Fixation and Processing

Specimens are usually received in the laboratory in formalin. Formalin is formaldehyde gas dissolved in water which produces a weak solution of methanol and the active component, carbonium ions. Formalin is a "fixative" and is probably the most widely used of these. Fixatives prevent degradation of tissue samples caused by putrefaction (rot caused by micro-organisms) and autolysis (a self-destruct system mediated by suicide bags called "lysozomes" contained within every cell). Aldehyde fixatives such as formalin cause the formation of cross-links between proteins resulting in a matrix which traps all the molecules present, thus preventing their interraction. No rot. For best results, specimens should be totally immersed in at least 15 times their own volume of formalin for 48 hours or more. In practice, this isn't always possible.

After the cut-up has been performed, cassettes are often left in formalin for a few more hours - or overnight - to allow fixation to complete before loading the cassettes into a tissue processing machine.

Tissue processors have the job of removing all the water from a tissue sample and replacing it with paraffin wax. This process must be done slowly to minimise damage to the tissue caused by dehydration and shrinkage. The process is usually begun either in formalin or in 50% or 70% alcohol. As the processing schedule progresses, the cassettes are passed through stronger solutions of alcohol (typically 50%, 70%, 96%, 99%) and afterwards may in some cases also be exposed to acetone to complete the dehydration process. After this the tissues must be "cleared".

Clearing is a stage in which the cellular material is rendered optically clear so that later, light will be more able to pass through the sections when they are viewed under the microscope. Clearing agents are usually dangerous organic solvents. The one most commonly used, Xylene, is no exception to this rule as it is both harmful and flammable. Xylene mixes well with both alcohol and paraffin which means it usefully bridges the gap between two otherwise immiscible substances.

At last, the cassettes are bathed in warm, therefore fluid, paraffin wax. Usually a processor has capacity for either two or four changes of paraffin as one is insufficient to remove excess xylene from the samples which would leave them soft.

The following morning, the cassettes are opened and the tissue from inside is embedded in fresh paraffin wax. These are cooled until they are hard. The results are known as blocks.

The blocks are usually made with the help of an embedding machine like this one.
The tissue is removed from the cassette...
...the tissue is embedded in cooling paraffin wax...
...and the cassette bearing the case number is added to the block which...
...is then left with others to cool.
When cool, the blocks are turned out of their moulds. The surfaces to be cut look like this.