Circulating cell-free DNA

What is cfDNA?

Many studies have shown that elevated levels of cfDNA also known as ctDNA, can be found in the blood of patients suffering from various diseases, especially cancer. cfDNA differs from normal cell-free DNA by the presence of somatic mutations, which can be used as genetic markers to determine therapeutic response and guide treatment decisions. Levels of mutant cfDNA can also be useful for following-up cancer patients and detecting minimal residual disease.

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 cfDNA. 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. cfDNA can then be purified manually, using the QIAamp Circulating Nucleic Acid Kit – or automatically, using the QIAsymphony PAXgene Blood ccfDNA Kit.

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.

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.

Which parameters do I need to consider to ensure cfDNA quality?

The following parameters should be considered when assessing the quality of cfDNA purified from cell-free biofluids:

• The efficiency of the cfDNA purification and the yield
• The size pattern of the extracted cfDNA
• The absence of any inhibitors of downstream enzymatic processes such as PCR or NGS library preparation
• The absence of nucleases
• The presence or absence of gDNA from cellular contamination or hemolysis

When do I need QC?

The amount of cfDNA that can be purified as well as the amount of residual inhibitors that remain after purification can vary from sample to sample. When analyzing cfDNA from standard human serum or plasma samples, we recommend quality control of all samples to monitor yields and absence of PCR inhibitors – and to identify any potential problems in the sample set prior to qPCR or NGS experiments.

For studies involving a very large number of samples, at least a subset of the samples should be quality controlled.

To ensure successful and high-quality urine collection and stabilization, analyze the size of the extracted cfDNA profile. cfDNA will be visible at a fragment size of about 200 bp.