The study protocol was approved by the Institutional Review Boards of the University of Maryland Baltimore. The surgical pathologic staging was determined according to the TNM classification of the International Union Against Cancer with the American Joint Committee on Cancer and the International Staging System for Lung Cancer [44 (link)]. Histopathologic classification was determined according to the World Health Organization classification [45 (link)]. Altogether, we recruited 39 patients with NSCLC and 32 cancer-free controls (Table 5 ). Blood samples were collected with the written informed consent from participants and obtained before therapeutic intervention using BD Vacutainer® Venous Blood Collection Tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The blood samples were immediately processed for plasma preparation via centrifugation at 3000 rpm (1900× g) for 10 min at 4 °C within less than 2 h after collection, as previously described in our published works [14 (link),15 (link),16 (link),17 (link),18 (link),19 (link),20 (link),21 (link)].
Vacutainer venous blood collection tubes
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BD Vacutainer Venous Blood Collection Tubes are used to collect and transport venous blood samples. These tubes are designed to ensure the integrity of the collected sample. They are available in various sizes and with different additive formulations to meet different laboratory testing requirements.
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14 protocols using vacutainer venous blood collection tubes
1
NSCLC Blood Sampling Protocol for Biomarker Analysis
2
Diagnosis of Tuberculosis: Integrated Workflow
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We collected the clinical symptoms of all participants. All participants underwent chest radiographs to diagnose tuberculosis in accordance with international standards and guidelines (8 (link)). Professional medical workers used BD Vacutainer Venous Blood Collection Tubes (Becton Dickinson, Sunnyvale, CA, USA) to collect venous blood while checked Mycobacterium bovis BCG vaccine scars.
The collected blood was centrifuged within 2 h at 2,500 rpm for 5 min. Then collect the upper serum, and next detected for IGRA through QuantiFERON-TB Gold Kit (QIAGEN,https://www.qiagen.com ). Suspected TB (IGRA seropositive with X-ray positive) sputum samples were collected for smear, bacterial culture and X-pert detection ① Sputum smear was examined under a microscope after acid-fast staining. ≥1 acid-fast bacilli/100 visual fields were found in both examinations, which was defined as positive. ② The bacteria are cultured on Löwenstein–Jensen (L-J) medium, incubated at 37°C with daily examinations for 8 weeks until the sample isolates at most minuscule one Mycobacterium tuberculosis complex colony, which is defined as positive. ③ After adding the corresponding reagent of GeneXpert MTB/RIF (Xpert, Cepheid, Sunnyvale, CA, USA), the sample was processed according to the manufacturer's instructions, and finally put into the machine to read the results (12 (link)).
The collected blood was centrifuged within 2 h at 2,500 rpm for 5 min. Then collect the upper serum, and next detected for IGRA through QuantiFERON-TB Gold Kit (QIAGEN,
3
Plasma Extraction from Venous Blood
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Venous blood samples (5 ml) were drawn from three FHCM patients and three healthy people to BD Vacutainer Venous Blood Collection Tubes containing EDTA, then turn tubes up and down many times to mixed blood and anticoagulant fully. Centrifuge 3000 rpm at 4 °C for 10 min, collected supernatants as plasma. The fresh plasma samples was frozen immediately at − 80 °C until using.
4
Gastric Cancer Biomarker Profiling
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In the present study, 5 patients with GC were recruited from the First Hospital of Jilin University between December 2018 and February 2019. All patients were pathologically diagnosed with gastric adenocarcinoma and were free of any other types of cancer. Patients who had received any type of anticancer treatment were excluded from the present study. In addition, 5 healthy donors with no history of cancer were enrolled as healthy controls (HC). The clinicopathological characteristics of patients and volunteers are presented in Table I . In total, 5 ml of peripheral blood samples were collected intravenously from each individual in BD Vacutainer® Venous Blood Collection Tubes containing EDTA, and plasma was isolated and frozen immediately at −80°C until use. This study was approved by the local independent Ethics Committee and the Institutional Review Board of the First Hospital of Jilin University (Changchun, China), and informed consent was obtained from all patients and volunteers. Blood collection and experiments were performed in accordance with the Declaration of Helsinki and relevant guidelines and regulations.
