Exeter Clinical and Health Research Standard Operating Procedure: Centrifuge Operation SOP Number: NIHRexe/019/GEN Version Number & Date: V2 13/06/2017 Review Date: 13/06/2018 Superseded Version Number & Date: V1.1 22/09/2016 Author: Name: Steven Spaull Position: CRF Technician Signature: Date: 13/06/2017 Approved by: Name: Diane Jarvis Position: CRF Technician Signature: Date: 13/06/2017 Senior Management Agreement: Name: Gillian Baker Role: CRF Manager Signature: Date: 13/06/2017 I agree that appropriate members of my workforce (as named above) have written and approved this SOP for use in clinical research. Page 1 of 8
1. BACKGROUND: A centrifuge is a common item of laboratory equipment that is designed to separate some of the component constituents of clinical samples and other fluids by the application of centrifugal force. Following centrifugation, either the supernatant or the sediment at the bottom of the container may be removed. The NIHR Exeter CRF and associated sites have a variety of centrifuge models, including three refrigerated bench-top models, in the RILD Sample Preparation laboratory General rules of operation will be common to all types, refer to individual manufacturers manuals for model specific details. 2. SCOPE: This SOP applies generically to clinical trials and research projects, clinical and health research in Exeter, unless a trial agreement specifically indicates that another organisations SOP should be used. 3. PURPOSE: To ensure standardised use and care of centrifuges required for research purposes. 4. DEFINITIONS AND ABBREVIATIONS RCF: Relative Centrifugal Force the acceleration in a centrifuge normalised to Earth s gravity. Measured in G s. RPM: Revolutions Per Minute Supernatant: The top fraction of a sample, after it has been centrifuged. The lightest fraction. Sediment: the bottom fraction of a sample, after it has been centrifuged. The heaviest fraction. Rotor: the section of the centrifuge that fits to the motor arm and carries the samples. Either a fixed angle rotor or a swing out bucket rotor. Bucket: the section of a swing out rotor that hold the samples. Usually fitted with an adaptor for the specific type of sample that is to be spun. Acceleration and braking: some centrifuges can have the acceleration and braking curve modified to provide gentle or hard acceleration and braking. Bucket lids: hard plastic lids that are either screwed or clipped on to a bucket and provide a hermetic seal Lubricant: only use lubricant that is supplied with the centrifuge. Page 2 of 8
5. ROLES AND RESPONSIBILITIES: It is the responsibility of staff undertaking clinical research to read and use this SOP when using centrifuges for the separation of blood or other substances in the CRF. 6. SKILL LEVEL: This procedure should only be carried out by personnel who have undergone the appropriate departmental training. All nurses, research practitioners and technicians must be familiar with the operation of the centrifuges they are going to use on a regular basis. This includes the bench top refrigerated units for the technicians and the portable centrifuges for nurses and practitioners (at minimum). 7. EQUIPMENT: Gloves, nitrile or latex Goggles (in case of spillage) Lab coat, disposable apron Centrifuge (either bench-top refrigerated or portable ambient) Blood spillage kit Sharps bin Clinical waste bag 8. PROCEDURE: Centrifuges can be dangerous if misused. Do not use a centrifuge unless you have received instruction. Before use ensure that the centrifuge is secure on the working surface and that one foot of free bench space surrounds the centrifuge where possible. Switch on the centrifuge. Allow the centrifuge to cool to the set temperature, if using a refrigerated centrifuge. Sample tubes MUST be balanced evenly across the rotor if in doubt, weigh the tubes in their holder. All types of centrifuge must be balanced before operation. In addition, the layout of the tubes must mirror each other across the rotor. Failure to correctly balance tubes will lead to damage to the centrifuge mechanism, or, in extreme cases, damage to the samples themselves. Most modern bench-top Page 3 of 8
centrifuges have an automatic cut off in case of unbalanced sample loading which stops the spin automatically. Rotor or bucket plastic covers MUST be used. Ensure these are correctly and securely fastened before spinning. Close the centrifuge lid firmly. Set the desired RCF, duration and temperature as described in your protocol, or select the appropriate program. Please note that RCF is the centrifugal force exerted. All protocols should specify centrifugation speed in RCF, not RPM. The advantage of quoting RCF is it is an absolute value, whereas RPM depends on the rotor size, and will vary from one centrifuge to the next. To calculate RPM or RCF manually, measure the radius of the centrifuge rotor from the centre of the turning axis to the bottom of the centrifuge (i.e. from the centre of the rotor to the bottom of one of the (swung out) buckets. Take this measurement and