Processing of Solid Tissue Specimens

Abstract


The protocol is derived from the WHO/IARC guideline “Common Minimal Technical Standards and Protocols”. The purpose of the document is to provide a framework for the development and coordination of Biological Resource Centers.
Careful and well-documented processing of tissue specimens is crucial to the overall usefulness of the repository as a resource for scientific research. This protocol to collect and freeze tissue samples was developed within TuBaFrost, a European project to come to a European virtual frozen tumor bank; it contains choices and recommendations for preserving solid tissue.

Security considerations

  • Carry out all procedures in accordance with the local codes of practice.
  • Working with liquid nitrogen and isopentane is hazardous and all procedure must apply with local safety rules specific to these chemicals.
  • All tissue must be handeled as if potentially infectious.

Reagents

  • Dependant on method of sampling and storage (see below)

Instructions

    Ward
    1. Make sure that consent is obtained from patient before surgery (if applicable – according to the law in the collecting country)
    Operating Theatre
    1. Deliver notification of tissue collection (and consent form if needed) to surgeon or flag up on operating list.
    Surgeon
    1. Perform operative procedure, record time of excision of specimen.
    2. Complete pathology form (if possible in advance).
    3. Place specimen in labeled sterile pot/bag and put on ice.
    Operating theatre staff
    1. Send fresh tissue specimen immediately to pathology department.
    Histopathology department
    1. Notify pathologist and tissue bank research technician (if not already present).
    2. Check paperwork and allocate pathology number to specimen as routine.
    Pathologist
    1. Macroscopically describe specimen as routine.
    2. Using clean instruments and on a clean surface (sterile foil or clean dissection board) dissect the tissue specimen – clean or change instruments between dissecting normal and tumor tissue.
    3. Take representative parts of tissue for routine diagnosis (for fixation and embedding) as priority and decide if there is sufficient material available for the tissue bank.
    4. Supply research technician with tissue sample(s) for cryostorage - representative parts of the lesion, normal tissue and pre-malignant conditions.
    Storage of tissue
    1. Options for storage
    2. Transfer the snap frozen sample from the isopentane to a pre-chilled storage container for transfer to either a locked –80°C freezer or liquid nitrogen storage facility in liquid or vapour phase. For storage longer than 5 years, liquid nitrogen is recommended.
    3. Place cryostraws in a designated visotube within a goblet (removable liquid nitrogen storage elements) and place within the locked liquid nitrogen repository.
    4. Store duplicate samples in a different storage facility if this is available.
    5. Check the back-up system for the storage repository - either a back-up freezer running constantly or adequate supplies of liquid nitrogen.
    6. Record storage details in the inventory book and check earlier data that was entered. At a minimum the information recorded will include: inventory number (local sequential code); location; pathology number; type of tissue (site and also whether the sample is tumor/unaffected (normal)/premalignant); lag time between excision and freezing; and date.
    7. Transfer details to the computerised database system.
    8. Update the database when samples are moved or depleted.
    Formalin Fixation
    1. Formalin fixation is standard practice in most routine histopathology laboratories. The following guidelines address specific issues related to preservation of formalin-fixed specimens in BRCs.
    2. Tissue specimens should not be bigger than 1.5 x 1 x 0.5 cm.
    3. Specimens will be fixed in fresh 10% neutral buffered formalin (NBF) Blue) for a minimum of 4 and a maximum of 48 hours, after which time they will be embedded in paraffin following conventional techniques.
    4. All reagents should be DNAse, RNAse free (e.g. prepared using DEPC water)
    5. Fixation media such as Bouin’s containing picric acid should be avoided as this compound interferes with subsequent PCR analysis of extracted nucleic acids.
    6. Alcohol fixation may be used as an alternative to formalin. For this, tissue is placed into 70% alcohol (diluted with DEPC water) for a minimum of 4 hours.
    7. Alternatives for formalin can be desirable to use as a routine fixative, due to the chemical hazards of formalin. The effect on long-term storage using these alternative fixatives on the desired macromolecules is however not always known and should be empirically established.
    RNAlater
    1. This substance protects RNA within biomaterials, whereas other macromolecules of interest and morphology are not.
    Tissue
    1. Cut tissue to be less than 0.5cm in at least one dimension
