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Radioactive PCR - Prior risk assessment for a technique involving

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					                                                                          MRC Clinical Sciences Centre
                                                                    Imperial College Faculty of Medicine
                                                                                 Hammersmith Campus
                                                                                         Du Cane Road
                                                                                      London W12 0NN




   RISK ASSESSMENT FOR A TECHNIQUE INVOLVING THE USE OF
                     RADIONUCLIDES

                                             Technique Title
       Performing a Radioactive PCR


                                              Staff involved

       Authorised radiation users in Room ………….


                                    Radionuclide and activity used
        32
            P dCTP, 0.37 MBq of activity per reaction, up to 80 reactions at a time.


                                        Other hazards involved
       Chemicals: MgCl, DMSO, Mineral oil, Decon90
       RSI from pipetting:
       Electric shocks and burns:
       Burns from heating blocks:



                                           Route of exposure
       Skin, eyes, ingestion, penetration, inhalation of aerosols, contact.


                                                  Harm
       Radioactivity: Burns to skin and eyes, permanent cell damage, cancers.
       Chemicals: The respective COSHH forms for the above substances must be read prior to
        this technique commencing so that their hazards and control measures are understood.
       RSI: Long term injury can result from using pipettes and other repetitive techniques.
       Burns: PCR blocks can burn if they are opened when the temperature is high.


                                    Receipt Transport and Storage
       Authorised users only can order isotopes through EBP. CSC Safety will approve and place
        the order and inform the likely delivery date. Pick-up point is CSC Safety, Room A10 Admin
        Building.
       Always open new pots of radioisotopes on a splash tray behind appropriate shielding or in a
        fume cupboard if it is volatile.
       Monitor all new pots of isotope upon receipt to ensure that the contents have not leaked.
        For 3H compounds swab and check for leaks in a scintillation counter
       Contact your RPS immediately if you find a leaking pot and follow the clean up procedure
        outlined below if you suspect that there has been a spill.
       Always employ “double containment” when transporting isotopes or radionuclide containing
        compounds to contain any possible spills.
       Only use one glove when transporting radionuclides between labs, floors or buildings to
        prevent door handles, lift buttons etc. from being contaminated.
       Radionuclides can only be stored in the designated freezers and fridges in the department.




Risk Assessment Form for a Technique                                               1
Involving the Use of Radionuclides
MRC Clinical Sciences Centre
Version 3 July 09
                                                                       MRC Clinical Sciences Centre
                                                                 Imperial College Faculty of Medicine
                                                                              Hammersmith Campus
                                                                                      Du Cane Road
                                                                                   London W12 0NN




 Identify any persons and any special risks associated with the technique and steps that
                            must be taken to minimise them

       Person carrying out the work: There is a reasonable risk of high contact doses to the
        body and hands as 32P is a high Beta emitter. Some risk of radioactive contamination to the
        body and hands. There is a possibility of aerosols being generated during this procedure as
        PCR has several heating and cooling steps in it. Ideally screw capped tubes should be used
        to minimise leaking and aerosols wherever possible. Food/drink etc are not consumed in
        any Haematology laboratory so the risks of ingesting isotope are negligible. All chemicals,
        reagents and associated techniques used in this protocol will have their associated risks
        and control measures so their relevant COSHH forms should be read in conjunction with
        this protocol. RSI can come from repeated pipetting and needs to be monitored. Risk of
        burns to the fingers from the heating blocks used.
       Other persons: Only at risk if personal contamination is transferred to other surfaces and
        equipment during the experiment and is not detected by monitoring. All surfaces,
        equipment etc. is monitored and checked for contamination after each experiment.

       Controls 1: Training and experience to monitor effectively, and to deal with spillage and
        decontamination. Personal protective equipment: fastened lab coat, two pairs of gloves in
        case the outer pair gets contaminated, eye protection. Spill tray and dedicated sharps bin
        for contaminated material should be used at all times. Finger TLD’s if issued should be worn
        at all times. There is a plentiful supply of Decon 90 available for cleaning up radioactive
        spillages.
       Controls 2: After the work has been completed the workstation and all equipment used is
        monitored using a Type E or EP15/EL contamination monitor to check for any
        contamination. Also, the room is monitored on a monthly basis by the RPS to act as a back
        up for local monitoring. Contaminated Gloves are removed before exiting from the
        laboratory, and hands are washed in the hand wash sink. All other locations and equipment
        used in this protocol will be monitored after every experiment and the results sent to the
        RPS. All laboratories have fully functional fume cupboards that are used when working with
        toxic or volatile chemicals. No eating and drinking is allowed in CSC labs either so there is
        no real risk of ingestion of chemicals.


                                  Emergency Spillage Procedure

       Contain large spills on floor/bench using towel roll.
       Avoid walking in spill area or it will be spread further.
       Notify others in area.
       Contact your RPS immediately. If they are unavailable, begin decontamination. Do not allow
        the spill to dry out if at all possible.

