SOP Title Procedure for operation of the FACSCalibur, including

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					SOP Title: Procedure for operation of the FACSCalibur, including start-up and shutdown
of the flow cytometer

SOP Type: Procedure


                              Hammersmith Hospitals NHS Trust
                                  Department of Haematology
                          Quality Management Policy and Procedures

Purpose: To provide a basic guide to operating the FACSCalibur, including start-up and
shutdown procedures necessary to ensure correct use of the flow cytometer

Scope: Applies to all laboratory staff involved in the use of the FACSCalibur

Departmental Responsibility: SCI Laboratory                    Date Effective: 05.Apr.07

Author:
Revised version: V Enright
Original version: J G Davis                                    Review Date: 04.Apr.08

Document Control
The Master Copy of the Quality Manual and SOP’s will be filed in the Quality Management
Office. Controlled copies are numbered and stamped “Controlled Copy” in red ink. Any copies
without this red stamp are uncontrolled and should be destroyed.

If this SOP appears inadequate or outdated it is the responsibility of all staff to bring this to the
attention of their Supervisor immediately.

Training Record
The training record for this SOP is filed with the master copy of the SOP in the Quality
Management Office.

Security Statement
This SOP is the intellectual property of the BMT Unit within the Division of Clinical Haematology
at the Hammersmith Hospital NHS Trust and as such, must not be circulated outside of the Trust
without written approval from the Quality Assurance Manager and the Author of this procedure.
_________________________________________________________________________

Contents
      Section          Subject                                                                 Page

       1.0             Personnel & Responsibilities                                            2
       2.0             Background                                                              2
       3.0             Definitions                                                             2
       4.0             Health & Safety                                                         2
       5.0             Equipment/Documentation                                                 2
                       5.1     Equipment                                                       3
                       5.2     Consumables                                                     3
                       5.3     Documentation                                                   3
       6.0             Procedure                                                               3
                       6.1     Setting up the flow cytometer                                   3
                       6.2     FACSCalibur start-up                                            3
                       6.3     FACSCalibur shutdown                                            4
       7.0             Training & Competency Assessment                                        5
       8.0             References                                                              5
       9.0             List of Appendices                                                      5

SOP No: BMTU-SCIL-016                       Issue No: 3.0                                Page 1 of 9
SOP Title: Procedure for operation of the FACSCalibur, including start-up and shutdown
of the flow cytometer

SOP Type: Procedure

      10.0           Revision History                                                     5
      11.0           Managerial Approval                                                  6

1.0   Personnel & Responsibilities

      All laboratory staff who have been properly trained and are deemed to be competent
      may carry out the procedures. Training must be carried out in compliance with the
      training record accompanying the SOP.

      Any deviations from this SOP must be documented and approved according to SOP
      BMTU-QMP-006.


2.0   Background

      The FACSCalibur is an automated flow cytometer, which analyses cells as they pass one
      at a time through a focused laser beam while moving in a fluid stream. The FACSCalibur
      system includes two lasers: a 488nm argon-ion laser and a ~635nm red-diode laser. A
      flow cytometer consists of a combined system of fluidics (to introduce and focus the cells
      for analysis), optics (to generate and collect light signals) and electronics (to convert the
      optical signals to proportional electronic signals and digitise them for computer analysis).

      The FACSCalibur should always be turned on before turning on the computer when
      acquiring data, as this enables the computer to recognise that the cytometer is
      connected. Before shutdown procedures are carried out, the FACSCalibur should
      always be cleaned to ensure that the instrument fluidics function consistently. Laser
      lifespan may be reduced if the flow cytometer is unnecessarily switched on and off
      throughout the day.


3.0   Definitions

      SIP – Sample Injection Port


4.0   Health & Safety

      All staff must follow safe practice for dealing with biological materials as stated in the
      Hammersmith Hospitals NHS Trust Infection Control Policy, Section 2:Universal Infection
      Control including Safe Handling and Disposal of Sharps, Sharps Injuries and Exposure to
      Blood & Body Fluids, Spillage & Waste. Refer to departmental COSHH assessments for
      reagents. All human derived products should be treated as a potential biohazard.

      All staff will need to have attended the Trusts Non-Clinical Manual Handling course and
      be aware of equipment that can be used to safely move or carry heavy materials such as
      20L FACSFlow containers.


