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  4. Contained use - Flow cytometry

Contained use - Flow cytometry

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Summary

Biosafety aspects in a flow cytometry (FCM) routine or research laboratory should be taken into account mainly when working with unfixed and infectious materials. Biohazards can theoretically arise either from sample handling or more specifically from aerosols and droplets generated by the flow itself. Biological specimens can contain documented or not documented pathogens. As a consequence, flow cytometer users are subjected to occupational exposure through accidental inoculation, droplets or aerosols. Because laboratory personnel is more exposed to pathogenic orgnaisms than the general population, occupational exposures can lead to laboratory-acquired infections.

Such exposure risk is even higher when flow cytometry is organized as a central service, the operator not always being aware of all the details concerning the safety status of the handled material. These considerations justify the following recommendations about sample handling, FCM procedures related to aerosol's containment, waste management and equipment maintenance.

Awareness about the origin of the sample, the potential presence of infectious agents (genetically modified or not) is a key to protection of the lab scientists. All unfixed materials (blood samples, various body fluids, cultured cells and environmental samples) are a priori considered as biohazardous. The use of a fixative is not always sufficient to eliminate all types of biohazards. The use of appropriate biosafety cabinet for sample preparation is highly recommended. Gloves and protective clothing should always be worn by flow cytometer operators. Only experienced and well-trained operators should perform potentially biohazardous cell sorting.

Cell-sorters are equipped with a nozzle to form a jet of microdroplets : this experimental step is likely to generate aerosols. Instrument failures such as clogged sort nozzle or air in the fluidic system can drastically increase aerosol formation. For this reason biohazardous materials should not be sorted unless supplementary suitable containment measures are applied. For instance a droplet containment module should be installed to reduce the risk of exposure to generated droplets and aerosols. All biohazardous material must be decontaminated in a separate room and placed in leak-proof containers which are sealed before evacuation.

In conclusion, all scientists and laboratory staff in the FCM field should be aware of the potential hazards associated with their discipline. Up to date guidelines on handling and proper disposal of biohazardous materials should be applied in all FCM laboratories. General recommendations approved by the International Society for Advancement of Cytometry (ISAC) should help to settle a basis for biosafety guidelines in FCM laboratories. Because cell sorting practices and standards are subjected to continuous improvement it is recommended to take into consideration the last updated version of the available Cytometry Cell Sorter Biosaefty Standards (Schmid et al., 1997, Schmid et al., 2007, Holmes et al., 2014).

A Biosafety Sheet should be filled out and approved by the FCM laboratory team before starting any new set of experiments. The establishment of a local biosafety committee including experts in FCM is strongly recommended in order to control the containement efficacy of the cell sorter.

Risk assessment and biosafety recommendations

During FCM (including cell sorting) experiments, when handled biological samples (potentially) contain pathogenic organisms and/or Genetically Modified Organisms (GMOs), biohazards can arise either from:

  1. Sample handling (opens in a new page)
  2. Flow generated aerosols and/or droplets (opens in a new page)
  3. Waste management and equipment maintenance (opens in a new page)

In many laboratories FCM is organized as a central service, and the operator is not necessarily aware of all the details concerning the safety status of the handled material. As a consequence, flow cytometer users are subjected to occupational exposure (according to European Directive 2000/54/EC).

Recommendations

All flow cytometer users should be aware of the potential biohazards associated with their discipline.

  • Guidelines on handling and proper disposal of biohazardous materials should be applied in all FCM laboratories.
  • A Biosafety Sheet should be filled in and approved by the FCM laboratory team before starting any new set of experiments.
  • It is strongly recommended to include FCM experts in the local Biosafety Committee in order to control the containment level of the analysed and/or sorted biological materials.

