Immunohistochemical Staining (IHC) Flashcards
What does immunohistochemistry (IHC) identify?
Biomarkers that guide treatment decisions
What are normal results in immunohistochemistry?
Cells appear normal with high levels of maturity and are appropriate for the tissue site
What are abnormal results in immunohistochemistry?
Cells appear immature or poorly differentiated, or are found in an inappropriate tissue for their cell type
What is a major role of an immunohistochemist?
Troubleshooting for variables introduced by fixation, processing, and tissue types
What is an epitope?
The specific site on an antigen where an antibody attaches
What is a polyclonal antibody?
A mixture of antibodies from many clones of lymphocytes
What is a limitation of polyclonal antibodies?
They are not as selective and can result in non-specific (background) staining
What is a monoclonal antibody?
A homogenous population of immunoglobulin directed against a single epitope
What are the advantages of monoclonal antibodies?
- High homogeneity
- Absence of nonspecific antibodies
- No batch-to-batch variability
- Unlimited supply
How are antibodies used in histology on a tissue section?
Antibodies against characteristic antigens are added to the sample, binding wherever the antigens are present
What is the purpose of fixation in immunohistochemistry?
To preserve antigenic reactivity in the tissue
What is the recommended method for antigen recovery?
Heat-induced epitope retrieval (HIER)
What factors govern the effectiveness of antigen retrieval?
- pH
- Volume of fluid
- Heating time and temperature
What are the common retrieval solutions used?
- Sodium citrate buffer (0.01M, pH 6.0)
- EDTA (1mM, pH 8.0)
What is the effect of overfixation by formaldehyde?
It can result in the antibody not having access to its epitope, leading to false negative results
What is the ideal thickness for tissue sections in IHC?
3-4 microns
What is the result of improper positioning of tissue sections on slides?
Areas of incomplete coverage or drying may cause false negative or nonspecific results
What is the purpose of automation in immunohistochemistry?
To perform steps of heating, deparaffinization, and antigen retrieval, reducing human error and ensuring consistent temperature and exposure time.
What does HIER stand for in immunohistochemistry?
Heat-Induced Epitope Retrieval.
What factor is important for nuclear and cell surface antigens during antigen retrieval?
pH.
Name three methods of heating used in immunohistochemistry.
- Lab microwave
- Modified pressure cooker
- Circulating water bath.
What does EIER stand for in immunohistochemistry?
Enzyme-Induced Epitope Retrieval.
What is a proteolytic enzyme used for in EIER?
To expose epitope sites.
List three enzymes that can be used in EIER.
- Pronase
- Trypsin
- Pepsin.
Fill in the blank: The enzymatic activity is stopped by placing the specimen in cold buffer at _______ prior to processing with antibody.
4°C.
What are some disadvantages of the EIER method?
- May increase non-specific staining if not properly used
- May weaken specific staining causing false negatives
- May cause fragmentation or loss of tissue sections.
What is the benefit of combining HIER and EIER?
To enhance the quality of morphology and minimize enzymatic pretreatment.
What is a fluorochrome?
A dye that absorbs light and emits its own light at a longer wavelength.
What is the principle of immunofluorescence?
Fluorochrome attached to an antibody allows visualization of the reaction sites between antigen and labeled antibody.
What are the most common fluorochromes used in immunofluorescence?
- Fluorescein isothiocyanate (FITC)
- Rhodamine.
True or False: Enzyme IHC is recommended for tumor differentiation.
True.
What is the role of chromogen in enzyme IHC?
It provides the indicator system to visualize the location of the antibody.
List two commonly used enzymes in enzyme IHC.
- Horseradish peroxidase (HRP)
- Alkaline phosphatase (AP).
What can happen if hematoxylin containing alcohol is used with AEC or AP chromogens?
The reaction product will be dissolved and removed, leading to false negative results.
Fill in the blank: The direct method of immunohistochemistry uses a _______ antibody to identify antigens.
labeled.
What is the advantage of the indirect method over the direct method?
More sensitive due to signal amplification and more versatile.
