DECALCIFICATION Flashcards
Injurious to the organic ground substance of the bone or other tissues
Acid solutions
How often are acid solutions changed
-once or twice until decalcifiyis completed
How often are acid solutions changed
-once or twice until decalcifiyis completed
Reagent volume and tissue ratio for decalcification
20:1
Ideal decalcification time
24-48 hrs
Decalcification time for dense bones
14 days or longer
Optimum temp for decalcification
18-30C
Examples of agitation
- low speed rotation
- stirring
- introduction of bubbles
Temp in which tissues will undergo complete digestion
55C
temp in which it impairs nuclear staining with van gieson’s
37C
temp in which it reduced effectiveness of trichrome and PAS
37C
percentage of preparing nitric acid
5-10%
most common and fastest decalcifying agent
nitric acid
components of nitric acid
formaldehyde or alcohol and chromic acid
causes yellow spontaneous yellow discoloration (HNO2)
nitric acid
remedy of yellow discoloration
-neutralize with 5% NaSO4 > wash for 12 hrs
- add 0.1% urea
decalcifying and water softener
EDTA
decalcifying and softener
perenyi’s
removal of calcium ions from a bone or calcified tissue through histological processes that makes them flexible and easier to cut
decalcification
3 main types of decalcifying agents
- strong mineral acids
- weaker organic acids
- chelating agents
10% aqueous nitric acid solution formula
- conc. HNO3
- dH2O
decalcification time of aq HNO3 sltn 10%
12-24 hrs
formol HNO3 formula
-concd. HNO3
- strong formaldehyde 40%
- dH2O
decalcification time of formol-HNO3
1-3 days
perenyi’s fluid formula
-HNO3
- chromic acid
- absolute ethyl alcohol
decal. time of perenyi’s
2-7 days
phloroglucin-HNO3
- conc. HNO3
- Phloroglucin
Nitric acid 10%
decal time of phloroglucin
12-24 grs
most rapid decalcifying agent so far, recommended for urgent cases
Phlorogucin _ HNO3
inferior compared to HNO3 in its role as a decal agent because of its slower action and greater distortion of tissue produced on the decalcified section
HCl
solution recommended for surface decalcification of tissue blocks
1% HCl, 70% alcohol
moderation of HCl recommended for teeth and small pieces of bones
von ebner’s
fixative and decalcifying agent for post mortem research tissues
Formic acid
for small and large pieces of bones and gentler on tissues
formic acid
decalcification time for formic acid
2-7 days
recommended for autopsy materials, bone marrow, cartilage and tissues studied for research purposes
Formic Acid - sodium citrate
decalcification time for Formic acid - sodium citrate
3-14 days
T/F FA-Sc requires neutralization with 5% sodium sulfate
T
decal time for TCA
4-8 days
does not require washing out and is a weak decalcifying agent, not used for dense tissues, and is suitable for only small spicules of bones
TCA
very weak decal sltn suitable for minute pieces of bones
sulfurous acid
fixative and decal agent for minute pieces of bones
chromic acid/ flemming’s
environmental toxin
flemming’s
permits excellent nuclear and cytoplasmic staining and it does not produce cell or tissue distortion. However, action is too slow for routine purposes
Citric acid - citrate buffer
substances that combine with Ca ions and other salts to form weakly dissociated complexes and facilitate removal of Ca salt
Chelating agents
most common chelating agent
EDTA
commercial name of EDTA
versene
used as anticoagulant and H2O softener
EDTA
pH of EDTA
7-8
T/F Edta takes longer decalcification time - weeks rather than days
T
how much EDTA is needed for 1g of bones
2-4 oz
decal time of edta for small samples
1-3 weeks
decal time of edta for dense cortical bones
6-8 weeks
hastens decalcification by removing Ca ions by removing Ca ions from formic acid-containing decalcifying solutions, thereby increasing solubility from the tissue
Ion exchange resin
resin used for ion exchange resin
ammonium-sulfonated polystyrene
end point of ion exchange resin
- x-ray
- physical method
reactivation of used resin
- N/10 HCl (2x)
- dH2O (3x)
volume of ion exchange resin
20-30
process whereby positively charged Ca ions are attracted to a negative electrode and subsequently removed from the decalcifying solution
electrophoresis
sltn used for electrolytic decal
- 88-90% Formic acid
- conc. Hcl
- dH2O
time of decal is shortened d/t heat and electrolytic rxn produced in the process
electrophoresis
temp for electrophoresis
30-45C
post-decalcification treatment for washing out
running water: 30 mins to 1-4 hrs
post-decalcification treatment for neutralization
- 2% Li2CO3
- 5-10% Na Bicarbonate
post-decalcification treatment for frozen sectioning
- acid- decalcified tissues —> water
- store in formal - saline with 15% sucrose or
- in phosphate-buffered saline with 15-20% sucrose
at what temp should frozen sectioning for post-decalcification done
4C
a method of dealing with small unexpected deposits of calcium that may be encountered in paraffin blocks
surface decalcification
may cause resistance of gratings sensation in surface decalcification
small foci
remedy for surface decal
- reome block
- place facedown on a pad of cotton of gauze saturated with 10% HCl for approx 1 hr
- rinse with HCl, chill, then section
other name for chemical test
Ca oxalate test
cannot be used when radiopaque metallic salts have been used
x-ray
x-ray cannot be used when _______ have been used
radiopaque metallic salts
reagents for chemical test
- 0.5 mL sat. aq. NH4 oxalate
- 1% Na oxalate
ppt of chemical testing
- Ca oxalate
- Ca hydroxide
tissue softener that causes tissue to swell and become soapy
molliflex
tissue softeners
- perenyi’s
- 4% aq. phenol
- molliflex
