W9L3 - Immunohistochemistry Principles and Techniques

Question 1

What is IHC?

Answer 1

Specialised technique in AP where immunological and biochemical techniques are used to identify discrete components in tissues by using appropriately labelled antibodies to bind specifically to their target antigens in situ
Makes it possible to visualise and document the distribution and localisation of specific cellular components within cells and within the histological context of the tissue

Question 2

Antibodies IgM and IgG

Answer 2

For immunoassays, IgG and IgM are the two important ones
IgM eliminates pathogens during the early stages of B cell mediated immunity before there is sufficient IgG
IgG molecule has two separate functions:
- to bind to the pathogen that elicited the response
- to recruit other cells and molecules to destroy the antigen

Question 3

Structure of IgG

Answer 3

MW of 150 kD
Composed of 2 heavy and light chains (50 kD and 25 kD respectively)
Each end of the forked portion of the ‘Y’ structure on the Ab is referred to as the Fab region
Ag specific Fab ‘arms’ are responsible for antigen binding
Fab fragments comprise of one light chain and the segment of heavy chain on the N-terminal side
The light and heavy chain segments are linked by interchain disulfide bonds
The Fc tail is a region of an Ab composed of two heavy chains on the C-terminal side
Fc has effector functions (bind complement) and serves to distinguish one class of Ab from another

Question 4

Structure of IgM

Answer 4

Accounts for ~10% of the Ig pool and is used for the production of antibodies, to a lesser extent than IgG
The IgM molecule contains 5 or 6 ‘Y’ shaped subunits that are covalently linked together with disulfide bonds
The individual heavy chains have a MW of approx. 65,000 and the entire molecule has a MW of 970,000
3rd most common serum Ig

Question 5

Polyclonal Antibodies

Answer 5

Polyclonal antibodies are produced by injecting an antigen into an animal
IgG specific for a particular Ag is then produced by B lymphocytes through activation of an immune response
These Ig are then extracted and purified from the animals serum
Specific IgG concentration of between 1-10mg/ml can be obtained
Ab obtained by this method are derived from different types of immune cells and are referred to as polyclonal

Question 6

Monoclonal Antibodies

Answer 6

Derived from a single cell line (clone)
Mab’s the tumour cells replicate endlessly and are fused to the mammalian cells that produce Ab’s resulting in a hybridoma, that will continually produce Ab’s
Mab’s are identical because they are produced by immune cells that are clones of a single parent cells
Production of Mab’s requires immunising an animal with immune cells from its spleen and fusing the cells with a cancer cell i.e. from a myeloma, to make them immortal

Question 7

Differences Between Polyclonal and Monoclonal Antibodies

Answer 7

Polyclonal
- raised in different animal species
- has more non-specific reactivity
- likely to be reactive in unknown application
- inexpensive
- minimal skill required to generate
- short time scale
- recognises multiple epitopes on one antigen
- produces large amount of non-specific antigen
Monoclonal
- raised in mouse or rabbit as a hybridoma
- often cleaner
- often generates false -ve results (if target epitope damaged/altered)
- expensive to produce
- requires training
- takes time to produce
- recognises only one epitope on an antigen
- produces a large amount of specific antigen

Question 8

Affinity vs Avidity

Answer 8

Affinity
- refers to the 3D fit of the Ab to the specific Ag
- it is a measure of the binding strength between the Ag epitope and the specific Ab
Avidity
- refers to the heterogeneity of the antiserum which contains various Abs, that react to different epitopes of the Ag
- a specific multivalent Ab is less likely to be removed by the washing process than the monovalent Ab
- so avidity can be described as the functional strength of an Ab to the Ag

Question 9

Antibody Specificity vs Sensitivity

Answer 9

Specificity
- refers to the characteristics of an Ab to bind selectively to a single epitope on an Ag
Sensitivity
- refers to the relative amount of Ag that is present in an IHC technique that is able to be detected

Question 10

Fixation in IHC

Answer 10

Appropriate fixation of biological probes will have a significant impact on the quality of IHC staining
Fixation should be sufficient to maintain the integrity of the section, but not so harsh to destroy the Ag being investigated
While most fixatives preserve tissues from degradation, some may destroy or mask the antigenic sites or molecular targets within specific tissues
3 main groups of fixatives are:
- those containing formalin
- those with alcohol
- those that have a combination of both
Fixation is by far the most significant factor that can impact Ag retrieval and epitope binding

Question 11

Criteria of Specimens Required for IHC Analysis

Answer 11

Cell and tissue preservation must be adequate to characterise the localisation of the component of interest
The antigenicity of the component of interest must be present and accessible to the Ab

Question 12

How does formalin penetrate tissue?

