Processing of blood specimens

Abstract


The protocol is derived from the WHO/IARC guideline “Common Minimal Technical Standards and Protocols”. The purpose of the document is to provide a framework for the development and coordination of Biological Resource Centers.
If serum and plasma are being collected, it is important to prioritize the separation of them so they can be frozen as soon as possible. This is critical for time sensitive samples for protein studies for example.

Reagents

  • Reagents and chemicals
  • EtOH , 70%
  • Liquid Nitrogen
  • DMSO
  • Tris-EDTA buffer

Equipment

  • Filter paper
  • Vacutainer , of type EDTA/ACD
  • Filter papers
  • Plastic pasteur pipette
  • sterile 15ml centrifuge tubes
  • sterile 50ml centrifuge tubes
  • Cryovial
  • Tris-EDTA buffer
  • Equipment
  • Pipette tips
  • Paper envelope
  • Pipette
  • Class II Biological Safety Cabinet
  • Centrifuge

Instructions

    Filter papers
    1. Always handle filter papers wearing gloves and only by the upper corner, marked out for labeling. Do not allow the card to come into contact with any unclean surface e.g. bench, base of hood. Use EDTA / ACD tubes to produce filter papers
    2. Mix anticoagulated blood containing vacutainers by inversion before starting.
    3. Wipe top of vacutainers with 70% ethanol before opening the lid.
    4. Make 2 filter papers by placing 40μl of blood in the circle using a micropipette.
    5. Dry air the filter papers thoroughly in the back of the Class II Biological Safety Cabinet.
    6. Store filter papers in a paper envelope (not plastic) at room temperature.
    Blood pellets (white cells)
    1. Blood pellets can be used for the isolation of DNA (from EDTA / ACD tubes)
    2. Transfer blood from the original tube to a labeled 50ml tube.
    3. Fill tube with Tris-EDTA buffer (formula) and mix vigorously. Place on ice for 5 to 10 minutes.
    4. Spin at 1200xg for 10 minutes.
    5. Carefully pour off supernatant into a beaker containing chlorine bleach. Briefly vortex the pellet and add 50ml Tris-EDTA buffer. Shake vigorously.
    6. If division of the sample is necessary, at this point pour 25ml of sample into another falcon tube.
    7. Spin both tubes at 1200xg for 10 minutes.
    8. Repeat washing if red cells persist.
    9. Carefully pour off supernatant.
    10. Using a swirling motion, remove the pellet with a pipette and transfer to labeled cryovial.
    11. Store in -80°C or liquid nitrogen until further use. As an alternative red cells can be lysed by using an ammonium-containing lysis buffer.
    Platelet Poor Plasma
    1. Platelet-poor plasma can be used for the isolation of plasma DNA (from EDTA tubes)
    2. Spin blood at 3200xg for 12 minutes at room temperature.
    3. Pipette off plasma using a plastic pasteur pipette. Transfer into tube.
    4. Spin plasma at 2000xg for 10 minutes at 4°C.
    5. Aliquot into 1ml aliquots in labelled cryovials.
    6. Store at -80°C.
    Buffy coat cells
    1. The buffy coat is a thin, greyish-white layer of white blood cells (leukocytes and lymphocytes) and platelets covering the top of the packed red blood cells after 450xg centrifugation (from EDTA / ACD containing blood tubes).
    2. After having spun the blood, take buffy coat off with about 100μl of plasma using a disposable sterile Pasteur pipette: be careful not to lift red cells.
    3. Lyse remaining red cells by addition of red cell lysis buffer at room temperature.
    4. Spin tube at 450xg for 10 minutes at room temperature
    5. Resuspend the pellet
    6. Aliquot as appropriate into labeled cryovials.
    7. Place in liquid nitrogen to snap freeze.
    8. Store in liquid nitrogen.
    Whole Blood
    1. To be prepared from EDTA tubes. The anti-coagulated blood can be snap frozen as it is. In case the blood cells are needed intact, DMSO is needed to keep them alive while freezing.
