Novel Impactor and Microsphere-based Assay used to Measure Containment of Aerosols Generated in a Flow Cytometer Cell Sorter

Abstract

Today’s state-of-the-art cell sorting flow cytometers are equipped with aerosol containment systems designed to evacuate aerosols from the sort chamber during a sort. This biosafety device is especially important when the sort operator is sorting infectious or potentially infections samples. Hence, it is critical to evaluate the performance for this system in normal operation and in “failure” mode to determine the efficacy of containment.

In the past decade, the most popular published method for evaluating containment has been the Glo-Germ bead procedure. These highly fluorescent and multi-size particles can easily be detected on a microscope slide and enumerated using a fluorescent microscope. Collecting particles on this slide is accomplished using an Aerotech impactor. This sampler collects potentially escaping aerosols from the sort chamber before enumerating any particles.

Although the Glo-Germ procedure has been adopted by many labs, there are several drawbacks with the procedure that have limited its adoption by cell sorter laboratories: The Aerotech impactor is a reusable device that requires rigorous cleaning between measurements. The surface area of the collection slide is large and difficult to scan on a fluorescence microscope. These beads produce a wide variation in sizes resulting in inconsistency in flow rates. Here, we describe a novel and replacement method utilizing a Cyclex-d impactor and Dragon Green beads. This method was compared for sensitivity of detection of escaped aerosols with a published method for aerosol detection which utilizes a UV-APS aerodynamic particle sizer and a UV-excitable dye. One of the advantages of the Cyclex-d system is the narrow-defined field of collection as compared to the standard Glo-Germ bead procedure, this means a smaller sampling area is used in the Cyclex-d impactor as compared to the AeroTech impactor. In addition, the sensitivity of detection was found to be better using the Cyclex-d collection device as compared to the standard Glo-Germ bead procedure.

Introduction

It is well established that stream-in-air cell sorters may produce high concentrations of aerosols during fail mode (i.e. partial nozzle obstruction and with stream deviations; FM) (1). Due to the possible exposure of the operator to these aerosols while sorting potentially infectious samples, cell sorting is considered a high-risk laboratory procedure. Instrument manufacturers have designed aerosol containment systems to mitigate this risk. Therefore, as recommended in the latest International Society for the Advancement of Cytometry (ISAC) Cell Sorter Biosafety Standards (2), aerosol containment testing of these systems must be performed at intervals determined by a risk assessment to validate containment. These published standards reference a method for containment testing measuring the release of Glo-Germ beads (melamine copolymer resin beads) collected in an Aerotech impactor (3). However, this method has several drawbacks and alternate methods have been actively pursued with the goal of establishing a sensitive assay for aerosol containment testing.

Several other methods for the evaluation of containment have been described previously. These containment assays can be broadly characterized as utilizing either active (e.g. impactor) or passive deposition methods (3–7) or real-time measurement methods (1,8,9). Real-time measurement methods offer the advantage of immediate determination of particle counts, but require fluorescence detection instruments to distinguish ambient air particles from cell sorter derived aerosols to prevent false positives (1). Such instruments (e.g. UV-APS, TSI, Inc.) represent significant additional expense and service considerations. In addition, some of these instruments were designed to measure solid particles such as fungal spores, dust particles, and other airborne allergens. Optical particle counters such as the Fluke 985 Air Quality meter used by Xie et al. (9), for example, determine particle size by measuring light scatter intensity and is calibrated using polystyrene latex beads. This instrument will therefore underestimate particle size if used to measure liquid particles (i.e. aerosolized sheath fluid particles that do not contain fluorescent beads) due to the lower index of refraction of liquid particles versus polystyrene beads (10,11). In addition, this instrument cannot distinguish ambient air particles from cell sorter generated aerosols.

Although the capture of aerosols containing highly fluorescent Glo-Germ beads seemed to provide a simple and sensitive assay for containment testing, there are several drawbacks with the assay that have forced a re-evaluation of current containment methods. These include the following: the size distribution of these particles is very large, resulting in inconsistency in cell sorter flow rates; the Aerotech collection device is not used as designed. Specifically, the Aerotech impactor is a viable impactor, which is designed to capture bio-aerosols onto an agar-coated plate for subsequent growth analysis, and has a cutoff diameter, or d₅₀, of 0.65μm (12). However, as used in cell sorter containment testing, the glass slide is positioned on an empty petri dish in the Aerotech impactor; this setup changes the jet-to-plate distance and will change the d₅₀, which is related to this distance(13). It is possible that the altered d₅₀ may not be within the desirable aerodynamic diameter (AD) range for cell sorter aerosol measurements causing an underestimation of escaped aerosols. In this regard, a preliminary experiment comparing yellow-green (YG) fluorescent beads, collected in a Cyclex-d impactor with Glo-Germ beads collected in the AeroTech impactor showed 3-fold higher collection for the Cyclex-d at identical flow rates (Figure 1). Finally, and perhaps the biggest drawback with the Aerotech impactor is that it must be cleaned thoroughly after every test to eliminate carry-over particles from being detected, which could lead to false positive results if not cleaned properly.