Helpful links

Associations

  • The Belgian Association for Cytometry, ABC/BVC, Belgium (link)
  • Association Française de Cytométrie, AFC, France (link)
  • Deutsche Gesellschaft für Zytometrie, DGfZ, Germany (link)
  • Danish Society for Flow Cytometry DSFCM, Denmark (link)
  • FlowcytometryUK , UK (link)
  • International Society for Advancement of Cytometry, ISAC (link)

Miscellanous

  • Purdue University, Cytometry laboratories (link)
  • Journal of ISAC: Cytometry (link)

References

  • Norden MA, Kurzynski TA, Bownds SE, Callister SM, Schell RF. Rapid suscptibility testing of Mycobacterium   tuberculosis (H37Ra) by flow cytometry. J Clin Microbiol 1995; 33 : 1231-1237.
  • Schmid I, Nicholson JKA, Giorgi JV et al. Biosafety Guidelines for Sorting of Unfixed Cells, Special reports, Cytometry 1997; 28 : 99-117.
  • Kirk SM, Schell RF, Moore A, Callister SM, Mazurek G. Flow cytometric testing of susceptibilities of Mycobacterium tuberculosis isolates to ethambutol, isoniazid, and rifampin in 24 hours. J Clin Microbiol 1998; 36 : 1568-1573.
  • Moore AV, Kirk SM, Callister SM, Mazurek GH, Schell RF. Safe determination of susceptibility of Mycobacterium   tuberculosis to antimicrobial agents by flow cytometry. J Clin Microbiol 1999; 37 : 479-483.
  • Alvarez-Barrientos A, Arroyo J, Cantan R, Nombela C, Sanchez-perez M. Application of flow cytometry to clinical microbiology. Clin Microbiol Rev 2000; 13 : 167-195.
  • Oberyszyn AS & Robertson FM. Novel rapid method for visualization of extent and location contamination during high-speed sorting of potential biohazardous samples. Cytometry 2001; 43: 217-222.
  • Burdz TVN, Wolfe J, Kabani A. Evaluation of sputum decontamination methods for Mycobacterium tuberculosis using viable colony counts and flow cytometry. Diagn Microbiol Infect Dis 2003; 47 : 503-509.
  • Schmid I, Merlin S, Perfetto SP. Biosafety concerns for shared flow cytometry core facilities. Cytometry 2003; 56A : 113-119.
  • Perfetto SP, Ambrozak DR, Koup RA, Roederer M. Measuring Containment of viable infectious cell sorting in high-velocity celle sorters. Cytometry 2003; 52A: 122-130.
  • Perfetto SP, Ambrozak DR, Roederer M, Koup RA. Viable infectious cell sorting in a BSL-3 facility. Methods Mol Biol. 2004; 263: 419-424.
  • Lennartz K, Lu M, Flasshove M, Moritz T, Kirstein U. Improving the biosafety of cell sorting by adaptation of a cell sorting system to a biosafety cabinet. Cytometry 2005; 66A: 119-127.
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  • Ghidoni D, Lockhart E, Eagleson D, Zarembo M. Performance testing of large biological safety enclosures. ABSA meeting 2005, Poster.
  • Schmid I, Lambert C, Ambrozak D, Marti GE, Moss DM, Perfetto P. International Society for analytical cytology: Biosafety standards for sorting of unfixed cells. Cytometry 2007; 71A: 414-437.
  • Wallace RG, Aguila HL, Fomenko J, Price KW. A method to assesse leakage from aerosol containment systems: testing a fluorescence-activated cell sorter (FACS) containment system using the radionuclide technetium-99m. Applied Biosafety Journal 2010; 15 (2): 77-85.
  • Perfetto SP, Ambrozak DR, Nguyen R, Roederer M, Koup RA, Holmes KL. Standard practice for cell sorting in BSL-3 facility. Methods Mol Biol 2011; 699: 449-469.
  • Holmes KL, Fontes B, Hogarth P, Konz R, Monard S, Pletcher CH, Wadley RB, Schmid I, Perfetto SP. International Society for the Advancement of Cytometry Cell Sorter Biosaefty Standards. Cytometry 2014; 85A: 434-453.
  • Xie M, Waring MT. Evaluation of Cell Sorting Aerosols and Containment by an Optical Airborne Particle Counter. Cytometry 2015; 00A: 00-00. doi: 10.1002/cyto.a.22684