5.0   Equipment/Documentation




SOP No: BMTU-SCIL-016                    Issue No: 3.0                              Page 2 of 9
SOP Title: Procedure for operation of the FACSCalibur, including start-up and shutdown
of the flow cytometer

SOP Type: Procedure

      5.1     Equipment

              FACSCalibur flow cytometer
              FACSFlow supply system
              Mac OS 9.2 computer
      5.2     Consumables                                         RA/COSHH assessment    risk


              FACSFlow        342003                              CA12           L
              FACS Clean 340345                                   CA51           M
              FACS Rinse 340346                                   CA27           L
              Bleach (sodium hypochlorite)                        CA28           H
              Distilled Water
              5ml Falcon Tubes

      5.3     Documentation

              FACSCalibur Daily Log          (Appendix 1)


6.0   Procedure

      6.1     Setting up the flow cytometer: sheath and waste reservoirs

      6.1.1   The FACSCalibur in the SCI laboratory is supplied with FACSFlow sheath fluid
              via an automated supply station located under the bench. FACSFlow is supplied
              to the flow cytometer secondary reservoir directly from a 20L FACSFlow
              container attached to the supply station. Waste is aspirated into an empty 20L
              FACSFlow container, also attached to the supply station.

      6.1.2   When the supply container is empty and/or the waste container is full, the supply
              station will emit an audible alarm, and a red warning light will activate.

      6.1.3   If the flow cytometer has run out of FACSFlow, the 20L supply container is
              disconnected from the automated supply station, removed and replaced with a full
              one. The system must be primed by depressing the prime button on the supply
              station to refill the secondary reservoir. Press the restart button on the supply
              station to reset the system. Always ensure that there is a spare FACSFlow next to
              the supply station.

      6.1.4   If the waste container is full, it must be disconnected from the automated supply
              station and emptied into the cleaning cupboard waste sink. Once the waste
              container has been emptied, it should be placed into a yellow bin bag and
              discarded as clinical waste.

      6.1.5   Replace the container with an empty 20L FACSFlow container and label with
              high-risk tape. Approximately 10ml undiluted bleach should be added to the
              waste container before attachment to the supply station. Press the restart button
              on the supply station to reset the system.

      6.2     FACSCalibur start-up

      6.2.1   To ensure proper connection between the flow cytometer and the computer, the
              FACSCalibur is switched on before the computer. Turn on the FACSCalibur by

SOP No: BMTU-SCIL-016                    Issue No: 3.0                             Page 3 of 9
SOP Title: Procedure for operation of the FACSCalibur, including start-up and shutdown
of the flow cytometer

SOP Type: Procedure

              pressing the power switch located on the lower right corner of the instrument.
              Wait approximately 15 seconds and then turn on the computer by pressing the
              circular power button on the front.

      6.2.2   Sign the FACSCalibur daily log located in the FACSCalibur user/cleaning logs
              folder that is kept on the SCI laboratory bench (see Appendix 1).

      6.2.3   Slide the fluidics drawer out and pressurise the FACSCalibur by flipping the vent
              valve toggle switch to the pressurised position. The system must then be primed
              by depressing the PRIME fluidic control button twice (on HI flow rate). Run a tube
              of distilled water on HI flow rate for 5 minutes to rinse the system. This will allow
              the laser to warm up after before running any samples.

      6.2.4   When not in use, the FACSCalibur should be left on standby by depressing the
              STANDBY button (on LO flow rate), with a tube of distilled water on the SIP. This
              lowers laser power to conserve sheath fluid and prolong laser life. Ensure that
              the level of the water does not touch the top of the SIP.

      6.2.5   Check the sheath filter to be sure that no air bubbles are trapped inside. If
              bubbles are visible, pressurise the flow cytometer and gently tap the filter body to
              dislodge them. Push the roller in the pinchcock forward to allow sheath fluid to
              force the bubbles out and return the pinchcock to closed position.

      6.2.6   In Appendix 2 a summary of start-up instructions can be found.

      6.3     FACSCalibur shutdown

      6.3.1   To prevent the sample injection tube from becoming clogged, run BD FACS
              Clean through the SIP at the end of each day. Install a tube containing
              approximately 3 ml FACS Clean on the SIP (with the support arm to the side) and
              let it run for 1 minute. Then run FACS Clean on HI flow rate for 5 minutes, with
              the support arm under the tube.

      6.3.2   FACS Rinse should then be run on HI flow rate for 5 minutes, followed by a
              distilled water rinse, also on HI flow rate for 5 minutes.

      6.3.3   Leave the tube of distilled water on the SIP. Depressurise the flow cytometer by
              flipping the vent valve to the depressurised position and leave the machine on
              STANDBY (LO flow rate) for 10 minutes to allow the laser to cool down.

      6.3.4   Choose Shut Down from the Special menu on the computer desktop to turn off
              the computer, and then shut off the power to the FACSCalibur by pressing the
              power switch located on the lower right corner of the instrument. Sign the
              FACSCalibur daily log located in the FACSCalibur user/cleaning logs folder that
              is kept on the SCI laboratory bench (see Appendix 1).