What is the purpose of the three-step indirect method?
To further amplify the signal for staining antigens with a limited number of epitopes.
What does PAP stand for in immunohistochemistry?
Peroxidase-Anti-Peroxidase.
What fixative is used in the basic PAP immunoperoxidase procedure?
10% NBF with epitope enhancement/retrieval.
What steps are involved in the basic PAP immunoperoxidase procedure?
- Deparaffinize
- Dehydrate
- Block endogenous reaction
- Apply primary antibody.
What is the purpose of the blocking serum in the Basic PAP procedure?
To prevent non-specific binding of antibodies
Blocking serum is crucial for reducing background staining.
What is the chromogen used in the Basic PAP procedure?
AEC
What color indicates a positive reaction in the Basic PAP procedure?
Brick red
True or False: If DAB is used as chromogen instead of AEC, a synthetic resin may not be used.
False
What are the two techniques used in the Avidin-Biotin method?
- Avidin-biotin complex method (ABC)
- Labeled avidin-biotin method (LAB)
What is the purpose of pre-treating tissue sections with H2O2 in the Avidin-Biotin method?
To remove any endogenous peroxidase enzyme activity
What does DAB reagent produce when it reacts with peroxidase?
An insoluble dark brown precipitate
True or False: Positive controls can be commercially prepared slides.
False
What should be used to block nonspecific background staining?
Non-immune serum from the same species as the primary antibody
What are the two main blocking reactions in most immunoperoxidase methods?
- H2O2 in absolute methanol
- Human serum from the same species
What is the best practice for daily quality control (QC) in staining?
Review and document results of antibody staining for each day
What may cause false positive results due to background staining?
Delayed fixation or inadequate fixation
What is the purpose of using a secondary or linking antibody in the Avidin-Biotin method?
To bind to the primary antibody and also to bind ABC
What type of antibody has a shorter shelf life and cannot be frozen?
Prediluted antibody
What is the ideal storage temperature for most antibodies?
4°C to 8°C
What should be done if antibodies are stored at -20°C to -70°C?
Avoid repeat freeze/thaw cycles
What is a common cause of overstaining?
Primary and/or secondary antibody concentration too high
This can lead to excessive background and false positives.
What is the recommended solution for high antibody concentration?
Reduce antibody concentration. Titrate antibodies for optimal dilution
Titration helps find the best dilution for accurate results.
What should be done if incubation temperature is too high?
Reduce temperature
High temperatures can denature antibodies and affect binding.
If sections have dried out, what is the solution?
Avoid drying out of tissues
Dried sections can lead to compromised staining.
What is a potential cause of high background in staining?
Sections inadequately washed at least 3X between steps
Insufficient washing can retain excess reagents.
What can be done if tissue contains endogenous enzymes like peroxidase?
Block endogenous enzymes prior to incubation of primary antibodies
This prevents false positives due to enzyme activity in the tissue.
What is the solution if tissue contains endogenous biotin activity?
Block endogenous biotin with avidin/biotin blocking reagent prior to incubating primary antibodies
This prevents non-specific binding of antibodies.
What technique can reduce non-specific binding of primary antibodies?
Use a higher dilution of primary antibodies
Higher dilutions can minimize background staining.
What is a recommended action if secondary antibodies are not stored properly?
Store secondary antibodies in the dark
Light exposure can degrade antibody effectiveness.
What might cause insufficient staining in control and specimen?
Improper epitope retrieval
Epitope retrieval is critical for antibody recognition.
What is a common issue leading to high background staining?
Absent or insufficient blocking of non-specific binding
Blocking agents like non-immune serum are essential.
What are some methods used in IHC?
Polyclonal and monoclonal antibodies, immunofluorescence, enzyme IHC
Various techniques enhance the visualization of antigens.
What does CD 3 identify?
T-Cell lymphoma
This antibody is specific for T-cells in lymphomas.
What does CD 20 identify?
B-Cell lymphoma
CD 20 is a marker for B-cells and is used in lymphoma diagnosis.
