Tissue Preperation
Before a physician can examine the tissue for diagnosis, it must first be prepped and turned into microscope slides for easy viewing.
Tissue Fixation
Tissue fixation is the first step in preparing the tissue for a microscope slide. The purpose of this step is to prevent tissue degradation. Immediately after the tissue is collected, it should be transferred into a fixative such as formalin. Other options are available, but most tissue specimens are fixed in a 10% neutral buffered formalin. After 24-48 hours, the tissue will be fixed and ready for the next step.
Specimen Transfer to Cassettes
Once the tissue has been fixed, it is then trimmed using a scalpel and fit into a labeled tissue cassette. A tissue cassette is a disposable plastic structure that is used to hold tissue samples. The cassettes fit in microtome chuck adapters and are designed to keep specimens safe during processing.
Tissue Processing
The first phase of tissue processing involves dehydration, in which the tissue is immersed in increasing concentrations of alcohol so that water and formalin can be removed. Next, the tissue is rinsed in an organic solvent to remove the alcohol and prepare the tissue for the embedding phase. During the embedding phase, the tissue specimen is covered in molten paraffin wax to create a formalin-fixed paraffin-embedded (FFPE) block. Once the block has solidified, the block is ready for sectioning.
Sectioning
Wax is removed from the surface of the block to expose the tissue, which is then chilled for 10 minutes. A microtome is used to create thin tissue slices, which are carefully transferred to a warm water bath. Next, they are placed on a slide under the water level. Slides are then placed upright and allowed to dry at 37℃ for a few hours to allow the excess paraffin wax to melt.
Staining
Staining is used to allow transparent cells to be easily viewed. A coverslip is then mounted over the tissue specimen and carefully glued down.
Once tissues are acquired, samples are carefully handled and stored to maintain quality. Failure to properly place tissue samples in storage may cause cell degradation, which alters research results. Preparation for storage usually depends on specific requirements, including tissue size, cut, or purpose. A medical pathologist ensures that the sample is placed in storage throughout preparation to ensure quality remains intact.
If a sample study is completed, the tissue is placed in a storage bank as a cassette, slide, or block. Records, including donor information, consent forms, and when the tissue was collected, are kept in case research teams need to access the tissue sample again.
Tissue Storage Conditions
Storage Containers and Specimen Size
Tissue specimens should be stored in storage containers that are structurally stable and ensure stability at freezing cold temperatures. The freezing and thawing of samples should be kept at a minimum to avoid introducing contaminants or damaging biological markers, which would make analysis unreliable. It is recommended that tissue specimens be divided into smaller portions and frozen in separate containers to reduce these possibilities. There is no “minimal size” that is recommended for storage. The best practice is that the specimen should be reasonably sized for the storage space and resources of the facility.
High-density shelves increase storage cabinet capacity for cassette, microscope slides, or paraffin block storage. Most laboratories require more cabinet space for tissue storage. Finding high-density cabinets tailored to leave little wasted space is excellent for storing tissue and paraffin blocks. High-density cabinets can be adjusted, so that paraffin blocks stack together and sit in cabinets with no added space between one another. Proper storage cabinets will extend to the top of a storage room to prevent wasted overhead space. Storing tissue through paraffin blocks in a cabinet makes it easy to handle increased storage without complications.
Temperature
Temperature storage must be consistent with maintaining cell integrity and preventing degradation. Most tissue banks store specimens at -70℃ or lower in ultracold freezers such as the Stirling Ultracold SU780XLE or SU105UE for longer terms. Tissue samples should never be stored in self-defrosting freezers as it will cause samples to get freezer burn. Specimens can be transported in either dry ice or a portable ultra-cold freezer such as the ULT25NEU.
