Decalcification

Procedure whereby calcium or lime salts are removed from tissues (most especially bones and teeth) to make them flexible and easier to cut.

Done after fixation and before impregnation.

Bones, teeth, tuberculous organs, arteriosclerotic vessels, calcified tumors or granulomas, and calcified heart valves.

Room Temperature (RT, 18-30°C).

24-48 hours.

20 times the volume of the tissue.

Increased temperature/heat, increased concentration and volume of solution, increased fluid access/slender specimen, smaller/softer tissue size, and increased agitation.

Complete digestion of the tissue specimen and poor staining capacity of the cell.

1. Acids, 2. Chelating agents, 3. Ion-Exchange Resin, 4. Electrical Ionization / Electrophoresis.

Nitric acid (almost 2x as fast as formic acid) and Formic acid (provides better tissue preservation and staining).

At least 1 ounce per gram of tissue.

Should be changed once or twice a day until decalcification is completed.

Most common and fastest decalcifying agent used so far; produces minimal distortion but spontaneously turns yellow due to HNO₂ formation (inhibits nuclear stains and damages tissues).

Mix Nitric acid with formaldehyde or alcohol.

12-24 hours; recommended for urgent biopsies, needle/small biopsy specimens, and large or heavily mineralized cortical bone.

1-3 days; less tissue destruction than 10% aq. nitric acid; rapid-acting with good nuclear staining; yellow nitrous acid color removed by neutralizing with 5% sodium sulfate and running tap water for 12 hours.

2-7 days; decalcifies and softens tissues at the same time; maceration is avoided due to the presence of chromic acid & alcohol.

12-24 hours; most rapid decalcifying agent (recommended for urgent works), but has poor nuclear staining and yellow color formation.

Inferior to nitric acid due to slower action and more tissue distortion, but provides good nuclear staining.

36% Sat. Aq. NaCl, Conc. HCl, Distilled water; recommended for teeth and small pieces of bones.

10% concentration; moderate-acting decalcifying agent; overall time range 2-14 days (a. 2-7 days, b. 3-14 days).

Not suitable for urgent examination because its action is moderate/slow.

Formic acid + 10% formol saline; recommended for small pieces of bones and teeth; suitable for routine surgical specimens and immunohistochemical staining.

20% Aq. Sodium citrate + 45% Formic acid; recommended for autopsy materials, bone marrow, cartilage, and research tissues.

4-8 days; permits good nuclear staining and does not require washing out.

Weakest decalcifying agent; suitable only for minute pieces of bone.

Used as fixative and decalcifying agent for minute bone spicules; environmental toxin, highly corrosive to skin/mucous membranes, and carcinogenic.

6 days; permits excellent nuclear and cytoplasmic staining without cell/tissue distortion, but too slow for routine purposes.

1. Wash in tap water for 30 minutes (small samples) or 1-4 hours (larger specimens); 2. Immerse in alkaline solution for several hours (sat. Lithium carbonate or 5-10% aq. Sodium bicarbonate).

1. Wash in water; 2. Store in frozen section media containing 15% sucrose or PBS with 15-20% sucrose at 4°C before freezing.

Substances which combine with calcium ions and other salts (iron and magnesium) to form weakly dissociated complexes and facilitate removal of calcium salt.

Nucleic acid & nuclear DNA preservation, enzyme histochemistry, IHC, FISH, and PCR.

Versene; EDTA (Ethylenediaminetetraacetic acid).

1. Rinse in running tap water; 2. Overnight immersion in Formol-saline / Phosphate buffered saline.

Ammonium form of Polystyrene resin; hastens decalcification by removing calcium ions from Formic acid, thereby increasing solubility.

Not recommended for fluids containing mineral acids such as Nitric acid or Hydrochloric acid.

Positively charged Calcium ions are attracted to a negative electrode (cathode) and removed; relies on direct current (DC).

88% Formic acid, Conc. HCl, and Distilled water.

Ideal thickness 2-4 mm (optimal 3-5 mm sections); maintains temperature at 37-55°C.

Faster than routine decalcification while providing good tissue preservation and staining efficacy.

Block removed from chuck and placed face down on a pad of cottonauze saturated with 10% HCl for approximately 1 hour to dissolve surface calcium.

1. Physical / Mechanical, 2. Radiological / X-ray, 3. Chemical.

Touching, bending, squeezing tissue with fingers; weighing specimen after rinsing; pricking/slicing tissue with a fine needle or probe.

Radiological / X-ray method.

Mercuric chloride-fixed tissues.

Litmus paper, Strong ammonia, Sat. Aq. Ammonium oxalate / Conc. Ammonium hydroxide.

Cloudiness indicates incomplete decalcification; Clear solution indicates complete decalcification.

1. Perenyi's fluid (12-24 hrs), 2. 4% Phenol (1-3 days), 3. Molliflex (swollen/soapy tissue), 4. 2% HCl, 5. 1% HCl in 70% Alcohol.

Process of removing intracellular and extracellular water from the tissue following fixation and prior to wax impregnation.

At least 10 times the volume of tissue to be dehydrated.

Dehydrates rapidly without distortion, does not evaporate too fast, dehydrates even fatty tissues, does not overharden tissue, does not remove stains, non-toxic, and not a fire hazard.

Beginning with 70% → 95% → 100% increasing / ascending grades of alcohol.

Causes shrinkage and hardening of tissues.

High concentrations (>80%) make tissue hard, brittle, and difficult to cut; low concentrations (<70%) tend to macerate/soften tissue.

May interfere with the staining properties of the specimen.

Ethanol (Ethyl alcohol).

Should be at least 99.7% pure.

Excellent substitute for good-grade absolute ethyl alcohol.

Used for blood and tissue films and for smear preparations; toxic dehydrating agent.

Utilized for plant and animal micro-techniques (recommended for tissues not requiring rapid processing).

Cheap, rapid dehydrating agent utilized for most urgent biopsies (1/2 to 2 hours); limited to small tissue pieces due to volatility, flammability, and tissue shrinkage.

Diethylene dioxide; excellent dehydrating and clearing agent miscible with water, melted paraffin, alcohol, and xylol (expensive and extremely toxic).

Ethylene glycol monoethyl ether; dehydrates rapidly without harming tissues, but toxic by inhalation, skin contact, and ingestion.

Tissues can be transferred directly after fixation & washing; used to dehydrate sections and smears following certain stains.

Dehydrates and clears tissues since it is miscible in water & paraffin; toxic if ingested or inhaled, causes eye/skin irritation and conjunctival irritation.