Peripheral blood samples were collected at treatment baseline in the overall cohort. A second blood drawn was performed at 12 (+/−6) weeks after cycle 1 of therapy in NSCLC patients treated with ICIs. Blood samples were harvested in sodium citrate tubes (Ref 454387, Becton Dickinson Biosciences (BD), San Jose, CA, USA,) and processed within 8 h from collection.
Sodium citrate tube
Manufactured by BD
Sourced in United States, United Kingdom
Sodium citrate tubes are laboratory equipment used for the collection and storage of blood samples. They contain a solution of sodium citrate, which acts as an anticoagulant, preventing the blood from clotting during the collection and transportation processes.
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Lab products found in correlation
Ethylene diamine tetraacetic acid (edta), by BD (3 mentions)
Facsverse, by BD (2 mentions)
Quant it picogreen dsdna reagent, by Thermo Fisher Scientific (2 mentions)
Edta tube, by BD (2 mentions)
Facsuite v 1, by BD (1 mentions)
Cytometer setup and tracking module, by BD (1 mentions)
Xe 2100 hematology analyzer, by Sysmex (1 mentions)
Fluoroskan ascent microplate fluorometer, by Thermo Fisher Scientific (1 mentions)
Cell death detection elisa kit, by Roche (1 mentions)
Human neutrophil elastase platinumelisa kit, by Thermo Fisher Scientific (1 mentions)
Enzyme linked fluorescent immunoassay, by bioMérieux (1 mentions)
Fluoroskan ascent, by Thermo Fisher Scientific (1 mentions)
Cell death detection elisa, by Roche (1 mentions)
Hemosil recombiplastin, by Werfen (1 mentions)
Acl 3000, by Werfen (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
100 um red nile beads, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Macs column, by Miltenyi Biotec (1 mentions)
Sodium citrate tubes, by Greiner (1 mentions)
33 protocols using sodium citrate tube
1
Longitudinal Blood Sampling for NSCLC ICI Therapy
2
Quantifying Extracellular Vesicles by Flow Cytometry
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Peripheral blood samples were drawn using sodium citrate tubes (Becton Dickinson Biosciences-BD, San Jose, CA, USA, Ref 454387) and analyzed within 4 h from venipuncture. The EV staining was carried out as previously described [21 (link),55 (link),56 (link),57 (link)]. Briefly, the reagent mix summarized in Table S1 was added to 195 μL of PBS 1X; then, 5 μL of whole blood were added to the mix. After 45 min of staining (RT, in the dark), 500 μL of PBS 1X were added to each tube and 1 × 106 events/sample were acquired by flow cytometry (FACSVerse, BD Biosciences, San Jose, CA, USA), placing the trigger threshold on the lipophilic cationic dye channel (LCD) [58 (link)]. To avoid immune complex formation and antibody aggregation, each reagent stock solution was centrifuged before its use (21,000× g, 12 min). For all measured parameters, the height (H) signals are shown.
Instrument performances, data reproducibility, and fluorescence calibrations were monitored by the Cytometer Setup and Tracking Module (BD Biosciences). The evaluation of non-specific fluorescence was obtained by acquiring FMO combined with the respective isotype control [59 (link),60 (link)]. Data were analyzed using FACSuite v 1.0.6.5230 (BD Biosciences) and FlowJo X v 10.0.7 (BD Biosciences) software. EVs concentrations were calculated based on the volumetric count function.
Instrument performances, data reproducibility, and fluorescence calibrations were monitored by the Cytometer Setup and Tracking Module (BD Biosciences). The evaluation of non-specific fluorescence was obtained by acquiring FMO combined with the respective isotype control [59 (link),60 (link)]. Data were analyzed using FACSuite v 1.0.6.5230 (BD Biosciences) and FlowJo X v 10.0.7 (BD Biosciences) software. EVs concentrations were calculated based on the volumetric count function.
3
Pulmonary Arterial Hypertension Evaluation
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Subjects underwent standard evaluation by means of medical history, clinical examination, electrocardiogram and pulmonary function tests. Pre-capillary PH was considered when mean pulmonary arterial pressure ≥20 mmHg, pulmonary artery wedge pressure of ≤15 mmHg and pulmonary vascular resistance ≥250 dyn·s·cm5, measured at rest by right heart catheterization [1 (link)]. Other groups with PH rather than PAH were ruled out according to the current diagnostic algorithm [7 (link)]. Severity of PAH was assessed by doppler echocardiography, pulmonary haemodynamics, New York Heart Association-World Health Organization functional class (NYHA-WHO FC), 6-min walking distance (6-MWD) and blood brain natriuretic peptide (BNP) level measured at baseline and at follow-up.
Venous blood samples were obtained from fasting subjects by peripheral venepuncture and placed into two 4.5 mL sodium citrate tubes (Becton Dickinson, UK) to measure circulating EVs, and into two 4 mL tubes with EDTA (Becton Dickinson, UK) to measure circulating PCs. Samples were obtained in a different day or if in the same day before right heart catheterization.
Venous blood samples were obtained from fasting subjects by peripheral venepuncture and placed into two 4.5 mL sodium citrate tubes (Becton Dickinson, UK) to measure circulating EVs, and into two 4 mL tubes with EDTA (Becton Dickinson, UK) to measure circulating PCs. Samples were obtained in a different day or if in the same day before right heart catheterization.
