Decalcification is a process of complete removal of calcium salt from the tissues like bone and teeth and other calcified tissues following fixation.
Aim of Decalcification
Decalcification is done to assure that the specimen is soft enough to allow cutting with the microtome knife. Unless the tissues is completely decalcified the sections will be torn and ragged and may damage the cutting edge of microtome knife.
- To ensure adequate fixation and complete removal of the calcium it is important that the slices are 4-5 mm thick. Calcified tissue needs (2-3) hours only, for complete decalcification to be achieved so it is necessary to check the decalcification after (2-3) hours.
- Fixative of choice for bone or bone marrow is Zenker formal or Bouin's fluid. Unfixed tissue tends be damaged 4 times greater during decalcification than a properly fixed tissue.
Methods of Decalcification
- Dissolution of calcium by a dilute mineral acid.
- Removal of calcium by used of dilute mineral and along with ion exchange resin to keep the decalcifying fluid free of calcium.
- Using Chelating agents EDTA.
- Electrolytic removal of calcium ions from tissue by use of electric current
The Criteria of a good decalcifying agents area :
- Complete removal of calcium.
- Absence of damage to tissue cells or fibers.
- Subsequent staining not altered.
- Short time required for decalcification.
Calcium may be removed by any of the following agents:
- Chelating agents
- Ion-Exchange resins
- Electrical Ionization (Electrophoresis)
Removal of Calcium by mineral acids
Acid decalcifies subdivided into:
Strong acid - e.g. Nitric acid and Hydrochloric acid
I - Nitric acid: This is the most common decalcifying agent used so far, utilized both as a simple solution or combined with other reagents. It is very rapid decalcifying agents. It is very rapid decalcifying agents, producing minimal distortion and therefore, recommended for routine process. It has, however, the disadvantages of inhibiting nuclear stains and destroying tissues, especially in concentrated solutions. Combining Nitric Acid with Formaldehyde or Alcohol may prevent this.
*Aqueous nitric acid
- 5-10% aqueous solution is used.
- They decalcify vary rapidly but if used for longer than 12-24 hrs, it will cause deterioration of stain ability specially of the nucleus.
Nitric acid 5-10 ml , Distilled water to 100 ml.
- Place calcified specimen in large quantities of nitric acid solution until decalcification is complete (change solution daily for best results).
- Wash in running water for 30 minutes.
- Neutralize for a period of at least 5 hours in 10% formalin to which excess of calcium or magnesium carbonate has been added.
- Wash in running water over night.Dehydrate, clear and impregnate in paraffin or process as desired.
Note: Overexposure to nitric acid impairs nuclear staining.
Nitric Acid is the solution of choice for decalcifying temporal bones.
- It is rapid in reaction.
- It produces minimum distortion of tissue.
- It is recommended for urgent biopsies.
- The acid may be easily removed by 70% alcohol.
- Prolonged decalcification may leads to tissue distortion.
10% nitric acid 40ml , Absolute alcohol 30 ml. 0.5% chromic acid. 30ml.
- All these ingredients may be kept in stock and should be mixed immediately before use.
- This solution may acquire of blue violet tinge after a short while but this will have no effect in the decalcifying property.
- It is slow for decalcifying hard bone but excellent fluid for small deposits of calcium e.g. calcified arteries, coin lesions and calcified glands. Also good for human globe which contains calcium due to pathological conditions. There is little hardening of tissue but excellent morphologic detail is preserved.
*Formalin Nitric Acid
Formalin 10 ml, Distilled water 80 ml, Nitric acid 10ml.
Nitric acid causes serious deterioration of nuclear stain ability which partially inhibited by formaldehyde. Old nitric acid also tends to develop yellow discoloration which may be prevented by stabilization with 1% urea.
*Aqueous Formic Acid
90% formic acid 5-10 ml, Distilled water to 100 ml.
II - Hydrochloric Acid
- 5-10% aqueous solution decalcification slower than nitric acid but still rapid. Fairly good nuclear staining.
