Analyzing circulating cell free DNA (ccfDNA) offers a non-invasive, dynamic window into the body’s physiological state and its use as a biomarker is gaining rapidly. However, multiple challenges remain with establishing its routine application. This is mainly due to its low levels, high rates of fragmentation, and preanalytical variabilities associated with sample collection, cfDNA extraction and quantification. Here, we go through some tips on how one can establish a reliable cfDNA workflow from plasma samples.
1 – Sample Collection
Cell free DNA is found in various body fluids and can be shed from cells undergoing apotosis or necrosis, or actively released from live cells1. Plasma is the most well-studied sample type and is recommended over serum as the latter tends to experience higher genomic DNA contamination attributed to white blood cell (WBC) lysis during the clotting process2. Depending on the application, sample volume required should be established beforehand. Striving to detect low frequency mutations in circulating tumour DNA (ctDNA) for example, may require higher plasma input volumes which may necessitate drawing a higher volume of blood.
To minimize genomic DNA contamination when isolating cfDNA from plasma,
- Minimize cell lysis during blood drawing e.g., use appropriate needle size, avoid prolonged tourniquet
- Avoid harsh temperature changes and excessive agitation when storing or transporting blood tubes
- Store isolated plasma at -80°C if not using immediately and avoid freeze thaw cycles
2 – Preparation of Plasma
It is recommended to prepare the plasma as fresh as possible to avoid contamination of cfDNA with genomic DNA which is released from ruptured blood cells. When using EDTA blood tubes3, isolate plasma within 6 h of blood collection. A later timepoint might be possible when using specialized cell free blood collection tubes containing stabilizers (please refer to tube manufacturer’s instructions).
A double centrifugation protocol is recommended during plasma preparation to minimize the potential for carry over of cells and genomic DNA. This involves centrifuging the blood sample twice at different speeds to separate the plasma fraction from the cellular fraction, which contains genomic DNA. The first centrifugation step is typically done at a lower speed to separate the red blood cells and white blood cells from the plasma, and the second step is done at a higher speed to further separate any remaining cellular material from the plasma. This can help reduce the risk of contamination of plasma with genomic DNA, which is important for downstream analysis of cell-free DNA, such as in the case of cfDNA analysis.
Our JANUS® G3 Blood iQ™ workstation is a state-of-the-art laboratory automation system designed to streamline the genetic analysis of cfDNA, cfRNA, and genomic DNA from fractionated blood.
The proprietary imaging technology used in the JANUS® G3 Blood iQ™ workstation allows for precise identification of the plasma and buffy coat layers in centrifuged blood tubes.
By automating the sample preparation process, one can:
- Increase of efficiency, reduce errors, and improve the traceability of samples throughout the workflow
- Accurately detection of the interface between the plasma and cellular components of the blood sample which is critical for effective fractionation of the blood and subsequent analysis of cell-free DNA
- Ensure reliable results and reduce the risk of contamination or loss of sample material during the fractionation process
- More efficient processing of larger numbers of samples, which can be particularly important for high-throughput applications
3 – Extraction of cfDNA from Plasma
Extraction of cfDNA typically relies on silica columns or magnetic beads to concentrate cfDNA from large plasma volumes, followed by several wash steps and elution in a smaller buffer volume for downstream processing. There have been studies comparing commercially available kits which report varying yields and quality4. However, these results are also influenced by variabilities in sample source, handling steps and quantification procedures across labs.
We tested the chemagic™ cfDNA extraction kits5, based on patented chemagic™ M-PVA Magnetic Beads, on the chemagic 360 instrument and obtained comparable and consistent yields to a competitor’s manual silica column and vacuum-based method.
As different extraction kits have reportedly variable extraction efficiencies based on DNA fragment length, we also analyzed fragmentation of obtained cfDNA by PCR using the KAPA™ Human Genomic DNA Quantification and QC Kit from Roche. Fragmentation/degradation scores with the chemagic extraction kits were in the range of (0.16 – 0.37) which was comparable to the results gained with the silica membrane-based competitor kit on the same samples (degradation scores 0.15 – 0.4).
cfDNA was isolated from 1 ml, 2 ml and 5 ml from two donors both with the chemagic kits on the chemagic 360 instrument and manually with competitor. For cfDNA analysis, a short fragment (115 bp) from a consensus sequence with abundant genomic ALU repeats was amplified. Exemplary data from donor 2 shows that the yield of cfDNA is scalable to sample input.
By automating cfDNA extraction with chemagic™ technology, one can:
- Increase sample throughput while maintaining comparable yields to manual methods
- Reduce hands-on steps and speed up workflow (< 2 h for up to 96 samples with on-board lysis and no heating required)
- Enable a wider range of sample inputs (0.5 to 18 ml applicable)
- Maintain sample integrity with sample tracking options (barcode reading and bidirectional LIMS communication capability)
- Reduce running cost per sample
- Increase downstream success with various assays such as digital droplet PCR (ddPCR), Next-Generation Sequencing (NGS), quantitative PCR (qPCR) etc. (see references)
As cfDNA levels vary widely between samples, one may also include an exogenous DNA control in samples to monitor extraction efficiency6.
