Standard Operating Procedure (SOP) SOP-WP5-002/Version 1.0
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1 Page 1 of 12 (SOP) SOP-WP5-002/Version 1.0 Title: Manual blood processing Validity: valid after: valid until: insert end date Replaced versions : insert previous version Modification note: insert modifications with respect to previous version Distribution list: Original: quality management / quality assurance representative Authorized copies (ed) 1. insert distribution pathway 2. insert distribution pathway 3. insert distribution pathway Written by: Prof. Thomas Illig Date: 07/14/2011 Name(s) Checked and approved by: Date Name(s) Content 1. Aim/field of application/tasks Terms, definitions, abbreviations... 3
2 Page 2 of Scope of application Affiliations to other regulations Responsibilities Work procedure Flow-chart of operating procedure Description of operating procedure Required materials and equipment: Required documents: Operating Procedure: manual sample processing Proceeding in case of discrepancies Attachments... 8 Attachment 1: flow chart for serum sample processing (adapt to corresponding study/biobank)... 9 Attachment 2: flow chart for plasma sample processing (adapt to corresponding study/biobank) Table 1: SOP instruction to staff Table 2 : Sample documentation protocol blood processing (BPP)... 12
3 Page 3 of Aim/field of application/tasks Processing and storage of blood-samples are important steps in the pre-analytical process of biosamples. Therefore the aliquoting and the storage of the samples should be done immediately according to the SOP. This is a description for the manual processing of blood samples according to the requirements and standards of the insert corresponding study/biobank here. 2. Terms, definitions, abbreviations BE Pb (SP) EDTA: Temp. RT x g min ml SN BPP Monovettes: Vacutainer: Mistakes: Blood extraction /Taking of blood-sample Study participant Ethylenediaminetetraacetic acid; chelating agent temperature room temperature Centrifuge rotor speed in g minute milliliter Supernatant Bioprobenprotokoll (sample documentation protocol) Tubes for bloodsamples, according to this system (e. g. Kabevette, Monovette ) comply with the structure of a syringe. Pulling out the plunger leads to an underinflation, which increased speed of blood extraction. Inside a sample-container of this type (e. g. Kabevette, Vacuette, Vacutainer ) is a negative pressure. When plugging the sample-container with the adapter, which is connected with the needle, the negative pressure inside leads to blood extraction. A disadvantage of this system compared with the aspirationsystem is that the negative pressure does not vary and that it is not adjustable to specific conditions of different veins. Errors in the correlation of participants and samples which lead to misclassification of samples, also the use of inadequate material in the sample preparation as source for serious non-conformity.
4 Page 4 of Scope of application Insert scope of application here. 4. Affiliations to other regulations WP5-001-blood withdrawal. 5. Responsibilities Insert responsible person here.
5 Page 5 of Work procedure 6.1. Flow-chart of operating procedure See attachments 1 and Description of operating procedure Required materials and equipment: a) Biological materials: Blood-filled monovettes according to WP5-001-blood withdrawal b) Equipment: adapt according to corresponding study/biobank Cool able table-centrifuge with a swing-out rotor for max. 4x4 tubes ul single-channel pipettes Tube holder with bowl Refrigerator (4 C) Freezer -20 Freezer -80 C Cold packs/dry ice and suitable containers for transport Scale (calibrated, weighing range: 1g, d=0,01) VisionMate ST / VisionMate SR Barcode-Scanner (Thermo; #3125 bzw. #3115) c) Consumable materials: adapt according to corresponding study/biobank conical and sterile tubes with caps, e.g. PP (Greiner BioOne, # or #188271; Note: not suitable for storage by -80 C) sterile, filtered pipette tips, ul (e.g. MBP ART, # ) sterile, disposable Pasteur pipettes (e.g. Sarstedt, # ) round bottom tubes (e.g. Sarstedt, #60.610, 92x15,3mm) -> for medical laboratory (tap) water ice dry ice cryoboxes for tubes (e.g. Roth, #Y055.2) divider for cryoboxes, (e.g. for 15ml tubes, Roth, #Y042.2 or for 50ml tubes, Roth, #Y038.2)
6 Page 6 of 12 d) Aliquots Serum / Plasma: 1.4 ml Micronic screw cap tubes with caps (Integra Biosciences, Tubes: #MP32062; cap: #MP53104) or similar cryobox, 133x133x50 mm (e.g. Kat. Nr , VWR International GmbH) grid for cryobox, 10x10, height 25 mm (e.g. Kat. Nr ; VWR International GmbH) Required documents: Adapt according to corresponding study/biobank Operating Procedure: manual sample processing Adapt according to corresponding study/biobank All times which arise during processing and transport of the samples have to be annotated in the sample documentation protocol (BPP) SerumMonovettes: Flow chart see attachment 1 1. Store filled serum monovettes after blood extraction in upright position for min at RT (BPP). 2. Centrifugation at 2000 x g for 15 min,15 C (BPP). 3. Weigh an empty, labeled tube and tare the scale (TARE). 4. Take up the serum with a sterile transferpipette within min after blood taking; supernatants should be completely taken up with the transferpipette, but without contact to the phase. Then collect the supernatant in the weighed, labeled tube (BPP). Close filled tube tightly. 5. Immediately place the filled tube in a tube-holder placed in ice water for cooling (BPP). 6. Dry the filled and labeled tube with a tissue and weigh the serum inside. Note the weight in the bio-sample protocol (BPP). 7. If immediate laboratory analysis is desired, transfer 1 ml of the serum in a labeled round bottom tube for analysis in the medical laboratory. Storage of the tube until dispatching at 4 C. 8. Transfer supernatant x 1.0 ml in labeled 1.4 ml micronic screw cap tubes or similar and freeze the filled tubes on dry ice. Local storage of all aliquots at the end of the day at -80 C in corresponding cryoboxes. 9. Note only completely filled aliquots in the BPP and storage of rest in tube at -20 C (if residual volume remains) (BPP).
