Minidex Trial Specific Sample Collection, Handling, Processing, Analysis and Storage Guidance
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1 16A: Microsampling protocol Minidex Trial Specific Sample Collection, Handling, Processing, Analysis and Storage Guidance EudraCT No: , REC Ref: 16/NW/0396 Guidance Sheet 16A Version /01/2018 Page 1 of 10
2 Table of Contents 1 Table of Contents 2 2 Introduction 3 3 Applicability 3 4 Blood Samples Preparation: Sampling: Sample Numbering and recording: Initial Sample Processing: Sample Storage: Sample transit: 5 5 Bronchoalveolar Lavage Samples Preparation Sampling Sample Numbering and recording Initial Sample Processing: Sample Storage Sample transit 6 6 Equipment maintenance 6 7 Temperature records 7 8 Appendices Appendix 1 Blood sample processing Appendix 2 - BAL sample processing 10 EudraCT No: , REC Ref: 16/NW/0396 Guidance Sheet 16A Version /01/2017 Page 2 of 10
3 Introduction This guidance document is for researchers collecting, handling, processing, storing and transporting trial specific samples for the Minidex trial within the Leeds Teaching Hospitals Trust (LTHT), Hull Royal Infirmary (HRI) and Bradford Royal Infirmary (BRI). The collection of blood and bronchoalveolar lavage (BAL, secretions) samples for the analysis of cytokine networks is an important aspect of the clinical trial. This guidance ensures quality by requesting that all samples are stored, handled, processed, stored and transported in a demonstrable manner such that high quality data generated can be confirmed by audit and inspection. Applicability This Guidance applies to the trial specific samples for cytokine estimation generated by the subset of consenting babies participating in the Minidex trial at either the Leeds Teaching Hospitals Trust (LTHT), Hull Royal Infirmary (HRI) or Bradford Royal Infirmary (BRI). This guidance is applicable to all members of research staff involved in running the Minidex trial who collect, process or analyse trial samples including but not restricted to Chief and Principal Investigators, Research Nurses and laboratory staff. The Principle Investigator has ultimate responsibility for ensuring that all applicable study site staff adheres to the guidance laid out in this document. Blood Samples Blood microsamples are to be collected on the day of randomization (prior to the administration of the first dose of dexamethasone) and in the 4 hours before the doses of dexamethasone given on the 4 th, 7 th, 10 th and 14 th days of the trial (equating to 4 hours prior to drug doses 4, 7, 10 and 12) on the subset of consenting babies participating in the Minidex trial at either the Leeds Teaching Hospitals Trust (LTHT), Hull Royal Infirmary (HRI) or Bradford Royal Infirmary (BRI). 4.1 Preparation: Label all aliquot tube lids with the baby s study number. Assemble the tubes needed for each patient in an individual rack 1 graded storage tube and 1 Eppendorf tube. Label tubes with sample number (generated by combination of study number, type of sample and date of sample). o Study number/blo/date o XXXX/Blo/DDMMYYYY 4.2 Sampling: Blood microsamples of microliters are to be collected at the time of clinically indicated blood sampling by a member of the clinical team who has received training in line with the local unit policy. Samples are to be collected in an EDTA 500 microliter microtainer (capillary, venous or arterial blood is acceptable so long as steps are taken to avoid heparin contamination). EudraCT No: , REC Ref: 16/NW/0396 Guidance Sheet 16A Version /01/2017 Page 3 of 10
4 Universal precautions and local precautions for infection prevention, infection control, analgesia and asepsis must be adhered to during sample collection Sample Numbering and recording: Samples are each allocated a unique identifier according to the baby s study number, the sample type and the day of the trial on which the sample was collected. o Study number/blo/date o XXXX/Blo/DDMMYYYY After the sample has been collected take it immediately to the designated laboratory area on the neonatal unit and note the patient initials, sample ID number and date and time the sample was taken in the lab log book and worksheets (kept in the trial sample folder). Then perform initial processing on NNU Initial Sample Processing: Members of the research team who have received Sample Processing Trial Specific Training and completed the training log (filed in the