Date of revision and approval

WIMR Document reference: WIMR-SWP-OP-WS-508.01

Date of approval: 9th November 2018

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

Functional unit: Flow Cytometry

Enquiries contact name: Xin Wang, Suat Dervish, Edwin Lau

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

Objective

This document describes the safe operation of the CytoFLEX flow cytometer located at the Westmead Research Hub Flow Cytometry Core Facility in the Westmead Institute for Medical Research (WIMR). This document includes starting up the system, basic operation, cleaning, and shutting down the instruments when necessary by both staff and researchers. The procedures apply to users operating the CytoFLEX in room J.2.06, level 2 of WIMR. All personnel require training prior to independent operation of the instruments. All instrument operation is to be conducted by trained operators. Any assay to be run on the analysers involving hazardous chemicals must have appropriate approval. 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.

Note: internal phones require a '0' to dial an outside line. Omit the listed '0' if calling from outside of WIMR. 

Reception / business hours

8.30am to 5.00pm

Monday to Friday

Advanced Specialist on 0-8627 3216

Cytometry/Imaging/EM Manager on 0-8627 1820

Scientific platforms Director on 0-8627 3210

Email: westmead.cytometry@sydney.edu.au

For POLICE, AMBULANCE or FIRE emergencies contact 0-000

Outside of business hours

After hours contact the WIMR Emergency Management System contact on 0-0467 818 730

For access or security related issue contact TRICORP security on 0-1300 456 321

For POLICE, AMBULANCE or FIRE emergencies contact 0-000

Research hours - WIMR and Hub

6.00am to midnight

Monday to Friday

8.00am to 8.00pm

Weekends and public holidays

Training and competency requirements

All new users must have completed CytoFLEX training with a relevant training record and competency testing completed.  

Westmead cytometry staff or superusers may provide training. Access will be provided once a training record and competency quiz have been completed. 

Instrument booking guidelines

The CytoFLEX is not a bookable instrument. The instrument will be run as a first in, first use instrument with a 15 minutes limit to be applied if another user is waiting. This is to facilitate the usage of the cytometer as a cell counter within the institute. 

The instrument will be initialised daily (unless due to unforeseen circumstances - where a user may need to perform the QC), and prepared for use as a cell counter for the day. 

All users will need to log in using their PPMS account and ensure to log off after each session.

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. DI water – Deionized water
2. EtOH – Ethanol
3. Sheath solution – CytoFLEX sheath fluid
4. SIP - Sample injection probe
5. QC beads - Quality control beads

List of resources 

(including personal protective clothing, chemicals and equipment needed)

• Flow cytometer systems
• BSC
• PPE including gloves, long sleeve gowns, P2 mask, safety glasses & enclosed shoes
• Bleach (12.5%) (Diluted 1 in 10 final – final available >1% bleach)
• Decon 90 (concentrated surface decontaminant) (Diluted 1 in 20 final)
• Ethanol (Diluted 70% w/w)

Biosafety considerations

• Acquisition of samples on the CytoFLEX must only 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 daily and upon renewing the waste container. 
• We recommend filtering samples prior to acquisition on the CytoFLEX to prevent sample acquisition issues arising from SIP clogging. 
  • Fixed samples may be filtered immediately before acquisition using the Westmead Cytometry provided 50um filters and plastic tweezers or tube top filters. 
  • Unfixed samples may be filtered before acquisition using the Westmead Cytometry provided 50um filters and plastic tweezers or tube top filters in a biological safety cabinet.

Procedure (Step by step instructions for undertaking the task)

PPE including gloves, long sleeve gowns, & enclosed shoes must be worn while working in the PC2 laboratory. 

Before and during instrument use ensure there are no visible leaks on the instrument as this may pose an electrical hazard.

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.

Initial instrument startup, initialisation, setup & quality control (normally performed by staff/superuser)

1. Turn on the CytoFLEX from the power switch located on the back of the instrument.
2. Log into windows if necessary. The all users account is password is cytoflex
3. CytExpert software and Westmead Cytometry confluence page should start automatically.
4. Click QC > start QC.
5. Initialise the instrument
6. Prepare QC beads if necessary. ~1 drop in ~10 drops of H20.
7. Choose the correct configuration/bead lot number and click start QC.
8. Exit the QC (file > close QC) and prepare the workspace for default cell counting by loading an appropriate workspace.

