scholarly journals Plasma free DNA

2018 ◽  
Vol 29 (1) ◽  
pp. 153-156 ◽  
Author(s):  
Dietmar Enko ◽  
Gabriele Halwachs-Baumann ◽  
Gernot Kriegshäuser
Keyword(s):  
Blood Collection ◽  
Plasma Samples ◽  
Cell Free Dna ◽  
Free Dna ◽  
Roche Diagnostics Gmbh ◽  
Dna Collection ◽  
Roche Diagnostics ◽  
The Impact ◽  
Time Point ◽  
Circulating Cell Free Dna

Introduction: Standardized pre-analytical blood sample procedures for the analysis of circulating cell-free DNA (ccfDNA) are still not available. Therefore, the present study aimed at evaluating the impact of storage conditions related to different times (24 and 48 h) and temperatures (room temperature (RT) and 4 - 8 °C) on the plasma ccfDNA concentration of blood samples drawn into Cell-Free DNA collection tubes (Roche Diagnostics GmbH, Mannheim, Germany). Materials and methods: Venous blood from 30 healthy individuals was collected into five 8.5 mL Cell-Free DNA Collection Tubes (Roche Diagnostics GmbH) each. Plasma samples were processed at time point of blood collection (tube 1), and after storage under the following conditions: 24 h at RT (tube 2) or 4-8 °C (tube 3), and 48 h at RT (tube 4) or 4 - 8 °C (tube 5). Circulating cell-free DNA concentrations were determined by EvaGreen chemistry-based droplet digital PCR (ddPCR). Results: No statistically significant differences between median (interquartile range) plasma ccfDNA concentrations (ng/mL) at time point of blood collection (3.17 (2.13 – 3.76)) and after storage for 24 h (RT: 3.02 (2.41 – 3.68); 4-8 °C: 3.21 (2.19 – 3.46)) and 48 h (RT: 3.13 (2.10 – 3.76); 4-8 °C: 3.09 (2.19 – 3.50)) were observed (P values from 0.102 – 0.975). Conclusions: No unwanted release of genomic DNA from white blood cells could be detected in plasma samples after tube storage for 24 and 48 h regardless of storage temperature.

scholarly journals Optimization of Preanalytical Variables for cfDNA Processing and Detection of ctDNA in Archival Plasma Samples

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Marijana Nesic ◽  
Julie S. Bødker ◽  
Simone K. Terp ◽  
Karen Dybkær
Keyword(s):  
Nucleic Acid ◽  
B Cell Lymphoma ◽  
Circulating Tumor Dna ◽  
Blood Collection ◽  
Plasma Samples ◽  
Cell Free Dna ◽  
Blood Samples ◽  
Free Dna ◽  
The Impact ◽  
Blood Collection Tubes

DNA released from cells into the peripheral blood is known as cell-free DNA (cfDNA), representing a promising noninvasive source of biomarkers that could be utilized to manage Diffuse Large B-Cell Lymphoma (DLBCL), among other diseases. The procedure for purification and handling of cfDNA is not yet standardized, and various preanalytical variables may affect the yield and analysis of cfDNA, including the purification kits, blood collection tubes, and centrifugation regime. Therefore, we aimed to investigate the impact of these preanalytical variables on the yield of cfDNA by comparing three different purification kits DNeasy Blood & Tissue Kit (Qiagen), QIAamp Circulating Nucleic Acid Kit (Qiagen), and Quick-cfDNA Serum & Plasma Kit (Zymo Research). Two blood collection tubes (BCTs), EDTA-K2 and Cell-Free DNA (Streck), stored at four different time points before plasma was separated and cfDNA purified, were compared, and for EDTA tubes, two centrifugation regimes at 2000 × g and 3000 × g were tested. Additionally, we have tested the utility of long-term archival blood samples from DLBCL patients to detect circulating tumor DNA (ctDNA). We observed a higher cfDNA yield using the QIAamp Circulating Nucleic Acid Kit (Qiagen) purification kit, as well as a higher cfDNA yield when blood samples were collected in EDTA BCTs, with a centrifuge regime at 2000 × g . Moreover, ctDNA detection was feasible from archival plasma samples with a median storage time of nine years.

scholarly journals Evaluation of pre-analytical factors affecting plasma DNA analysis

2017 ◽  
Author(s):  
Havell Markus ◽  
Tania Contente-Cuomo ◽  
Winnie S. Liang ◽  
Mitesh J. Borad ◽  
Shivan Sivakumar ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Fragment Size ◽  
Digital Pcr ◽  
Clinical Samples ◽  
Blood Collection ◽  
Plasma Samples ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Free Dna ◽  
The Impact

