Chemical or Physical Agents which are used to prevent and preserve the tissue are called fixatives.

Types of Fixatives:

Physical Agents:

1. Heat
2. Ultra Violet Rays
3. Microwaving
4. freeze-drying

Chemical Agents:

1. Coagulants
2. Non Coagulants

Classification of Fixative:

On the basis of working condition

• 95% ethyl alcohol best fixative for cytology
• Formalin best fixative for histopathology

Hopwood Classification of Fixatives:

1.  Aldehyde Fixatives:

• Formaldehyde Fixatives
• Gluteraldehyde Fixatives
• Acrolein
• Glyoxal

2.  Oxidizing Agents:

• Osmium tetroxide
• Potassium permanganate
• Potassium dichromate

3.  Protein Denaturing Agents:

• Acetic Acid
• Methyl Alcohol
• Ethyl Alcohol

4.  Cross Linking Agents:

• Carbodiimides
• Dimethyl Subrimidate

5.  Unknown Mechanism/Miscellaneous:

• Mercuric Chloride
• Picric Acid (Explosive when dry or when complexes with metal and metallic salts)

Aldehydes:

• Aldehydes include formaldehyde (formalin) and gluteraldehyde.
• Tissue is fixed by cross-linkages formed between the proteins particularly between lysine residues.
• Only those lysine residues which are on the exterior of protein molecule react.
• The reaction with formaldehyde is largely reversible by excess of water especially over the first 24 hour of reaction time.
• With gluteraldehyde the reaction is rapid and irreversible.
• Reaction between aldehydes and proteins is pH dependent proceeding more rapidly at high pH.
• Fixation denatures proteins to some extent which is acceptable and satisfactory for routine pathology work.
• For enzyme histochemistry, immunocytochemistry and electron microscopy this issue should be considered.
• Enzyme molecules and antigenic sites must not be altered or destroyed by chemical fixation.
• Shapes of DNA and RNA should not be changed.
• Gluteraldehyde causes loss of up to 30% of αhelix structure of protein and formaldehyde markedly less.
• Formaldehyde does not give as good morphological picture as gluteraldehyde.
• Cross linkage does not damage the arrangement of proteins significantly therefore antigenicity is not vanished.
• Therefore, formaldehyde is good for immunoperoxidase techniques. Formalin penetrates tissue well, but is relatively slow.
• Normal solution is about ten (10%) percent neutral with buffered formalin solution.
• A buffer stops acidity which stimulate autolysis and reason for precipitation of formol haem pigment in the tissues.
• Formaldehyde in its 10% neutral buffered form.
• Neutral Buffered Formalin (NBF) is the best fixative in diagnostic pathology labs.
• Pure formaldehyde is a vapor which when completely dissolved in water forms a solution containing 37–40% formaldehyde; this aqueous solution is known as ‘formalin’.
• The typical 10% formalin used in the fixation of different histopathological. This may consists of four (4%) percent weight/volume of formal.

Formaldehyde Fixatives:

• 37% Formaldehyde is available commercially and should be stored at room temperature in close and air tight container
• Clear, colorless and no precipitates
• Storage should not be more than 1 year
• Already opened bottles should not be used more than six months
• 10% formalin should not be used more than 3 months.

Other variants of formaldehyde containing fixatives:

• Mostly in lab formaldehyde buffered to pH 7 with phosphate buffer is used.
• Use of neutral buffered formalin stops formation of formalin pigment which forms in non-buffered formaldehyde solutions.
• For lipids formol calcium is used.
• Alcoholic formalin has the advantage of rapid fixation and especially recommended for glycogen.
• They are carcinogenic.
• PPE should be wear during working with formalin.

Gluteraldehyde:

• Gluteraldehyde causes deformation of alpha-helix structure in proteins so it is not good for immunoperoxidase staining.
• Though, it fixes very rapidly and excellent for electron microscopy.
• It infiltrates/penetrate very poorly and unwell, but provides finest cytoplasmic and nuclear features.
• The standard solution is a 2% buffered Gluteraldehyde
• Rapid irreversible reaction have led to its use as a sterilizing agents of fiberoptic endoscope

Mercuric Chloride Containing Fixative:

• These fix tissue by an unknown mechanism.
• Examples include fixatives as Zenker, Susa and Helly’s fluid.
• These fixatives penetrate poorly and cause some tissue shrinkage so mostly used in combination of other fixatives or as secondary fixatives.
• Their best application is for fixation of hematopoietic and reticuloendothelial tissues and fetal brain
• They comprise mercury so should be dispose of sensibly.
• Mercuric chloride is significantly preferred for its abilities of improving the staining qualities of tissues, mostly for trichrome stains.
• Tissues fixed with mercuric chloride fixatives (except susa) contain black precipitate
• This is removed by treating in alcoholic iodine soln. and then decolorizing in sodium thiosulfate.

Alcohol Containing Fixative:

• Alcohols, as well as methanol and ethanol, are protein denaturants and are not suitable for daily basis for tissues because they create too much brittleness/fragility and hardness/rigidity in the tissues.
• They are excellent for cytological smears because their action is rapid and provide maximum nuclear details and features.
• Spray cans of alcohol fixatives are promoted to labs perform PAP smears but inexpensive hairsprays do fair as well.
• Recommended for preservation of glycogen
• Example include carnoy’s fixative

Oxidizing Agents Fixatives:

• Oxidizing mediators/agents contain permanganate fixatives for example potassium permanganate, dichromate fixatives such as potassium dichromate, and osmium tetroxide.
• Few of them have specific applications but are used very rarely.
• Some of them are identified according to their appliance of action but osmium tetroxide make crosslinking with proteins.

Picric Acid Containing Fixative:

• They have an unknown mechanism of action.
• Examples include Bouin’s, gendre’s and rossman’s fluid.
• Picric acid has ability of burst/explosive danger in dry its form.
• Yellow color may be removed but another acid dye or lithium carbonate or washing with changes of 50-70% alcohol

Reaction of fixatives of proteins:

• In fixation of tissues for routine histopathology the most important reactions are those which stabilize proteins
• Proteins comprise more than 50% of dry body weight
• The principle involved is that the fixative have the property of forming crosslinks with proteins forming a gel which provides strength and keeping everything as close to their in vivo state as possible
• Soluble(globular) proteins are fixed to structural(fibrous) proteins and rendered insoluble and whole structure is given mechanical strength allowing subsequent preparations to take place
• Fixation normally takes place in aqueous solution. Occasionally vapors can be used.

Reaction of fixatives with Nucleic Acids:

• Fixation bring about changes in physical and chemical state of DNA & RNA
• In their native state DNA & RNA do not react with formaldehyde at room temperature
• Reaction mixture are heated 450C for RNA and 650C for DNA
• High temperature relates to the uncoiling of DNA & RNA
• Up to 30% of nucleic acids are lost during fixation
• Alcoholic fixatives like ethanol, methanol and carnoy’s fixative are commonly used
• DNA will collapse at 65% conc. Of ethanol

Reaction of fixatives with Lipids:

• They are largely removed during tissue preparation
• If lipid are to be demonstrated specifically frozen sections or cryostat should be used
• Formol calcium is the choice of fixative

Reaction of fixatives with Carbohydrates:

• Alcoholic fixatives are the fixative of choice for glycogen.