Cell counting 

*All data collected will be synced to the WIMR server. If you would like to copy your data elsewhere copy it from this location. Your export folder can be easily accessed from the Scientific Platforms folder under My computer. Your data should be able to be accessed from the Scientific Platforms networked drive within WIMR, and via a username/password for non-WIMR users if desired (email westmead.cytometry@sydney.edu.au to setup).

*Data should follow and be stored in the below listed format/location.

1. Log in to PPMS screen locker. 
2. CytExpert should already be open (if it isn't start the software).
3. Click new experiment from template.
   
4. Navigate to the template folder and choose a template.
   
5. Make a new experiment in the CytExpert Data Folder using the provided syntax.
6. Initialise the instrument by clicking 'Initialise' if needed (if needed the button will be activated). 
7. Provide 50uL (minumum) of sample to be counted (facility provided DAPI - 5uL can be added). Samples can be provided in eppendorf tubes or 5mL FACS tubes. Templates are set to record 40 seconds of sample @30uL/min. this means 20uL of sample will be used. These may be changed/adapted for different cell types.
8. Click 'Run' to start acquisition. Adjust the detector sensitivities if needed using the hand tool. 
9. Click 'Record' to record 20uL of sample (alternatively the 'Run' data is saved and can be used also).
10. Adjust the gate to exclude debris. New gates can be drawn if needed. 
11. Extract the events per uL statistic from the template. 
12. If desired export the PDF - final workflow to be determined. 
13. Save your experiment.
14. Log out of PPMS.

*the instrument is not shutdown by users. The instrument will enter a standby state after 10 minutes of inactivity

Cleaning the instrument

*The instrument automatically rinses the SIP after each sample and therefore we have not setup a 'per use' cleaning procedure. However if there is a suspected instrument clog the following cleaning procedure (that will be run as a preventative maintenance schedule) can be run. 

1. Select Daily Clean from the Cytometer menu.
   
2. Follow the Daily Clean procedures, which consist of:
3. Run FlowClean cleaning fluid for 3 minutes.
4. Run DI water for 5 minutes.
5. If necessary, empty the waste container.
6. Remove the sample tube and store according to your laboratory procedures.
7. Close the CytExpert software.
8. Shut down the Workstation.
9. Turn off the main power on the Cytometer.

1. 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 ensure data has been transferred to a secure location.
• 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 Westmead.Cytometry@sydney.edu.au and it is the users responsibility that all ethics approvals are complied with. 

Notes

• Filtering your sample beforehand can prevent clumps and erroneous data. 
• Violet SSC provides better resolution for smaller particles and hence many templates trigger off violet SSC.
• Abort rate should be kept below 2.5% for accurate cell counting. 

Additional Information

Clean Up and Waste Disposal Requirements

Complete PPE, including gowns, gloves and enclosed shoes is to be worn. Inactivated waste (including 100mL bleach) from the waste tank is poured down the sink gently under a running tap, minimising splashing. All laboratory biohazardous waste is to be removed via the biohazardous waste disposal bins located in the lab.

Instrument failure

In the event of instrument failure, do not attempt to fix the instrument. Report all incidents and instrument failures to the instrument specific emergency contacts listed above.

Training and Competency Requirements

Training is conducted by a super user or the scientific platforms staff with competency demonstration necessary before authorisation of access. Competency is assessed via completion of a competency quiz.

Emergency procedures

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

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.

Bleach

Eye exposure:

1. Wash out immediately with saline from the emergency eye wash bottle.
2. Ensure complete irrigation of the eye for at least 15 minutes by keeping eyelids apart and away from eye and moving the eyelids by occasionally lifting the upper and lower lids.
3. Seek medical attention immediately.
4. Removal of contact lenses should only be done by medical personnel.
5. Notify emergency contacts

Skin exposure:

1. Remove all contaminated clothing immediately.
2. Flush affected area under running water.
3. Seek medical attention if necessary.
4. Notify emergency contacts

Emergency power off button activation if an electrical, or laser hazard is present

1. Note that the emergency power shutdown will only isolate the red powerpoints. 

2. If there is an electrical hazard and the instrument is plugged into a power point that is not on the uninterruptible power supply (blue power point), pressing the emergency power off button will stop electricity to the instrument. Note that if an electrical device is plugged into the blue power point it will continue to be live. In this event leave the room, ensure a message is placed on the door alerting others and seek assistance.

3. In an electrical emergency press the red power shutdown button in the room.
4. Leave the room and place signage that the room is not to be entered due to a major electrical problem.
5. Notify emergency contacts

Emergency high pressure gas shutdown

If there is an issue with the high-pressure gas, the gas supply can be shut down by closing the valve on the wall and or the secondary shut off valve located in the main corridor outside of room J2.06.