trial master file) are able to perform the preliminary processing, recording and short term storage of the samples. Ensure universal precautions are adhered to at all times. Blood samples must be processed immediately to obtain optimum results, if delays are unavoidable they can be stored in a fridge at <6 C for a maximum of 90 minutes. Invert gently to ensure sample is mixed. Pulse spin (turn on, wait 2 seconds, and switch off) the microtainer on a IKA mini G centrifuge. Remember to balance with an empty microtainer. Transfer the blood using a plastic Pasteur pipette (0.5ml in size) into an Eppendorf tube (0.5ml these are smaller than the ones used for BAL). Using the adaptors for the smaller tubes, centrifuge the Eppendorf at room temperature for 10mins at 2,000 rcf (Fig. 1; back of sheet). Remember to balance the rotor (Fig. 2). Once spun, gently remove the top plasma layer into a fresh Eppendorf tube (1.5ml) using a plastic Pasteur pipette (0.5ml in size). Avoid collecting any of the red bottom layer with the pipette tip (Fig. 3). Should this happen, return all contents to the tube and spin again. Haemolysed samples (with red/orange plasma) are still acceptable for analysis. Aliquot equal volumes of approximately 50µl each into fresh Eppendorf tubes. Use the coloured fluid as a visual guide (Fig. 4). Store all aliquots into the pre-frozen Isofreeze rack in the -20 C freezer until they are transferred into the -80 C freezer (transport them in the Isofreeze rack transfers should take place weekly). Never allow samples to thaw after being frozen. Record details of the time of freeze, location, number of aliquots of each sample, date of transfer to -80 C, etc. in the log book and individual worksheet. 4.3 Sample Storage: All samples must be frozen within 2 hours. Short-term storage of serum samples at -20 C can be done on the on the neonatal unit. All samples must be labeled with sample ID and logged in the sample log/database before being placed into the freezer. Record details of time of freeze, location, number of aliquots of each sample etc. in the lab log book and individual worksheet. EudraCT No: , REC Ref: 16/NW/0396 Guidance Sheet 16A Version /01/2017 Page 4 of 10
5 4.4 Sample transit: Longer-term storage at -80 C will be undertaken centrally at UoL and in the HRI, BRI or LTHT pathology laboratory. Keep the samples in the Isofreeze rack whilst transferring into the -80 C freezer. Bronchoalveolar Lavage Samples 5.1 Preparation Label all aliquot tube lids with the baby s study number. Assemble the tubes needed for each patient in an individual rack 2 graded storage tubes and 2 Eppendorf tubes. Label tubes with sample number (generated by combination of study number, type of sample and date of sample). o Study number/ BAL/date o XXXX/BAL/DDMMYYYY 5.2 Sampling BAL (secretions) samples are to be collected on the day of randomization (prior to the administration of the first dose of dexamethasone) and in the 4 hours before the dose of dexamethasone given on the 4 th, 7 th, 10 th and 14 th days of the trial (equating to drug doses 4, 7, 10 and 12) on the subset of consenting babies participating in the Minidex trial at either the Leeds Teaching Hospitals Trust (LTHT), Hull Royal Infirmary (HRI) or Bradford Royal Infirmary (BRI). BAL samples are to be collected at the time of clinically indicated endotracheal tube suctioning by a member of the clinical team who has received training in line with the local unit policy. BAL fluid collection is performed using a standardised technique. Universal precautions and local precautions for infection prevention, infection control, analgesia and asepsis must be adhered to during sample collection. Babies are positioned supine. Cardiorespiratory monitoring is continued throughout the procedure. Inspired oxygen concentration is adjusted just before performing BAL to maintain oxygen saturations >90% throughout the lavage procedure. A procedure should be performed using a clean inline suction set. A 1.0 ml/kg (up to a maximum volume of 1.0 ml) aliquot of normal saline is instilled gently via the side port, followed by a 0.5 ml air bolus to clear the dead space. The suction catheter is then connected to a suction trap and inserted into the ET tube mm Hg of suction pressure is applied while simultaneously withdrawing the catheter from the endotracheal tube. The infant is reconnected to the ventilator and monitored to ensure a stable state. The whole process is then repeated using a second identical volume aliquot of normal saline to obtain a sample of up to 1 ml. Note the sample ID number, patient initials, and time sample was taken in lab log book and worksheets. (Kept in the trial sample folder) Sample Numbering and recording Samples are each allocated a unique identifier. Samples are each allocated a unique identifier according to the baby s study number, the sample type and the day of the trial on which the sample was collected. o Study number/ BAL/date EudraCT No: , REC Ref: 16/NW/0396 Guidance Sheet 16A Version /01/2017 Page 5 of 10
