By : Maria Wynnchuk, Pathology Department, Women's College Hospital, Toronto, Ontario, Canada
A theoretical and historical overview of paraffin wax processing is presented. Processing affects chemical and physical changes in each step of the procedure, therefore an appropriate choice of reagents and processing times can minimize microscopic artifacts. Solutions to processing problems are offered.(The J Histotechnol 16:71, 1993)
Key words: artifact, paraffin tissue processing
Most contemporary surgical pathology laboratories use automated paraffin tissue processors to process surgical specimens. Each customizes the schedule to meet specific needs. Positive or negative pressure and heat are now commonly used to accelerate reactions and facilitate more
efficient reagent exchange. Microwave stimulated diffusion has recently been applied to processing with favorable results, and it significantly reduces the time required to process tissue compared to conventional methods (1). This method has yet to receive wide acceptance for routine use. Because various sizes and types of tissue specimens are processed during each cycle, it is all but impossible to devise a processing schedule with optimum times for each reagent. Most schedules err on the side of overprocessing. The excessive times in reagents, together with the resulting physio-chemical changes, can cause tissue to become hard and brittle, contributing to microscopic artifacts during sectioning and staining. The size and type of tissue specimen, processing times, and processing reagents used will determine the severity of such artifacts. Chemical and physical changes occur in tissue during the routine paraffin processing steps of fixation, dehydration, dealcoholization, and infiltration with paraffin wax. Each of these steps plays a significant role in preserving and influencing how the tissue charge sites will react in a staining procedure. The sectioning quality of paraffin infiltrated tissue is also affected. Formaldehyde is the fixative most commonly used in routine procedures to preserve and harden tissue. Its use as a fixative was first described by Blum in 1893 (2). Although he had used formaldehyde as an antiseptic, it was not until he accidentally left an anthrax-infected mouse in a dilute solution overnight that he realized its hardening effect. He then investigated the effects of a simple 4% aqueous solution on a variety of tissues with celloidin methodology. Increasing grades of alcohol, usually ethyl alcohol, have
been used to dehydrate tissues since the middle of the last century (2). This process has not changed significantly in current practice. Lockhart Clarke established the use of clearing agents in 1851 by treating sections with turpentine before mounting them in balsam (2). Today's clearing agents, such as xylene and toluene, are used on tissues to penetrate and remove alcohol, allowing infiltration with paraffin wax. Paraffin wax was discovered by Karl Von Reichenbach during his studies of industrial wood-coke and iron manufacture in 1830 (3). His investigations were designed to yield greater amounts of harder charcoal from the available wood with simultaneous recapture of the liquid products of the dry distillation. One of these byproducts was paraffin wax. Its first use as an embedding medium for processed tissue is credited to Professor Edwin Klebs in 1867.
During fixation with 10% neutral buffered formalin the formaldehyde (in equilibrium with carbonyl formaldehyde and methylene glycol) penetrates rapidly (as methylene glycol) and fixes slowly (as carbonyl formaldehyde) through covalent bonding (4). As fixation proceeds, the equilibrium favors the formation of more formaldehyde from the dissociation of methylene glycol. Proteins, glycoproteins, nucleic acids, and polysaccharides are fixed by formaldehyde in the fixation process in a crosslinking additive manner at sites that contain reactive hydrogen (5). These sites occur in primary and secondary amines, amides, sugars, fats, aromatic amino acids, and sulfur containing amino acids. The nature of these sites is altered as methyl groups are added to tissue molecules. Cationic tissue groups are softened and anionic tissue groups are hardened. Even though the physical characteristics are also altered considerably during the intra- and intermolecular crosslinking of the macromolecules, the amount of tissue shrinkage is minimal (4). A study by Fox with histomorphometric methods indicates that the concentration of formaldehyde is not critical to fixation, as adequate results were obtained with concentrations varying from 2-20% (4). Significantly more nuclei were preserved when temperature was increased to 37C during fixation; lowering the temperature to 4C increased the number of intracellular spaces. He recommended fixation with formaldehyde for at least 24 hr at room temperature or 16 hr at 37C even though "the process of fixation, as judged by purely histological standards, is not complete until at least
7 days have elapsed" (6). Boon demonstrated that the fixation time for tissue blocks less than 5 mm thick can be significantly reduced by microwave irradiation (7). Tissues immersed in formalin for 4 hr were irradiated for 1.5 min. Consistent microscopic results were evident throughout the block, even in the central portion. Reducing atmospheric pressure to increase the rate of fixation with formaldehyde contradicts the generally accepted rule that reactions increase with increases in pressure. The penetration rate of formaldehyde into tissue increases under the effect of a vacuum (8).
