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Date of revision and approval
WIMR Document reference: WIMR-SWP-OP-WS-508.01
Date of approval: 6th 9th November 2018
Approval authority: Scientific Platforms Manager, Workplace Health and Safety Manager, Advanced Specialist Flow Cytometry
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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 (WRHFlow) 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.
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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
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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.
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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
| 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. | low | ||
| Contact/exposure with biohazardous materials | Exposure to biohazardous material can cause health issues | PPE while emptying waste tank and adding bleach to waste tank. 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 for all room users and signage for existing projects that require SIP removal) Users empty waste after completion of operation) with running water gently down the sink 100ml of bleach is added to the instrument waste container after emptying the waste (refer to SWP for detailed instruction) Biological spill kit - Access to emergency biological spill kit and/or cleaning equipment. Bleach decontamination of sample lines on cytometers done by all users of the cytometers. Project approval process. Handling samples (e.g. transferring, pipetting) in biological safety cabinet. | 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 | 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. | low | ||
| Operating instrument | Manual handling – pinch hazard | Injury to hand | Education – Providing information, demonstrating and observing proper instrument use while loading samples on the CantoII during training to avoid pinching Instrument hardware monitoring for failed tube load. | low | ||
Handling hazardous chemical including Bleach, cleaning solutions, Decon/Contrad, Ethanol, QC beads | 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. 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. | 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 | ||
| Exposure to sodium azide while performing QC | Exposure 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. | low | usage of nitrile gloves if possible - added protection | low |
Procedure
Definitions
In these procedures the following terms have the meaning set out below
- DI water – Deionized water
- EtOH – Ethanol
- Sheath solution – CytoFLEX sheath fluid
- SIP - Sample injection probe
- 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 flow cytometry analysers to CytoFLEX to prevent sample acquisition issues arising from SIP clogging.
- Fixed samples may be filtered immediately before acquisition using the WRHFlow Westmead Cytometry provided 50um filters and plastic tweezers or tube top filters.
- Unfixed samples may be filtered before acquisition using the WRHFlow 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.
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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)
- Turn on the CytoFLEX from the power switch located on the back of the instrument.
- Log into windows if necessary. The all users account is password is cytoflex
- CytExpert software and Westmead Cytometry confluence page should start automatically.
- Click QC > start QC.
- Initialise the instrument
- Prepare QC beads if necessary. ~1 drop in ~10 drops of H20.
- Choose the correct configuration/bead lot number and click start QC.
- 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 wrhflow@sydneywestmead.cytometry@sydney.edu.au to to setup).
*Data should follow and be stored in the below listed format/location.
- Log in to PPMS screen locker.
- CytExpert should already be open (if it isn't start the software).
- Click new experiment from template.
- Navigate to the template folder and choose a template.
- Make a new experiment in the CytExpert Data Folder using the provided syntax.
- Initialise the instrument by clicking 'Initialise' if needed (if needed the button will be activated).
- Provide 50uL (minumum) of sample to be counted (with or without facility provided DAPI - 5uL to can be added is used). Samples can be provided in eppendorf tubes or 5mL FACS tubes. Templates are set to record 30 40 seconds of sample @30uL/min. this means 15uL 20uL of sample is will be used. These may be changed to suit your experiment/adapted for different cell types.
- Click 'Run' to start acquisition. Adjust the detector sensitivities if needed using the hand tool.
- Click 'Record' to record 15uL 20uL of sample (alternatively the 'Run' data is saved and can be used also).
- Adjust the gate to exclude debridebris. New gates can be drawn if needed.
- Extract the events per uL statistic from the template.
- If desired export the PDF - final workflow to be determined.
- Save your experiment.
- Log out of PPMS.
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- Select Daily Clean from the Cytometer menu.
- Follow the Daily Clean procedures, which consist of:
- Run FlowClean cleaning fluid for 3 minutes.
- Run DI water for 5 minutes.
- If necessary, empty the waste container.
- Remove the sample tube and store according to your laboratory procedures.
- Close the CytExpert software.
- Shut down the Workstation.
- Turn off the main power on the Cytometer.
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 wrhflow@sydney 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.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
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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.
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- 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.
- 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.
- Review the SDS and make sure you understand the hazardous properties of the spilled material before you attempt to clean it up.
- 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.
- 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.
- For minor spills alert lab manager, safety officer and others in the lab and cordon off the affected area. Isolate the spill.
- 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.
- Identify the spill - there is a pH strip tester that can be used, also can refer to SDS, labels, supervisors.
- Select the agent to clean up the spill.
- For acid spills: Spill-X-A® acid neutraliser
- For caustic spills: Spill-X-C ® caustic neutraliser
- For solvent spills: Spill-X-S ® solvent adsorbent
- For formaldehyde spills: Spill-X-FP ® formaldehyde
polymeriser - In some instances combinations of adsorbents may be necessary.
- Encircle spill, cover with adsorbent
- Mix adsorbent into spill
- Using the absorbent pads from the spill kit, carefully wipe up the
spilled liquid, again working from the outside in. - Beware of any neutralising reactions
- Collect powdery residue using spatula, deposit in waste bags
- 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. - Label and dispose of waste according to building policies.
- Remove PPE and thoroughly wash hands.
- Report the spill using the WIMR incident notification form.
Associated Documents
This Standard Operating Procedure should be read in conjunction with:
- Australian/New Zealand Standard - Safety in Laboratories Part 3: Microbiological safety and containment (AS/NZS 2243.3:2010)
- Australian/New Zealand Standard - Management of clinical and related wastes (AS/NZS 3816:1998)
- 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.
- “A Guide to the WIMR Tissue Culture Facilities” – WIMR laboratory guideline
- WIMR – Management of Hazardous Materials Policy & Procedure
- WIMR – Critical Risk Management Plan for biosafety
- WIMR – Critical Risk Management for Waste Management
- WIMR – Critical Risk Management Plan for chemical safety+
- OGTR Guidelines - http://www.ogtr.gov.au/
- CytoFLEX Setup Guide [B53767AC 2015].pdf
- CytoFLEX Quick Start Guide [B49008AC FEB 2015].pdf
Appendix
Chemical and biological spill kit location
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