Lecture 3 Flashcards
What are the 4 components of pathology?
- Aetiology - Defining the cause
- Pathogenesis - The mechanism of action behind the disease
- Morphology - The structural changes caused
- Clinical significance - The functional changes
What are the 3 ways in which tissue can be acquired to start the histopathological examination?
- Surgery
- Biopsy
- Autopsy
What is the problem with postmortem tissue that you have to take into consideration when examining it?
Post-mortem interval (delay) can lead to changes in the integrity of the tissue and proteins within that tissue, also the RNA and DNA in the tissue
Why is a tissue preserved?
To prevent decay from Autolysis or putrefaction
What are the 2 ways that tissue is preserved for histopathology?
It can be fixed or frozen
What is used to section a frozen piece of tissue and what is frozen tissue good for?
A machine called a crystal is used to section the frozen samples
- Can be very useful for rapid procedures however the quality of the morphology compared to fixed tissue is a lot poorer
What are the 2 ways in which viable tissue is fixed?
- Perfusion (very invasive, injecting fixative into the left ventricle through the circulatory system fixing the internal organs whilst the animal is still alive)
- Immersion (samples are submerged into fixative and left for a couple of months to soak and reach all of the tissue)
What are the 4 key factors that need to be controlled when fixing tissue?
- Concentration
- pH
- Temperature
- Duration
What does a tissue sample have to be embedded in to be able to be cut on a microtome?
They are embedded in wax (most commonly paraffin wax)
This is because the wax is liquid at around 58c and can therefore permeate the tissue and is cooled and solidifies allowing the sample to be sectioned on the microtome
What is the process of embedding a sample in wax ready for the microtome?
- The tissue sample is placed into a ‘cassette’
- A correctly sized mould is selected
- Wax is dispensed into the mould
- Need to make sure that the tissue sample is in the mould in the correct anatomical orientation
- The wax is then allowed to solidify
What size sections does a microtome cut?
- Typically for diagnostic purposes 3-10 micrometers
However thicker sections can be cut for other purposes such as research
What are the aims of staining tissue samples?
- Make the cell structure visible
- Show variation in the structure of the sample
- Shows the pathological changes in the tissue sample
What some of the types of staining that can be used?
- Non-vital - Staining of dead tissue that has been fixed and processed
- Vital/ Phagocytic - The colouring of living tissue using dyes or by phagocytes ingesting dyes
- [True] Histochemical - A chemical reaction that matches what would also happen in a test tube doing the same thing
- Lysochrome - [Alcohol based dye] The dye molecules leave the solvent they are in an enter lipids (hydrophobic)
What are some more of the types of staining that can be used on tissue?
- Impregnation - The formation of a coating around individual fibres (neurones)
- Injection - Inject dye straight into vessels and organs to view
- Fluorochromes - Combines a fluorochrome with tissue and is visualised using fluorescent microscopy
- Immunostaining - Using antibody-antigen reactions to stain and differentiate tissue
What do dyes contain that allow them to interact with the sample they are staining?
They contain charges (+/-) which allows bonding and attractions between the the sample and the stain
What charge does and acidic element in tissue have and what kind of dye needs to be used in order to stain it?
The acidic element has a -ve charge and so you need a basic dye that is +vely charged to complement and stain it
What charge does a basic element in tissue have and what type of dye is needed to stain it?
The basic element has a positive charge and so an acidic dye which has a negative charge is used to complement and stain the tissue
Why is knowing the tissue pH important in histopathology?
So you can find the right type of stain that can be used and is going to work with the tissue you have got
What is the isoelectric point in staining?
When the tissue sample is neither acidic or basic, it is neutral
How does the process of indirect attachment staining work?
- The process uses a intermediate (mordant) to link the tissue and the dye
- The mordant and the dye make what is called a ‘dye lake’ and the colour of this lake is dependant on the mordant used
- This ‘dye lake’ is then differentiated by some form of acid
- The use of the mordant allows for stronger attachment of the dye to the tissue sample
What happens during differentiation?
The excess staining is removed and this then differentiates between the structures we want to see in the tissue and the background of the sample
What is haematoxylin and what does it do?
- It is a naturally derived stain that has been used for hundred of years in labs
- It stains the nuclei of cells blue/black
What is Eosin and what does it do?
- Eosin is the acidic counterstain to haematoxylin
- It stains cytoplasm, muscle, collagen etc red
What is immunohistochemistry?
The microscopic study of tissues with the aid of antibodies that bind to tissue components to reveal their presence
What do the antibodies in IHC bind to?
They bind to the antigens in the target tissue
How does the word immunohistochemistry break down?
