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Date of revision and approval
WIMR Document reference: WIMR-SWP-OP-WS-503.02
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Enquiries contact: weatmead.cytometry@sydney.edu.au
Objective
This document describes the operational setup and operation for the BD Influx flow cytometer, a high-speed fixed-alignment benchtop cell sorter located at the Westmead Research Hub. This document includes starting up the system, setting up the stream, checking cytometer performance, sorting, cleaning, and shutting down the system. It also includes safety considerations and emergency procedures.
The procedures apply to users operating the BD Influx in room J.2.03, level 2 of WIMR. All personnel require training prior to independent operation of the instrument. Training is conducted by a trained operator or the scientific platform manager (if appropriate) with competency demonstration necessary before authorisation of access. Competency is assessed via demonstration of independent instrument operation, in conjunction with verbal explanation of all aspects of operation of the instrument and troubleshooting common and simulated faults. All instrument operation is to be conducted by trained operators. OGTR requirements for safe work in a PC2 laboratory apply.
Hours of operation and emergency contacts
The following hours of operation are valid from July 2nd 2018 unless otherwise updated.
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Weekends, public holidays and shutdown periods.
Training and Competency Requirements
All users must have completed training.
Training is conducted via an initial theoretical introduction to the components and safety aspects of the instrument and laboratory followed by assisted sessions with a trained operator followed by independent operation until competency in all aspects of safety and operation are demonstrated via independent operation, including dealing with emergency situations and performing all applicable tasks. Description and purpose of functional components used while independently operating the instrument are also required.
List of hazards and risk controls as per risk assessment
Task or scenario | Hazard/s | Associated harm | Existing risk controls | Current risk rating | Additional risk controls? | Residual risk rating? |
Instrument operation | Electrocution | Contact with electricity can cause electric shock and burns | Routine instrument maintenance - to ensure instrument is in good condition and cabling is not damaged. Electrical equipment annual testing. Educate users to check for visible liquid leaks. Safety circuit breakers and fuses on instrument to prevent general electrocution due to instrument failure, especially in the presence of liquid. Emergency power off button located in laboratories to disconnects power to the red power points and not the blue uninterruptible power points. Routine maintenance - to ensure instrument is in good condition Bright LED - lit to notify of high voltage deflection plates - educate users about importance of ensuring high voltage is off before accessing plates and surrounding area Instrument covers - when sorting all covers are to be in place as physical access is restricted High resistance installed on HV plates - to limit current draw Circuit breakers and fuses on instrument - to prevent general electrocution due to instrument failure, especially in the presence of liquid | low | ||
Contact/exposure with biohazardous materials | Exposure to biohazardous material can cause health issues | Electrostatic droplet cell sorter creates thousands of drops per second. Droplets can be small and readily enter airways. Therefore containment of aerosols is critical. This is achieved via having the cell sorter in a BSC, operating with covers, ensuring instrument aerosol management option is properly utilised and following emergency procedures in the case of sort stream failure. BSC - Instrument is inside a biosafety safety cabinet for increased protection. Software - Software controls to maintain a stable stream Filtration - Ensure samples are filtered prior to loading on the instrument, avoid blockage to minimise aerosol generation during sorting Visual check - Check sample for visible clumps that can cause nozzle clogs Signage - Emergency sort failure procedure in SWP and in room Signage - Signage on door during sorting to prevent unauthorised access during sort. Aerosol management option installed and in use as per SWP. PPE while emptying waste tank and adding bleach to waste tank (bleach decontamination of waste material). Ensure users and support technicians are familiar with risk assessment and SWP for the material used. PPE – gloves, gown & enclosed shoes (P2 mask and safety glasses in sort failure). Users empty waste upon setting up of instrument with running water gently down the sink. 100ml of bleach is added to the instrument waste container after emptying the waste. Biological spill kit - Access to emergency biological spill kit and/or cleaning equipment. Bleach / decon / ethanol decontamination of sample lines. Project approval process. Handling samples (e.g. transferring, pipetting) in biological safety cabinet. Running the sample dry can introduce air bubbles resulting in unstable stream and rogue aerosol formation. SWP states to not run the sample dry. | low/medium | air bubble detector in sample line to prevent running sample dry. | low | |
Manual handling (i.e. lifting, transferring) heavy weight such as waste tank | Manual handling can result in injuries of the back, neck, shoulders, arms or other body parts | Providing information and training to workers on manual handling tasks and request for assistance options Maximum possible weight for tanks is <10kg Trolley/pallet jack - to transfer more than 1 box of saline/water – lifting only 1 box at a time Education - Providing information and training to workers on manual handling tasks Planning - Organising manual handling tasks in a safe way, with loads split into smaller ones, and proper rest periods provided | low | |||