Answer 12

Penetrates the tissue by forming cross linkages between reactive amino groups in proteins

Question 13

Section Preparation for IHC

Answer 13

Sections required for IHC or ISH are cut at 3-5um
Occasionally, thicker sections may cause difficulty due to multilayering of cells
Sections can be picked up using adherent slides to prevent sections lifting during the staining process

Question 14

Control Material for IHC

Answer 14

Control material must be fixed and processed in the same manner as the test sample to assure accuracy of the results
Tissue with known protein expression of the target are used as controls
Cell lines can also be used as control tissue

Question 15

Optimal Concentration of Antibodies

Answer 15

While automated systems have TGA approved concentration, when new Ab’s are being optimised in a lab, the optimal working concentration is important
Optimal Ab’s conc^ is important to determine;
- Ag density
- loss of Ag activity in specimen
- Ab avidity
- Ab sensitivity of the IHC procedure.
For a large range of Poly and Monoclonal 1° Abs in routine IHC already have a known working conc^ between 1-5ug/ml
2° Ab are usually applied at the working conc^ approx. 5-10ug/ml

Question 16

Antigen Retrieval - Drawbacks of Formalin

Answer 16

Induces molecular modification of proteins (Ag) resulting in a loss in the ability and sensitivity of the Ab to react with Ag
This loss can only be restored by treating the section with an Ag retrieval step
Ag retrieval methods include:
- enzyme digestion - improves IHC staining only for limited Ag
- heat inactivation - to break the cross linkages between formalin and protein >100°C
- microwave treatment - another form of heating
- strong alkaline treatment

Question 17

Factors that Effect Ag Retrieval - Heating

Answer 17

Most important factor - high temp heating of formaldehyde fixed proteins in FFPE tissue sections results in hydrolysis that contributes to break down cross links
For many Ag, any type of heating tx will give similar results
There is an inverse correlation between heating temp (T) and heating time (t) expressed by the formula:
- AR = T x t
For many Ag, higher temp heating such as boiling FFPE tissue sections for 10-20mins may be optimal
To preserve tissue morphology, a lower temp (90°C) with an inc time may be optimal
For some Ag, the extreme conditions of temp and time (e.g. pressure cooker for hours) gives the greatest staining, but at the expense of morphology

Question 18

Factors that Effect Ag Retrieval - pH Solution

Answer 18

Tris-HCl buffer produces better results at higher pH compared with other buffers
Optimisation of Ag retrieval should include optimisation of the pH solution
Higher pH solution such as Tris-HCl or sodium acetate buffer at pH 8-9 may be suitable for most Ag
Low pH solutions are useful for nuclear Ag but may give weak focal false positive nuclear staining

Question 19

Factors that Effect Ag Retrieval - Chemical Composition of the Ag Retrieval Solution

Answer 19

Chemical composition of the Ag retrieval solution incl;
- citrate buffer
- metal salts
- urea
- citraconic anhydride
These usually have a high pH (pH 9), a low-mid pH method (pH 6) and an enzyme based method for a small number of Abs

Question 20

Immunostaining Methods

Answer 20

Direct
Two step indirect
Three step indirect
PAP
ABC
Polymer/Direct

Question 21

Direct Immunostaining Method

Answer 21

Primary Ab is directly conjugated to a label
This is suitable for detecting highly expressed antigens
Benefit is that additional incubation step with a 2° reagent is not necessary
The labelled Ab reacts directly with Ag in the tissue section or cytological preparation
This is a quick and easy to use technique however, it provides minimal signal amplification and lacks sensitivity compared to other techniques.
Mainly used to demonstrate Ig and complement in frozen sections of skin and renal bx
Low level Ag identified in certain tumours may not be demonstrated by this method

Question 22

Indirect Immunostaining Method

Answer 22

The 1° Ab is bound by a labelled 2° Ab that has been raised against the host species of the 1° Ab
May include amplification steps to increase signal intensity

Question 23

Two Step Indirect Immunostaining Method

Answer 23

Where a labelled 2° Ab is directed against the Ig of the animal species in which the 1° antibody was raised in, is an unlabelled 1° Ab
HRP is the most common method used together with an appropriate chromogen substrate
Method is more sensitive than the direct method as multiple 2° antibodies may react with different Ag sites on the 1° Ab which increases signal amplification
This method offers versatility because the same labelled 2° Ab can be used against a variety of 1° Ab raised in different animal species

Question 24

Polymer Chain 2 Step Indirect Immunostaining Method

Answer 24

This method uses an unconjugated 1° Ab, followed by a 2° Ab that is conjugated to an enzyme (HRP) which has been labelled by a polymer sugar backbone/chain
The sugar chain contains up to 70 molecules of enzyme and 10 molecules of Ab attached
Conjugation of both anti-mouse and anti-rabbit 2° Ab enables the same reagent to be used both monoclonal (rabbit and mouse) and polyclonal (rabbit) 1° Ab
The method does not react with endogenous biotin, is quick, reliable and easily reproducible
Good sensitivity