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Table of Contents

Date of revision and approval

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The procedures apply to users operating the BD FACSAria III in room J.2.04, 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

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Monday to Friday

Advanced Specialist on 0-8627 3216

Cytometry/Imaging/EM Manager on 0-8627 1820

Scientific Platforms Manager platforms Director on 0-8627 3210

Email: westmead.cytometry@sydney.edu.au

For POLICE, AMBULANCE or FIRE emergencies contact 0-000

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Task or scenarioHazard/sAssociated harmExisting risk controlsCurrent risk ratingAdditional risk controls?Residual risk rating?
Instrument operationElectrocution

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).Engineering control - SIP sheath cover installed to minimise aerosol exposure from sample upon unexpected sample unloadof 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/mediumair 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

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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.

Laser safety shielding - to prevent laser exposure

Automatic shutters – pressure driven or mechanical when cover is open

low

Operating AriaIII – pinch hazardManual handling – pinch hazard while sample loading

Injury to hand

Education – Providing information, demonstrating and observing proper instrument use while loading samples on the ARIAIII during training to avoid pinching. Including closing of cover before loading of sample occurs. 

Instrument hardware monitoring for failed tube load.

Physical cover - Cover to prevent accidental contact with the stage during sample load up

Emergency stop button – easily accessible button to stop instrument in an emergency situation

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Manual handling – pinch hazard while unscrewing/screwing tankInjury to hand

Education – Providing information, demonstrating and observing proper instrument use while loading samples on the ARIAIII during training to avoid pinching. Including removing lid completely before filling tank and removing air supply line while depressurising lid for removal. 

mediumRemoval of air line until lid is being removed. low

Handling hazardous chemicals including Bleach, Decon/Contrad, Ethanol, CST beads (Sodium Azide)

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 disconnected tube supplying high pressure air

Tubing connectors may degrade resulting in escape of air

Auto shutting off connections if disocnnected.

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

Exposure to sodium azide while performing CSTExposure to toxic chemical

Acute toxicity

Introduce relevant hazards with SDS.

Ensure PPE, i.e. Gloves are worn while handling the sample.

Sodium azide is used for a minimal time during the quality control procedure.  Ensure only minimum quantity (1 drop in 300uL) made each time quality control solution is made.

Dual barrier protection (gloves, tube) & lid.