Figure 1.

Cyclex-d cassette collection of YG beads (red bars) compared to the collection of Glo-Germ beads using Aerotech impactors (blue bars). Data shows the increase in collection of both particles with increase sample rate. In addition, the data shows the collection of YG beads by the Cyclex-d cassette is 3x more efficient as compared to the Aerotech impactor and the collection of Glo-Germ particles.

This work was undertaken to develop a novel cell sorter aerosol containment assay that met the following criteria: 1) the assay must be performed and results available within the same day and before cell sorter operation, 2) the sensitivity must be high, and the assay must be validated using an alternative aerosol testing procedure using other established instruments, 3) equipment and supplies must be affordable and utilize commonly available flow cytometry laboratory equipment, 4) accuracy and specificity must be high, with little false positives or ambient air background readings for both BSC enclosed and non-enclosed cell sorters, 5) efficiency of aerosol collection must be high within the AD range of cell sorter aerosol production.

This paper describes the use of a non-viable disposable impactor and uniform fluorescent microspheres as an alternative to the currently recommended containment assay. The results of this work describe the development of a rapid and efficient method for testing containment of aerosols generated by cell sorters. The sensitivity of this method was evaluated by utilizing the UV-APS particle sizer (TSI Inc.) and sample containing UV excitable dye.

Materials and Methods

Beads and Impactor:

Internally fluorescent 1.0 μm Dragon Green (DG) beads (Excitation (Ex) 480nm; Emission (Em) 520nm; Bangs Laboratories) and 0.75, 1.0 and 2.0 μm Fluoresbrite yellow green (YG) beads (Ex 441nm; Em 486nm; Polysciences, Inc.) were used.

Cyclex-d setup and particle detection:

Cyclex-d impactor and sampling cassettes, MegaLite pump and Rotameter (Environmental Monitoring Systems, Charleston, SC) were used to collect aerosol samples Figure 2a shows the location and distance of the Cyclex-d impactor (15 cm) relative to the sorting chamber of a FACSAria. Note the distance used in the collection for this paper is different than the collection distance used in the routine procedure (see Appendix: Standard Operating Procedure: Aerosol Containment Measurement), which is set at 5cm for maximal sensitivity. The impactor was connected to a vacuum regulator (Figure 2b) and the vacuum set at a constant vacuum equal to 20L/min. Figure 2c show an example of the histograms generated after 10 min of collection of Dragon Green beads at a rate of 50,000 beads/sec. Appendix: Standard Operating Procedure: Aerosol Containment Measurement outlines a complete description of the routine procedure for aerosol measurement using the Cyclex-d impactor and Dragon Green beads.

Figure 2.

The location and angle of the Cyclex-d impactor in front of the cell sorting chamber of the FACSAria cell sorter was placed at 15cm from the sort chamber as shown in 2a. The tubing from the impactor is attached to the MegaLite pump and Rotameter as pictured in 2b. Figure 2c is an example of the collection of dragon green beads after a 10 min collection at 50,000 beads/sec as measured in FITC detector using a 515/20nm bandpass filter (labelled as the 515-A detector).

Microscope and Slide Measurement:

Dragon Green microspheres were collected by Cyclex-d impactor sampling cassettes and counted using a Nikon Eclipse E400 Epi-Fluorescent microscope with a 450–490nm excitation filter (Figure 3a). To optimize visualization and quantification of DG beads after collection, the cover slip inside the cassette was removed and placed (adhesive side down) onto a gridded microscope slide (Electron Microscopy Sciences #63405–02). As a critical note, it is important to have the gridded side up before attaching the cover slip from the Cyclex-d impactor (Figure 3b). This technique ensured the focal plane of the DG beads is the same as the grid lines, facilitating viewing of slides with few or no beads. (Figure 3c).

