Fixation Of Histology Samples: Principles, Methods, And Types Of Fixatives

Fixation of histology sample is the first step of any histological and cytological laboratory technique. It is the process by which the cells in the tissue are fixed in a chemical and physical state, and all the biochemical and proteolytic activities within the cells are prevented so that the cells or tissues can resist any morphological change or distortion, or decomposition after subsequent treatment with various reagents. The fixation helps to maintain the tissue nearest to its original state in the living system.

Aims Of Fixation

The basic aims of fixation are the following:

• To preserve the tissue nearest to its living state
• To prevent any change in shape and size of the tissue at the time of processing
• To prevent any autolysis
• To make the tissue firm too hard
• To prevent any bacterial growth in the tissue
• To make it possible to have clear stain
• To have the better optical quality of the cells

Ideal Fixative

An ideal fixative should have the following qualities:

1. Prevention of autolysis of the cells or tissue.
2. Prevention of decomposition of the tissue by bacteria.
3. Maintaining the volume and shape of the cell as far as possible.
4. Consistently high-quality staining particularly routine stains such as hematoxylin and eosin stain and Papanicolaou’s stain.
5. Rapid action.
6. Cheap.
7. Non-toxic.

A large number of fixatives are available in the market. Each fixative has its own advantages and disadvantages. In fact, it is difficult to find universally accepted ideal fixatives.

Tissue Changes In Fixation

The following changes may occur in tissue due to

fixation:

1. Volume changes: Fixatives may change the volume of the cells. Some fixatives such as osmium tetroxide cause cell swelling. The

the exact mechanism of the change in volume is not properly understood.

However, the volume change may be due to (a) altered membrane permeability, (b) inhibition of the enzymes responsible for respiration, and (c) change of transport Na + ions. Formaldehyde may cause shrinkage of the volume by 33%. In an experiment, Bahr et al. noted that the shrinkage of tissue is inversely proportional to the formaldehyde concentration.

Similarly, glutaraldehyde also causes significant tissue shrinkage. However when glutaraldehyde and osmium tetroxide is used

as fixations in epoxy resin then a 70% increase of cell size is noted.

2. Hardening of tissue: The fixation changes the consistency of the tissue, and some amount of hardening occurs due to fixation.

3. Interference of staining: Fixation may cause hindrance of staining of enzymes. Formaldehyde inactivates 80% of the ribonuclease enzyme. It has been noted that osmium tetroxide inhibits hematoxylin and eosin staining.

4. Changes of optical density by fixation: The fixation may cause the change of optical density of the nuclei, and the nuclei may look condensed and hyperchromatic.

Types of Fixation

The fixative can be classified on the basis of the

following criteria:

A. Nature of fixation

B. Chemical properties

C. Component present

D. Action on tissue protein

Nature of Fixation

1. Immersion fixation: This is the commonest way of fixation in laboratories. In this technique, the whole specimen is immersed in the liquid fixative such as tissue samples are immersed in 10% neutral buffered formalin or cytology smear in 95% ethyl alcohol.

2. Coating fixation: This is commonly used in cytology samples. The fixative spray is used for easy transportation of the slide.

The main advantages of spray fixatives are:

(a) Fixation of the cells

(b) To impart a protective covering over the smear

(c) No need to carry liquid fixative in bottle or jar

The spraying over the smear should be smooth and steady, and the optimum distance of 10–12 inches should be maintained between the nozzle of the spray and the smear. The fixative spray usually consists of alcohol and wax. Therefore, this wax should be removed before the staining procedure.

3. Vapor fixation: In this type of fixation, the vapor of a chemical is used to fix either a smear or tissue section. The commonly used chemicals for vapor fixation are formaldehyde, osmium tetroxide, glutaraldehyde, and ethyl alcohol.

The vapor converts the soluble material to insoluble material, and these materials are retained when the smear comes in contact with the liquid solution.

4. Perfusion fixation: This is mainly used for research purposes. In this technique, the fixative solution is infused into the arterial system of the animal, and the whole animal is fixed. The organ such as the brain or spinal cord can also be fixed by perfusion fixation.

