Biosafety

Shared research facilities face several challenges when working with clients whose research stem from a broad variety of biological research interests. Samples may have varying biological risk factors associated with their use and must be considered on an individual basis. For example, samples can originate from human, non-human primate, immortalized cell cultures, infectious animal models and can contain known human pathogens such as Hep, HIV, CMV or oncogenic viruses such as EBV, HTLV, SV-40 or Retroviral agents. Fixation is often good enough to neutralize most agents, but in some circumstances this is not an option, especially when viable cells are needed for further experimentation, which is true for cell sorting.

Laboratory safety during cell sorting:

The safety requirements for cell sorting are different than those for standard flow cytometry. The particles most responsible for the spread of infectious disease are on the order of 0.1 – 60 microns (µM). These particles are primarily transmitted via inhalation into nasal passages and subsequent absorption through mucous membranes (see table below). The droplets generated during sorting are about 80 µM, but actual sizes are variable and are influences by operating conditions such as pressure and nozzle vibration.

Sorters also generate satellites drops that range between 0.5 and 7 µM that can be easily caught up by air currents and remain suspended for extended periods. These droplets could quickly spread infectious agents to the area around the instrument and the whole laboratory if operated without proven and effective containment measures. This is especially true when clogs happen as the directionality of the stream is uncontrolled.

For this reason, all unfixed human specimens and primary cell cultures are treated as infectious in our laboratory. In addition, our sorters have been placed inside Class II, A2 biological safety cabinets to protect our operators while also protecting samples from atmospheric contamination. Sorting is considered a BSL-2 activity and as an additional measure of safety, our technicians are the only persons authorized to operate the sorters since they have been trained to troubleshoot breakdowns and in procedures for handling aerosol containment during failure situations.

 

Agent Source of Infection Species Route of Infection Biosafety Level Practices
Hepatitis B,C,D Blood

Cerebrospinal fluid

Human

Infected primates

Inoculation, mucous membrane, exposure to aerosols, broken skin 2 / 3 (when aerosols produced or large concentrations
HSV ½, Varicella, CMV, EBV Blood

Tissues

Transformed cell lines

Human Inoculation, mucous membrane, exposure to aerosols, broken skin 2 / 3 (when aerosols produced or large concentrations
HIV Blood

Body fluids

Tissues

Human Inoculation, mucous membrane, exposure to aerosols, broken skin 2 / 3 (when aerosols produced or large concentrations
Influenza Brochoalveolar lavage Respiratory  tissues Human

Naturally or experimentally infected animals

Aerosol  inhalation 2
Chlamdydia  psittaci Blood,

Tissues

Infected animals

Human

Exposure to infectious aerosols and droplets 2  / 3 (when aerosols produced or large concentrations
Bacillus  anthracis Blood, cerebrospinal fluid, pleural fluid Naturally and experimentally infected animals Exposure of intact and broken skin, inoculation 2 / 3 when aerosols produced, large concentrations

Table 1:  A small sample of known human pathogens commonly used in research laboratories.  Adapted from US HHS Publication: Biosafety in Microbiological and Biomedical Laboratories, 4th Ed., 1999

Working in our Lab:

The Faculty of Medicine Flow Cytometry Facility operates at biosafety containment level 2+ (BSL2+).   Aside from our sorters, all other instruments are treated as level 1 resource and therefore all Risk 2+ agents must be fixed prior to bringing them to the lab.

When working in the Facility, proper attire is mandatory, i.e. no sandals, shorts, skirts etc.  You may be asked to leave the lab if your clothing does not meet published guidelines. Also, when in the lab, lab coats must be worn at all times, in addition to any other personal protective equipment deemed necessary for your safety and that of others.    For more information please see the University’s Biosafety Policies and Procedures Manual.

At the completion of your work in the lab, please use the disinfectant wipes provided to clean with work area.

In the event of a biological spill, please notify the technical staff on duty of the nature of the spill so they can attend to it.  If working after hours and a spill occurs, please refer to spill containment procedures posted at both sinks in the lab.  A copy of the forms may be downloaded here.

 

Biosafety Links and Papers:

U of T Environmental Health and Safety
Canadian Biosafety Standards and Guidelines
Pathogen Safety Data Sheets and Risk Assessment
Human Pathogens and Toxins Act
Laboratory Safety Guidelines: 3rd Edition 2004

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