bone marrow biopsies.

  Chromate Fixatives
  Chromic Acid

  • 1-2% Chromic Acid

  Potassium Dichromate

  • 3% Potassium dichromate

  Regard’s (Muller’s) Fluid

  • Recommended for demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies, red blood cells (RBC), and colloid-containing tissues.

  Orth’s Fluid

  • Recommended for study of early degenerative processes and tissue necrosis.

  • Demonstrates rickettsiae and other bacteria.

  • Preserves myelin better than buffered formalin.

Lead Fixatives

• Concentration: 4% aqueous solution

• Purpose: Recommended for acid mucopolysaccharides

• Function: Fixes connective tissue mucin

• Chemical Reaction: Takes up CO2 to form insoluble lead carbonate

• Removal Process: This precipitate can be removed by filtration or by adding acetic acid.

Picric Acid Fixatives

• Function: Excellent fixative for glycogen demonstration.

• Color: Characterized by a yellow color.

• Removal: Yellow color can be removed by treatment with another acid dye or lithium carbonate.

Bouin’s Solution

• Recommended for: Embryos & pituitary biopsies.

• Function: Excellent fixative for preserving soft & delicate tissues.

• Preferred for: Tissues to be stained by Masson’s trichrome for collagen, elastic, or connective tissue.

Brasil’s Alcoholic Picroformol Fixative

• Comparison: Better and less "messy" than Bouin’s solution.

• Function: Excellent fixative for glycogen.

Glacial Acetic Acid

• Usage: Normally used in conjunction with other fixatives.

• Physical Property: Solidifies at 17°C.

• Function: Very useful in the study of nuclear components of the cell.

• Effect on Tissue: Causes tissue to swell, which is used to counteract the shrinkage produced by other components (e.g., mercury).

• Contraindication: Contraindicated for cytoplasmic fixation.

Alcohol Fixatives

• Function: May be both a fixative and dehydrating agent.

• Demonstration: Excellent for glycogen demonstration.

• Contraindication: Contraindicated when lipids are to be studied.

• 100% Methyl Alcohol: Excellent for fixing dry & wet smears, blood smears, and bone marrow tissues.

• 95% Isopropyl Alcohol: Used for fixing touch preparations and for certain special staining procedures such as Wright-Giemsa.

• 70%-100% Ethyl Alcohol: Used as a simple fixative but more frequently incorporated into compound fixatives.

Carnoy’s Fluid

• Recommended for: Fixing chromosomes, lymph glands, and urgent biopsies.

• Function: Considered to be the most rapid fixative.

• Usage: Also used to fix brain tissue for the diagnosis of rabies.

Newcomer’s Fluid

• Recommended for: Fixing mucopolysaccharides and nuclear proteins.

• Function: Acts both as a nuclear and histochemical fixative.

Osmium Tetroxide (Osmic Acid)

• Function: Fixes conjugated fats & lipids permanently.

• Usage: Used extensively for neurological tissues.

• Application: Adequately fixes for electron microscopy.

• Penetration: Has poor penetration; effective only for samples 2-3 mm thick.

• Addition: The addition of saturated aqueous mercuric chloride will prevent its reduction, avoiding the formation of black deposits.

Flemming’s Solution

• Type: Most common chrome-osmium acetic acid fixative.

• Function: Excellent for preserving nuclear structures.

• Property: Permanently fixes fat.

Flemming’s Solution without Acetic Acid

• Usage: Recommended for preserving cytoplasmic structures, particularly mitochondria.

Trichloroacetic Acid

• Function: Precipitates proteins and is a poor penetrating agent.

• Decalcifying Agent: Serves as a weak decalcifying agent, with a softening effect on dense fibrous tissues.

• Effect on Tissues: Produces a marked swelling effect on tissues, which counteracts the shrinkage caused by other fixatives; sometimes incorporated into compound fixatives.

Acetone

• Usage Temperature: Used at ice cold temperatures from -5°C to 40°C.

• Function: Study of water-diffusible enzymes, especially phosphatases & lipases.

• Application: Fixing brain tissues for the diagnosis of rabies.

• Property: Dissolves fat.

• Preservation: Preserves glycogen poorly.

Heat Fixation

• Function: Thermal coagulation of proteins.

• Suitability: Suitable for frozen tissue sections and preparation of bacteriologic smears.

• Effects: Produces considerable tissue shrinkage & distortion, destroys RBC, and dissolves starch and glycogen.

Secondary Fixation

• Definition: Placing an already fixed tissue into a second fixative before dehydration, and on deparaffinized sections before staining.

• Purpose: Facilitates & improves demonstration of particular substances and special staining techniques; ensures further & complete hardening and preservation of tissues.

Post-Chromatization

• Definition: A form of secondary fixation.

• Process: A primarily fixed tissue is placed in 2.5 to 3% potassium dichromate for 24 hours.

• Benefits: Acts as a mordant for better staining effects and aids in cytologic preservation of tissues.

Washing-Out

• Purpose: Removing excess fixative to improve staining and remove artifacts.

• Solutions Used: Tap water, 50-70% alcohol, and alcoholic iodine.

Improper Fixation

• Causes:

  • Autolysis: Delay in fixation or insufficient fixative.

  • Removal of substances: Loss or inactivation of enzymes due to wrong choice of fixative.

  • Artifact pigments: Incomplete washing.

  • Soft & feather-like tissues: Incomplete fixation.

  • Brittle & hard tissues: Over fixation.

Fixation Artifacts

• Mitigation: Eliminated or reduced by fixation in phenol formalin.

• Recommendation: Use neutral buffered formalin and phenol formalin to almost completely stop the formation of formalin pigment.

• Example: "Crush artifact" refers to partial coagulation of partially fixed protein by ethanol or incomplete wax impregnation.

Microwave Technique

• Usage: Used to accelerate staining, decalcification, immunohistochemistry (IHC), and electron microscopy (EM).

• Process: Blocks are placed in a microwave at 450 watts and 55°C for 1.5 to 4 minutes.

• Benefits: Useful in preserving neurochemical substances, such as acetylcholine; rapid fixation that reduces time taken for IHC and ISH.

Immunofluorescence and Immunoperoxidase Techniques

• Enzyme Histochemistry: Aims to preserve maximum enzyme activity at its original localization while maintaining structural integrity. Common fixatives include 4% Formaldehyde or Formal saline; Acetone or Formaldehyde for fresh frozen cryostat sections.

• Electron Microscopy: Common fixatives include Osmium tetroxide, Glutaraldehyde, Paraformaldehyde, and Karnovsky’s paraformaldehyde-glutaraldehyde.

Electron Microscopy Fixatives

• Osmium Tetroxide: A fixative known for preserving conjugated fats and lipids, widely used in neurological tissues.

• Glutaraldehyde: Effective for electron microscopy, it often undergoes secondary fixation with osmium tetroxide. Concentrations of 2-5% are used for smaller samples and 4% for larger specimens.

• Paraformaldehyde: Commonly used in various fixation protocols; it is effective given its ability to penetrate tissues well, but it may form pigment granules.

• Karnovsky's Paraformaldehyde-Glutaraldehyde Solution: A mixture specifically tailored for improved electron cytochemistry.

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