Week 2 Flashcards
What happens when there is Calcium in tissues bone, kidney stone or calcified blood vessels
-calcified tissues contain Calcium salts called hydroxyapatite crystals which makes the tissues hard and brittle
-this makes the tissues hard to cut and can damage the microtome blade
-need to be decalcified
what is decalcification, what determines the type of method to use and how long does it take
- removing calcium from calcified tissues for paraffin infiltration
-Uses acid methods, ion exchange resins, electrolyte method and chelating agents
-bone size determines if you need a strong or weak acid
-the method you use to decalcify determines the time
-over decalcification causes loss of nuclear morphology
-under decalcification will make sectioning harder
What is the acid method of decalcification and how do they work
Strong acids
- if you use very strong acids for an extended time on tissue, you wont be able to stain it properly.
-no nuclear staining
-calcium salts are dissolved at pH 4.5 , decal agents are usually 0.5 - 3.5 but are buffered before use
-Use 5-10% HCl, Nitric acids, formic acid (slower acting) or a mix of formic and formaldehyde (to fix and decalcify together)
-when exposed to these acids Ca ions will come out of the tissues and into the solution until it is saturated with Ca ion - making a barrier
-the solution must be changed frequently and agitated
-heat must not be used as it will cause swelling and maceration of tissues
what is the ion exchange resin method of decalcification
-using formic acid over ammoniacal salt of sulfonated resins
-the ammonium ions from the resin will exchange for calcium ions therefore the solution will remain free of calcium ions making decalcification faster and you wont have to change solutions so much
-this type of staining will not harm the tissue, one of the best methods
What is the electrolytic method of decalcification
-Using a electroplating device place in formic acid and hydrochloric acid
-the anode (+ charged) has bone attached to it and passes a current through the solution making calcium ions attracted to the cathode (- charge)
-bones decalcify in 2-6 hours
-generates heat may cause loss of tissue morphology
-acid solutions are neutralized with 1% sodium bicarbonate before disposal
What are chelating agents
- organic compounds that bind to certain metals
-only EDTA can bind to ionized calcium
-slow but do not damage tissue
how is the end point of decalcification determined using what three methods
Physical /mechanical method - test how flexible the specimen is, probe the sample with needle or pin - can create artifact
Radiography - most accurate when trying to determine when complete demineralization has been completed (zero opaqueness (no white) = complete decalcification)
-metallic based fixatives should not be used
Chemical testing -depends on precipitation of calcium of oxalate from tissues
- addition of ammonium hydroxide, if turbidity is seen due to calcium oxalate then solution can be changed if not then add ammonium oxalate, sit for 30 mins and observe for turbidity if you see then change solution if not then decalcification is complete
what is surface decalcification
-if there is micro calcification it can cause nicks on the blade so you need to expose the tissue block to decal fluid, rinse and section gently
What is cryotomy - frozen sectioning
-method of processing what is it used for
-Must wear full PPE-handling of fresh tissue specimen- N95
-used when you need to do rapid testing (surgery) or when staining will not work on tissue that was processing routinely
-used for demonstration of fat ; because all fixative dissolve fat from tissue except osmium tetroxide
-used for enzyme and direct immunohistochemical techniques since fixation and heat inactivates most enzymes and AG
-therefore used for immunofluorescence and lymphocyte surface marker studies
what do frozen sections use and what are the associated hazards:
Cyrostat
-microtome in refrigerated cabinet at -20
-slow freezing artifacts can be formed because freezing in slow and ice crystals can form in tissues . When the tissue thaws those crystals can make holes . A heat extractor and flattening surface is used to reduce this
-frozen sections are tissues embedded in cyrotechnique medium and frozen in isopentane (-150)
-Hazards would be:
flammable liquid
extreme cold temp
cutting unfixed tissues
what are some artifacts, faults, with decalcification
-bone dust
-under decalcification
-over decalcification
what is bone dust artifact and how can it be prevented
created when duct pressed on the bone surface
can be prevented by :
-using diamond blade saw
-trimming bone surface after decalcification but before tissue processing
what is under decalcification and how can it be prevented
-can occur due to rapid turn around time leading to grossly decalcified tissue
prevented by:
choosing a decal reagent with short turn around time
-proper decal protocol
-ensuring endpoint with radiography
What is over decalcification and how can it be prevented
- when the decalcification endpoint is missed
can be prevented by
-choosing better decal with appropriate procedure
-ensuring endpoint with radiography
What are the artifacts associated with frozen sectionining
-freezing artifacts
-block loosens from chuck while sectioning
-tissue not embedded flat on chuck
-tissue curling or rolling at blade edge
