HTL Immunohistochemistry & Special Stains 2 — Questions and Answers
Question 1: Which antigen retrieval method uses a microwave or pressure cooker with citrate buffer to unmask epitopes?
- Enzymatic digestion
- Heat-induced epitope retrieval (HIER) (Correct answer)
- Cold acetone fixation
- Trypsin pretreatment
Correct answer: Heat-induced epitope retrieval (HIER)
HIER uses heat applied in citrate buffer (pH 6) or EDTA buffer (pH 9) to reverse formalin cross-links and unmask epitopes.
Question 2: In the ABC (avidin-biotin complex) IHC detection method, which molecule bridges the primary antibody signal to the enzyme?
- Streptavidin directly conjugated to enzyme
- Biotinylated secondary antibody plus preformed avidin-biotin-enzyme complex (Correct answer)
- Protein A linked to peroxidase
- Alkaline phosphatase conjugated primary antibody
Correct answer: Biotinylated secondary antibody plus preformed avidin-biotin-enzyme complex
The ABC method uses a biotinylated secondary antibody that binds the preformed avidin-biotin-enzyme complex, amplifying signal.
Question 3: A Masson's trichrome stain produces which color combination for collagen versus muscle?
- Collagen red, muscle blue
- Collagen blue/green, muscle red (Correct answer)
- Collagen yellow, muscle purple
- Collagen orange, muscle green
Correct answer: Collagen blue/green, muscle red
Masson's trichrome stains collagen blue or green and muscle (cytoplasm) red, with nuclei staining dark.
Question 4: Which special stain is used specifically to demonstrate fungi, including Pneumocystis jirovecii, in tissue sections?
- Ziehl-Neelsen
- Grocott methenamine silver (GMS) (Correct answer)
- Giemsa
- Gram stain
Correct answer: Grocott methenamine silver (GMS)
GMS (Gomori methenamine silver) stains fungal cell walls black by reducing silver ions, and is the preferred stain for Pneumocystis.
Question 5: Endogenous biotin activity is a common source of background in IHC. Which tissue type is most problematic?
- Skeletal muscle
- Kidney and liver (Correct answer)
- Spleen and lymph node
- Smooth muscle
Correct answer: Kidney and liver
Liver and kidney contain high levels of endogenous biotin, causing non-specific background in biotin-based detection systems.
Question 6: The Periodic Acid-Schiff (PAS) stain highlights which tissue components?
- Lipids and myelin
- Glycogen, mucins, and basement membranes (Correct answer)
- Amyloid and fibrin
- Nucleic acids and ribosomes
Correct answer: Glycogen, mucins, and basement membranes
PAS oxidizes vicinal diols in polysaccharides to aldehydes, which react with Schiff reagent to produce a magenta color in glycogen, mucins, and basement membranes.
Question 7: Which IHC marker is characteristically positive in Merkel cell carcinoma and has a distinctive paranuclear dot pattern?
- S100 protein
- CK7
- CK20 (Correct answer)
- Synaptophysin
Correct answer: CK20
CK20 shows a paranuclear dot pattern in Merkel cell carcinoma, distinguishing it from small cell lung carcinoma (CK20-negative).
HTL Immunohistochemistry & Special Stains 2
Which antigen retrieval method uses a microwave or pressure cooker with citrate buffer to unmask epitopes?