W9L3 Part 2 - Immunohistochemistry Principles and Techniques Flashcards
Blocking Steps
During the IHC detection process there are wash steps and blocking to prevent non specific binding
Endogenous peroxidase - 1% H2O2 in methanol for 30mins at RT
This removes peroxidases from lysosomes, Hb etc.
Normal serum - made up in 10% PBS from the same animal in which 2° Ab is produced - 10 mins at RT
The main cause of non specific background staining is a non immunological binding of specific immune sera by hydrophobic and electrostatic forces to certain sites
Detection Methods Used in IHC - Enzyme Labels
Includes:
- Enzymes - most widely used for IHC, and usually incubation with a chromogen will produce a stable, coloured reaction as the end product
- HRP - the most commonly used in IHC in combination with the chromogen, 3,3α diaminobenzidine tetrahydrochloride (DAB) which results in a ‘brown’ colour on all tissue
- Colloidal metal labels - may appear pink under light microscopy
- disadvantage is, a silver precipitation reaction is developed resulting in background deposit - Fluorescent labels
- Radio labels
Biotin-Avidin Procedure
Biotin-avidin procedure involves the high affinity binding between biotin and avidin
Steps:
1. Biotin is linked to the 1° Ab
2. Produces biotinylated conjugate that localises to the sites of the Ag
3. Avidin is then conjugated to HRP
4. Avidin binds to the Biotinylated Ab
5. Peroxidase is released at the site of the Ag in the FFPE
Chromogens
In order to detect Ag using a specific Ab, a chromogen is required to visualise the target Ag
Chromogens are specific chemical compounds used in IHC labelling procedures to produce a coloured product
E.g. HRP and AP
HRP
- DAB (yields crisp dark brown end-point, also carcinogen)
- aminoethyl carbazole (red)
Alkaline phosphatase (AP)
- fast red
- permanent red
Dual Label IHC Staining
Is a method that can be useful however, should only be performed in IHC Ag that are not located on the same structure i.e. nucleus and membrane is OK
The success of double staining is based on the development of the first IHC stain which should mask the structure stained first
Can use two Ab’s raised in the same species, but better results are obtained if 2 different species are used
- e.g. p16/Ki67 dual stain method
Advantages and Disadvantages of Direct and Indirect Immunofluorescence
Direct IF: Pros - simpler - Ab from same species Cons - lower signal - higher costs - less flexibility Indirect IF: Pros - higher signal (amplified) - flexibility - low cost Cons - more steps - Abs from same species can't be used together - background may be amplified
Merits of Techniques
Immunofluorescence - sensitive, non-permanent, dedicated microscopy
Confocal microscopy - sensitive, fine definition
Micro-arrays - large screening capacity
Labelled primary antisera - avoid aberrant binding by secondary
Labelled polymer - simple, large signal per binding site
Enhancement - increases signal, may increase background
Immunoblotting - qualitative and quantitative analysis
Flow cytometry (cell biology) - cell population studies
Applications of IHC - Tumour Pathology
Classification of neoplasms - e.g. carcinoma vs B or T cell lymphomas Prognostic markers - e.g. proliferation markers Ki-67 Dx malignancy Predict response to tx - e.g. ER, PR receptors, HER2/neu Detection of metastases Screening of inherited cancer syndromes
Applications of IHC - Non-Tumour Pathology
Neurodegenerative diseases
Brain tumours
Muscle disorders
Identification of extracellular components e.g. amyloidosis
Dementias
Identification of infectious agents e.g. HPV
Slides Required for IHC
Tissue may be lost during the staining process
Coated slides are useful;
- poly L lysine
- albumin
- gelatin
To ensure the entire section is exposed to the Ab, some automated systems have a liquid coverslip that makes sure there is even spread and avoids drying
Factors Affecting Immunogenicity
Type of fixative used - some may destroy antigenic sites (e.g. keratin lost in B5) - time of fixation may cause protein x-linking whereby decreasing antigenicity Decalcification - reduces antigenicity for Ab such as ER, PR, Ki-67 Length of time since the FFPE was cut - can reduce antigenicity - Ag retrieval is OK here Freezing tissue - may also show inconsistency Ag retrieval procedures Type of Ab Incubation time/temperature Dilution/concentration of Ab Methods of signal amplification - e.g. fluorochrome, DAB, polymer
Handing of Antibodies - Basic Rules
Receiving
- need a log of receipt, identifying the expiry date
- need to store as per manufacturers instructions
Storage
- use non protein absorbing containers
- temperature - monitor for accuracy, usually store at 4-8°C.
Handling and use
- changes T°
- excessive light
- contamination with other reagents
- bacterial growth
Positive and Negative Controls for Immunofluorescence
Positive controls
- internal i.e. Ag to show that the system is working
- e.g. S100 (melanocytes, nerve), Vimentin (BV’s, stroma)
- external i.e. a similar tumour known to express Ag of interest
Negative control
- primary Ab is replaced with non immune animal serum diluted to the same concentration as the primary Ab
Expected Sites of Reaction Product
Nuclear - ER, TTF-1, P63, Ki-67, S100, Calretinin
Membranous - EMA, HER2neu
Cytoplasmic - keratin markers
Stromal - amyloid
Rare cases where IHC reaction is unusual e.g. TTF-1 is cytoplasmic positivity in HCC
ID of Immunoreactive Cells
Immunoreactivity of tumour cells must be distinguished from reaction in normal entrapped cell
- e.g. macrophages and plasma cells are +ve with many ab’s
Intensity of the reaction
- is the reaction real or non specific?
Number of reacting cells
- can be an important criteria
- e.g. Ki-67 markers of cell proliferation
Criteria for a positive result
- depends on the lab but the more cells +ve usually easier for patient management and Dx