4
Blood Collection and Analysis Protocol
5
Biomarker Profiling of Vascular Health
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Venous blood samples were obtained after fasting overnight peripheral venipuncture into two 4.5mL sodium citrate tubes (Becton Dickinson, Plymouth, UK) to measure circulating EMPs, into two 4mL tubes with EDTA (Becton Dickinson, Plymouth, UK) to measure circulating PCs and into two 4mL tubes with EDTA for other biochemical determinations. The latter were centrifuged immediately 10min (800g, 4°C) and, after centrifugation, plasma was aliquoted and stored at -80°C until analysis. Plasma levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), ultra-sensible C-reactive protein (hsCRP), transforming growth factor beta (TGF-β), brain natriuretic peptide (BNP), fibrinogen, hepatocyte growth factor (HGF), letpine, adiponection, cyclic guanosine monophosphate (cGMP), soluble tumor necrosis factor-a receptor type I (sTNFa-RI), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble tyrosine kinase receptor Axl (sAXL) were determined by ELISA using commercially available kits (DuoSet, R&D Systems, Abingdon, UK).
6
Endurance Exercise Training and Circulating EVs
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Venous blood samples were obtained at rest before cardiopulmonary exercise and before and after the 8-week endurance exercise training; they were obtained by peripheral venipuncture and placed into two 4.5 mL sodium citrate tubes (Becton Dickinson, Plymouth, UK) to measure circulating EVs, and into two 4.5 mL tubes with EDTA (Becton Dickinson, Plymouth, UK) to measure circulating PCs.
7
Multiparametric Flow Cytometry Protocol
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From each enrolled subject, two sodium citrate tubes (Becton Dickinson Biosciences (BD), San Jose, CA, USA, Ref 454387) were used to collect the peripheral blood samples, using 21 G needles. Samples were processed within 4 hours from bleeding. The first harvested tube of PB was discarded to minimize venepuncture-induced vascular damage effects [40 (link), 41 (link)]. Of note, phalloidin was added to the reagent mix to stain events characterized by damaged membranes, given its binding to F-actin [10 (link), 42 (link)]. The staining was performed following an already described protocol [10 (link)]. In detail, as the first step, the reagent mix was prepared by adding FITC-conjugated phalloidin (when needed) and LCD (BD Biosciences–Catalogue, #626267, Custom Kit), and all reagents detailed in Table 1 (Panel 1 or Panel 2 or Panel 3) were added to 195 μl of PBS 1X, and then 5 μl of whole blood was added to the mix. After 45 min of staining (RT, in the dark), 500 μl PBS 1X was added to each tube, and 1 × 10 [6 (link)] events/sample were recorded by flow cytometry (FACSVerse, BD Biosciences).
To avoid the immune complex formation and the unspecific background linked to the antibody aggregation, each antibody stock solution was centrifuged before its use, at 21,000 g for 12 minutes.
To avoid the immune complex formation and the unspecific background linked to the antibody aggregation, each antibody stock solution was centrifuged before its use, at 21,000 g for 12 minutes.
8
Peripheral Blood Sampling for Therapy
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A baseline peripheral blood sample was collected at the time of enrollment and an additional blood sample was drawn at 12 (+/−6) weeks after cycle 1 of therapy. Peripheral blood was collected (21 G needles) in two sodium citrate tubes (Becton Dickinson Biosciences-BD, San Jose, CA, USA). Peripheral blood samples were processed within 4 h from venipuncture. The first harvested tube was discarded to minimize the EV release induced by vascular damage effects.
9
Biomarker Analysis of Plasma Samples
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Peripheral blood samples were collected in sodium citrate tubes (Becton Dickinson, San Jose, CA, USA). After centrifuging the whole blood samples for 15 min at 1550 × g, the plasma was aliquoted and stored at -70°C, with subsequent thawing for further analyses.
The histone-DNA complex levels were measured using a cell death detection ELISA kit (Roche Diagnostics, Indiana, USA). Cell-free DNA levels were measured using the Quant-iT PicoGreen dsDNA reagent (Molecular Probes, Eugene, Oregon, USA) and Fluoroskan Ascent microplate fluorometer (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). Neutrophil elastase levels were measured using a human neutrophil elastase platinum ELISA kit (eBioscience, Vienna, Austria). Prekallikrein levels were determined using an ELISA kit (Cloud-Clone, Houston, Texas, USA). Initial serum CA-125 levels were evaluated using an immunoradiometric assay kit (Institute of Isotopes, Budapest, Hungary).
The histone-DNA complex levels were measured using a cell death detection ELISA kit (Roche Diagnostics, Indiana, USA). Cell-free DNA levels were measured using the Quant-iT PicoGreen dsDNA reagent (Molecular Probes, Eugene, Oregon, USA) and Fluoroskan Ascent microplate fluorometer (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). Neutrophil elastase levels were measured using a human neutrophil elastase platinum ELISA kit (eBioscience, Vienna, Austria). Prekallikrein levels were determined using an ELISA kit (Cloud-Clone, Houston, Texas, USA). Initial serum CA-125 levels were evaluated using an immunoradiometric assay kit (Institute of Isotopes, Budapest, Hungary).
10
Biomarker Collection and Evaluation
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Blood samples were collected at the time of inclusion. Sodium citrate tubes (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA) were used for the HBP test using the Axis-Shield HBP microtitre plate ELISA (Axis-Shield Diagnostics, Dundee, UK).6 (link) Coagulant tubes with a silica clot activator, polymer gel, silicone-coated interior (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA) were used to evaluate PCT and CRP levels. PCT levels were detected by enzyme-linked fluorescent immunoassay (bioMérieux, Marcy, France) and CRP levels were measured by latex immunoturbidimetry (Abbott Laboratories, Chicago, Illinois, America) (detection limit, 0.05 ng/mL). These blood samples were centrifuged at 3000 rpm for 10 min immediately, and separate aliquots of the plasma supernatants were stored at −80°C until analysis. Plastic whole blood tubes with spray-coated K2EDTA (Axis-Shield Diagnostics, Dundee, UK) stored whole blood for cell counting detected within an hour.
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