5 th Lecture

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The majority of tissue fixatives are made up in aqueous solution and the function of the dehydrating agent is to remove free and bound water molecules from the tissue specimen. The most commonly employed dehydrating agent, for routine paraffin processing, is 99.85% ethanol which, due to its expense, is purchased by laboratories in the form of the less expensive 99% industrial methylated spirit (IMS) containing 2% methanol. Certain lipids and water-soluble proteins are removed from the tissue during this stage.

Dehydration time is dependent on the size and type of tissue specimen. In general, tissue blocks of 1 mm thickness require 30 minutes in each graded alcohol and tissue blocks of 5 mm thickness require up to 90 minutes in each graded alcohol.

The clearing agent is a reagent that is miscible with paraffin wax and acts as a link between the dehydrating agent (non-miscible) and the paraffin wax itself. The most commonly used clearing agents for routine tissue processing are hydrocarbons such as xylene, toluene, chloroform and petroleum solvents. These reagents are all controlled substances under the COSHH regulations, are flammable, cause skin degreasing and have varying degrees of toxicity.

Toluene is a possible carcinogen, chloroform has a narcotic effect and xylene, although not proved to be a carcinogen, may cause headaches as a side-effect to inhalation of the vapour where adequate extraction facilities are not provided (usually in cover slipping and manual staining areas). For the reasons stated previously, xylene is probably the most widely used clearing agent. It is compatible with all the major enclosed tissue processors and is relatively cheap.

Paraffin wax is the infiltration and embedding medium of choice for routine histopathology. Paraffin wax is categorized by its melting point.
Melting points, although not wholly accurate, are in the range 39 –68 oC and, as a general rule, the higher the melting point the harder the wax.
Softer tissues benefit from infiltration by lower melting point waxes and harder tissues from infiltration by those with a higher melting point.
However, routine histopathological processing is partly a compromise, due to the variation in size and hardness of the tissue samples to be processed and the preferred clearing agent. To this end, paraffin wax of melting point 56 –58 C is normally employed.

Infiltration times are greatly reduced by the application of a vacuum. This is essential for specimens of lung, where the vacuum forces out trapped air molecules from the tissue.
Additives have been included in some paraffin waxes to improve their crystalline consistency by reducing their hardness and brittleness and, hence, improve their cutting (microtomy) characteristics.
These substances include, among others, thermoplastic resins, plastic polymers and dimethyl sulphoxide
(DMSO), which aids infiltration and allows thin sectioning.

(Table.1): ‘Routine overnight’ and ‘rapid’ schedules for tissue processing

Paraffin wax for tissue embedding should be clean, filtered and held at
2 –4 oC above its melting point. the tissue basket is transferred to a heated reservoir or free-standing bath of paraffin wax. Tissues are removed and placed face down in a prefilled mould of molten paraffin wax. The mould is placed on a refrigerated surface or ice tray to facilitate the clamping of the tissue to its base. Moulds are left on the cold plate until the wax solidifies or, where a form of refrigeration is not available, can be immersed in cold water once a thick enough crust has formed on the surface of the wax.

Commercial embedding centers, which combine a heated mould store, a molten wax reservoir/dispenser and a cold plate, are readily available (Figure.1). Once the wax has solidified, the tissue blocks may be gently removed from their moulds and prepared for microtomy.

(Figure.1): Shandon Histocentre 3 tissue embedding center

Carousel-type ‘open’ tissue processors (Figure.2a) have mostly been replaced by ‘enclosed’ (Figure.2b), Health and Safety-compliant processing machines with larger cassette capacities. Open processors release reagent vapours into the atmosphere as the tissue basket moves between stations and, therefore, should be contained within a well-ventilated area/extraction facility. Tissues are processed through the solvents at room temperature. Enclosed processors are standalone/modular units which fully contain reagent fumes during tissue processing by means of water/vapour traps and charcoal adsorption filters.

Microtome is the production of thin sections, 1 –5 microns (mm) in thickness, from paraffin wax-embedded tissue blocks. This process utilizes a microtome in conjunction with a microtome knife or disposable microtome blade and blade holder. Microtomes are of the ‘rotary’ or ‘base sledge’ variety and are commercially available from a large number of laboratory equipment suppliers.
The microtome knife has largely been superseded, in routine histopathology departments, by the disposable microtome blade. Microtome knives need to be specially sharpened, either manually (a highly skilled procedure) or by machine, which can be extremely timeconsuming in an age where workload activity demands the streamlining of histopathology services.

The rotary microtome (Figure.3) is a general-purpose machine for the production of semi-thin tissue sections for light microscopy. The block moves up and down against the knife in a vertical plane with the production of flat sections on the downward stroke, usually 3 –5 mm thick for routine histological purposes. The section thickness range of these microtomes is usually 0.5
–60 mm.
The sledge microtome, although best suited for the sectioning of hard tissues and some softer resins, is equally suitable for the sectioning and ribboning of soft tissues and biopsies.

Conventional steel microtome knives have, in the main, been replaced by disposable blades for routine histopathology. Steel knives are manufactured from a high-grade carbon or tool-quality steel that should be free from impurities and rust-resistant. Knives are tempered (hardened) from the tip inwards and the degree of hardness measured using the Vickers scale
(usually 400 –900 on the scale).

The degree of hardness can vary between manufacturers and between individual knives from the same manufacturer.
A hardness of 700 is desirable, 400 being soft and 900 being considered brittle, for routine use. Steel knives come in a variety of profiles, dependent on their intended use.

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Profile A:
 is biconcave and It is rarely used in routine histopathology.
Profile B:
 is plano-concave.
 Profiles A and B produce the sharpest edges.
Profile C:
 is wedge-shaped and is the most commonly utilized steel knife for routine histopathology. It is a more rigid knife than profiles A and
B and is used for paraffin wax sectioning of all tissues.
 This profile cannot be ground as sharp as profiles A and B.
Profile D:
 is tool-edge or plane-shaped and is used for sectioning hard materials, such as resins and extremely hard paraffin wax tissue blocks.
 This knife produces the least sharp edge when ground.

Disposable blades are modified, thickened razor blades produced from high-quality stainless steel and give reproducible, compression-free, good quality sections. The thin blade is held rigidly in its own special holder to minimize vibration during microtomy. The blades may be coated with platinum or chromium for a longer cutting life.
These blades appear to be the first choice, nowadays, for routine histopathology.