Handling Prions

Recommended Biosafety Practices for Handling Prions and Prion-Infected Tissues

Introduction

Research-related activities involving prions or tissues containing prions have been on the rise at MSU in both the animal health and human health arenas. Because the infectious nature of prions is not well characterized and destruction of these particles goes beyond the techniques typically required for biohazard inactivation, work with these agents requires special considerations for biocontainment to minimize both occupational and environmental exposure risk.

Prions & General Biosafety Recommendations

Prions (proteinaceous infectious particles, an abnormal isoform of a normal cellular protein) cause Creutzfeldt-Jakob disease (CJD), scrapie and other related human and animal neurodegenerative diseases. Human prions are manipulated at Biosafety Level (BSL) 2 or 3, depending on the activity, with most human prions treated as BSL-3 under most experimental conditions.In many instances, BSE prions can also be manipulated at BSL-2, however due to the high probability that BSE prions have been transmitted to humans, certain circumstances may require the use of BSL-3 facilities.All other animal prions are considered BSL-2 pathogens.However, when a prion from one species is inoculated into another the resultant infected animal should be treated according to the guidelines applying to the source of the inoculum. Please see the following table adapted from the BMBL for a list of common mammalian prions and general BSL recommendation.

Note: Biosafety level assignment should be established using a risk assessment that accounts for the nature and host range of the agent, as well as the nature of the procedures and concentration and quantity of the agent. 

The Prion Diseases 

Disease (abbreviation)	Natural Host	Prion	Pathogenic PrP Isoform	Biosafety Level
Scrapie	sheep, goats and mouflon	scrapie prion	Sc OvPrP	2
Transmissible mink encephalopathy (TME)	mink	TME prion	Sc MkPrP	2
Chronic wasting disease (CWD)	mule deer, elk and white tail deer	CWD prion	Sc MdePrP	2
Bovine spongiform encephalopathy (BSE)	cattle	BSE prion	Sc BoPrP	2/3
Feline spongiform encephalopathy (FSE)	cats	FSE prion	Sc FePrP	2
Exotic ungulate encephalopathy (EUE)	nyala, greater kudu and oryx	EUE prion	Sc UngPrP	2
Kuru	humans	kuru prion	Sc HuPrP	2/3
Creutzfeldt-Jakob disease (CJD)	humans	CJD prion	Sc HuPrP	2/3
Gerstmann-Sträussler- Scheinker syndrome (GSS)	humans	GSS prion	Sc HuPrP	2/3
Fatal familial insomnia (FFI)	humans	FFI prion	Sc HuPrP	2/3

BMBL, 5th ed., 2007

The highest concentration of prions is found in the central nervous system (CNS), and extreme caution must be exerted when handling CNS samples. However prions can also be found in the CSF, lung, liver, kidney, spleen/lymph nodes, placenta. Unfixed samples of brain or spinal cord, as well as other tissues known to contain human prions should be handled at BSL-3. With regards to BSE prions, it is also recommended that animal tissue samples (e.g., brain, spinal cord) known or strongly suspected to contain prions be handled at BSL-3 (BMBL 2007). For other samples, the level of containment will depend on the type of tissue handled, the nature of the manipulation and the amount of material handled (SDS 1997).

Formaldehyde or formalin-fixed, glutaraldehyde-fixed and paraffin-embedded tissues, particularly of the brain, remain infectious for long periods, if not indefinitely (BMBL 2007, WHO 2000). They should be handled cautiously as fresh materials from fixation through embedding, sectioning, staining and mounting on slides, unless treated with 95% formic acid (WHO 2000).

Although there are no documented laboratory-acquired prion infections, the primary hazard is from accidental parenteral inoculation or ingestion. Cuts and punctures should be avoided and the use of sharp knives, scalpels, blades and needles should be minimized. If the use of sharps cannot be avoided, cut-resistant gloves should be worn (CFIA 2005).

