National Health System Support Services
Tumour Bank
Annexe 1: General protocol for tissue treatment
- Transfer of tissue from surgeons to the pathologist
- First examination of the biopsy or surgical specimen
- Non-neoplastic tissue
- Fixation and Processing
- Freezing
- Identification of samples
- Conservation of fixed tissue
- Conservation of frozen tissue
- Security measures for frozen tissue
- Non-standard methods
1. Transfer of tissue from surgeons to the Pathology Department
The surgical specimens must be collected in the operating theatre immediately after their removal and transported in an unfixed state to the Pathology department. This transfer must be carried out as quickly as possible in order to minimise action of hypoxic phenomena on genetic expression. This protocol will be applied for all biopsies and surgical specimens whose possible diagnosis may involve any type of malignant neoplasia.
The best method for transferring the specimens is not to immerse them in liquid of any kind but simply to place them in a closed container and to transport them with the urgency as with frozen section diagnosis.
2. First examination of the biopsy or surgical specimen
The pathologist will decide whether it is possible to separate one or more fragments for freezing without this operation affecting the correct pathological diagnosis of the process.
The material selected for freezing must comprise, as far as possible, one or two fragments measuring 1.5 x 1.5 x 0.4 cm. Those areas with massive ischaemic and/or necrotic phenomena must be avoided. For each frozen fragment a fragment of similar dimensions will be selected in continuity with the frozen material. This second fragment will be selected for fixation in formaline will provide information surrounding the amount of tumour present in the block and will serve for additional immunohistochemical and molecular studies.
This entire process must be carried out in the most aseptic conditions possible.
3. Non-neoplastic tissue
Whenever possible, another pair of non-neoplastic fragments will be selected for freezing, fixation and paraffin embedding. These will be comprised of as many cellular components as possible.
Some studies will require the collection of buccal mucous as a source of fresh tissue. In such cases, a cytological brush or swab will be passed across the internal surface of the cheek following rinsing of the buccal cavity with an antiseptic liquid. After taking the sample, the end of the swab will be cut off, introduced into a cryotube with distilled water, and left well stoppered for 2-3 hours so that the cells dissolve in the medium. After this time, the end of the swab is discarded and the cryotube with the distilled water and cellular component is centrifuged for 10 minutes at 3 000 rpm. As much distilled water as possible is discarded in order to isolate the resulting cell pellet. The cryotube is thoroughly stoppered, frozen by immersion in liquid nitrogen or isopentane, and stored as if it were a conventional tissue sample.
4. Fixation and processing
Normal and neoplastic tissue, selected for fixation and paraffin embedding will be divided up into fragments no greater than 1.5 x 1 x 0.5 cm. These will be fixed in 10% buffered formalin for a minimum of 16 hours and a maximum of 48 hours after which time, they will be embedded in paraffin following conventional techniques. These specially selected fragments will be included in a duly identified histological cassette of characteristic colour.
5. Freezing
Samples will be frozen and duly identified in plastic cryomoulds (e.g., Cryomold standard, 25 x 20 x 5 mm, Tissue-Tek 4557, Distributor Bayer S.A) embedded in a cryosolidifiable medium (OCT-Compound, Tissue-Tek 4583, Distributor Bayer S.A) through immersion in a highly cryogenic medium that allows for rapid freeze.
The ideal medium for rapid freezing is isopentane that has been cooled to its freezing point (-160ºC). To achieve this the vessel containing the isopentane must be introduced in another container of liquid nitrogen. The freezing point approximately corresponds to the moment when opaque drops begin to appear in the isopentane. The process of cooling the isopentane is facilitated by keeping it in a chest freezer at -80ºC.
Direct immersion in cooled isopentane at –80ºC (kept in a chest freezer) or at temperatures around –50ºC (Histobath®, and similar) are equally valid and simpler.
Freezing by direct immersion in liquid nitrogen is a valid method, although it is more damaging to the tissue and hinders subsequent microscopical examination, especially when microdissection techniques are required. Liquid nitrogen vaporises when it comes into contact with the tissue, giving rise to irregular freezing. On the other hand, isopentane remains in a liquid state and thus avoids these artefacts, and as it is a very good cryoconductor, allows particularly rapid freezing.