6 o XXXX/BAL/DDMMYYYY After the sample has been collected take it immediately to the designated laboratory area on the neonatal unit and note the patient initials, sample ID number and date and time the sample was taken in the lab log book and worksheets (Kept in the trial sample folder). Then perform initial processing on NNU Initial Sample Processing: Members of the research team who have received Sample Processing Trial Specific Training and completed the training log are able to perform the preliminary processing, recording and short term storage of the samples. Ensure universal precautions are adhered to at all times. Samples must be processed immediately to obtain optimum results, if delays are unavoidable they can be stored in a fridge at <6 C for a maximum of 90 minutes. Mix the BAL gently by inverting the tube, and by using a plastic Pasteur pipette (3ml) transfer in an Eppendorf tube (1.5ml). Centrifuge the Eppendorf at room temperature for 10mins at 2,000 rcf in a IKA mini G centrifuge (Fig. 1; back of sheet). Remember to balance the rotor (Fig. 2). Once spun, gently remove the supernatant into a fresh Eppendorf tube using a plastic Pasteur pipette (0.5ml in size). Avoid touching the bottom of the tube with the pipette tip so as to not disturb the cell pellet (Fig. 5). Aliquot equal volumes of approximately 100µl each into fresh Eppendorf tubes. Store all aliquots into the pre-frozen Isofreeze rack in the -20 C freezer until they are transferred into the -80 C freezer (transport them in the Isofreeze rack transfers should take place weekly). Never allow samples to thaw after being frozen. Record details of the time of freeze, location, number of aliquots of each sample, date of transfer to -80 C, etc. in the log book and individual worksheet. 5.3 Sample Storage All samples must be frozen within 2 hours. Short-term storage of serum samples at -20 C can be done on the on the neonatal unit. All samples must be labeled with sample ID and logged in the sample log/database before being placed into the freezer. Record details of time of freeze, location, number of aliquots of each sample etc. in the lab log book and individual worksheet. 5.4 Sample transit Longer-term storage at -80 C will be undertaken centrally at UoL and in the HRI or BRI pathology laboratory. Keep the samples in the Isofreeze rack whilst transferring into the -80 C freezer. Samples should be transferred from -20 C to -80 C weekly. Equipment maintenance All equipment and software used should have servicing, maintenance, validation and calibration records available for review. EudraCT No: , REC Ref: 16/NW/0396 Guidance Sheet 16A Version /01/2017 Page 6 of 10
7 Temperature records Regular temperature readings of all fridges and freezers used to store samples should be recorded on temperature logs. Temperature alarms should be in place to mitigate power failures Any deviations in the recorded temperatures should be managed according to the laboratory SOPs. EudraCT No: , REC Ref: 16/NW/0396 Guidance Sheet 16A Version /01/2017 Page 7 of 10
8 Appendices 8.1 Appendix 1 Blood sample processing Fig. 1 - Press the button 10 (10 minutes spin) Fig.2 - Balance the rotor with the empty tube opposite Fig.3 - Collecting plasma without disturbing the red cell layer EudraCT No: , REC Ref: 16/NW/0396 Guidance Sheet 16A Version /01/2017 Page 8 of 10
9 Fig.4 Volume=50µl Fig.5 Pipetting of the supernatant EudraCT No: , REC Ref: 16/NW/0396 Guidance Sheet 16A Version /01/2017 Page 9 of 10
10 8.2 Appendix 2 - BAL sample processing Fig. 1 Press the button 10 (10 minutes spin) Fig.2 Balance the rotor with the empty tube opposite Fig.3 Pipetting of the supernatant Fig.4 Volume = 100 µl EudraCT No: , REC Ref: 16/NW/0396 Guidance Sheet 16A Version /01/2017 Page 10 of 10
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