Date of revision and approval

WIMR Document reference: WIMR-SWP-OP-WS-501.02

Date of approval: TBD 12 Dec 2018 

Approval authority: Scientific Platforms Manager, Workplace Health and Safety Manager, Advanced Specialist Flow Cytometry

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Enquiries contact: weatmead.cytometry@sydney.edu.au

Objective

This document describes the operational setup and operation for the BD FACSAria III 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 with Cytometer Setup and Tracking (CS&T) beads, sorting, cleaning, and shutting down the system. It also includes safety considerations and emergency procedures. 

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.

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

Procedure

Definitions

In these procedures the following terms have the meaning set out below

1. BD – Beckton Dickinson
2. DI water – Deionized water
3. EtOH – Ethanol
4. Sheath solution – saline, 0.9% NaCl
5. SIP sheath - outer SIP steel cover
6. BSC – Biological safety cabinet (class 2 or above)
7. CST Beads – Cytometer Setup and Tracking Beads
8. SIP - Sample injection probe
9. FACS - fluorescence activated cell sorting 

List of resources 

(including personal protective clothing, chemicals and equipment needed)

• FACSAriaIII 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. 

Procedure (Step by step instructions for undertaking the task)

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.

Starting up the FACSAria III system

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. 
18. Spray the sheath tank lid with ethanol.
19. Unscrew the sheath tank lid completely.
20. 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).
21. Fill sheath tank to the weld mark (~10cm from top of tank).
22. Reseal sheath tank - only holding the screw mechanism to keep fingers clear of the lid. 
23. Empty waste container slowly into the sink under running water. Put 100ml of bleach into the waste tank.
24. Reconnect waste container.
25. 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 vent. 
26. Start the stream again. The stream should be aligned in the centre of the waste catch. If needed adjust. 
27. Open the front cover of the BSC hood and close the front of the ARIAIII lid. 
28. Close the front cover of the BSC.
29. 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. 
30. Allow ARIAIII stream/lasers to warm / stabilise for at least 30 min.
31. Adjust frequency and amplitude as needed with sweet spot off, updating sweet spot values if necessary.
32. Carefully check the instrument for wet areas indicating any leaks in the tubing or failing valves.
33. Do not continue the sort with an unstable stream or leaks. 

Checking Cytometer Performance with CS&T beads in DIVA.

1. Uncheck sweet spot. Select Cytometer > CS&T.
2. Once complete click 
   ‘Menu’ > ‘Cytometer’ > ‘CS&T’. Select 'Check Performance', and ensure the correct bead lot number is selected. Each instrument will have a bead lot number displayed on the front of the instrument. Load CS&T beads from the WRHFlow fridge (normally premade, but can be made with 1 drop from the stock bottle in the flow fridge with 300uL saline in a 5mLs FACS tube – refer to MSDS. Ensure tube is labelled appropriately. ) and click the run button. Take note of CST result. If successful, the instrument alignment is right and good to use, and the CS&T window can be closed. If CS&T fails, note the error and report it to flow staff before using.
   
3. Select performance check. Ensure correct bead lot CST beads are loaded onto the instrument (Correct bead lot is displayed on asign on the instruement) and click run.
4. If the beads fail, inspect the report and determine whether the stream and sample are as expected. If CS&T continues to fail call BD service.
5. If QC passes, view report to check any warnings.
6. Quit the CS&T window. Click use CS&T settings in Diva.

Checking drop delay (the time between the cytometer seeing an event and the event going into the last connected droplet) with Accudrop beads

1. Use prepared or if needed prepare Accudrop beads by adding 1 drop of beads into ~250ul of saline in a 5mL FACS tube.  Diluted beads are OK to use within ~1 week. Ensure tube is labelled appropriately. 
2. Open the Accudrop experiment template. Bring out sort window from global worksheet.
3. Load Accudrop beads. Click sort, then click cancel on the dialog that appears to keep the waste drawer closed.
4. Turn on high voltage plates.
5. Make sure sweet spot is on and the stream has been optimised. Using Accudrop beads, toggle optical filter on and adjust the Accudrop laser focus to obtain the brightest bead spot on the side stream.
6. Adjust drop delay to give the cleanest side streams by incremental increases/decreases.
7. You may need to increase / decrease sample flow rate. You may need to sweep the accudrop laser to intensify the accudrop beads visible on the camera. 
8. Check, adjust if necessary and type in drop breakoff and gap values. Drop delay is now set.
9. Unload Accudrop beads.
10. Turn on test sort. Check if the side streams are clean and directed to indicated marks on the screen (i.e. going into the collection tubes intended to be used for the sort). If the side stream is slightly out of the centre of the collection tube, use the slider controls to adjust the side stream voltage. Turn off test sorts and high voltage plates.

