Cell-free DNA illustrationOptimum plasma sample collection and handling for cell-free DNA liquid biopsies

A recent publication indicates the best way to preserve intact cell-free DNA for any minimally-invasive genomic assay

Liquid biopsy, whether examining cell-free DNA or circulating tumor cells (CTCs), is a very promising approach for non-invasive cancer management. In particular, cell-free DNA approaches in plasma are expanding their repertoire of analytes to include MSI immunotherapy markers from blood plasma, to include a measurement of Tumor Mutational Burden (TMB) from circulating tumor DNA, and even a "'Blood First' Cancer Testing Paradigm" as applying liquid biopsy as a replacement for invasive lung cancer biopsy.

There are practical pre-analytical challenges to the implementation of new 'liquid biopsy' assays that examine circulating cell-free DNA. Here we will address post-collection sample handling and plasma preparation; in a later article we will address purification of cell-free DNA from collected plasma samples.

Standard sample handling for cell-free DNA

The importance of standardized sample handling in the context of cell-free DNA analysis cannot be overstated. The amount of circulating tumor DNA (and circulating tumor methylated DNA) in the bloodstream is only a small fraction; indeed at the 2017 FDA/AACR Liquid Biopsy Meeting both Foundation Medicine and Guardant Health indicated the median allele-frequency of the mutations they detect are on the order of 0.5%. One-half of one percent is 1 in 200 parts; this approaches the accuracy of the sequencing itself, and special techniques such as molecular barcoding and error-correcting schemes have to be utilized.

If the samples are not handled correctly post blood collection, the amount of leukocyte lysis will occur, effectively diluting the rare signal present, making detection all the more difficult.

The practicalities of a clinical setting

In a hospital environment where blood samples are collected and processed, the clinical laboratory may be accustomed to running different kinds of requirements for different kinds of blood-based tests. For protein analytes, the sample is serum (blood allowed to clot at room temperature for 15-30 minutes, followed by centrifugation). For plasma, different preservatives are commonly found, including heparin (green top), citrate (light blue top), or EDTA (lavender top), depending on the specific downstream test to be performed.

For plasma preparation, centrifugation removes cells at 1,000 - 2,000 xg for 10 minutes while a 15 minute spin at 2,000 xg depletes platelets, and samples frozen at -20C afterward.

There are no 'standard' procedures for EDTA tubes

In the hospital environment, while there are rules and limitations, if there aren't rules you can expect plenty of variation. For example, in plasma preparation you can use one of the aforementioned collection tubes: citrate, heparin or EDTA, and all can be processed to obtain plasma. However, only one is suitable for cell-free DNA work, and that is EDTA ("lavender top").

In addition, how long is allowed before centrifugation, given the often-irregular nature of blood collection? What effect would refrigeration have on the quality of the cell-free DNA? Is a second high-speed centrifugation (at 16,000 xg) absolutely required, in an environment where a centrifuge that can pull higher speeds may not be readily available?

Recent publication in the November 2018 Journal of Molecular Diagnostics

A review of the literature (going back to 2001) indicates whole-blood samples be processed as soon as possible after collection, and then centrifuged twice with the second time at high-speed in order to clear cellular debris.

A recent paper published in the November 2018 Journal of Molecular Diagnostics, titled "Effects of Collection and Processing Procedures on Plasma Circulating Cell-Free DNA from Cancer Patients" answered several questions about these pre-analytical variables. Using digital PCR, targeted amplicon sequencing, and low-pass whole-genome sequencing, they assessed the effect of different room-temperature and 4C incubation times before centrifugation, the effect of lower-speed centrifugation for the second spin, and the use of a proprietary Streck collection tube.

Findings and recommendations

By using actual cancer patient samples and digital PCR to measure the absolute amount of mutant allele fractions, they were able to generate a robust set of statistics with distributions; a total of n=231 samples from a total of 62 cancer patients were used for this study.

For EDTA tubes, processing within 6 hours of blood collection is recommended; a detectable rise in background genomic DNA was observed past that point, significantly so 48 and 96 hours post-collection. Interestingly the absolute amount of ctDNA molecules did not decrease over that timeframe.

A second finding, on the second centrifugation spin speeds (14,000 xg versus 3000 xg), the optimim centrifugation was a 10 minute first spin at 820 xg followed by a 10 minute second spin at 14,000 xg. (Refer to Figure 4 in the paper for details.)

A third finding, on the usage of Streck Blood Collection Tubes, indicated that use of such tubes for room-temperature stabilization maintained integrity and levels of ctDNA and background DNA for up to a week.

These recommendations would apply for circulating cell-free DNA assays for minimally-invasive cancer monitoring and therapy guidance, as well as for early detection assays including Singlera's PanSeer and ColonES assays, in addition to non-invasive prenatal testing (NIPT) assays offered for maternal health.


  1. Chiu RW and Lo YM et al. Clin Chem (2001) Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma. PMID: 11514393
  2. Risberg B and Gale D et al. J Mol Diagn (2018) Effects of Collection and Processing Procedures on Plasma Circulating Cell-Free DNA from Cancer Patients. PMID: 30165204