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Tissue Fixation

Mr. Aamir Rashid Khan BMLT

Fixation is the process to reduce damage to the biopsy material / tissue from external or internal factors. It is done in order to maintain morphology and contents of tissue. Complete Fixation is key to good quality section and staining.

Common fixatives
Formalin 10% NBF Bouins formula Carnoys fluid Hellys fluid Heat Freezing / cryo preservation


10% NBF
forms bonds with the amino and peptide groups of proteins very effective in stabilizing nucleic acids and nucleoproteins effective in stabilizing carbohydrates, glycogen and mucoproteins. Reacts with lipids but does not stabilize them.

How to fix
Tissues are fixed in a fixative solution by immersing it in sufficient quantity of fixative. Volume should be 15-20 times. Simple fixative or compound fixative. Conventional method, time consuming. Microwave fixation. Latest technology, quick.

Duration & factors

It depend on various factors Size 24-48 hrs for big tissue 4-8 hrs for very tiny biopsy. Type of tissue Type of fixative Common Factors affecting fixation are Agitation Temperature vacuum Microwave

Ideal fixative
There is no single fixative which is ideal. Compound fixative 10% NBF Close to perfect Good fixative qualities Easily available, cheap, easy to prepare, long lasting, storage at RT. Allows routine HE, special stains & IHC.

How to prepare
Formalin is available commercially as 3740% formaldehyde gas dissolved in water. In order to prepare optimal solution, 10%, its further diluted in 1:10 ratio. So the actual and final concentration of gas in 10% NBF is 3.7-4.0% only. Phosphate salts are added to increase buffering power, and avoid formation of formic acid.

Formalin (37-40%)- 100ml Disodium hydrogen phosphate- 6.5 gm Sodium dihydrogen phosphate 4.0 gm Distilled water- 900ml

Preparation steps
Weigh both the salt separately. Measure water in a glass or plastic container. Dissolve the salts in pre measured water, keep stirring the solution. Add formalin and mix thoroughly. Wait for 30 mnts. Check the pH (7.2-7.6) and use. Store at RT in close lid containers.

Problems & artefacts

Under fixation- poor processing, no cutting, no staining. Over fixation- hardening of tissue, difficulties in cutting, not good for IHC. Formalin pigment- brown black deposits, can be removed by treating sections in alkaline alcohol or saturated picric acid.

Formalin is Skin & eye irritant, long exposure may cause allergy. Wear gloves, mask, goggles while preparation. If possible prepare under fume hood. For further safety related issues refer osha website or read MSDS.

Thank you.