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 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 Genome-wide study of the effect of blood collection tubes on the cell-free DNA methylome

2020 ◽  
Author(s):  
Ruben Van Paemel ◽  
Andries De Koker ◽  
Christa Caggiano ◽  
Annelien Morlion ◽  
Pieter Mestdagh ◽  
...  
Keyword(s):  
Time Delay ◽  
Fragment Size ◽  
Cost Effective ◽  
Methylation Pattern ◽  
Blood Collection ◽  
Liquid Biopsies ◽  
Blood Draw ◽  
Cell Free Dna ◽  
Free Dna ◽  
The Impact

AbstractBackgroundThe methylation pattern of cfDNA, isolated from liquid biopsies, is gaining substantial interest for diagnosis and monitoring of diseases. We have evaluated the impact of type of blood collection tube and time delay between blood draw and plasma preparation on bisulfite-based cfDNA methylation profiling.Methods15 tubes of blood were drawn from three healthy volunteer subjects (BD Vacutainer K2E EDTA spray tubes, Streck Cell-Free DNA BCT tubes, PAXgene Blood ccfDNA tubes, Roche Cell-Free DNA Collection tubes and Biomatrica LBgard blood tubes in triplicate). Samples were either immediately processed or stored at room temperature for 24 or 72 hours before plasma preparation. DNA fragment size was evaluated by capillary electrophoresis. Reduced representation bisulfite sequencing was performed on the cell-free DNA isolated from these plasma samples. We evaluated the impact of blood tube and time delay on several quality control metrics.ResultsAll preservation tubes performed similar on the quality metrics that were evaluated. Furthermore, a considerable increase in cfDNA concentration and the fraction of it derived from NK cells was observed after a 72-hour time delay in EDTA tubes.ConclusionThe methylation pattern of cfDNA is robust and reproducible in between the different preservation tubes. EDTA tubes processed as soon as possible, preferably within 24 hours, are the most cost effective. If immediate processing is not possible, preservation tubes are valid alternatives.

scholarly journals A New Method for Improving Extraction Efficiency and Purity of Urine and Plasma Cell-Free DNA

Diagnostics ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 650
Author(s):  
Selena Y. Lin ◽  
Yue Luo ◽  
Matthew M. Marshall ◽  
Barbara J. Johnson ◽  
Sung R. Park ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Amplification Efficiency ◽  
Normal Donor ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Free Dna ◽  
Pcr Product ◽  
Cleanup Procedure ◽  
Dna Profiles ◽  
The Impact

This study assessed three commercially available cell-free DNA (cfDNA) extraction kits and the impact of a PEG-based DNA cleanup procedure (DNApure) on cfDNA quality and yield. Six normal donor urine and plasma samples and specimens from four pregnant (PG) women carrying male fetuses underwent extractions with the JBS cfDNA extraction kit (kit J), MagMAX Cell-Free DNA Extraction kit (kit M), and QIAamp Circulating Nucleic Acid Kit (kit Q). Recovery of a PCR product spike-in, endogenous TP53, and Y-chromosome DNA was used to assess kit performance. Nucleosomal-sized DNA profiles varied among the kits, with prominent multi-nucleosomal-sized peaks present in urine and plasma DNA isolated by kits J and M only. Kit J recovered significantly more spike-in DNA than did kits M or Q (p < 0.001) from urine, and similar amounts from plasma (p = 0.12). Applying DNApure to kit M- and Q-isolated DNA significantly improved the amplification efficiency of spike-in DNA from urine (p < 0.001) and plasma (p ≤ 0.013). Furthermore, kit J isolated significantly more Y-chromosome DNA from PG urine compared to kit Q (p = 0.05). We demonstrate that DNApure can provide an efficient means of improving the yield and purity of cfDNA and minimize the effects of pre-analytical biospecimen variability on liquid biopsy assay performance.

scholarly journals Preferred end coordinates and somatic variants as signatures of circulating tumor DNA associated with hepatocellular carcinoma

