Handling cfDNA

Preanalytical handling and cfDNA stability

Various biofluids, including blood serum or plasma and urine, can be used for cfDNA analysis. The choice of sample type depends on the research objective and disease context. 

Analysis of cfDNA in serum and plasma samples is vital to many research applications, including the evaluation of the fetal DNA fraction in maternal blood or the examination of circulating tumor DNA.

Avoiding cellular DNA contamination

Obtaining accurate results requires appropriate collection and isolation methods that ensure preservation of the sample’s actual cfDNA profile. Since cfDNA is only present in blood in low amounts, its level can dramatically change if it becomes diluted with genomic DNA from apoptotic or lysed cells from improper handling.

Therefore, you should take measures to prevent lysis of cells in all steps, from collecting whole blood to preparing serum and plasma. Failure to do so may prevent the subsequent detection of low-abundance cfDNA.

Cellular DNA contamination will influence all quantifications of cfDNA yield, except for capillary electrophoresis analysis and similar methods that assess DNA fragment sizes. Cellular contamination can be indirectly quantified by quantitative PCR using both a short and a long target amplicon, where the long assay will only detect cellular DNA (1).

Serum or plasma – which is preferred?

Both serum and plasma samples have been used successfully for biomarker discovery. However, with some serum preparation tubes, significant contamination of the cfDNA fraction with genomic DNA can occur.

Therefore, plasma is the preferred sample type for cfDNA analysis, and most ctDNA studies to date have also been carried out with plasma samples (2).

Collection and stabilization

Using dedicated, high-quality blood collection tubes increases cell-free DNA stability and minimizes negative impacts on downstream experiments. At QIAGEN, we recommend PAXgene Blood ccfDNA Tubes, which stabilize cfDNA in whole blood samples and reduce hemolysis and variability from different shipping or storage conditions (Figures “PAXgene Blood ccfDNA stabilization minimizes hemolysis compared to alternative solutions“ and „PAXgene Blood ccfDNA stabilization reagent helps prevent release of gDNA into plasma and leaves cfDNA chemically unmodified“). 

PAXgene Blood ccfDNA Tubes are compatible with our offering of cfDNA purification kits to enable a seamless and standardized preanalytical workflow.

Sample storage and transportation

Blood samples should be kept within the optimal temperature range from the time of collection through to cfDNA purification. However, temperature control throughout transport is often impossible, and fluctuations can occur, even with cooling packs. 

If you use PAXgene Blood ccfDNA Tubes, blood samples can be stored for up to 10 days at temperatures up to 25°C, seven days at temperatures up to 30°C or three days at temperatures up to 37°C. Do not store blood-filled tubes below 2°C.

Sample handling and pretreatment

It is essential to handle all samples consistently to avoid introducing any technical variation. The blood draw is a critical first step in plasma preparation, and hemolysis can even occur at the time of phlebotomy. Therefore, blood should only be collected by those highly experienced in venipuncture and according to a standardized procedure. Where possible, samples should also be collected at the same time.

To achieve optimal results from archived samples, select only samples collected and processed according to the same protocol. For multi-center studies, bear in mind that different institutions may use different equipment and procedures for sample collection. The sampling method must be consistent throughout the study to minimize any preanalytical variables.

Plasma preparation

Whole blood should be processed immediately after collection into either serum or plasma. If the whole blood is not centrifuged immediately following collection (or after the clotting time, in the case of serum), the samples should be stored at 2–8°C for no longer than 4 hours. Prolonged storage may result in lysis of thrombocytes and contamination of the cfDNA profile.

Standard plasma preparation generates three fractions: red blood cells, an intermediate buffy coat layer containing white blood cells, and plasma. The optimum centrifugation speed, time and temperature depend on the blood collection tube used. We recommend a second high-speed centrifugation step to reduce cellular contamination (3).

Our recommended plasma pre-processing workflow is as follows for blood collected in EDTA/citrate tubes or PAXgene Blood ccfDNA Tubes.

Plasma preparation using EDTA/citrate tubes

  1. Store for up to 4 h at 2-8°C:
  2. Centrifuge for 10 min at 4°C and 1900 x g.
  3. Carefully aspirate the supernatant.
  4. Centrifuge for 10 min at a minimum of 3000 x g., maximum 16,000 x g.
  5. Tip: 3000 x g will remove platelets, cell fragments and apoptotic bodies, while 16,000 x g will remove some of the larger extracellular vesicles.

