Individuals who work in a laboratory that handles infectious or hazardous
substances are at risk of exposure to the substances they handle.
Laboratory-acquired infections (LAIs) are not uncommon — over 5,000 cases and 190 deaths were reported up to 1999 (1), although these figures are believed to be significantly underestimated because of underreporting (2,3). Additionally, only about 20% of infections can be attributed to any known, single exposure event (4).
There are a number of ways in which infectious substances can enter the body and cause infection, including ingestion, inhalation, or contact with mucous membranes, including conjunctivae (transfer of microorganisms to the eyes by contaminated hands), or with nonintact skin. The types of events that can lead to an infection include the following: exposure to infectious aerosols; spills and splashes; accidental needlestick injuries; cuts from sharp objects and broken glass; bites and scratches from animals or ectoparasites; oral pipetting (which is prohibited); centrifuge accidents; secondary spread of infectious materials to nonlaboratory areas. Exposure to aerosols may be the greatest biohazard facing laboratory workers (5). Aerosols can present a risk in terms of inhalation, ingestion, mucous membrane contact, etc. Operational practices and techniques must be used to minimize the creation of aerosols associated with common laboratory procedures. (6)
Cell Sorting Service
FCF Analyzer Rooms
Biohazard potential of unfixed cells
ALL primary human cells are at least considered as BSL-2!
Biological specimens can harbor known and unknown pathogens that may be transmitted through droplets and aerosols that are generated either through laboratory manipulations or the cell sorting process. Samples may also consist of genetically manipulated cells, which contain genomic sequences of potentially infectious organisms. Although most pathogens encountered when sorting clinical or research samples are transmitted by inoculation, by direct exposure of broken skin or mucous membranes, or by ingestion, some may be transmitted by inhalation of organism-containing particles (see table 1.). For instance, commonly known blood-borne infectious agents, e.g., the Human Immunodeficiency Viruses (HIV-1, -2) and the Hepatitis viruses are primarily transmitted through inoculation. However, infection through aerosolization of virus particles has at least been documented for Hepatitis B . Biological particles of 0.1 µm to 60 µm sizes (i.e., aerosols) are considered important in the spread of infectious diseases. Droplets that fall out of suspension in air will fall onto surfaces and can be transmitted by exposure to broken skin or mucous membranes, or by ingestion. Both HIV and HBV have been shown to be transmitted through mucous membrane exposure. Therefore, it is important that all laboratory personnel, and in particular the sorter operator, be protected from exposure to aerosols, droplets and accidental splashes.
Creation of droplets and aerosols during the sorting process
In general, cell sorters produce droplets in the size range of 40-200 µm and micro droplets (satellite droplets, 3-7 µm) during their normal operation. Usually droplets larger than 80 µm will fall out of the air rapidly; smaller droplets may be aerosolized, particularly when they are elevated by air currents. Cell sorters create secondary aerosols of various droplet sizes when the undeflected center stream and the side streams splash into receptacles. During failure operation of the cell sorter, e.g., a clogged sort nozzle or air in the fluidic lines, aerosol production may increase substantially. For instance, a partially clogged nozzle will cause deflections in the fluid streams that are then likely to strike an obstacle and create considerable amounts of aerosols. Usually sorter generated aerosols are contained within the sorting chamber when the door is properly closed and the interlock is engaged. Because of the potential health risk to sorter operators if aerosols escape into the room, it is strongly recommended that aerosol containment of a cell sorter be verified.
Sorting of BSL-2 classified samples
The FACSAria cell sorter at LICR@UNIL in Epalinges is housed in a separate, lockable room within a certified, class-II biological safety cabinet making it possible to process samples considered as BSL-2.
However aerosol containment of the cell sorter may be breached during a sort for any number of reasons, thus exposing the operator to potentially biohazardous aerosols. Therefore, to compensate for the fact that aerosols cannot be contained within a biosafety cabinet during cell sorting, it is recommended that BSL-2 containment be combined with BSL-2+ work practices as described below.
Awareness of the origin of the sample, the potential presence of infectious agents or genetically engineered material is essential to protect the operator and lab.
For proper risk assessment, it is critical that relevant biohazard information about the samples be transmitted to personnel before cell sorting experiments. A biosafety assessment cell sorting registration form must be completed and submitted at least 48 hours prior to the cell sorting experiment. This will allow personnel to determine whether the design of the facility and cytometer is appropriate for the planned experiments. Lists of different biological specimens and their Biosafety levels, together with the 'Statement of the Swiss Expert Committtee for Biosafety on the classification of work with genetically modified viral vectors' can be found in the download section of this website.
Guidelines for agents that must be processed in the BSL-2 FACSorter at LICR@UNIL
- Fresh or fixed human cells from patient blood or a blood bank, body fluids, or other unfixed/fixed tissues obtained from sources not thought to be infected with any risk group <1 reagents
- Fresh or fixed murine cells, body fluids, or other unfixed/fixed tissues obtained from sources not thought to be infected with any risk group <1 reagents
- murine or human cells which have been infected with recombinant retroviral or lentiviral vectors which have been IBC (Institutional Biosafety Committee)-approved for less than or equal to BSL-2 containment
- murine or human cells which may have been exposed to bacteria from Risk Group 2 or lower
- other biological specimen considered to less than or equal to BSL-2
Samples known to contain BSL-3 pathogens and some BSL-2 pathogens (considered as BSL-3 in practice as described in "International Society for Analytical Cytology biosafety standard for sorting of unfixed cells", Ingrid Schmid et al, Cytometry A. 2007 Jun;71(6):414-37 will not be accepted for sorting. These include samlples known to contain Hepatitis viruses B and C, HIV-1, HIV-2, HTLV-1, HTLV-2, tuberculin bacillus, and SIV (Simian Immune deficiency virus). The facility Operator and Director will make decisions concerning sorting of other known infectious agents on an individual basis in consultation with the Biosafety Officer.
To assure the assignment of proper safety practices and procedures, it is necessary to collect detailed information about all samples prior to receipt. A Cell Sorting Registration Form must be completed and submitted to the Flow Cytometry Operator at least 48 hours prior to the start of each project. Samples must be properly packaged prior to transport to the facility. Failure to comply with the following packaging requirement may lead to the rejection of the sample. Samples should be transported in sealed leak-proof primary and secondary containment. The secondary (outer) container should have a biohazard sticker. Samples should be provided in sealed or capped tubes placed in larger conical tubes, sealed specimen bags, Tupperware or test tube racks placed inside of a larger sealed container. Samples must be clump free and in an appropriate tube for sorting (12x75mm snap-cap tube or 15ml conical tube). Sample must not have contaminated the outside of the tube.
All instrument rooms of the Flow Cytometry Facility must minimally follow BSL-1 guidelines. Biosafety Level 1 represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended. ALL primary human cells are at least considered as BSL-2 and must be fixed with a proven method (paraformaldehyde etc) before being processed on any of the flow cytometers!
Basics of BSL-1 practice
- public access to work areas must be limited
- eating, drinking, smoking or handling contact lenses in the work area is not permitted at any time
- use sharps only when no alternative exists, handle and dispose of them carefully
- minimize activities that are likely to create splashes, sprays or aerosolswash hands after removing gloves, and before leaving the laboratory