Fresh Frozen Tissue Protocol – Appendices


Appendix 1

Introduction

To describe the procedure for collecting specimens from surgical resections of the colon or rectum, and preparing specimens for storage.

Supplies

  1. Liquid nitrogen in appropriate LN2 transport carrier
  2. Safety glasses or face shield
  3. Freezer gloves
  4. Disposable sterile latex or nitrile surgical gloves
  5. Surgical mask
  6. Clean laboratory coat
  7. Clean protective shoes
  8. Sterile cryovials, labeled and unlabeled extras
  9. Racks for vials/containers (separate for tumor and normal)
  10. Sterile disposable scalpels or scalpel blades (#10, 21, 22, 60 or single edge razor blade)
  11. Sterile disposable forceps
  12. Sterile 21 G needles
  13. Sterile disposable towels or drapes
  14. Sterile dishes or plates, such as Petri or cell culture dishes
  15. Sterile saline (PBS)
  16. Sterile gauze
  17. Marking pen or pencil
  18. Chipped ice
  19. Flat container or tray for chipped ice, such as an autoclavable Pyrex cake dish
  20. Sharps container for disposal of biohazardous sharps

Procedures

  1. Arrive at the pathology receiving room prior to the expected time of completion of the surgical resection. Wear a clean lab coat, and bring personal protective equipment and all supplies required for the procedure.
  2. Bring a copy of the signed consent form for the patient.
  3. While waiting for the specimen to arrive, set up supplies needed for the procedure. Arrange pre-labeled vials in a manner that allows for easy differentiation between vials intended for storage of tumor specimens and those for normal specimens. Place the pre-labeled sterile culture dishes on ice, but do not uncover. The liquid nitrogen transport container should be placed in a location in the pathology room where it can be easily accessed. Ensure that the vessel is secured in a manner that will prevent accidental spillage.
  4. The surgical specimen should be transported from the surgical suite to the pathology receiving room in a sterile bowl that has been covered with a sterile towel. It is absolutely imperative that the OR be reminded NOT to submerge the specimen in formalin.
  5. In order to minimize changes secondary to autolysis, specimens must be placed on ice and/or processed as quickly as possible after transport to the pathology receiving room. Ideally, specimens should be snap frozen or placed in appropriate reagent within 5 minutes of surgical removal.
  6. In the pathology suite, request that the specimen be placed onto a sterile surface for sectioning. If necessary, place the specimen on a sterile towel that has been placed onto a surface appropriate for sectioning.
  7. Provide the pathologist with sterile scalpel blades or scalpels or razor blades and sterile forceps for bisecting samples from the resection specimen. Separate blades and forceps should be used for specimens from each anatomic site to avoid cross-contamination of tissue.
  8. Using a sterile needle or forceps, transfer the specimens provided by the anatomic pathologist to separate pre-labeled sterile dishes. Use separate forceps/needles for each specimen to avoid cross-contamination. Place the forceps or needle in the dish or dish lid for further use. It is imperative to avoid any contact of the ice with the dish or specimen, as this will result in contamination of the specimen. Check that each dish is correctly labeled with the anatomic site and tissue type (normal/tumor).
  9. Using a sterile blade, bisect the specimen into 0.5 x 0.5 x 0.5 cm or smaller samples. Samples should not exceed 0.5 cm on one dimension, as larger sizes may impair freezing or perfusion rates. Size ranges from 0.5 to 0.3 cm are optimal. The forceps/needle can be used to hold the specimen while bisecting. Avoid crushing artifacts by gently but firmly fixing the specimen with the needle/forceps while cutting.
  10. Tumor tissue:
    1. The tumor specimen should include only tumor tissue. Trim any normal submucosa if possible.
    2. Site priority (in order of decreasing priority)
      1. Central area of the tumor
      2. Tumor margin (leading edge if known)
    3. Size requirements
      1. Maximum storage size: 0.5 x 0.5 x 0.5 cm
      2. Minimum storage size: none
      If surplus tissue is available, request specimens that are 1.0 x 1.0 x 1.0 cm. A specimen with these dimensions can be bisected along the horizontal and vertical axis into 4 specimens of 0.5 x 0.5 x 0.5 cm.
    4. Storage priority
      After cutting tissue from each anatomic site to the appropriate size, specimens are placed into separate sterile labeled vials or containers for snap freezing, OCT media embedding, RNA stabilizing reagent, or fixative (see appendices 2-5).
      Priority Specimen Collection
      First priority: Flash freezing in liquid nitrogen
      Second priority: Formalin fixation
      Third priority: OCT embedded sample
      Fourth priority: RNA stabilizing reagent
  11. Normal tissue:
    1. Normal colon specimens should include the full thickness of the colon wall, e.g., mucosa plus underlying submucosa.
    2. Site priority (in order of decreasing priority)
      1. Distant, grossly uninvolved colon
      2. Perilesional uninvolved colon (normal tissue adjacent to the tumor)
      3. Surrounding stroma
      4. Polyps
    3. Size requirements:
      1. Maximum storage size: 0.5 x 0.5 x 0.5 cm
      2. Minimum storage size: none
      If surplus tissue is available, request specimens that are 1.0 x 1.0 x 1.0 cm. A specimen with these dimensions can be bisected along the horizontal and vertical axis into 4 specimens of 0.5 x 0.5 x 0.5 cm.
    4. Storage Priority:
      After cutting tissue from each anatomic site to the appropriate size, specimens are placed into separate sterile labeled vials or containers for snap freezing, OCT media embedding, RNA stabilizing reagent, or fixative (see appendices 2-5).
      Priority Specimen Collection
      First priority: Flash freezing in liquid nitrogen
      Second priority: Formalin fixation
      Third priority: OCT embedded sample
      Fourth priority: RNA stabilizing reagent
  12. Polyps: If possible, collect tissue from each polyp removed during the procedure, and any polyps that will be discarded as surplus tissue. Make sure to record the site of each polyp. As above, separate sterile supplies should be used for each separate polyp.

