What is Fixation in Histopathology?

Fixation

Fixation is a preservations after death of the shape structures and chemical constituents of tissues and cells. Soon after death, tissues and cells begin to undergo change leading to their breakdown and ultimate destruction. Such change may be due to the actions of enzyme normally present in the tissues themselves and so the change are called autolytic actions or self-destruction. The change can also be due to external influence such as bacteria which causes decomposition and putrefaction. These bacteria maybe does that constitute the normal flora of the body or pathogens that are present due to a disease process.

This change can be slowed down by low temperature or prevented by fixation.

Adequate and complete fixation is the foundations of all good histological preparation. Fault in fixation cannot be rectified at any later stage and the finished section can only be as good as its initial fixation.

The stabilization of the protein part of the framework of a cell is an important functions of fixation.

In addition to preserving the tissues and cells, the fixatives (i.e. the fixing fluid or vapour) also make them resistant to the subsequent treatment during processing.

It is essential that tissues are fixed as soon as possible after death or removal from the body.

The tissues should never be allowed to dry as a it will shrink.

The length of time required for adequate fixation varies according to the size and constituency of the issues and The fixative employed.

If delay information cannot be avoided, the tissue should be kept moist and chilled.

The aim of fixation is to prevent the tissue undergoing adverse change and to preserve the tissues in as life-like manner as possible.

The idea fixative in order to fulfill this you should meet the following requirements:

1. Prevent autolysis and putrefaction.

2. should not add or remove from the tissues constituent.

3 should not cause any shrinkage or swelling.

4. should penetrate the tissues and cells rapidly evenly and deeply.

5. prevent distortions by any reagent used subsequently.

6. Should impact a suitable hardness and texture to allow for easy sectioning.

7. should render the tissues receptive of the stains

8. should be non-toxic non corrosive and non inflammable.

9. should be cheap and easy to prepare.

10. should be stable.

11. should allow for long-term storage of the specimen.

12. should allow restoration of some natural colours for museum work and photography.

So far in the search for diagnostic perfection, there is no single fixative that fulfill all of the above requirements. Many substance on their own have individual properties that make them fairly good fixatives.

These substance are referred to as simple fixatives.

So in order to obtain a fixative which will comply as closely as possible with the conditions requirement for fixation it become necessary to combine several simple fixatives of to achieve the desired effect, the resulting solutions is referred to as compound fixative.

Compound fixative

Compound fixative is therefore by definition a solutions of two or more simple fixative mix together in order to obtain the combined effect of their individual actions upon the cells and tissues constituent.

Choosing a fixative

The choice of a fixative is the determined by the nature of the investigation required.

Several mixture have become established as routine general fixative, but most of them have been devised for specific purpose and are not suitable for routine use.

Fixative are usually grouped according to the actions upon the cells and tissues come to constituents. Those which preserved the tissues as well as allow the general microscopical studies of the tisues structures and also allow the various layers of the tissues and cell to retain their formal relationship with each other are called microanatomical fixative.

Those fixative which are devise for specific actions upon specific areas of the cells are known as cytological fixatives.

This group of fixative are further subdivided into nuclear and cytoplasmic fixative.

It is obvious from the attachment which part of the cell each act upon.

Practically does cytological fixatives with a pH of less than 4.6 or those which contain glacial acetic acid Nuclear fixative and the reverse is true for cytoplasmic fixative.

Secondary fixation is a Technique where by considerable improvement over the result obtained by the use of 10% formalin alone is achieved.

All specimens are collected in 10% formalin and the block selected by the pathologist for processing are then treated with the second, more specialised fixative for a shorter length of time:

Thus the functions of formalin for bulk use distribution outside The laboratory, frozen section work, storage of gross specimen etc.

Is enhanced and supplemented by the secondary fixative. The method is however not without some shortcoming.

It complicate the processing schedule and for preparation such as saturated mercuric chloride solution, extreme caution is needed in handling and it is also necessary to remove pigment from the section. This consideration has missed a techniques less popular.

Whatever method of fixation is used, a large volume of fixative is required.

It is recommended that the volume of the fixative should be about 20 to 40 times that of the tissues. In case of large specimen such as whole spleen, this is not practicable.

Such large specimen should be slices at interval of 2cm or less to allow adequate penetration of the fixative.

For the same reasons, Hallow specimen like intestine and stomach should be slit open.

Credit:

medical laboratory science theory and practical by

J Ochei.

And A Kolhatkar