Decalcification

MR G.P. TIWARI Technologist Tata Memorial Hospital

Bones
Bones are dense connective tissue and rigid organs that form part of the endoskeleton of vertebrates. They function to move, support, and protect the various organs of the body, produce red and white blood cells and store minerals.

Purpose of bone biopsy 
Confirm the diagnosis of a bone disorder determine if a bone tumor is malignant (cancerous) or benign evaluate bone pain or tenderness . Check bone problems seen on an X-ray determine the cause of an unexplained infection or inflammation Find the cause of ongoing bone pain.

Purpose of bone biopsy cont.
‡ Bone disorder may include infection, cancer, or bone disorder (including Paget & apos disease, osteomyelitis, a bone cyst, or a benign bone growth called an osteoma). The bone tissue may also show osteoporosis or osteomalacia, Most cancer of the bone spreads to the bone from another part of the body, such as the breast, lungs, prostate, or other organs. But bone cancer can also start in the bone itself (such as osteosarcoma or multiple myeloma)

Decalcification

The process of removing the calcium from bony hard tissues which are difficult to cut for sectioning is known as decalcification

Principle of decalcification
Insoluble calcium salts are converted into soluble calcium salts by the action of decalcifying agent so that the tissue become soft. Chelating agent binds to calcium ions present in the bone and decalcification is carried out.

Purpose of Decalcification
‡ Decalcification is done to assure that the specimen is soft enough. To allow cutting with the microtome knife. If the tissues is completely decalcified ‡ Failure to decalcification results in torn, ragged sections and damage to the cutting edge of the microtome knife.

Criteria of a good decalcifying agents
‡ 1 Complete removal of calcium.
‡ 2 Less of damage to tissue , cells, fibers ‡ 3 Subsequent staining should not altered. ‡ 4 Short time required for Decalcification.

Specimens for decalcification µJ¶ Needle Biopsy 
Bone Marrow trephine biopsy  Sections from the bone tumors or Tumors producing osteoid/ chondroid . Sections from underlying bone in tumors of jaw or chest - mandible, rib assessment of bone involvement needs to be done in these cases.

Different techniques 
    

Acid decalcification Chelating agents Ion exchange resin method Electrophoresis removal method Microwave decalcification Surface decalcification

Acid Decalcifiers
acid Decalcifiers subdivided Strong acid / weak acid. Strong acid - e.g. Nitric and hydrochloric acid. weak acid- e.g. Formic acid Trichloroacetic acid

Acid Decalcifiers Cont. 
Nitric acid - 5-10% aqueous solution are widely used  HCL - 5-10% can be used. It is slower then nitric acid but gives Fairly good nuclear staining.  Weak acid e.g. formic, acetic and picric acid of these formic acids is extensively used as acid Decalcifier. 5-10% aqueous solution with additives like formalin or buffer are used.

Acid Decalcifiers Cont.
Formic acid
‡ 1. Brings out fairly rapid decalcification. ‡ 2. Nuclear staining in better. ‡ 3. But requires neutralization and thorough washing prior to dehydration.

CHELATING METHOD
Chelating agents are organic compounds that are capable of binding with certain metals. They are typically very slow acting and gentle, making them good fixatives for electron microscopy and immunohistochemistry studies. Ethylenediaminetetraacetic acid (EDTA) is the most common chelating agent used for decalcification. It binds with calcium ions and gradually depletes the crystal size of the outer layer of the hydroxyapatite crystal.

Advantage/disadvantage of EDTA
Advantage ‡ Good morphology ‡ Improve staining Disadvantage ‡ Time consuming ‡ costly

Use of Ion exchange resins
Ion exchange resins consist of a polymeric matrix and a functional group with a mobile ion which can be exchanged with other ions present in the solution to be treated. The most common synthetic structures are Cross-linked polystyrene Cross-linked polymethacrylate Phenol-formaldehyde

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 in this fluid 

The resins are an ammoniated salt of sulfonated resin along with various concentrations of formic acid are used

It has advantage of (ii) faster decalcification (ii) tissue preservation and iii) cellular details better preserved.

Electrolytic method
This is based on the principle of attracting calcium ions to a negative Electrode in decalcifying solution Decalcify with electrolyte apparatus with the below mentioned decalcifying solutions.

Decalcifying solution HCL (Conc.) 80ml Formic acid 90% 100 ml Distilled water 1000 ml.

