HISTOPATHOLOGY

 HISTOPATHOLOGY

Decalcification

This is a process of removing calcium from bone or other mineralized hard tissue to make it soft enough for sectioning.

Method of Decalcification

         i.            Acid method

       ii.            Ion exchange method

     iii.            Chelation

     iv.            Eletrical ionization.

Acid method

This is the common method done in most laboratory.

Procedure

Calcification hard tissue is cut in to small pieces (2-6mm in size) with a thin blade, or sharp knife. The cut pieces are fixed in buffered formalin or natural formalin for 8-10 minutes.

Tissue is washed thoroughly in water to remove fixative.

Tissue is kept in decalcified solution which may be one of the following type

a.       5% HNO3

b.       5% Formic acid

c.       A mixture of formic acid & HCL (5% formic acid 10ml+HCL 8ml

d.       D/W 82ml)

The tissue is suspended in decalcified solution by mean of gauze bag tied by string which has been dipped in matted paraffin.

The bag is suspended in large quantity of decalcified solution (more then 20 times the volume of the tissue) string of the bag is tied with a support in the cover of the beaker so that tissue can be regularly. The decalcification takes few days usually 2-4 day, But it can be done rapidly if the beaker is kept in an incubator at 56 ̊c Decalcify solution is change every day until the process decalcification is completed.

 

Test for complete Decalcification

Roughly it can be done by mechanically bending the tissue if the tissue can be bent like a soft tissue. It is suggestive of complete decalcification. It can also be done by the tissue with fine needle if the needle the tissue easily it means there is no calcium in it. But final test for decalcification is done by chemical method.

 

Chemical is done ib following ways.

Take 5ml of decalcified fluid which was in contain, with tissue in a clean test tube. The fluid is take in a test tube A piece of litmus paper turns blue color. If the fluid turns turvid during this process it means calcium is present in the solution.

If the fluid does not turn turvid it means there is no calcium in it.

The tissue is again suspended in fresh decalcified solution for few hours (2-4 hours). The decalcified solution of this is also tested for presence of calcium it means decalcification is completed.

The decalcified tissue is removed from the bag & is washed running tap water for 4 hours to remove decalcify solution.

To neutralized the acid in decalcified tissue. The tissue is treated with magnesium carbonate to neutralize the intracellular acid in the decalcified cells. The tissue is now ready for dehydration.

 

 

 

 

Dehydration

This is a process through which water from cells & tissue is removed so that space is subsequently taken up by wax. The dehydration is done by passing the tissue through ascending grades of alcohol 50%. Alcohol, 70% alcohol, 80% alcohol, 90% alcohol, absolute alcohol  1hrs in each. Ideal in ethyl alcohol, if this not available isopropyl alcohol of methyl alcohol may be used.

 

 

Clearing

It is process of making the dehydration tissue transparent to necked eye by removing alcohol. So that tissue can be made for paraffin embedding. Usual chemical used for this process is xylene for a tissue of 5 mm thickness it takes one hour. Prolonged stain xylene makes the tissue brittle. Other chemical which may be used as clearing agents are.

         i.            Cedar wood oil :- It is expensive viscus and needs removal by xylene.

       ii.            Benzene :- It causes less shrinkage by xylene but it is carcinogenic.

     iii.            Chloroform :- It does not change refractive of the tissue and hence and point of tissue can not be made up.

     iv.            Toluene :- It is poisonous and hence should not be used.

 

 

 

Paraffin infiltration

Infiltration is process of removal of xylene from the tissue by diffusion in the surrounding melted way. This is done by placing the clear tissue in to paraffin bath. Counting molten wax. After xylene has be removed the molten wax replaces the clearing agent. This process is known as impregnation.

Paraffin wax is maintain is condition by temperature of 55-60 ̊c.  This temperature is maintain it temperature is known as paraffin bath. The tissue kept in paraffin bath for 2-3 hours if temperature of paraffin bath becomes too hot (75 ̊c) the tissue will get cooked which becomes unfit for histopathological Examination.