AbstractPre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci (mean amplicon size: 71 bp and 471 bp respectively). Using this assay, we compared performance of 7 cfDNA extraction kits and found cfDNA yield and fragment size varies significantly between them. We also compared 3 blood collection protocols used to collect plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA BCT tubes at ambient temperature processed within 24 hours and 72 hours of collection). To assess whether cell-stabilizing preservative in BCT tubes introduced noise in cfDNA, we performed digital targeted sequencing. We found no significant differences in cfDNA yield, fragment size and background sequencing noise between these protocols. In 219 clinical samples tested for quality using the ddPCR assay, cfDNA fragment size was significantly shorter in plasma samples immediately processed for ctDNA analysis compared to archived samples, suggesting background DNA contributed by lysed peripheral blood cells. In summary, we describe a multiplexed ddPCR approach that enables cfDNA quality assessment and could inform the design of future circulating tumor DNA studies.Gene namesNone

scholarly journals The influence of biological and lifestyle factors on circulating cell-free DNA in blood plasma

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Nicole Laurencia Yuwono ◽  
Kristina Warton ◽  
Caroline Elizabeth Ford
Keyword(s):  
Acute Exercise ◽  
Blood Collection ◽  
Occupational Hazard ◽  
Biological Factors ◽  
Cell Free Dna ◽  
Biological Sex ◽  
Hazard Exposure ◽  
Free Dna ◽  
Complete Reporting ◽  
Circulating Cell Free Dna

Research and clinical use of circulating cell-free DNA (cirDNA) is expanding rapidly; however, there remain large gaps in our understanding of the influence of lifestyle and biological factors on the amount of cirDNA present in blood. Here, we review 66 individual studies of cirDNA levels and lifestyle and biological factors, including exercise (acute and chronic), alcohol consumption, occupational hazard exposure, smoking, body mass index, menstruation, hypertension, circadian rhythm, stress, biological sex and age. Despite technical and methodological inconsistences across studies, we identify acute exercise as a significant influence on cirDNA levels. Given the large increase in cirDNA induced by acute exercise, we recommend that controlling for physical activity prior to blood collection is routinely incorporated into study design when total cirDNA levels are of interest. We also highlight appropriate selection and complete reporting of laboratory protocols as important for improving the reproducibility cirDNA studies and ability to critically evaluate the results.

scholarly journals Detection of Colorectal Cancer in Circulating Cell-Free DNA by Methylated CpG Tandem Amplification and Sequencing

2019 ◽  
Vol 65 (7) ◽  
pp. 916-926 ◽  
Author(s):  
Jingyi Li ◽  
Xin Zhou ◽  
Xiaomeng Liu ◽  
Jie Ren ◽  
Jilian Wang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Early Stage ◽  
Plasma Samples ◽  
Dna Hypermethylation ◽  
Cell Free Dna ◽  
Noncancerous Tissue ◽  
Clinical Sensitivity ◽  
Free Dna ◽  
A Genome ◽  
Circulating Cell Free Dna

Abstract BACKGROUND Aberrant DNA hypermethylation of CpG islands occurs frequently throughout the genome in human colorectal cancer (CRC). A genome-wide DNA hypermethylation analysis technique using circulating cell-free DNA (cfDNA) is attractive for the noninvasive early detection of CRC and discrimination between CRC and other cancer types. METHODS We applied the methylated CpG tandem amplification and sequencing (MCTA-Seq) method, with a fully methylated molecules algorithm, to plasma samples from patients with CRC (n = 147) and controls (n = 136), as well as cancer and adjacent noncancerous tissue samples (n = 66). We also comparatively analyzed plasma samples from patients with hepatocellular carcinoma (HCC; n = 36). RESULTS Dozens of DNA hypermethylation markers including known (e.g., SEPT9 and IKZF1) and novel (e.g., EMBP1, KCNQ5, CHST11, APBB1IP, and TJP2) genes were identified for effectively detecting CRC in cfDNA. A panel of 80 markers discriminated early-stage CRC patients and controls with a clinical sensitivity of 74% and clinical specificity of 90%. Patients with early-stage CRC and HCC could be discriminated at clinical sensitivities of approximately 70% by another panel of 128 markers. CONCLUSIONS MCTA-Seq is a promising method for the noninvasive detection of CRC.

scholarly journals Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows

2019 ◽  
Vol 66 (1) ◽  
pp. 149-160 ◽  
Author(s):  
Rita Lampignano ◽  
Martin H.D Neumann ◽  
Sabrina Weber ◽  
Vera Kloten ◽  
Andrei Herdean ◽  
...  
Keyword(s):  
Clinical Decision Making ◽  
Clinical Decision ◽  
Digital Pcr ◽  
Blood Collection ◽  
Routine Practice ◽  
Acid Extraction ◽  
Cell Free Dna ◽  
Free Dna ◽  
Analytical Work ◽  
Circulating Cell Free Dna

Abstract BACKGROUND In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making. METHODS We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID (http://www.cancer-id.eu) for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites. RESULTS We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT. CONCLUSIONS This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows.

