Applied Cellular Pathology (Sarah Hughes)

Histology

the study of tissues at the microscopic level, focusing on the structure, composition, and function of cells and their organization in various organisms.

Fixation

is the process of preserving tissue samples by using chemicals or physical methods to maintain their structure and allow for accurate microscopic examination.

Types of fixative

include formaldehyde, buffered formalin concrete, neutral buffered formalin

properties of formalin

• 40% formaldehyde

• buffered formalin concentrate

• 10% Neutral buffered formalin

Routes of a specimen

fixation, dissection, processing, embedding, microtome,staining

processing

the method of preparing a specimen for microscopy after fixation, including dehydration, clearing, and infiltration with embedding medium.

microtomy

the process of cutting thin sections of a specimen for microscopic examination.

staining

the technique used to enhance contrast in specimens, making specific structures more visible under a microscope.

Fresh tissue

that has not undergone any processing or preservation methods, often requiring immediate fixation for analysis. clinically urgent usually intraoperative

How are fresh tissue frozen

• Standard freezing- Cryobar

  • Most common

  • Quick

  • Cheap and easy

  • Usually -20C to -40C
    Used in cryostat (cryobar set at -50C)

• Liquid N2

  • -196 C rapid freezing on contact

  • Used to store some fresh specimens

  • Boil on contact

  • Risk- asphyxiation and freeze burns

• Cryospray- Mohs

  • Different technique for Mohs

  • Tissue is frozen onto a glass
    slide

  • Allows for orientation
    purpose

  • This is inverted onto a chuck to embed onto
    freezing medium

Explain the effects frozen tissue can have on the tissue

• Slow freezing give large hexagonal ice crystals

• Intracellular ice crystals damage cell membrane

• Rapid freezing gives small cubic ice crystals

• Less distortion of cell membrane

• Ice crystal artefact
  

Rate of cooling depends upon the rate of conduction. This depends upon the temperature difference between the tissue and the coolant

What are inter-operative frozen sections

Inter-operative frozen sections are quickly prepared tissue samples that are frozen and sliced during surgery to provide immediate pathological information, helping surgeons make real-time decisions.

What different techniques can be performed on fresh tissue

What steps are involved in the production of a frozen section

1. Specimen received in lab and booked in

2. Pathologist examines specimen

3. Sample area of suspicion

4. Rapid frozen onto a freezing medium

5. Sections cut

6. Section stained

7. Reported by pathologist

8. Specimen fixed and processed to paraffin

Freezing

is a preservation method where fresh tissue is rapidly cooled to maintain cellular structure and prevent degradation, often used for intraoperative

What are the advantages of frozen sections

• Quick

• Cost-effective

• Compatible with other techniques

• Only method for demonstration of some methods e.g. oil red o

• Diagnostic sections

What are the disadvantages of frozen sections

• Morphology is not as good

• Difficult to cut

• Artefact

• Storage

• Time pressure

• High-risk specimens

what are the risks and hazards associated of  working in the lab with frozen sections

• Burns - exposure to cryogenic temperatures can cause burns; potential

• Sharps - handling sharp instruments poses injury risks;

• Biological - exposure to biological hazards from pathogens;

• Chemical - and risk of chemical exposure from reagents used in processing.

What is Immunohistochemistry (IHC)

• A laboratory technique used to identify specific antigens in cells or tissue sections by utilising antibodies tagged with a detectable marker.

• Aiding in the diagnosis of diseases such as cancer.

What are the uses of IHC

• Define cell types in tumours

• Cell types in inflammatory cells

• Identify infectious pathogens

• Prognostic indicators

• Predicative indicators in targeted therapy

What two ways to expose the antigen

Common methods include using heat or enzymatic digestion to enhance antigen retrieval for better staining.

Direct IHC

A method where the primary antibody is directly conjugated to a detectable marker, allowing for immediate visualization of antigen-antibody binding.

Indirect IHC

A technique where an unlabeled primary antibody binds to the target antigen, followed by a labelled secondary antibody that binds to the primary antibody, amplifying the signal for improved visualization.

What is the process of IHC

1. Fixation

2. Take to water

3. Antigen retrieval/Wash

4. Block/wash

5. Primary antibody/wash

6. Detection system/wash

7. Chromagen/wash

8. Counterstain wash

9. Quality control

What is the purpose of the positive controls

• confirming the reliability of the assay results to validate the IHC process

• contains relevant tissue antigen

• confirms antibody is working

• low expression

What is the purpose of negative controls

• Not routinely used ely

• Detects non-specific interactions

• Known to not contain target antigen

Name the positive controls used in IHC labs

• Positive

  • Beta catenin

  • Melan-A

• Negative

What is the importance of fixation in IHC

• IHC secondary investigation

• Fixation is optimised for routine H&E

• Fixation alteration can affect IHC and cause

• Can lead to antigens being in other cellular compartments that is not expected e.g. oestrogen receptor can be seen on the outside of the nucleus in poorly fixed breast tissue.

• If inadequately fixed can lead alterations in architecture and in highly cellular tissue can lead to specimen being unusable to IHC.

