Histology Basics and Techniques

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Questions and Answers

What is histology primarily concerned with?

The analysis of body fluids

The study of genetic material in cells

The examination of skeletal structures

The study of tissues and their organization in organs (correct)

Which of the following biopsy types is the method of choice for obtaining a histological diagnosis?

Core needle biopsy (correct)

Excision biopsy

Incisional biopsy

Fine-needle aspiration cytology (FNAC)

What is the primary purpose of fixation in tissue processing?

To stain the tissue for better visibility

To preserve the structure and protect from microorganisms (correct)

To remove any excess cellular debris from the sample

To enhance the mechanical strength of the tissue

Which fixative is commonly used for tissue preservation?

10% formaldehyde Signup and view all the answers

What is a key characteristic of proper tissue processing?

Embedding the tissue in a medium that maintains its rigidity while allowing for thin sectioning Signup and view all the answers

What is the maximum thickness of tissue fixation penetration typically achievable?

1 cm Signup and view all the answers

Which of the following is NOT a step involved in tissue processing?

Electrophoresis Signup and view all the answers

What commonly results from the fixation process?

Loss of biological function but preservation of structure Signup and view all the answers

What is the main action of autolysis during cell death?

It releases lysosomal enzymes that hydrolyse tissues. Signup and view all the answers

Which fixative is known to cross-link proteins due to its two aldehyde groups?

Glutaraldehyde Signup and view all the answers

In immunohistochemistry, why is it important for fixatives not to destroy amine groups?

To allow for antibody binding to antigens. Signup and view all the answers

What is the primary purpose of dehydration in tissue processing?

To facilitate penetration of embedding medium. Signup and view all the answers

Which reagent is commonly used for the clearing process in tissue preparation?

Xylene Signup and view all the answers

What thickness of sections is typically produced by a microtome in routine histopathology?

3-5 μm Signup and view all the answers

What is the main goal of applying a glass coverslip over a tissue section?

To protect the section. Signup and view all the answers

Which staining method preferentially colors acidic components like DNA in cells blue?

Hematoxylin Signup and view all the answers

What does the term metachromasia refer to in histology?

Staining that changes color based on concentration of tissue components. Signup and view all the answers

What is the purpose of using a vibratome in sectioning?

To cut thicker sections from fresh or fixed tissue. Signup and view all the answers

Which fixing agent is likely to degrade the secondary structure of proteins due to its rapid action?

Glutaraldehyde Signup and view all the answers

What does the immediate frozen section technique offer compared to traditional histological preparation?

Faster evaluation with quicker results. Signup and view all the answers

What is a potential drawback of using alcohols as fixatives compared to aldehydes?

They do not preserve tissue structure as well. Signup and view all the answers

What is the purpose of the condenser lens in a microscope?

To focus light onto the specimen. Signup and view all the answers

What is the primary reason for tissue fixation during the preparation process?

To preserve the structure of the tissue for analysis Signup and view all the answers

What is a key difference between excisional and incisional biopsies?

Excisional biopsy removes the entire lesion while incisional biopsy only takes a portion Signup and view all the answers

Which step in tissue processing allows for the preservation of biological structure?

Fixation Signup and view all the answers

What is the maximum thickness of tissue that can be effectively fixed by common methods?

1cm Signup and view all the answers

In which method of biopsy is a larger tissue sample obtained than with Fine Needle Aspiration Cytology (FNAC)?

Core needle biopsy Signup and view all the answers

What is the goal of tissue processing in histology?

To embed tissue in a medium that allows thin sections to be cut Signup and view all the answers

What type of biopsy is typically performed when a lesion is not suspected to be malignant?

Excisional biopsy Signup and view all the answers

Which of the following processes is NOT part of tissue processing for light microscopy?

Immunohistochemistry Signup and view all the answers

Which statement about the effect of aldehyde fixatives on tissue is correct?