Wherever possible, the laboratory and equipment used for work with prions should be dedicated to that task alone. All employees should be informed and aware that prion research is being conducted in the lab. The entrance to the lab should allow for the separation of PPE/lab clothing and staff clothing. An exposure protocol should be developed, posted and communicated to all employees (CFIA 2005, UCSD 2002). Procedures should be in place for the effective decontamination of all waste, re-usable equipment, surfaces and other lab space (CFIA 2005, UCSD 2002).

Working with Prion-Risk Materials at MSU

At this time, work with prion-risk materials at MSU is limited to research and diagnostic laboratory applications. Therefore, this guidance document applies to these procedures only. Guidelines for use of prion-risk materials in conjunction with live animals will be developed if needed. Therefore, if future project plans call for use of live animals and prion-risk materials, please notify the MSU Biosafety Officer at the proposal-writing stage to perform a risk assessment and identify containment requirements.

Procedures involving the manipulation of animal tissues that are from known or suspected scrapie or CWD cases must be handled under BSL-2 conditions as a minimum standard. Procedures involving manipulation of human tissues that are known or suspected cases of CJD must typically be handled at BSL-3 conditions, unless a risk assessment completed in conjunction with an EHS Biosafety Professional allows for BSL-2 facilities and procedures. In general, procedures that involve aerosolization or vigorous disruption of the material (i.e., centrifugation, sonication, laser dissection) bear the greatest risk to personnel and the environment and will require special consideration for containment at both biosafety levels.

A summary of BSL-2 and BSL-3 facility and procedural requirements as outlined in the BMBL is attached at the end of this document. Additionally, the following specific measures should be implemented for all work with prion-risk materials:

1. Access to the laboratory must be restricted to trained personnel when work is being conducted on tissue.

2. Personnel working with prion-risk materials must complete Biosafety Principles for Animal Users through EHS, as well as complete on-site training relative to the nature of the prion in use, routes of transmission, and specific hazards of the tissue handling process. Written procedures and training records should be kept as outlined in the BMBL.

3. Personnel must wear gloves and gowns while handling tissues that are potentially contaminated. All protective clothing must be removed before leaving the laboratory.

4. All fixed, non-fixed, or frozen tissues must be contained within watertight containers. Containers must be individually labeled with the universal biohazard symbol or placed in a secondary container (i.e., a tray with sides) that is labeled with the universal biohazard symbol.

5. Sonication or homogenization of tissues must be performed in a properly certified Class II biosafety cabinet.

6. Microtome blades and knives used for cutting tissue must be cleaned with an instrument that does not put the hand or finger of the operator in or near contact with the blade.

7. Disposable, absorbent pads or disposable trays should be used whenever possible to help confine contamination and to facilitate cleanup and disinfection.

8. The following practices should be followed when using reusable instruments:

• Instruments should be kept wet until cleaned and decontaminated;
• Instruments should be cleaned as soon as possible to prevent drying of material;
• Do not mix instruments used on materials potentially infected with prions with those instruments used for other purposes;
• Instruments that will be cleaned in a dishwasher must be decontaminated first and the washer must be run through an empty cycle before being used for other instruments

9. The following provisions for decontamination of wastes, reusable instruments and contaminated surfaces must be followed to assure effective inactivation of prions:

Liquid waste

Liquid waste may be treated in the following ways:

• Mix with NaOH for a final concentration of 1.0 N NaOH and hold at room temperature for 1 hour; or
• Mix with bleach for a final concentration of 20,000 ppm available chlorine and hold at room temperature for 1 hour

This waste should be stored in a chemical fume hood for the duration of the treatment period. After the treatment period, liquid waste may be neutralized and discharged to the sewer by way of the lab sink, or disposed of through EHS as liquid chemical waste. 

Contaminated surfaces 

Contaminated surfaces may be treated in the following ways:

• Bleach solution (20,000 ppm available chlorine) for 1 hour; or
• 1N NaOH for 1 hour

After treatment, surfaces should be thoroughly rinsed with clear water.

