Unit 3.1. Decalcification

Decalcification

removal of calcium salts from the organic extracellular matrix

20:1

fluid to tissue ratio for decalcification

24-48 hrs (1-2 days)

ideal time for decalcifying tissue

14 days

dense bones require up to ________ to complete decalcfication

1. Concentration of Decalcifying Agent

2. Ratio of Decalcifying agent to tissue

3. Temperature

4. Mechanical Agitation

5. Ideal time required

5 FACTORS INFLUENCING THE RATE OF DECALCIFICATION

True

T or F

↑ Concentration of decalcifying agent = ↑ Rate of infiltration

18-30℃

Ideal temp for decal

mechanical agitation

Constant ________ decalcifies tissues rapidly

• stirring

• rocking

• shaking

• rotating

mechanical agitation examples

1. Acid decalcifying agent

2. Chelating agent

3. Ion exchange resin

4. Electrophoresis
   
   

[ACIE]

4 METHODS OF DECALCIFICATION

Acid decalcifying agent

4 METHODS OF DECALCIFICATION

• most widely used agent for large amounts of bony tissues

1. stable

2. easily available

3. relatively inexpensive

3 characteristic of acid decalcifying agent

• nitric acid (5-10%)

• hydrochloric acid

• formic acid sodium citrate

• tricarboxylic acid

• sulfurous acid

• chromic acid/ flemming’s acid with acetic acid

• citric acid citrate buffer

Enumerate acid decalcifying agents

Nitric acid (5-10%)

ACID DECALCIFYING AGENTS

• most common

• fastest decal agent

• 10% aqueous Nitric acid

• Formol nitric acid

• Perenyi's Fluid

• Phloroglucin–Nitric Acid

Enumerate examples of nitric acid

24 hrs

ideal time for routine decalcification using 10% aqueous NA

1-3 days

ideal time for decalcification using Formol NA

Nitric acid + Formaldehyde

Formol Nitric acid compostion

Perenyi’s fluid

NITRIC ACID

• decalcifies and softens the tissue at the same time

2-7 days

ideal time for decalcification using Perenyi’s fluid

Nitric acid

Absolute ethyl alcohol

Chromic acid

Perenyi’s fluid composition

Phloroglucin-Nitric acid

NITRIC ACID

• most rapid and specific nitric acid decal agent

12-24 hrs

ideal time for decalcification using Phloroglucin-NA

yellow color

NITRIC ACID

• NItric acid can impart __________ to the tissue

addition of 0.1% urea to pure nitric acid

NITRIC ACID

• how to remove the yellow color imparted by NA to the tisssue

Hydrochloric acid (HCl)

ACID DECALCIFYING AGENTS

• recommended for surface decalcification

Von Ebner's Hydrochloric Acid

ACID DECALCIFYING AGENTS

• the only decal agents that contains 36% HCl

• weak and slow ; not routinely used

sodium chloride

HCl

distilled water

Von Ebner’s composition

Formic acid-sodium citrate

ACID DECALCIFYING AGENTS

• for cartilage, bone marrow, research and autopsy spx

Tricarboxylic acid

Sulfurous acid

ACID DECALCIFYING AGENTS

• weak and slow

Chromic acid/ Flemming’s with Acetic acid

ACID DECALCIFYING AGENTS

• not common

• toxic and corrosive

Citric acid-citrate buffer

ACID DECALCIFYING AGENTS

• uses chloroform as preservative and a decalcifying agent

1. 5% Formic acid

2. Chromic acid

3. Trichloroacetic acid (TCA)

4. Sulfurous

USED BOTH AS FIXATIVE & DECAL AGENT

5% formic acid

USED BOTH AS FIXATIVE & DECAL AGENT

• best general decalcifying agent

Chromic acid

USED BOTH AS FIXATIVE & DECAL AGENT

• Carcinogenic, corrosive to skin and mucus membranes

TCA

USED BOTH AS FIXATIVE & DECAL AGENT

• a “weak” decal agent

Sulfurous acid

USED BOTH AS FIXATIVE & DECAL AGENT

• a “very weak” decal agent

Chelating agents

METHODS OF DECALCIFICATION

• combine calcium ions and other salts to form weak dissociated complexes and facilitate removal of calcium salts

• EM

• enzyme and immunohistochemical staining

Chelating agents are best for

• EDTA (Versene)

• CAL-EX

CHELATING AGENTS examples

EDTA (Versene)

CHELATING AGENTS

• most common chelating agent in the market

• slow decal agent

EDTA (Versene)

