ASCP Hematoxylin and Eosin Staining 2 — Questions and Answers
Question 1: What does 'over-differentiation' of hematoxylin produce in an H&E section?
- Deep blue cytoplasm
- Pale or unstained nuclei (Correct answer)
- Excessive eosin uptake
- Green nuclear staining
Correct answer: Pale or unstained nuclei
Over-differentiation removes too much hematoxylin, resulting in pale or ghost nuclei that are difficult to visualize.
Question 2: What type of hematoxylin is most commonly used in routine histology laboratories in the US?
- Delafield's hematoxylin
- Harris hematoxylin (Correct answer)
- Mayer's hematoxylin
- Weigert's hematoxylin
Correct answer: Harris hematoxylin
Harris hematoxylin is the most widely used progressive/regressive hematoxylin in US histology labs for routine H&E staining.
Question 3: Which method of hematoxylin staining requires a differentiation step?
- Progressive staining
- Regressive staining (Correct answer)
- Vital staining
- Metachromatic staining
Correct answer: Regressive staining
Regressive staining over-stains tissue and then differentiates to remove excess dye from non-target sites, requiring a controlled acid-alcohol step.
Question 4: What is a common cause of eosin staining appearing too pale in H&E sections?
- Over-fixation in formalin
- Insufficient eosin concentration or exhausted eosin solution (Correct answer)
- Excessive differentiation with acid-alcohol
- Over-dehydration during processing
Correct answer: Insufficient eosin concentration or exhausted eosin solution
Diluted or exhausted eosin solution lacks sufficient dye concentration to adequately stain cytoplasmic proteins.
Question 5: Which reagent is used to mount coverslips on H&E sections stained using an alcohol-based final dehydration?
- Aqueous mounting medium
- Xylene-based mounting medium (e.g., Permount) (Correct answer)
- Canada balsam only
- Glycerin jelly
Correct answer: Xylene-based mounting medium (e.g., Permount)
Xylene-based mounting media like Permount are miscible with the dehydrated, xylene-cleared sections and provide permanent, clear mounts.
Question 6: What staining result indicates that sections were not completely deparaffinized before H&E staining?
- Overly pale nuclei
- Unstained or poorly stained areas with paraffin residue (Correct answer)
- Excessive eosin background
- Green cytoplasm
Correct answer: Unstained or poorly stained areas with paraffin residue
Residual paraffin acts as a barrier to aqueous dyes, producing unstained patches or areas with poor dye penetration.
ASCP Hematoxylin and Eosin Staining 2
What does 'over-differentiation' of hematoxylin produce in an H&E section?