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 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 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 Comparison of Blood Collection Tubes from Three Different Manufacturers for the Collection of Cell-Free DNA for Liquid Biopsy Mutation Testing

2017 ◽  
Vol 19 (5) ◽  
pp. 801-804 ◽  
Author(s):  
Christina Alidousty ◽  
Danielle Brandes ◽  
Carina Heydt ◽  
Svenja Wagener ◽  
Maike Wittersheim ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Mutation Testing ◽  
Blood Collection ◽  
Cell Free Dna ◽  
Free Dna ◽  
Blood Collection Tubes

Abstract 3110: In depth comparison of cell free DNA blood collection tubes

2020 ◽  
Author(s):  
Joel Desharnais ◽  
Jacob M. Vasquez ◽  
Katya J. Reshatoff ◽  
Quyen Bui ◽  
Han-Mei Chen ◽  
...  
Keyword(s):  
Blood Collection ◽  
Cell Free Dna ◽  
Free Dna ◽  
Blood Collection Tubes

scholarly journals Specialized Cell-Free DNA Blood Collection Tubes Can Be Repurposed for Extracellular Vesicle Isolation: A Pilot Study

2020 ◽  
Vol 18 (5) ◽  
pp. 462-470 ◽  
Author(s):  
Jessica Heatlie ◽  
Vanessa Chang ◽  
Sandra Fitzgerald ◽  
Yohanes Nursalim ◽  
Kate Parker ◽  
...  
Keyword(s):  
Pilot Study ◽  
Extracellular Vesicle ◽  
Blood Collection ◽  
Cell Free Dna ◽  
Specialized Cell ◽  
Free Dna ◽  
Blood Collection Tubes

Zero delay plasma vs. Streck cfDNA tubes: Does immediately centrifuging blood at the point of draw improve cell-free DNA signal-to-noise?

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14529-e14529
Author(s):  
Greg Sommer ◽  
Laura Fredriksen ◽  
Gabriella Iacovetti ◽  
Kyungjin Hong ◽  
Ulrich Schaff
Keyword(s):  
Room Temperature ◽  
Blood Collection ◽  
Signal To Noise ◽  
Blood Draw ◽  
Plasma System ◽  
Cell Free Dna ◽  
Clinical Evaluations ◽  
Free Dna ◽  
Collection Tube ◽  
Blood Collection Tubes

e14529 Background: Sample quality is a critical consideration for high fidelity cell-free DNA (cfDNA) testing. Oncological cfDNA tests used for liquid biopsy typically employ specialty blood collection tubes containing chemical preservatives to minimize degradation of samples prior to lab testing. Here we describe a newly developed device, Zero Delay Plasma– a portable centrifuge and disc system designed to immediately isolate cell-free plasma at the point of blood draw – and evaluate its performance against the Streck cfDNA collection tube. Methods: Whole blood was collected, processed, and stored at room temperature for up to 7 days with both the Zero Delay Plasma system and the Streck cfDNA blood collection tube. Sample hemolysis was measured via cell-free hemoglobin. Genomic contamination and cfDNA signal-to-noise were evaluated by qPCR and electrophoresis, comparing signal from target 150-200bp cfDNA to contaminating longer length genomic sequences in the sample. 2 sets of hemolysis experiments, 2 sets of electrophoresis experiments and 4 sets of qPCR experiments were conducted. Results: Plasma processed with the Zero Delay Plasma system yielded ~4X lower hemolysis levels, ~10X lower genomic contamination, and ~20X higher cfDNA signal-to-noise compared to the Streck cfDNA collection tube after 7 days of storage at room temperature. Conclusions: The Zero Delay Plasma system minimizes sample degradation and analytical background signal for cfDNA testing by immediately removing cells and other contaminants at the point of blood collection. Clinical evaluations are in process.

scholarly journals Cell-Free DNA Blood Collection Tubes Are Appropriate for Clinical Proteomics: A Demonstration in Colorectal Cancer

2018 ◽  
Vol 12 (3) ◽  
pp. 1700121 ◽  
Author(s):  
Juhura G. Almazi ◽  
Peter Pockney ◽  
Craig Gedye ◽  
Nathan D. Smith ◽  
Hubert Hondermarck ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Clinical Proteomics ◽  
Blood Collection ◽  
Cell Free Dna ◽  
Free Dna ◽  
Blood Collection Tubes

scholarly journals Efficient Detection of BRAF Mutation in Plasma of Patients after Long-term Storage of Blood in Cell-Free DNA Blood Collection Tubes

2015 ◽  
Vol 61 (6) ◽  
pp. 886-888 ◽  
Author(s):  
Marc G Denis ◽  
Anne-Chantal Knol ◽  
Sandrine Théoleyre ◽  
Audrey Vallée ◽  
Brigitte Dréno
Keyword(s):  
Braf Mutation ◽  
Blood Collection ◽  
Cell Free Dna ◽  
Long Term Storage ◽  
Free Dna ◽  
Efficient Detection ◽  
Blood Collection Tubes ◽  
Term Storage

scholarly journals Total and endothelial cell-derived cell-free DNA in blood plasma does not change during menstruation

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250561
Author(s):  
Nicole Laurencia Yuwono ◽  
Claire Elizabeth Henry ◽  
Caroline Elizabeth Ford ◽  
Kristina Warton
Keyword(s):  
Endothelial Cell ◽  
Menstrual Cycle ◽  
Repetitive Sequences ◽  
Modern Medicine ◽  
Blood Collection ◽  
Comprehensive Understanding ◽  
Cell Free Dna ◽  
Physiological Processes ◽  
Free Dna ◽  
The Impact

Assays measuring cell-free DNA (cfDNA) in blood have widespread potential in modern medicine. However, a comprehensive understanding of cfDNA dynamics in healthy individuals is required to assist in the design of assays that maximise the signal driven by pathological changes, while excluding fluctuations that are part of healthy physiological processes. The menstrual cycle involves major remodelling of endometrial tissue and associated apoptosis, yet there has been little investigation of the impact of the menstrual cycle on cfDNA levels. Paired plasma samples were collected from 40 healthy women on menstruating (M) and non-menstruating (NM) days of their cycle. We measured total cfDNA by targeting ALU repetitive sequences and measured endothelial-derived cfDNA by methylation-specific qPCR targeting an endothelium-unique unmethylated CDH5 DNA region. CfDNA integrity and endothelial cfDNA concentration, but not total cfDNA, are consistent across time between NM and M. No significant changes in total (ALU-115 p = 0.273; ALU-247 p = 0.385) or endothelial cell specific (p = 0.301) cfDNA were observed, leading to the conclusion that menstrual status at the time of diagnostic blood collection should not have a significant impact on the quantitation of total cfDNA and methylation-based cancer assays.

Abstract 3145: Clinical evaluation of streck cell-free DNA blood collection tubes for liquid profiling in oncology

2016 ◽  
Author(s):  
Inga Medina Diaz ◽  
Stefan Holdenrieder ◽  
Annette Nocon ◽  
Makbule Kobilay ◽  
Dirk Skowasch ◽  
...  
Keyword(s):  
Clinical Evaluation ◽  
Blood Collection ◽  
Cell Free Dna ◽  
Free Dna ◽  
Blood Collection Tubes
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