Processing and Embedding Flashcards
steps of tissue processing
dehydration, clearing, infiltration
dehydration step in tissue processing
series of alcohol in increasing concentrations to remove water to avoid excessive distortion of the tissue
most commonly used dehydrating reagent
ethanol
dehydrating reagent used to fix blood smears
methanol
dehydrating reagent that causes excessive shrinkage
acetone
dehydrating reagent that does not harden or shrink tissue as fast as ethanol but cannot be used in staining as it dissolves eosin
isopropyl alcohol
displaces ethanol in tissue
clearing
intermediate solvent that is miscible with ethanol and paraffin wax
clearing reagent
most common clearing reagent
xylene
helps remove fat from tissue which may act as a barrier to wax infiltration
clearing
process in which tissue is infiltrated with a supporting medium
infiltration
infiltrative medium
paraffin wax
processing schedule is dependent on these 5 things
-size of specimen
-volume of lab
-type of processor
-time constraints
-reagent choice
microwave processing can _______ fixation prior to tissue processing
speed up
sources of poor processing (4)
-poor nuclear staining causing cloudy and washed out appearance of tissue
-water in tissue before clearing
-ethanol condensation
-mechanical problem
this occurs between fixation and processing to remove calcium salts
decalcification
criteria for a good decalcifying agent (4)
-takes the least amount of time
-little or no damage to the cells by acid
-little or no disruption to cell morphology
-does not alter staining properties
slow acting decalcifying agent that is gentler in action and less likely to interfere with nuclear staining; can be used alone and good for large tissues
formic acid
rapid decalcifying agent that can cause cell shrinkage so you shouldn’t expose tissue to it too long; good for small samples; need to wash formalin before placing in this agent to avoid formation of bischloromethyl ether
hydrochloric acid
rapid decalcifying agent but if end-point is exceeded, it will impair staining
nitric
slowly binds and removes calcium ions from bone
chelating agents
common chelating agent
EDTA
gentle to tissue producing minimal artifacts and good staining results
chelating agents
chelating agents are not appropriate for this size specimens
large or urgent speciments
good when molecular elements need to be preserved for techniques such as IHC, FISH, or PCR (bone marrow biopsies)
chelating agents
determining the end point of decal (3)
chemical, radiographic, physical
relies upon detection of calcium in the decal solution
chemical method
equal parts of ammonium oxalate and ammonium hydroxide and the decal solution
how to use the chemical method to determine when decal is done
decalcification is complete if test aliquot is
colorless or clear
decalcification is incomplete if the test aliquot is
cloudy or turbid
why does undecalcified/overdecalcification occur (3)
-TAT
-poor end point determination
-wrong decal
the longer a bone marrow biopsy is in decal, it can start to remove this
iron
bone marrow biopsies should be in decal no longer than _______
45 min to an hour
the end point of a bone marrow biopsy decalcification is
hard to determine
blocking of tissue section which involves enclosing the tissue in infiltration medium
embedding
multiple tissue pieces are aligned this way
across the long axis of the mold
tissues with an epithelial surface are embedded to provide sections in a plane _______ to the surface
right angle/perpendicular