      6.3.5   In Appendix 2 a summary of shutdown instructions can be found.




SOP No: BMTU-SCIL-016                    Issue No: 3.0                               Page 4 of 9
SOP Title: Procedure for operation of the FACSCalibur, including start-up and shutdown
of the flow cytometer

SOP Type: Procedure

7.0     Training and Competency Assessment

Type of Training                    New staff to read SOP and go through the procedure step by
                                    step with a trained member of staff. Staff previously trained in
                                    SOP CGTL016/02 are to read SOP but no additional training
                                    is required unless no routine procedure has been carried out
                                    for twelve months.
Method of competency                Staff to be supervised until fully competent. Competency
assessment for “procedure”          assessment by observation.
List Staff required for Training    All SCI Laboratory staff and research staff working in the
and Competency Assessment           laboratory.
Who is to perform this training     Laboratory Director, Head of Processing Dept, Head of Quality
                                    Dept and Specialist BMSs.
Evidence Log of Training &          Complete the Training Record (Appendix 3).
Competency Assessment

8.0     References

        8.1 BD Biosciences FACSCalibur Training Manual

        8.2 SOPs:     BMTU-QMP-006 Deviating from an SOP


9.0     List of Appendices

        Appendix 1    FACSCalibur Daily Log
        Appendix 2    Summary of Instructions
        Appendix 3    Record of Training and Competency Assessment


10.0    Revision History

Issue       Change
1.0         First Issue
2.0         Refer procedure for actual details
3.0         JACIE format




SOP No: BMTU-SCIL-016                    Issue No: 3.0                              Page 5 of 9
SOP Title: Procedure for operation of the FACSCalibur, including start-up and shutdown
of the flow cytometer

SOP Type: Procedure


11.0      Managerial Approval

          Name Printed          Signature                Title            Date
Author:
                                                 Specialist BMS
V Enright                Victoria Enright                           26/2/07
Reviewers
                         John Davis
J. G. Davis                                      SCI Lab Director   21/2/07
                                                 Head of
S. Loaiza                Sandra Loaiza                              21/2/07
                                                 Processing Dept.
                                                 Head of Quality
P. Elsey                 Penny Elsey                                21/2/07
                                                 Dept.
                                                 SCI Lab Medical
A. Rahemtulla            Amin Rahemtulla                            27/3/07
                                                 Director
                         Kirtash Patel
K. Patel                                         QA Manager         28.Mar.07




SOP No: BMTU-SCIL-016                    Issue No: 3.0                          Page 6 of 9
SOP Title: Procedure for operation of the FACSCalibur, including start-up and shutdown
of the flow cytometer

SOP Type: Procedure

Appendix 1   FACSCalibur Daily Log




SOP No: BMTU-SCIL-016                Issue No: 3.0                         Page 7 of 9
SOP Title: Procedure for operation of the FACSCalibur, including start-up and shutdown
of the flow cytometer

SOP Type: Procedure

Appendix 2    Summary of Instructions


FACSCalibur Start-up

•   Switch instrument on – wait approximately 15 seconds

•   Switch computer on

•   Pressurise the instrument and Prime x 2 (HI flow rate) with distilled water

•   Run on HI for 5 minutes with distilled water

•   Sign the FACSCalibur daily log

•   Leave instrument on STANDBY (LO flow rate)

•   Instrument ready for use



FACSCalibur shutdown

•   Install a tube of FACS Clean on the SIP (with support arm to the side) and run for 1
    minute

•   Run FACS Clean on HI flow rate for 5 minutes, with support arm under the tube

•   Run FACS Rinse on HI flow rate for 5 minutes

•   Run distilled water on HI flow rate for 5 minutes

•   Leave the tube of distilled water on the SIP

•   Depressurise the flow cytometer and leave the instrument on STANBY (LO flow rate)
    for 10 minutes

•   Shutdown computer and switch off instrument

•   Sign the FACSCalibur daily log




SOP No: BMTU-SCIL-016                  Issue No: 3.0                              Page 8 of 9
SOP Title: Procedure for operation of the FACSCalibur, including start-up and shutdown
of the flow cytometer

SOP Type: Procedure

Appendix 3    Training Record and Competency Assessment

SOP No: BMTU-SCIL – 016             Version: 3.0           SOP Type: Procedure

SOP Title: Procedure for operation of the FACSCalibur, including start-up and shut down of the flow cytometer

The following table constitutes the Record of Training - Training Status and Competency, in this procedure, for the personnel detailed below.


 Date        Trainee        Trainee            Date          Supervisor /     Supervisor / Trainer     Competency Assessment Method.
 Training    Name           Signature          Training      Trainers         to sign, if Trainee is
 Started                                       Completed     Signature        Competent in SOP.




This completed Record Training & Competency Assessment form must be forwarded to the QA Manager for archiving, by the trainer.



SOP No: BMTU-SCIL-016                    Issue No: 3.0                            Page 9 of 9

				
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