Figure 3.

This figure shows the removal of coverslip from the Cyclex-d impactor (3a and 3b) and the examination of the beads measured in the fluorescent microscope (3c). Note the grid lines on the microscope slide provides a focal plane to ensure the focus of the dragon green beads, which is helpful when scanning slides with very few or no beads collected.

UV-APS and Aerosol concentrations and aerodynamic diameter (AD) measurements:

Aerosol concentrations and the AD measurements were conducted on a BD FACSAria II model cell sorter (BD biosciences, San Jose, CA) operating at 70 psi (482,633 pascals), using an Aerosol Particle Sizer (APS: UV-APS Model 3314; TSI, Shoreview, MN) equipped with a UV laser (350nm). Sorter generated aerosols were distinguished from ambient particles with a UV-excitable dye (Clear Blue Fluorescent Water Tracer Dye [CBD]; Risk Reactor, Santa Anna, CA) that was added to the sample tube. In some experiments, aerosol measurements were conducted on a FACSAria enclosed in a Class II Biological Safety Cabinet (BSC) abrogating the use of the UV dye. Use of the UV dye, methods and analysis of data were performed as previously described (1). For containment testing, large number of aerosols were created by covering the waste trough with a small piece of tubing while running the waste stream as previously described (14). This was also known as the Fail Mode (FM) which also simulated the event of clogged flow cell tip.

Evacuation airflow Restriction Tests:

The Buffalo filter AMS (Medtec Devices Inc, Buffalo NY) was operated at the manufacturer’s recommended setting of 20% of maximum vacuum. AMS airflow reduction experiments utilized a PVC valve connected inline of the AMS hose just prior to its connection to the sorter AMS port (Figure 4). Prior to containment tests, airflow (ft/min) was measured at the end of the AMS hose using a hot wire anemometer (VelociCal Air Velocity Meter Model 9535; TSI, Shoreview, MN). Measurements were in ft/min and then were converted to CFM (ft³/min) using the standardized airflow calculation, as based upon the AMS tubing diameter of 1.25 inches (3.18 cm) at the point of measurement.

Figure 4.

This figure shows the installation of the restriction valve (yellow arrow) in the vacuum lines of the Aerosol Management System (FACSAria) used in airflow reduction experiments. As the valve is turned, the airflow is reduced, which was accurately measured at the opened end of the tube attached to the sort chamber using a hot wire anemometer as described in the methods section.

Results

Determination of Optimal Microsphere Diameter

The goal of any cell sorter containment assay is to accurately and reliably detect aerosols that have escaped from the point of generation, i.e. sorting or collection chambers. Since it has been determined that a high concentration of aerosols with an AD in the range of 1–3μm are produced by cell sorters in FM (1), detection of aerosols in this range is essential. The initial use of the Cyclex-d cassettes and fluorescent microspheres in a cell sorter containment assay, as first described (15) utilized YG microspheres of graded sizes 0.5, 1.0, 2.0, 6.0,10.0 and 20.0μm. Since microspheres can never occupy an aerosol smaller than its own physical size, the physical size is the starting point for AD and therefore, it was reasoned that a smaller, single diameter microsphere alone would be sufficient. However, physical size of microspheres does not necessarily equate with AD, since this is dependent upon size, shape and density of the particle. To determine the size of the microsphere that occupied the largest range of aerosols generated by a sorter, YG microspheres of 0.75, 1.0 and 2.0μm were run separately as samples on a FACS Aria enclosed in a BSC in FM with AMS off, and resulting aerosols were measured with the UV-APS. Although the excitation maximum of the YG microspheres is 441nm, the 351nm UV laser of the UV-APS was capable of excitation of the microspheres due to the very broad excitation spectrum of the microspheres. The results (Figure 5) showed that the smallest size of YG microsphere tested occupied the largest range of AD aerosols and that the microspheres were always contained in aerosols of AD greater than the physical bead size. However, the ability to reliably detect microspheres when visualized on a slide is critical and it was found that the 0.75μm microspheres were more difficult to detect at low magnification. Since 1.0μm microspheres are easier to detect, and were found in aerosols of AD of interest, i.e. 1 to 3μm, 1.0μm microspheres were used for subsequent experiments.

Figure 5.

