decalcification

Question 1

decalcification

Answer 1

removing calcium from tissue

calcium in a tissue block causes tears during cutting

Question 2

when is decalcification done

Answer 2

after fixation before processing

Question 3

alternatives to decalcification

Answer 3

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

Question 4

two basic choices

Answer 4

acid methods

chelation

Question 5

decal fluid used is determined by

Answer 5

urgency

degree of mineralization

extent of investigation

staining techniques required

Question 6

acids affect on tissue

Answer 6

any acid will affect tissue staining

stringer acid or the longer the acid is used the more damage is done to the tissue

Question 7

over decalcification

Answer 7

lack of nuclear staining *

nuclear staining is the main thing we want to access

Question 8

acid methods

Answer 8

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

Question 9

hydrochloric & Nitric acids

Answer 9

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) *

Question 10

formic acid

Answer 10

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

Question 11

other weal organic acids

Answer 11

picric acid & acetic acid

used as part of carnoys & bouins
they act as incidental agents

Question 12

when to use which acid

Answer 12

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

Question 13

simple decalcification

Answer 13

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

Question 14

what happens if you apply heat to speed up decal

Answer 14

NEVER apply heat

specimen swells, becomes digested & damaged

Question 15

proprietary decalcifiers

Answer 15

trade secrets

rapid is usually HCL
slow is usually formic

advantages is the avoidance of concentrated acid handling

Question 16

what is considered the best method for decalcification

Answer 16

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

Question 17

electrolytic method

Answer 17

formic acid & HCL
NOT recommended - heat & tissue damage

Ca attracted to cathothe

fast ( 2-6 hrs) *

Question 18

chelating agents

Answer 18

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 )

Question 19

washing

Answer 19

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

Question 20

factors for decal

Answer 20

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

Question 21

end point of decal

Answer 21

most critical step in process

bone should stay in decal fluid no longer than necessary to avoid damage

frequently ignored in labs :(

Question 22

three methods of endpoint decal

Answer 22

mechanical - physical

chemical

radiographic

Question 23

chemical

Answer 23

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

Question 24

endpoint testing

Answer 24

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

Question 25

after decal

Answer 25

wash with water, saturated lithium carbonate, 5-10% sodium bicarbonate or weak ammonia

to neutralize acid & stop decal

then rinse with water

Question 26

surface decal

Answer 26

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 )

Question 27

when using acids

Answer 27

aprons 
gloves
eye protection
acid carrier
fume hood 
always add Acid to Water

Question 28

bone dust

Answer 28

may be pressed on surface of tissue by bone saw

• use a saw with a diamond blade
• trim surface after decal but before processing

Question 29

under decal

Answer 29

choose a rapid decal
develop a good endpoint testing
check endpoint with radiography when in doubt

Question 30

over decal

Answer 30

choose a decal that fits needs

develop good endpoint method

check with radiography

Question 31

what solution will appear white if decal isn’t complete

Answer 31

ammonium oxalate mixed in with decal solution (usually forming acid)