Immunohistochemistry principles

7 DOH Abu Dhabi (HAAD) lab licensing practice questions on Immunohistochemistry principles, part of Histopathology and Cytology. The first 7 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

1. Question 1 · Hard

   A manual cytokeratin (AE1/AE3) stain is done on a sentinel lymph node section using heat-induced epitope retrieval, an HRP polymer detection system and DAB. Each section is circled with a hydrophobic pen and covered with 100 µL of reagent, and the slides lie flat on an open tray near an air-conditioning vent for the 60-minute primary incubation. Results: the patient section shows a band of intense brown along every tissue edge, with the expected pattern elsewhere; the negative reagent control, with antibody diluent in place of the primary, shows the same brown rim; the tonsil positive control shows correct epithelial staining plus the same rim. What is the most likely cause?

   1. ASections drying at the edges during incubation
   2. BEndogenous peroxidase not blocked before staining
   3. CHeat retrieval that was too long or too hot
   4. DPrimary antibody used at too high a concentration

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   Answer: A · Sections drying at the edges during incubation

   The brown rim also appears on the negative reagent control, which received no primary antibody, so the cause lies outside the primary antibody step. Staining confined to tissue edges on every slide is the typical pattern of drying: a small volume on an open tray in moving air evaporates first at the margins, where reagents concentrate and bind nonspecifically, and DAB then deposits there. Incubating in a closed humid chamber, with enough reagent to cover the whole section and no drying between steps, prevents it. Unblocked endogenous peroxidase marks red cells and granulocytes throughout the section, and over-retrieval tends to cause diffuse background or damaged tissue rather than an edge band. The run should be repeated on fresh sections before any result is reported.

   • B: Endogenous peroxidase stains red cells and granulocytes throughout the section, not a rim at the edges.
   • C: Over-retrieval tends to cause diffuse background or damaged sections, not an edge band.
   • D: An over-concentrated primary could not stain the negative control, which had no primary antibody.

2. Question 2 · Easy

   How does heat-induced epitope retrieval improve immunostaining of formalin-fixed tissue?

   1. ARaising the affinity of the primary antibody
   2. BReversing formalin cross-links that hide epitopes
   3. CRemoving the last traces of paraffin wax
   4. DBlocking endogenous peroxidase activity in red cells

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   Answer: B · Reversing formalin cross-links that hide epitopes

   Formaldehyde forms methylene bridges between proteins, and these can hide or change the shape of the epitope the antibody recognizes. Heating sections in a buffer such as citrate at pH 6 or Tris-EDTA at pH 9 reverses much of this masking. Time, temperature and buffer pH are set for each antibody during validation. Too much retrieval can damage morphology or lift sections off the slide.

   • A: Retrieval changes the tissue, not the antibody's affinity.
   • C: Paraffin is removed with xylene or a substitute before retrieval.
   • D: Peroxidase is blocked with hydrogen peroxide, a separate step.

3. Question 3 · Medium

   Before the primary antibody in a peroxidase-based method, sections are treated with 3% hydrogen peroxide. This step prevents:

   1. AFalse staining from the tissue's own peroxidase
   2. BLoss of the antigen during heat-induced retrieval
   3. CNonspecific binding of antibody to Fc receptors
   4. DBackground staining from endogenous tissue biotin

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   Answer: A · False staining from the tissue's own peroxidase

   Red cells, neutrophils and eosinophils have peroxidase activity that would turn DAB brown wherever they lie. Excess hydrogen peroxide inactivates this endogenous enzyme before the labeled reagents are added. Methanol is sometimes used as the solvent, but it can damage some epitopes. Endogenous biotin and Fc binding are handled by separate blocking steps.

   • B: Peroxide does not protect antigens during retrieval.
   • C: Fc and protein binding are reduced with a protein or serum block.
   • D: Endogenous biotin needs an avidin-biotin block or a biotin-free polymer system.

4. Question 4 · Hard

   An estrogen receptor (ER) stain on a woman's breast carcinoma gives these results. Tumor cells: no staining. External positive control (tissue with known moderate ER expression): no staining. Normal ducts in the patient's section: no staining. Negative reagent control: clean. What should be done?

   1. ARepeat only the negative reagent control
   2. BRepeat the stain, because the run failed
   3. CReport ER as equivocal and add a comment
   4. DReport the tumor as ER negative

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   Answer: B · Repeat the stain, because the run failed

   Both the external positive control and the internal control (normal ducts, which normally contain some ER-positive cells) failed, so the run did not detect antigen known to be present. A negative tumor result cannot be reported from a run with failed positive controls. The stain should be repeated after checking the reagents, retrieval and instrument steps. A false-negative ER result could deny the patient endocrine therapy.

   • A: The negative reagent control was clean; the problem is the missing positive staining.
   • C: Failed controls make the result invalid, not equivocal.
   • D: A negative result is valid only when the positive controls stain as expected.

5. Question 5 · Hard

   A liver biopsy from a 50-year-old with chronic hepatitis B is immunostained with an avidin-biotin complex (ABC) method after heat retrieval. Fine granular brown staining fills the hepatocyte cytoplasm, and the negative reagent control (no primary antibody) shows the same. The most likely cause is:

   1. AOver-retrieval damaging the target epitope
   2. BCross-reaction of the primary antibody with liver proteins
   3. CIncomplete removal of the paraffin wax
   4. DEndogenous biotin detected by the avidin complex

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   Answer: D · Endogenous biotin detected by the avidin complex

   Liver, kidney and some other tissues are rich in biotin, and heat retrieval exposes more of it. Avidin in the ABC reagent binds this endogenous biotin directly, so staining appears even without the primary antibody. An avidin-biotin blocking step, or a biotin-free polymer detection system, removes the problem. Staining in the negative reagent control rules out the primary antibody as the cause.

   • A: Over-retrieval tends to weaken specific staining, not add granular background.
   • B: The primary antibody was left out of the control, yet it stained the same.
   • C: Residual wax gives patchy weak or absent staining, not granular brown deposits.

6. Question 6 · Medium

   Which control shows that brown staining comes from the primary antibody rather than from the detection system?

   1. AA tissue known to express the antigen, run in parallel
   2. BA second patient section stained with H&E
   3. CNormal cells within the patient's own section
   4. DA section run with diluent in place of the primary

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7. Question 7 · Easy

   Which chromogen gives a brown, alcohol-insoluble product with horseradish peroxidase?

   1. ANBT with BCIP substrate
   2. BDAB (3,3'-diaminobenzidine)
   3. CFast Red with naphthol phosphate
   4. DAEC (3-amino-9-ethylcarbazole)

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