Healthcare research has been moving towards finding new, noninvasive procedures – commonly referred to as “liquid biopsies” – to replace our current protocols for diagnosis and treatment monitoring. Current protocols have tended to be overly invasive, posing health risks to the patient and decreasing quality of life. This movement has been very prevalent in the areas of noninvasive prenatal testing (NIPT), cancer therapy and disease monitoring. Two important players in noninvasive testing are circulating cell-free DNA (ccfDNA) and circulating tumor cells (CTCs), both of which offer potential as valuable biomarkers.
We’ve already discussed some of the most frequently asked questions about CTCs in a previous post, which you can read here. Now, we want to talk about some of the most frequently asked questions on ccfDNA research.
During our liquid biopsy webinar series, some of QIAGEN’s resident liquid biopsy experts, Dr. Phoebe Loh and Dr. Daniel Groelz, have discussed different blood collection and stabilization systems, such as the PAXgene Blood ccfDNA Tubes. We collected our top 9 most common questions from the webinar and have answered them for you here.
- What is the minimum volume of blood to draw?
With most kits on the market, 8–10 ml of blood is required from each draw to further process the plasma and ccfDNA. Similarly with the PAXgene Blood ccfDNA Tubes, a blood draw of 10 ml is required. This measure is to ensure a 0.15 ml ratio of cell stabilization additive per ml of blood when the evacuated tube is filled correctly.
- When you talk about blood collection, does that mean full blood, serum or plasma?
For sample collection, the PAXgene Blood ccfDNA Tubes are for the collection of whole human blood. Subsequently, you can use a protocol with the tubes to process plasma. ccfDNA can then be purified manually, using the QIAamp Circulating Nucleic Acid Kit – or automatically, using the PAXgene Blood ccfDNA Kit. For more information, please refer to the PAXgene Blood ccfDNA Tube product circular.
- Is it possible to determine the sex of a fetus by taking a maternal blood sample using your technology?
This is something that we have tested, although it is important to note that the PAXgene Blood ccfDNA system is for research use only (RUO). A field tester used the PAXgene Blood ccfDNA Tubes in combination with the QIAamp Circulating Nucleic Acid Kit and the QIAsymphony PAXgene Blood ccfDNA Kit. Maternal blood was collected and the fetal fraction was analyzed in 43 samples. Sex was successfully determined using NGS-based PrenaTest from Lifecodexx. The results were also compared to those from Streck BCT tubes. Results from this analysis showed that the results from the PAXgene Blood ccfDNA procedure were consistent with the current methods. For more details, you can check out our poster: A New Blood Collection Tube and Automated Extraction Method for Stabilization and Analysis of ccfDNA.
- What kind of blood stabilization should be used for DNA extraction and analysis via PCR?
It is preferable if you can prevent DNA modification from crosslinking reagents. The PAXgene Blood ccfDNA Tubes use an alternative, proprietary solution for stabilization that does not include crosslinking. This solution lets you decrease the release of gDNA and minimize hemolysis – effectively preserving the integrity of your sample – while also preventing DNA modification, which can complicate PCR results.
- Is the ccfDNA isolation protocol manual or automated?
Both! The kit to be used would depend on whether you want to automate your isolation or do it manually on the bench, as each method uses different chemistries for ccfDNA extraction. The QIAamp Circulating Nucleic Acid Kit is a manual method using silica membrane-containing columns to extract ccfDNA from plasma samples – either from the PAXgene Blood ccfDNA Tubes or from other alternative collection tubes. This manual procedure calls for a flexible input of 1–5 ml plasma and involves 4 steps: lyse, bind, wash, elute.
For the automated isolation, the QIAsymphony PAXgene Blood ccfDNA Kit is used in conjunction with the PAXgene ccfDNA Tubes. The QIAsymphony ccfDNA Kit includes protocols for using either 2.4 ml or 4.8 ml plasma input. This automated protocol employs a magnetic bead technology that binds the ccfDNA to the bead surface, ensuring a rapid isolation and highly pure ccfDNA sample.
- How can isolated ccfDNA be quantified?
Due to the nature of ccfDNA, ccfDNA can be difficult to quantify. The yield can vary in healthy donors, ranging from 1 to 100 ng per 4 ml plasma; ccfDNA concentration can vary even beyond this range during various disease states. For total quantification, we recommend using quantitative or digital PCR.
- Can you use butterfly needles for blood draw with the PAXgene Blood ccfDNA tubes?
Yes, the PAXgene Blood ccfDNA Tubes can be used with standard blood collection equipment. You can also refer to the PAXgene Blood ccfDNA Tube handbook for more details on sample collection procedures.
- Are DNA methylation states maintained in blood?
Yes, ccfDNA purified with the PAXgene Blood ccfDNA System is compatible with methylation-specific assays that require treatment with bisulfite. In this poster, we also compared the performance of EDTA, Streck and PAXgene Blood ccfDNA Tubes at two time points – 0 days and 6 days – using the Agilent Bioanalyzer. We saw after 6 days that the Streck samples had a 7–8 bp shift in the main ccfDNA peak, which indicates DNA modification by formaldehyde-releasing reagents.
- What downstream applications can you use to analyze ccfDNA?
There are a lot of technologies available to analyze ccfDNA, including: next-generation sequencing, PCR, qPCR, digital PCR and SNP genotyping.
To date, ccfDNA from blood samples collected and processed using the PAXgene Blood ccfDNA System have been successfully tested in NGS applications and in PCR, including multiplex qPCR and ddPCR.