Lecture 6 - Immunocytochemistry in Diagnostic Pathology Flashcards
In diagnostic pathology, what does immunohistochemistry assess, and when is it typically requested in conjunction with H&E staining?
Immunohistochemistry assesses the presence of a cell marker on the cell membrane, cytoplasm, or nucleus. It is often requested as further work following H&E staining.
List three applications of immunocytochemistry in diagnostic pathology.
Defining cell types in tumour pathology, defining cell types in inflammatory cell infiltrates, evaluating prognostic indicators.
Define antigen and epitope in the context of immunocytochemistry.
Antigen is the protein that interacts with an antibody complex, and epitope indicates binding components involved in the interaction with the antibody.
How is formalin fixation addressed in tissue preparation for immunocytochemistry, and why is it necessary?
Formalin fixation involves covalent bonds, and antigen retrieval is performed to remove cross-linking. This is essential for antibody access and epitope unmasking.
What are the two main methods for antigen retrieval in immunocytochemistry, and describe the heat-induced epitope retrieval process.
The methods are heat-induced epitope retrieval (HIER) and proteolytic digestion. HIER involves removing formaldehyde through heat, with options like pressure cooker, microwave, or water bath.
What are the two categories of antibodies used in immunocytochemistry, and how do they differ in terms of specificity and production?
Polyclonal antibodies recognize multiple epitopes, are simpler and cheaper to produce, and tend to have more non-specific reactivity. Monoclonal antibodies recognize a single epitope, are more complicated and expensive, and have less non-specific reactivity.
Explain the basic principles of direct immunocytochemistry and the two common methods of visualization.
In direct ICC, the antibody binds directly to the target antigen, and visualization can be through immunofluorescent labeling or using a reporter enzyme like HRP or AP with a substrate.
What is the advantage of the two-step method in indirect immunocytochemistry, and how does it enhance sensitivity?
The two-step method involves using an enzyme conjugated to a secondary antibody, allowing more than one secondary antibody to bind to the primary antibody, enhancing sensitivity and amplifying the signal.
Describe the three-step method involving the PAP complex in immunocytochemistry.
The PAP complex is attached to the primary antibody via a linking antibody, and the complex and primary antibody must be from the same species. HRP substrate DAB is applied for visualization.
What is the significance of avidin-biotin methods in immunocytochemistry, and what is a potential issue that needs to be addressed?
Avidin-biotin methods offer increased sensitivity. However, endogenous biotin binding must be blocked to ensure low background signal.
How do polymer-based methods in immunocytochemistry work, and what are their advantages and limitations?
Polymer-based methods use a polymer backbone to which multiple antibodies and enzymes can be conjugated. They are quick, reduce background noise, but the size of the polymer can affect penetration, and they can be expensive.
Why is quality control essential in immunocytochemistry, and what are the types of controls used?
Quality control is crucial to ensure validity. Reagent controls include positive/negative controls, and tissue controls involve using normal tissue for positive/negative/internal control.
How are HER2 staining controls utilized in immunocytochemistry, and why are they important for quantitative assessment?
HER2 staining controls from cell lines provide robust quantitative assessment of HER2, aiding in quality control and accuracy.
What is the significance of the Cluster of Differentiation (CD) classification system in immunocytochemistry?
The CD classification system allows cells to be defined based on their surface antigens. It includes over 350 CD numbers used in immunocytochemistry for cell typing.
In diagnostic practice, what antibody panel is commonly used in immunocytochemistry to identify the tumor type for a cancer of unknown origin?
A panel of four antibodies is used, including broad-spectrum cytokeratin, vimentin, S100, and CD45/LCA.