    2. Dispense 50μl DMSO into two 1ml sterile cryovials.
    3. Invert EDTA tube twice then add 450μl of blood to each cryovial.
    4. Invert cryovial to mix the whole blood with the DMSO. Note: DMSO is cytotoxic at room temperature, therefore as soon as it is mixed with blood it should be placed in a controlled rate freezer.
    5. Transfer to -80°C after at least 4 hours.
    Plasma
    1. Plasma collected in EDTA and ACD tubes, can be used for bioassays, plasma DNA isolation, proteomic analysis, and biomarker discovery.
    2. If serum and plasma are being collected, it is important to prioritize the separation of them so they can be frozen as soon as possible. This is critical for time sensitive samples for protein studies for example.
    3. Separate plasma from blood cells
    4. Spin vacutainer (about 9ml) at 815xg for 10 minutes at 4°C to separate plasma from blood cells.
    5. Extract plasma
    6. After wiping each tube with 70% alcohol, remove about 3ml plasma.
    7. Tube can be retained for white blood cell extraction.
    8. Centrifuge again
    9. Transfer to a labeled 15ml tube and centrifuge at 2500xg for 10 minutes at 4°C.
    10. The purpose of double spinning the plasma is to remove all cellular contaminants so that the plasma is suitable for plasma DNA analysis. It is extremely important, therefore, not to disturb the buffy coat after the first spin, and any pellet after the second spin.
    11. Aliquot into cryovials
    12. Aliquot plasma into 1ml labeled cryovials (3 to 4 aliquots).
    13. Snap Freeze
    14. Place in liquid nitrogen Dewar to snap freeze.
    15. Centrifuge again
    16. Transfer to a labeled 15ml tube and centrifuge at 2500xg for 10 minutes at 4°C.
    17. Store cold
    18. Store at -80°C or in liquid nitrogen.
    Serum
    1. The protocol describes the collection of blood without added anticoagulants.
    2. The blood is collected into tubes without addition of anticoagulants. Then two phases are originating, a solid phase, containing fibrin and cells, and a fluid phase, containing the serum.
    3. This process should be completed after 30 minutes at room temperature, and then the process described below starts.
    4. Centrifuge blood
    5. Spin blood in 15ml sterile tubes at 1500xg for 10 minutes at room temperature.
    6. Aliquot into cryovials
    7. Aliquot 1ml portions of supernatant into labeled cryovials.
    8. Snap freeze
    9. Place ryovials into liquid nitrogen Dewar or dry ice to snap freeze.
    10. Store cold
    11. Transfer to -80°C freezer or liquid nitrogen storage.
    White Blood Cells
    1. White blood cells collected in EDTA and ACD tubes, can be used for DNA extraction and the creation of cell lines.
    2. Instead of a separation based on Ficoll, a Percoll separation can be used alternatively.
    Transfer of blood
    1. Transfer the remaining blood from the plasma spin to a labeled 50ml tube containing 10ml RPMI 1640. After alcohol swabbing the lid of this tube, aliquot 3ml Ficoll into each of two clearly labelled 15ml tubes. Carefully layer 9ml diluted blood onto each tube of Ficoll. Treat gently, do not mix, but spin as soon as possible.
    Preparation for storage
    1. Pour off the supernatant into a waste container containing chlorine bleach. Add 3ml of cold freezing mix (10% DMSO, 20% FCS, RPMI 1640) and resuspend.
    2. Dispense the white blood cells into 3 x 1ml labeled cryovials, which have been sitting on ice. Place vials in a rate-limiting freezer as to cryopreserve cells in conditions that maintain cell viability. This should be done as soon as possible as DMSO is toxic at room temperature.
    3. INFO: Transfer on a weekly basis to liquid nitrogen tanks.
    Separation of sample