Detection of EGFR alterations in circulating cell-free DNA of lung tumor patients: Higher sensitivity obtained with plasma compared to serum.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e19046-e19046
Author(s):  
Marc G. Denis ◽  
Marie Marcq ◽  
Paul Hofman ◽  
Acya Bizieux-Thaminy ◽  
Jaafar Bennouna ◽  
...  
Keyword(s):  
Egfr Mutation ◽  
Lung Tumor ◽  
Blood Collection ◽  
Molecular Testing ◽  
Alternative Source ◽  
Cell Free Dna ◽  
Free Dna ◽  
Tki Treatment ◽  
Nsclc Patients ◽  
Circulating Cell Free Dna

e19046 Background: Detection of EGFR alterations is critical for predicting the response to tyrosine kinase inhibitors (TKI) in patients with non-small-cell lung cancer (NSCLC). In clinical practice, molecular testing is performed on tumor tissues when available. We investigated the use of circulating cell-free DNA for the detection of EGFR alterations in patients with NSCLC. Methods: Serum and plasma were obtained by centrifugation (10 min, 2,000 g, 20°C) performed within 3 hours following blood collection. Cell-free DNA was extracted using the QIAamp Circulating Nucleic Acid kit (Qiagen). Detection of EGFR alterations was performed using the approved Therascreen EGFR RGQ kit (Qiagen). Samples were tested positive for EGFR mutation when the ΔCt value (Ct of the mutation specific PCR – Ct of the control PCR) was lower than 12 for exon 19 deletions, and below 14 for L858R mutation. Results: No EGFR alteration was detected in samples collected from healthy donors (n=6), NSCLC patients with a wild type EGFR (n=60), and early stages NSCLC patients presenting an EGFR mutation in their tumor (n=11). Thirteen metastatic patients presenting an EGFR mutation in their tumor were tested before initiation of TKI treatment. When available, both serum and plasma were analyzed. Nine of these patients (9/13; 69.2%) were tested positive in their serum. The ΔCt values obtained were lower for plasma than for serum in most cases, and more patients (10/11; 90.9%) were EGFR mutation positive when plasma was tested. Finally we tested 3 patients during TKI treatment on a monthly basis. For 2 patients we were unable to detect the mutation initially found in pretreatment samples. Clinically, both patients were partial responders. The third patient did not respond to TKI, and we detected the EGFR mutation with stable ΔCt values at all points tested. Conclusions: EGFR alterations can be found in patients presenting an EGFR mutation in a metastatic NSCLC. Plasma samples allowed a better detection rate. Our results suggest that DNA circulating in plasma is a useful alternative source of tumor DNA that could be used for determining EGFR mutation status, and for follow-up of treatment. Supported by a grant from Astra-Zeneca.

scholarly journals Correction: Versatile hybrid acoustic micromixer with demonstration of circulating cell-free DNA extraction from sub-ml plasma samples

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Alvaro J. Conde ◽  
Ieva Keraite ◽  
Alfredo E. Ongaro ◽  
Maïwenn Kersaudy-Kerhoas
Keyword(s):  
Dna Extraction ◽  
Plasma Samples ◽  
Cell Free Dna ◽  
Free Dna ◽  
Circulating Cell Free Dna

Correction for ‘Versatile hybrid acoustic micromixer with demonstration of circulating cell-free DNA extraction from sub-ml plasma samples’ by Alvaro J. Conde et al., Lab Chip, 2020, 20, 741–748, DOI: 10.1039/C9LC01130G.

scholarly journals Versatile hybrid acoustic micromixer with demonstration of circulating cell-free DNA extraction from sub-ml plasma samples

Lab on a Chip ◽  
2020 ◽  
Vol 20 (4) ◽  
pp. 741-748 ◽  
Author(s):  
Alvaro J. Conde ◽  
Ieva Keraite ◽  
Alfredo E. Ongaro ◽  
Maïwenn Kersaudy-Kerhoas
Keyword(s):  
Dna Extraction ◽  
Low Cost ◽  
Plasma Samples ◽  
Cell Free Dna ◽  
Free Dna ◽  
Circulating Cell Free Dna

A low-cost and easy to implement acoustic micromixer compatible with multiple fabrication technologies that can provide efficient and vigorous mixing.

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