Blood sample collection and processing
All experimental protocols were reviewed and approved by the Oregon Health & Science University Institutional Review Board. All methods were carried out in accordance with relevant guidelines and regulations. Blood samples from healthy individuals were obtained from the Cancer Early Detection Advanced Research center (CEDAR) at Oregon Health and Science University. All samples were collected under institutional review board (IRB) approved protocols with informed consent from all participants for research use. Whole blood was collected from healthy individuals in 10 ml in K2EDTA tubes (BD Vacutainer, Becton Dickinson, catalog number: 36643), 4.5 ml in 3.2% Sodium Citrate (NaCit) tubes (BD Vacutainer, Becton Dickinson, catalog number: 369714), and 10 ml in sodium heparin tubes (BD Vacutainer, Becton Dickinson, catalog number: 367874) via antecubital vein puncture using a 21G butterfly needle (BD Vacutainer, Becton Dickinson, catalog number: 367281). For NaCit/ACD tubes, we first made acid-citrate-dextrose (ACD) by adding 12.5 g of sodium citrate (Sigma-Aldrich, catalog number: S4641), 10 g of D-glucose (Sigma-Aldrich, catalog number: G7528), and 7.5 g of citric acid (Sigma-Aldrich, catalog number: C0706) in 500 ml of distilled water. 500 ul of ACD solution was injected into each commercial 3.2% NaCit tube before the blood draw. Tubes were transported vertically at room temperature before processing. Within 1 hour of blood withdrawal, plasma was prepared by centrifugation (Eppendorf 5810-R centrifuge, S-4-104 Rotor, Eppendorf) at two different processing conditions. The highest acceleration and deceleration setting was used, setting ‘9’. 10 ml of whole blood was spun at 1,000 × g for 10 minutes at 23°C (S1). Supernatant was collected until 10 mm above the buffy coat. The second centrifugation was done at 15,000 x g for 10 minutes at 23°C in 1.5 ml microcentrifuge tubes (VWR, catalog number: 89126-714). Resulting supernatants of platelet-depleted plasma were aliquoted to new 1.5 ml microcentrifuge tubes (VWR, catalog number: 89126-714) and immediately stored at −80 °C. Age and gender status were recorded for all individuals. Unless indicated, plasma was processed at RT in EDTA.
(i) Platelet isolation from peripheral blood
For platelet marker studies, 10ml of citrated whole blood was centrifuged at 200 × g for 20 min at room temperature without any brakes. 10 µl of 10 mg/ml prostaglandin I2 (PGI2) was added to the upper two thirds of platelet-rich plasma (PRP). PRP was centrifuged at 1,000 × g for 10 min at room temperature to pellet the platelets. 1 ml of Tyrode’s buffer (TB) was added with 150 ml of acid citrate dextrose (ACD) to resuspend the platelets. To wash the platelets, extra TB was added up to a total of 25 ml followed by 3 ml of ACD and 10 µl of PGI2. The platelet suspension was centrifuged at 1,000 × g for 10 min and resuspended in 1 ml of TB. The washed platelets were counted using a haemocytometer and resuspended to final concentration of 109 platelets/ml.
(ii) Platelet activation to induce platelet-derived EV formation
For platelet derived EV studies, whole blood collected in EDTA was centrifuged at 200 × g for 20 min without any brakes to collect platelet-rich plasma (PRP). PRP was collected and centrifuged again at 1,000 × g for 10 min at RT to pellet resting platelets. The platelet pellet was resuspended in Tyrode’s buffer (TB) pre-warmed to 37°C and centrifuged at 200 × g for 1 min to remove any residual red blood cells or platelet aggregates. Platelet suspension was incubated at RT, frozen at -80°C, or treated with 13 mM ionomycin in 8 mM CaCl2 for 10 min. After treatment, the platelet suspension was centrifuged at 1,000 × g for 10 min to pellet any residual platelets, and the supernatant was collected for platelet-derived EV studies. For TEM studies, the platelet suspension was recentrifuged at 1,000 × g for 10 min. Isolated platelets were kept at either RT or -80°C for 10 min and resuspended before imaging.
For counting platelets in differentially processed plasma, blood samples from three healthy individuals were obtained in 10 ml K2EDTA tubes (BD Vacutainer, Becton Dickinson, catalog number: 36643). Plasma was processed using single spin at 1,000 x g (S1) and double spin at 15,000 x g (S2) both at either room temperature or at 4°C to compare with the standard platelet-rich plasma (PRP) processing protocol at 200 x g for 20 min at room temperature. The platelet count was measured by the improved Neubauer haemocytometer (VWR Scientific Products, Piscataway, NJ) by two independent, experienced researchers. The total number of platelets were counted from central 1 x 1 mm area consisting of 25 groups of 16 squares separated by closely ruled triple lines, equivalent to a volume of 0.1 μl.