ELECTROLYTIC DECALCIFICATION
THIS METHOD IS CARRIED OUT UNDER THE INFLUENCE OF ELECTRIC FIELD THE CALCIUM ION LIBRATED BY THE DECALCIFYING FLUID IS MORE RAPIDLY THEN ANY NORMAL METHOD (RICHMAN, GELFAND, AND HILLS. 1947) IT IS OBSERVED THAT THE INCREASED SPEED OF REACTION IS DUE TO RISE IN TEMPARATUREDUE TO PASSAGE OF THE ELECTRIC CURRENT(MOLENAAR)

Role of Microwave in decalcification
‡ microwave decalcification provides an efficient and reliable means of processing bony tissue for histological and histochemical examination. Decalcification time is significantly reduced with no apparent adverse effects on structural preservation or antigenicity (Keithley et al., 2000; Madden and Henson, 1997; Louw et al., 1994; Faria et al., 1992; Hellstrom and Nilsson, 1992; Ng and Ng, 1992; Roncaroli et al., 1991).

MICROWAVE DECALCIFICATION

Surface Decalcification
When cutting a tissue block the feeling of scraping and tears seen in the section means incomplete Decalcification to overcome this problem surface decalcification is carried out in this method cotton pad or sponge soaked with decalcified solution is used to wrap the tissue block for 15- 30 minutes Be certain to rinse the block before it is placed in the microtome.

Factor influencing the rate of decalcification
‡ ‡ ‡ ‡ ‡ Type of tissue Concentration of decalcifying agent Temperature Agitation Suspension

Procedure for Dense bone
‡ Dense bone may take days ± Often Decalcification is done in incubator to hasten the process ‡ Usual decalcification process - 10% formal nitric acid and kept overnight to fix and decalcify simultaneously ‡ Next day morning every section is checked for completion of decalcification with the help of a sharp pin. ( not ideal but practical)

Procedures for light decal tissue
‡ The tissue are kept in 5 % formal nitric acid in grossing room. ‡ Next day morning they are checked with sharp pin for the end of decalcification ‡ If the decalcification is over then they are processed as elaborated in procedure ‡ If the decalcification is incomplete then tissue are transferred in 5 % Nitric acid. ‡ For bone marrow ± AZF used as fixative so that decalcification starts at fixation level but slowly to preserve morphology. Also preserves immunogenicity of tissues

Decalcifiers

‡ Determination of end point of decalcification ‡ 1. Flexibility method ‡ Bending, needling or by use of scalpel if it bends easily that means ‡ decalcification is complete. ‡ Unreliable, causes damage and distortion of tissue.

‡ 2. 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. 

         

3. Chemical Method It is done to detect calcium in the decalcifying fluid when no further calcium is detected, decalcification in considered complete. Procedure 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.

‡ Neutralization : It has been said that following immersion in mineral acids, ‡ tissues should be deacidified or neutralized, before washing by treatment ‡ with alkali. This may be effected by treatment over night in 5% lithium or ‡ sodium sulphate.

Acetic acid zinc formalin (AZF)-for bone marrows
‡ ‡ ‡ ‡ Zinc chloride Conc. Formalin Glacial acetic acid Distilled water 12.5grams 150ml 7.5ml 1000ml

Troubleshooting
‡ If paraffin embedded bones were not decalcified completely : ‡ one can soak the paraffin blocks in the same decalcification solution for a few minutes before cutting. ‡ One can soak the paraffin blocks in the soap solution for a few minutes before cutting. ‡ One can overcool the paraffin bocks before cutting.

SAFTY
‡ Acids used to make decalcifying solutions should be handled carefully, concentrated acid is very hazardous. ‡ Nitric acid: Corrosive to skin mucous membranes and most metals, toxic by inhalation. ‡ Wear apron, gloves and goggles for handling however small the quantity. ‡ Always add acid to water never water to acid to avoid severe splattering. ‡ Explosive mixtures may be formed with hydrogen peroxide, diethyl ether and anion exchange resins. ‡ Handle Concentrated Acid very carefully with personal protective devises, as it is very corrosive 

Adequate washing is necessary as incomplete washing interferes with routine staining of decal sections. Such sections fail to take up nuclear stain i.e. haematoxylin.  Prior to processing, always keep decalcified tissues in 70% alcohol for 30 minutes & again wash under running tap water.  Incomplete decalcification interferes in cutting. Therefore this tissue needs to be reprocessed and then same tissues should be put in 10% Nitric acid or 5% Nitric acid depending upon amount of calcification and proceed in the usual manner.  Excessive heat during decalcification should be avoided as over decalcification deteriorates the morphology of the tissues.  ³Cardboard´ labels should be put in cassettes as the regular ordinary papers are torn (charred) due to action of strong acid.  Tissues should be handled delicately while checking the endpoint of decalcification. Care should be taken not to spoil the tissues by piercing the pin too frequently in the tissue.

Points to remember
‡ Adequate washing is necessary. ‡ Incomplete

Conclusion
‡ Decalcification is a challenging task in histotechnology ‡ Awareness of different methods and variations can ³save the day´ for a technician and a pathologist

A QUANTITATIVE STUDY OF DECALCIFICATION METHODS IN HISTOLOGY H. H. W. Verdenius and L. Alma

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