UV-APS analysis of YG 0.75μm (5a), 1.0μm (5b) and 2.0μm (5c) microspheres run separately as samples on a FACS Aria enclosed in a Class II Biosafety Cabinet showing that the smallest microsphere occupied the largest AD range of aerosols. Fig, 1a. represents 0.75μm beads with a minimum AD=0.9μm; mean AD=3.3μm and % positive fluorescence = 0.20. Fig. 1b. represents 1.0μm beads with a minimum AD = 1.1μm; mean AD = 3.4μm and % positive FL = 0.13. Fig. 1c. represents 2.0μm beads with a minimum AD = 2.1; mean AD = 3.5 and % positive FL = 0.30.

The frequency of aerosols occupied by the fluorescent microspheres was measured to be in the range of 0.13 to 0.3% of total (Figure 5a, 5b and 5c). However, it was hypothesized that the frequency of occupied aerosols will vary dependent upon Poisson statistics, concentration of microspheres, and the sample flow rate (16). This was verified by measuring aerosols with the UV-APS, generated by running YG beads as a sample at two different event rates, with the instrument in FM, aerosol management system off, and sort chamber ajar. Figure 6 shows the frequency of occupied drops ranged from a mean of 0.16% at 75000 events/sec to 0.21% at 82000 events/sec. Also shown are measurements under the same conditions (75000 events/sec) but with the sort chamber door closed

Figure 6.

Aerosols containing YG beads were generated while the sort chamber door open (ajar) at two event rates; 75,000 and 82,000 beads/sec. Aerosols containing YG beads were also generated with the sort chamber door closed (CL) as a control test. The data shows the increase in aerosols containing YG beads as measured by the UV-APS, as a function of event rate; the higher the rate the greater the number of aerosols containing YG beads.

Equally critical to a robust containment assay, is the ability to wash or remove beads subsequent to containment testing. Unfortunately, the ability to easily remove the YG beads from the fluidics of the cytometer proved problematic. Microspheres manufactured by Bangs Laboratories (Fishers, IN, USA), hard dyed with Dragon Green (DG) fluorescent dye were found to be more hydrophilic and much easier to clean and remove completely from the instrument fluidics system, in addition, these were also much brighter than the YG microspheres when viewed under 20× magnification as described in the methods section. Therefore, due to the brightness factor and the completeness of the wash/removal of these microspheres for containment testing procedures, all subsequent experiments were performed using 1.0μm DG microspheres.

Development and Validation of Containment Assay

As detailed in the published containment assay using Glo-Germ beads (3), the procedure for determining the containment efficiency of a cell sorter AMS uses an impactor placed in close proximity to the sort chamber to collect aerosols during fail mode while running the fluorescent microspheres as a sample. Use of the Cyclex-d cassettes and DG microspheres showed that under standard operating procedures (routine method) as described in the methods section, greater than 250 beads were detected when the AMS (positive control test) is off and the instrument is set to FM as compared to zero particles detected under the same conditions (negative control test, data not shown). However, it was important to establish the level of sensitivity of detection in this assay or the lowest level of concentration of aerosols escaping from the cell sorter. Experiments were conducted to correlate the aerosol concentration (using the UV-APS) with DG microsphere counts, under conditions where the AMS airflow was reduced using a restriction device (see methods section). In these experiments, at each AMS airflow condition, FM was initiated with CBD (UV tracer dye) as a sample and concentration of UV (non-ambient) aerosols was determined using the UV-APS at 10 min. collection time. Subsequently, DG beads were substituted as a sample, and collection with the Cyclex-d cassettes was performed over the same time period. This permitted the concentration of the escaped aerosols (UV+ aerosols) from the sort chamber to be correlated with DG bead detection. The results (Figure 7) showed that the lowest aerosol concentration in which DG beads were detected ranged from 0.026 to 0.04 aerosols/cm3. However, since there were experiments in which no DG beads were detected within this aerosol concentration range and DG beads were always detected above 0.04 aerosols/cm3, this concentration was established as the level of sensitivity. It is important to note that this level sensitivity was measurable only when the AMS airflow was reduced to 4.43 CFM (0.13 m³/M). This represents a 70% reduction in normal AMS airflow (i.e. 15.34 CFM or 0.43 m³/M). In addition, it was shown that, similar to experiments with YG beads, there was a direct correlation to event rate and the detection of DG beads, indicating that at higher flow rates more aerosols containing particles can be captured by the Cyclex-d (Figure 8).

Figure 7.

