Tissue processing begins with fixation. The fixative of choice in most histology laboratories is 10% neutral buffered formaldehyde (NBF). That is: this solution is made by diluting stock formaldehyde to a 10% concentration with phosphate buffer in the pH range of 7.0 to 7.4. Since stock formaldehyde is actually a 40% solution, the final chemical concentration of formaldehyde used in fixation is 4% formaldehyde.
Formaldehyde fixes by crosslinking the proteins present in the tissue. Fixation times will vary, depending upon tissue type and size. The general “rule of thumb” is to prepare tissue such that it is no thicker than 2 mm (i.e. the size of a nickel), and it should be in a specimen jar containing formaldehyde equalling 25 times the volume of the tissue. It should be noted that extended periods of fixation may alter antigenicity of proteins and inhibit enzymes. Conversely, if tissue is not fixed long enough, subsequent processing may cause artefacts such as “bubbling nuclei”, which result when nuclei begin the fixation process in formaldehyde – but finish the process in alcohol, which “twists’ the nuclear material within cells, causing the bubbling artefact.
Substitute fixatives for formaldehyde are available. Laboratories must be certain to validate any substitutes selected for fixation and processing, as the appearance and color balance in the final microscope slide may be affected. The subsequent standard tissue processing procedure dehydrates the tissue with alcohol, clears with xylene and infiltrates with molten paraffin wax. The tissues can then be embedded in paraffin to make blocks that can be cut on a microtome, generating sections to be affixed to microscope slides and stained. Substitutes can be made using different alcohols/ alcohol blends, xylene substitutes and varying blends of paraffin wax and polymers. The same validation rules apply as detailed above.
Standard tissue processing may be carried out on any number of open and closed tissue processors, although closed processors are preferred due to safety concerns, both for the tissues and laboratory personnel. Closed system processors are “smart’ enough to prevent tissues from drying out in the event of a power failure, and the chemical fumes are kept inside the processor; released through filters and/or vented to the outside of the laboratory space.
Another tissue processing option is the use of microwave assisted processors that use conventional heat and microwaves to adjust and maintain temperature control during processing. Specimens are dehydrated through ethanol and isopropanol. Then, after vacuum vaporization, specimens are infiltrated with molten paraffin. The specimens are then ready for embedding. There are also ancillary units that will perform automated embedding of the tissues, if the proper cassettes are used.
A major advantage of microwave assisted tissue processors is the rapidity of processing of biopsy specimens. Small tissue biopsies of skin, prostate and gastrointestinal tissue can be processed into paraffin in approximately one hour. This is extremely valuable for cases requiring “rush” status. Another advantage is that, since no xylene is used, the tissues are generally much softer in the paraffin block, and therefore much easier to cut during mictrotomy, resulting in fewer cutting artefacts.
Processing procedures using microwave assisted tissue processors must be clearly and accurately defined, with much attention during the validation process. The fixation and dehydration steps must be complete to ensure proper infiltration with molten paraffin. Like routine tissue processors, the basic stages of tissue processing must accomplished:
- (a) Fixation of tissue to stabilize proteins and harden the tissue.
- (b) Dehydration of tissue to remove all unbound water.
- (c) Clearing of tissues to remove the dehydrant
- (d) Infiltration of tissue with molten paraffin, to ensure the embedding process is successful.
There are many factors involved in tissue processing, which provide many opportunities for things to go awry. Carry over of fixative into the processing alcohol can inhibit subsequent dehydration. If the absolute alcohol stations prior to the clearing stations contain water, this will result in incomplete dehydration as well. When water is left in the specimen, it cannot be removed by the clearant, and becomes trapped within the tissue during paraffin infiltration. The resulting paraffin blocks will be soft and difficult to cut during microtomy.
Conversely, tissue can become “over dehydrated” if the processing times in alcohol are too long. Tissues contain an amount of “bound water’ within the nuclei and some other tissue elements. If this water is removed during extended dehydration, the resulting paraffin blocks may be dry / scratchy / hard to cut during microtomy. Soaking in cold ice water, once the block is faced off, may sometimes be used as a remedy. This is common in laboratories that use only one tissue processor to process all of their tissues, regardless of size and type. In this case, smaller tissues (i.e. biopsies) may become dry and brittle for cutting.
- Theory and Practice of Histological Techniques. JD Bancroft, A Stevens ed. Churchill Livingstone, NY. Fourth edition. 1996