The containment of high-pressured biological sample streams generated by flow cytometer cell sorters is biologically imperative; the risk of exposure to (potentially) infectious agents must be mitigated. Subsequently, evaluating levels of aerosol containment are imperative for biosafety assurance.
A team of researchers from the National Biodefense Analysis and Countermeasures Center (NBACC) have developed a robust assay to determine cell sorter aerosol containment that is rapid, affordable, and efficient. The new method challenges the industry status quo – the Glo‐Germ bead procedure – which, although widely used, has several drawbacks.
Stephen Perfetto and team succeeded in developing a new method utilizing Cyclex‐d impactor and Dragon Green fluorescent microsphere beads. Its smaller sampling requirement and sensitivity strongly positions it as replacement assay for the standard Glo-Germ bead procedure.
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Is Glo-Germ missing the mark as an effective containment test?
Modern cell sorting flow cytometers are fitted with an important biosafety device: aerosol containment systems. Sorters use a stream-in-air sorting method achieved by the creation of microdroplet jets from a nozzle. The blockage of the sorting nozzle tends to increase the incidence of aerosol formation, necessitating the containment of aerosolized samples to mitigate the risk of operator exposure to infectious agents.
The production of aerosolized samples in this way occurs during fail mode (FM), which describes the aberrant operation of the cell sorter due to partial nozzle obstruction and deviations in the sample stream. According to the latest International Society for the Advancement of Cytometry (ISAC) Cell Sorter Biosafety Standards, aerosol containment must be validated by assays performed at several intervals.
The most popular measure for containment testing is the Glo-Germ bead procedure; the assay measures the release of melamine copolymer resin beads which are highly fluorescent. Any beads escaping the sort chamber are collected on a microscope slide for enumeration via fluorescent microscopy.
Despite its popularity, the method suffers several drawbacks. Firstly, the impactor is incorrectly used - typically it is designed to collect bio-aerosols on agar plates for growth analysis. This establishes a cutoff diameter, or d50, of 0.65. When used for containment testing purposes, the glass slide set-up alters the d50 subsequently causing an underestimation of escaped aerosol. Finally, the biggest limitation posed by the Aerotech impactor is the need to be thoroughly cleaned to avoid carry-over particles that could generate false positives.
Is there a better way to contain the aerosol spray during sorter failure?
To address these drawbacks, the team developed a novel cell sorter to overhauls the Glo-Germ procedure. The article, published in Cytometry Part A: Journal of Quantitative Cell Science, outlines a replacement procedure utilizing a Cyclex‐d impactor and Dragon Green (DG) beads conducted on a BD FACSAria II model cell sorter, using an Aerosol Particle Sizer equipped with a UV laser.
The Cyclex-d impactor was placed near the cell sorter to collect aerosolized samples during FM. The team compared this procedure to UV‐APS aerodynamic particle sizer and a UV‐excitable dye. This type of containment evaluation is a real-time method that provides the advantage of immediate determination of particle counts but suffers the drawback of expense.
The advantages of the Cyclex-d Dragon bead procedure
The team determined that 1μm of DG microspheres were representative of the size range released. The use of the Cyclex-d impactor and use of disposable cassettes and fluorescent microspheres ensured low background noise; further, the collection efficiency of the impactor approaches 100% as the median AD (1.6 μm)> the d50 of the Cyclex‐d (1 μm).
When measuring the collected aerosol, the procedure reflected a high sensitivity; the lowest level of detection reading 0.04 aerosols/cm3, a figure approaching the background aerosol measurements. High sensitivity was achieved despite the low frequency of microsphere-occupied droplets – an occurrence attributed to the higher sampling volume of air captured by the Cyclex‐d versus the UV‐APS over identical time periods. Subsequently, the group posture that sensitivity could be increased by increasing the collection time.
The Aerosol Management System (AMS) was demonstrably robust, as up to 70% of air can be lost before containment is compromised. This finding provides an effective method of monitoring containment during cell sorter operation and serves as a supplementary means of containment testing as recommended by the SAC Cell Sorter Biosafety Standards.
The Cyclex-d/ DG method is applicable to cell sorters other than the BD FACSAria II model. To do so et al., suggest that operators should consider (1) distance from the Cyclex‐d, to the sorting chamber (recommended to be as short as possible); (2) optimizing the method for creating a FM that is representative of true partial nozzle obstruction; (3) determining the best form of positive control i.e. when the AMS is off and the instrument is set to FM (4) means to prevent false positives or background readings for both enclosed and non-enclosed cell sorters in order to ensure accuracy and specificity and (5) high efficiency of aerosol collection, within the AD range of the cell sorter.
The work of Perfetto and colleagues has provided an improved biosafety device for cell sorters that sort both innocuous and infectious samples. Despite the recommended use of the Glo‐Germ procedure for the purpose of cell containment monitoring, its adoption by cell sorter labs is poor.
The drawbacks – extensive cleaning and widely variable bead size – are the culprits. The Cyclex-d / DG procedure presents a rapid, robust and efficient containment test for aerosols produced during cell sorting and the team ‘recommended as a replacement assay for the previously published Glo‐Germ/Aerotech impactor collection method’.
The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the DHS or S&T.
This work was funded under Contract No. HSHQDC‐15‐C‐00064 awarded by the Department of Homeland Security (DHS) Science and Technology Directorate (S&T) for the operation and management of the National Biodefense Analysis and Countermeasures Center (NBACC), a Federally Funded Research and Development Center.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was supported by the Intramural Research Program of the Vaccine Research Program, NIH.
Perfetto, S.F. et al. (2019) Novel Impactor and Microsphere‐Based Assay Used to Measure Containment of Aerosols Generated in a Flow Cytometer Cell Sorter. Cytometry Part A: Journal of Quantitative Cell Science. https://doi.org/10.1002/cyto.a.23680