Decontamination:
     Wear gloves and lab coat. Place contaminated towel roll into a double layer of plastic bags.
     Working from the outside of the area toward the centre, swab with aqueous acid phosphate or
      with commercial cleaner e.g. Decon or Lipsol (5% solution) using disposable towel roll.
     Wash contaminated gloves before removing them. Dispose of along with contaminated paper
      towel into double layer of plastic bags.
     Monitor the area, repeat until contamination has gone, then monitor the clean up personnel.
     The RPS must be informed.

Contaminated personnel: remove contaminated clothing, then wash yourself immediately for 3 - 4
minutes with plenty of water, then wash again with soapy water. Dry on disposable towel, monitor skin
then wash again if necessary to reduce counts to safe level. Do not wash more than 3 -4 times, do
not spread contamination to uncontaminated areas.

Risk Assessment Form for a Technique                                            1
Involving the Use of Radionuclides
MRC Clinical Sciences Centre
Version 3 July 09
                                                                      MRC Clinical Sciences Centre
                                                                Imperial College Faculty of Medicine
                                                                             Hammersmith Campus
                                                                                     Du Cane Road
                                                                                  London W12 0NN



                                  Emergency Spillage Procedure

The RPS must be informed. To decontaminate fixed spills or glassware, use acid phosphate:
Make up an aqueous phosphate solution using any phosphate source (sodium phosphate will do) by
taking 50 g into 500 ml of water, and acidify it by adding HCl x 10 ml. Rub the solution into the
contaminated area using a cloth. Continue applying until the count drops to safe level.
             USE 5 % lipsol or Decon90 liquid to decontaminate radioactive spills

       All accidents must be reported to the Safety Supervisor / Radiation Protection
        Supervisor or the departmental first aid personnel, and CSC Safety via an accident
        report form:
                     http://safety.csc.mrc.ac.uk/CSCAccidentReportForm.pdf


                     Include a brief stepwise description of the technique
       Put on your laboratory coat, TLD and two pairs of gloves and prepare your work area.
       Monitor the workstation using a Type E or EP15/EL contamination monitor before
        commencing work to ensure that there is no contamination left behind from a previous
        experiment. Ensure that the battery in the monitor has sufficient charge.
       If you find contamination, contact the previous user and inform the RPS
       Sign the log for that workstation and commence work.
       Thaw out the required PCR solutions and 32P dCTP
       Book the radioactive PCR machine and enter/alter the PCR programme if required
       Plan the experiment and calculate the number of samples including controls
       Set up the appropriate PCR tubes
       Aliquot out 1l of each DNA sample into each PCR tube
       Sign into a radioactive work station
       Make the master mix for the PCR experiment without the radioactivity as follows, add the
        radioactivity and Taq at the last minute in the hot room (room 4S7) following the usual
        radioactive safety procedures and carefully mix well:

                        Solutions                   1x mix                  Final
                                                                        Concentration
                      10x Buffer                     2.5l                    1x
                      25mM MgCl                       2l                   25M
                100M or 100ng/l F & R           1l of each             4M or 4ng
                        primers
                     10mM dNTPs                      0.75l                  0.3mM
                       5U of Taq                      0.1l                  0.02U
                         dH2O                       16.55l                     -
                                                   (14.05l)
                      32P dCTP                       0.1l                  0.4%
               (100% DMSO if necessary)             (2.5)l                  10%
                    Final volume                      24l                     -

       Carefully aliquot 24l of the radioactive master mix into each PCR tube
       Add mineral oil if required
       Place the radioactive PCR tubes into the PCR machine
       Run the PCR programme
       Store at 4C until ready to run on a SSCP gel
       Complete appropriate TAD form, recording the amount of Isotope used and the disposal of
        both solid and liquid waste.
       Monitor all work areas, equipment used, spill tray etc. with a Type E or EP15/EL
        contamination monitor. Clean up any contamination found immediately with Decon90 and
        monitor again to ensure that the contamination has been cleared.
       Get another registered user to confirm that there is no contamination left behind on the
Risk Assessment Form for a Technique                                          1
Involving the Use of Radionuclides
MRC Clinical Sciences Centre
Version 3 July 09
                                                                         MRC Clinical Sciences Centre
                                                                   Imperial College Faculty of Medicine
                                                                                Hammersmith Campus
                                                                                        Du Cane Road
                                                                                     London W12 0NN



                        Include a brief stepwise description of the technique
          work station.
         When disposing of liquid waste down the designated radioactive waste sink please use
          copious amounts of water to flush it all away. Failure to do this can lead to high background
          dose rates and an increased risk of contamination.
         Remove gloves after checking for contamination and wash hands before exiting the lab.




Due consideration should be made in this assessment for women of child-bearing years, and a full
              re-assessment should be carried out upon notification of pregnancy.

   This assessment has been carried out by the appointed Radiation Protection Supervisor (RPS).
                     A copy of this assessment has been sent to CSC Safety.




RESPONSIBLE PERSON                          NAME                      SIGNATURE             DATE

  Radiation Protection
      Supervisor


Principal Investigator


        Annual Review




Risk Assessment Form for a Technique                                              1
Involving the Use of Radionuclides
MRC Clinical Sciences Centre
Version 3 July 09

				
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