    1. Spin at 450xg for 30 minutes. When centrifuging, do not use brake.
    2. Remove most of the top layer (RPMI 1640) using a 1ml Eppendorf tip and discard (≈3-4ml) into waste container containing chlorine bleach. Collect white blood cells with the same Eppendorf tip using a swirling motion to ‘vacuum up’ white blood cells.
    3. Do not take too much Ficoll (third layer), as it is toxic to the cells. Place the white blood cells in a labeled 15ml tube containing 10ml RPMI.
    4. Spin at 450xg for 10 minutes.
    Plasma
    1. Plasma collected in EDTA and ACD tubes, can be used for bioassays, plasma DNA isolation, proteomic analysis, and biomarker discovery.
    2. Spin vacutainer (about 9ml) at 815xg for 10 minutes at 4°C to separate plasma from blood cells.
    3. After wiping each tube with 70% alcohol, remove about 3ml plasma. Tube can be retained for white blood cell extraction.
    4. Transfer to a labeled 15ml tube and centrifuge at 2500xg for 10 minutes at 4°C.
    5. Aliquot plasma into 1ml labeled cryovials (3 to 4 aliquots).
    6. Place in liquid nitrogen Dewar to snap freeze.
    7. Store at -80°C or in liquid nitrogen.
    8. The purpose of double spinning the plasma is to remove all cellular contaminants so that the plasma is suitable for plasma DNA analysis. It is extremely important, therefore, not to disturb the buffy coat after the first spin, and any pellet after the second spin.
    Serum
    1. The blood is collected into tubes without addition of anticoagulants. Then two phases are originating, a solid phase, containing fibrin and cells, and a fluid phase, containing the serum. This process should be completed after 30 minutes at room temperature, and then the process described below starts.
    2. Spin blood at 1500xg for 10 minutes at room temperature.
    3. Aliquot 1ml portions of supernatant into labeled cryovials.
    4. Place into liquid nitrogen Dewar or dry ice to snap freeze.
    5. Transfer to -80°C freezer or liquid nitrogen.
    White Blood Cells
    1. White blood cells collected in EDTA and ACD tubes, can be used for DNA extraction and the creation of cell lines.
    2. Transfer the remaining blood from the plasma spin to a labeled 50ml tube containing 10ml RPMI 1640.
    3. After alcohol swabbing the lid of this tube, aliquot 3ml Ficoll into each of two clearly labelled 15ml tubes.
    4. Carefully layer 9ml diluted blood onto each tube of Ficoll. Treat gently, do not mix, but spin as soon as possible.
    5. Spin at 450xg for 30 minutes. Note: when centrifuging, do not use brake.
    6. Remove most of the top layer (RPMI 1640) using a 1ml Eppendorf tip and discard (≈3-4ml) into waste container containing chlorine bleach.
    7. Collect white blood cells with the same Eppendorf tip using a swirling motion to ‘vacuum up’ white blood cells. Do not take too much Ficoll (third layer), as it is toxic to the cells. Place the white blood cells in a labeled 15ml tube containing 10ml RPMI.
    8. Spin at 450xg for 10 minutes.
    9. Pour off the supernatant into a waste container containing chlorine bleach. Add 3ml of cold freezing mix (10% DMSO, 20% FCS, RPMI 1640) and resuspend.
    10. Dispense the white blood cells into 3 x 1ml labeled cryovials, which have been sitting on ice.
    11. Place on ice. Place vials in a rate-limiting freezer as to cryopreserve cells in conditions that maintain cell viability. This should be done as soon as possible as DMSO is toxic at room temperature.
    12. Transfer on a weekly basis to liquid nitrogen tanks. Instead of a separation based on Ficoll, a Percoll separation can be used alternatively.

    Citation:
    IARC - International agency for research on cancer. Processing of blood specimens. The Molecular Methods database. Fri, 03/08/2013 - 10:10. Acc.nr uugLt10.