Date of revision and approval

Document reference: WIMR-SWP-OP-WS-506.07

Date of approval: To be approved

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

Functional unit: Flow Cytometry

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

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

Objective

This document describes the safe operation of the GentleMACS Octo Dissociator located at the Westmead Research Hub Flow Cytometry Core Facility. This document includes starting up the system, basic operation, cleaning, and shutting down the instruments. All personnel require training prior to independent operation of the instruments. 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 1820

Scientific Platforms Manager on 0-8627 3210

Email: wrh.flow@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 require instrument training with a relevant training record completed. Initial access is not limited to business hours. Initial usage of the instrument is to be with a trained user.

Instrument booking guidelines

Bookings can be made on PPMS.

List of hazards and risk controls as per risk assessment

Task or scenario	Hazard/s	Associated harm	Existing risk controls	Current 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 prevent liquid spill into the instrument. 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
	Mechanical hazard	Moving parts can cause injury to body parts	Educate users to keep the Trough, Sleeves, and Safety Shield attached when operating. When the instrument’s rotors are rotating, do not touch them or load or remove tubes from the instrument. Always stop or abort a procedure before any adjustments. Warning signs on instrument moving parts.	low
	Exposure to bio-hazardous material	Exposure to biohazardous material can cause health issues.	PPE – gloves, gown & enclosed shoes. Biological spill kit - Access to emergency biological spill kit and/or cleaning equipment. Ethanol decontamination of instrument performed during shutdown. Project approval process.	low
	Hot surface hazard	The heating foil of the gentleMACS heaters may become hot enough to cause burns if touched.	Educate users not to touch the heating foil, while the instrument is in operation. Warning signs on heating parts	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. 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

Procedure

Definitions

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

C tubes- Purple-cap C Tubes to get viable single-cell suspensions for cell separation, culture, or analysis.
M tubes - Orange-cap M Tubes to achieve thorough sample homogenization for subsequent molecular and microbiology analysis.

List of Resources Required

(including personal protective clothing, chemicals and equipment needed)

GentleMACS® Octo Dissociator with heaters
GentleMACS kit
GentleMACS C or M Tubes
PPE including gloves, long sleeve gowns & enclosed shoes

Biosafety considerations

Sample isolation on the GentleMACS® Octo Dissociator with heaters must only occur after the approval of an associated project in PPMS.

Procedure (Step by step instructions or order for undertaking the task)

PPE including gloves, long sleeve gowns, & enclosed shoes must be worn before operating the instrument.

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

Samples that are to be isolated are required to be transported to the flow cytometry labs in accordance with PC2 lab requirements in an appropriate container.

Starting up the instrument

Ensure that the device is plugged in before setting the switch to the ON position (“ ” indicates “ON”, “ ” indicates “OFF”).
The instrument starts within a few seconds and displays the Main menu.
The gentleMACS Octo Dissociator with Heaters is operated using the touchscreen display. Items that can be selected are displayed in yellow, while items that cannot
be selected are displayed in white.

Different folders can be accessed by pressing a folder icon. The arrow indicates scroll function. The Miltenyi & Template folder contains gentleMACS Programs
provided by Miltenyi Biotec. You can also store userdefined programs in the user folder.

Menu tabs

Performing dissociation

Tips and hints for the processing of samples:

• The tubes are for single use only
• Only use C Tubes with the gentleMACS Heaters.
• Sample sizes range from 300 μL to maximal 10 mL total volume and 20 to 4,000 mg of tissue.
• The maximal amount of tissue depends on the consistency of the tissue as well as on the respective gentleMACS Protocol.
• Dependent on the consistency of the tissue it might be recommended to cut the sample into smaller pieces before processing on the gentleMACS Octo Dissociator with Heaters.
• The tubes are suitable for a broad range of tissues, for example, spleen, liver, or brain. However, very hard material, such as bone, may damage thetubes.

Up to eight samples can be run simultaneously using the same or different programs. In this example, eight samples will be processed using the same program.

Attach the tubes upside down to the Sleeves of the free tube positions. The status of the tube positions will change from Free to Selected.
Refer to the kit for the appropriate program for dissociation.
To select a dissociation program, press the Folder icon to select the required folder. The list of gentleMACS Programs in the respective folder will be displayed.
Highlight the desired gentleMACS Program touching the screen or using the scroll buttons.
Select the required program and press OK to confirm. The program will be assigned to all positions with tubes. The status of the tube positions will change to Ready.
Press Start to commence the program. The status of the tube positions will change to Running. The progress bar and timer indicate the time remaining.
On completion of the program, the status of the tube positions will change to Done. The progress bar and timer display the elapsed time since completion of the program.
Remove the tubes from the tube positions. The status of the tube positions will change to Free.

Cleaning & switching the instrument off

In case of spillage or contamination, clean the instrument appropriately.
For decontamination, wipe the instrument with 70% ethanol. Clean the instrument only with cleaning agents that are non-corrosive and not damaging to the plastic
housing of the gentleMACS Octo Dissociator with Heaters. Suitable cleaning agents include household detergents and 70% ethanol.
Power off the instrument

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.

Biological spill

Ensure all PPE is on.
Contain the spill if necessary using absorbent pads available in the flow cytometry cupboards.
Add 50mL undiluted bleach (~12.5% sodium hypochlorite) into the biological spill bottles located around the laboratory that contain 450mL of H20.
Add this solution to the biological spill (and absorbent pads) and allow 10 minutes contact time.
Use a second towel to pick up the first absorbent pads and place in a yellow biological hazards container.
Repeat the process until the spill has been cleaned and the area decontaminated.

Clean Up and Waste Disposal Requirements
Complete PPE, including gowns, gloves and enclosed shoes. Bleached waste 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.

Training and Competency Requirements
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 safe independent instrument operation, in conjunction with verbal explanation of aspects of operation of the instrument.

Emergency shutdown if an electrical, or laser hazard is present

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.

Additional Information

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.

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
GentleMACS Octo Dissociator user manual
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

Appendix
Instrument diagrams depicting main components of gentleMACS Dissociator.