Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Informed patient consent and full compliance with relevant national ethics requirements in each jurisdiction e.g., the Human Tissues Act (HTA – United Kingdom, 2004) and the Health Insurance Portability and Accountability Act (HIPAA – United States, 1996) are mandatory. Be certain to have fully documented ethics approval before beginning any work on human-derived materials. Blood used in the optimization of this protocol was provided with appropriate consent from the Volunteers Advancing Medicine Panel (VAMP) (ethics reference 16/EE/0459, study CRF494, sub study 001) run by the NIHR Cambridge Clinical Research Facility, Cambridge, United Kingdom, under a Human Biological Samples supply agreement with the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre. The AstraZeneca Biobank in the UK is licensed by the Human Tissue Authority (License No. 12109) and has National Research Ethics Service Committee (NREC) Approval as a Research Tissue Bank (RTB) (REC No 17/NW/0207).

1. General preparation guidance

NOTE: All work with unfixed human material such as the blood, plasma and PBMCs in this protocol must operate under the assumption that these materials may carry potentially infectious agents and so must be performed under suitable biosafety precautions. For patients that have been tested as negative for known pathogens, do not assume samples are non-infectious and so suitable safety precautions must still be applied. Waste products generated from these materials should be treated with the same biosafety precautions and disposed according to local rules.

1. Choose between the two types of mononuclear cell preparation tubes that utilize either sodium citrate or sodium heparin as anticoagulants. For many downstream applications these two anticoagulants can be used interchangeably but both anti-coagulants should be tested before selecting one for the trial.
2. Label one 8 mL mononuclear cell preparation tube to be used for the blood collection with the patient’s coded ID. Keep the tubes at room temperature (18-25 °C).
3. Store 1x PBS at room temperature (18-25 °C). Use 30 mL per preparation.
4. Make sure tubes for separate plasma samples and the cryovial for the PBMC sample are accurately labeled with unique identifiers, as specified in the appropriate section of the lab manual.
5. If PBMCs are going to be used to monitor phosphorylated proteins, prepare PBS supplemented with phosphatase inhibitors: mix 5 mL of PBS with 50 µL of phosphatase inhibitor cocktail 2 + 50 µL of phosphatase inhibitor cocktail 3. Prepare fresh and keep on ice until use.
6. If PBMCs are going to be cryopreserved, prepare 1 mL freezing mixture per sample by mixing 90% FBS + 10% DMSO.

2. PBMC collection (Figure 1A)

1. Draw 8 mL blood into the mononuclear cell preparation tube using the standard technique described by the manufacturer. Invert the tube gently 8 to 10 times to mix the anticoagulant additive with blood. Do not shake to avoid hemolysis. Record the time at which the blood was drawn.
2. After collection, store the tube upright at room temperature until centrifugation. Process the samples as soon as possible, ideally within one hour of this blood collection but not later than 4 h post collection. Record the timing when starting the processing of the blood.
3. Immediately prior to the centrifugation remix the blood sample by gently inverting the tube 8 to 10 more times.
4. Centrifuge the tube/blood sample tubes in a horizontal rotor (swing-out head) at 1,500 – 1,800 x g for 30 min at room temperature (18-25 °C). Ensure that all the tubes are balanced properly.

Figure 1: General overview of the protocol and representative images of the centrifuge. (A) Schematic overview of the protocol for the preparation of PBMCs and plasma. *If there are time constraints, step 2.4 can be shortened to 20 min and steps 3.3 to 3.6 can be removed. ** Take an aliquot to count cells, if required. *** 2 extra centrifugation steps required for ctDNA analysis/metabolomics (B) image of a swing-out head rotor centrifuge set for step 2.4. (C) Image of the rotor, which includes two buckets containing the adaptors to spin mononuclear cell preparation tubes. Please click here to view a larger version of this figure.

NOTE: x g (also referred to as RCF, relative centrifugal units) and RPM (revolutions per minute) are different units. Be sure to set the centrifuge for x g (RCF). Use the online calculator (see Table of Materials) for the conversion. If only fixed-angle rotor is available, perform this step for 10 min at 1,500 – 1,800 x g.

5. After centrifugation, check for the presence of a dark red layer under the barrier (containing mostly red blood cells), and 2 layers above the barrier. The top layer is the plasma (straw-colored) and the whitish layer underneath is the buffy coat containing the PBMCs. These layers can be easily distinguished when viewing the tube against a dark/black background.

