Prior risk assessment for a technique involving the use of

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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 Pseudouridylation Assay


                                             Staff involved

       Authorised radiation users in Room……


                                    Radionuclide and activity used
        33
            P orthophosphate, 7.5 MBq of activity per reaction, 2 reactions at a time


                                        Other hazards involved
       Chemicals: Gelatin, PBS, FCS, Qiagen RLT buffer,       captoethanol, Sodium acetate,
        SDS, EDTA, Isobutyric acid, Sodium hydroxide, Agarose, Isopropanol, HCl, Decon 90
       Cuts from gel plates, sharps etc.
       Electric shock from gel power packs
       Fires caused by HFC’s
       Unbalanced/ malfunctioning centrifuges.
       Manual handling issues with centrifuge rotors


                                           Route of exposure

       skin, eyes, ingestion, penetration, inhalation of aerosols


                                                  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.
       Centrifuges: Can be badly damaged and in extreme cases cause injury if not used
        correctly. If you are unsure as to the safe use of a centrifuge, contact your Safety
        Supervisor or the Lab Manager.
       Manual Handling: Centrifuge rotors can be extremely heavy and awkward to lift into and
        out of large refrigerated centrifuges. Seek appropriate training to do this safely from either
        the Laboratory Manager or the Safety Unit.




                                    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 the 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.
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



                                  Receipt Transport and Storage
       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.


 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 contact doses to the body
        and hands as 33P is a Beta emitter. Food/drink etc are not consumed in CSC laboratories so
        the risks of ingesting isotope are negligible.

       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.

       Controls 2: After the work has been completed the workstation and all equipment used is
        monitored using a Type 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.




                                  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
Day one:
     Put on your laboratory coat, TLD and gloves and prepare the area.
     Monitor the workstation using a type 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.
   
     Pre-gelatinise a 6-well plate with 0.1% Gelatin in PBS for at least 1hr at 37C
     Collect, count and plate out HeLa cells at 30x105/well
     Grow at 37C and 5%CO2 overnight

Day two:
     Grow in 2mls of 10% FCS in inorganic phosphate-free media for 1hr
     Drain and wash in PBS
     Working on a spill tray, add 2mls pre-warmed media with 33P orthophosphate at 100uCi/m.
     *Take a 5ul sample of this for counting on a scintillation counter.*
     The dishes will be placed into a 1cm thick Perspex box with the lid cracked open to allow
      CO2 into them. This box will be transported on a trolley to 4N8 and then put onto a spill
      tray in the incubator and a sign will be placed on the incubator informing all staff that a
      radioactive incubation is taking place in there.
     Incubate the cells at 37C for 3 hours @ 5%CO2.
     Return the cells to 4N5 on the trolley and collect and resuspend the cells in 350l of
      (Qiagen RLT buffer & ME)
     *Take a 5ul sample of this for counting on a scintillation counter.*
     Extract total RNA (see “RA for preparing RNA from cell pellets.doc and RA for RNA clean
      up~ Qiagen method.doc”)
     *Take 5ul samples from several steps of these protocols for counting on a
      scintillation counter.*
     Run on 0.8% agarose gel and carefully cut out the 18S and 28S rRNA bands.
     Monitor all equipment and work areas for radioactive contamination.

Day three:
      Gel extract 18S and 28S rRNA bands
      Digest in a final volume of 5ul containing RNase T2 in (50mM Na Acetate pH4.5, 0.05%
       SDS, 1mM EDTA) at 37C for 90mins
      Scintillation count an aliquot of the sample
      In the fume cupboard in room 4N2 run 20,000cpm of each sample on first dimension of a
       TLC gel in isobutyric acid/NH4OH/H2O (577:38:385 by volume)
      *Take a 5ul sample of this for counting on a scintillation counter.*
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
         Again, in the fume cupboard in room 4N2, run on second dimension of a TLC gel in
          isopropanol/HCl/H2O (70:15:15 by volume)
         *Take a 5ul sample of this for counting on a scintillation counter.*
         Dry and expose
         Carefully cut out the Up and Cp bands and count on a scintillator
         Analyse Up:Cp ratios for each rRNA molecule by count and by densitometry.
         Monitor all areas and equipment used with radioisotopes and clean up any contamination
          found.
         Ensure the TAD form for that pot of isotope has been completed fully.
         Send a monitoring report to the RPS of all areas and equipment that was used outside of
          4N6

    * samples of buffers etc taken for monitoring need only be done once or twice. This is
    carried out to quantify where all the radioactive waste is generated and will assist in
    formulating the most efficient and safe way of disposing of it all. There is the
    possiblilty of generating radioactive solvents and these steps might show that this is
    not actually the case. Records of this will be attached to this risk assessment after it
    has been carried out and will be reviewed should there be any changes to the
    protocol.*



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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