Question

Describe the steps that one should follow when using a tissue processor when performing pre-embedding process in histopathology.

Answer 1

Microscopic analysis of cells and tissues requires the preparation of high quality sections that is appropriately strained to demonstrate normal and abnormal structures . Mainly there are two strategies :

• We can freeze the tissue and keep it frozen while we cut our sections and called frozen sections.
• Alternatively we can infiltrate our tissue specimen with a liquid agent that can be subsequently converted into a solid that has appropriate physical properties which will allow thin sections to cut from it.Paraffin wax is such an agent.This produces so called "paraffin sections".

Steps in tissue processing for paraffin sections.

1.Obtaining a fresh specimen

Fresh tissue specimens will come from various sources. They can be easily damaged during removal so they should be handled carefully and appropriately fixed as soon as possible after dissection.

2.Fixation

The specimen is placed in a liquid fixing agent such as formaldehyde solution. 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. Specimen should remain fixative for between 6 and 24 hrs 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.

Following fixation the specimens may require further dissection to select appropriate areas for examination. Specimens that are to be processed will be placed in suitable labelled cassettes to segregate them from other specimens. The duration of 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.

3.Dehydration

Because melted paraffin wax is hydrophobic, most of th water in the 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 solutions 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 should used to avoid excessive distortion of the tissue.

A typical dehydration sequence for specimens more than 4 mm thick would be:

1.70% ethanol -15 min

2.90%ethanol -15 min

3.100%ethanol -15 min

4.100% ethanol -15 min

5.100%ethanol -30 min

6.100% ethanol -45 min

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

4.Cleaning

Wax and ethanol are largely immiscible we should use an intermediate solvent that is fully miscible with ethanol and paraffin wax.This solvent will replace the ethanol in the tissue, then this in turn replaced by molten paraffin wax.This stage is called "clearing "and reagent used is called "clearing agent ".Clearing agent also removes substantial amount of fat from the tissue otherwise presents a barrier to wax infiltration.

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

1.xylene 20 min

2.xylene 20 min

3.xylene 45 min

5.Wax infiltration

The tissue can now be infiltrated with suitable histological wax, parafgin wax based histological wax are more 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.

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

1.wax 30 min

2.wax 30 min

3.wax 45 minmin

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 "embedded centre " where a mould is filled with molten wax and the specimen placed into it.The specimen is very carefully oriented in the mould because its placemtwill determine the "plane of section " an important consideration in both diagnostic and research histology. A cassette is placed on top of the mould, 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 mould 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 archive material.