Fluorescent antibody labeling of whole blood, isolated platelets, and EVs for flow cytometry
To fluorescently label myeloid cells, leukocytes, and platelets, 100 ml of whole blood was stained with 2 ml of each antibody: Brilliant Violet-650 conjugated anti-CD45 (clone HI30, Biolegend Catalog number 304042), FITC-conjugated anti-CD41a (Biolegend, catalog number 303730), Phycoerythrin (PE)-conjugated anti-CD9 (Biolegend, catalog number 312106), AlexaFluor 488-conjugated anti-CD63 (Thermo Fisher Scientific, clone MEM-259, catalog number MA5-18149), and PE-conjugated anti-CD81 (Thermo Fisher Scientific, clone M38, catalog number A15781). Prior to flow cytometric analysis, red blood cells (RBCs) were lysed by the addition of 1 ml of 1X RBC lysis buffer (Invitrogen, catalog number 00433357). To fluorescently label purified platelets, 40 ml of resuspended isolated platelets were stained with 2 ml of each respective antibody and incubated with 50 ml of TB for 15 min at room temperature on a 200 rpm shaker. For thrombin activation, bovine thrombin was added to a final concentration of 0.1 U/ml. After incubation, platelets were fixed by adding 100ml of 2% Paraformaldehyde (PFA) for 10 minutes. Fixed platelets were diluted with 300ml of D-PBS prior to flow cytometric analysis.
To fluorescently label EV surface proteins, 5 µl of plasma, isolated extracellular vesicles and buffer were incubated with 5µl of antibody mix prepared after established dilution series. CD41a BV421 (Biolegend, clone: HIP8, catalog number: 303730, concentration: 0.1mg/ml) was diluted to a final concentration of 0.0005 mg/ml - 0.001 mg/ml for staining. CD81 PE (Thermofisher scientific, clone: M38, catalog number: A15781, concentration: 0.3 mg/ml) was diluted to a final concentration of 0.012 mg/ml for staining. CD63 alexa fluorophore 488 (Thermofisher scientific, clone: MEM-259, catalog number: MA5-18149, concentration 0.26 mg/ml) was diluted to a final concentration of 0.0026 mg/ml for staining. CD9 alexa fluorophore 647 (Thermofisher scientific, clone: #209306, cat. number: FAB1880R-100μg) was diluted to a final concentration of 0.001 to 0.002 mg/ml for staining. Incubation was done for 3 hours at room temperature in the dark. Prior to flow cytometry, the stained EV samples were further diluted with 0.1 mm filtered D-PBS without calcium and magnesium (Thermo Fisher Scientific, catalog number: 14190250) to limit the “swarming” effect using an abort rate of < 5% and keeping the threshold rate below 20,000 events per second. A buffer-only control of 0.1 µm-filtered DPBS without calcium and magnesium (Thermo Fisher Scientific, catalog number: 14190250) was recorded at the same flow cytometer acquisition settings as all other samples, including triggering threshold, voltages, and flow rate. The buffer-only control had a count of <500 events per second.
Flow cytometry set-up for platelets, microvesicles, and exosomes
For whole blood and isolated platelet marker studies, a Becton-Dickinson FACSymphony equipped with 200 mW 488 nm, 561 nm, 405 nm and 640 nm lasers was used. For each sample, the SSC trigger threshold value was set to 200. The sample flow rate was set at low, approximately 12 ml/minute, and the data was collected for 60 seconds. For submicron size detection of smaller platelets, microvesicles, and exosome-sized nanoparticles, Beckton-Dickinson FACSAria Fusion equipped with 488 nm (60 mW), 561 nm (100 mW), 405 nm (100 mW) and 640nm (100 mW) lasers was used. For optimal configuration of submicron size detection, 0.1 µm size filter was applied to the sheath fluidic system to reduce sheath fluid noise. Data collection was set using the SSC trigger threshold value of 200 and analyzed using a log scale SSC-A to adjust for the geometric increase in light scatter related to particle diameter and refractive index. Voltage was set based on aligning 200 nm Megamix beads at SSC intensity of 104. All the fluorescent voltages were set by aligning fluorescent intensity from buffer alone control to be below 2 x 102 for each channel. The sample flow rate was set at 1, which is approximately 10 ml/minute. Timed collections were recorded for 60 seconds in repeated experiments with high reproducibility within and between BD flow cytometers (FACSAria and FACSymphony). To account for the electronic abort rate due to nanoparticle coincidence within nano-droplets being imaged (also known as “swarming”), stained samples were diluted to retain the threshold event rates below 20,000 events per second with less than a 5% abort rate and validated via consistent median fluorescent intensity across plasma dilutions (Supplementary Fig. 3A). Megamix-plus FSC and SSC (BioCytex, catalog number 7802 and 7803) polystyrene calibration beads (100, 160, 200, 240, 300, 500, and 900 nm) conjugated to green fluorescence were measured using the 488 nm laser. All experiments were standardized to the 200 nm bead SSC-A and 900 nm bead 488 nm fluorescence (similar to using MESF beads). SSC signal intensity for the 200 nm beads was set at 104 for every experiment. This ensured comparable fluorescence and light scatter measurements between experiments. We analyzed plasma stained with CD41a, CD63, CD81, and CD9 markers using FSC/SSC/FL dot plots. All measurements were analyzed using FlowJo software in bi-exponential scale for forward, side-scatter, and fluorescence intensities. Events whose SSC intensity was less than 104 were labelled as exosome-sized (EXO) populations. Events whose SSC intensity was greater than 104 were back-gated onto SSC/FSC dot plot, further differentiating into FSC+ and FSC- subsets defined as platelet (PLT/MV) and microvesicles (MV), respectively.
