Decalcification

Decalcification • the procedure whereby Calcium or lime salts are

removed from tissues (most especially bones & teeth)

• done by immersion of the specimen into a decalcifying solution after fixation, & before impregnation

• good decalcifying agents must remove Ca completely from tissues without destroying or distorting the cell & tissue components or adversely affecting staining capacity (esp. of the nucleus)– Acids – Ion exchange resins – Chelating agents – Electrophoresis

Procedure

• fix specimen as usual: 10% Buffered Neutral Formalin for up to 5 days.

• cut tissues into small pieces (to ensure complete penetration of the decalcifying solution)

• Specimens should NOT be crowded together to provide for complete penetration of the decalcifying agent and should NOT contact the bottom of container to avoid possible precipitates

Decalcification Solutions for Bone

• Choice of decalcification solution will depend on the type of sections to be cut, type of staining to be performed, speed of process required, and it may be necessary to experiment with different solutions to obtain the best results with your material and staining method. The following decalcification solutions have been found useful for various purposes.

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Decalcifying Agents

• Acids – Nitric acid– Hydrochloric acid– Formic acid– Trichloroacetic acid– Sulfurous acid– Chromic acid– Citric acid

• Chelating agent– EDTA

• Ion Exchange Resin– Ammonium form of

Polystrene • Electrical

Ionization/ Electrophoresis– Formic acid-HCl

solution

Acid Decalcifying agents• most commonly used for routine

decalcification; are stable, easily available, relatively inexpensive;

• Nitric acid – used as simple solution or combined with other agents; rapid-acting, produces minimal tissue distortion but tends to inhibit nuclear stains &, if used as pure solution, destroy tissues– Aqueous Nitric acid 10% solution: decal time = 12-24 hrs.

• Good nuclear staining; acid easily removed by 70% alcohol– Formol(Formaldehyde)-Nitric acid: decal time = 1-3 days

• Relatively good nuclear staining– Perenyi’s fluid: Chromic acid-EtAl: decal time = 2-7 days

• Decalcifies & softens tissues at the same time; good staining– Phloroglucin-Nitric acid: decal time = 12-24 hrs

• Most rapid, for urgent decal; poor nuclear staining; yellow color

Acid Decalcifying agents• Hydrochloric acid – slower action, greater

tissue distortion (than Nitric acid); but good nuclear staining; – 1% sol’n with 70% alcohol suitable for surface decal of tissue

blocks– Von Ebner’s fluid: conc. HCl + sat. Aqueous NaCl 36% sol’n in

distilled H2O – good cellular staining, moderately rapid, doesn’t require washing out before dehydration; for teeth, small bones

• Sulphurous acid – very weak agent, suitable only for minute bone pieces– H2SO3

Acid decalcifying agents

• Formic acid – produces better nuclear staining & less tissue distortion, but slow-acting; recommended for postmortem research tissues, not suitable for urgent examinations; requires neutralization by 5% Sodium Sulfate then washing.– Formic acid-Formol saline 10%: decal time= 2-7 days

• Both fixative & decalcifying agent; excellent staining;

– Formic acid-Sodium citrate: decal time= 3-14 days• Better nuclear staining than Nitric acid, but slow; for autopsy research

materials, bone marrow, cartilage

Acid decalcifying agents• Trichloroacetic acid – good nuclear staining

but very slow-acting & weak, suitable only for small pieces– TCA + Formol Saline: decal time= 4-8 days

• Not for urgent examinations or dense tissues; good nuclear staining; suitable for small bone spicules

• Chromic acid – Flemming’s fluid – may be used as both fixative & decalcifying agent but nuclear staining is inhibited– Chromic acid + Osmium tetroxide + glacial Acetic acid

• recommended for minute bone spicules; nuclear staining is inhibited

Acid decalcifying agents

• Citric acid – no cellular/tissue distortion, permits excellent nuclear & cytoplasmic staining but very slow-acting– Citric acid + Ammonium citrate + Zn sulfate + Chloroform– decal time = 6 days (not for routine exams)

Chelating Agents• chemicals that bind Calcium ions,

extracting them from tissues to be decalcified; usually combine with other salts also, e.g. Iron & Magnesium

• EDTA – Ethylene Diamine Tetraacetic Acid – most common; very slow; causes slight tissue hardening– excellent staining; minimal cell & tissue distortion; minimal

artefact (bubble) formation– tissue is placed in the solution for 1–3 wks. (solution is changed

every 3 days initially, then every day until completed – extent of decalcification can be measured by routine chemical

testing

Ion Exchange Resin

• reusable accelerator of decalcification in formic acid solutions; removes Calcium ions from the solution to “renew” it, hastening extraction of Calcium from the tissue & eliminating the needed for changing the decalcifying solution– Ammonium form of Polystrene Resin: decal time= 1-14 days

• cellular detail is preserved • cannot be used with Nitric & Hydrochloric acid solutions• degree of decalcification cannot be measured by chemical

means (use physical method or X-ray)• may be reactivated after use by immersion in N/10 HCl twice

& washing with distilled water thrice

Ion Exchange Resin

• the bottom of the container is lined by a ¼ in thick layer of Resin

• the specimen is placed atop the resin• Formic acid solution (20–30x the tissue

volume) is added • the tissue is allowed to stay in

solution for 1-14 days• degree of decalcification is measured

by X-ray or physical method

Electrophoresis / Electrical Ionization

• process whereby positively-charged Calcium ions are attracted to a negative electrode & subsequently removed from the decalcifying solution; uses electricity, produces heat & electrolysis– Formic acid + conc. HCl in distilled H2O

Evaluation of Decalcification

• Overdecalcification can also permanently damage a specimen. The following procedure help determine the correct end-point of decalcification. 

• End-Point of Decalcification: X-ray (the most accurate way) Chemical testing (accurate) Physical testing (less accurate and potentially damage

of specimen)

Assessment of Decalcification

• Radiology / X–ray– ideal method, most reliable– can detect even the smallest residual Calcium (opaque)– very expensive

• Physical / Mechanical method– inaccurate, unreliable– touching/bending the tissue or inserting a pin, razor,

probe or scalpel directly into the tissue

Assessment of Decalcification

• Chemical tests - simple, reliable, convenient, suitable for routine testing; performed on the discarded decalcifying fluid– Blue litmus paper in 5 ml. discarded fluid (becomes red)

• Add strong ammonia sol’n drop by drop til litmus paper becomes blue: cloudiness indicates (+) Ca

• If sol’n remains clear, add Ammonium oxalate & stand for 30 min: cloudiness=(+)Ca

– 10 ml. Ammonium hydroxide/Ammonium oxalate working sol’n is added to 5 ml. of discarded fluid, mixed well & allowed to stand overnight

• no precipitate for 2 consecutive days = complete decal

Softening of Tissues

• Perenyi’s fluid• 4% aqueous Phenol solution• Molliflex : 2% HCl + 1% HCl in 70%

Alcohol

Happy Week-end!!!