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4.21 Flow Cytometry

Flow cytometers are technically sophisticated instruments that measure quantitative properties of single cells, such as:

  • Cell size
  • Total DNA
  • Newly synthesized DNA
  • Messenger RNA
  • Specific surface receptors
  • Intracellular proteins
  • Transient signaling events in living cells

Measurements/counts are taken by passing a suspension of unclumped cells, in single file, through an electrified aperture causing changes in voltage, or across a laser beam where scattered light is measured. In some instances, the cell suspension is then discarded; in other situations, cells are actually sorted by this method into separate containers.

Instrument Types

  • Benchtop analytical flow cytometers – Utilized in an individual PI’s lab, or in a core facility.
  • High-speed cell sorter flow cytometers – Typically utilized in a core facility; this type of instrument aerosolizes samples via a nozzle that forms a jet of micro-droplets, and thus cannot be used for sorting infectious or potentially infectious samples unless contained in a BSC, or by another approved containment method. Fixed materials may be sorted outside of containment.

Laser Hazards

  • In most instances, flow cytometers contain safety devices that prevent user exposure to laser beams. Safety covers would have to be intentionally removed, or interlocks intentionally inactivated for laser exposure to be hazardous.
  • Users must be trained to avoid staring at the objective/ specimen while the instrument is scanning.

Biohazards

  • Instrument malfunctions such as a clogged nozzle or air in the fluidic system can drastically increase aerosol formation, thus routine maintenance and preventive maintenance on equipment is essential for laboratory safety.
  • Exposures can occur from sample handling as well as from aerosols. Best practice: Handle all live samples for submitted for cell cytometry as biohazardous material.  
  • Cell sorter operators who handle and process specimens without being aware of all details concerning the nature of the specimens are placed at increased risk. Thus, a completed questionnaire should be submitted with samples for flow cytometry which would supply the operator with information about sample origins, whether they are live or fixed, and if chemicals, nanoparticles, potential pathogens or genetically modified material are present in samples. It is incumbent upon the person requesting cell analysis/ sorting to thoroughly divulge information on all potentially hazardous materials (biological or chemical) in specimens submitted.
  • Samples that must be treated as potentially biohazards.

The following samples must be treated as potential biohazards include:

  • Samples containing recombinant nucleic acid material (e.g., rDNA, rRNA, viral vectors, transgenes, etc.)
  • Samples containing lentivirus, adenovirus, or any genetically engineered amphotropic virus
  • Unfixed human or NHP specimens
  • Unfixed cells from primary or immortalized cultures of human/ NHP origin
  • Unfixed cells from primary and immortalized cultures from animal donors that could be potentially infectious with zoonotic agents, and from transgenic animals
  • CD34 stem cells, tumor cells, transfected tumor cells
  • Samples containing nanoparticles

Risk Assessment for Cell Cytometry

  • A laboratory conducting flow cytometry must identify its specific biohazardous risks from every angle – specimen processing/handling, aerosol containment, waste management, equipment maintenance, operator training level, proficiency, experience, and PPE – and develop mitigating practices and procedures for each. The following chart provides general guidelines per BSL for some sample types and agents.  

Learn more about the Biosafety Level Guidelines for Cell Sorting (2014 ISAC Standards)

Requirements for Flow Cytometry Using Biohazardous Materials (BSL-2)

  • Lab facilities must demonstrate negative air pressure, and possess required design and equipment features of a BSL-2 lab.  
  • The lab must operate with closed doors and restricted entry (with appropriate signage posted) when potentially biohazardous materials are being analyzed or sorted.
  • A BSC must be used for biohazardous sample handling.
  • Aerosol containment must be achieved when biohazardous materials are analyzed/sorted, whether that is provided by BSC containment for equipment, by use of a portable aerosol management system (provides a negative pressure environment for a cell sorter by drawing air from the unit through a HEPA filter, then releasing the air into the environment) or by another method.  
  • Aerosol control measures on instruments must be tested periodically, and test results documented.  Laboratories must develop and implement such testing methods (e.g., “Glo Germ”™ is a rapid and inexpensive product that can be used to provide good qualitative aerosol containment data.)
  • Operators must wear appropriate PPE when handling and analyzing samples; at a minimum, this would include disposable gloves, lab coat or disposable gown, and eye protection.
  • Work with certain materials may also require respiratory protection in the form of N95 respirators or PAPRs in addition to using a BSC for containment.

Personnel Requirements

  • Personnel who operate flow cytometry equipment must be well trained on the instrument, as well as on safe handling of specimens, and use of safety equipment. They must also demonstrate a sound biological understanding of the types of materials handled/ processed, and the hazards, exposure potentials, routes of transmission, etc. associated with each.
  • Only fully trained personnel must be allowed to use this equipment.
  • Personnel who operate this equipment and may be exposed to biohazardous material must complete a Medical Questionnaire, and update it yearly.

Waste Management

  • Effluent must be collected from the instrument into containers with fresh full-strength bleach or other suitable disinfectant, in sufficient quantity to achieve a 10% final concentration.
  • Fluid lines must be decontaminated routinely with freshly diluted (1% - 10%) bleach or other suitable disinfectant.