Figure 2: Tubes to isolate PBMCs. (A) Image of the tubes before centrifugation (step 2.4), either empty (left) or containing blood (right). (B) Successful separation of the PBMC layer after centrifugation (step 2.5). (C) Image of the PBMC layer after centrifugation and a bit of plasma left to ensure all PBMCs are collected (step 2.7). Please click here to view a larger version of this figure.

NOTE: If these layers are not visible, this probably indicates an error in setting up the centrifugation units. Ensure the right centrifuge adapter is used and the correct “x g” are set. Repeat step 2.4.

6. Immediately following centrifugation, use a serological pipette to transfer approximately half of the plasma into a labeled 15 mL size conical centrifuge tube with cap, while being careful to not disturb the PBMC layer (approximately 4-5 mL). Temporarily store this tube with plasma on wet ice to be used later in the step 4. Set aside for now.
7. Collect the entire PBMC layer with a Pasteur pipette by placing the pipette within the layer of cells and transfer to a different 15 mL size conical centrifuge tube with cap. It is acceptable to also take a small amount of plasma, if necessary, to completely get all the PBMC layer. The volume is usually 1-2 mL. Immediately continue with the step 3 below.

3. PBMC washing steps

NOTE: The purpose of the wash steps is to dilute out and remove residual platelets and plasma from the PBMC pellet. All centrifugation steps should be performed at room temperature (18-25 °C).

1. Add room temperature PBS to the PBMC tube to bring the volume to 15 mL. Cap the tube. Mix cells by gently inverting the tube 5 times.
2. Centrifuge for 15 min at 300 x g. Note that this is a much gentler centrifugation than the initial centrifugation.
3. Visualize the pellet by viewing the tube against a dark/black background. Remove the supernatant by vacuum aspiration or with a pipette (Figure 3A). Discard the supernatant leaving a volume of approximately 500 μL above the whitish colored PBMC pellet.

Figure 3: PBMC pellets. (A) Pellet obtained in the first wash step 3.3. (B) Pellet isolated in the second wash (step 3.7). (C) Pellet with high level of hemolysis obtained from blood processed later than 4 h from the blood draw isolated in the second wash (step 3.6). Please click here to view a larger version of this figure.

4. Resuspend the cell pellet by gently pipetting up and down in the residual supernatant.
5. Add additional 1x PBS to bring the volume to 10 mL. Cap the tube. Mix the cells by inverting the tube gently 5x. If cell counting is required in the study protocol go to step 3.5.1, if not required move directly to step 3.6.
   1. Take a 40 μL aliquot of the re-suspended cells and mix with 40 μL of trypan blue. Count the viable cells using a hemocytometer or an automatic cell counter.
6. Centrifuge the suspension from step 3.5 for 10 min at 300 x g. Remove as much supernatant as is reasonably possible without disturbing the cell pellet.
7. Carefully remove and discard any remaining supernatant above the cell pellet by pipetting using a fine tip Pasteur pipette or a micropipette without disturbing the cell pellet. Leave little to no liquid above the pellet after completing this step. If the endpoint of this protocol is a PBMC pellet move to step 3.8; if the endpoint is cryopreserved PBMCs go to step 3.10.
8. Add 50 µL of new PBS (or the supplemented PBS prepared in step 1.5 if applicable to your endpoint) to the pellet and pipette up and down gently using a micropipette to homogenously resuspend all the cells. Transfer the entire cell suspension to a labeled 1.5 mL cryovial and immediately place on wet ice until freezing the samples.
9. Freeze the labeled samples by placing them in liquid nitrogen or directly in dry ice. Cells can also be frozen at -80 °C freezer using cell freezing boxes. In this case, prechill the boxes completely in -80 °C before adding the cell tubes. Record the time at which cell pellets were frozen. Once cells freeze, store at -80 °C, ship frozen on dry ice.
10. Optionally, to cryopreserve the PBMCs re-suspend the pellet in 1 mL of the freezing mixture (step 1.6) and transfer to a labeled cryovial. Continue as described in step 3.9, but, in this case, it is only acceptable to use cell freezing boxes to freeze down the samples. Transfer to liquid nitrogen for shipping and storage after being in the freezing box for a minimum of 24 h.