- Weak acid e.g. formic acid, acetic acid and picric acid of these formic acids is extensively used as acid decalcifier. 5-10% aqueous solution or with additives like formalin or buffer are used
III - Formic Acid
- Brings out fairly rapid decalcification.
- Nuclear staining in better.
- Requires neutralization and thorough washing prior to dehydration.
IV - Trichloroacetic Acid
Decalcification: 4- 8hr
- It permits good nuclear staining.
- It does not require washing out; the excess acid may be removed by several.
- It is a weak decalcifying agent, not used for dense tissues, and is suitable only for small spicules of bone.
- It is a very slow- acting; hence is not recommended for urgent examinations.
- The surface of the block to be decalcified is trimmed with scalpel. The block is then placed in acid solution at 1%hydrochloric acid face downwards so that acid bathes the cut surface for 15-60 min.
- As penetration and decalcification is only sufficient for a few sections be cut the block shall be carefully oriented in microtome to avoid wastage of decalcified tissue.
Decalcification of Bone marrow biopsy
- Tissue after fixation in Bouin's or Zenker's fixative is decalcified for (2½) hours followed by an hour of washing. The tissue is then dehydrated beginning with alcohol.
Use of Ion exchange resins
Ion exchange resins in decalcifying fluids are used to remove calcium ion from the fluid. Therefore ensuring a rapid rate of solubility of calcium from tissue and reduction in time of decalcification. The resins an ammoniated salt of sulfonated resin along with various concentrations of formic acid are used. The resin is layered on the bottom of a container to a depth of = ½ inch, the specimen is allowed to rest in it. After use, the resin may be regenerated by washing twice with dilute N/10HCL followed by three washes in distilled water. Use of Ion exchange resin has advantage of
- faster decalcification
- tissue preservation and
- cellular details better preserved
Chelating agents are organic compounds which have the power of binding certain metals. Ethylene-diamene-tetra-aceticacid (EDTA), disodium salt called Versenate has the power of capturing metallic ions. This is a slow process but has little or no effect on other tissue elements. Some enzymes are still active after EDTA decalcification. Versenate 10 gm.Distilled water 100 ml(pH 5.5 to 6.5)Time 7-21 days.
This is based on the principle of attracting calcium ions to a negative electrode into a decalcifying solution.
Washing after Decalcification:
Through washing of the tissue before processing is essential to remove acid (or alkali if neutralized has been carried out) which would otherwise interfere with staining)
Determination of end point Decalcification
- Flexibility method: Bending, needling or by use of scalpel if it bends easily that means decalcification is complete . This method is Unreliable, causes damage and distortion of tissue.
- X-ray method: Best method for determining complete decalcification but very costly. Tissue fixed in mercuric chloride containing fixatives cannot be tested as they will be radio opaque.
- Chemical Method: It is done to detect calcium in the decalcifying fluid, when no further calcium is detected, decalcification is considered complete.
- Take 5 ml of decalcifying fluid from the bottom of container which has been in contact with the tissue for 6-12 hrs.
- Add 5 ml each of 5% ammonium oxalate and 5% ammonium hydroxide.
- Mix and let it stand for 15-30 min.
- A cloudy solution caused by calcium oxalate indicates that specimen is not thoroughly decalcified.
- Absence of turbidity indicates completeness of decalcification.
Softening of Tissues
Unduly hard tissues which are liable to damage the microtome knives may require tissue softneres, aside from decalcification.
Treatment of Hard Tissues
Keratin and chitin are softened by use of concentrated sulphuric and with that aid of heat keratin is completely dissolved from the tissue sections. But much tissue distortion will also occur.
Immersing hard tissues in this solutions for 12-24 hours will make sectioning easier and excellent preparation of calcified arteries, thyroid and calcified glands is possible.
It is very useful for tissues which became hard at the time of fixation. Following washing out of the fixative, tissue is immersed in a 4% aqueous solution of phenol for 1-3 days.