visit the online calculator here. Formulae for calculation are listed in the appendices. Rotors have a maximum speed at which they may be used. Check the manual to ensure you are not spinning above recommended speed. Press the button to start the centrifugation process but do not leave the vicinity until the centrifuge has reached the desired velocity and is operating normally. After the centrifugation period and when all movement has ceased, open the centrifuge lid and inspect the samples tubes to ensure that they are intact before removing them from the centrifuge. Remove the samples and continue with the next stage of your protocol. Remember to close the lid if the centrifuge is refrigerated and is set colder than room temperature. USE OF A CENTRIFUGE COULD BE HAZARDOUS IF STANDARD PROCEDURES ARE NOT ADHERED TO AT ALL TIMES. Consequently it is important that: Materials are not used in centrifuges unless specifically designed for that purpose. Centrifuges are never operated with an imbalance that could cause damage to the centrifuge or make it unstable. ROUTINE CLEANING / CHECKING: Centrifuges should be inspected visually on a regular basis, with any problems reported ASAP to a senior technician or manager. If in doubt do not use the centrifuge, close the lid and display a note to that effect. Clean the outside of the centrifuge once a week with multi-surface wipes, and/or alcohol wipes. Clean tube holders by soaking in an appropriate detergent solution for a minimum of 2 hours. Rinse and dry all holders before reuse. Page 4 of 8
Clean and grease trunnions, no more than once per week, depending on usage. Record maintenance on the wall list in the sample prep laboratory. Centrifuges should only be repaired by the manufacturers. Do not remove any part of the casing yourself, or attempt any repairs. Centrifuges need to be inspected regularly and should be tagged with the future date to be inspected. SAMPLE TUBE BREAKAGE / LEAK: Plastic tubes are in routine use so breakages should be infrequent, although manufacturing faults can lead to hairline fractures within the tubes. There are however, a small number of glass tubes in circulation, so extra care will be required if a breakage occurs. Most bench-top centrifuges have an emergency pull cord to open the lid, if there is any power interruption and / or damage to the lid. These pull cords should be located by operators before use of a centrifuge, in case a problem occurs. Detection of Breakage If the centrifuge is spinning and the sound of a breakage is heard, turn off the centrifuge and leave the lid closed for 60 minutes to allow settlement of any aerosols. If a breakage is discovered when the centrifuge has already stopped and before any samples have been removed, similarly, close the lid and allow 60 minutes to elapse. If a tube breakage or leakage is not noted until the tube holders have been removed, follow the cleaning protocol without the 60 minute delay. Page 5 of 8 Cleaning Following the 60 minute delay, wear two pairs of disposable gloves, and then carefully open the centrifuge, remove the affected bucket (if possible) and take off the plastic lid. Remove the tube holders and then remove any intact samples, if they are visibly contaminated wipe the outside of the tube with an alcohol wipe. For sealed rotor centrifuges remove the rotor and place at a sink area for ease of working. Using forceps and/or swabs carefully remove any broken tube fragments and place them in a sharps bin. If it is possible to identify the sample from the fragments without risk then note these details. Dispose of the remainder of the broken sample in a sharps bin. Using a thick wad of tissue soaked in warm water carefully wipe out any excess organic matter and dispose of it in a yellow waste bag.
Wipe the inside of the rotor (sealed rotor centrifuge) with multi-surface wipes, then alcohol impregnated wipes or, if available use an alcohol spray disinfectant. For non-sealed rotor centrifuges wipes all surfaces with water soaked tissue, multi-surface wipes, and then disinfect all surfaces with alcohol wipes or spray. Check all tube holders for fragments and remove debris as above. Soak in a suitable detergent solution for a minimum of 2 hours. Rinse and dry all holders before reuse. 9. DESIRED OUTCOME: When followed, this SOP should result in safe and effective centrifugation of samples. If a spillage or damage to a sample does occur, following the relevant instructions will result in safe cleaning and decontamination of the effected centrifuge. 10. REFERENCES: http://www.hettweb.com/mobile-app http://insilico.ehu.es/mini_tools/rcf_rpm.php 11. APPENDICES: A: Centrifuge speed calculation B: Weekly centrifuge maintenance Page 6 of 8
Appendix A Formulae for calculating RCF / RPM (if no internet connection) RCF = ( RPM 1000 ) 2 r 1.118 RPM = RCF r 1.118 1000 Page 7 of 8
Appendix B Weekly centrifuge maintenance Please initial each column as task is carried out Date Clean bowl Clean buckets and adaptors Clean and regrease trunnions Page 8 of 8