Failure to adhere to SWP | Exposure to laser | High power lasers used in instruments can cause skin/eye damage/burns | Use laser safety shielding at all times - to prevent avoid laser exposure. Do not disengage automatic shutters – electronically or mechanically activated when certain covers are open. Educate users not to circumvent shutters and to avoid looking into any exposed lasers or reflections as laser light can be invisible. Laser safety shielding - to prevent avoid laser exposure. Automatic shutters – pressure driven or mechanical when cover is open | low | ||
Handling hazardous chemicals including Bleach, Decon/Contrad, Ethanol | Hazardous substance exposure | Eye exposure causing eye damage
Contact with skin can cause irritation or burn | Safety goggles are provided for researchers and recommended for use in the lab. Emergency showers & eye wash stations available in shared J.2.06 laboratory. Chemical spill kit available in shared lab J.2.06. PPE – gloves, gown & enclosed shoes are necessary for working in the laboratory. SDS available to users to ensure awareness of relevant chemical hazards and emergency procedures. Required to read and understand before using chemicals. | low | ||
Sample handling | Contact with bio-hazardous material | Exposure to bio-hazardous material | Handle samples in biosafety cabinet PPE – gloves, gown & enclosed shoes are necessary for working in the laboratory. Access to emergency biological spill kit and materials to clean up spills. | low | ||
High pressure gas | Physical injury caused by high pressure air | Tubing connectors may degrade resulting in escape of air | Auto shutting off connections if disconnected. Emergency shut off valves - Education of user to the location and operation of the emergency shut off valve. Venting ports - Education of users to depressurise any tank/fluidics line before accessing or opening the sheath tank and other components. | low |
Procedure
Definitions
In these procedures the following terms have the meaning set out below
BD – Beckton Dickinson
DI water – Deionized water
EtOH – Ethanol
Sheath solution – saline, 0.9% NaCl
SIP sheath - outer SIP steel cover
BSC – Biological safety cabinet (class 2 or above)
SIP - Sample injection probe
FACS - fluorescence activated cell sorting
List of resources
(including personal protective clothing, chemicals and equipment needed)
FACSInflux system
Biosafety Safety Cabinet
Sonicator
PPE including gloves, long sleeve gowns, P2 respirator, safety glasses, enclosed shoes
Bleach (12.5%) (Diluted 1 in 10 final)
Decon 90 (concentrated surface decontaminant) (Diluted 1 in 20 final)
Ethanol (70% w/w)
Trigene (Diluted 1:100) or F10SC (Diluted 1:250) if Trigene isn’t available.
Biosafety considerations
Samples can only be run on the fluorescence activated cell sorters occur after the approval of an associated project in PPMS.
100mL of 12.5% sodium hypochlorite (undiluted from the provided containers) must be added to the emptied waste containers at the start of the sorting session.
Sheath tank must be filled prior to stream startup to prevent cell sort failure midrun.
Biological samples can only be run on the fluorescence activated cell sorters occur after filtration.
Follow the WIMR essential laboratory safety practices.
PPE including gloves, long sleeve gowns, safety glasses, enclosed shoes must be worn before operating the instrument.
Samples that are to be acquired on the instrument are required to be transported to the flow cytometry labs in accordance with PC2 sample transport requirements.
PPE including gloves, long sleeve gowns, safety glasses, enclosed shoes and a P2 respirator must be worn before operating the instrument in any event where there may be aerosol generation.
Procedure (Step by step instructions for undertaking the task)A. Initial set-up
10. Log into the computer as operator (if needed the username is .\operator) 11. Log into the PPMS account as operator. 12. Open Sortware and check whether the collection chamber is clear and click continue 13. Press RINSE for a few seconds: bubbles should be removed from the fluidics. Shake the filter and release the bubbles B. Debubble
C. QC
D. Laser Alignment
10. Adjust the stream focus knob (back silver knob) and then side knob on the blue laser systematically to achieve the highest signal, compare the previous data to double check 11. Click the dot plot for the next laser, find the right pinhole on the oscilloscope, adjust the top knob first to get symmetry and highest signal of the peak and then adjust the side knob to achieve the highest signal, adjust the laser delay to move the peak to the right side of the pinhole (it is normally the optimal position for this laser) 12. Close shutter for the first two lasers, change camera to FSC and align FSC to best signal. 13. Continue for other lasers 14. Find the QC folder and save a new file in there using todays date ‘YYYYMMDD_P3s_##um’ and 2000 events as default. E. Stream Stability
F. Reload User’s Workspace
G. Data Transformation (compensated data only)
H. Pause the sorting for topping up the sheath tank
I. Device position
J. Sort on 96-well plate
K. Instrument shutdown
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Emergency procedures
Emergency procedure folder can be found here in the sample prep lab:
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If a stream failure occurs, the Influx may detect this and shut off the stream and sample automatically. If this occurs during sample being put through the instrument, the instrument must be given time for any potential aerosols to settle or be removed via the biological safety cabinet before accessing the sort block.If the instrument does not automatically shut down the stream upon a stream failure, the stream sample and stream need to be stopped via the software or via the red emergency button located on the instrumentstart button. Then after a period of 3 minutes, and with the appropriate PPE (gloves, gown, enclosed shoes and safety respirator), the nozzle may be removed if necessary, decontaminated and sonicated if necessary to remove the clog and reinstalled if needed. If the stream failure was due to a clogged sample, the sample may need to be re-filtered or a larger nozzle used. The primary objective is to prevent potentially biohazardous aerosols being exposed outside of the biological safety cabinet. The procedure is outline below.
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