Biohazards in a flow cytometry laboratory can either arise from sample handling or from the use of the flow cytometry analyzer or the cell sorter. Although most samples that are analyzed in the flow cytometry laboratory are fixed, some protocols require that cells are run through the instruments unfixed, e.g., certain apoptosis assays, measurement of calcium flux, and most importantly viable cell sorting. Specimens can contain either known or unknown pathogens. Most of the infectious agents encountered in the specimens that are analyzed on analytic and sorting flow cytometers are transmitted by percutaneous or mucous membrane exposure; however, some may also be transmitted by inhalation of aerosols. Also, many dyes used for staining in flow cytometric protocols are toxins, mutagens or carcinogens.Factor 12-953 and Factor 12-393 both house flow cytometry analyzers and are classified as BSL2. All Users who run samples on the instruments in these rooms must wear gloves and a laboratory coat. Please note that laboratory coats must not be worn outside the laboratory, but can be carried from one laboratory space to the next. We strongly prefer users to bring their own coat, but have disposable coats available for purchase for $5 each. Gloves must not be worn outside the laboratory and are to be disposed of only into biohazard waste containers, even when they are unsoiled. All analyzers in the UCLA Flow Cytometry Core Facility have biosafety features for reduced risk of operator exposure to instrument-generated sample droplets and aerosols, e.g., enclosed flow cells, droplet containment modules. However, operators of these instruments need to take care to securely place each sample tube into the sample introduction port, otherwise it could be blown off once it is pressurized and splash sample onto the operator. For improved splash protection, it is recommended that safety glasses be worn during acquisition. The waste needs to be collected into a waste container which contains fresh concentrated household bleach in sufficient quantity to achieve a final concentration of 10% when the flask is full. Routine decontamination of instrument fluid lines with freshly diluted (7%-10%) bleach solution is standard laboratory practice. For protection of core laboratory personnel and users of the facility from exposure to occupational hazards it is imperative that all users of the flow cytometry core facility comply with this policy, irrespective of the type of samples that are run, and with all applicable UCLA safety regulations.
Our sort laboratories (Factor 12-671, 12-639, and 12-637) are classified as BSL2+, because in contrast to flow cytometry analyzers, jet-in-air, deflected droplet cell sorters like the FACSVantage and BD FACSAria sorters generate droplets and aerosols during their normal operation increasing the hazard potential of the experiments performed on these instruments. Aerosol production can increase substantially during instrument failures, e.g., a partial clog in the nozzle tip. Therefore, only personnel trained in safety protocols is permitted to run these flow sorters and the efficiency of aerosol control measures on the instruments needs to be tested periodically, particularly when unfixed human cells and known biohazardous samples are acquired or sorted. Users are required to fill out the Biosafety Questionnaire before a sort appointment can be made (please print it, fill it out completely, and return it to Ingrid Schmid). Users are also required to fill out a Sorting Protocol Sheet before each sort to provide hazard information about the sample and details about the experiment that are relevant for instrument set-up.Access is restricted during ongoing sort experiments to essential personnel and investigators involved in the sort. Please note that Users wishing to monitor the sort must wear the same personal protective gear as the operator of the cell sorter. This includes wearing N-95 respiratory masks which have to be fit tested by EH&S specifically for each user. Re-testing is required annually. Please see the EH&S website for details.For the official safety standard of the International Society of Analytic Cytology (ISAC) for cell sorting of potentially and know biohazardous samples, refer to "International Society for Analytical Cytology Biosafety Standard for Sorting of Unfixed Cells," Schmid et al., Cytometry Part A71A: 414, 2007. Other publications relevant for safety precautions for cell sorting are: Schmid I, Hultin LE, and Ferbas J.: Testing the efficiency of aerosol containment during cell sorting. In: Robinson JP, Darzynkiewicz Z, Dean P, Dressler L, Tanke H, Rabinovitch P, Stewart C, Wheeless L (eds.), Current Protocols in Cytometry, Suppl. 1, Unit 3.3, Wiley & Sons, New York, 1997 or to Schmid et al. Biohazard Sorting in: Methods in Cell Biology, Cytometry 4th ed., Darzynkiewicz Z, Roederer M, Tanke H, eds., Elsevier Inc., San Diego, CA, 2004, pp 221-240.
Cell separations of unfixed samples using the RoboSep are performed in a class II biosafety cabinet for splash protection. The instrument is operated with the cover closed to prevent escape of any fluid and aerosolized sample into the room in case of a fluid line detachment.
For protection against biohazardous spills (in the case of unfixed samples) and a chemical spill (in the case of samples fixed in formaldehyde and/or stained with toxic dyes) it is mandatory that for transport to and from the Flow Cytometry Core Facility all samples are placed into a second leak-proof container labeled with the biohazard symbol. The secondary container, such as a plastic bag with a leak-proof seal or a plastic container with a secure lid must be able to contain the sample(s) in case of breakage of the primary container. Acceptable containers include plastic buckets with a screw cap or Playmate chests with lockable lids. Unacceptable transport containers include sample racks, ice buckets, and aluminum foil wrappers. Users bringing samples to the Core in unacceptable containers will not allowed entrance.
For further information on occupational safety issues and safety issues related to flow cytometry, please refer to the web pages from the Laser Institute of America, Occupational Health and Safety, National Institute of Health, Clinical and Laboratory Standards Institute , and the Centers for Disease Control.
Information related to practical biosafety issues for HIV immunophenotyping such as survival and inactivation of HIV under laboratory conditions, sample shipping, fixation, sample disposal, laser safety, flow cytometer fluid lines disinfection, and post-exposure management is available in the Q&A document "Biosafety Concerns for Flow Cytometric HIV Immunophenotyping" taken from the Adult AIDS Clinical Trial Group Website (Adobe Acrobat required) and from the publication by Schmid et al., "Biosafety Consideration for Flow Cytometric Analysis of Human Immunodeficiency Virus Infected Samples," Cytometry (Communications in Clinical Cytometry) 38:195-200, 1999.
Please note that the CFAR Virology Core Laboratory at UCLA provides testing services, blood and blood cell products for research labs working with HIV and related viruses.