2018 ◽  
Vol 115 (46) ◽  
pp. E10925-E10933 ◽  
Author(s):  
Peiyong Jiang ◽  
Kun Sun ◽  
Yu K. Tong ◽  
Suk Hang Cheng ◽  
Timothy H. T. Cheng ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Peripheral Circulation ◽  
Sequence Information ◽  
Sequencing Analysis ◽  
Plasma Samples ◽  
Dna Molecules ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Free Dna ◽  
Tumor Dna

Circulating tumor-derived cell-free DNA (ctDNA) analysis offers an attractive noninvasive means for detection and monitoring of cancers. Evidence for the presence of cancer is dependent on the ability to detect features in the peripheral circulation that are deemed as cancer-associated. We explored approaches to improve the chance of detecting the presence of cancer based on sequence information present on ctDNA molecules. We developed an approach to detect the total pool of somatic mutations. We then investigated if there existed a class of ctDNA signature in the form of preferred plasma DNA end coordinates. Cell-free DNA fragmentation is a nonrandom process. Using plasma samples obtained from liver transplant recipients, we showed that liver contributed cell-free DNA molecules ended more frequently at certain genomic coordinates than the nonliver-derived molecules. The abundance of plasma DNA molecules with these liver-associated ends correlated with the liver DNA fractions in the plasma samples. Studying the DNA end characteristics in plasma of patients with hepatocellular carcinoma and chronic hepatitis B, we showed that there were millions of tumor-associated plasma DNA end coordinates in the genome. Abundance of plasma DNA molecules with tumor-associated DNA ends correlated with the tumor DNA fractions even in plasma samples of hepatocellular carcinoma patients that were subjected to shallow-depth sequencing analysis. Plasma DNA end coordinates may therefore serve as hallmarks of ctDNA that could be sampled readily and, hence, may improve the cost-effectiveness of liquid biopsy assessment.

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.

scholarly journals Single-molecule sequencing reveals a large population of long cell-free DNA molecules in maternal plasma

2021 ◽  
Vol 118 (50) ◽  
pp. e2114937118
Author(s):  
Stephanie C. Y. Yu ◽  
Peiyong Jiang ◽  
Wenlei Peng ◽  
Suk Hang Cheng ◽  
Y. T. Tommy Cheung ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Analysis ◽  
Large Population ◽  
Maternal Plasma ◽  
Dna Molecules ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Dna Molecule ◽  
Free Dna ◽  
Monogenic Diseases

In the field of circulating cell-free DNA, most of the studies have focused on short DNA molecules (e.g., <500 bp). The existence of long cell-free DNA molecules has been poorly explored. In this study, we demonstrated that single-molecule real-time sequencing allowed us to detect and analyze a substantial proportion of long DNA molecules from both fetal and maternal sources in maternal plasma. Such molecules were beyond the size detection limits of short-read sequencing technologies. The proportions of long cell-free DNA molecules in maternal plasma over 500 bp were 15.5%, 19.8%, and 32.3% for the first, second, and third trimesters, respectively. The longest fetal-derived plasma DNA molecule observed was 23,635 bp. Long plasma DNA molecules demonstrated predominance of A or G 5′ fragment ends. Pregnancies with preeclampsia demonstrated a reduction in long maternal plasma DNA molecules, reduced frequencies for selected 5′ 4-mer end motifs ending with G or A, and increased frequencies for selected motifs ending with T or C. Finally, we have developed an approach that employs the analysis of methylation patterns of the series of CpG sites on a long DNA molecule for determining its tissue origin. This approach achieved an area under the curve of 0.88 in differentiating between fetal and maternal plasma DNA molecules, enabling the determination of maternal inheritance and recombination events in the fetal genome. This work opens up potential clinical utilities of long cell-free DNA analysis in maternal plasma including noninvasive prenatal testing of monogenic diseases and detection/monitoring of pregnancy-associated disorders such as preeclampsia.

scholarly journals A new method for improving extraction efficiency and purity of urine and plasma cell-free DNA

2021 ◽  
Author(s):  
Selena Y. Lin ◽  
Yue Luo ◽  
Matthew M. Marshall ◽  
Barbara J. Johnson ◽  
Sung R. Park ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Amplification Efficiency ◽  
Normal Donor ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Free Dna ◽  
Pcr Product ◽  
Cleanup Procedure ◽  
Dna Profiles ◽  
The Impact

