Tissue can be studied under the following headings

• 1. Examination of fresh cells
• 2. Dissociation of teasing method
• 3. Smear technique

Overview of the steps in tissue processing for paraffin sections

1. Obtaining a fresh specimen

• Collection of tissue

Fresh tissue specimens will come from various sources. It should be noted that they can very easily be damaged during removal from the patient or experimental animal. It is important that they are handled carefully and appropriately fixed as soon as possible after dissection. Ideally, fixation should take place at the site of removal, perhaps in the operating theatre, or, if this is not possible, immediately following transport to the laboratory.

2. Fixation

• Tissue fixation

The specimen is placed in a liquid fixing agent (fixative) such as formaldehyde solution (formalin). This will slowly penetrate the tissue causing chemical and physical changes that will harden and preserve the tissue and protect it against subsequent processing steps.2 There are a limited number of reagents that can be used for fixation as they must possess particular properties that make them suitable for this purpose. For example, tissue components must retain some chemical reactivity so that specific staining techniques can be applied subsequently.3 Formalin, usually as a phosphate-buffered solution, is the most popular fixative for preserving tissues that will be processed to prepare paraffin sections. Ideally, specimens should remain in fixative for long enough for the fixative to penetrate into every part of the tissue and then for an additional period to allow the chemical reactions of fixation to reach equilibrium (fixation time). Generally, this will mean that the specimen should fix for between 6 and 24 hours. Most laboratories will use a fixative step as the first station on their processor.

Following fixation, the specimens may require further dissection to select appropriate areas for examination. Specimens that are to be processed will be placed in suitably labeled cassettes (small perforated baskets) to segregate them from other specimens. The duration of the processing schedule used to process the specimens will depend on the type and dimensions of the largest and smallest specimens, the particular processor employed, the solvents chosen, the solvent temperatures, and other factors. The following example is based on a six-hour schedule suitable for use on a Leica Peloris™ rapid tissue processor.

3. Dehydration

Because melted paraffin wax is hydrophobic (immiscible with water), most of the water in a specimen must be removed before it can be infiltrated with wax. This process is commonly carried out by immersing specimens in a series of ethanol (alcohol) solutions of increasing concentration until pure, water-free alcohol is reached. Ethanol is miscible with water in all proportions so that the water in the specimen is progressively replaced by the alcohol. A series of increasing concentrations is used to avoid excessive distortion of the tissue.

A typical dehydration sequence for specimens not more than 4mm thick would be:

• 70% ethanol 15 min
• 90% ethanol 15 min
• 100% ethanol 15 min
• 100% ethanol 15 min
• 100% ethanol 30 min
• 100% ethanol 45 min

At this point, all but a tiny residue of tightly bound (molecular) water should have been removed from the specimen.

4. Clearing

Unfortunately, although the tissue is now essentially water-free, we still cannot infiltrate it with wax because wax and ethanol are largely immiscible. We, therefore, have to use an intermediate solvent that is fully miscible with both ethanol and paraffin wax. This solvent will displace the ethanol in the tissue, then this, in turn, will be displaced by molten paraffin wax. This stage in the process is called “clearing” and the reagent used is called a “clearing agent”. The term “clearing” was chosen because many (but not all) clearing agents impart an optical clarity or transparency to the tissue due to their relatively high refractive index. Another important role of the clearing agent is to remove a substantial amount of fat from the tissue, which otherwise presents a barrier to wax infiltration.

A popular clearing agent is xylene, and multiple changes are required to completely displace ethanol.

A typical clearing sequence for specimens not more than 4mm thick would be:

• xylene 20 min
• xylene 20 min
• xylene 45 min

5. Wax infiltration

The tissue can now be infiltrated with a suitable histological wax. Although many different reagents have been evaluated and used for this purpose over many years, the paraffin wax-based histological waxes are the most popular. A typical wax is liquid at 60°C and can be infiltrated into tissue at this temperature then allowed to cool to 20°C, where it solidifies to a consistency that allows sections to be consistently cut. These waxes are mixtures of purified paraffin wax and various additives that may include resins such as styrene or polyethylene. It should be appreciated that these wax formulations have very particular physical properties which allow tissues infiltrated with the wax to be sectioned at a thickness down to at least 2 µm, to form ribbons as the sections are cut on the microtome, and to retain sufficient elasticity to flatten fully during flotation on a warm water bath.

A typical infiltration sequence for specimens not more than 4mm thick would be:

• wax 30 min
• wax 30 min
• wax 45 min

6. Embedding or blocking out

Now that the specimen is thoroughly infiltrated with wax, it must be formed into a “block” which can be clamped into a microtome for section cutting. This step is carried out using an “embedding centre” where a mold is filled with molten wax and the specimen placed into it. The specimen is very carefully orientated in the mold because its placement will determine the “plane of section”, an important consideration in both diagnostic and research histology. A cassette is placed on top of the mold, topped up with more wax, and the whole thing is placed on a cold plate to solidify. When this is completed, the block with its attached cassette can be removed from the mold and is ready for microtomy. It should be noted that, if tissue processing is properly carried out, the wax blocks containing the tissue specimens are very stable and represent an important source of archival material.