Contaminated reusable instruments  

Contaminated reusable instruments may be treated in the following ways:

• Immerse in 1N NaOH or sodium hypochlorite (20,000 ppm available chlorine) for 1 hour, transfer to water, autoclave (gravity displacement) at 121°C for 1 hour (BMBL 2007, WHO 2000);
• Immerse in 1N NaOH or sodium hypochlorite (20,000 ppm available chlorine) 1 hour, rinse with water, autoclave at 121°C for 1 hour (gravity displacement) or at 134 °C for I hour (porous load) (BMBL 2007, WHO 2000); or
• Immerse in sodium hypochlorite solution with 20,000 ppm available chlorine (preferred) or 1N NaOH (alternative) for 1 hour (WHO 2000)

Contaminated dry waste  

All contaminated dry waste should be picked up for incineration.Prioncontaminated sharps waste must be identified as “prion contaminated sharps- for incineration only” on the hazardous waste pickup request to assure incineration of these materials. Contact the EHS Biosafety Staff for further assistance regarding treatment and disposal.

10. Intact skin exposure to prion-risk materials should be followed by washing with 1N NaOH or 10% bleach for two to three minutes, followed by extensive washing with water. For needle sticks or lacerations, gently encourage bleeding, wash with warm soapy water, rinse, dry and cover with a waterproof dressing. In the event of a splash to the eye, rinse the affected eye with copious amounts of water or saline only. In the instance of a splash or puncture, the exposed individual should then report to Olin Primary Care Clinic for follow-up through MSU Occupational Health.

11. The Principal Investigator (PI) must assure that all spills or exposures involving prion- risk materials are managed with the proper procedures. Additionally, these eventsshould be reported to the MSU Biosafety Officer as soon as possible for follow-up and assistance with actions to reduce future occurrences.

12. Prion-risk materials may be subject to permit requirements for shipment and receipt. USDA permits apply to interstate and international shipment of animalrelated materials capable of transmitting infection. CDC permits apply to import of materials that are potentially infectious to humans. Additionally, shipment of these materials requires specific training for the shipper. Contact the EHS Biosafety Staff for further information.

Notes on chemical disinfection

Sodium Hydroxide (NaOH, or soda lye):Be familiar with and observe safety guidelines for working with NaOH.1N NaOH is a solution of 40 g NaOH in 1 liter of water.1 N NaOH readily reacts with CO2 in air to form carbonates that neutralize NaOH and diminish its disinfective properties.10 N NaOH solutions do not absorb CO2, therefore, 1N NaOH working solutions should be prepared fresh for each use either from solid NaOH pellets, or by dilution of 10 N NaOH stock solutions.

Sodium hypochlorite (NaOCl solution, or bleach): Be familiar with and observe safety guidelines for working with sodium hypochlorite.Household or industrial strength bleach is sold at different concentrations so a standard dilution cannot be specified.Efficacy depends upon the concentration of available chlorine and should be 20,000 ppm available chlorine. 

These solutions are corrosive and appropriate personal protective equipment must be worn when preparing and using them. 

 

Please Refer to Section IV—Laboratory Biosafety Level Criteria (PDF) of the CDC/NIH BMBL.

 

References

AAVLD. 2004. Practices for Handling Suspect Biosafety Level 2 Animal TSE. Veterinary Laboratory Diagnosticians’, Waste disposal and Pathology Committee, p. 3-4

BMBL. 2007. Section VIII-H. Prion Diseases. Biosafety in Microbiological and Biomedical Laboratories, 5 ed. Centers for Disease Control and Prevention, National Institute of Health, U.S. Department of Health and Human Services

CFIA. 2005. Containment Standards for Laboratories, Animal Facilities and Post Morten Rooms Handling Prion Disease Agents. Biohazard Containment and Safety Unit, Canadian Food Inspection Agency, http://www.inspection.gc.ca/english/sci/bio/anima/consult/prionse.shtml 

SDS. 1997. Creutzfeldt-Jakob agent, Kuru agent. Material Safety Data SheetInfectious Substances. Office of Laboratory Security, Population and Public Health Branch, Health Canada, http://www.hc-sc.gc.ca/pphb-dgspsp/msds-ftss/msds45e.html

UCSD. 2002. Prion Research Guidelines. University of California, San Diego, http://www.neurosci.ucsd.edu/Safety_Docs/UCSD_Prion_FactSheet.htm

WHO. 2000. 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

Updated May 2015