CHELATING AGENTS

• only for detailed microscopic studies

• small spx = 1-3 weeks

• dense cortical bone = 6-8 weeks or longer

EDTA (Versene) decalcifies small spx for _______, while dense cortical bone may take ________

3 days ;

7 - 7.4 pH

Replace EDTA every ______ and its pH must be adjusted to _______

alkaline phosphate;

magnesium chloride

EDTA inactivate _______________, then add _____________

CAL-EX

CHELATING AGENTS

• HCl + EDTA

Ion exhange resin

METHODS OF DECALCIFICATION

• ammonium form of polystyrene resin that hastens decalcification by removing calcium ions from formic acid containing decalcifying solutions

calcium ions ;

formic acid

Ion exhange resin removes __________ from ________ containing decalcifying solutions

mineral acids

urgent biopsies

Ion exchange resin is not suitable for tissues containing ____________ and ___________

20-30:1

Ion exchange resin to tissue ration

Electrophoresis

METHODS OF DECALCIFICATION

• process whereby calcium ions which are (+) charged are attracted to a (-) charged electrode and removed

Electrophoresis

METHODS OF DECALCIFICATION

• most rapid method due to electricity

• Disadvantage: dependent on the electric current

small bone fragments

Electrophoresis is satisfactory for

• 88% formic acid

• concentrated HCl

• distilled water

• sodium chloride

[FoHCDiSC = fuck this]

SOLUTIONS USED FOR ELECTROLYTIC DECAL

TISSUE SOFTENERS

last resort if after all decalcifying agent you have tried is still unsuccessful (tissue still hard)

• Perenyi’s fluid

• Lendrum’s method

• Molliflex

• 2% HCl

• 1% HCl in 70% alcohol

TISSUE SOFTENERS examples

place spx on a 4% aqueous phenol for 1-3 days

Explain Lendrum’s method

1. Physical/ Mechanical test

2. X-Ray/ Radiological test

3. Chemical Test (Calcium oxalate test)

4. Bubble test

TESTS TO MEASURE COMPLETENESS OF DECAL

Physical/ Mechanical test

TESTS TO MEASURE COMPLETENESS OF DECAL

• done by touching, probing, bending resistance to finger nail or by needling

• not reliable

pricking using fine needle/probe

In Physical/ Mechanical test, needling is done by

X-ray / Radiological test

TESTS TO MEASURE COMPLETENESS OF DECAL

• most ideal, sensitive, and reliable way of determining extent of decalcification

X-ray / Radiological test

TESTS TO MEASURE COMPLETENESS OF DECAL

• measures the opaqueness of the spx

• opaque = incomplete decalcification

mercuric chloride fixative

X-ray / Radiological test is not sutable for ________________ fixative

Radiography

TESTS TO MEASURE COMPLETENESS OF DECAL

• most sensitive test for detecting calcium in bone or tissue calcification

Chemical test (calcium oxalate test)

TESTS TO MEASURE COMPLETENESS OF DECAL

• simple, reliable, convenient

• recommended for routine purposes

• detect calcium in decalciying solution

calcium oxalate / calcium hydroxide

Chemical test (calcium oxalate test) is done by the detection of calcium by precipitation of insoluble ______________________

1. Ammonium hydroxide = screening of calcium salts

2. Ammonium oxalate = confirmation of calcium salts

Solutions used in Chemical test (calcium oxalate test)

strong ammonia;

clear

Chemical test (calcium oxalate test) Process:

Step 1: add ________________ to the discarded decalcifying solution

• if the solution is _______, proceed to step 2

ammonium oxalate ;

complete decalcification

Chemical test (calcium oxalate test) Process:

Step 2: Discarded solution + __________________

• clear solution means _______________

Bubble test;

bubbles

TESTS TO MEASURE COMPLETENESS OF DECAL

• addition calcium carbonate to the solution

  • formation of _____ = incomplete decal

Post-Decalcification

a process where a decalcified tissue is neutralized and where excess decal agents are removed

1. Immersing in saturated lithium carbonate or 5-10% sodium carbonate

2. Rinsing in tap water

3. Storing in Formol saline with 15% sucrose or phosphate buffered saline at 15-20% sucrose at 4 ℃

3 Ways of Post-Decalcification

Rinsing in tap water

most common method of Post-Decalcification

12-24 hrs

Decalcification time of 10% Nitric acid

1-3 days

Decalcification time of Formol nitric acid

2-7 days

Decalcification time of Perenyi’s fluid

12-24 hrs

Decalcification time of Phloroglucin–Nitric Acid

2-7 days

Decalcification time of formic acid

4-8 days

Decalcification time of trichloroacetic acid