5: Decalcification

Question 1

What is used to decalcify tissues ?

Answer 1

Since calcium salts dissolve at pH<4.5:

1. Weak acid: Formic acid + formalin (3-10 days)
2. Strong acid: HCl (<48 hours)

Question 2

T or F: Decalcification can be done without fixation

Answer 2

FALSE; tissue MUST BE FIXED BEFORE DECALCIFICATION
- acids will destroy delicate tissue components and alter basophilia (cationic staining)

Question 3

Why should formalin-fixed tissue be thoroughly washed before decalcifying with HCl ?

Answer 3

Formalin can react with HCl = bis-chloromethyl ether (CARCINOGEN)

Question 4

Decalcification method: Simple Acid

Answer 4

• tissue submerged and agitated in acid
• replace acid as it becomes saturated with calcium ions

Question 5

Decalcification method: Ion-exchange resins

Answer 5

• used alongside formic acid decalcification
• resin exchanges ammonium ions for calcium ions = prevents saturation/ need to replace solution = faster decalcification

Question 6

Decalcification method: EDTA

Answer 6

• selectively bind metal ions without affecting other components
• used when acids might destroy labile elements that must be demonstrated (enzymes, molecular targets)
• TAKES WEEKS

Question 7

Decalcification Endpoint Detection: Chemical

Answer 7

• aliquot is neutralized with ammonium hydroxide > react with equal volume of ammonium oxalate > existing calcium ions will react to form a white precipitate
• if no precipitate forms = decalcification is complete (all calcium has been removed from tissue)
• smaller volumes = increased sensitivity

Question 8

Decalcification Endpoint Detection: Radiography

Answer 8

calcium is solid white on X-rays (highly accurate)

Question 9

Decalcification Endpoint Detection: Physical

Answer 9

• bending, probing, cutting the sample (NOT RECOMMENDED)
• unreliable, and small deposits of calcium can be missed
• physical forces can also create artifacts

Question 10

Regardless if tissue is under- or over-decalcified, the root cause is almost always __.

Answer 10

Regardless if tissue is under- or over-decalcified, the root cause is almost always POOR ENDPOINT DETECTION

Question 11

Troubleshoot: Under-decalcified

Answer 11

• generally recognized IN MICROTOMY= scores and tears, handwheel resistance,
• SEVERE= melt wax off, “reverse process” (xylene>100% alc>95% alc>70% alc>water), place back in decalcifying solution
• Sectioned= surface decalcification; place in acid cut-side down for 30 min max. Wash well in water, chill, cut, repeat.

Question 12

Troubleshoot: Over-decalcified

Answer 12

• MARKED REDUCTION in NUCLEAR STAINING = pale red (loss of basophilia)
• difficult to reverse; place slide in 5% sodium bicarbonate overnight. Wash slides in the morning, and re-stain.