Slow freezing by placing tissue in a refrigerator, chest freezer or cryostat must be completely avoided, since it greatly hinders the production of subsequent sections with a good quality cryostat due to the formation of ice crystals.
6. Identification of samples
Each sample to be processed by the tissue bank will be immediately identified in accordance with the tissue identification protocol, with clear reference to the tissue type (normal or pathological). This identification must be mechanised, whenever possible, for greater clarity of references.
The final identification of the samples will be carried out using a double system of barcodes and alphanumeric characters for generating labels for identifying preparations, blocks, reports, etc. In order to ensure as far as possible the correct identification of any sample and to avoid human errors in the transcription of references, these operations will be carried out mechanically using a barcode reader/generator.
7. Conservation of fixed tissue
Blocks of fixed tissue embedded in paraffin previously selected for their availability by the CNIO, containing neoplastic and non-neoplastic tissue will be appropriately identified with the aim of not being used for diagnosis, except when this is absolutely necessary.
They may be conserved in archives specifically set aside for this purpose until they are required for a specific project or in the Hospital’s own diagnostic material archives.
Fixed tissues will be conserved in the Pathology Departments belonging to the collaborating Hospitals.
8. Conservation of frozen tissue
The blocks of neoplastic and non-neoplastic tissue frozen and included in cryomoulds with OCT, previously selected on the basis of their availability by the CNIO, will be suitably identified with the aim of not being used for diagnosis, except when this is absolutely necessary.
Tissue will be stored at temperatures below -75ºC in chest freezers with the necessary security measures to avoid thawing and/or exposure to large changes in temperature.
The duly identified cryomoulds will be stored in specially designed boxes until they are required for a specific project.
Frozen tissues will be conserved in the Pathology Departments of the collaborating Hospitals, except when a Centre prefers them to be kept temporarily or permanently on the premises of the Molecular Pathology Programme at the CNIO.
9. Security measures for frozen tissue
The maintenance of frozen tissue in refrigerated chests at –80ºC has numerous advantages compared with conservation in containers of liquid nitrogen, such as its greater accessibility, greater ease of installation and lesser maintenance requirement. For its part, liquid nitrogen guarantees a lower and more stable temperature. However, the main advantage of storage at –80ºC is the subsequent use of the tissue for pathological studies in which it is necessary to make frozen sections and to maintain the quality of conservation of tissue morphology.
As the refrigerated freezers depend on the electrical power supply network they will require appropriate security measures to minimise the risk of large interior temperature fluctuations.
These security measures may be summarised as follows:
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Inclusion in the Hospital's secure electricity supply in such a way that, in the case of deficiencies in the electrical supply, emergency generators will ensure continuity of supply.
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Triple alarm system. Any temperature increase of more than 65-70º must trigger a progressive series of alarms:
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Local alarm (acoustic and visual), in the room where the freezer is located and in a nearby room.
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Distant alarm. An acoustic and visual stimulus will be activated in a distant room in the same building or complex (Maintenance, security, etc.) where there must be a staff presence 24 hours a day. This alarm will not be effective unless clear and concise written instructions are left indicating what must be done.
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Remote alarm. If 15 minutes have passed without action being taken over the alarm system, a system of telephone localisation will be activated to contact a series of pre-programmed numbers.
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Contingency plans in particular the location of other freezers of similar dimensions and characteristics into which the tissue may be transferred in the event of breakdown or when the main equipment is being cleaned. These freezers should be located on the premises of the Hospital itself or of Complexes belonging to other clinical or teaching Services or Units of the same institution.As a precaution against the lack of availability of alternative deposits, the CNIO will maintain spare space in the form of –80ºC chest freezers for unforeseen circumstances.
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Temperature control by weekly graph.
10. Non-standard methods
The aforementioned methods must be considered as standard for the permanent and systematic collection of cases. When other methods of selection, fixation and storage are necessary in order to carry out a specific project, the central office of the TBN and the group named in the project will draw up a specific protocol adjusted to the aims of the research. These protocols will be distributed between the associated hospitals for the correct handling of these cases, following a favourable report, where necessary, of the Monitoring and Research Committee of each Hospital. Once the previously established number of cases have been collected the participation of the TBN will no longer continue.