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Appendix 2

Introduction

Flash freezing in liquid nitrogen provides excellent specimen integrity and a wide array of options for tissue analysis. The specimen is placed into a sterile 1-2 ml cryovial, which is then tightly capped and submerged in liquid nitrogen for "flash freezing". The vial is transferred from the temporary LN2 transport container to a liquid nitrogen storage tank for long-term storage.

Supplies

  1. Liquid nitrogen in approved LN2 transport vessel
  2. Safety glasses or face shield
  3. Freezer gloves
  4. Disposable sterile latex or nitrile surgical gloves
  5. Surgical mask
  6. Clean laboratory coat
  7. Clean protective shoes
  8. Sterile 1-2 ml cryovials, labeled and unlabeled
  9. Rack for cryovials (one for normal and one for tumor)
  10. Disposable sterile forceps
  11. Sterile 21 G needles
  12. Marking pen or pencil

Procedure

  1. Prior to the arrival of the resection specimen, set up supplies in the pathology receiving room. Arrange cryovials in a manner that will allow for easy differentiation of those intended for tumor and those intended for normal specimens.
  2. Ensure that the liquid nitrogen transport vessel is accessible, but is secure against accidental spillage.
  3. Once the surgical specimen has been trimmed, use a sterile forceps to gently place the specimen in a sterile cryovial. Avoid crushing artifacts. Use a separate forceps for each type of specimen to avoid cross contamination.
  4. Secure the lid, and then place the vial directly into the liquid phase of nitrogen. Use freezer gloves and a face shield during this procedure. Take precautions to avoid accidental spillage or spattering of liquid nitrogen that may affect others in the pathology receiving room, and make sure that the room is well ventilated.
  5. Properly dispose of all biohazardous waste and other waste before leaving the pathology receiving room.
  6. After transport to the Family Registry lab, transfer the specimens to a liquid nitrogen freezer for storage. Optimally, specimens should be stored in the liquid phase of nitrogen. However, storage at -190 C in the vapor phase of nitrogen is acceptable.
  7. Record all specimen information into the laboratory log and in the Family Registry tracking system.

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Appendix 3

Introduction

Embedding in OCT compound followed by flash freezing not only preserves DNA, RNA, and protein integrity, but also allows for sectioning of the frozen tissue. In this method, the OCT compound is poured into a small plastic tray, the tissue sample is submerged in the compound, and the plastic tray is placed on dry ice or is held with a forceps over the mouth of the liquid nitrogen transport vessel for freezing in the vapor phase of nitrogen. After hardening of the OCT compound, the tray is placed in a biopsy bag and transferred to a liquid nitrogen storage tank or -80 C freezer for storage.