5. Freeze-drying: In this technique, the tissue is cut into thin sections and then rapidly frozen to a very low temperature. Subsequently, the ice within the tissue is removed with the help of the vacuum chamber at a higher temperature (-30 ° C).

6. Microwave fixation: Basic principle: Microwave is a type of electromagnetic wave with frequencies between 300 MHz and 300 GHz, and wavelength varies from centimeter to the nanometer. Scientific and medical microwave ovens operate with a frequency of 2.45 GHz and 0.915 GHz, respectively.

The electromagnetic field is created by the microwave, and the dipolar molecules such as water rapidly oscillate in this electromagnetic field.

This rapid kinetic motion of these molecules generates uniform heat. The generated heat accelerates the fixation and also other steps of tissue processing. The most important characteristic of microwave heat generation is a homogeneous increase of temperature within the tissue, and every part of the tissue is heated.

Essential Precautions For Fixation in General

Certain essential precautions are necessary for

proper fixation:

• The tissue should be free from excessive blood before putting it into a fixative.
• The tissue should be thinly cut in 3–5 mm thickness.
• The amount of fixative fluid should be 20 times more than the volume of the tissue.
• The tissue with fixative should be in a tightly screw-capped bottle.

Mechanism Of Fixation

Dehydration and coagulation of protein: Methanol and ethanol are commonly used coagulative fixatives. These two alcohols remove water from the tissue and causing destabilization of the hydrogen bonds and thereby disruption of the tertiary structure of the protein. 

However, the secondary structure of the protein is maintained. Ethanol is a relatively stronger dehydrating agent than methanol. The ethanol and methanol start work from 60–80% concentration, respectively.

The dehydrating fixative has two disadvantages:

1. Shrinkage of the cells
2. Removal of the soluble substances from the tissue

Cross-linking fixatives: Formaldehyde: Formaldehyde in an aqueous solution combines with water to form methylene hydrate, a methylene glycol.

Glutaraldehyde: It has two aldehyde groups that are separated by three methylene bridges. The aldehyde group of glutaraldehyde reacts with the amino group of the protein predominantly lysine. When one aldehyde group reacts with the amino group, the other free aldehyde group may help to cross-link.

Glutaraldehyde rapidly and irreversibly cross-links the protein. The penetration of glutaraldehyde is slower than formaldehyde.

Osmium tetroxide: Osmium tetroxide (OsO4) is mainly used as a fixative in electron microscopy. Item is used alone or as a combination with another agent.

Factors Affecting Fixation

The following factors may affect the fixation:

1. Hydrogen ion concentration (pH): Most of the fixatives work better in neutral pH. In fact, good fixation occurs when pH remains 6–8, and no morphological distortion is seen in that range of pH. 

There may be changes in the ultrastructure when the pH is too low or too high. In very low pH, the NH2 group of amino acid is converted to NH3, and the reaction between aldehyde groups of the fixative is reduced.

Usually, the buffer solution is added to maintain the pH of the fixative. The commonly used buffers in the fixatives are phosphate, bicarbonate, Tris, and acetate. The buffers should be chosen in such a way that they should not react with the fixative.

2. Temperature: Room temperature is alright for tissue fixation, and there is no difference in cell morphology from 0 to 45 ° C. However, the fixation time may be reduced at higher temperatures (60–65 ° C). At higher temperatures, the vibration and movement of the molecules are increased.

This increases the rate of penetration of the fixative within the tissue and accelerates the process of fixation. In case of a very high temperature, the antigen within the tissue may be destroyed. The enzymes are better preserved in lower temperatures, and for enzyme histochemistry 0–4 ° C is suitable.

3. Duration of fixation: The depth of penetration of fixative is directly proportional to the square root of time of fixation.

4. Osmolarity of the fixative solution: Osmolality of the fixative has a considerable effect on fixation. The hypertonic fixative solution causes shrinkage of the cell, whereas the hypotonic fixative solution induces swelling of the cells.

