HTL Tissue Processing & Fixation 1 — Questions and Answers
Question 1: Which fixative is considered the gold standard for routine histological tissue processing?
- Bouin's solution
- 10% neutral buffered formalin (Correct answer)
- Zenker's solution
- Carnoy's fixative
Correct answer: 10% neutral buffered formalin
10% neutral buffered formalin (NBF) is the standard fixative because it preserves tissue morphology, is compatible with most staining techniques, and is cost-effective.
Question 2: What is the primary chemical mechanism by which formalin fixes tissue?
- Protein denaturation by acid precipitation
- Cross-linking of proteins via methylene bridges (Correct answer)
- Coagulation of proteins by dehydration
- Lipid saponification
Correct answer: Cross-linking of proteins via methylene bridges
Formalin fixes tissue by forming methylene bridges (cross-links) between adjacent protein molecules, stabilizing cellular structures.
Question 3: The recommended fixation time for a core needle biopsy specimen before processing is:
- Less than 1 hour
- 6–72 hours (Correct answer)
- More than 96 hours
- Exactly 24 hours only
Correct answer: 6–72 hours
CAP/ASCO guidelines recommend 6–72 hours of formalin fixation for core needle biopsies to ensure adequate fixation without over-fixation artifacts.
Question 4: Which reagent is used in the clearing step of tissue processing, replacing the dehydrant before paraffin infiltration?
- Ethanol
- Acetone
- Xylene (Correct answer)
- Chloroform only
Correct answer: Xylene
Xylene (or xylene substitutes) is used as the clearing agent because it is miscible with both alcohol and paraffin, making tissue transparent and ready for wax infiltration.
Question 5: What is the purpose of the dehydration step in tissue processing?
- To remove formalin from the tissue
- To remove water so the clearing agent can penetrate (Correct answer)
- To harden the tissue before embedding
- To stain the tissue with eosin
Correct answer: To remove water so the clearing agent can penetrate
Dehydration removes water from the tissue using a graded series of alcohols, allowing the non-aqueous clearing agent to penetrate in the next step.
Question 6: Over-fixation in formalin most commonly results in:
- Poor nuclear staining and masked antigens (Correct answer)
- Autolysis of cytoplasmic components
- Excess basophilic staining
- Loss of lipid content only
Correct answer: Poor nuclear staining and masked antigens
Over-fixation creates excessive protein cross-linking that masks antigenic epitopes and reduces nuclear hematoxylin staining quality, affecting both morphology and IHC results.
Question 7: Which fixative is preferred for preserving glycogen in tissues for PAS staining?
- 10% NBF
- Zenker's solution
- Alcoholic formalin (Correct answer)
- Glutaraldehyde
Correct answer: Alcoholic formalin
Alcoholic formalin preserves glycogen better than aqueous formalin because the alcohol prevents glycogen diffusion and dissolution that can occur in aqueous fixatives.
HTL Tissue Processing & Fixation 1
Which fixative is considered the gold standard for routine histological tissue processing?