    2. Submerge tissue in 5 volumes of RNAlater (e.g. a 0.5g sample requires about 2.5ml of RNAlater).
    3. Cells Resuspend pelleted cells in a small volume of PBS before adding 5-10 volumes of RNAlater.
    Storage
    1. RNAlater-treated tissue and cell samples can be stored at 4°C for one month, at 25°C for one week or at -20°C for indefinite time.
    2. For RNA isolation, simply remove the tissue from RNAlater and process.
    Histopathology department
    1. Notify pathologist and tissue bank research technician (if not already present).Then check the paperwork and allocate pathology number to specimen as routine.
    Pathologist
    1. Macroscopically describe specimen as routine.
    2. Using clean instruments and on a clean surface (sterile foil or clean dissection board) dissect the tissue specimen – clean or change instruments between dissecting normal and tumor tissue.
    3. Take representative parts of tissue for routine diagnosis (for fixation and embedding) as priority and decide if there is sufficient material available for the tissue bank.
    4. Supply research technician with tissue sample(s) for cryostorage - representative parts of the lesion, normal tissue and pre-malignant conditions.
    Technician
    1. Prepare tissue sample for snap-freezing on a clean surface and using clean instruments - change instruments between preparing normal and tumor tissue. The minimum size of tissue for snap freezing is approximately 0.5cm3 though the amount of tissue available will differ depending upon the sample site. INFO: Smaller fragments should still be snap-frozen and stored in the tissue bank. INFO: If there is sufficient material, freeze multiple samples.
    2. Pre-cool the freezing medium isopentane (2-methyl butane) to the moment when opaque drops begin to appear in the isopentane and the solution becomes misty; this will bring the isopentane towards its freezing point (-160°C), the optimal freezing point for the tissue. Options:
    3. Liquid Nitrogen; Suspend a vessel of isopentane in liquid nitrogen
    4. Dry ice: Add dry ice (cardice) to the isopentane until a slush is formed, or by suspending a vessel of isopentane in dry ice. Label cryovials, cryomolds or cryostraws with a bar-code and/or sequential code (depending upon local laboratory practice). Use waterproof pen able to withstand long-term storage at low temperatures. The sequential code is the local inventory code and must not relate to the pathology number or other identifiers. If a bar-code is used readable recognition must also be included to make the sample identifier readable at institutes where there are no bar-code readers.
    5. Record the local sequential code, pathology number, date, lag time from excision to freezing, the type of tissue (site and whether the sample is tumor/normal/premalignant) in the inventory book. If a bar-code system is in use this can be scanned into the Laboratory Information Management System and the above data recorded.
    6. Freeze directly in isopentane. Do not remove the tissue from the isopentane until freezing is complete (only 5 seconds or less is needed depending on size) but ensure sample does not crack. Remove sample from isopentane and enclose in the labeled cryovial. Strive to snap freeze all tissue within 30 minutes of excision from patient. Tissue subject to a delay of up to 2 hours should still be collected and the delay noted within the local inventory database. Options for freezing:
    7. Embed the tissue samples in O.C.T. (optimal cutting temperature) compound and freeze in isopentane or freeze directly in isopentane. The isopentane used is either cooled by suspension in liquid nitrogen or through addition of dry ice. Do not remove the tissue from the isopentane until freezing is complete (5 seconds or less depending on size) but ensure sample does not crack. Remove sample from isopentane and enclose in the labeled cryovial.
    8. Orientate the tissue on a piece of cork and an equally sized piece of Whatman paper soaked in physiologic salt solution. The isopentane used is either cooled by suspension in liquid nitrogen or through addition of dry ice. Do not remove the tissue from the isopentane until freezing is complete (5 seconds or less depending on size) but ensure sample does not crack. Remove sample from isopentane and enclose in the appropriate labeled storage vessel.
    9. Embed samples in a cryosolidifiable medium (e.g., Tissuetec) in plastic cryomolds and immerse in the pre-cooled isopentane. The isopentane used is either cooled by suspension in liquid nitrogen or through addition of dry ice. Do not remove the tissue from the isopentane until freezing is complete (5 seconds or less depending on size) but ensure sample does not crack
    10. If the cryostraw system is used introduce a carrot of tissue into the straw, thermically seal each extremity and place in liquid nitrogen.

    Citation:
    IARC -International agency for research on cancer. Processing of Solid Tissue Specimens. The Molecular Methods database. Wed, 12/19/2012 - 11:27. Acc.nr IegLt10.