Aldehydes destroy amine groups while maintaining tissue structure. Signup and view all the answers

What is the primary disadvantage of using ethanol as a fixative?

It maintains amine groups but poorly preserves tissue structure. Signup and view all the answers

In immunohistochemistry, why might antibodies fail to bind their targets?

Lysine residues may be altered by fixation. Signup and view all the answers

Which component is essential in creating a section from embedded tissue?

Tissue block Signup and view all the answers

What role does the microtome play in histology?

It cuts thin sections from embedded tissue. Signup and view all the answers

Which process directly follows the dehydration of tissue?

Clearing Signup and view all the answers

What happens to the structure of proteins when glutaraldehyde is used as a fixative?

The alpha-helix structure is deformed. Signup and view all the answers

How does toluidine blue demonstrate metachromasia?

It polymerizes in the presence of specific polyanions. Signup and view all the answers

What can result from inadequate dehydration during tissue preparation?

A lack of staining contrast. Signup and view all the answers

What is a common characteristic of sections produced using a cryostat?

They are produced from unfixed tissue blocks. Signup and view all the answers

What is the main purpose of staining in histology?

To provide contrast and distinguish cellular details. Signup and view all the answers

In what scenario are cell smears preferred for examination?

For examining blood and bone marrow. Signup and view all the answers

Which type of sectioning device is best for thicker sections up to 200 μm?

Vibratome Signup and view all the answers

What is the primary purpose of a core needle biopsy?

To obtain a larger tissue sample for histological diagnosis Signup and view all the answers

In tissue processing, what role does dehydration serve?

To prepare tissues for embedding in a solid medium Signup and view all the answers

What is a disadvantage of using formaldehyde for fixation?

It may not penetrate tissues thicker than 1cm effectively Signup and view all the answers

Which step in tissue processing follows fixation?

Dehydration Signup and view all the answers

What is the ultimate goal of staining in histology?

To enhance the visibility of tissue components for analysis Signup and view all the answers

Why is fixation necessary before histological examination?

To stabilize the tissue structure against decay Signup and view all the answers

What type of biopsy is performed primarily when a lesion is large and suspicious for malignancy?

Incisional biopsy Signup and view all the answers

What is one of the key steps in the preparation of tissue for light microscopy?

Embedding in a medium for support and rigidity Signup and view all the answers

Which of the following statements accurately describes a characteristic of formalin as a fixative?

It covalently binds to amine groups on amino acids. Signup and view all the answers

What is the primary reason for using ethanol in tissue fixation?

It removes water from the tissue without destroying amine groups. Signup and view all the answers

Which fixative is known for causing deformation of the alpha-helix structure in proteins?

Glutaraldehyde Signup and view all the answers

During the dehydration step of tissue processing, how is ethyl alcohol typically applied?

Graduated strengths are routinely applied in increasing percentages. Signup and view all the answers

What is the primary aim of the clearing process in tissue preparation?

To replace alcohol with a solvent that is miscible with paraffin. Signup and view all the answers

Which of the following is characteristic of hematoxylin staining?

It stains acidic components, particularly DNA, blue. Signup and view all the answers

What equipment is essential for sectioning fixed, embedded tissue?

Microtome Signup and view all the answers

What inherent limitation exists with the use of glutaraldehyde for immunohistochemical staining?

It alters protein structures, affecting antibody binding. Signup and view all the answers

Which of the following best describes the function of toluidine blue in histology?

It demonstrates metachromasia by staining polyanion-rich structures a different color. Signup and view all the answers

What is the typical thickness of tissue sections when using a microtome for routine histopathology?

3-10 μm Signup and view all the answers

In which situation is immunohistochemistry particularly beneficial?

To demonstrate specific antigens in FFPE tissues. Signup and view all the answers

Why are sections mounted on glass slides during histology preparation?

To prevent air bubble formation and protect the section. Signup and view all the answers

What is the immediate benefit of using the freezing method for tissue sections?

