Tissue Processing Overview: Steps & Techniques for Histopathology

1. Obtaining a fresh specimen

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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 theater, or, if this is not possible, immediately following transport to the laboratory.

2. 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:

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. 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.

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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:

1. xylene   20 min
2. xylene   20 min 
3. 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:

1. wax      30 min
2. wax      30 min
3. wax      45 min

  6. Embedding or blocking out

Tissue processing is an essential and first step in anatomic pathology laboratory techniques. It is designed to remove all extractable water from the tissue, replacing it with a support medium that provides sufficient rigidity to enable tissue sectioning without damage or distortion.

There are two types of tissue processing machines in the histology world. They are a carousel-type processor (traditional tissue transfer) and a microwave tissue processing machine. The former was the first automated tissue processor used in the histology laboratory that transports tissues in cassettes through a series of steps known as fixation, dehydration, clearing, and impregnation by using chemicals of formalin, alcohol, xylene, and paraffin wax, respectively. It requires 12 hours to complete the whole processing, mostly histology laboratory professional process overnight, to receive the completed, processed tissue the next morning. The latter, microwave processing, in principle uses previously fixed tissue that is dehydrated rapidly using microwave energy to heat the reagent alcohol to just below its boiling point. Isopropanol further dehydrates the tissue and prepares it for paraffin infiltration. The residual isopropanol is effectively “boiled out” using microwave energy to heat. The heat uses for the faster penetration of the chemicals through the tissues resulting in rapid processing. The tissue should be thoroughly fixed before being placed in the microwave for tissue processing. It uses only three reagents: ethanol, isopropanol, and paraffin wax, and it takes a  maximum of two and half hours to complete the whole processing. When we look at the differences of these two tissue processing machines, the differences are mainly: in the chemical they use, the quality of microscopic tissue, the time taken to the whole process, and safety.

As mentioned above, the microwave uses only three types of chemicals: ethanol, isopropanol, and paraffin wax, whereas a carousel-type processor uses formalin, ethanol, xylene, and paraffin wax. Overall, the quality of microscopic tissues from traditional and microwave processing methods was highly similar. It was not possible to distinguish between the two techniques by studying the tissue sections. The time taken to complete the whole tissue processing by microwave and traditional processing is two and half hours and 12 hours, respectively. This shows the microwave method is better in shortening turnaround time (TAT) to complete the diagnosis. Turnaround time has been an important issue for many years. It has become increasingly important in this age of managed care and commitments to reducing healthcare services costs.

As far as safety is concerned, it is essential to mention that microwave tissue processing uses high temperatures for a rapid chemical reaction to penetrate the tissue for rapid processing. The histology laboratory professional must be careful when removing the cassettes in the beaker from the microwave to avoid burns and be far from the beaker when opening the cover lid as high vapor comes from the heated alcohol. Avoiding xylene by microwave tissue processing machine can be an advantage over the traditional one.

References:

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1. L Carson, C. Cappellano, Histotechnology, A self-Instructional text, America Society for Clinical Pathology press, 4th ed. Page 48,  
2. L. Ralph Rohr, Lester J. Layfield, D. Wallin, D. Hardy, A Comparison of Routine and Rapid Microwave Tissue Processing in a Surgical Pathology Laboratory, America Society for Clinical Pathologists, Am J Clin Pathol,  ;115:703-708
3. Experience With an Automated Microwave-Assisted Rapid Tissue Processing Method, America Society for Clinical Pathologists, Am J Clin Pathol, ;121:528-536
4. D. Bancroft, M. Gamble, Theory and Practice of Histological Techniques, Churchill Livingstone, 6th edition, page 84,