decalcification Flashcards
decalcification
removing calcium from tissue
calcium in a tissue block causes tears during cutting
when is decalcification done
after fixation before processing
alternatives to decalcification
GMA or resin embedding
grinding of bone with waterproof sandpaper
small cone samples may sometimes be cut through frozen section
special blades or adhesive tape methods may be helpful
two basic choices
acid methods
chelation
decal fluid used is determined by
urgency
degree of mineralization
extent of investigation
staining techniques required
acids affect on tissue
any acid will affect tissue staining
stringer acid or the longer the acid is used the more damage is done to the tissue
over decalcification
lack of nuclear staining *
nuclear staining is the main thing we want to access
acid methods
dissolve and then ionize calcium salts
calcium salts dissolve at a pH of 0.5- 3.0
if buffered to a pH 4.5 some enzymes can be demonstrated
hydrochloric & Nitric acids
strong acids
they are fast & must be closely monitored
- nitric acid causes serious staining problems after 48hrs
- formalin fixed tissues must be washed before & after decal
- avoid reaction of HCL with formalin bc it releases bis-chlormrthyl ether ( KNOWN CARCINOGEN) *
formic acid
weak acid
slow
rarely hampers staining
- aqueous 5-10%
- buffered ( sodium formate or sodium citrate)
- mixed with formalin
said to simultaneously fix & decal but pre-fix is better
other weal organic acids
picric acid & acetic acid
used as part of carnoys & bouins
they act as incidental agents
when to use which acid
use strong acid for urgent cases
use weak acids for routine
ideally bone should be removed just as all the calcium is removed - no longer
simple decalcification
allow calcium to migrate out of tissue and into surrounding solution
solution around the bone becomes saturated with calcium slowing the process:
- agitate
- change solutions
- suspend bone in biopsy bag
vacuum may be used at beginning of decal to aid in infiltration
specimen should be suspended in glaze tallow all surface to be exposed
what happens if you apply heat to speed up decal
NEVER apply heat
specimen swells, becomes digested & damaged
proprietary decalcifiers
trade secrets
rapid is usually HCL
slow is usually formic
advantages is the avoidance of concentrated acid handling
what is considered the best method for decalcification
ion-exchange resin *****
formic acid over a layer of ammoniated salt of suffocated resin
calcium is exchanged fro ammonium
- speeds reaction
- time not so critical
- less changing of solutions
electrolytic method
formic acid & HCL
NOT recommended - heat & tissue damage
Ca attracted to cathothe
fast ( 2-6 hrs) *
chelating agents
ethylene- diaminetetracidic acid (EDTA)
will not bind calcium below pH 3
best results the acid side of pH 5.0 -7.2
slow but far less damaging than acid ( 4-6 weeks )
washing
generally wise to do so before decal
phosphates from NBF will oppose action of acid
dichromate is reduced by formic acid
mercury or zinc will chelate with EDTA
wash after HCL decal to avoid release of carcinogen
factors for decal
concentration - increase rate with increase concentration
temperature
microwave to 55 degrees 10x increase in speed
age of patient - mature bones decalcify faster than immature bones
type of bone - compact bones decal slower than spongy bones
size of specimen
agitation - gentle agitation facilitates fluid exchange
suspension - all surfaces make contact with fluid
volume - 20x bone volume
change fluid daily - to prevent saturation of fluid with calcium
end point of decal
most critical step in process
bone should stay in decal fluid no longer than necessary to avoid damage
frequently ignored in labs :(
three methods of endpoint decal
mechanical - physical
chemical
radiographic
chemical
precipitation of calcium oxalate
take 5mL of decal fluid
use litmus paper , add concentration ammonium hydroxide* until solution is neutral pH
add 5 m of ammonium oxalate*, mix
let stand for 30min, observe for persistant turbidity
change solution after testing
endpoint testing
HCL, formic or Nitric acid
- test daily
- if incomplete replace
- near endpoint test every 5 hrs
- last anticipated change use 5x bone volume for more accurate test
minimal calcification or Jamshidi biopsy -acid decalcifier
- test once
- jamshidi is a tubular needle with cine shaped tip use to obtain bone marrow core
EDTA
- test weekly
after decal
wash with water, saturated lithium carbonate, 5-10% sodium bicarbonate or weak ammonia
to neutralize acid & stop decal
then rinse with water
surface decal
if decal is incomplete or mineral deposits
- place exposed block surface in 1% HCL or 10% formic acid or commercial prep
for 15-30 min
rinse & resection ( limited penetration repeat as necessary )
when using acids
aprons gloves eye protection acid carrier fume hood always add Acid to Water
bone dust
may be pressed on surface of tissue by bone saw
- use a saw with a diamond blade
- trim surface after decal but before processing
under decal
choose a rapid decal
develop a good endpoint testing
check endpoint with radiography when in doubt
over decal
choose a decal that fits needs
develop good endpoint method
check with radiography
what solution will appear white if decal isn’t complete
ammonium oxalate mixed in with decal solution (usually forming acid)
