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Table of Contents

Date of revision and approval

WIMR Document reference: WIMR-SWP-OP-WS-503.02

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

Objective

This document describes the operational setup and operation for the BD Influx 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, sorting, cleaning, and shutting down the system. It also includes safety considerations and emergency procedures. 

The procedures apply to users operating the BD Influx in room J.2.03, 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. 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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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

 

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.

Routine maintenance - to ensure instrument is in good condition

Bright LED - lit to notify of high voltage deflection plates - educate users about importance of ensuring high voltage is off before accessing plates and surrounding area

Instrument covers - when sorting all covers are to be in place as physical access is restricted

High resistance installed on HV plates - to limit current draw

Circuit breakers and fuses on instrument - to prevent general electrocution due to instrument failure, especially in the presence of liquid

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Contact/exposure with biohazardous materials

Exposure to biohazardous material can cause health issues

Electrostatic droplet cell sorter creates thousands of drops per second. Droplets can be small and readily enter airways. Therefore containment of aerosols is critical. This is achieved via having the cell sorter in a BSC, operating with covers, ensuring instrument aerosol management option is properly utilised and following emergency procedures in the case of sort stream failure. 

BSC - Instrument is inside a biosafety safety cabinet for increased protection.

Software - Software controls to maintain a stable stream

Filtration - Ensure samples are filtered prior to loading on the instrument, avoid blockage to minimise aerosol generation during sorting

Visual check - Check sample for visible clumps that can cause nozzle clogs

Signage - Emergency sort failure procedure in SWP and in room

Signage - Signage on door during sorting to prevent unauthorised access during sort. 

Aerosol management option installed and in use as per SWP. 

PPE while emptying waste tank and adding bleach to waste tank (bleach decontamination of waste material).

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 in sort failure).

Users empty waste upon setting up 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.

Bleach / decon / ethanol decontamination of sample lines.

Project approval process. 

Handling samples (e.g. transferring, pipetting) in biological safety cabinet.

Running the sample dry can introduce air bubbles resulting in unstable stream and rogue aerosol formation. SWP states to not run the sample dry. 

low/medium

air bubble detector in sample line to prevent running sample dry.

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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

Trolley/pallet jack - to transfer more than 1 box of saline/water – lifting only 1 box at a time

Education - Providing information and training to workers on manual handling tasks

Planning - Organising manual handling tasks in a safe way, with loads split into smaller ones, and proper rest periods provided

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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.

Automatic shutters – pressure driven or mechanical when cover is open

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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 in shared J.2.06 laboratory.

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. Required to read and understand before using chemicals.

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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.

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High pressure gas

Physical injury caused by disconnected tube supplying high pressure air

Tubing connectors may degrade resulting in escape of air

Auto shutting off connections if disconnected.

Emergency shut off valves - Education of user to the location and operation of the emergency shut off valve.

Venting ports - Education of users to depressurise any tank/fluidics line before accessing or opening the sheath tank and other components.

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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. SIP - Sample injection probe

  8. FACS - fluorescence activated cell sorting 

List of resources 

(including personal protective clothing, chemicals and equipment needed)

  • FACSInflux 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.

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

PPE including gloves, long sleeve gowns, safety glasses, enclosed shoes and a P2 respirator must be worn before operating the instrument in any event where there may be aerosol generation.

A.    Initial set-up

  1. Turn on the CAS hood, chiller and aerosol management system (AMS).

  1. Turn on the lasers and computer.

  1. Sonicate the nozzle for 5min if a different size nozzle is required.

  1. Turn on the air pressure and vacuum to pre-set levels.

  1. Top up the sheath tank to the top weld line. Ensure the scales are accurately tared to provide an accurate reading of when the tank is near empty.

  1. If needed, increase the sheath tank pressure to make the sheath tank easier to be sealed.

  1. Empty waste container slowly into the sink under running water. Put 100ml of bleach into the waste tank.

  1. Turn on the waste camera feed, waste illumination, and all other video feeds.

  1. Tighten the waste tank lid to achieve the vacuum to 5-10psi

10.  Log into the computer as operator (if needed the username is .\operator)

11.  Log into the PPMS account as operator.

12.  Open Sortware and check whether the collection chamber is clear and click continue

13.  Press RINSE for a few seconds: bubbles should be removed from the fluidics. Shake the filter and release the bubbles

B.    Debubble

  1. Install the nozzle, waste bucket and half-cut transfer pipette with water in there

  1. Make sure the plates are open and not charged

  1. Press RUN to wet the line

  1. Purge the nozzle, continue purging and pulsing to remove bubbles. Ethanol, and blocking vacuum lines temporarily can be used to increase vacuum if needed. Residual micro bubbles generally can be reabsorbed once the sheath lines are pressurised, therefore, ensure all bubbles from the nozzle assembly are removed, but micro bubbles are okay.

  1. Remove half-cut pipette and stream catch gently and the waste bucket. Using a kimwipe tip to remove any residue on the nozzle.

  1. Monitor the waste video feed and make sure the waste is getting into the waste holder and the stream is centered and focused

C.    QC

  1. Restore the workspace for the correct nozzle size under the QC folder with the laser delay restored.

  1. Adjust the Sheath pressure to the right pressure, if the pressure used is 6.0psi, adjust the tank to the interface just below 6.1 for a more accurate and reproducible stream.

