K2 edta tube

We used blood samples from 4 healthy volunteers to understand the baseline expression level of biomarkers (Supplementary Table S4). These donors gave written informed consent for the use of their blood samples in accordance with IRB protocol number 2021-01033. The volunteers underwent an overnight fast and were subsequently subjected to a blood test (referred to as “fasting samples”). After the fasting blood samples were collected, the participants ate a nutritious breakfast. Following this, they were instructed to avoid any food or drinks except water. Additional blood samples were collected from the individuals three hours and five hours after the breakfast (termed “non-fasting samples”). Blood samples were collected into BD K2 EDTA tubes. Plasma samples were isolated right after the blood collections via centrifuging at 1500× g for 15 min. Total RNA, including small RNA, was extracted from 200 μL of whole plasma using miRNeasy Serum/Plasma Kit (Cat. No.217184, Qiagen, Hilden, Germany), following the manufacture protocol.

Blood was collected in BD K₂EDTA tubes (BD, cat. no. 367525), 10 ml draw volume, from healthy volunteers. After approval by Norwegian south east regional committee for medical and health research ethics (REC South East), all participants signed a written informed consent before participating in the study in accordance with the Helsinki declaration. As soon as possible after the first blood tube collection, EDTA blood from each volunteer was transferred to and PAXgene® Blood RNA Tubes (PAXgene) (PreAnalytiX) to maintain gene expression, incubated at room temperature for 2 hours, and then stored at −80°C.

All 20 mL blood samples (collected at baseline and during treatment) were divided into two 10 mL BD k2-EDTA tubes and centrifuged at 300× g for 10 min. The upper plasma layer from each tube was transferred into a new 15 mL tube, then centrifuged again at 2000× g for 10 min. Then, 1 ml volumes of the top plasma layer were aliquoted into 1.5 mL tubes and stored at −80 degrees C for analysis at some future time. The two 15 mL samples of CSF collected at baseline and on day 60 were each aliquoted into 1.5 mL tubes, then frozen and stored at −80 °C until analysis of the same AD markers as indicated for plasma. At the end of the 31-month study, the plasma/CSF samples were thawed completely on ice, then mixed well on a vortex and centrifuged at 2000× g for 10 min to precipitate any debris for the determination of the following AD biomarkers in duplicate: soluble/monomeric Aβ1-40 and Aβ1-42, oligomeric Aβ, total tau (t-tau), and p-tau.

Prior to the psychiatric interviews, blood samples were obtained in the morning after fasting. All participating subjects were summoned at 8:30 a.m. when the extraction was performed. Venous blood was extracted into 10 mL K2 EDTA tubes (BD, Franklin Lakes, NJ, USA) and immediately processed to obtain plasma for 8:30–12 h. Blood samples were centrifuged at 2200× g for 15 min (4 °C) and individually assayed to detect infectious diseases by four commercial rapid tests for HIV, hepatitis B, and hepatitis C (Strasbourg, Cedex, France) and SARS-CoV-2 (Bio-Connect, Huissen, the Netherlands). Finally, plasma samples were individually characterized, registered, and stored at −80 °C until further analyses.

Whole blood was collected in 6 mL K2-EDTA tubes (BD, Franklin Lakes, NJ, USA) and stored at room temperature for no more than one hour prior to plasma isolation. For MI patients, blood was taken within a period from several hours to 7 days after MI (mean value: 2.22 days). To obtain platelet-free plasma (PFP) for miRNA isolation, the whole blood sample was processed in two steps—whole blood was centrifuged at 2130× g for 10 min at room temperature (Step 1), and then the crude plasma was repeatedly centrifuged in sterile 15 mL conical tubes at 2130× g for 10 min at room temperature (Step 2). The upper two-thirds of the plasma layer was stored at −20 °C in 500 μL portions in sterile RNAse-free 1.5 mL tubes in the clinical facility. Within 1 month, frozen plasma samples were transported to the laboratory facility without thawing and then stored at −80 °C. Isolation of miRNA was performed within one month after blood collection. Before miRNA isolation, plasma samples were thawed on ice and centrifuged at 16,000× g for 15 min at 4 °C to pellet down any residual cell debris (Step 3). The supernatant in a volume of 300 µL was used immediately for miRNA extraction and 10 µL was aliquoted and stored at −20 °C for further hemolysis assessment.

Plasma sample collection was based on previous studies about lipid mediators in addiction and comorbid disorders^{41 (link)}. Blood samples were obtained in the morning after fasting for 8–12 h (prior to the psychiatric interviews) by experienced nurses. Venous blood was extracted into 10 mL K₂ EDTA tubes (BD, Franklin Lakes, NJ, USA) and immediately centrifuged at 2200×g for 15 min (4 °C) to obtain plasma. The plasma samples were individually assayed to detect infectious diseases using commercial rapid tests for HIV, hepatitis B, and hepatitis C (Strasbourg, Cedex, France). Plasma samples were individually stored at – 80 °C until further analyses.

Prostate cancer patients (n = 3) and healthy female blood donors (n = 4) provided written informed consent to participate in the study. The study was approved by the South Western Sydney Local Healthy District Ethics Committee, Australia (HREC/13/LPOOL/158; 02/09/2013). Peripheral blood was drawn by venipuncture into blood tubes with the initial 3 mL discarded to prevent keratinocyte contamination and false positive CTCs being present. For the main comparison experiment, a total of 192 mL blood from each healthy donor (n = 3) was drawn into a total of 24 blood tubes including: 4 × 9 mL K₃EDTA tube (Greiner Bio-One, Kremsmünster, Austria), 4 × 9 mL acid citrate dextrose-B tube (Greiner Bio-One, Kremsmünster, Austria), 4 × 10 mL Cell-free DNA BCT (Streck, Omaha, NE, USA), 4 × 10 mL Cell-free RNA BCT (Streck, Omaha, NE, USA), and 4 × 2 × 5 mL Cyto-Chex BCT (Streck, Omaha, NE, USA). In the follow-up experiment to test increased proteinase K treatment, 38 mL blood from two healthy donors was drawn into one set of EDTA, Citrate, and DNA BCT and RNA BCT tubes. In the experiment with prostate cancer patients, blood was drawn into 3 × 6 mL K₂EDTA tubes (BD, Franklin Lakes, NJ, USA) available in the clinic.