In the Rotterdam Study, fasting blood samples were collected during the fourth study visit. Plasma was isolated and immediately put on ice and stored at −80°C. In the Amsterdam UMC cohort, serum was collected in 5 mL BD Vacutainer SST II Plus plastic serum tubes (Becton, Dickinson and Company, New Jersey, USA). Blood was centrifuged for 5 min at 1900 g at 20°C after clotting to obtain serum, which was stored in cryovials at –80°C. In both cohorts, Lp(a) was measured on a Cobas 8000 analyser using the KIV-2 number independent Randox immunoassay (Randox Laboratories, UK) with an Lp(a) concentration range of 0–187 mg/dL.15 (link)
Vacutainer sst 2 plus plastic serum tube
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The BD Vacutainer SST II Plus plastic serum tubes are primary blood collection tubes designed for the separation of serum from whole blood. They contain a gel barrier that facilitates the separation of serum from cellular components during centrifugation.
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4 protocols using vacutainer sst 2 plus plastic serum tube
1
Lp(a) Measurement in Cohort Studies
2
Hemolysis Reduction in ED Blood Samples
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This study was based on the systematic identification of hemolysis in serum samples received in the clinical laboratory of the University Hospital of Parma from the local ED, a large urban facility with approximately 92.000 visits per year. In this ED, blood samples are typically drawn using intravenous catheters (1.0 x 3.2 mm, 20-gauge catheter; Neo DELTA VEN, Viadana, MN, Italy). According to the study protocol, the investigation was divided in two observational periods of 4 months each.
In the former period (i.e., from the 1st of January to the 30th of April, 2014), blood was collected from intravenous catheters using 13x100 mm x 5.0 mL BD Vacutainer® SST II Plus plastic serum tubes (Becton Dickinson Italia S.p.A, Milan, Italy). According to the positive outcome of a previous local investigation (8 (link)), the Direction of the University Hospital of Parma authorized the complete replacement of these serum tubes with 4.0 mL S-Monovette serum tubes (S-Monovette, Sarstedt AG & Co., Nümbrecht, Germany) on May 15th, 2014. After 15 days of familiarization with the new blood collection device, the second observational period started on the 1st of June, 2014, and was concluded on the 30th of September, 2014. In this second phase of the study, blood was collected from intravenous catheters by the ED personnel always using S-Monovette blood tubes in aspiration mode.
In the former period (i.e., from the 1st of January to the 30th of April, 2014), blood was collected from intravenous catheters using 13x100 mm x 5.0 mL BD Vacutainer® SST II Plus plastic serum tubes (Becton Dickinson Italia S.p.A, Milan, Italy). According to the positive outcome of a previous local investigation (8 (link)), the Direction of the University Hospital of Parma authorized the complete replacement of these serum tubes with 4.0 mL S-Monovette serum tubes (S-Monovette, Sarstedt AG & Co., Nümbrecht, Germany) on May 15th, 2014. After 15 days of familiarization with the new blood collection device, the second observational period started on the 1st of June, 2014, and was concluded on the 30th of September, 2014. In this second phase of the study, blood was collected from intravenous catheters by the ED personnel always using S-Monovette blood tubes in aspiration mode.
3
Serum RNA Isolation Protocol
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Serum was obtained from peripheral blood samples (5-ml BD Vacutainer SST II Plus plastic serum Tube, REF 368159) by centrifugation at 4000 rpm at 4 °C for 20 min. The serum was collected and stored at -80 °C until analysis. Isolation of total RNA from serum pools was performed by a combination of Trizol LS (Invitrogen) and miRNeasy kit (Qiagen) according to the manufacturer’s instructions. RNA profiles and quantification were assessed on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and the Small RNA chip.
4
Extracellular Vesicle Isolation and Characterization
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Cells were cultured in EV-depleted medium, prepared by ultracentrifugation of 50% FBS (120,000g, 16 h) (Théry et al, 2006 (link); Soares et al, 2015 (link)). Conditioned medium was subjected to differential centrifugation at 4°C (10 min, 300g; 20 min, 16,500g). Supernatants were filtered (0.22 μm) and ultracentrifuged (70 min, 120,000g). Unless stated otherwise, WB analysis were performed in EVs obtained from 12 × 106 secreting cells, during the indicated conditioning times and treatments. Intracardiac EVs were isolated after tissue mincing in 0.9% NaCl and centrifugation (5 min, 400g; 15 min, 2,000g; and 45 min, 16,500g). Supernatants were incubated with Total Exosome Isolation Reagent (from serum; Thermo Fisher Scientific) overnight at 4°C and centrifuged (60 min, 10,000g; 70 min, 120,000g). Blood from humans and mice was collected into non-heparinized tubes (BD Vacutainer SST II Plus plastic serum tube; BD Biosciences), allowed to cloth for 30 min (room temperature) before serum retrieval by centrifugation (15 min, 1,000g). Samples were centrifuged (30 min, 2,000g) and EVs isolated with Total Exosome Isolation Reagent (from serum), according to the manufacturer. EVs were kept at −80°C until further analysis.
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