• Ideal fixative: preserve morphology, preserve antigen immunoreactivity, prevent antigen extraction during IHC and not interfere with antigen antibody interactions

What is antigen retrival

The process of unmasking antigens in paraffin-embedded tissue sections to enhance antibody binding during immunohistochemistry.

What are the two types of antigen retrival

• Enzyme digestion

• Heat-mediated antigen retrieval (HMAR)

What are three enzymes seen in enzyme digestion

1. Trypsin: most common. It removes the cross-links as well as aiding antigenic precursors by digesting protein aggregates which can block access to antigens.

2. Chymotrypsin: slower agent, many commercial agents use a combined chymotrypsis and trypsin component.

3. Protease: ready-made for commercial used on automated stainers

How and why are enzyme degestion is used

• Selectively breaking the protein links to reveal antigen epitopes without disrupting the protein structure.

• Water bath at 37 degrees in a controlled manner

What are the advantages and disadvantages of enzyme digestion

• Advantages

  • Only way to demonstrate some epitopes e.g. renal

• Disadvantages

  • Has to be expertly controlled

  • Can destroy some epitopes

What is monoclonal antibodies

• Derived from a single clone of B
  cells

• Bind to single epitope—highly
  specific

• Higher production times and cost
  due to complex manufacturing
  processes

• Commonly used in precision-
  focused research applications like
  diagnostic assays (ELISA, western
  blot, etc.), therapeutics (e.g.,
  targeted cancer therapies

What is monoclonal advantages and disadvantages

• ADVANTAGES

  • Precise, improves effectiveness and reduces side effects

  • No batch to batch or lot to lot variation

  • Inexpensive to produce

• DISADVANTAGES

  • Targeted epitope must survive fixation

  • Target epitope must be unique to one antigen cross reactivity cannot be removed

  What is polyclonal antibodies

• Derived from multiple clones of B
  cells

• Recognize multiple epitopes—
  broader specificity

• Versatile and widely employed in
  various research applications

• Quicker and more cost-effective to
  produce

• Commonly used in research
  applications where broad
  specificity is needed like
  immunohistochemistry (IHC),
  immunofluorescence (IF), Western
  blot, and early diagnostic tests

What is polyclonal advantages and disadvantages

• ADVANTAGES

  • Less batch to batch variation than with larger animals

  • Recognizes more than one epitope on an antigen

• DISADVANTAGES

  • Recognises more than one epitope on an antigen

  • Cross reaction

What are the advantages and disadvantages of DAKO (automated IHC)

• ADVANTAGES

  • Complete kit

  • Ready-to-use reagents

  • Increased sensitivity

  • Faster than other techniques

  • No wastage

  • Reduction in operator error

  • Elimination of endogenous biotin

  • Can be used with automation

• DISADVANTAGES

• COST!! Approx £800 per kit

What are chromagens

Chemical compounds Interacts with the final antibody-antigen complex to produce a visualised colour

commonly used in immunohistochemistry.

• Horseradish peroxidase converts DAB (3,3-diaminobenzidine)- To brown

• Alkaline phosphatase converts AEC (3-amino-9-ethyl carbazole)- To red

Liver panel

PAS/DPAS, reticulin, orcein, perls

renal panel

Mehtenanime silver, EVG, PAS

Special stains

regressive staining vs progressive staining

Progressive staining refers to techniques where staining increases over time

while regressive staining involves removing excess stains to achieve the desired visualization of specific cellular components.

What are the types of speical stains

• Histochemical

  • Utilises a true chemical reaction as if it would happen in a test tube e.g. Periodic Acid Schiffs (PAS) for demonstration of carbohydrates

• Lysochrome

  • Staining of neutral lipids/fats

  • Elective solubility- hydrophobic nature of the lipid allows the dye to penetrate the lipid

• Impregnation

  • Deposition of silver in a tissue section

  • Argyrophil/Argentaffin/Ion exchange

• Injection

  • Introduction of coloured compounds into tissue to highlight structures

• Fluorochrome

  • Adding a fluorochrome with the tissue and then visualised under fluorescent microscope e.g. amyloid

• Trapping agents

  • Prevents the dye from escaping once entered e.g. Gram stain

what are the disadvantages of using automated staining

• Cannot adapt stain

• Staff being de-skilled

• Cost of capital purchase

• Preservatives in staining reagents limits sensitivity

• Kits can go to waste

• Limited number of stains

what are the advantages of using automated staining

• Closed system

• Reproducibility

• Amount of slides

• Multiple stains run at the same time

• Free up lab staff time

• Standard protocols

What is the advantage of using hand staining

• Adaptability to changes

• Cheaper

• Easy to train staff on singular methods

• Controlled by eye

What is the disadvantage of using hand staining

• Level of interpretation

• Variability

• In-house solution cost

• Labour intensive

• Exposure to hazardous chemicals

What are some of the controls for special stains

• PAS- positive for fungi

• Acid fast bacilli in ABPAS

• Mycobacterium positive in Ziell Nielson

Name some more examples of special stain controls