Decalcification — Exam 3

When can decal take place?

After or in conjunction with fixation

Simultaneous decal and fixation:

Decalcifying acids (mainly formic acid) can be directly added to formalin or purchased

Criteria of good decal solution

1. Complete removal of calcium

2. No damage to morphology or staining

3. Reasonable speed of decal

Specimen prep

• Fix specimen first

• Remove all soft tissue from bone bc acidic solution can damage morphology

• Tissue pieces should be thin and well fixed (except w/simultaneous decal and fixation

• Bone saw may be needed

• EX: bone marrow bx, bone resections

Simple acids are…

Hydrochloric, nitric, formic acids

Simple acids - general info

• Ca dissolve into solution

• [5-10%]

• Solution needs changed frequently to avoid saturation

Decal is enhanced by…

• Agitation

• Vacuum

• Exposing all surfaces
  — Precipitated Ca will sink = more [ ]ed at bottom

• Heating will sped up BUT damages tissue

Concentration and Decal

5-10% is ideal

Volume and Decal

Acid depletes as it reacts so large volume to tissue is best

Solution depletion and Decal

bc acid depletes, solution needs replaced w/fresh if specimen is being decaled more than 1 day

Time and Decal

Strong acids negatively affect tissue if left in for long periods of time

Strong Inorganic Acid is…

HCl → pH is 1.5-3.5

Most common

Advantages: rapid acting

Must completely rinse formalin-fixed with water before placing is HCl and vice versa

HCl Disadvatages

• Swelling and destruction of nuclear detail

• Rxn w/ formaldehyde → forms carcinogen

• DO NOT use with formalin for simultaneous decal/fixation!!!

Nitric Acid

pH = 3.0

Advantages: rapid acting

Disadvantages:

• Deterioration of staining results occurs if tissue left in >48 hrs

• some tissue Ag and enzymes lost

• increased chance of tissue damage and negative staining effects

Weak organic acid is…

Formic acid, ph is 3.47

Formic acid advantages

• gentler than strong inorganic acids

• recommended for advanced staining bc it does not destroy morphology if tissue is left in it up to 2 weeks

• Can be used with formalin for simultaneous fix/decal

Formic acid Disadvantage

Works much more slowly than HCl and Nitric acid

Other acids that decal small calcifications…

Picric acid, pH is 2.0 = Bouin, Hollande

Acetic acid, pH is 2-3 = Gendre, Zenker, Davidson, Carnoy, Methacarn, Clarke

Chelating agents

EDTA pH is 7-10
Chelates Ca gradually depleting the outer layer of the calcium apatite crystal

EDTA advantages

Safest methods, gentler than weak acids

Preserves tissue morphology with minimum artifacts

EDTA disadvantages

Slow acting, much slower than formic acid

Cartilage is damaged if overexposed → staining is affected

Ion-Exchange Resin

• Formic acid is placed over a layer of ammoniated polystyrene resin in a container + tissue

• Ammonium ions exchanged for Ca ions, solution lasts longer

• Volume should be 20-30 times to tissue

Ion-Resin advantages

• Low tissue damage

• Exposure time not critical

• Does not need changed daily

Ion-Resin disadvantages

• degree of decal cannot be measured

• Large volume required

Electrolytic method

Formic or HCl is used with electroplating device

Anode and cathode probes in acid

Bone is on Anode (+)

Ca ions drawn from bone to cathode (-)

Electrolytic method advantages

fast method: most decal within 2-6 hrs

Electrolytic method disadvantage

Strong potential for tissue damage bc of heat

→ loss of cellular detail and stainability

Decal end point methods

1. physical/mechanical

2. chemical

3. radiographic

Physical/Mechanical

Flexibility is tested by manually bending tissue

If it bends → it sends

AVD: Quick, easy, inexpensive

DSV: subjective to each person, can damage tissue if not done correctly

Chemical (measuring)

Calcium oxalate test: detects Ca in decal solution at top of fluid

5% ammonium hydroxide + 5% ammonium oxalate are added to a small amount of decal solution

Cloudiness/turbidity after 30 mins indicates Ca in solution (calcium ocalate precipitation) = INCOMPLETE decal

Clear = complete decal

Chemical advantages and disadvantage

Adv:

• less invasive

• more accurate

• less damaging

Dsv:

• subjective in interpretation of solution and following procedure

Radiography

X-ray is taken of tissue

After decal… (steps)

Rinse w/water to remove acids

Lithium carbonate can also be used

Further trim as needed → diamond saw blades can cut thin sections w/o introducing bone dust into spec

Surface Decal

Decals remaining Ca or unsuspected mineral deposits

After processing or at microtome

NEVER decal breasts

Surface decal steps

1. Float block in decal solution

2. rinse with water and chill

Time depends on type of decal solution and extent of calcifications (5-30 mins.)

Does not have to be the same decal solution orig used

Teeth specimens

Contain both mineralized and soft tissues

• enamel, cementum (what we try to decal), dentin, pulp soft tissue

Decal is NOT appropriate bc enamel will dissolve completely

Nails/heavily keratinized skin

Looking for fungal infections

After fixation and before processing, place specimen in sodium/potassium hydroxide or commercial nail softening solution