MLT GlobeHistopathology: Immunohistochemistry & molecular
28 Histopathology MCQs on Immunohistochemistry & molecular with answers and explanations.
In immunohistochemistry, what colour does DAB produce where the antigen is present?
Peroxidase oxidises DAB to an insoluble brown polymer; AEC or Fast Red give red products.
After DAB immunostaining, which counterstain is usually applied to show the nuclei?
A light haematoxylin stain gives blue nuclei that contrast with brown DAB without masking it.
When grossing tissue for IHC, which practice is correct?
Crushed, necrotic or calcified tissue gives false or uninterpretable staining; clean cuts from viable tumour are best.
Before IHC, paraffin sections are dewaxed by immersion in:
Xylene dissolves paraffin; alcohol then removes the xylene before rehydration and retrieval.
An IHC technologist wants to unmask formalin-hidden epitopes. Which procedure would NOT achieve antigen retrieval?
Antigen retrieval uses heat (HIER) or proteolytic enzymes (PIER) to reverse formaldehyde cross-links. Freezing a section does not break these methylene bridges.
In an alkaline phosphatase IHC detection system, alkaline phosphatase serves as the:
Alkaline phosphatase is the enzyme label that acts on a substrate–chromogen such as Fast Red or BCIP/NBT to give coloured product.
Which marker is expressed on essentially all mature T cells?
CD3 is part of the T-cell receptor complex and serves as the pan-T marker. CD20 marks B cells, CD15 granulocytes and Reed–Sternberg cells, and CD68 macrophages.
CD68 immunostaining is used to identify:
CD68 is a lysosomal glycoprotein abundant in macrophages. Plasma cells are shown with CD138 and T cells with CD3.
Which IHC result directly guides the choice of targeted therapy in breast carcinoma?
ER/PR and HER2 IHC predict response to hormonal and anti-HER2 therapy; cytokeratin only confirms epithelial origin.
Which treatment is routinely used to unmask formalin-masked antigens before IHC?
Heat-induced epitope retrieval (or protease digestion) reverses cross-link masking; mordants play no part in antigen retrieval.
A poorly differentiated tumour needs confirming as a carcinoma. Which IHC marker is most useful first?
Cytokeratins mark epithelial cells; desmin marks muscle, CD45 lymphoma and S-100 melanoma or nerve sheath tumours.
Why should prolonged fixation in formalin be avoided for tissue destined for IHC?
Over-fixation creates heavy methylene cross-linking that hides epitopes; guidelines give fixation windows (e.g. 6–72 h for breast).
How does formaldehyde fixation mask antigenic epitopes?
Formaldehyde reacts with protein groups, forming methylene cross-links that alter epitope shape; retrieval breaks these links.
Which heating device is widely used for heat-induced epitope retrieval?
HIER needs sections immersed in buffer near or above 95–100 °C; pressure cookers, microwaves, steamers or water baths achieve this.
DAB is favoured in IHC because its brown product is insoluble in alcohol and xylene. This allows:
Alcohol-stable DAB can be dehydrated, cleared and mounted in DPX; alcohol-soluble chromogens like AEC need aqueous mountants.
Why are slides often coated with APES (silane) before IHC?
Silane or charged coating bonds sections firmly so they do not float off during HIER and repeated buffer washes.
Streptavidin, used in many IHC detection systems, is obtained from:
Streptavidin is a biotin-binding protein produced by the bacterium Streptomyces avidinii; it has less non-specific binding than avidin, which comes from egg white.
In pressure-cooker heat-induced epitope retrieval, sections reach approximately what temperature?
At full pressure a domestic or laboratory pressure cooker reaches about 120 °C, higher than a water bath or steamer (95–100 °C), allowing shorter retrieval times.
Direct immunofluorescence on a fresh renal biopsy is used to demonstrate:
Fluorescein-labelled antibodies against immunoglobulins and complement localize deposits in glomerular disease. Amyloid and calcium are shown by other methods.
In IHC, nonspecific binding of the secondary antibody is reduced by incubating sections with:
Normal serum or protein block occupies nonspecific sites. Hydrogen peroxide blocks endogenous peroxidase, and proteolytic enzymes are used for antigen retrieval.