  1. Loose the sample offset to the end (the value is not going down anymore)

  1. Adjust the Sample pressure to 1 psi below the sheath pressure

  1. Tight the sample offset to 1 psi above the sheath pressure

D.    Laser Alignment

  1. Adjust the top silver nozzle knob to move the nozzle tip to just disappear from the field on the monitor

  2. Adjust the front black knob to achieve the brightest waste spot on the monitor

  3. Adjust the side black knob to move the waste line to the centre of the waste holder

  4. Using side silver knob to move the stream to the centre of the pinholes

  5. Repeat black knobs/silver knobs until both nozzle and waste streams are aligned.

  6. Adjust the back silver knob to focus the stream

  7. Make sure all laser shutters are open. Close the front stream shield and touch the top right to turn on the lasers

  8. Run single peak rainbow beads at approximately 500 events/sec and display 500 events.

  9. When the signal appears, adjust top knob of the blue laser and watching the oscilloscope to achieve the symmetrical peak

10.  Adjust the stream focus knob (back silver knob) and then side knob on the blue laser systematically to achieve the highest signal, compare the previous data to double check

11.  Click the dot plot for the next laser, find the right pinhole on the oscilloscope, adjust the top knob first to get symmetry and highest signal of the peak and then adjust the side knob to achieve the highest signal, adjust the laser delay to move the peak to the right side of the pinhole (it is normally the optimal position for this laser)

12.  Close shutter for the first two lasers, change camera to FSC and align FSC to best signal.

13.  Continue for other lasers

14.  Find the QC folder and save a new file in there using todays date ‘YYYYMMDD_P3s_##um’ and 2000 events as default.

E.    Stream Stability

  1. Close the HV plate and turn it on, allow stream to stabilise for 30min.

  1. Since AMO can affect the stream, leaving a gap in the sort chamber will solve this problem.

  1. Adjust the frequency to achieve the highest break off points. It may have a couple of break off points.

  1. Adjust the amplitude to achieve a viewable and desirable breakoff point. However, if the amplitude is too low, it won’t have enough energy on the drop. If the amplitude is too high, there is no space to move and for smaller nozzles may increase the noise.

  1. Turn on the Test Stream and adjust the amplitude to achieve the farthest and most stable side stream. Fine tune with finer tuning the amplitude using the short flash test streams.

  1. Run Accudrop at < ¼ of the frequency or at >1000 events/sec, sort (Non-P1) and adjust the drop delay to achieve the minimal signal in the waste

  1. Save the workspace, with the same QC name. Amend the information if stream camera, pressure, or frequency has changed.

F.    Reload User’s Workspace

  1. Find the workspace for each user and reload the workspace without “restore the laser delay”, “restore data” and “fluidics set up”

  1. Record user data

  1. Once the users decided the sort gate, save a pdf file as a record.

  1. At the end of sorting, save a new workspace for this user with the format FirstnameLastinitial YYYYMMDD Experimentdescription

G.    Data Transformation (compensated data only)

  1. Duplicate the plot

  1. Change DPS to ADP for both parameters

  1. Inspector: change to logical 

  1. Tick visualise

  1. Adjust logical scaling value to scale up or down of the transformation

  1. Remember gates cannot be used on logicle plots

H.    Pause the sorting for topping up the sheath tank

  1. Remove all samples and run clean the lines.

  1. Put waste under the stream nozzle.

  1. Put one the holder gently and not touching the nozzle: let the liquid fill part of the tip, but not reach to the black part.

  1. Stop Run.

  1. Depressurise tank.

  1. After topping up the sheath tank and press run and remove the holder and waste line

  1. Allow pressure and stream to re-equalise and stabilise

I.      Device position

  1. Move up (Y direction forward), one CTL+click is about 1 mm

J.     Sort on 96-well plate

  1. Put the plate to back left

  1. Select 96 well plate in sort window

  1. Set deflection, best to test sort and ensure stream is on desired target.

  1. Choose appropriate sort mode, i.e. 1.0 drop single for single cell.

  1. Click sort ready and select all the wells, right click to select Not-P1

  1. Sort Accudrop beads at 10/well and run the entire plate to check the position of sorted cells.

  1. Re-adjust plate, set new home position and repeat until centers of wells are the location of sorted cells/beads.

K.    Instrument shutdown

  1. Place waste catcher block under the stream. Ensure aerosols are not created (this can happen on the 70 or 86um nozzles). If this occurs, put the half-cut pipette reservoir under the nozzle to prevent any aerosol generation.

  1. Fill, to 15mm below the line three 5mL tubes with 1% bleach, H20 and ethanol. Run bleach for 5 minutes, then 5% Decon, then H20, then load ethanol onto the SIP.

  1. Place the half-cut pipette reservoir under the stream and allow the liquid to just touch the tip of the nozzle.

  1. Stop running the instrument.

  1. Fill the reservoir with 70% ethanol. Purge the instrument until the flow cell is filled with ethanol, or until just before air is allowed to enter from the tip. Top off the amount of ethanol.

  1. Shutdown UTOPEX through Sortware

  1. Ensure HV plates are off and wipe plate with ethanol, clean waste catcher with ethanol also, and spray and clean sort chamber, and sample input area with 1:50 Trigene.

  1. Ensure sort chamber is closed as UV light is set on a 24hr timer at approx.. ~3am. 

  1. Turn off lasers. Shut down computer and turn off lasers and then power supplies. Turn off biosafety hood and light.

Emergency procedures

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

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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 Influx 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 Spill Kit Instructions (located in J.2.06 - not Cell Sorter Lab)

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