Investigator Exposed to Infectious Material in Cryotube Explosion
Key Instruction Points:
Be aware of the potential for pressurization when working with cryogenic liquids.
A researcher at a university reported that a vial of potentially infectious materials "exploded" when she removed it from liquid nitrogen.
As you may have guessed, the "explosion" occurred when the liquid N2 that has leaked into a vial expands when removed from the cold. This used to be a fairly common problem with heat-sealed glass ampules, because it was difficult to obtain perfectly fused glass with no microscopic holes. This problem was largely resolved with laboratories began using plastic cryovials with a silicone seal. Nunc* makes a sleeve called CryoFlex that slips over the vial and then is heat-sealed to keep the liquid out. However, even with this type of product an explosion infrequently occurs.
There are several ways to prevent this from happening:
Cryogenic storage vials are designed for VAPOR PHASE STORAGE in liquid nitrogen freezers. This means that they are designed to sit in the cloud of extremely cold nitrogen gas that sits just above a small reservoir of liquid nitrogen in the bottom of the freezer. Leakage of liquid nitrogen into the vial occurs with the freezer is overfilled and the vials are immersed in liquid nitrogen. This problem can be avoided by not overfilling the freezers with liquid nitrogen.
Visually check each cryovial prior to filling to ensure there are no defects around the rim. Cryovials should never be re-used.
When removing samples, pause for a moment in the neck of the dewar before bringing them into the room atmosphere - if one is going to pop, it will usually do so early in the warm-up process.
The importance of gloves and face shield can not be overemphasized. Tubes stored in liquid phase dewars, where the ampules are in canes is especially hazardous. Since nitrogen freezers tend to be located separate from the labs, full face shields and gloves should be available near the nitrogen freezers so no one is tempted to pull a vial without protection because they forgot to bring a shield with them.
Following the outbreak of foot and mouth disease (FMD) in Surrey on 3 August, the UK government asked the Health and Safety Executive (HSE) to lead an investigation into biosecurity issues at the Pirbright facility a site occupied by the Institute of Animal Health (IAH) and also by two private companies called Merial Animal Health Ltd (Merial) and Stabilitech Ltd (Stabilitech). The Department of the Environment, Food and Rural Affairs (Defra) had established that the virus strain causing FMD in the first infected herd of cattle at a farm in Normandy, Surrey was O1 BFS67 (also known as 01 BFS1860 and hereafter referred to as O1 BFS). This is a laboratory strain not naturally found in the environment and was one upon which work was being carried out by all three occupants of the Pirbright site ahead of the first outbreak. HSEs job was to lead a team to investigate:
- potential breaches of biosecurity at the Pirbright site;
- whether such breaches may have led to a release of any specified animal pathogen;
- whether any such breaches had been rectified to prevent future incidents.
Sabia Virus in Centrifuge Incident
Key Instruction Points
- Vacate room and wait at least 30 minutes for infectious aerosols to settle before attempting a clean-up
- Use sealed rotors or safety cups when centrifuging blood, pathogens or other potentially infectious materials.
- Written emergency procedures should be clearly defined for potential accidents/incidents that can occur in the lab.
- All personnel should be trained in emergency procedures when using hazardous materials.
A few years ago the newly discovered arenavirus, Sabia, was being studied by a visiting senior virologist, who was purifying it from 1 litre of tissue culture fluid. At the time of the incident, the virologist was working alone in the biosafety level3 laboratory (negative pressure with HEPAfiltered exhaust system). The researcher used a high speed centrifuge with a large angle head centrifuge rotor with six new plastic bottles in the purification process. During the centrifugation cycle there was a nasty rattling sound that indicated a problem. As soon as the unit stopped, he opened the chamber and removed the bottles from the rotor and placed them in a biological safety cabinet..Only then did he realize that one of the plastic bottles had leaked about 100 ml of infectious tissue culture into the centrifuge rotor. (Subsequently a hairline crack was found.)
The researcher was probably exposed to aerosolized Sabia virus when he opened the chamber. He immediately disinfected the centrifuge rotor, the defective bottle and the centrifuge using 10% Clorox solution. He cleaned the spilled material from the centrifuge while wearing a gown, surgical mask, and gloves. After disinfecting the centrifuge and bottles, he continued to work in the laboratory for 3-4 more hours. He did not inform anyone or report the incident.
About 10 days later he went to the doctor with a 4 day history of fever, malaise, backache, stiff neck, and myalgias that he attributed to another cause. The diagnosis of Sabia Virus was made and confirmed. Although Sabia virus infection is potentially fatal, the researcher recovered after 2 weeks of treatment.
Persons who came into contact with the researcher or with his biological specimens in the hospital laboratories were notified and enrolled in a surveillance program. None of these persons developed the virus.
After this incident, the CDC classified Sabia Virus as a BL4 pathogen, which requires use of a glove box or containment suit.
Personnel who work with BL2 or higher mateirals must receive annual training in biosafety which should include how to recognize a potential exposure incident and procedures to follow in an emergency. Personnel working with higher level organisms need additional training.
Blood, other potentially infectious material and pathogens must only be centrifuged using a sealed rotor or safety cups. These must be opened in a biological safety cabinet.
If there is any incident which creates aerosols of an infectious material, the room should be evacuated and the material allowed to settle for at least 30 minutes before any clean-up activities are undertaken. The incident should be reported as an emergency to the Health and Safety Office. Possible personnel exposures should be treated as a medical emergency. The Health and Safety Office must evaluate the need for respiratory protection and other personal protective equipment before clean-up is performed.