Our study included a total of 52 individuals aged 18–68 (mean 36 years) from Goiânia city, state of Goiás (GO), Brazil. All individuals tested negative for dengue and chikungunya. Of these 52 participants (Supplementary Table S1 ) 25 were potential blood donors recruited from the Center of Serology and Immunohematology of Goiânia-GO–Brazil, aged 27–53 (mean 36.4 years) and displaying negative blood tests for several infectious diseases (CONTROL). The remaining 27 participants showed Zika fever-like acute symptoms during the outbreak of ZIKV infection in Brazil between January and May 2016. All 27 patients were positive for ZIKV at real-time RT-PCR test (ZIKV+). A first plasma collection of ZIKV-infected subjects was done at enrollment, 2–9 days after symptom onset. A second collection was done from 6 ZIKV-infected subjects 2–3 weeks after the first sample, for evaluating the recovery phase (RECZIKV+) (Table 1 ). From these, 18 samples, we randomly selected a total for miRNA profiling: 6 control individuals (CONTROL), 7 ZIKV+ and 5 RECZIKV+. Plasma was isolated from blood samples collected into EDTA-coated Vacutainer tubes (Becton & Dickinson, United States), centrifuged at 1,200x g for 10 min, and stored at −80°C freezer until the analysis.
Edta coated vacutainer tubes
Manufactured by BD
Sourced in United States, Canada
EDTA-coated vacutainer tubes are laboratory blood collection tubes designed to collect and store blood samples. The tubes are coated with the anticoagulant ethylenediaminetetraacetic acid (EDTA), which prevents the blood from clotting during the collection and storage process. These tubes are commonly used in clinical and diagnostic settings to obtain blood samples for various laboratory analyses.
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Lab products found in correlation
Ficoll density gradient, by Cytiva (2 mentions)
Accuri c6, by BD (2 mentions)
Ficoll paque, by GE Healthcare (2 mentions)
Guava viacount, by Merck Group (2 mentions)
Collagenase type 2, by Merck Group (2 mentions)
Mirvana paris isolation kit, by Thermo Fisher Scientific (1 mentions)
Universal cdna synthesis kit, by Qiagen (1 mentions)
C elegans mir 39, by Thermo Fisher Scientific (1 mentions)
Ficoll hypaque, by Merck Group (1 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus solution, by GE Healthcare (1 mentions)
Rnalater solution, by Thermo Fisher Scientific (1 mentions)
Sepmate tube, by STEMCELL (1 mentions)
Cellsave preservative tube, by Menarini Silicon Biosystems (1 mentions)
Rosettesep ctc enrichment cocktail containing anti cd56, by STEMCELL (1 mentions)
Lymphoprep, by STEMCELL (1 mentions)
Cellsearch system, by Menarini Silicon Biosystems (1 mentions)
Red blood cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
Taq polymerase, by New England Biolabs (1 mentions)
40 protocols using edta coated vacutainer tubes
1
Profiling Zika Virus Infection Biomarkers
2
Plasma RNA Extraction and Profiling
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Plasma samples were acquired by standard venipuncture and centrifugation in EDTA-coated vacutainer tubes (Becton Dickinson) and frozen at -80 °C until use. RNA was extracted from plasma using the mirVana PARIS Isolation Kit (Life Technologies), according to the manufacturer’s instructions with one modification; prior to the addition of acid-phenol:chloroform, 150Amoles of c-elegans miR 39 (Life Technologies) was added as an internal standard and RNA carrier. RNA was extracted from 100 μl plasma and eluted in 100 μl nuclease free water. A 4 μl aliquot of RNA elute was reverse transcribed in 20 μl reactions using the Universal cDNA Synthesis Kit (Exiqon), according to the manufacturer’s instructions.
3
Blood Collection and PBMC Isolation
4
Oxidant Burden Evaluation Protocol
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After an overnight fast (>10 h), venous blood was collected into EDTA-coated vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ, USA) for centrifugation (3000× g for 15 min at 4 °C). The plasma supernatant was stored as aliquots (500 μL) at −80 °C until analysis.
Plasma samples were used to evaluate the oxidant burden which served as the present study’s primary outcome. Secondary outcomes included the participant’s plasma carotenoids concentration and SCS, as well as their corresponding dietary carotenoids intake, body composition and anthropometric measurements.
Plasma samples were used to evaluate the oxidant burden which served as the present study’s primary outcome. Secondary outcomes included the participant’s plasma carotenoids concentration and SCS, as well as their corresponding dietary carotenoids intake, body composition and anthropometric measurements.
5
Healthy Blood Sample Collection
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Healthy blood was obtained from consented donors at the Penn State General Clinical Research Centre according to an Institutional Review Board (IRB) approved protocol. Samples were drawn into EDTA-coated vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) from peripheral venepuncture. Blood samples were processed immediately upon receipt to facilitate optimal filtration conditions.
