Prions, also referred to as “unconventional” infectious agents or “agents of transmissible spongiform encephalopathies,” are comprised of protein only. They can cause Creutzfeldt-Jakob disease in humans, scrapie in sheep, bovine spongiform encephalopathy in cattle, etc. These infectious agents are unusually resistant to inactivation by the normal processes of laboratory disinfection and sterilization and materials suspected of containing them require special processing before reuse or disposal. Therefore, it is important to stress the use of disposable instruments whenever possible and the use of a disposable protective covering for the work surface of a BSC when working with materials known to contain or suspected to contain prions.
Laboratory transmission of prions can occur through ingestion of contaminated materials or puncture of the researcher’s skin. The following precautions should be taken when working with prions or material suspected of containing prions:
- Use dedicated equipment, e.g., equipment not shared with other laboratories.
- Wear disposable laboratory protective clothing (gowns, aprons) and gloves (steel mesh gloves between rubber gloves for pathologists).
- Use disposable plastic ware that can be treated and discarded as dry waste.
- Do not use tissue processors due to the problems with disinfection; jars and beakers should be used instead.
- Perform all manipulations in a BSC.
- Take great care to avoid aerosol production, ingestion, and cuts and punctures of the skin.
- Regard formalin-fixed cultures as still infectious, even after prolonged exposure to formalin.
To date, the World Health Organization suggests the following for the inactivation of prions:
- All non-disposable instruments, including steel mesh gloves, must be collected for decontamination.
- Infectious liquid waste should be treated with sodium hypochlorite containing available chlorine at 20 gram/liter (2%) (final concentration) for one hour.
- Bench waste, including disposable gloves, gowns, and aprons, should be autoclaved using a porous load steam sterilizer at 134-137oC for a single cycle of 18 minutes, or six successive cycles of 3 minutes each, followed by incineration.
- Instruments that cannot be autoclaved can be cleaned by repeated wetting with sodium hypochlorite containing available chlorine at 20 gram/liter (2%) over a one hour period. Washing to remove residual sodium hypochlorite is required.
- Instruments that can be autoclaved should be soaked in sodium hypochlorite containing available chlorine at 20 gram/liter (2%) for one hour and the rinsed well in water before autoclaving.
- Contaminated BSC and other surfaces can be decontaminated with sodium hypochlorite containing available chlorine at 20 gram/liter (2%) for one hour.
- Paraformaldehyde vaporization procedures do not diminish prion titres and prions are resistant to ultraviolet irradiation. However, BSCs must continue to be decontaminated by standard methods (e.g., formaldehyde gas) to inactivate other agents that may be present.
- HEPA filters should be incinerated at a minimum temperature of 1000oC after removal. Recommended additional steps prior to incineration include:
- Spraying the exposed face of the filter with lacquer hairspray prior to removal;
- “Bagging” of filters during removal; and
- Removal of the HEPA filter from the working chamber so that the inaccessible plenum of the cabinet is not contaminated.
- Histological samples containing prions are substantially inactivated after exposure to 96% formic acid for one hour.
The BMBL, 5th Edition, suggests the following prion inactivation methods:
- For reusable instruments and surfaces immerse in 1N NaOH or sodium hypochlorite (2% free chlorine concentration) for 1 hour followed by steam autoclaving at 121oC for 1 hour. Clean and sterilize by conventional means.
- Dry waste, animal carcasses and other tissue should be incinerated with a minimum secondary temperature of 1000oC.
- BSCs must be decontaminated with 1N NaOH and rinsed with water.
- HEPA filters should be bagged out and incinerated.