Tissue Fixation and Decalcification Flashcards
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What is the primary purpose of decalcification in histological tissue preparation?
Answer: To remove calcium mineral deposits so calcified tissue can be sectioned
Decalcification removes calcium salts from hard tissues such as bone and teeth, making them soft enough to be sectioned on a standard microtome without damaging the blade.
Which decalcifying agent is most commonly used for routine bone processing in histology?
Answer: Hydrochloric acid (HCl)
Hydrochloric acid (5–10% HCl) is widely used for routine decalcification because it is effective, relatively fast, and provides acceptable tissue preservation compared to other strong acids.
What is the primary advantage of using EDTA for decalcification compared to acid methods?
Answer: It preserves tissue morphology and antigenicity better, enabling IHC and molecular studies
EDTA chelates calcium ions slowly and gently, preserving cellular morphology, antigenic epitopes for immunohistochemistry, and nucleic acids for molecular pathology testing.
Which endpoint test uses a chemical reaction to confirm that decalcification is complete?
Answer: The ammonium oxalate test, where no white precipitate in the used decal fluid confirms completion
A few drops of ammonium oxalate solution added to the spent decalcification fluid will form a white precipitate (calcium oxalate) if calcium ions remain; a clear solution indicates complete decalcification.
When is formic acid decalcification preferred over hydrochloric acid?
Answer: When nuclear morphology and DNA preservation for molecular studies are important
Formic acid (5–10%) is a weaker organic acid that decalcifies more slowly but causes less tissue damage, better preserving nuclear morphology and DNA integrity for molecular pathology.
What preparatory step should be performed on tissue BEFORE beginning acid decalcification?
Answer: Fix in 10% neutral buffered formalin before decalcification
Tissue must be adequately fixed in 10% NBF before acid decalcification to preserve the protein components of the tissue, since acid solutions alone do not adequately fix proteins.
Which decalcification agent is the fastest but causes the most tissue damage, reserved for cases where speed is critical?
Answer: Nitric acid (5–10%)
Nitric acid is the most rapid decalcifying agent but causes significant tissue damage including poor nuclear staining and reduced antigenicity, limiting its use to situations where speed outweighs quality concerns.