Graded alcohols are commonly used to remove water from tissue before dealcoholization with a solvent that is miscible with paraffin (5). Free water is removed in a simple exchange process by diffusion. Bound water, which is attached to tissue molecules by hydrogen bonds, may be removed during excessive dehydration. Remember that additional fixation occurs during dehydration by denaturation, and bound water is removed in a non-additive reaction (5). Such excessive dehydration causes tissue to become hard, brittle, and difficult to section. In addition, some lipids are also dissolved. The efficiency of the dehydrating agent depends on its hydrogen bonding strength and molecular weight. A powerful hydrogen bonding dehydrant that competes with tissue molecules, will more readily remove bound water, and a smaller molecular size penetrates the tissue more readily. Vacuum and increased temperature also speed up the dehydration process (5). However, if either of these are used, it is important to reduce the dehydration time. Microwave irradiation can also be used to enhance diffusion, penetration, and the exchange rate (1). Ethyl alcohol is the traditional dehydrating agent. Others are methanol and acetone. Isopropanol (99%) is probably the best substitute for ethyl alcohol. Isopropanol has long been successfully used as a substitute with few deleterious effects on tissue. "The tolerance of its mixtures with various paraffin solvents for small amounts of water at least equals on the average that of corresponding ethanol mixtures," according to Lillie (9).
The aromatic hydrocarbons xylene and toluene are, perhaps, the most commonly used reagents to remove alcohol from tissue before infiltration with paraffin. Some lipids may be dissolved during this process, which is advantageous because lipids and paraffin are not very miscible. Hydrogen bonding parameter, molecular weight, negative pressure, increased temperature, and microwave irradiation all accelerate clearing in a similar fashion as that described for dehydration(1,5). The time of tissue exposure to these clearing agents is critical as excessive dealcoholization can further denature tissue molecules in the same manner as excessive dehydration to cause difficulties in sectioning. Although xylene and toluene are in common use, toluene is more suitable in humid environments; it is more tolerant of atmospheric water contamination of tissue processing reagents (10). Commercial substitutes such as Histoclear (National Diagnostics, Sommerville, NJ), Micro-Clear (CZ Scientific Instruments Canada Inc, Markham, ON), and Safety-Solv (Esbe Laboratory Supplies, Markham, ON) are now available. The change to these less toxic clearing agents must be considered, especially if results are shown to be comparable to those obtained with traditional clearing agents.
Infiltration and Embedding with Paraffin Wax
Infiltration with wax provides physical support for the tissue during sectioning but necessitates the use of heat. Excessive heat denatures tissue molecules, coagulating protein and making tissues hard and brittle. As with all the steps in processing, negative pressure may be used to advantage to accelerate paraffin infiltration. The melting point and crystalline structure of paraffin wax influences the section quality (8). Although a low melting point wax is less brittle when solidified than a wax with a high melting point, it will compress more during sectioning. Higher melting point waxes provide better support for hard, firm tissue specimens than lower melting point waxes do. Paraffin wax of small crystalline structure fits closely to the embedded tissue, providing good support for sectioning, whereas wax of larger crystalline structure adheres poorly to tissue, providing less support during sectioning. In the latter case, the degree of curvature of the paraffin section may be more pronounced and difficult to flatten after sectioning (8). The speed at which the molten paraffin is cooled influences the crystalline structure and its pattern. The faster a paraffin is cooled, the smaller will be the size of crystals formed. In the deeper portions of the block, the paraffin crystals are larger and looser, resulting in poorer cutting texture than in the superficial portion (8). It is imperative, therefore, that paraffin blocks be rapidly cooled to promote the uniform formation of small paraffin crystals.
Numerous physical and chemical changes occur in tissue during the paraffin processing technique. Some of these, such as removal of bound water, may contribute to microscopic artifacts during sectioning and staining. The severity of artifacts can be minimized if appropriate reagents are carefully selected and processing times reduced to compensate for accelerated reactions when heat and vacuum are used. Rapid cooling of paraffin blocks is also important.
Sincere thanks to Mrs Susan Cromwell, Miss Karen Chiba and Miss Michelle Nadin for excellent secretarial support. Special thanks to Mr Patrick D. Horne, RT, BSc for his expert advice and helpful criticisms throughout the preparation of this manuscript.
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