Decalcification

Question 1

General Considerations for
Routine Acid Techniques

Thickness of the Specimen
1. dense/hard bone:
2. softer tissue:

Duration
1. ideal:
2. dense cortical bone (dense tissues):

Temperature & Heat
1. required temp
2. impairs nuclear staining w/ Van Gieson’s
3. tissues will undergo complete digestion within 24-48hrs

Answer 1

Thickness of the Specimen
1. 2-5mm thick
2. 4-6mm thick

Duration
1. 24-48hrs
2. 14 days

Temperature & Heat
1. 18-30C
2. 37C
3. 55C

Question 2

General Considerations for
Routine Acid Techniques

Solution Used
1. conc. of solutions: directly proportional to rate of ________
2. _____: affects antigenicity of cells & tissue components

Agitation & Partial/Complete Vacuum
1. vigorous agitation:
2. low speed rotation, rocking, mechanical
stirrer, bubbling air into the solution

1. protect tissues but slows down decalcification
2. changing of the solution
3. ____ volume of the tissue

Answer 2

Solution Used
1. decalcification
2. strong acids

Agitation & Partial/Complete Vacuum
1. sonication
2. gentle fluid agitation

1. Additives
2. once or twice a day
3. 20x the volume of the tissue

Question 3

General Decalcifying Methods

______: injurious to the organic ground substance of tissue
- principle

______: most common & fastest; rapid decalcifying agent: may inhibit nuclear stains & damage tissues
1. recommended conc. when used as a simple solution
2. washing of tissue: acid removed by 3 changes of
3. washing slide: brought to water & placed in
4. causes causes _______ discoloration
5. if present in tissues: neutralize w/ ______
6. if present in solution: add ____ to pure conc nitric acid

Nitric Acid Formulations:
1. for silver impregnation of nerve fibers
2. slow-acting decalcifying agent
3. most rapid decalcifying agent (for urgent work)

Answer 3

Use of Acid Solutions/ Acid Decalcification
- Chemical Substitution

5-10% Nitric Acid
1. 5 to 10%
2. 70-90% ethanol
3. 1% aq. lithium
4. spontaneous yellow
5. 5% NaSO4
6. 0.1% urea

1. De Castro’s Fluid
2. Perenyi’s Fluid
3. Phloroglucin-
   Nitric Acid

Question 4

Use of Acid Solutions/ Acid Decalcification other agents

1. for small & large pieces of bones & teeth
   - concentrated reagent:
   - washing: neutralize w/
2. slower action, w/ greater distortion

other acids
1. two for minute samples
2. for excellent nuclear and cytoplasmic staining
3. for very minute samples since it is a very weak decalcifying agent

Answer 4

Use of Acid Solutions/ Acid Decalcification other agents

1. Formic Acid
   - 90%
   - 5% sodium sulfate
2. Hydrochloric Acid

other acids
1. TCA & Fleming
2. Citric Acid-Citrate Buffer
3. Sulfurous Acid

Question 5

Use of Chelating Agents

1. acid but does not work as w/ organic acids
2. available in 2 formulations:
   - salt at 7.0-7.4 (buffered since pH affects
   the removal of calcium)
   - pH adjusted to 7.4 using conc hac
3. duration
   - small specimens
   - dense cortical bone
4. pH
   - inhibits calcium binding
   - optimum binding (alkaline environment)
   - allows binding & does not destroy tissue components

Answer 5

1. EDTA
   2.
   - 5-10% EDTA Disodium
   - EDTA Tetrasodium
2. duration
   - 1-3 weeks
   - 6-8 week
3. pH
   - pH 3
   - pH 8
   - pH 7.0-7.4

Question 6

Ion Exchange Resin

1. uses ____ + _____
2. volume of acid solution:
3. 10 % and 20 % RAF:
   - 2-3hrs:
   - 4-8hrs:
4. reactivation of used resin
   - 2washings w/
   - final washing w/
5. end-point of decalcification is measured using

Answer 6

1. ammonium-sulfonated polysterene + formic acid
2. 20-30
   3.
   - 2-3mm thick
   - 5-6mmthick
   4.
   - N/10 HCl
   - distilled water
3. Physical/ X-Ray Methods

Question 7

Electrophoresis
1. (+) charged _____ ions are attracted to a (–) electrode
2. temp
3. uses _____ and _____ as acid solution

Answer 7

1. calcium
2. 30 to 45 degrees Celsius
3. 90%/88% formic acid and conc. HCl

Question 8

Tests for Complete Decalcification
1. vague and not a reliable method
2. very expensive, most ideal, most sensitive & most reliable method
- DO NOT use for _____ fixed tissues
3. simple, reliable, & convenient method for routine purposes
- + ___ = Incomplete Decalcification

Answer 8

1. Physical Test
2. Radiologic Test (X-Ray)
   - mercuric chloride fixed tissues
3. Chemical Test
   - CALCIUM

Question 9

Samples That Require Decalcification

Bones & Other Calcified Samples
1. cut into small pieces using _____, trimmed w/ hand razor and fixed w/ ______

_______: partial/ complete decalcification is required before cutting samples

______: detected during sectioning/ examination
- remedy if detected during sectioning: surface decalcification using a pad of cotton/gauze soaked w/ ______ for 1hr
- commonly found in

Answer 9

Bones & Other Calcified Samples
1. FRET SAW; 10% NEUTRAL BUFFERED FORMALIM

TEETH

MICROCALCIFIED SAMPLES
- 10% HCL
- MALIGNANCY