AbstractThis study assessed three commercially available cell-free DNA (cfDNA) extraction kits and the impact of a PEG-based DNA cleanup procedure (DNApure) on cfDNA quality and yield. Six normal donor urine and plasma samples, and specimens from four pregnant (PG) women carrying male fetuses underwent extractions with the JBS cfDNA extraction kit (kit J), MagMAX Cell-Free DNA Extraction kit (kit M), and QIAamp Circulating Nucleic Acid Kit (kit Q). Recovery of a PCR product spike-in, endogenous TP53, and Y-chromosome DNA was used to assess kit performance. Nucleosomal-sized DNA profiles varied among the kits, with prominent multi-nucleosomal-sized peaks present in urine and plasma DNA isolated by kits J and M only. Kit J recovered significantly more spike-in DNA compared with kit M or Q (p<0.001) from urine, and similar amounts from plasma (p=0.12). Applying DNApure to kit M- and Q-isolated DNA significantly improved the amplification efficiency of spike-in DNA from urine (p<0.001) and plasma (p≤0.013). Furthermore, kit J isolated significantly more Y-chromosome DNA from PG urine compared to kit Q (p=0.05). We conclude that DNApure provides an efficient means of improving the yield and purity of cfDNA and minimizing effects of pre-analytical biospecimen variability on liquid biopsy assay performance.

scholarly journals Use of Magnetic Beads for Plasma Cell-free DNA Extraction: Toward Automation of Plasma DNA Analysis for Molecular Diagnostics

2003 ◽  
Vol 49 (11) ◽  
pp. 1953-1955 ◽  
Author(s):  
Christine Stemmer ◽  
Michèle Beau-Faller ◽  
Erwan Pencreac’h ◽  
Eric Guerin ◽  
Anne Schneider ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Plasma Cell ◽  
Molecular Diagnostics ◽  
Magnetic Beads ◽  
Dna Analysis ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Free Dna

scholarly journals Predominant Hematopoietic Origin of Cell-free DNA in Plasma and Serum after Sex-mismatched Bone Marrow Transplantation

2002 ◽  
Vol 48 (3) ◽  
pp. 421-427 ◽  
Author(s):  
Yanni YN Lui ◽  
Ki-Wai Chik ◽  
Rossa WK Chiu ◽  
Cheong-Yip Ho ◽  
Christopher WK Lam ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Marrow Transplantation ◽  
Buffy Coat ◽  
Plasma Samples ◽  
Plasma Dna ◽  
Serum Samples ◽  
Cell Free Dna ◽  
Dna Concentration ◽  
Free Dna

Abstract Background: Despite current interest in the biology and diagnostic applications of cell-free DNA in plasma and serum, the cellular origin of this DNA is poorly understood. We used a sex-mismatched bone marrow transplantation model to study the relative contribution of hematopoietic and nonhematopoietic cells to circulating DNA. Methods: We studied 22 sex-mismatched bone marrow transplantation patients. Paired buffy coat and plasma samples were obtained from all 22 patients. Matching serum samples were also obtained from seven of them. Plasma DNA, serum DNA, and buffy coat were quantified by real-time PCR of the SRY and β-globin gene DNA. To investigate the effects of blood drawing and other preanalytical variables on plasma DNA concentrations, blood samples were also collected from 14 individuals who had not received transplants. The effects of blood sampling by syringe and needle, centrifugation, and time delay in blood processing were studied. Results: The median percentage of Y-chromosome DNA in the plasma in female patients receiving bone marrow from male donors (59.5%) differed significantly (P &lt;0.001) from that in the male patients receiving bone marrow from female donors (6.9%). This indicated that plasma DNA in the bone marrow transplantation recipients was predominantly of donor origin. Compared with paired plasma samples, serum samples had a median 14-fold higher DNA concentration, with the additional DNA being of donor origin. Control experiments indicated that none of the three tested preanalytical variables contributed to a significant change in cell-free DNA concentration. Conclusions: After bone marrow transplantation, the DNA in plasma and serum is predominantly hematopoietic in origin. Apart from the biological implications of this observation, this finding suggests that plasma and serum can be used as alternative materials for the study of postbone marrow transplantation chimerism.

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