Lab Med Online.  2019 Apr;9(2):45-56. 10.3343/lmo.2019.9.2.45.

Effects of Pre-analytical Variables on Cell-free DNA Extraction for Liquid Biopsy

• Jeong TD
• Kim MH
• Park S
• Chung HS
• Lee JW
• Chang JH
• Huh J

• KMID: 2441823
• DOI: http://doi.org/10.3343/lmo.2019.9.2.45

Abstract

BACKGROUND
Extraction of cell-free DNA (cfDNA) is a key step for determining the quality of cfDNA-related molecular diagnostics. We evaluated the effect of sample containers and sample storage conditions on cfDNA extraction.
METHODS
The cfDNA extraction using the MagMAX Cell-Free DNA Isolation Kit from five healthy controls and five lung cancer patients was evaluated according to the type of sample container and storage conditions: K2-EDTA container, <1, 6, 24, and 48 hr storage at 4℃ after immediate plasma separation; and Cell-Free DNA BCT container, <1, 3, 7, and 14 days stored at room temperature. Mutation analysis of EGFR exons 18-21 was performed. To assess the effect of a delay in centrifugation, EDTA whole blood samples from five healthy individuals were stored at 4℃ for 6, 12, and 24 hr before plasma separation.
RESULTS
There was no significant difference in the amount and nucleic acid size of cfDNA in both controls and patients with cancer when EDTA plasma was stored at 4℃ up to 48 hr. The amount and size of cfDNA in the BCT container were not different up to 7 days; however, the 14-day sample showed an increase in cfDNA concentration due to genomic DNA contamination. EGFR mutations were detected on EDTA containers up to 48 hr and with BCT containers up to 14 days. When EDTA whole blood was stored at 4℃ and plasma separation was delayed, the cfDNA concentration increased from 24 hr.
CONCLUSIONS
The cfDNA extraction was affected by the sample containers and storage conditions.

Keyword

Cell-free DNA; Extraction; Storage; Container; EGFR

MeSH Terms

Biopsy*
Centrifugation
DNA Contamination
DNA*
Edetic Acid
Exons
Humans
Lung Neoplasms
Pathology, Molecular
Plasma
DNA
Edetic Acid

Figure

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  Fig. 1. Schematic flow of the study design. (A) Venous whole blood (WB) was drawn from 10 subjects (healthy controls, N=5; lung cancer patients, N=5). The cell-free DNA (cfDNA) was extracted under different specimen storage conditions in K2-EDTA at 4°C and Cell-Free DNA BCT containers at room temperature (RT). (B) Venous blood was drawn from five healthy subjects in K2-EDTA containers, and the cfDNA was extracted under different centrifugation conditions.

• 

  Fig. 2. Comparison of cell-free DNA (cfDNA) concentrations according to specimen container in healthy controls and patients with lung cancer. The cfDNA concentration of patients with lung cancer was significantly higher than that of healthy controls. In the healthy control group, the mean concentration of cfDNA in the K2-EDTA container was significantly higher than that of the Cell-Free DNA BCT containers.

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  Fig. 3. Comparison of cfDNA concentration and size according to storage conditions in healthy controls and patients with lung cancer. (A) Changes in cfDNA concentration over time in EDTA plasma. There were no significant differences in cfDNA concentration up to 48 hr in EDTA plasma stored at 4˚C. ∗P >0.05 (one way repeated measures ANOVA or Friedman test, N=15). (B) Example of size distribution of nucleic acids in EDTA plasma in a healthy control (case number: HC04). No significant differences were observed according to time. (C) Example of size distribution of nucleic acids on EDTA plasma in a patient with lung cancer (case number: LC09). No significant differences were observed according to time. (D) Changes of cfDNA concentrations over time in BCT containers. There were no statistically significant differences in cfDNA concentration up to 14 days in BCT stored at room temperature (RT). On day 14, an increase in cfDNA concentration was observed. ∗P >0.05 (one way repeated measures ANOVA or Friedman test, N=15). (E) Example of size distribution of nucleic acids in BCT in a healthy control (case number: HC04). (F) Example of size distribution of nucleic acids in BCT in a lung cancer patient (case number: LC09).

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  Fig. 4. Comparison of cell-free DNA (cfDNA) in EDTA containers depending on time of centrifugation. X-axis represents centrifugation condition (A, immediate: immediate plasma separation from EDTA whole blood and the plasma stored at 4˚C; B, delayed: stored EDTA whole blood at 4˚C and delayed plasma separation). (A) The concentration of cfDNA increased when EDTA whole blood was stored at 4˚C for more than 24 hr. (B) Example of size distribution of nucleic acids in EDTA plasma over time in healthy control (case number: HC13). No significant differences were observed according to storage time. (C) Example of size distribution of nucleic acids in EDTA whole blood over time in healthy control (case number: HC13). Large-sized nucleic acids were observed with increasing storage time.

Reference

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