4. Plasma preparation steps (Figure 1A)

1. Following -80 °C storage of the PBMC pellets, now prepare the plasma aliquot that was temporarily stored on wet ice in step 2.6. Perform the following centrifugation steps to clarify the plasma if the purpose of the sample is ctDNA analysis, otherwise move directly to step 4.2.
   1. Centrifuge the plasma for 10 min at 1,600 – 2,000 x g at 4 – 8 °C in a fixed-angle rotor (or 15 min in a swing-out rotor).
   2. Carefully pipette off the plasma supernatant, taking care not to disturb any pellet and transfer to a new 15 mL tube. Discard the tube containing the pelleted material.
   3. Centrifuge the plasma for 10 min at 1,600 – 2,000 x g at 4 – 8 °C in a fixed-angle rotor (or 15 min in a swing-out rotor).
   4. Carefully transfer the plasma supernatant to a new 15 mL tube, being sure not to disturb any pelleted material. Discard the tube containing the pelleted material.
      NOTE: If a refrigerated centrifuge is not available, keep cells on ice for 5 min between each spin, or centrifuge samples in a cold room.
2. Transfer plasma in 1 mL aliquots into 5 fresh 2 mL microtubes. Use fewer than 5 vials if there is not enough plasma to fill 5 vials with 1 mL aliquots and it is expected that the last vial will contain less than 1 mL plasma. If there is more than 5 mL plasma, discard the remainder. Record the total volume of plasma in each tube.
3. Immediately freeze the plasma aliquots upright, by storing them at -80 °C.

5. Sample shipment

1. Ship both PBMC pellets and plasma samples on dry ice.
2. Ensure that the sample is not thawed before and during shipment. Pack sufficient dry ice with the samples to ensure they remain frozen for the entirety of the shipping process, considering possible delays that may occur in transit.

6. Whole blood irradiation and western blot analysis of DDR biomarkers (Figure 4A)

NOTE: This is an ex vivo treatment that will not be required in most cases in the clinic, but these experiments are a valuable exploratory strategy to find suitable clinical biomarkers. Blood samples should be treated as soon as possible upon collection to ensure best results.

1. Warm up the X-ray cabinet. Label three 15 mL tubes as 0, 0.2 and 7 Gy, respectively.
2. Take three mononuclear cell preparation tubes containing freshly drawn blood from a single individual and after gently inverting the tubes 8 to 10 times transfer the bloods to the three 15 mL tubes.
3. Place the 0.2 Gy tube in the X-ray cabinet, close the door and apply 0.2 Gy dose to the tube by selecting the shelf number and dose. Apply 7 Gy radiation dose to the 7 Gy tube by adjusting the dose selected in the cabinet.
4. Incubate the three tubes at 37 °C for one hour.
5. Transfer the blood back to the mononuclear cell preparation tubes and carry out the PBMC preparation protocol as for a clinical setting from step 2.4 to step 3.8.
6. Add a volume of lysis buffer supplemented with protease and phosphatase inhibitors equal to the cell pellet volume (in this case 70 μL of RIPA buffer) to each of the cell pellets, pipette up and down and incubate on ice for 10 min. Sonicate the samples if a sonicator is available (3 cycles, 30 s ON / 30 s OFF, 4 °C), alternatively syringe the samples to break the nucleic acids to eliminate viscosity in the sample.
7. Centrifuge the samples for 10 min at ≥ 15,000 x g, at 4 °C. Transfer each supernatant to a new 1.5 mL tube, qualitatively assess the level of hemolysis (visual inspection) and measure the protein concentration by any preferred method.
8. Mix 40 μg of total lysate with sample loading buffer containing SDS and sample reducing agent. Boil samples for 5 min in a heat block.
9. Load 20 μg of each sample per lane in duplicate in a 4-12% bis-tris protein gel and run an SDS-PAGE.
10. After separation, transfer the proteins to a nitrocellulose membrane using a commercially available system (20 V, 10 min) and block with 5 % milk in TBST.
11. Cut the membrane at the relevant molecular sizes and incubate with the primary antibodies overnight at 4 °C (see Table of Materials for antibodies and dilutions).
12. Remove the primary antibodies and wash the membranes 3 times with TBST for 5 minutes at room temperature. Incubate with the HRP-conjugated secondary antibodies for 45 minutes at room temperature.
13. Wash 3 times with TBST for 5 min.
14. Apply the ECL reagent and analyze the images obtained relative to HRP signal.