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TJUHBB

Snap Freezing

Standard Operating Procedure (SOP 06.004)

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PURPOSE

Tissue samples are collected from patients that have been through the informed consent process and agreed to participate in the brain biorepository program. Fresh frozen tissue collections are a valuable resource for research purposes. Tumor tissues are only suitable for proteomic and genomic studies if frozen in a timely and appropriate manner. The purpose of this document is to outline standardized procedures for the TJUH Brain Biorepository to follow during snap freezing tumor tissue.

SCOPE

This standard operating procedure (SOP) describes how tissues are snap frozen. The SOP does not cover detailed safety procedures for handling Human Biological Materials (HBMs) or hazardous chemicals and it is recommended that personnel follow institutional safety guidelines.

REFERENCE TO OTHER TJUHBB SOPS OR POLICIES

  • TJUHBB SOP 06.001 Tissue Collection and Transportation
  • TJUHBB SOP 06.002 Tissue Harvesting
  • TJUHBB POL 02.001 Chemical Hygiene Plan
  • TJUHBB POL 02.002 Laboratory Safety Policy

ROLES AND RESPONSIBILITIES

This SOP applies to all qualified tumor biobank personnel and clinical staff at the collection centers that are involved in recruiting patients and the acquisition of informed and voluntary consent. This may include the following personnel:

Tumor Biobank PersonnelResponsibility
Tumor Bank MangerCollecting, harvesting, processing and storage of biospecimens

MATERIALS, EQUIPMENT AND FORMS

The materials, equipment and forms listed in the following list are recommendations only and may be substituted by alternative/equivalent products more suitable for the site-specific task or procedure.

Materials & EquipmentQuantity
Insulated Ice Box1
Sterile Specimen ContainerDepends on Quantity
Sterile Disposable Forceps1
Sterile Disposable Scalpel2
Sterile Disposable Petri Dish1
Pre-filled, Pre-labelled 2.0 mL CryotubesDepends on Quantity
Small Liquid Nitrogen Container1
Insulated Gloves for handling liquid nitrogen cooled material1 Pair

DEFINITIONS

See the TJUHBB Glossary.

PROCEDURES

This procedure is intended to ensure that tissue samples collected from consented participants will be frozen in a safe and efficient manner while eliminating the risks of contamination and loss of molecular integrity. To facilitate the use of genomic and proteomic techniques, banked tissue that has been adequately frozen is vital to obtaining products with high integrity and quality.

Snap Freezing of Biospecimens

  • Treat all tissue as potentially infectious.
  • Freezing is performed by the laboratory technician/technologist or trained personnel designated by the biorepository.
  • Have materials and equipment for ready. Have as many cryovials as needed labelled and ready.
    • 1 white/clear cryovial with 2 mL of 10% neutral buffered formalin
    • 2 red cryovials with 1 mL of RNALater each
    • 6 blue cryovials for flash freezing in liquid nitrogen
  • Priority of collection should be to collect a formalin-fixed sample and at least 6 flash-frozen samples before collecting samples for RNALater fixation. Additional flash frozen pieces may be collected depending on amount of sample available.

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Attention

Unless intended for another method of preservation fresh tumor tissue should be frozen as soon as possible. Optimally, tissue should be frozen within 30 minutes from resection.

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Sample rejection

In order to maintain the quality of the bank, samples may be rejected due to a failure of the following:

  • Do not freeze the tissue directly on ice. Any sample that was not snap frozen in the vapor phase of liquid nitrogen should be rejected.
  • Any frozen specimen which exceeds 10 mm in diameter may be rejected due to inadequate flash freezing.
  • Snap frozen tissue is suitable for preparation of DNA, RNA and protein. Do not place the sample in contact with formalin at any point in the process.
  • Place the tissue specimen into an empty cryovial, close the cryovial, and immediately submerge the cryovial into liquid nitrogen. The specimen should freeze within 30-60 seconds.

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Attention

It is not recommended to snap freeze large samples (>10 cubic millimeters) directly in liquid nitrogen, as longer freezing time will result in ruined morphology. For samples larger than ten cubic millimeters, an alternative protocol using liquid nitrogen cooled isopentane should be used.

  • Once snap frozen, transfer the sample to liquid nitrogen storage container (preferred) or to an -80º C (or colder) freezer.
  • If storing the samples in liquid nitrogen, it is recommended that the samples be placed in the vapor phase of liquid nitrogen. Note: the glass transition temperature of water is -134º C. As such, samples intended for indefinite long-term storage should be kept at temperatures lower than -135º C.
  • Record the storage location.

APPLICABLE REFERENCES, REGULATIONS AND GUIDELINES

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