Supplies

  1. Safety glasses or face shield
  2. Disposable sterile latex or nitrile surgical gloves
  3. Freezer gloves
  4. Surgical mask
  5. Clean laboratory coat
  6. Covered, protective shoes
  7. Tissue Tec OCT cryo-compound at room temperature
  8. Tissue cryomolds, pre-labeled and extra unlabeled
  9. Biopsy nylon bags, pre-labeled
  10. Disposable sterile polystyrene forceps
  11. Pencil or marking pen
  12. Liquid nitrogen transport container
  13. Sharps biohazardous waste container

Procedure

  1. While awaiting arrival of the tissue from the OR, arrange cryomolds on a tray in a manner that will allow for easy differentiation of those intended for tumor tissue vs. those for normal tissue.
  2. Fill each cryomold with OCT by slowly and carefully filling the mold to the top. It is important to avoid formation of air bubbles, and to ensure that the top surface of the OCT compound is completely level. Avoid any uneven surfaces.
  3. Using a sterile forceps or needle, transfer the specimen to the OCT-filled cryomold and gently submerge the tissue into media until it is completely covered. None of the tissue should remain exposed.
  4. The OCT can be hardened by holding the cryomold with a forceps over the opening of the liquid nitrogen transfer container. Freezer gloves and a face shield should be used during the procedure. Alternatively, the molds may be placed on grate that has been placed over the opening of the LN2 container for hardening, or placed into a cryomicrotome for hardening. Placing the cryomold on dry ice is another option.
  5. After the OCT has hardened, place the mold into a pre-labeled nylon biopsy specimen bag.
  6. Place the specimen bag into the liquid phase of nitrogen in the transport container.
  7. Properly dispose of all biohazardous waste and other waste before leaving the pathology receiving room.
  8. After transport back to the Family Registry lab, the OCT specimens are placed into a freezer box, preferable one that has inserts dividing the box into 8 sections.
  9. The specimens are placed into a liquid nitrogen freezer for storage.
  10. Record all specimen information into the laboratory log and in the Family Registry tracking system.

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Appendix 4: Processing of Surgical Resection Specimens in RNA Preserving Reagent

Introduction

As specific and non-specific RNA degradation begins immediately after tissue harvest, prompt transfer of fresh specimens into an RNA stabilizing reagent is helpful to minimize changes in gene expression, and ensure accurate quantitative analysis of gene expression. This protocol is intended for use with RNA later, but similar products (RNAzol, Trizol) are widely used. Frozen specimens are not appropriate for use with this protocol.

Supplies

  1. Safety glasses or face shield
  2. Disposable sterile latex or nitrile surgical gloves
  3. Surgical mask
  4. Clean laboratory coat
  5. Covered, protective shoes
  6. Disposable sterile polystyrene forceps
  7. RNAlater (Qiagen, Valencia CA, 800-426-8157 phone)
  8. Sterile labeled vials or containers pre-filled with appropriate volume of RNAlater (ambient temperature)
  9. Rack for vials/containers
  10. Pencil or marking pen
  11. Sharps Biohazardous waste container

Procedure

  1. Prior to the procedure, transfer RNAlater to sterile specimen containers. A minimum of 10 volumes of RNAlater reagent is required for each 1 mg of tissue - e.g., 10 l reagent per 1 mg of tissue. For colon specimens that approximate 0.5 cm3, place 5 ml of reagent into a sterile container that is of sufficient size to allow for complete submersion of the specimen. A 15 ml sterile centrifuge or conical tube is sufficient. Prefilled tubes can also be purchased from the manufacturer. If in doubt about quantifying the appropriate volume of reagent, pre-filled tubes can be weighed before and after specimen collection, and for tissue samples exceeding 5 mg, additional reagent can be added to the collection tube.
  2. Set up supplies in the pathology receiving room before the tissue arrives from the OR.
  3. Efforts must be made to expedite the transfer of harvested tissue to the sterile pre-filled containers of RNAlater as quickly as possible after harvest.
  4. It is imperative that height and width of the specimen not exceed 0.5 x 0.5 cm, as this tissue perfusion of the RNA stabilizing reagent is significantly impaired in larger specimens. However, specimens can exceed 0.5 cm in length. (See appendix 1).
  5. Using sterile forceps, place the tissue into the tube containing the RNAlater, and ensure that the specimen is completely submerged. Use a separate forceps for each specimen to avoid cross contamination.
  6. Place the tube into a rack to keep it upright.
  7. Properly dispose of all biohazardous waste and all other waste before leaving the pathology receiving room.
  8. During transport to the Family Registry lab, keep the tubes in an upright position into order to keep specimens submerged. Even transient exposure of the specimens to air can result in DNA degradation.
  9. Incubate the specimens in the RNAlater reagent overnight at 2-8 C.
  10. After a minimum of 12 hours of refrigeration, transfer the specimen using a sterile forceps to a sterile labeled cryovial. This procedure should be done in a biological hood under sterile conditions using protective personal equipment.
  11. Specimens are transferred to a liquid nitrogen freezer or a mechanical -80 C freezer for long-term storage.