  6. The supernatant is plasma without cell debris or vesicles.
  7. Extract cfDNA immediately or store plasma at 4-8°C for up to 14 days or at -20°C to -80°C for up to 2 years.

Plasma preparation using PAXgene Blood ccfDNA Tubes

  1. Store for up to 10 days at up to 25°C; up to 7 days at up to 30°C or up to 3 days at up to 37°C.
  2. Centrifuge for 15 min at room temperature and 1600-3000 x g.
  3. Carefully aspirate the supernatant.
  4. Centrifuge for 10 min at room temperature and 1600-3000 x g.
  5. Tip 3000 x g will remove platelets, cell fragments and apoptotic bodies, while 16,000 x g will remove some of the larger extracellular vesicles.

    Tip PAXgene Blood ccfDNA Tubes can also be centrifuged once at 3000 x g only, when used directly for the primary tube handling workflow on the QIAsymphony SP instrument.

  6. The supernatant is plasma without cell debris or vesicles.
  7. Extract cfDNA immediately or store plasma at 4-8°C for up to 14 days or at -20°C to -80°C for up to 2 years.

Screening for hemolysis

A quick and low-cost method to screen serum and plasma samples for hemolysis is to perform spectrophotometric measurements of oxyhemoglobin absorbance at 414 nm. Usually, optical density is scanned from around 200 to 700 nm, and you can use the presence of distinct absorbance peaks at 414 nm to disqualify samples affected by hemolysis from further analysis.

Our recommendations for blood plasma or serum samples:

 ✔ Only use blood collected by highly trained staff

 ✔ Use appropriate blood collection tubes, such as PAXgene Blood ccfDNA Tubes

 ✔ Minimize the time between blood draw and serum or plasma generation

 ✔ Keep sampling methods consistent for multi-center studies or when using archived samples

 ✔ Maintain proper and consistent shipping and storage conditions

 ✔ Screen for hemolysis

Urine is an interesting sample material for cfDNA analysis. In contrast to blood plasma, samples are easier to obtain and sample volume is not usually limiting.

You can use larger volumes of urine for cfDNA extraction increasing the amount of cfDNA available for downstream assays. But, keep in mind that urine has the risk of containing concentrated metabolites that could interfere with PCR- or NGS-based analysis. Stabilization of urine samples after collection is also important.

Urine sample pre-processing for cfDNA analysis

When collecting urine from animals, such as for preclinical studies, consider possible sources of contamination and variation due to sample collection procedures. We recommend standardizing sample handling and storage protocols.

The number of different cell types present in urine may vary between individuals or disease states, or following certain medical treatments, and this can affect the cfDNA analysis of whole urine samples. Therefore, urine samples should be centrifuged to remove cells and other debris.

The resulting supernatant can be used directly for cfDNA extraction or stored at –80°C. It is essential to perform the centrifugation step before freezing, as freezing the urine sample first will lead to lysis of cells and the release of genomic DNA upon thawing.

To analyze cfDNA from urine, it is best to stabilize samples immediately after collection using appropriate collection devices – to prevent DNase activity and release of gDNA from cells.

If no stabilizing agent is available, centrifuge the cells immediately after sample collection to prevent genomic DNA release. We recommend adding Buffer ATL, which contains SDS, to the saved supernatant at a ratio of 1:10 to eliminate DNase activity. After a second low-speed centrifugation step, the cell-free supernatant can be used for cfDNA extraction.

Sample pre-processing of non-stabilized urine

  1. Centrifuge at 1900 x g to remove cells.
  2. Carefully save the supernatant.
  3. Pre-treat with Buffer ATL.
  4. Centrifuge at 1900 x g and remove the pellet.
  5. Extract cfDNA.

Sample pre-processing of stabilized urine

  1. Centrifuge at 1900 x g to remove cells.
  2. Carefully save the supernatant.
  3. Extract cfDNA.
  4. Tip: you can also store the supernatant at room temperature, 4–8°C; –20°C or –80°C.

  1. Johansson, G. et al. (2019) Considerations and quality controls when analyzing cell-free tumor DNA. Biomol. Detect. Quantif. 17.
  2. Keller, L., Belloum, Y., Wikman, H., and Pantel, K. (2021) Clinical relevance of blood-based ctDNA analysis: mutation detection and beyond. Br. J. Cancer 124 (2).
  3. Trigg, R.M., Martinson, L.J., Parpart-Li, S., and Shaw, J.A. (2018) Factors that influence quality and yield of circulating-free DNA: A systematic review of the methodology literature. Heliyon 4 (7).