How to optimize the preanalytical portion of your circulating cell-free DNA (cfDNA) studies


Advances in liquid biopsy are allowing new strides in studies involving circulating cell-free DNA (cfDNA).

With initial use in the prenatal testing field, cfDNA began to tell its story in fetal DNA genetic testing and has offered an alternative to the invasive and risk-prone amniocentesis.

Now, it is breaking barriers in the field of cancer research with an increasing number of studies accessing biomarkers through non-invasive blood draw rather than traditional biopsy methods (e.g., those requiring tissue or lung lavage).

Developing technologies have the potential to revolutionize cancer and transplant care through the analysis of cfDNA for screening and monitoring purposes. However, as these new cfDNA methods filter into the laboratories, sifting through the available technologies to set up a workflow for a new type of analyte can pose a new set of challenges and requires some effort and learning.

Workflow challenges for circulating cfDNA studies

Circulating cell-free DNA analysis comes with its own set of challenges. As with the study of any analyte, one of the most important steps in the cfDNA workflow is taking good care of your starting material. This not only promotes success of your analysis (e.g., good DNA yield for your downstream application) – but it also ensures the results you report are accurate.

Recovery of cfDNA requires appropriate sample collection processing and effective isolation methods made specifically for circulating DNA, as cfDNA is present in low abundance in the blood. In addition, the cfDNA fraction can change dramatically after blood collection, as apoptotic white blood cells release genomic DNA and further dilute the cfDNA profile.

Blood collection for circulating cfDNA analysis: your workflow options

There are generally two options when considering blood collection for cfDNA analysis:

1. Using EDTA blood collection tubes (general purpose anti-coagulant tubes)
2. Using specialized blood collection tubes made to stabilize circulating cfDNA levels in whole blood

Significant workflow differences exist between these two options.

Using EDTA tubes requires cooling at 4°C after collection, and plasma must be separated within about 4 hours to avoid any changes in the cfDNA profile. This option can work if the blood collection site is in the same facility as the laboratory – or if a tightly regulated logistics system is possible for transport and processing of blood samples.

In many cases, however – such as when samples must be collected from multiple satellite physician offices – these conditions cannot be met, and the use of EDTA tubes is not an option.

In such cases, the only alternative option is to use a cfDNA stabilization tube, which allows room temperature transport and storage of blood samples for several days until plasma can be separated.

How to choose circulating cfDNA stabilization tubes

Currently, there is a small selection of cfDNA stabilization tubes available on the market. Tube selection can impact the number of samples you must collect, your workflow time and the overall cost of processing.

When choosing the right cfDNA stabilization tubes for your needs, there are a few key attributes to keep in mind:

• The material of the collection tube
• The stabilization reagent’s performance
• The stabilization reagent’s chemistry compatibility with your downstream application
• The ease of integrating the generated plasma into your chosen isolation protocols

Material of the collection tube

When comparing glass and plastic blood collection tubes, keep in mind the frequency of potential tube breakage – not only in transport but also in centrifugation steps, where cleaning up a broken blood tube can require significant efforts. Most who use glass tubes collect two tubes from each donor in order to have a backup, in the event of any lost samples during sample transport and processing.

Since the amount of blood collected relies on the vacuum in the collection tube, the vacuum technology of various tube types can vary and may result in faster or slower collection times. This variance can translate into more incidences of inadequately filled tubes if blood flow near the end of collection is so slow that the phlebotomist stops the collection prematurely.

Also take into consideration the type of closure on the tube. Modern blood collection tube technology now provides the option of a stopper containing an outer shield. This option helps protect personnel working with the samples and minimizes cross-sample contamination due to blood splatter when opening the tubes.

Performance of the stabilization reagent

Stabilization performance of the reagent inside the tube is also of great importance. Most tubes offer a similar window of room temperature stabilization with limited tolerance of higher temperatures – although this temperature range can vary slightly.

Compatibility with downstream application

It’s important to also consider what type of downstream analysis are you planning. Does the reagent contain a formaldehyde releaser that may cause cross-linking of nucleic acids? Tubes containing this type of reagent chemistry are likely not suitable for methylation-based assays, such as those used in cancer research.

The ability to prevent hemolysis (rupture of red blood cells) also varies between stabilization tubes. Higher hemolysis levels result in less defined separation of plasma, buffy coat and hematocrit. This effect could cause inaccurate separation of plasma and deposit background gDNA in your downstream analysis.

Hemolysis levels will also determine how many centrifugation steps are required to achieve adequate phase separation for plasma removal. A tube with effective hemolysis prevention could result in the ability to cut out a second centrifugation step and could expedite your workflow. For those wishing to also analyze genomic DNA in the hematocrit after removal of plasma, a tube allowing effective plasma separation is recommended.

Integration with your isolation protocols

Finally, keep in mind how well your tube selection will integrate into cfDNA isolation protocols.  Some tube/isolation kit combinations may require a pretreatment step – such as a proteinase K step, for example. Other combinations may require a slight variation to core protocols – such as doubling the incubation time during the lysis step.

While pretreatments and protocol alterations can add time and cost to the core isolation procedure, there are collection tubes on the market that integrate smoothly into companion manual and automated cfDNA isolation without any required add-ons for optimal results. Be sure to keep these options in mind when choosing your pre-analytical workflow.

As technology advances, there are more and more options for selecting workflows that are tailored to suit your specific needs. By paying attention to product features and protocol details, you have the opportunity to save on time and costs in your cfDNA workflow, and to ensure your ability to deliver accurate results.

Where do I go from here?

Just getting started in your studies involving circulating cfDNA? The new PAXgene Blood ccfDNA Tubes are plastic collection tubes that provide stabilization and transport of your whole blood samples. To stabilize blood cells, the tubes use a non-crosslinking solution to prevent the release of any gDNA into your sample and stabilize circulating cfDNA levels. This solution is applicable to a variety of downstream applications, whether you are doing NGS, PCR or SNP genotyping.

Once samples are ready to process, you can use the QIAamp Circulating Nucleic Acid Kit or the automated QIAsymphony PAXgene Blood ccfDNA Kit to isolate out your circulating cfDNA.

If you’re looking to improve the analysis and interpretation of your circulating cell-free DNA studies, QIAGEN is presenting a four-part webinar series on “Circulating cell-free DNA (cfDNA) as liquid biopsy” this month. Part 4 in this series will be presented on August 22 and will focus on analysis and interpretation of cell free DNA. Held by one of our experts at the QIAGEN Bioinformatics Clinical Program, this webinar is not to be missed!

Learn more and register for the webinar today!

The applications presented here are for research use only. Not for use in diagnostic procedures.

Phoebe Loh

Phoebe Loh received a degree in Biochemistry from the University of Oklahoma where she began her career in genomics, taking part in the sequencing effort of the Human Genome Project. Her research involvement centered on sequencing and identifying genes of the cat eye syndrome region of human chromosome 22q and further continued in comparative genomic studies and oligonucleotide synthesis. She moved onto to work on siRNA synthesis working at Dharmacon, and then studying the mechanisms of cellular trafficking and gene transport at the University of Rochester Medical Center. Upon first joining QIAGEN she managed an RNA isolation portfolio and now currently oversees product management and marketing of PAXgene products which offer stabilization and preservation of biomolecules and their profiles in blood, tissue, and bone marrow specimens.

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