Sorting of live, infectious cells by BSL-3 laboratory
Sorting of live, infectious cells by BSL-3 laboratory
As the need for high-speed sorting of viable, infectious and therapeutic cell samples increases, so does the importance of protecting the flow cytometer operator. Ensure sample containment and protection prior to sorting live, infectious cells with the BSL-3 Laboratory. This procedure includes aerosol control, physical barriers, environmental controls and personal protection. The aerosol handling system generates negative pressure inside the sorting chamber allowing the aerosol to pass directly through the vacuum unit to the high efficiency particulate air filter. Physical barriers include the instrument manufacturer's standard plastic shields and shielding panels. For maximum environmental control reasons, the BSL-3 laboratory flow cytometer is protected from operators by a respiratory safeguard system. Aerosol protection was determined by sorting through strongly fluorescent Glo-GermTM particles under the same conditions as the sorter was calibrated to produce a large amount of aerosol. These aerosols were collected by vacuum sampling system at three separate locations for 10 min on slides and then examined by microscopy. This system enables rapid and efficient cell sorting for aerosol protection and reduces the risk to operators when sorting active, infectious cells.
Principle
The basic principle for sorting live, infectious cells by the BSL-3 Laboratory is that aerosol protection can be determined by using strongly fluorescent particles ( Glo-GermTM ), which emit the strongest fluorescence under UV light. An efficient procedure is established to collect and measure the aerosol, and if poor control produces an aerosol containing Glo-GermTM particles, a slide containing the particles can detect the fluorescent particles under a fluorescence microscope. Thus, the efficiency content of the control is related to the number of particles added to each slide, with an acceptable deviation of no detectable particles on the outside of the sorting chamber.
Operation method
Sorting of live, infectious cells by BSL-3 laboratory
Principle
The basic principle of sorting live, infectious cells by the BSL-3 Laboratory is that aerosol protection can be determined by using strongly fluorescent particles (Glo-GermTM), which emit the strongest fluorescence under UV light. An efficient procedure is established to collect and measure the aerosol, and if poor control produces an aerosol containing Glo-GermTM particles, a slide containing the particles can detect the fluorescent particles under a fluorescence microscope. Thus, the efficiency content of the control is related to the number of particles added to each slide, with an acceptable deviation of no detectable particles on the outside of the sorting chamber.
Materials and Instruments
Equipment: Move The basic process of sorting live, infectious cells through the BSL-3 laboratory can be divided into the following steps: Sorting live, infectious material (infectious cells) under high pressure conditions must follow the guidelines and operating procedures listed below to ensure that the aerosol is well controlled. 1.1 The AMS system must be set up and used in accordance with the manufacturer's instructions. 1.2 The allowable range of the vacuum unit monitor must be 1.5 to 2 ft-hr water column. If not within this range, replace the HEPA filter and tubing. Autoclave the replacement HEPA filter and tubing. 1.3 Filter life range indication must be above 40%. If below 40%, replace HEPA filter and tubing. Autoclave the replacement HEPA filter and tubing. 1.4 The Accudrop system on the FACSDiVa is used to determine droplet delay and must be operated properly according to the manufacturer's instructions. In addition, a camera system is used to monitor the sorting airflow and alert the operator to potential increases in aerosols that may occur. If an increase in aerosols occurs, the operator should immediately correct the sorting airflow to minimize aerosol contamination.
(1) AeroTech 6
TM
active microbial particle sampler (Aerotech Laboratories, Inc., Phoenix, AZ [www.aerotechlabs.com], catalog no. 6
TM
).
(2) Matheson Flow Meter: vacuum gauge (Thomas Scientific, Swainsboro, NJ, catalog no. 5083R60).
(3) Aerosol handling system (AMS) with HEPA filtration system (BD Biosciences, San Jose, CA).
(4) Dupuy Biohazard Respiratory System (Chesapeake Surgical, Ltd., Stirling, Fayetteville): photovoltaic helmet and battery, catalog no. 5430-01-000; Batteries, Catalog No. 5430-35-000;4 Charging Stations, Catalog No. 5430-48-000; Hytrel General Purpose Protective Clothing, 5430-32-000; Knee-High Boots, Catalog No. 5409-55-000.