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Appendix 5: Processing of Surgical Resection Specimens in Neutral Buffered Formalin

Introduction

Neutral buffered formalin is used to stabilize protein in fresh tissue, and prevent autolysis and putrefaction. Fresh surgical resection specimens which have been fixed in 10% buffered formalin and embedded in paraffin blocks can be sectioned with a microtome, mounted on slides, and stained with Hematoxylin and Eosin (H&E). It is not possible to prepare high quality H&E sections from snap frozen sections; hence paraffin-embedded tissue (PET) tissue provides an opportunity to evaluate the morphological characteristics of the tissue obtained from the surgical resection, while unstained PET sections can be used for immunohistochemical analyses of protein expression.

Supplies

  1. Safety glasses or face shield
  2. Disposable sterile latex or nitrile surgical gloves
  3. Surgical mask
  4. Clean laboratory coat, disposable lab coat, and surgical scrubs
  5. Covered, protective shoes
  6. Disposable sterile polystyrene forceps
  7. 10% buffered formalin
  8. Labeled vials or containers pre-filled with appropriate volume of 10% buffered formalin (ambient temperature)
  9. Rack for vials/containers
  10. Pencil or marking pen
  11. Sharps container for biohazardous sharps

Procedure

  1. Prior to the procedure, aliquot formalin to specimen containers. This can be done in the Family Registry laboratory up to several days ahead. The volume of formalin should be a minimum of 15-20 times the volume of the tissue sample - e.g., 20 ml of formalin per 1 cm3 of tissue. A 15 ml sterile centrifuge or conical tube can be used, or alternatively, a urine container or other capped, leak-proof container.
  2. While awaiting arrival of the surgical specimen, set up supplies needed for the procedure. Arrange labeled vials in a manner that allows for easy differentiation of those designated for tumor collection from those designated for normal tissue.
  3. Specimens intended for formalin fixation should be processed after the completion of other fresh tissue procedures, such as flash freezing, embedding in OCT compound, and submersion in RNA stabilizing reagent.
  4. The tissue sample should be trimmed such that it is approximately 0.5 cm in thickness. Specimens that exceed this dimension may not allow for adequate perfusion of the formalin.
  5. After trimming, transfer the specimen to a container using a forceps. To avoid cross-contamination, a separate forceps should be used for each tissue sample.
  6. Ensure that the specimen is completely submerged in the formalin fixative. Securely tighten the lid to avoid any spillage.
  7. Properly dispose of all biohazardous waste and all other waste before leaving the pathology receiving room.
  8. The tissue should be fixed at room temperature for a minimum of 24 hours, at which point the tissue can be transferred to a histology lab for embedding in paraffin.
  9. Formalin should not be discarded down the drain. Refer to the chemical disposal plan at your institution for appropriate method of disposal.

Safety Precautions

  1. Formaldehyde, also called formalin, is a clear, colorless solution that readily vaporizes and has a pungent odor. It is the most commonly used fixative in pathology. It is toxic by inhalation and if swallowed, and causes irritation to eyes, the respiratiory system, and to skin. Exposure to formalin poses a cancer risk, and repeated or prolonged exposure increases this risk. Precautions should be taken to avoid inhalation, and contact with skin and other body surfaces.
  2. Protective personal equipment should always be used while working with formalin, including disposable gloves, face shields, protective covered shoes, and protective laboratory coat. Avoid contamination of street clothes.
  3. Adequate ventilation is mandatory while using formalin. Avoid breathing the vapors.
  4. Formalin is flammable. Avoid all sources of heat, sparks or flame while working with formalin.
  5. Remove disposable gloves and wash hands prior to leaving the pathology receiving room and after working with formalin-fixed tissue. Do not eat, drink, apply cosmetics or engage in any other behavior that could result in self-contamination while working with formalin.
  6. Refer to product MSDS for additional information on use of formalin. Keep MSDS posted in an accessible area of the laboratory.

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