Histopathology

Fixative : Types, Classification, Reaction

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

Types of Fixatives:

Physical Agents:

Heat
Ultra Violet Rays
Microwaving
freeze-drying

Chemical Agents:

Coagulants
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