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Optimal Fixation & Processing, Part 2 – Tissue Processing

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The preceding article (Optimal Fixation), discussed fixation as the foundation of tissue processing, upon which the success of all other steps is dependent. In this article we will discuss the post fixation steps in routine histological tissue processing (dehydration, clearing, infiltration), and how they must be administered and maintained to achieve complete and optimal processing. After all enzymatic activity in the cell has been suppressed or ‘fixed’ to prevent cellular degradation, the three successive steps previously mentioned are applied to first remove all water from the tissue with dehydrants (primarily graded alcohols); secondly to clear the alcohol from the tissue (typically with xylene), in preparation for the next step/solution; and third, to infiltrate the tissue with an embedding medium (infiltration wax) that will give the tissue/cells the structural support needed to withstand the thin sectioning of microtomy.

With all the transition of solutions that has been discussed, a vital action that must be observed is optimal fluid exchange from one reagent to the next. Because of the complete penetration of a solution down to the cellular level, it is unlikely that one solution can be totally drained from fixed tissue before the next solution is administered. Thus, there must be a gradual process of exchange from former to new solution. The image to the right demonstrates the gradual change of thermal convection where heat is slowly introduced to change the environmental temperature of a colder element.
These are the same dynamics involved with fluid exchange throughout tissue processing. In the first step, dehydration, placing tissue directly into pure dehydrant can and mostly likely will result in cellular disruption where the cell membrane rapidly shrinks forming folds, wrinkles and distorting the natural morphology. A series of graded alcohols (70%, 80%, 95%, 100%) is necessary to gradually remove water without affecting structure or cellular components. Time and multiple changes of solutions are key factors in not only the dehydrant but also all subsequent solutions to follow. As a standard there should be 45 minutes to an hour time in each solution, and a minimum of 1-2 changes of solution depending on the solution. As previously mentioned, how solutions are administered is critical.
Under-dehydration can result in residual bound water in tissue blocking subsequent reagents, inconsistency in tissue profile, and soft and mushy center. The image to the right was used in the previous article and again now to demonstrate incomplete penetration of fixative and/or dehydrant. The tissue in the soft center will block the effectiveness of the next solutions and result in an under processed, undiagnosable area, and lead to reprocessing steps. Over-dehydration can result in fragmentation of the molecular bond in the tissue, disintegration of the protein molecule, and brittle fragmentation (flakes) during microtomy.
Alcohol (dehydrant) is immiscible with the final solution (infiltration wax), so there must be a solution that can clear out the alcohol and prepare the tissue for wax. This solution must be miscible with both alcohol and wax. Xylene is the best solution for basic tissue processing, and since we are discussing this interstitially at the cellular level, time and multiple solution changes are again critical factors for complete removal of dehydrant. Typically, the same time/changes standard as in the dehydrant are also applied to xylene. Xylene is a known carcinogen and must be used in observance of safety standards in relation to skin and respiratory exposure.
The final step, infiltration, serves to give structure and support to cells and tissue stroma to keep the tissue in proper relationship during microtomy. Most embedding waxes are fairly quick infiltrates that can accommodate various sizes and density in tissue specimen (breast, bone, etc.). Paraffin at the molecular level is a simple straight chain hydrocarbon, as seen in the image to the right.

There are various paraffin embedding waxes in the market, each with their own proprietary blend of ingredients. A primary classification distinguishing infiltration waxes can be looked at by the amount and type of additives manufactured into the wax and can be classified as high polymer and low polymer. Polymers and other additives (plastics, resin, etc.) are large polymers with repeating units, which when added to the original paraffin molecule create an enhanced polymer designed for a specific purpose and use. With respect to microtomy, polymer additives give more ribboning strength to the wax and typically allow you to achieve thinner sections during microtomy. However, the addition of these elements to the original molecule subsequently create a larger molecule, and ‘in theory’ create a slower infiltration medium. Thus, a high polymer wax ‘in theory’ is a slower infiltration wax and would be more desirable for microtomy; while a low polymer wax, ‘in theory’ may be more desirable for initial wax infiltration during processing.

This ‘theoretical’ discussion above is the source of why some laboratories choose to use one type of low polymer wax medium in their tissue processors for infiltration, and a high polymer wax in their embedding centers for microtomy. Thus, the Histologist has various choices of high/low polymer waxes to choose from in the market. In reality, most paraffin embedding waxes in the market today are designed for universal application for processing and/or microtomy. Using a high polymer wax for microtomy does not necessarily mean that the same wax will not perform successfully in infiltration. The decision as to whether one works better than the other depends on the personal preference of the user. This author personally prefers a high polymer wax because the added polymers do give a higher performance during microtomy. Ideally, if I could choose two waxes for my lab, I would ‘personally’ choose a high and a low, but if only given the choice of one, I would choose a high polymer wax for both embedding and microtome.

Having discussed the three elements of post-fixative tissue processing, it must again be emphasized that optimal processing can only be achieved through the administration and maintenance of principles such as: the integrity of solutions (using fresh reagents), time in solutions (mandatory processor start times), and daily monitoring of end results. Daily maintenance of processor machines and consistency in start and run times of tissue are the backbone of achieving optimal tissue processing. For more technical information contact your Customer Relations representative for Lab Storage Systems today.

References:

  1. Brown, HS, “The Dynamics of Fluid Transfer in Fixation & Processing”, Technical Development Series, Lab Management Consultants, 2017.
  2. Carson, FL, ‘Histotechnology-A Self-Instruction Text’, Chicago, Il., ASCP Press, 1997.
  3. PaperNerd Contributor, “Heat Transfer”, Science Essays, Wikipedia, 2001.
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