It results in rapid preparation time for urgent cases. Signup and view all the answers

Which component of the microscope is primarily responsible for focusing light onto the specimen?

Condenser Lens Signup and view all the answers

What typically happens to tissues subjected to inadequate dehydration during histology preparation?

They may not retain their structural integrity. Signup and view all the answers

Study Notes

Histology

The study of tissues and how they form organs.
Also called microscopic anatomy or microanatomy.
There are four basic tissue types: epithelial, connective, muscle, and nerve.

Why Histology

Processing biological material from living or dead bodies (biopsies, necropsies) can help determine the diagnosis, pathological process, or cause of death.
Vital for therapeutic management.

Sampling Biological Material

Biopsies: Excision biopsies can be diagnostic or therapeutic.
- Incisional biopsies are performed when the lesion is large or located in a hazardous location.
- Core biopsies provide a larger tissue sample compared to fine needle aspiration cytology (FNAC).
Cytology:
- Exfoliative cytology involves collecting cells that spontaneously shed from the body or are mechanically removed with a brush.
- Interventional cytology involves needle aspiration.

Tissue Processing

Process of preparing tissue for light microscopy:
- Fixation: preserving tissue structure with chemical compounds called fixatives.
  - Common method is using 10% formaldehyde which cross-links proteins.
  - Fixation prevents autolysis by blocking lysosomal enzymes.
  - Glutaraldehyde is an alternative fixative with similar properties.
  - Fixation for immunohistochemistry (IHC) requires specific fixatives that do not destroy amine groups.
- Dehydration: Removing extractable water from the tissue using graduated strengths of ethyl alcohol.
- Clearing: Replacing alcohol with a solvent miscible with paraffin (e.g., xylene or chloroform).
- Embedding: Encasing tissue in a solid medium (e.g., paraffin wax) for sectioning.
- Sectioning: Cutting thin tissue sections using a microtome (3-10 μm thickness).
- Sliding: Mounting the section on a glass slide.
- Staining: Coloring tissue components for visualization using dyes.
  - Hematoxylin stains acidic components blue (basophilic) and eosin stains basic components pink (acidophilic).
  - Metachromasia occurs when some stains change color in the presence of polyanions in tissue.
- Covering: Applying a thin glass coverslip to protect the section.

Immediate Frozen Section

Fast method of tissue preparation (approx. 5 minutes) for immediate results (e.g., during tumor surgery).
Less accurate than standard paraffin-embedded tissue sections.

Cell Smears

Examining tissues as thin layers or smears, such as blood, bone marrow, or epithelial cells.

Immunohistochemistry

Demonstrates specific antigens in tissues using antibodies.
Visualized with light microscopy, providing both morphology of tissue and location of specific antigen.
Can provide diagnostic and prognostic information, often semi-quantitatively.
Visualization methods include fluorescent detection (immunofluorescence).

Histology

Microscopic anatomy focused on tissues and their arrangements within organs.
Four primary tissue types: epithelia, connective tissue, muscle, and nerve.

Why Histology?

Processing of biological material from living or deceased individuals (biopsies, necropsies).
Diagnosis of disease or pathological process.
Cause of death determination.
Direct impact on therapeutic management.

Sampling Biological Material

Biopsy: Excision of tissue for diagnosis or treatment.
- Excisional biopsy: Removal of the entire lesion.
- Incisional biopsy: Removal of a portion of the lesion.
- Core cut (core needle biopsy): Removal of a cylindrical tissue sample using a needle.
Cytology: Examination of cells from various sources.
- Exfoliative cytology: Collection of cells that have shed from a tissue.
  - Spontaneous: Shedding from body fluids.
  - Mechanical: Collection using a brush.
- Interventional: Collection of cells through needle aspiration.

Excision Biopsy

Used when the lesion is large, located in a hazardous area, or suspected malignancy requires further surgery.

