Introduction to histology
Introduction to histology

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Introduction to histology

2.1 Fixation

It is important that the original structure of the tissue is preserved before it reaches the histopathology laboratory. As they die, cells break down releasing enzymes from their lysosomes and other intracellular organelles, which start to hydrolyse components of the tissue - a process termed autolysis. The degree of breakdown depends on the tissue and what has happened. For example, post mortem tissue is not usually taken until several hours or days after the person has died, and this would have undergone much more autolysis than a surgical specimen. Different components of tissue vary in their sensitivity to enzymatic digestion - RNA is particularly sensitive, most proteins less so and DNA may remain intact for very long periods. Cells and soft tissues tend to be more susceptible to breakdown than bone, cartilage, tendons and proteins of the extracellular matrix. A histologist has to be aware of all these processes and distinguish between changes that are due to a disease and those due to tissue autolysis.

Hydrolysis is the breakdown of proteins and nucleic acids, by reaction with water. These reactions are generally slow, but occur quickly if catalysed by enzymes. Proteins are hydrolysed by proteases; DNA and RNA are hydrolysed by nucleases.

SAQ 3

Why might you expect DNA to be more stable than RNA?

Answer

DNA is the genetic material, which must essentially remain stable for generations. RNA is an intermediate required in the formation of proteins. It is unstable and it breaks down in the cell when synthesis of the proteins is no longer required.

To minimise tissue breakdown, samples are often placed in a solution of fixative. However, since different fixation procedures are appropriate for each staining technique, it is important to know what technique will be applied to a tissue when it is taken. In addition to preventing autolysis, fixation may serve to retain the structure of the tissue and limit microbial growth. The most widely used fixative for light microscopy is 4% formaldehyde (formalin). The aldehyde group reacts covalently with amine groups (NH2) on amino acids such as lysine which blocks the activity of proteins, including lysosomal enzymes. Glutaraldehyde acts in a similar way, but because it has two aldehyde groups it can cross-link proteins and tends to harden tissue and preserve morphology (anatomical structure and cell shape). The two fixatives may be used in combination for particular purposes (Figure 5).

Figure 5 Fixation reaction of glutaraldehyde. The amine groups (-NH2) on amino acid residues in the protein, react with aldehyde groups (-CHO) in the fixative.

Another approach is the use of reagents such as ethanol, methanol or acetone, which disrupt hydrophobic bonds and protein structure and remove water from the tissue. There are critical differences between these two classes of fixative; aldehydes destroy amine groups, but tend to maintain tissue structure well: alcohols usually result in poorer preservation of structure (because they dehydrate cells) but do not destroy amine groups and they can preserve some secondary structure in proteins.

These considerations are important if immunohistochemistry (IHC) is to be used on the tissue. Antibodies bind to groups of amino acids within their specific target (antigen). If the target includes lysine, then the ability of the antibody to bind to the antigen may be destroyed. Histologists try to use antibodies that will work using a variety of fixatives; however, this is not always possible. This is the reason why specific fixation procedures may be recommended or required for particular applications.

Of course, if it is intended to derive live cells from tissue, then it must not be fixed. Freezing damages cells because of the formation of ice crystals, so tissue for cell culture is generally put into a culture medium, at 4ºC, and processed as quickly as possible.

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