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Scrapie Prion Research Procedures

(These procedures are for Scrapie only. Contact the university's Biosafety Officer at 626-6002 for work with other prion diseases - including Bovine Spongiform Encephalopathy and Chronic Wasting Disease.)

All protocols for working with prion diseases must be approved by the Institutional Biosafety Committee (IBC) before work is started.

There is no known effective treatment for prion disease making it necessary to handle prions with extreme caution. The highest concentrations of prions in infected animals are in the central nervous system and its coverings. Studies indicate that it is likely that high concentrations of prions may also be found in spleen, thymus, lymph node, and lung tissue. There is no evidence that scrapie can be transmitted to humans.

Exposure Control Methods

• The principle investigator or lab supervisor must train all workers in specific handling procedures for infectious material. Written Standard Operating Procedures (SOPs) must be available for worker reference.
• Lab access must be limited to trained individuals. If non-trained individuals need to enter the lab they must be accompanied by a trained individual.
• One of the main precautions to be taken when working with scrapie or other prion diseases is to avoid puncture of the skin. Therefore, sharps and glass should not be used unless it has been determined that there is no other alternative.
• All work surfaces should be protected with disposable bench covers that are handled as low level prion waste.
• All manipulations with prions should be done in a biological safety cabinet. Cabinet needs to be certified annually.
• Open centrifuge rotors in a biological safety cabinet.
• Transport of all prion materials outside a biological safety cabinet requires secondary containment.
• All workers should wear appropriate personal protective equipment:
  • Double gloves
  • Lab coat (disposable back closing preferred)
  • Goggles to prevent touching of face
  • Full face protection if it is necessary to handle prion material outside the biological safety cabinet

Spill Clean-up and Disinfection

Prion disease infectivity is characterized by extreme resistance to conventional inactivation procedures including irradiation, boiling, dry heat, and many chemicals (formalin, betapropiolactone, alcohols). Fixation with alcohol, formalin, or glutaraldehyde actually fixes the infectivity of the infectious agents and makes them more difficult to inactivate. Note: Be sure to decontaminate all equipment prior to maintenance or service work.

Effective Disinfectants

• Environ LpH from Steris (other LpH formulations are not approved for prion use) is effective and not as corrosive to surfaces as bleach or NaOH. This was formerly known as Canadian LpH. All staff using LpH must be trained in its proper use.
   Note: Thoroughly mix the treatment solution until uniform. Minimize breathing LpH fumes. Use squirt bottles and saturated lab wipes rather than spray bottles that create a mist. Containers with LpH should have tight covers. Wear eye protection if using outside a biological safety cabinet or chemical fume hood including pouring in drain for disposal. User must observe the precautions and safety requirements on the registered product label.
• 2% free chlorine by making a final concentration of 2.5% sodium hypochlorite solution (freshly made 40% household bleach, per USDA requirements) for one hour
• Freshly made 1N NaOH for one hour

In addition to the above disinfectants, prions can be inactivated by:

• Steam autoclaving at 132°C for 4.5 hours or
• Incineration

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Spill Clean-up

• Notify other lab workers that a spill has occurred.
• It is important to keep contaminated surfaces moist until decontamination is complete as the infectious agents become even more resistant to chemical inactivation when dry.
• Cover spill with paper towels or other absorbent material.
• Saturate with Environ LpH or other disinfectant listed above and let sit for at least 30 minutes. Use sufficient Environ LpH to have a final concentration of 5-9% (7-12 ounces of product to 1 gallon of water). If possible, leave the lab to avoid prolonged breathing of fumes. Direct other workers not to enter the lab.
• Dispose of absorbent material and any other clean-up material in yellow waste bag.
• Report all spills to lab supervisor.

Decontaminating Non-Disposable Items

Remove gross filth mechanically from the treatment solution before treatment begins.