lowusage of nitrile gloves if possible - added protectionlow

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  1. Turn on the CAS BSC hood, chiller, high-pressure gas and aerosol management system (set at 20% - for operational velocity evacuation).
  2. Turn on the main switch to the ARIAIII located on the left of the instrument. 
  3. Log into Windows using provided credentials. 
  4. Log into the PPMS account with provided credentials. 
  5. BD FACSDiva should launch automatically as should Google Chrome (contains the sorting log).
  6. Log in to BD FACSDiva, the software will detect and connect to the instrument. If it does not do it automatically, click Instrument in the main menu and connect the instrument manually.
  7. If a CST mismatch dialog appears always click ‘Use CST Settings’
  8. Open the BSC cabinet.
  9. Open the front cover of the AriaIII and prop it open.
  10. Determine if the correct nozzle is in place in the flow cell.
    1. If the cleaning nozzle is in place perform a fluidics startup (as this indicates the fluidics was shutdown).
    2. If the incorrect nozzle is in place in the flow cell, sonicate the appropriate nozzle in DI water for 5 minutes in a 5mL FACS tube. Ensure sonicator has sufficient liquid in it to conduct cleaning power to the nozzle.
      1. If needed - unlock and remove the previous nozzle or waste nozzle from the flow cell. Place the nozzle into an empty slot on the nozzle storage block.
      2. Use a Kimwipe to remove any residue liquid on the sonicated nozzle to be installed if necessary.
      3. Clean any salt residues around the nozzle assembly area using ethanol. Wipe and dry clean. 
      4. Insert sonicated nozzle with the O-ring facing upwards. Lock the nozzle using the nozzle clip.
      5. Since the nozzle was changed, also change nozzle size settings in the software by setting the correct cytometer configuration - >Cytometer>View Configurations>Select correct nozzle>Set Configuration. 
      6. Check the stream video feed is not impeded by any liquid/debris on the lens. If this is the case it can be cleaned with a lens cleaning tip.
    3. If the correct nozzle is in place in the instrument, ensure the correct instrument configuration is selected and applied in the software by looking at the window title bar. 
  11. The ARIAIII flow cell cover should be open.
  12. Open the sort chamber (we need to view whether the stream is aligned or not. 
  13. Close the front cover of the BSC.
  14. Be ready to change the waste catch position, loosen the screws on both sides of the collection assembly, and then start the stream.
  15. Check the stream angle. If the stream flows away from the centre of the waste catch, rotate the sort block to adjust. Tighten screws.
  16. Close the sort block and thumb screw it shut.
  17. Stop the stream. This still results in air into the sheath tank which is used to minimise contamination of the sheath tank when refilling. to view whether the stream is aligned or not). 
  18. Close the front cover of the BSC.
  19. Spray the sheath tank lid with ethanol.
  20. Unscrew the sheath tank lid completely.
  21. Relieve all pressure from the tank by venting using the venting valve, and breaking the air-pressure seal by pushing down on the lid. Ensure you keep fingers clear of the edges of the lid and remove it. It can be stored temporarily on the fluidics cart (keeping the inner face not touching anything).
  22. Fill sheath tank to the weld mark (~10cm from top of tank).
  23. Reseal sheath tank - only holding the screw mechanism to keep fingers clear of the lid. 
  24. Empty waste container slowly into the sink under running water. Put 100ml of bleach into the waste tank.
  25. Reconnect waste container.
  26. Check the filter attached to the sheath and at the front of the fluidic cart for air bubbles. If needed, ethanol spray and then gently bleed out any air bubbles if required from the finger screw vent on the filter. Tighten the ventif required from the finger screw vent on the filter. Tighten the vent. 
  27. Be ready to change the waste catch position, loosen the screws on both sides of the collection assembly, and then start the stream.
  28. Check the stream angle. If the stream flows away from the centre of the waste catch, rotate the sort block to adjust. Tighten screws.
  29. Close the sort block and thumb screw it shut.
  30. Stop the stream. 
  31. Start the stream again. The stream should be aligned in the centre of the waste catch. If needed adjust. 
  32. Open the front cover of the BSC hood and close the front of the ARIAIII lid. 
  33. Close the front cover of the BSC.
  34. Inspect the stream for stable droplet generation, symmetrical droplets and similar breakoff position to the last time the stream was started. Check for satellite droplet merging typically within 3 drops of breakoff. Click the sweet spot button and inspect the stream. 
  35. Allow ARIAIII stream/lasers to warm / stabilise for at least 30 min.
  36. Adjust frequency and amplitude as needed with sweet spot off, updating sweet spot values if necessary.
  37. Carefully check the instrument for wet areas indicating any leaks in the tubing or failing valves.
  38. Do not continue the sort with an unstable stream or leaks. 

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  • Under no circumstances should there be any patient/person identiable data recorded on the instruments as the data is not privately held and is accessible to all researchers. 
  • User data is deleted after 7 days off the instrument computers, it is the responsibility of the user to have transferred data to a secure location.
  • WRHFlow recommends exporting data from BD FacsDiva as FCS3.0 files in linear format to D:\BDExport\FCS. This folder is automatically synced to the WIMR Scientific Platforms Drive that all WIMR active directory users can access. For the sync to work properly your experiment must be in a folder with "FirstnameLastinitial" syntax. Please note this folder is accessible to all researchers.
  • If you would like your data accessible from outside of WIMR, a user specific share can be setup. This works by creating a copy of a specified folder on university provisioned storage that is shared with the user. This requires an email to wrhflow@sydneywestmead.cytometry@sydney.edu.au and it is the users responsibility that all ethics approvals are complied with. 

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  • Spray the sort chamber and other appropriate internal chambers specific to the instrument with 1:100 TRIGENE and wipe clean. Spray again with 1:100 TRIGENE and allow a contact time of at least 10 minutes.
  • Wipe the areas and follow with spraying and wiping clean with 70% ethanol.
  • Spray the exposed appropriate external surfaces specific to the instrument with 1:100 TRIGENE and wipe clean. Spray again with 1:100 TRIGENE and allow a contact time of at least 10 minutes.
  • Wipe the areas sprayed and follow with spraying and wiping clean with 70% ethanol.
  • No further biological samples are to be run on the instrument until maintenance is carried out.

Emergency procedures

Emergency procedure folder can be found here in the sample prep lab:

Image Added

You can also access all these information via WIMR intranet:

https://intranet.westmeadinstitute.org.au/workingatwmi/HandS/Pages/Sarah%20Johnston.aspx


All emergencies need to be reported to the emergency contacts listed above. Specific chemical exposure procedures are below.

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