6
Blood Sample Collection and Lysis for Breast Cancer
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Blood samples were obtained from a total of 73 samples from 55 breast cancer patients (tables S2 and S3) enrolled in various anticancer therapeutic trials. This study was approved by our institutional review board and local ethics committee (DSRB reference 2012/00105, 2012/00979, 2010/00270, and 2010/00691). All patients gave their informed consent for inclusion in this study. Samples were collected from each patient either once or several times before and after treatment. Blood samples were stored in EDTA-coated vacutainer tubes (Becton-Dickinson). Blood samples were lysed within 10 hours after withdrawal using RBC lysis buffer (Life Technologies) for 3 to 5 min with mixing at room temperature and washed once with sterile PBS.
7
Isolation of Human PBMCs from Blood
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Human blood sample was collected from a healthy donor in EDTA-coated Vacutainer tubes (Becton Dickson, #367525). Blood sample was diluted 1:1 with PBS. Equal volume of diluted blood was carefully layered over Ficoll-Paque PLUS solution (GE Healthcare, #17144002) in a 50 mL Falcon tube. The tubes were centrifuged with acceleration and brakes turned off, at 400 g 20 °C for 30 min. The upper layer of the resulting gradient containing plasma and platelets was removed using a sterile pipette. The buffy coat containing the peripheral blood mononuclear cells (PBMCs) was carefully isolated using a pipette. The PBMCs were treated with ACK lysing buffer (Gibco, #A1049201) to remove any red blood cells. The final PBMCs were washed in PBS and resuspended in assay medium.
8
Liquid Biopsy Workflow for CTC Enrichment
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Two EDTA-coated vacutainer tubes (Becton Dickinson) of 7.5 ml of peripheral blood and one additional 10 mL CellSave Preservative tube (Menarini-Silicon Biosystems) were collected per patient at different time points: 20 samples before the start of systemic therapy (visit 1, V1), 20 samples after 1 cycle of chemotherapy (visit 2, V2), and 13 samples after clinical progression (visit 3, V3) determined by CT-Scan. Blood samples were processed within two hours after withdrawal. All samples were anonymised and encoded before the analysis.
In total, 53 samples were included in this study. Blood samples were analysed in parallel with the label-independent antibody cocktail RosetteSep™ CTC Enrichment Cocktail Containing Anti-CD56 (STEMCELL Technologies) (V1, n = 20; V2, n = 20 and V3, n = 13) and with the EpCAM-based CellSearch® System (Menarini Silicon Biosystems) as previously described [22 (link)] (V1, n = 19; V2, n = 20 and V3, n = 13).
In addition, the PBMCs of the patients were isolated from one 7.5 mL EDTA tube of peripheral blood by density gradient centrifugation protocol (Lymphoprep™, STEMCELL Technologies) in SepMate™ tubes (STEMCELL Technologies) according to manufacturer’s instructions.
Enriched cells isolated with RosetteSep™ and PBMCs were placed in RNAlater™ Solution (Invitrogen, ThermoFisher Scientific) and kept at -80ºC until further analyses.
In total, 53 samples were included in this study. Blood samples were analysed in parallel with the label-independent antibody cocktail RosetteSep™ CTC Enrichment Cocktail Containing Anti-CD56 (STEMCELL Technologies) (V1, n = 20; V2, n = 20 and V3, n = 13) and with the EpCAM-based CellSearch® System (Menarini Silicon Biosystems) as previously described [22 (link)] (V1, n = 19; V2, n = 20 and V3, n = 13).
In addition, the PBMCs of the patients were isolated from one 7.5 mL EDTA tube of peripheral blood by density gradient centrifugation protocol (Lymphoprep™, STEMCELL Technologies) in SepMate™ tubes (STEMCELL Technologies) according to manufacturer’s instructions.
Enriched cells isolated with RosetteSep™ and PBMCs were placed in RNAlater™ Solution (Invitrogen, ThermoFisher Scientific) and kept at -80ºC until further analyses.
9
ZIKV Outbreak Specimen Collection
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Collection of specimens from subjects during the ZIKV outbreak in 2016 has been previously described.30 Briefly, 65 patients who were RT‐PCR‐positive for ZIKV in whole blood or urine, and negative for DENV RT‐PCR were enrolled.28 Whole blood specimens were collected in EDTA‐coated vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ, USA) after peripheral venipuncture and were centrifuged at 240 g for 10 min. Plasma was collected and heat‐inactivated for 30 min at 56°C before storage at −80°C. Specimens were obtained over a period of six time points: (1) acute [2–7 days post‐illness onset (pio)], (2) early convalescent (10–14 days pio), (3) late convalescent (1 month pio), (4) early recovery (3 months pio), (5) late recovery (5–6 months pio) and (6) full recovery (1 year pio) phases.
10
Measuring HIV Viral Load and CD4+ T-cells
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