Pathology & Oncology Research

, Volume 25, Issue 3, pp 915–923 | Cite as

Blood Collection and Cell-Free DNA Isolation Methods Influence the Sensitivity of Liquid Biopsy Analysis for Colorectal Cancer Detection

  • Barbara Kinga BartákEmail author
  • Alexandra Kalmár
  • Orsolya Galamb
  • Barnabás Wichmann
  • Zsófia Brigitta Nagy
  • Zsolt Tulassay
  • Magdolna Dank
  • Péter Igaz
  • Béla Molnár
Original Article


During colorectal cancer (CRC) development tumor-derived cell-free DNA (cfDNA) can be released into the bloodstream. Many different cfDNA isolation methods and specific blood collection tubes preventing the release of genomic DNA and stabilizing cfDNA with preservative reagents became available. These factors may affect greatly on the further liquid biopsy analyses. Our aim was to test different blood collection tubes and cfDNA isolation methods to determine whether these factors influence the cfDNA amount and the promoter methylation of four previously described hypermethylated biomarkers. Three manual isolation methods (High Pure Viral Nucleic Acid Large Volume Kit; Epi proColon 2.0 Kit; Quick-cfDNA™ Serum & Plasma Kit) and automated sample preparation systems (InviGenius and InviGenius PLUS) were examined. Furthermore, K3EDTA Vacuette tubes and Streck Cell-Free DNA BCT® tubes were compared. After cfDNA isolation and bisulfite conversion of samples, the methylation level of SFRP1, SFRP2, SDC2, and PRIMA1 were defined with MethyLight assays. We have ascertained that there are differences between the cfDNA amounts depending on the isolation methods. Higher cfDNA yield was observed using InviGenius system than column-based manual isolation method; however, InviGenius PLUS has produced lower cfDNA amounts. No remarkable variance could be found between K3EDTA and Streck tubes; slightly higher cfDNA quantity was detected in 60% of plasma samples using Streck tubes. In point of methylation level and frequency, manual column-based isolation produced more consistent results. Automated cfDNA extraction systems are easy-to-use and high-throughput; however, further improvements in the isolation protocols might lead to the increase of the sensitivity of further methylation analysis.


Liquid biopsy Plasma cfDNA isolation Blood collection Colorectal cancer DNA methylation 



This study was supported by the ÚNKP 16-3 New National Excellence Program of The Ministry of Human Capacities (ÚNKP-16-4-DSZ-2016-0010), by the National Research, Development and Innovation Office (KMR-12-1-2012-0216 grant) and by the Hungarian Scientific Research Fund (OTKA-K111743 grant).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no competing interests.


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Copyright information

© Arányi Lajos Foundation 2018

Authors and Affiliations

  • Barbara Kinga Barták
    • 1
    Email author
  • Alexandra Kalmár
    • 1
  • Orsolya Galamb
    • 2
  • Barnabás Wichmann
    • 2
  • Zsófia Brigitta Nagy
    • 1
  • Zsolt Tulassay
    • 1
    • 2
  • Magdolna Dank
    • 3
  • Péter Igaz
    • 1
    • 2
  • Béla Molnár
    • 2
  1. 1.2nd Department of Internal MedicineSemmelweis UniversityBudapestHungary
  2. 2.Molecular Medicine Research GroupHungarian Academy of SciencesBudapestHungary
  3. 3.Department of Clinical OncologySemmelweis UniversityBudapestHungary

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