(5) Flow cytometric analyzer with sorting capability (e.g., FACSDiVa, BD Biosciences).
(6) Fluorescence microscope (e.g., Nikon Micro Image Analysis System, Columbia, MD).
(7) Laminar flow cabinet (e.g., Baker Corporation, Sanford, Maine).
Reagents:
(1) Glo-Germ
TM
Granules: 5 ml of ethanol with 5 μm beads of melamine heteromolecular polymerization resin. If Glo-Germ is a powder, dissolve in 5 ml of 100% ethanol to make a suspension prior to use;
② 100% ethanol;
PBS.
Wash solution: PBS containing 10% fetal calf serum (FCS), 1% Tween-20, 1 mg/ml sodium azide.
When cells are sorted, the AMS must be tested under similar sorting conditions and the relevant indicators of AMS operation must be within the permissible range to ensure proper functioning of the AMS before sorting live, infectious material. However, in order to provide the greatest possibility of measuring aerosol protection, the sorter may be set to test in the "Failure Mode" described in this section.
2.1 Place a clean slide into a clean, covered petri dish and place it into the AeroTech6TM Active Microbial Particle Sampler. Close the top lid and carefully tighten the tabs on each side. The unit remains under the laminar flow frame until the end of the aerosol protection test.
2.2 Place a clean slide, which is used as a positive control, into the sorting chamber located on top of the automated arm.
2.3 Adjust the vacuum of the AeroTech6TM Microbial Particle Sampler to 45 L/min (measured by a Matheson flow meter).
2.4 Check all aerosol vacuum connections and turn the AMS vacuum switch on. Record the vacuum pressure in inches of water column in Table 1.
2.5 Adjust the Glo-Germ particulate flow rate to 20,000 particles/second at 25 pounds per square inch (psi).
2.6 Close the door and deflector plate and adjust the flow rate to the waste collector to produce the largest possible aerosol. This condition is referred to as the 'failure mode' of operation.
2.7 Close all protective barriers according to instructions.
2.8 Move the Aero Tech 6TM Reactive Microbial Particle Sampler to 3 positions, placing it in each position for 10 min. It is recommended that it be placed directly in front of the door of the sorting chamber or on top of the sorting chamber, 2 feet from the front of the sorting chamber (usually on the operator's chair).
2.9 Stop sorting; remove the sample and positive control slides from the Aero Tech 6TM Active Microbial Particle Sampler and from inside the sorting chamber. Prevent back-dropping of sample tubes. To remove excess particles from the sample tube, add 70% ethanol to the sampler and run for 5 minutes under sorting conditions.
2.10 Examine the entire slide by scanning with a fluorescence microscope with a 10x objective and a fluorescein isothiocyanate radiopaque filter (520-640 nm). Both slides were examined.
2.11 Record the results in Table 24-1. All permissible range determinations must be completed before starting live infectious sorting of cells. Repeat the procedure if there is any deviation in the results.
2.12 Permissible range of particles outside the sorting chamber: 0 Permissible, no particles on the entire slide. Any positive results must be checked and resolved, and retesting is required prior to infectious sorting.
2.13 Permitted range of particles in the sorting room (positive control): above 50 per field of view (10x objective, results vary with slide position).
3 Personal safety features (basic shielding): Dupuy HEPA filtered protective clothing and respiratory systemThis system is to be used prior to any potentially infectious sorting operation, as infectious aerosols can be formed during the sorting process.
3.1 Connect the batteries to the protective helmet; turn on the switch to make sure that the fan of the protective helmet is in normal condition and then turn it off.
3.2 Put on protective boots
3.3 Remove the Hytrel Universal Protective Suit from the sterile plastic bag and attach it to the top of the face shield using the Velcro strap on the front of the shield.
3.4 Make sure that the HEPA filter on the Hytrel Universal Protective Suit is located above the power fan of the helmet.
3.5 Adjust the Hytrel Universal Protective Clothing, helmet and sleeves for a better fit. The battery pack should be attached to the belt inside the suit. If done correctly, the airflow will cause the jacket to puff up.
3.6 Wear double gloves and sleeves. The air stream is filtered through a HEPA filter at the top of the helmet and out the bottom of the suit. Final adjustments should be made before entering the BSL-3 lab.
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