Core Needle Biopsy

Larger tissue sample compared to fine-needle aspiration cytology (FNAC).
Fast and easy, allowing for histological diagnosis.
Performed under palpation, stereotactic, or ultrasound guidance.
Preferred method for histological diagnosis.

Preparation of Tissue for Light Microscopy (Tissue Processing)

Embedding tissue in a solid medium for sectioning and preserving morphology.
Steps include fixation, dehydration, clearing, embedding, sectioning, sliding, staining, and covering.

Collecting the Sample

Clinical Details: Important information about the patient and the sample.
Adequate Specimen: Ensuring the tissue sample is suitable for analysis.

Fixation

Preservation of tissue structure using chemical compounds (fixatives).
Common methods include 10% formaldehyde.
Formalin protects tissue from microorganisms, but results in loss of biological function.
Cross-linking of proteins.
Denaturation of proteins (altering tertiary structure) without affecting antigenicity.
Formalin does not penetrate tissues thicker than 1cm.
Fixation speed: 1mm/hour.

Why Fixation?

Cell death triggers the release of lysosomal enzymes that degrade tissues (autolysis).
Cellular components have varying susceptibility to digestion (RNA >> Protein >> DNA).
The aldehyde group in formalin reacts with amine groups (NH2) in amino acids, blocking enzyme activity.
Glutaraldehyde acts similarly but cross-links proteins, hardening tissue and better preserving morphology.

Glutaraldehyde

Fixation reaction involves the reaction of amine groups on amino acid residues with aldehyde groups in the fixative.
Deforms alpha-helix structure in proteins making it less suitable for immunohistochemistry.
Rapid fixation properties make it ideal for electron microscopy.

Fixation for Immunohistochemistry (IHC)

Ethanol, methanol, or acetone disrupt hydrophobic bonds and protein structure, removing water.
Aldehydes destroy amine groups, but preserve tissue structure well.
Alcohols result in poorer structural preservation due to dehydration but do not destroy amine groups, preserving secondary protein structures.
In IHC, antibodies bind to specific amino acid sequences (antigens), so the presence of lysine may hinder antibody binding.

Dehydration

Removal of extractable water from the tissue.
Graduated ethyl alcohol solutions (30, 50, 70, 95, and 100%) are used, typically for 20-30 minutes each.

Clearing

Also known as dealcoholation.
Replacing alcohol with a solvent miscible with paraffin, such as xylene or chloroform.

Embedding

Before sectioning, tissue must be embedded in a material that hardens to facilitate cutting.
Paraffin wax is commonly used and heated to 60 degrees, then cooled to form a firm tissue block.

Sectioning

Thin tissue sections for microscopic examination are cut using a machine called a microtome, which produces sections of ~3μm.
Vibratome cuts thicker sections (up to 200μm) from fresh or fixed tissue using a vibrating blade.
Cryostat cuts sections from deep-frozen blocks of unfixed tissue.

Sliding (Mounting)

Placing the section onto a clean microscopic glass slide, ensuring no air bubbles are trapped.

Staining

Tissue staining enhances contrast for detailed microscopic examination.
Basophilic: Structures stained by basic dyes (color property in the basic radical).
Acidophilic: Structures stained by acidic dyes (color property in the acidic radical).

Hematoxylin and Eosin (H&E) Staining

Hematoxylin: Basic dye staining acidic components (DNA and RNA) blue, highlighting the nucleus and ribosome-rich regions of the cytoplasm.
Eosin: Acidic dye staining basic components (cytoplasm) pinkish, highlighting the remaining cellular components.

Metachromasia

Certain stains, like toluidine blue, can polymerize when exposed to high concentrations of polyanions.
The aggregates display a different color than individual molecules.
For example, toluidine blue stains tissues blue except for polyanion-rich areas like cartilage matrix and mast cell granules, which stain purple.
Structures staining purple are considered metachromatic.

