Date of revision and approval

Document reference: WIMR-SWP-OP-WS-506.02

Date of approval: 3rd October 2018

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 autoMACS® Pro Separator 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 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 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

It is advised that bookings are made to maximise the 2 week recommended life of the columns.

List of hazards and risk controls as per risk assessment

Associated risk assessment reference: WIMR-TSA-OP-WS-506.02

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 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
	Manual handling - pinch / crush / shear hazard	Moving parts can cause injury to body parts	Educate users to not open the front access covers while device is in operation. Always stop or abort a procedure before any adjustments. Warning signs on instrument moving parts. Manually controlled access cover.	low
	Exposure to bio-hazardous material	Exposure to biohazardous material can cause health issues.	PPE – gloves, gown & enclosed shoes. PPE is used while emptying waste tank and adding bleach to waste tank. Users empty waste (bleach decontaminated) after shutdown 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. Ethanol decontamination of sample lines on instrument performed during shutdown. Project approval process. Aerosol generating procedures (e.g. transferring, pipetting) to be performed in a biological safety cabinet. Instrument is located in a BSC for sample/user benefits.	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
	Exposure to strong magnetic force	Risk of severe personal injury to persons carrying pacemakers or electronic medical devices	Education - keep electronic equipment and magnetizable tools at least 20cm from the magnet cover. Magnet cover in place to prevent direct contact. Magnet only activated during operation.	med
	Visible & invisible laser radiation	Risk of eye damage	Education - Do not look directly at laser or LED Warning sign on instrument laser parts Hazard exists from barcode scanner - low level of risk due to low level of laser power.	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

Running buffer - PBS with 0.5% FBS & 2uM EDTA final
Shutdown solution - 70% EtOH w/w, analytical grade, diluted with distilled water
BSC – Biological safety cabinet (class 2 or above)

List of Resources Required

(including personal protective clothing, chemicals and equipment needed)

autoMACS® Pro Separator
MicroBead kit
BSC
PPE including gloves, long sleeve gowns & enclosed shoes
Running buffer
Washing solution
Shutdown solution
Chill rack
separation columns

Biosafety considerations

Sample isolation on the autoMACS® Pro Separator must only occur after the approval of an associated project in PPMS.
100mL of 12.5% sodium hypochlorite must be added to the emptied waste containers at the start and end of your session.

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 leaks 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.

Setting up & priming the autoMACS® Pro Separator

Check that all bottles are filled with the appropriate solutions. Running buffer can be found in the 4^oC fridge, and wash solution can be found in the cupboard underneath the biobank workbench. Empty the waste bottle and fill with 100mL of bleach.
Switch on the autoMACS® Pro Separator, the instrument will initialise automatically (note the BSC power point switch needs to be activated also).
After initialisng, the instrument will display the status menu. Confirm the instrument status. Green indicates ready and red needs attention.

Changing a separation column if needed (check with staff before changing, to ensure we keep a count on used columns)

If the column needs to be replaced, under "Option", select "Special" and then "Col_ex", press run.
When prompted, gently pull column out of the magnet housing and remove. Column can be removed by unscrewing top connector followed by the bottom connector. You only need to exchange 1 column at a time.
Screw new column on. New columns can be found in the cupboard underneath the biobank workbench.
Write down the change date. A new column is good to use for 2 weeks or 100. We recommend extending the life through to 3-4 weeks per column if appropriate.
After exchanging column select "Done", the instrument will prime automatically with running buffer. The instrument is now ready to start a separation.

Performing automated separation

Place an appropriate chill rack on the sampler, it'll be automatically detected by the autoMACS. Racks are located in the 4^oC fridge next to the instrument.
Place labelled samples into the original slots on the rack.
Under the separation tab, enter the correct volume, and program settings. Ensure the instrument is not to go to sleep after the last separation as there is a cleaning procedure that needs to be performed.
Place empty tubes into corresponding position into row B (negative fraction) & row C (Enriched fraction).
Start the separation.

Performing auto-labelling and separation

Place an appropriate chill rack on the sampler, it'll be automatically detected by the autoMACS. Racks are located in the 4^oC fridge next to the instrument.
Dilute single-cell suspension according to the table above.
Place sample tubes into row A, and empty tubes into corresponding position into row B (negative fraction) & row C (Enriched fraction).
Insert the MACS Reagent Rack (R1-4) onto the MACS mini-sampler.
Scan reagent vials. on the "Reagent" menu, select "Read Reagent" and present the reagent vial in front of the code reader.
After a successful scan, the software will automatically highlight the next available reagent rack position. Insert the reagent vial into the corresponding position.
1. Reagents can also be entered manually if required.
Under the separation menu, enter the sample rack template for separation. Select the desired positions and assign auto-labeling protocols as well as sample processing volume.
It is possible to change the rinse mode between samples, or go into sleep mode after the last sample.
Select run and then OK to start the separation protocol.

Positive selection programs:

Possel—Isolation of cells with normal antigen expression and frequencies higher than 5%; select if purity is the highest priority.
Possel_s—Isolation of cells with low antigen expression and frequencies higher than 5%; select if yield is the highest priority.
Posseld—For isolation of rare cells in low elution volume.
Posselds—For isolation of rare cells with low antigen expression.
Posseld2—For isolation of rare cells if purity is the highest priority.
Posselwb—For isolation of cell subsets from whole blood. Cell samples are automatically diluted with Running Buffer.

Depletion programs:

Deplete—For removal of cells with normal to high antigen expression and results in better target cell yield.
Depletes—Removal of cells with low antigen expression and results in better target cell purity.
Depl05—Removal of cells with low antigen expression and results in stringent depletion of cells.
Depl025—Removal of cells with low antigen expression and results in stringent depletion of cells.

Monitoring cell separation

Cleaning & switching the instrument off

After separation, disconnect probe from running buffer, spray and wipe down probe with 70% EtOH.
Insert probe into shutdown solution (70% EtOH w/w), under "Separation" select "Wash now" and then "Rinse". This will take ~5min.
On the panel press the power icon on the top right, then select shutdown. Shutdown procedure will take ~5min.
Remove probe and hang onto hook.
You can now power off the instrument with the main switch.
Empty the waste tank and discard unused single use running buffer.
Wipe down hood and shut down. Start UV procedure.

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
AutoMACS Pro Separator 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 autoMACS analyser.