Sorting

1. Ensure any disinfection solutions (1% bleach, 70% EtOH, 5% Decon, 1:50 Trigene, and any others necessary) are prepared before starting the sorter.
2. Create a new experiment or duplicate an existing one. Follow appropriate experiment creation workflow, i.e. syntax YYYYMMDD DescriptionSetup in a folder with the following syntax ‘FirstnameLastnameinitial’. Check the FSC-W, FSC-H, SSC-W, SSC-H parameters for recording in the Inspector tab as below to allow identification of doublets. Setup experiment using controls provided by the user. Filter biological samples before acquiring on a cytometer.
3. Install collection tubes into tube holder and slide into the sort block.
4. Filter the sample if needed
5. Load the filtered sample onto machine. Initially run the sample with a low flow rate in the acquisition window and record an appropriate number of events. Draw or adjust gates if needed.
6. Acquire, record and sort data/cells from each of your samples.
7. Confirm approval for sorting strategy.
8. If needed create a new sort layout form the >Sort menu. Input the population being sorted in the Sort Layout window. If doing a 4-way sort, use outer tubes for the lower frequency populations with the higher frequency populations in the middle two tubes. This is to reduce the chance of larger populations contaminating the smaller populations. There are some caveats to this, i.e. number of cells to be sorted, cell size, and side stream stability need to be also considered. Consider appropriate sort mask settings to be used. On the AriaIII, purity or 4way purity modes are commonly used and recommended. 
9. Install the correct and appropriate collection tubes in the sort collection chamber to collect sorted events. 
10. Click Sort and turn on agitation to an appropriate value. Adjust flow rate and side streams if necessary. Threshold rate should not exceed approximately ¼ of the frequency to ensure events are spaced out along the stream. Monitor sort efficiency and adjust parameters if necessary.
11. Ensure the 'Sort in Progress' signage is on the door. When the sort is finished this sign can be removed.
12. Avoid running the sample dry to prevent bubbles in the system. If it’s a precious sample add more saline close to the end. When the sample level is low, reduce or halt agitation of the sample to minimise the chance of drawing in air. Monitor sample continuously and stop the sample before it is at a critically low level to prevent drawing air into the sample line.
13. The sort chamber can only be opened when there is no sample running. Remove collection tube when sort is finished. 
14. Remove sample from instrument. 
15. Unless your data should not be synced to the WIMR server, export the data as FCS3.0 files in linear format to D:\BDExport\FCS (as this folder is automatically synced to the WIMR server). If you would like to copy your data elsewhere copy it from this location. Your data should be able to be accessed from the Scientific Platforms networked drive within WIMR.
    
    

Purity Check

1. Choose Instrument > Cleaning Modes > Sample Line Back flush. Click Start at prompt a few times to clean the valve, and wait for some time (~10-30sec) to clean the sample line, click Stop.
2. Load a full tube of DI water and change flow rate to 11. Clean the tubing for at least 1 minute or until no sample looking events are displayed. Unload water tube and change flow rate to 2-4.
3. Dilute ~5uL of sorted sample into 100uL of diluent (4 drops of saline) in a new tube. Ensure sample agitator is off. Load the purity check tube and record sample for approximately 30-60 seconds.
4. Between running purity check tubes, perform sample line back flush.

Data export

1. Export FCS files & batch analyse samples when appropriate into default locations. Move the batch analysis into the FCS files folder ready for syncing or user transfer as setup for data management.

Daily shutdown of instrument

1. Load 10% bleach. Change flow rate to 11 and run for 5 min.
2. Repeat with 5% Decon 90.
3. Repeat with DI water.
4. Stop stream.
5. If the next sort booked on the instrument in that week uses the same nozzle
   1. Load a full tube of 70% EtOH and initiate clean flow cell command. 
6. If the next sort booked on the instrument in that week uses a different nozzle
   1. Remove current nozzle. 
   2. Replace with cleaning nozzle.
   3. Load a full tube of 70% EtOH and initiate clean flow cell command. 
   4. When complete - remove cleaning nozzle and remove nozzle clip.
7. If there is no other sort booked for the week. 
   1. Remove current nozzle. 
   2. Replace with cleaning nozzle.
   3. Initiate fluidics shutdown program from cytometer menu and follow prompts.
   4. Complete fluidics shutdown. 
   5. Remove cleaning nozzle and nozzle clip and replace to default locations.
8. Spray the sort chamber and other appropriate internal chambers specific to the instrument as well as exposed appropriate external surfaces with 70% w/v ethanol and wipe clean. Spray again with 70% w/v ethanol and allow a contact time of at least 10 minutes.
9. Wipe the areas and follow with spraying and wiping clean with 70% ethanol
10. System is now ready to be shut down. Turn off instrument, gas, chiller, AMO & BSC afterwards. Log off computer.
11. Relieve any pressure from the tanks by venting using the venting valve

Notes

Detectors

• Remember there is a range of optimal PMT voltages that will provide similar resolution between dim and negative cells.
• PMT voltages that are too high will not affect the resolution of dim and negative cells, but can unnecessarily cause spreading of the negative population.
• Any bright populations with very large CVs (very broad distributions) must have PMT voltages lowered so that the brightest events or MFIs remain within the linear range of the detector.
• Remember your compensation controls should be representative of your samples, ensuring they are as bright or brighter than your sample (but not log folds brighter preferentially).
• Remember to leave enough room above the positive signal, and in the linear range of the detector, to allow for increases in fluorescence of certain epitopes.
• We recommend your samples are filtered beforehand.

