Date of revision and approval

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

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: Xin Wang, Suat Dervish, Edwin Lau

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.

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

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

Research hours - WIMR / non-WIMR

6.00am to midnight

Monday to Friday

8.00am to 8.00pm

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

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.

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.

Emergency procedures

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

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. 

1. 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.

1. In an electrical emergency press the red power shutdown button in the room.

2. Leave the room and place signage that the room is not to be entered due to a major electrical problem.

3. 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 J2.04.

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 who can for DNIR spill, double bag the waste in autoclavable bags and loosely seal with autoclave tape for removal for autoclaving.

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.

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 many 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 buildings incident notification form.

Emergency shutdown for stream failure:

If a stream failure occurs, the Influx may detect this and shut off the stream and sample automatically. If this occurs during sample being put through the instrument, the instrument must be given time for any potential aerosols to settle or be removed via the biological safety cabinet before accessing the sort block.

If the instrument does not automatically shut down the stream upon a stream failure, the stream sample and stream need to be stopped via the software or via the red emergency button located on the instrument. Then after a period of 3 minutes, and with the appropriate PPE (gloves, gown, enclosed shoes and safety respirator), the nozzle may be removed if necessary, decontaminated and sonicated if necessary to remove the clog and reinstalled if needed. If the stream failure was due to a clogged sample, the sample may need to be re-filtered or a larger nozzle used. The primary objective is to prevent potentially biohazardous aerosols being exposed outside of the biological safety cabinet. The procedure is outline below.

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 and biological spill kit location

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

Biohazard Spill Procedure Poster

Health Risk Matrix used in risk assesment

Sort failure procedure for operators

Instrument and room images depicting main components of Influx system and room.