Covering

Applying a thin glass coverslip to protect the section.

Different Parts of a Microscope

Eyepiece lens: Magnifies the image.
Tube: Connects eyepiece to objective lens.
Arm: Supports the tube and stage.
Base: Supports the entire microscope.
Illuminator: Light source.
Stage: Platform holds the slide.
Revolving nosepiece: Holds objective lenses and allows switching between them.
Objective lenses: Provides primary magnification.
Condenser lens: Focuses light onto the specimen.
Coarse & fine focus: Controls magnification.
Diaphragm: Regulates the amount of light passing through the condenser.

Immediate Frozen Section (IFS)

Minimizes tissue preparation time compared to standard histology (approximately 5 minutes vs. 16 hours).
Provides quick results for immediate diagnosis, particularly in tumor surgery.
Less accurate than formalin-fixed paraffin-embedded (FFPE) sections.

Cell Smears

Examination of thin layers of cells from sources like blood, bone marrow, or epithelial cells from the cervix or mouth.

Immunohistochemistry (IHC)

Demonstrates specific antigens within FFPE tissues.
Visualizes antigen-antibody complexes under a light microscope, revealing tissue morphology around the target antigen.
Semi-quantitative results provide crucial diagnostic or prognostic information.
Fluorochrome-labeled antibodies can be used for fluorescence detection (immunofluorescence).

Histology

The study of tissues and their arrangement in organs
Also known as microscopic anatomy or microanatomy
Four basic tissue types: Epithelia, Connective tissue, Muscle, Nerve

Why is Histology Important?

Processing of biological material from living or dead bodies (biopsies, necropsies)
Used for diagnosis, determining pathological processes, and identifying causes of death
Directly relevant to therapeutic management

Sampling Biological Material

Biopsy
- Excision (diagnostic or therapeutic)
  - Involves removing the entire lesion or a sizeable portion
- Incisional
  - A smaller portion of the lesion is removed for examination
- Core cut (core needle)
  - A cylindrical sample of tissue is extracted using a hollow needle.
Cytology
- Exfoliative cytology
  - Cells are collected from body fluids (e.g., sputum, urine, or fluid from a cyst)
- Mechanical (brush)
  - Cells are scraped from a surface using a brush
- Interventional
  - Needle aspiration is used to collect cells from a mass or lesion

Excision Biopsy

Used when a lesion is large, located in a hazardous area, or suspicious of malignancy requiring further surgery

Core Needle Biopsy

Provides a larger tissue sample compared to fine needle aspiration
Quick and easy, allowing for rapid histological diagnosis
Can be performed under palpation, stereotactic, or ultrasound guidance
Often the preferred method for histological diagnosis

Preparation of Tissue for Light Microscopy (Tissue Processing)

Embedding the tissue in a solid medium enables the creation of thin sections for examination.
The aim is to preserve tissue structure with minimal alteration.
Steps:
- Fixation
- Dehydration
- Clearing
- Embedding
- Sectioning
- Slides
- Staining
- Covering

Collecting the Sample

Clinical details and an adequate specimen are essential for accurate diagnosis.

Fixation

Preserves tissue structure using chemical compounds called fixatives.
Common method: 10% formaldehyde
- Protects tissue from microorganisms.
- Results in loss of biological function.
- Cross-links proteins.
- Denatures proteins by altering the tertiary structure.
- Does not affect antigenicity.
- Limited penetration (1mm/hour), requiring several hours for most specimens.

Why Fixation is Important

Cell death releases lysosomal enzymes that digest tissues - autolysis.
Different cellular components have varying susceptibility to digestion (RNA > Protein > DNA).
Formaldehyde reacts with amine groups (-NH2) in amino acids, blocking the activity of proteins including lysosomal enzymes.
Glutaraldehyde acts similarly but forms cross-links in proteins, hardening tissue and preserving morphology.