Dragon Green beads were collected with the Cyclex-d, subsequent to aerosol concentration measurements determined with the UV-APS and CBD, under the same conditions of air flow restriction. Data shows that at an aerosol concentration of 0.04 number/cm³ (or above), corresponding to an airflow restriction of 4.43 CFM (0.13 m³/M), Dragon Green microspheres were detected on the slide of the impactor (numbers beside dots; experiments with no beads detected are indicated in red). This demonstrated that the sensitivity of the Cyclex-d/DG bead assay to be at an aerosol concentration of 0.04 number/cm³.

Figure 8.

This figure shows the linear relationship (r = 0.99) of aerosols containing Dragon Green beads as a function of increased event rate. Dragon Green microspheres were captured using the Cyclex-d cassettes when the cell sorter was placed in FM and the impactor was used under standard collection procedures as described in the methods section.

Discussion

As stated earlier, this work was undertaken to develop a novel cell sorter aerosol containment assay that met specific criteria. In brief, these criteria are: 1) Same-day analysis and results, 2) High sensitivity with independent validation, 3) Affordable equipment and supplies, 4) High accuracy and specificity with low background, 5) High efficiency of aerosol collection within the desired AD range.

An assay to verify cell sorter aerosol containment may utilize any of the widely available aerosol collection methodologies of impaction, filtration and impingement that meet the above criteria. It was determined that collection of 1μm DG microspheres provided an appropriate surrogate of aerosols within the size range released from the sorter (Figure 5). Capture of these fluorescent microspheres with a non-viable impactor, such as the Cyclex-d, in which counting is easily performed on a microscope is appropriate since culturability and viability are not a concern. Use of fluorescent microspheres and disposable cassettes also meets the requirement for low background measurements, suitable for both BSC-enclosed and non-enclosed cell sorters. In addition, for non-viable impactors of this type, collection efficiency approaches 100% when the AD is greater than the impactor d₅₀ (17). The d₅₀ of the Cyclex-d is 1μm (18) and the median AD of the aerosols in validation experiments was 1.6μm (data not shown), in agreement with previously published reports of 1.7μm (1) and meeting the criteria of high collection efficiency of cell sorter derived aerosols.

Sequential measurements of cell sorter derived aerosols with the UV-APS/UV dye and the Cyclex-d/microspheres showed the lowest level of detection of the Cyclex-d to be 0.04 aerosols/cm³ (Figure 7). This concentration approaches background aerosol measurements of 0.022 UV+ aerosols/cm³ measured in these same experiments (data not shown), demonstrating the high sensitivity of the DG microsphere/Cyclex-d assay.

In addition, this high level of sensitivity was measured in spite of the low frequency of microsphere-occupied droplets (Figures 5). This is most likely due to the higher sampling volume of air (200L vs. 50L; Table 1) captured by the Cyclex-d vs. the UV-APS over the same collection time. In this regard, sensitivity could likely be increased with the Cyclex-d and DG microspheres by increasing the collection time. For bioaerosols, increasing collection time can be problematic due to desiccation of the sample, but this is not an issue with the plastic DG microspheres. Figure 6 shows that sensitivity can be increased by increasing the event rate due to an increase in the percentage of microsphere-occupied droplets. However, increasing sensitivity by increasing the event rate will eventually plateau due to an increase in coincident events and Poisson distribution statistics.

Table 1.

Collection			Total Volume Sampled
Device	Kate	Time
Cyclex-d	20L/min	600 sec	200 L
UV-APS	5L/min	600 sec	50 L

These results also illustrate the large amount of airflow loss (70% reduction of normal) required on this model of cell sorter before containment is compromised. This highlights the robustness of the AMS but also suggests that real-time monitoring of AMS airflow could be an effective method of monitoring containment during operation of the cell sorter and could be an important adjunct to containment testing as recommended by the ISAC Cell Sorter Biosafety Standards (2).

The assay described here was validated on a BD FACSAria, but it could be easily adapted to other sorters. The main considerations in adapting this assay to other instruments are the following: 1) Determine the measurement location of the Cyclex-d, which should be as close as possible to the sort chamber, 2) Determine the optimal method for creating a FM, mimicking partial nozzle obstruction with subsequent stream deviation, 3) Determine the best procedure for a positive control, and 4) Perform assay at the highest sheath pressure that will be used, which will result in the largest concentration of aerosols.

In summary, the Cyclex-d/DG microsphere assay fulfills the criteria for a robust cell sorter aerosol containment test and is recommended as a replacement assay for the previously published Glo-Germ/Aerotech impactor collection method (2).

