Why do we change the wax during tissue infiltration
Answers
1. DEHYDRATION
The first stage in tissue processing is dehydration (the removal of water). In tissues, water is present in both free and bound forms and needs to be removed before processing can continue. Dehydration is usually carried out using alcohols (such as ethanol) but these can dissolve certain cellular components such as lipids. Although dehydration can also cause tissue shrinkage, the stage is necessary in all infiltration methods, except where tissues are supported by an aqueous embedding medium (such as water-soluble waxes).
In paraffin wax processing, dehydration from aqueous fixatives such as formalin is usually initiated in 70% alcohol before progressing through 90%-95% to absolute alcohol before proceeding to the clearing stage. However, direct transfer to 95% alcohol is often performed if tissues are adequately fixed. Duration of dehydration is dependent on tissue thickness; the thicker the block, the longer the time. Generally, blocks 1 mm thick should receive up to 30 minutes while blocks 5 mm thick require up to 90 minutes or longer in each change.
2. CLEARING
Clearing is the transition step between dehydration and infiltration with the embedding medium. Although tissues are water-free following dehydration, infiltration with wax cannot be carried out because wax and ethanol are largely immiscible. Many dehydrants are immiscible with paraffin wax and a solvent (clearing agent or ante medium) miscible with both the dehydrating agent and the embedding medium is used to assist the transition between these steps. The term clearing arises because some solvents have a high refractive index. When dehydrated tissues are placed into these reagents, they are rendered transparent. This property is used to determine the endpoint and duration of the clearing step since the presence of opaque areas indicates incomplete dehydration. Clearing agents are fat solvents and therefore remove fat from the tissue. It must be noted that shrinkage occurs when tissues are transferred from the dehydrating agent to the clearing agent and from the clearing agent to wax. In the final stage shrinkage may result from the extraction of fat by the clearing agent. Xylene is the most popular clearing agent and several changes of it are required to completely displace the ethanol. The choice of a clearing agent depends upon the type of tissue processor used, the processing conditions such as temperature, safety factors and cost.
3 INFILTRATION AND EMBEDDEING
Infiltration
This is the saturation of tissue cavities and cells by a supporting substance which is generally the medium in which they are finally embedded. The most common agent of choice is paraffin wax which is molten when hot and solid when cold. An infiltrating and embedding medium should ideally be molten between 30°C and 60°C and suitable for sectioning. Additionally, the properties of the medium should be similar to those of the tissues to be sectioned with regard to density and elasticity. Various substances have been used to infiltrate and embed tissues in readiness for eventual section cutting or microtomy.
The use of vacuum infiltration is often used to help complete impregnation of tissues with wax. This is carried using a molten wax or other medium under reduced pressure. Vacuum assistance helps to not only reduce the time tissues are subjected to heat but it also assists in the complete removal of any remaining solvent. Modern tissue processors are equipped to deliver vacuum and pressure during tissue processing.
Embedding
Paraffin embedding is the standard method used in histology laboratories to produce blocks of tissue for section cutting (microtomy). This process is usually carried out using an embedding centre and involves surrounding the tissues by a medium such as paraffin wax which when cooled and solidified will provide sufficient support for section cutting or microtomy (see stages below). The production of properly oriented and accurately labelled blocks is one of the essential skills of trained histologists and includes knowledge and understanding in areas such as tissue sampling, identification and human anatomy.