• For small items such as animal cages, glassware, and instruments:
  • Submerge in 0.9% LpH (1.5 oz of product per gallon of water) for at least 16 hours or
  • Submerge in 5-9% LpH for at least 30 minutes
    Note: Container should be placed in a chemical fume hood or have a tight top to reduce exposure to fumes. Discard treatment solution daily or when it becomes dirty.
• Wipe down floors, equipment, lab surfaces, and other non-submersible low level scrapie contaminated items with 5-9% LpH. Re-treat as necessary to keep wet throughout the 30 minute time period.
• Following the contact time, surfaces or items may be rinsed with water and allowed to air dry.

Waste Handling

The following procedures are to be followed for disposing of prion waste. Other methods of waste disposal must first receive IBC approval.

Non-Tissue Low Level Solid Scrapie Waste (Includes Animal Bedding)

• Place waste in yellow bag.
• Place in yellow barrel for incineration.
• Call Waste Management (5-6481) to arrange to have a yellow barrel delivered to the lab and for pick-up / replacement of yellow barrels.

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Liquid Scrapie Waste

• Low titer waste: treat with sufficient Environ LpH to have a final concentration of 0.9% for at least 16 hours or 5-9% for a minimum of 30 minutes.
  Note: Container should be placed in a chemical fume hood or have a tight top to reduce exposure to fumes.
• High titer waste: follow above LpH treatment by autoclaving at 132°C for 4.5 hours.
• Dispose treated liquid waste down the sink. Wear gloves, disposable lab coat, and full face protection. If possible, dispose in a vented sink area. Rinse sink with copious amounts of water (at least 2.5L per ml of LpH).
  Note: LpH waste disposed in sink must not contain other chemicals and must have a pH between 5.0 and 9.0. Do not put concentrated LpH in sink.

Sharps & Histology Slides

Place sharps in a puncture proof red sharps container. When container is no more than 3/4 full, close and seal the container. Place container in a yellow waste bag for incineration.
Note:  Small quantities of sharps may be placed in other small, sealed, puncture-proof containers and put in yellow bag.

Infected Animal Carcasses and Tissue

Dispose of all infected carcasses and tissue in the tissue digester adjacent to Vet Diagnostics on the St. Paul campus. Call 625-0255 to make arrangements.

Accident Response

• Needle sticks or laceration: gently encourage bleeding, wash with warm soapy water, rinse, dry and cover with a waterproof dressing. Seek medical attention and report incident to the Biosafety Officer, 626-6002.
• Accidental contamination of skin: wash with warm soap and water for 5 minutes. (May clean briefly, 1 minute, with 0.1N NaOH, 10% bleach, or 5% LpH followed by washing with warm soap and water for 5 minutes.)
• Splashes to the eye or mouth: irrigate with copious amounts of water for 15 minutes. Seek medical attention.
• Report all incidents to supervisor as soon as possible. Report any incident requiring medical treatment to Biosafety Officer, 626-6002.

References

WHO infection control guidelines for transmissible spongiform encephalopathies. Report of a WHO consultation, Geneva, Switzerland, 23-26 March 1999, http://www.who.int/csr/resources/publications/bse/whocdscsraph2003.pdf

Section VIII-H. Agent Summary Statements-Prion Diseases. Biosafety in Microbiological and Biomedical Laboratories, 5th Edition. http://www.cdc.gov/od/ohs/biosfty/bmbl5/sections/SectionVIIIH-PrionDiseases.pdf

BL2 TSE Laboratory Standard Operating Procedures, U.S. National Institutes of Health (NIH), Rocky Mountain Laboratories (RML). Hamilton, Montana

Inactivation of Transmissible Spongiform Encephalopathy (Prion) Agents by Environ LpH, Richard E. Race and Gregory J. Raymond, Journal of Virology, Feb. 2004, p. 2164-2165

LpH Exemption for Use Letter Issued to Minnesota Department of Agriculture on 5-25-2006 by the United States Environmental Protection Agency.

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