Immuno - The antibodies used
Histo - The use of tissue
Chemistry - The detection of the reaction taken place
How is IHC visualised?
The visualisation is done through ‘tagging’ using either chromogens or fluorophores
What is an antigen?
A substance that induces a detectable immune response
What is an antibody?
A host protein produced in response to the presence of an antigen e.g. a foreign molecule
What is the variable region of the antibody called and what is it used for?
The region is called the paratope and it is where the specific antigen to the antibody will bind
Where are antibodies present?
They are seen on the B lymphocyte surface as surface antibodies
What do B cells do with these antibodies?
They secrete them into the bloodstream where they are called secreted antibodies
The binding site on the antibody is a paratope, but what is the binding site on the antigen called?
An epitope
What are the 5 major types of immunoglobulins found within mammals?
IgA (alpha chains), IgD (delta chains) , IgE (epsilon chains) , IgG (gamma chains) and IgM (mu chains)
How are the antibodies used in IHC produced?
They are ‘raised’ in animals, exploiting the animals ability to create and immune response. The antigen is injected into the animal, B cells are activated producing antibodies. Serum that contains the antibodies created is collected (Antisera)
What are the 2 major types of antibody?
- Polyclonal
- Monoclonal
How are polyclonal antibodies produced?
- Inject antigen into animal
- Antigen activates the B cells (differing epitomes on the antigen are binding to differing paratopes on the antibody as they have multiple different P’s and A’s on their surface at a time)
- Plasma B cells produce antibodies with varying paratope regions on them (polyclonal antibodies)
- The antibodies are secreted into the blood stream (antibodies with many varying paratopes)
- The antiserum is then obtained and centrifuged to collect the serum in the blood
What are polyclonal antibodies?
Anitbodies that are secreted by different B cell lineages within the body
How are monoclonal antibodies produced?
- The animal (usually mouse) is injected with the antigen
- The animal automatically produces the B cells that produce the antibody that is complementary to the antigen being injected
- The spleen cells are then removed (because they produce the B cells)
- The spleen cells are then fused with white blood cell lines from humans creating hybridomas (immortalised cell line)
- Hybridomas can then divide to produce millions of monoclonal anitbodies as the cells are specific to the original antigen that was given to the animal
- The MCA are then harvested from the animal
What are monoclonal antibodies?
They are anitbodies that are made by identical B cells which bind to the same epitope on an antigen
How are Antibody-antigen interactions used within IHC?
The antibody binds to a protein/carbohydrate etc epitope, exploiting the principles of antibodies binding specifically to antigentic sites in biological tissue
What part of the method of Histochemical staining and IHC staining is the very similar?
The fixation and processing of the sample is very similar, however the use of formulin creates cross linkages between proteins ‘masking’ them
What is the process of unmasking the proteins of a fixed sample to uncover the epitopes of the antigens known as?
Antigen retrieval
What are the 2 methods of Antigen retrieval?
- Through chemicals - (proteolytic-induced epitope retrieval) using things such as trypsin and pepsin
- Through heat - (heat-induced epitope retrieval) using things such as a microwave, pressure cooker or autoclave
What is a primary antibody and where does it bind to?
The primary antibody is one that is specific to the antigen found in the tissue which binds directly to that antigen in the tissue
What is on top of the primary antibody that allows the antiobody to be seen?
The Fc domain
What is attached to the Fc domain in order for the anitbody to be detected more easily?
A secondary antibody
Why is the secondary antibody not specific to a particular antigen?
Because it binds to the Fc domain of a primary antibody which is consistent across all animal species
What does the secondary antibody do to increase the signal and amplification of the primary antibody and allow it to be more easily visualised?
It conjugates with enzymes and leaves a precipitate e.g. a chromogen to show where the primary antibody has bound to the antigen in the tissue sample
Give an example of the ABC method of immunolabeling?
- Primary antibody binds to antigen
- Secondary antibody binds to primary antigen
- Secondary antibody conjugates with biotin
- Biotin binds to the ‘avidin peroxidase enzyme complex’
- A DAB (3,3’-Diaminobenzidine) reaction then visualises the binding of the antibody
What is a common issue in IHC staining ?
- Non-specific labelling using polyclonal antibodies will not give a clear picture as although the subject of want to investigate is dyed so is the background of the sample
What can be used to counterstain an IHC stain and why would this be used?
Conventional histochemical stain like H and E can be used. It allows architecture as well as markers to be visible, however must be stained quickly and lightly as to not mask the immunoreactivity
What else can you use to tag antibodies and what is the advantage of doing this?
Fluorophores can be used to tag the primary or secondary antibody of an IHC stain, the advantage is that multiple antigens can be labelled with different anitbodies on the same slide