Electrolytes (0.9% NaCl) or sucrose may be added in the fixative to maintain the osmolarity of the fixative. Mildly hypertonic fixative (400–450 mm) is preferable for routine use in the laboratory.

5. Concentration: Very low concentration of fixative prolongs the time of fixation, and higher concentration causes rapid fixation. However, higher concentrations of fixative may cause tissue hardening, tissue shrinkage, and artefactual changes.

A mildly lower concentration of fixative with neutral pH is needed for proper fixation. The optimal concentration of formaldehyde is 10%.

6. Agitation: Agitation increases the rate of penetration and therefore rapidity of fixation. Optimum agitation is needed as slow agitation may have no effect on fixation, whereas rapid Agitation may have a detrimental effect on delicate tissue.

Preparation Of Different Formalin Solution

Glutaraldehyde is used as a fixative for electron microscopy because it fixes and preserves the ultrastructure. The fixation occurs due to the extensive cross-linking of the proteins.

The penetration power of glutaraldehyde is poor and therefore only a small piece of tissue should be used for fixation. Glutaraldehyde does not react with lipid or carbohydrates, and therefore it should

be used in combination with the other fixative.

Advantages:

1. Better fixation of ultrastructure.

2. Less cell shrinkage.

3. Preservation of protein is better.

4. Good cross-linking with collagen.

5. Less irritating.

Disadvantages;

1. Poor penetration and tissue should be less than 0.5 mm thick.

2. Less stable compound.

3. No lipid fixation.

4. Glutaraldehyde polymerizes above pH 7.5.

5. Costly.

Osmium Tetroxide

Osmium tetroxide is used for fixation in electron microscopy. It reacts with unsaturated bonds in the lipid molecules and fixes them. The penetration of the osmium tetroxide in the tissue is poor, and if it is used alone, then a good amount of protein and carbohydrate may be lost during fixation.

Advantages:

1. This is a very good fixative for lipid.

2. It preserves cytoplasmic organelles such as Golgi bodies and mitochondria,

3. Does not make the tissue hard,

Disadvantages:

1. It does not fix the proteins and carbohydrates and therefore it should be used in combination with another fixative.

2. Osmium tetroxide may react with the ribose group and may cause clumping of DNA. Este can be prevented by pretreatment with potassium permanganate or post-fixation with uranyl acetate.

3. Poor penetration in the tissue.

4. Tissue swelling may occur.

5. Toxic and volatilizes at room temperature producing harmful vapor. This vapor is toxic to the eye and respiratory tract.

6. Expensive.

Methyl and Ethyl Alcohol

Methyl alcohol (methanol) and ethyl alcohol (ethanol) are used as dehydrating agents, and these two alcohols are used mainly as fixatives of cytology smears.

The tissue or smear containing water should not be put directly in the higher concentration of alcohol as it may cause distortion of the cells due to the rapid rush of fluid from the cell. Therefore graded alcohol should be used for dehydration.

Acetone

It is mainly used for enzyme study and immunocytochemistry. It is a poor fixative for morphological preparation as it causes significant cell shrinkage. Acetone works by dehydration of cells. Cold acetone is used at 4 ° C for fixation.

Bouin’s Fixative

Bouin's solution contains picric acid. This is an excellent fixative for glycogen. It reacts with protein and forms protein picrate. The tissue penetration rate of picric acid is high, and it fixes small tissue biopsy within 3–4 h. Bouin’s fixative is not suitable for DNA quantitative study as it damages the cell membrane and causes hydrolysis of nucleic acid.

Advantages:

1. It is a good fixative for connective tissue and glycogen.

2. Rapid penetration rate.

Disadvantages:

1. It produces a yellow stain on the tissue.

Mercury Salt-Containing Fixatives

Among the heavy metals, mercury is commonly used as a fixative. This is a rapidly acting fixative. Mercury is a poisonous substance and should be used carefully. Mercury-containing fixatives may corrode the metal so the fixative should be kept in a glass container.