University of Cambridge

NIHR Cambridge BRC Immunophenotyping Hub

e6, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 0QQ

Risk Assessment Form

This form is to be filled in if hazard ratings/quantities/dilutions make University procedures and/or “Good Laboratory Practice” insufficient control.

Group:
BRC Cambridge NIHR Immunophenotyping Hub / Personnel involved:
Anna Petrunkina, Simon McCallum, Natalia Savinykh, Valeria Radjabova, Alex Hatton,
Activity being assessed:
Immunophenotyping of Human Blood
This procedure comprises 4 steps, some of which may be omitted in any given protocol.
(1) Receiving blood samples from various internal and external hospital departments
(2) Separation of cellular populations via density gradient centrifugation and Magnetic Activated Cell Sorting (MACS)
(3) Processing MACS purified samples for RNA analysis
(4) Staining and running PBMC (Peripheral Blood Mononuclear Cells) through a flow cytometer.
Hazards identified:
(i) Exposure to cat2 infectious material (covered in document 3_cat-2-analysers.docx)
(ii) Needlestick injury from layering blood onto Histopaque 1077 gradients
(iii) Exposure to infectious material via aerosol whilst using centrifuges and flow cytometers
(iv) Chemical exposure during RNA isolation
(v) Exposure to infectious material from fixing blockages to flow cytometers
(vi) Exposure to infectious material from fixing blockage to the AutoMACS pro
Control measures to reduce level of risk
(i) [covered in document 3_cat-2-analysers.docx]
(ii)The initial blood layering step can be carried out via a syringe-needle system (with the barrel of the syringe removed). This step must take place in the tissue culture hood. All needles must be disposed of immediately after use, leaving the hood free of sharps for the next user of the hood.
(iii)Flow Cytometers are pressurised systems that can generate aerosols, (dealt with in document 3_cat-2-analysers.docx). Our main centrifuge is a sealed unit that would detect any breakage through imbalance. On such an eventuality, the centrifuge must be left for half an hour for the aerosol to settle before opening and decontaminating. Decontamination must involve disassembly of the buckets, rotor and main unit and thorough cleaning with trigene.
(iv) RNA isolation utilises ß-mercaptoethanol and guanidine isothiocyanate (both contained in buffer "RLTplus" from the Qiagen Qiashredder kit). BME can cause irritation to the nasal passageways and respiratory tract, GT can cause burns by any exposure route and should be handled with care in a tissue culture cabinet.
(v) [covered in document 3_cat-2-analysers.docx]
(vi) On the event of a autoMACS blockage, the sample block must be removed, prior to disassembly and unblocking, the machine must pass 2 clean cycles before resuming the experiment, all samples run though the machine after a blockage must be re-filtered through a 50µm cell strainer filter or smaller immediately prior to loading.
Emergency procedures
First Aid:
Refer to: Appendix XIII of the Institutes ‘Health and Safety Manual’
Name and status of assessor:
Anna Petrunkina / Simon McCallum
/ Date of assessment:
21st October 2015
Signature of assessor: / Revision due date:
1st September 2016