Glutaraldehyde

Forms bonds with amine groups (-NH2) in amino acid residues.
Can deform the alpha-helix structure of proteins, which makes it less suitable for immunohistochemical staining.
Fixes quickly and is preferred for electron microscopy (EM).

Fixation for Immunohistochemistry (IHC)

Ethanol, methanol, or acetone can be used to fix tissue by disrupting hydrophobic bonds and protein structure while removing water.
Aldehydes (e.g., formaldehyde) do not destroy amine groups and maintain tissue structure well.
Alcohols generally result in poorer structure preservation due to dehydration but do not destroy amine groups and can preserve some secondary protein structures.
Antibodies in IHC bind to specific amino acid sequences (antigens) within their targets.
If the target includes lysine, formaldehyde fixation may hinder antibody binding due to amine group modification.

Dehydration

Removes water from the tissue using different concentrations of ethyl alcohol (30%, 50%, 70%, 95%, and 100%).
Typically takes 20-30 minutes in each solution.

Clearing

Also called dealcoholation.
Replaces alcohol with a solvent miscible with paraffin (e.g., xylene or chloroform).

Embedding

Tissue is embedded in a hard material (e.g., paraffin wax) for sectioning.
Paraffin wax is heated to 60 degrees Celsius and solidifies with the tissue upon cooling, forming a firm block.

Sectioning

Cutting thin sections (3-10 μm) of tissue using a microtome.
Other devices include:
- Vibratome: Cuts thicker sections (up to 200 μm) from fresh or fixed tissue.
- Cryostat: Cuts sections from frozen unfixed tissue blocks.
Routine histopathology uses microtomes for thin wax-embedded sections.
Vibratomes and cryostats are used for unfixed sections, often preferred for antibody staining.

Sliding (Mounting)

Thin sections are mounted on clean glass slides, ensuring no air bubbles are trapped.

Staining

Tissues are naturally colorless, making staining essential for detailed examination.
Staining techniques enhance contrast and differentiate tissue components.
Basic dyes (e.g., hematoxylin) stain acidic components blue (e.g., DNA, RNA). These are called basophilic structures.
Acidic dyes (e.g., eosin) stain basic components pink (e.g., cytoplasm). These are called acidophilic structures.

Metachromasia

Some stains (e.g., toluidine blue) can polymerize when exposed to high concentrations of polyanions in tissue.
These aggregates have a different color than individual dye molecules.
Toluidine blue stains tissues blue, except for polyanion-rich areas (e.g., cartilage matrix, mast cell granules) which stain purple.
Tissues that stain purple with toluidine blue are called metachromatic.

Covering

A thin glass coverslip is applied to protect the section.

Different Parts of a Microscope

Eyepiece lens
Tube
Arm
Base
Illuminator
Stage
Revolving nosepiece
Objective lenses
Condenser lens
Coarse and fine focus knobs
Diaphragm

Immediate Frozen Section

Used for rapid diagnosis, eliminating the need for standard histology preparation (approximately 16 hours).
Frozen section takes approximately 5 minutes.
Used for immediate results (e.g., during tumor surgery).
Less accurate than FFPE (Formalin-Fixed Paraffin-Embedded) sections.

Cell Smears

Used for examining tissues as thin layers or smears.
Examples: blood, bone marrow, epithelial cells from the cervix or mouth.

Immunohistochemistry

Detects specific antigens in FFPE tissues using antibodies.
The antigen-antibody complex is visualized through light microscopy, enabling examination of tissue morphology around the specific antigen.
Results can be semi-quantitatively reported and provide diagnostic or prognostic information.
Visualisation can be achieved through fluorescent detection (immunofluorescence).

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Description

Explore the fundamental concepts of histology, including the study of tissues and their types. Learn about the importance of sampling biological material and the processing techniques essential for diagnosis and therapeutic management. This quiz will enhance your understanding of microscopic anatomy and its applications.