HTL Immunohistochemistry & Special Stains 3 — Questions and Answers
Question 1: Congo red staining of amyloid, when viewed under polarized light, produces which characteristic appearance?
- Blue birefringence
- Apple-green birefringence (Correct answer)
- Yellow-orange fluorescence
- Red dichroism without birefringence
Correct answer: Apple-green birefringence
Amyloid stained with Congo red exhibits apple-green birefringence under polarized light due to its beta-pleated sheet configuration.
Question 2: Which enzyme is used in the enzymatic antigen retrieval method to unmask antigens in formalin-fixed tissue?
- Amylase
- Protease K or pepsin (Correct answer)
- Lipase
- Hyaluronidase
Correct answer: Protease K or pepsin
Proteinase K or pepsin digest protein cross-links formed during formalin fixation to unmask antigenic sites.
Question 3: The Warthin-Starry stain is used to demonstrate which organisms in tissue?
- Mycobacteria
- Helicobacter pylori and spirochetes (Correct answer)
- Cryptococcus
- CMV inclusions
Correct answer: Helicobacter pylori and spirochetes
Warthin-Starry silver impregnation stains spirochetes (including Treponema pallidum) and Helicobacter pylori black against a yellow-brown background.
Question 4: In IHC, DAB (3,3'-diaminobenzidine) chromogen produces which color at the site of peroxidase activity?
- Blue
- Red
- Brown (Correct answer)
- Purple
Correct answer: Brown
DAB is oxidized by horseradish peroxidase to produce an insoluble brown precipitate at the antigen site.
Question 5: Which IHC panel best differentiates mesothelioma (CK5/6+, calretinin+) from lung adenocarcinoma (TTF-1+, napsin A+)?
- CD45, CD20, CD3, CD138
- CK5/6, calretinin, TTF-1, napsin A (Correct answer)
- ER, PR, HER2, Ki-67
- S100, SOX10, HMB-45, Melan-A
Correct answer: CK5/6, calretinin, TTF-1, napsin A
CK5/6 and calretinin support mesothelioma, while TTF-1 and napsin A support lung adenocarcinoma, making this the standard differentiating panel.
Question 6: Alcian blue at pH 2.5 stains which type of mucin?
- Neutral mucins only
- Sulfated and carboxylated acid mucins (Correct answer)
- Gastric surface mucins exclusively
- Lipid-rich mucins
Correct answer: Sulfated and carboxylated acid mucins
Alcian blue at pH 2.5 stains both sulfated and carboxylated (sialylated) acid mucins blue, while neutral mucins require PAS.
Question 7: Which fixative artifact can cause strong false-positive nuclear staining with proliferation markers like Ki-67?
- Mercuric chloride fixation
- Prolonged formalin fixation over 72 hours
- Cold ischemia time over 6 hours (Correct answer)
- Zenker's fixation
Correct answer: Cold ischemia time over 6 hours
Prolonged cold ischemia time before fixation can cause autolysis and non-specific antibody binding, leading to false-positive nuclear staining.
HTL Immunohistochemistry & Special Stains 3
Congo red staining of amyloid, when viewed under polarized light, produces which characteristic appearance?