Appendix: Standard Operating Procedure: Aerosol Containment Measurement

1. Cyclex-d impactor and Dragon Green Beads Collection

   1. Aerosol Management System (AMS)

      1. The AMS must be on and functioning according to the manufacturer guidelines. Appendix A shows the aerosol flow and the locations of the vacuum gauge and monitor. The vacuum monitor should be set to 20% and the vacuum gauge must read between 1.0 and 2.5 inches of water (250–622 pascals). If an increased percentage (vacuum) is needed to achieve this range, then it is possible that the HEPA filter unit and tubing are faulty and should be replaced. The expiration of the HEPA filter should not exceed 6 months.
   2. Measurement of Containment and Tolerances

      1. The AMS must be tested under simulated worse case failure mode. In this mode the instrument is set to 70 psi (482,633 pascals), and particles are concentrated to approach speeds of approx. 40,000–50,000 particles/second. The stream is forced to glance off of waste catcher shield to create a large plume of aerosols. This is accomplished by covering the waste catcher with a small piece of rubber tubing (appendix B).
      2. Attach Cyclex-d to vacuum pump and place Cyclex-d in front of the sort chamber approximately 2 inches (5 cm) from sort door. Close door to deflection plates but do not install tube holders (appendix C). The main sort chamber should also be closed.
      3. Turn AMS on (20%) and check for proper vacuum function (1.0–2.5 inches of water or 250–622 pascals).
      4. Place Dragon Green beads (Bangs Laboratories, 1.01μm, (catalog code = FS04F, catalog number = FSDG004, see appendix D, left panel) onto the sample station and adjust either the particle concentration or the flow rate to achieve a particle rate of 40,000 to 50,000 beads per second. Note: It is recommended that the operator wear respiratory protection while performing this test since a large volume of aerosols potentially can be generated. Appendix D (right panel) shows an example of the histograms during collection.

         1. To prepare Dragon beads add 20μl of beads to 2 ml of buffer (PBS with 0.01% sodium azide and 0.5% tween 20).
         2. Sample should be run around a flow rate of 5 or 6 to achieve a particle rate of 40,000 −50,000 bead per second. Note: trigger on fluorescence (FITC channel) signal.
      5. Turn on the Cyclex-d vacuum pump and adjust rate to 20 l/min.
      6. Click on sort drawer to retract, which will begin creating aerosols as the stream hits the rubber tubing covering the waste catcher.
      7. Collect aerosols for 10 minutes.
      8. Turn off vacuum and remove Cyclex-d unit. Mark unit as “AMS test”. Put on a fresh Cyclex-d cassette and continue collecting aerosols in “failure mode” with the AMS turned off for another 2 minutes (positive control). Stop sample acquisition and return waste catcher to normal position. Remove rubber shield from waste catcher and make sure to turn AMS back on.
      9. Remove the glass coverslip from inside Cyclex-d (see appendix E, fig. 1) and place onto a gridded microscope slide using following procedure below.

         1. Place the coverslip glue side down on a gridded microscope slide. Note: it is critical to invert the microscope slide to ensure the beads and the grid are in the same focal plane (back to back).
      10. Scan the test slide and positive control slide with the 10x or 20x objective and count all Dragon Green beads using a fluorescent microscope equipped with a FITC filter (520–640nm, see appendix E, fig. 2). Record all data using an electronic report file or paper record. It is recommended to examine the positive control slide first to the correct focal plane of the deposited beads.

          1. Note: Since the positive control slide will contain many beads it is not necessary count all beads on slide and for this reason this is usually reported as greater than a tolerance limit of 100 beads.
      11. Acceptable Tolerance: The acceptance tolerance are zero Dragon Green beads detected after 10 minutes of active air sampling in front of sort chamber door with no tube holder in place and the AMS turned on. The positive control coverslip must contain greater than 100 particles after 2–5 minutes of active air sampling with the AMS turned off and no tube holder in place.

          1. If test coverslip is negative for Dragon beads, the operator can proceed with sort
          2. If test coverslip is positive for any Dragon beads, the operator should check all vacuum tubing, re-seat filter in AMS and then repeat the test.
          3. If the test fails for a second time, infectious cell sorting must be ABORTED until the instrument containment can be verified.

Appendix A: Aerosol Collection Flow

Appendix B: Sort Chamber Modes

Appendix C: Cyclex-d collection location and vacuum source

Appendix D: Dragon Green Particles

Appendix E: Cyclex-d Device and collected Dragon Green Beads