4 – Quantification and Quality Control of Extracted cfDNA
Before cfDNA is subject to downstream sequencing, quality control checks are often performed to evaluate the quality of extracted cfDNA. As cfDNA yields isolated from human plasma samples are typically low in the range of 1 – 30 ng/mL of plasma, this may lie outside the detection parameters of common spectrophotometric assays and the use of cfDNA dedicated assay with high detection sensitivity is required. Hence, if quantification of the extracted cfDNA is required, a PCR-based method (e.g. qPCR or ddPCR) or an electrophoretic method dedicated to cfDNA measurement is recommended.
When using common fluorometric quantification methods, such as the Thermo Fisher® Scientific Qubit® fluorometer, the addition of Poly(A) RNA is essential for reliable performance. Fluorometric analysis of eluates extracted without the use of Poly(A) RNA may lead to varying and decreased quantification data. Additionally, care should be taken in using fluorometric methods for quantification as not only cfDNA but total DNA is measured.
To assess fragment distribution and quantitation of the extracted cfDNA samples, electrophoresis-based separation and quantitation systems, such as LabChip® GX Touch™ HT Nucleic Acid Analyzer may be used. On running the LabChip® cfDNA assay, a major peak at around ~170 bp is expected for high-quality mono-nucleosomal cfDNA, and in some cases, a smaller peak at around ~350 bp representing di-nucleosomal cfDNA smear can be seen in the electropherogram. PerkinElmer’s cfDNA assay uses 50 base pair DNA fragment that migrates close to the cfDNA peaks as a reference for quantification and can be used for reliable quantitation and Quality control of the cfDNA samples.
Our recommended cfDNA quantitation procedure involves:
- qPCR using an ALU115 primer set where a short fragment (115 bp) from a consensus sequence with abundant genomic ALU repeats was amplified
- other qPCR/ddPCR-based assays targeting “housekeeping” genes found in cfDNA which have been experimentally validated7
- Electrophoresis based assay dedicated to cfDNA quantitation and Quality control such as LabChip® GX Touch cfDNA assay
- Fluorometric approaches may be used for qualitative assessment of cfDNA but may lead to variable quantification results
Extracted cfDNA from chemagic cfDNA extraction kits have been successfully put through library preparation with the NEXTFLEX® Cell Free DNA-Seq Library Prep Kit 2.0. The option to automate this process can further contribute to a reliable cfDNA workflow by minimizing the risk of errors while increasing operational capacity8.
- van der Vaart, M., & Pretorius, P. J. (2007). The Origin of Circulating Free DNA. Clinical Chemistry, 53(12), 2215–2215. https://doi.org/10.1373/CLINCHEM.2007.092734
- Wong, F. C. K., Sun, K., Jiang, P., Cheng, Y. K. Y., Chan, K. C. A., Leung, T. Y., Chiu, R. W. K., & Lo, Y. M. D. (2016). Cell-free DNA in maternal plasma and serum: A comparison of quantity, quality and tissue origin using genomic and epigenomic approaches. Clinical Biochemistry, 49(18), 1379–1386. https://doi.org/10.1016/J.CLINBIOCHEM.2016.09.009
- Merker, J. D., Oxnard, G. R., Compton, C., Diehn, M., Hurley, P., Lazar, A. J., Lindeman, N., Lockwood, C. M., Rai, A. J., Schilsky, R. L., Tsimberidou, A. M., Vasalos, P., Billman, B. L., Oliver, T. K., Bruinooge, S. S., Hayes, D. F., & Turner, N. C. (2018). Circulating tumor DNA analysis in patients with cancer: American society of clinical oncology and college of American pathologists joint review. Journal of Clinical Oncology, 36(16), 1631–1641. https://doi.org/10.1200/JCO.2017.76.8671
- Lampignano, R., Consortium, for the I. M. I. C.-I., Neumann, M. H. D., Consortium, for the I. M. I. C.-I., Weber, S., Consortium, for the I. M. I. C.-I., Kloten, V., Consortium, for the I. M. I. C.-I., Herdean, A., Consortium, for the I. M. I. C.-I., Voss, T., Consortium, for the I. M. I. C.-I., Groelz, D., Consortium, for the I. M. I. C.-I., Babayan, A., Consortium, for the I. M. I. C.-I., Tibbesma, M., Consortium, for the I. M. I. C.-I., Schlumpberger, M., … Consortium, for the I. M. I. C.-I. (2020). Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows. Clinical Chemistry, 66(1), 149–160. https://doi.org/10.1373/CLINCHEM.2019.306837
- Technical Note: Automated Circulating Cell-Free DNA Purification with the chemagic™ 360 Instrument. Technical Note: Automated Circulating Cell-Free DNA Purification with the chemagic™ 360 Instrument
- Pallisgaard, N., Spindler, K. L. G., Andersen, R. F., Brandslund, I., & Jakobsen, A. (2015). Controls to validate plasma samples for cell free DNA quantification. Clinica Chimica Acta, 446, 141–146. https://doi.org/10.1016/J.CCA.2015.04.015
- Lefèvre, A. C., Pallisgaard, N., Kronborg, C., Wind, K. L., Krag, S. R. P., & Spindler, K. L. G. (2021). The clinical value of measuring circulating hpv dna during chemo-radiotherapy in squamous cell carcinoma of the anus. Cancers, 13(10). https://doi.org/10.3390/cancers13102451
- Application Note: High-Throughput, End-to-End Cell Free DNA Analysis Workflow from Plasma. https://chemagen.com/wp-content/uploads/2022/01/cfDNA-App-Note-AG012112_10_AP-v4.pdf