Data management

• 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@sydney.edu.au and it is the users responsibility that all ethics approvals are complied with. 

Application Settings

Application settings are useful in ensuring longitudinal studies (studies measured over multiple days/weeks/months) maintain consistent MFIs over the course of the experiment. We have successfully implemented and assisted many users in creating and applying application settings to their experiments.

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• Open the original experiment (for this protocol, do not open an experiment that has already had application settings restored, only open the original experiment).
• Right click experiment > ‘Duplicate experiment without data’.
• In the newly created duplicated experiment, right click the duplicated experiments top-level cytometer settings ‘Cytometer Settings’ > ‘Application Settings’ > ‘Apply’ > Apply saved application settings
• As the copied experiment will have a compensation matrix that is non-zero from when the experiment was setup, click ‘keep the compensation values’ (you can re-record compensation control tubes and recalculate the compensation matrix if desired).
• If a dialog appears asking to ‘Confirm Cytometer Changes’ – in regards to FSC Area Scaling values have changed – overwrite them by clicking YES.
• If you are not recording new compensation controls, proceed to record samples (adjusting FSC and SSC detector voltages to those noted if necessary).
• If you are recording new compensation controls (recommended), right click the compensation tubes ‘Cytometer Settings’ > ‘Application Settings’ > ‘Apply’ > Apply saved application settings. Record compensations (only adjusting FSC and SSC detector voltages (as noted) to bring populations onto scale, then calculate compensation. Clicking apply and save.
• Check the voltages have been slightly updated.
• Proceed to record samples (adjusting FSC and SSC detector voltages to those noted if necessary)

Additional Information

Contamination testing sample collection – For contamination testing samples from the tanks, stream and sample tubing are collected for downstream contamination testing. In addition to the above procedures the following need to be undertaken when obtaining these samples.

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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

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

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1. Ensure BSC is on
2. Ensure PPE is in use
1. Gloves, gown, safety glasses & P2 respirator (provided)
3. Stop the stream if the instrument hasn’t automatically. The stream can be stopped via the software or the emergency red button on the instrument
4. Minimise BSC turbulence
5. Keep sort chamber closed and sample cover closed to contain aerosols
6. Increase AMO to maximum flow rate to facilitate the evacuation of aerosols
7. Leave the room. 
8. Allow 3 minutes for the AMO & BSC to evacuate any aerosols
9. Remove collection tubes and sample aseptically.
10. Spray the sort chamber, sort collection block 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.
11. Determine cause of sort failure if possible.
12. Remove nozzle and sonicate if necessary.
13. Restart the stream if possible.
14. If nozzle clog occurred – refilter sample or use larger nozzle before loading sample.
15. Log the failure in the sort log.

Associated Documents

This Standard Operating Procedure should be read in conjunction with:

1. Australian/New Zealand Standard - Safety in Laboratories Part 3: Microbiological safety and containment (AS/NZS 2243.3:2010)
2. Australian/New Zealand Standard - Management of clinical and related wastes (AS/NZS 3816:1998)
3. University of Sydney Safety Health & Wellbeing: Guideline for the Decontamination of Clinical/Biological Waste and Spill Management, http://sydney.edu.au/whs/guidelines/biosafety/decontamination_guidelines.shtml#2.2.1.
4. “A Guide to the WIMR Tissue Culture Facilities” – WIMR laboratory guideline
5. BDFACS ARIA III User Manual – Scientific Platforms Network Drive
6. Australian/New Zealand Standard - Safety in Laboratories Part 3: Microbiological safety and containment (AS/NZS 2243.3:2010)
7. Australian/New Zealand Standard - Management of clinical and related wastes (AS/NZS 3816:1998)
8. University of Sydney Safety Health & Wellbeing: Guideline for the Decontamination of Clinical/Biological Waste and Spill Management, http://sydney.edu.au/whs/guidelines/biosafety/decontamination_guidelines.shtml#2.2.1.
9. “A Guide to the WIMR Tissue Culture Facilities” – WIMR laboratory guideline
10. Holmes KL, Fontes B, Hogarth P, et al. International Society for the Advancement of Cytometry Cell Sorter Biosafety Standards. Cytometry Part A : the journal of the International Society for Analytical Cytology. 2014;85(5):434-453. doi:10.1002/cyto.a.22454.
11. MSDS - CST beads
12. WIMR – Management of Hazardous Materials Policy & Procedure
13. WIMR – Critical Risk Management Plan for biosafety
14. WIMR – Critical Risk Management for Waste Management
15. WIMR – Critical Risk Management Plan for chemical safety
16. OGTR Guidelines - http://www.ogtr.gov.au/

Appendix

Chemical and biological spill kit location

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Sort failure procedure for oepratorsoperators

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Image AddedInstrument and room images depicting main components of ARIAIII system and room.

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