7 Page 7 of 12 EDTA-blood: flow chart see attachment 2 1. Place EDTA monovettes after blood extraction between 30 sec to 5 min at a swivel shaker (BPP). 2. If applicable, processing for: a) Medical laboratory: No further processing necessary Storage at 4 C (refrigerator) until transportation (also at 4 C; use of cold packs ( - 20 C), but without contacting the tube) to medical laboratory for haematological examination, which should take place within a maximum of 6h after blood extraction (BPP) b) storage: Centrifugation at 2000 x g, 15 min, 15 C (BPP) Weigh an empty, labeled tube and tare the scale (TARE) Take the maximal volume of the plasma with a sterile disposable Pasteur pipette without contact of the buffy coat Pool the plasma of one sample in a weighed (tared) and labeled tube. Filled tube has to be tightly closed and immediately cooled in a tube-holder, placed in a bowl filled with ice water (BPP). Dry the filled, tared and labeled tube with a tissue and weigh the plasma. Note the weight in the sample documentation protocol (BPP). Transfer supernatant x 1.0 ml in labeled 1.4 ml micronic screw cap tubes or similar, and freeze the filled tubes on dry ice; Local storing of all aliquots at the end of the day at -80 C in corresponding cryoboxes Note the obtained and completely filled aliquots in the BPP and the storage of residual volume in tube at -20 C (if a residual volume remains) (BPP). 7. Proceeding in case of discrepancies Aliquotation: If possible, all aliquots should be completely filled, if not possible please keep tubes empty and note the of aliquots in the BPP. Haemolysis of the sample: Errors might occur in blood extraction (too fast) or in processing (excessive shaking).
8 Page 8 of 12 Processing of samples: Record the chronological steps of the processing. Hold durations short, cooling of the sample should occur at 4 C. Contamination: Use sterile disposable materials; for work protection use personal protective equipment (disposable gloves, safety glasses, lab coat). Spillage of samples: Cover the spilled sample with disposable tissues and distribute surface-disinfectants on area. Please pay attention to specific application times and close area if necessary, then take up tissues (use disposable gloves), disinfect again and dispose tissues in biomedical waste. 8. Attachments
9 Page 9 of 12 Attachment 1: flow chart for serum sample processing (adapt according to corresponding study/biobank) Serum monovette e Upright storage, RT, min xg, 15min, 15 C Weighing of empty tube (TARE) Transfer the supernatant max min after BE, pooling of one sample in labeled and tared tube, close tube with cap 4 Storage of sample rest at -20 C 12 optional Note of weight / volume in BPP 7 Weighing of filled tube 6 Cooling of the tube in ice water 5 optional STOP 8 Transfer 1.0 ml from sample in a tube for for medical laboratory Preparing of 1.0 ml aliquots (micronic tubes or similar), note in BPP 10 9 Transport to hospital for laboratory analysis at 4 C Freezing of tubes on dry ice, local storage at -80 C 11 STOP
10 Page 10 of 12 Attachment 2: flow chart for plasma sample processing (adapt according to corresponding study/biobank) EDTA monovette swivel shaker, RT, 30sec - 5 min xg, 15min, 15 C Weighing and labeling of empty tube (TARE) 5 transfer of SN in weighed and labeled tube, pool of one sample, close with cap 6 Cooling the tube in iced water 7 optional swivel shaker, RT, 30sec - 5 min 1 10 Preparing of 1.0 ml aliquots (micronic tubes or similar), annotation in BPP Note of weight / volume in BPP 9 Weighing of filled tube 8 optional Transport at 4 C to medical laboratory max. 6h after BE Freezing of the tubes on dry ice,local storage at - Storage of rest of 80 C sample in tube at -20 C STOP STOP
11 Page 11 of 12 Table 1: SOP instruction to staff Name of staff-member Date of instruction Instructed and understood (signature)
12 Page 12 of 12 Table 2 : Sample documentation protocol blood processing (BPP) Serum, sample # Procedure Step (flow chart) Number of aliquots: n= time (from-to) [min:sec] volume [ml] EDTA, sample # Procedure Step (flow chart) Number of aliquots: n= Time (from-to) [min:sec] Volume [ml]
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