Biohazard spill 

1. Obtain biohazard spill kit - located in all flow cytometry labs - see appendix for location.
2. Remove contaminated PPE and place in biohazard waste bag; and
3. Put on waterproof gown, gloves and mask with eye protection before proceeding.
4. Sprinkle CliniSorb powder over the spill; or
5. Cover the area of the spill with disposable cloth soaked in freshly prepared disinfectant suitable for the material in use (1% sodium hypochlorite - 100mL of 12.5% bleach in 1L bottle, or 1%Virkon - 2 tablets in 1L bottle); and
6. Leave it for at least 10 minutes.
7. Apply second cloth soaked in disinfectant on top of the first;
8. Use a third cloth (dry) to pick up and transfer the cloths to waste bags. If CliniSorb powder was used, collect the spilt material and Clinisorb powder mixture using spatula/scraper. Discard to waste bags;
9. Decontaminate the area thoroughly with suitable disinfectant;
10. Dispose of all waste including gloves in waste bags;
11. Notify emergency contact for DNIR spill, double bag the waste in autoclavable bags and loosely seal with autoclave tape for removal for autoclaving.
12. Report the spill using the WIMR incident notification form. 

Chemical spill 

1. If you are in doubt of your ability to clean a chemical spill safely then evacuate the area and seek help. It is better to ask for help if you are not sure of how to clean it up properly.
2. If there is risk to the rest of the building contact the emergency contacts to initiate building evacuation or activate the emergency evacuation that is located on the exit doors at the end of the corridor as you are exiting the flow labs. 
3. Review the SDS and make sure you understand the hazardous properties of the spilled material before you attempt to clean it up.
4. First aid is always the top priority. If a hazardous material is spilt on yourself, remove any potentially contaminated clothing immediately and use the emergency shower. If a chemical spill has entered your eyes flush for at least 15 minutes at the eyewash station. Seek appropriate medical treatment.
5. Determine whether it is a major or minor spill. A major spill is one that involves a large amount of hazardous material, poses a risk of fire/explosion, a respiratory hazard exists, or when dealing with unknown chemical spills. Seek assistance from emergency contacts for major spills. 
6. For minor spills alert lab manager, safety officer and others in the lab and cordon off the affected area. Isolate the spill.
7. Retrieve the spill kit (there are also adequate instructions in the chemical spill kit). Located in the shared prep lab J2.06 - see appendix for location. Stop and think about your plan to clean the spill. Remove the gloves and goggles and from the kit, put them and all appropriate PPE on before approaching the spill.
8. Identify the spill - there is a pH strip tester that can be used, also can refer to SDS, labels, supervisors. 
9. Select the agent to clean up the spill.
   1. For acid spills: Spill-X-A® acid neutraliser
   2. For caustic spills: Spill-X-C ® caustic neutraliser
   3. For solvent spills: Spill-X-S ® solvent adsorbent
   4. For formaldehyde spills: Spill-X-FP ® formaldehyde
      polymeriser
   5. In some instances combinations of adsorbents may be necessary. 
10. Encircle spill, cover with adsorbent
11. Mix adsorbent into spill
12. Using the absorbent pads from the spill kit, carefully wipe up the
    spilled liquid, again working from the outside in.
13. Beware of any neutralising reactions
14. Collect powdery residue using spatula, deposit in waste bags
15. Place all waste materials in a plastic bag. Once the spill has been
    fully cleaned, place the waste bag with in the fume hood temporarily.
16. Label and dispose of waste according to building policies. 
17. Remove PPE and thoroughly wash hands.
18. Report the spill using the WIMR incident notification form. 

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. WIMR – Management of Hazardous Materials Policy & Procedure
6. WIMR – Critical Risk Management Plan for biosafety
7. WIMR – Critical Risk Management for Waste Management
8. WIMR – Critical Risk Management Plan for chemical safety+
9. OGTR Guidelines - http://www.ogtr.gov.au/
10. CytoFLEX Setup Guide [B53767AC 2015].pdf
11. CytoFLEX Quick Start Guide [B49008AC FEB 2015].pdf

Appendix

Chemical and biological spill kit location

Chemical Spill Kit Instructions

Biohazard Spill Procedure Poster

Health Risk Matrix used in risk assesment

Instrument diagrams depicting main components of CytoFLEX

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