A Brief Guide to Tissue Fixation and Its Importance in Pathology Analysis

Fixation is the first key factor to ensure the scientific value and quality of biological specimens. It is important for at least 3 purposes:
1) To stop tissue autolysis by hydrolytic enzymes released from cells, and thus to better preserve cellular morphology for analysis.
2) To immobilize tissue and cellular antigens for immunolabeling of these antigens, and
3) To offer better preparation of samples for histological sections.

1. A Brief Guide to Tissue Fixation Fixation is the first key factor to ensure the scientific value and quality of biological specimens. It is important for at least 3 purposes: 1) To stop tissue autolysis by hydrolytic enzymes released from cells, and thus to better preserve cellular morphology for analysis. 2) To immobilize tissue and cellular antigens for immunolabeling of these antigens, and 3) To offer better preparation of samples for histological sections.

2. The Types of Fixation Physical Methods Chemical Fixation Physical methods include heating, microwaving and cryopreservation. Heat fixation is rarely used on tissue specimens, its application being confined to microorganism smears. Microwave fixation is now widely practiced in routine laboratories. Cryopreservation, usually in the form of freeze drying, has some applications in histochemistry but is not usually applied to diagnostic tissue specimens. Chemical fixation include immersion fixation, perfusion fixation, and vapor fixation. Chemical fixation is usually achieved by immersing the specimen in a fixative (immersion fixation) or by perfusing the vascular system with a fixative (perfusion fixation). For some specialized histochemical procedures, fixatives are occasionally applied in vapor form.

3. Non-coagulant fixing agents chemically react with proteins and other cell and tissue components, becoming bound to them by addition and forming intermolecular and intramolecular cross-links. Cross-Linking Denaturation This effect is induced by dehydrants such as the alcohols or acetone. These reagents remove and replace free water in cells and tissues and cause a change in the tertiary structure of proteins by destabilizing hydrophobic bonding.

4. Time Interval Specimen Dimension Concentration of Fixative Volume Ratio Buffering pH Temperature Duration Factors Involved in Tissue Fixation

5. The Classification of Fixatives Aldehydes: Formaldehyde/10% Formalin/Glutaraldehyde Oxidizing agents: Permanganate fixatives/Dichromate fixatives/Osmium tetroxide Alcohols: Carnoy’s /Methanol/Ethanol Picrates : Bouin's Mercurials : Mercuric chloride/B-5/ Zenker's

6. Which is the Better Fixative? Fixatives act by denaturing or precipitating proteins which then form a meshwork due to cross linking of proteins. This meshwork tends to hold the other cell constituent in vivo relation to each other and insoluble proteins provide mechanical strength for subsequent procedures.

7. Formalin Different cell types have highly specific growth requirements, therefore, the most suitable media for each cell type must be determined experimentally. Advantages It is cheap, relatively stable and easy to prepare. Allows most routine staining. Does not make tissues very hard or brittle. Frozen section is possible with formalin fixed tissues. Natural color may be restored. Most commonly used fixative and is the best fixative for the nervous system. Tissue penetration is good. Disadvantages It may cause dermatitis and asthma in allergic individuals. There may be formation of dark brown artefact granules especially in tissues containing much blood (liver, spleen). It is slow to act (less tissue penetration). Gradual loss of basophilic staining or nucleus and cytoplasm. So, prolonged fixation is not advisable. Loss of myelin reactivity when Weigert iron hematoxylin stain is used.

8. Glutaraldehyde Advantages Disadvantages Formation of more cross linkages with better preservation of cellular & fluid proteins. Expensive. Resists acid hydrolysis. Less stable. Causes less shrinkage than formalin. Penetrates tissue more slowly from formalin. More pleasant & less irritant. Inferior formalin for PAS satin. Does not corrode metal.   Does not cause dermatitis.  

9. Advantages Disadvantages Methyl alcohol (80-100%) is excellent fixative for smears. Causes marked shrinkage, unless used at 0 ℃ or cooler. Ethyl alcohol is used as a fixative for enzymes. Distorts morphology and hardens the tissue. Carnoy’s fluid is used for urgent biopsy (paraffin processing within 5 hours). Contraindicated for lipid study. Good fixative to demonstrate glycogen. Although glycogen can be demonstrated it causes polarization, as the streaming of glycogen granules to one pole of the cell. Alcohols

10. Zenker’s Fixative Advantages Disadvantages Better staining of nuclei and connective tissue. Recommended for fixing fetal brain. Solution rapidly deteriorates. Gives best results for metachromatic staining. Corrodes most metals except nickel alloy. Cytoplasmic staining with acidic dyes is enhanced. Zenker’s solution removes iron of hemosiderin and causes RBC lysis.   Tissues become very hard and brittle if left for 1-2 days in fixation.   Reduces the demonstrable glycogen in tissues. Better staining of nuclei and connective tissue. Recommended for fixing fetal brain. Solution rapidly deteriorates.

11. Bouin’s Solution Advantages Disadvantages It is a good fixative except mammalian kidney and penetrates rapidly. Shrinkage is minimal. Prolonged fixation causes tissue hard and brittle. Good fixative to demonstrate glycogen. It lyses RBCs and reduces the iron content. Small fragments easily visualized because of yellowish color after fixation. Lipids are both altered & decreased. Solution is stable.   Best for testicular biopsies.  

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