Transmission electron microscopy
Ultrathin carbon film on lacey carbon support with 400 mesh on copper (Ted Pella, catalog number: 01824) was glow discharged for 30 seconds using PELCO easiGlow glow discharger (Ted Pella). Isolated platelet samples (RT vs. -80°C) were put on charged grids for 1 min, washed for 30 seconds with MilliQ water, and fixed with 1% uranyl acetate for 30 seconds. Grids with stained samples were air dried at least 30 minutes before imaging. Prepared samples were imaged at 120 kV using FEI Tecnai™ Spirit TEM system. FEI- Tecnai™ Spirit TEM system was interfaced to a bottom mounted Eagle™ 2K TEM CCD multiscan camera and to a NanoSprint12S-B cMOS camera from Advanced Microscopy Techniques (AMT) fast side mounted TEM CCD Camera. Images were collected at 8,000-80,000x magnification under 1-2μm defocus. Regions shown in Figure 3 were representative of each sample, which were uniformly observed from larger fields of view across the grids. Images were acquired as 2048 × 2048 pixel, 16-bit gray scale files using the FEI’s TEM Imaging & Analysis (TIA) interface on an Eagle™ 2K CCD multiscan camera.
RT-qPCR profiling of cell free mRNA
For characterizing the effect of freeze thaw on cell free mRNA expressions, RNA was extracted using plasma processed with S1, S2, S1FR, S2FR, and S1FRS2 conditions. Cell free mRNA was isolated by using plasma/serum circulating and exosomal RNA purification Kit (Norgen Biotek) followed by 10X Baseline-ZERO DNase treatment (Epicentre). DNase treated RNA samples were purified and further concentrated using RNA clean and concentrator (Zymo Research). The purified RNA samples were assayed by RT-qPCR using 16 primers (MTND2, PPBP, B2M, PF4, ACTB, CORO1C, GSE1, GAPDH, SMC4, HBG1, NUSAP1, MIKI67, FGB, APOE, FGG, and ALB). Template RNA was mixed with Superscript III One-step RT-PCR system with Platinum Taq DNA polymerase (Invitrogen) to generate cDNA according to the protocol. PCR amplification products were treated with Exonuclease I (New England Biolabs) to digest single stranded primers at 37°C for 30 min followed by inactivation of enzymes at 80°C for 15 min. For RT-qPCR, cDNA from preamplification was diluted 1:80 and set-up in 96-well plates with SsoFast EvaGreen supermix with low ROX (BioRad) with above primers at 10 M. QuantStudio 7 Flex (Applied Biosystems) was used to run RT-qPCR assay according to manufacturer’s recommended cycling conditions.
To determine the impact of overall preanalytical factors including spin, temperature, and types of anticoagulant tubes, statistical analysis was performed on marker specific events from the flow cytometry data. The significance of individual preanalytical factor comparisons were determined using pair-wise Wilcoxon rank sum test. To compare average platelet-derived EV counts associated with ionomycin or temperature (-80°C) induced platelet activation, pair-wise Wilcoxon rank sum test was performed on marker specific events from flow cytometry analysis. To determine the effect of freeze thaw on extracellular vesicles, flow cytometric counts of exosomes per different stained surface markers (CD41a+, CD63+, and CD41a+/CD63+) were normalized across pre/post-thaw processing conditions. To determine the significance of freeze-thaw effects in pre/post-thaw processing conditions, Tukey’s multiple comparison test was performed on normalized counts. Analyses were conducted using GraphPad Prism and R package.