5
Collection and Storage of Human Blood Samples
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All samples were prospectively collected and analyzed according to a standard IRB protocol (Yale University: 6/17/2013) in accordance with the World Medical Association Declaration of Helsinki regarding the ethical conduct of research involving human subjects (22) (link). All individuals from whom blood was obtained were present (6/2012–12/2013) at the School of Medicine out-patient clinics following an informed consent. The blood samples (5 ml) were collected in 9 mg K2EDTA tubes (BD Vacutainer Venous Blood Collection Tubes, BD Diagnostics, Franklin, NJ, USA). The aliquots of whole blood were stored at −80 °C within 2 h of collection (samples immediately stored on ice at 4 °C after sampling) per standard molecular diagnostics protocols for PCR-based studies (31) (link). A second aliquot (2 ml) was spun (600 g , 10 min) and the plasma collected for CgA ELISA using the DAKO Kit as described previously (22, 23, 32) (link).
6
Gallbladder Adenocarcinoma Exosome Isolation
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All patients provided written informed consent to participate in the study. The study protocol was approved by the Ethics Committee of Ruijin Hospital of Shanghai Jiao Tong University. In total, five patients with GBC were recruited from Ruijin Hospital, Shanghai Jiao Tong University between January 2019 and December 2019. All patients were pathologically diagnosed with gallbladder adenocarcinoma, and were free of any other form of cancer. The patients included did not receive any type of anticancer treatment during the study. In addition, five patients with XCG with no history of cancer were enrolled as negative controls. For exosome isolation, peripheral blood specimens (4 ml) were collected intravenously in BD Vacutainer® Venous Blood Collection Tubes containing ethylenediaminetetraacetic acid (EDTA). Plasma was isolated and frozen immediately at −80°C until use. Blood collection and experiments were performed in accordance with the Declaration of Helsinki.
7
NETest and CgA Biomarker Collection
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Blood samples for the NETest were collected in 10.8mg K2EDTA tubes (BD Vacutainer Venous Blood Collection Tubes, BD Diagnostics). Aliquots of whole blood were stored at -80°C within 2 hrs of collection (samples immediately stored on ice/4°C after sampling) per standard molecular diagnostics protocols [24 (link)]. Blood samples for CgA were collected at the same time point in PPT plasma preparations tubes. Matched tumor tissue samples were available in seven cases. Tissue (n = 7) were collected at the time of surgery [25 (link)]. Samples were snap frozen in liquid nitrogen. Deidentified samples were sent to Wren Laboratories for RNA isolation and NETest PCR.
8
Serum Collection for Endometrial Biopsy
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Peripheral blood samples were collected using BD Vacutainer® Venous Blood Collection tubes containing clotting activator (BD Company, Franklin Lakes, New Jersey, U.S.) before anaesthesia. Samples were processed within 1 h after collection. Serum was isolated from the peripheral whole blood samples by centrifugation at 2000×g/10 min/4 °C (cohort 1) or two centrifugations at 1600×g for 10 min and at 16,000×g for 10 min at 4 °C (cohort 2). Separated serum samples were stored at − 80 °C until use. Endometrial biopsies were collected at laparoscopy or in the mid-luteal phase for those not undergoing laparoscopy.
9
Whole Blood Collection for NETest
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Whole blood for NETest measurement was collected at baseline (the day before surgery) and thereafter at clinically defined points during the follow-up. Blood samples (10 ml) were collected in ethylenediaminetetraacetic acid (EDTA) tubes (BD Vacutainer Venous Blood Collection Tubes, BD Diagnostics, Franklin, NJ). Aliquots of whole blood were stored at −80 °C within 2 h of collection (samples immediately stored on ice/4 °C after sampling) for PCR-based studies.
10
Establishing LCLs from Breast Cancer Patients
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Ten milliliters of blood were drawn from each patient through venous puncture into BD Vacutainer® venous blood collection tubes (Lavender top), and inverted 8–10 times. Blood samples were processed immediately (for all University of Chicago samples) or shipped on ice overnight to University of Chicago for processing. Peripheral blood mononuclear cells (PBMCs) were isolated using Accuspin™ System-Histopaque®-1077 tubes as instructed by the manufacturer (Sigma-Aldrich®, St. Louis, MO) with some modifications.
A previously-described Epstein-Barr virus (EBV) transformation protocol [22 (link)] was adapted to establish LCLs from breast cancer patients enrolled into our study. When the cells reached a total viability of 80% the flasks were sub-cultured and further expanded to a total viable cell count of 3 × 107. Two independent LCL colonies were established from each patient's peripheral blood sample.
A previously-described Epstein-Barr virus (EBV) transformation protocol [22 (link)] was adapted to establish LCLs from breast cancer patients enrolled into our study. When the cells reached a total viability of 80% the flasks were sub-cultured and further expanded to a total viable cell count of 3 × 107. Two independent LCL colonies were established from each patient's peripheral blood sample.
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