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

Tae-Dong Jeong; Mi Hwa Kim; Sholhui Park; Hae-Sun Chung; Jin Wha Lee; Jung Hyun Chang; Jungwon Huh

doi:10.3343/lmo.2019.9.2.45

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Jeong, Kim, Park, Chung, Lee, Chang, and Huh: Effects of Pre-analytical Variables on Cell-free DNA Extraction for Liquid Biopsy

Original Article

Lab Med Online 2019;9(2):45-56.

Published online: 20 April 2019

DOI: https://doi.org/10.3343/lmo.2019.9.2.45

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

Tae-Dong Jeong, M.D.^1,, Mi Hwa Kim, M.T.¹, Sholhui Park, M.D.¹, Hae-Sun Chung, M.D.¹, Jin Wha Lee, M.D.², Jung Hyun Chang, M.D.², Jungwon Huh, M.D.¹

¹Department of Laboratory Medicine, College of Medicine, Ewha Womans University, Seoul, Korea

²Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, College of Medicine, Ewha Womans University, Seoul, Korea

Corresponding author: Tae-Dong Jeong, M.D., Ph.D. https://orcid.org/0000-0002-3873-3117 Department of Laboratory Medicine, College of Medicine, Ewha Womans University, 260 Gonghang-daero, Gangseo-gu, Seoul 07804, Korea Tel: +82-2-6986-3386, Fax: +82-2-6986-3389, E-mail: tdjeong@ewha.ac.kr

Received 23 March 2018 Revised 5 June 2018 Accepted 18 June 2018

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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˚C 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˚C 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˚C 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˚C 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.

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Keywords: Cell-free DNA, Extraction, Storage, Container, EGFR

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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.

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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. 3.

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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.

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Table 1.

Clinical characteristics of five patients with lung cancer

Case No.	Age at diagnosis	Sex	Histology of lung cancer	EGFR mutation analysis (date)
Case No.	Age at diagnosis	Sex	Histology of lung cancer	Tissue biopsy∗	Plasma^†
LC06	59	F	NSCLC (adenocarcinoma)	L858R (Aug/2016)	L858R (Oct/2017)
LC07	65	F	NSCLC (adenocarcinoma)	Exon 19 deletion (Oct/2014)	No mutation detected (Oct/2017)
LC08	80	F	NSCLC (adenocarcinoma)	Exon 19 deletion (Oct/2017)	Exon 19 deletion (Oct/2017)
LC09	69	F	NSCLC (adenocarcinoma)	Exon 19 deletion, T790M (Mar/2017)	No mutation detected (Oct/2017)
LC10	58	F	NSCLC (adenocarcinoma)	Exon 19 deletion (Sep/2015)	Exon 19 deletion (Oct/2017)

^∗ EGFR mutation was determined using PNAClamp

^TM EGFR Mutation Detection Kit (PANAGENE Inc., Daejeon, Korea);

^† EGFR mutation was determined using cobas® EGFR Mutation Test v2 (Roche Diagnostics, Indianapolis, IN, USA). Abbreviations: F, female; NSCLC, non-small cell lung cancer.

Table 2.

Cell-free DNA yield according to the specimen container and storage duration

Case No.	K2-EDTA (ng/μL)				Cell-Free DNA BCT (ng/μL)
Case No.	<1 hr	6 hr	24 hr	48 hr	1 day	3 days	7 days	14 days
Healthy controls (N₌5)
HC01	0.202	0.181	0.193	0.144	0.149	0.143	0.247	1.150
HC02	0.188	0.184	0.225	0.202	0.160	0.141	0.152	0.386
HC03	0.177	0.175	0.194	0.175	0.166	0.161	0.159	0.232
HC04	0.175	0.188	0.181	0.178	0.161	0.142	0.238	0.436
HC05	0.122	0.094	0.102	0.127	0.105	0.108	0.128	0.111
Mean	0.1728	0.1644	0.1790	0.1652	0.1482	0.1390	0.1848	0.4630
SD	0.0304	0.04	0.046	0.03	0.025	0.019	0.054	0.405
Shapiro-Wilk test for normal distribution	W=0.8600	W=0.6650	W=0.8508	W=0.9567	W=0.7588	W=0.8651	W=0.8545	W=0.8344
	accept normality	a reject normality	accept normality a	accept normality	a reject normality	a accept normality	accept normality a	accept normality
	(P ₌0.2283)	(P ₌0.0040)	(P ₌0.1970)	(P ₌0.7849)	(P ₌0.0358)	(P ₌0.2470)	(P ₌0.2093)	(P ₌0.1501)
Patients with lung cancer (N₌5)
LC06	0.545	0.438	0.489	0.541	0.460	0.470	0.391	1.080
LC07	0.310	0.286	0.277	0.289	0.265	0.245	0.438	1.040
LC08	0.776	0.618	0.826	0.566	0.716	0.590	0.460	7.690
LC09	1.120	1.320	1.190	1.080	0.806	1.010	0.702	1.350
LC10	0.396	0.480	0.457	0.467	0.372	0.393	0.237	0.411
Mean	0.6294	0.6284	0.6478	0.5886	0.5238	0.5416	0.4456	2.3142
SD	0.3263	0.404	0.362	0.295	0.229	0.29	0.168	3.025
Shapiro-Wilk test for normal distribution	W=0.9324	W=0.8164	W=0.9226	W=0.8661	W=0.9299	W=0.9175	W=0.9430	W=0.6595
	accept normality a	a accept normality	accept normality a	accept normality	accept normality a	accept normality a	accept normality	reject normality
	(P ₌0.6125)	(P ₌0.1096)	(P ₌0.5471)	(P ₌0.2509)	(P ₌0.5954)	(P ₌0.5141)	(P ₌0.6872)	(P ₌0.0034)

Abbreviations: BCT, cell-free DNA BCT container; SD, standard deviation.

Table 3.

Cell-free DNA yield according to centrifugation conditions in K2-EDTA containers

Case No.	Immediate plasma separation from EDTA whole blood and stored at 4˚C			EDTA whole blood stored at 4˚C with delayed plasma separation
Case No.	6 hr	24 hr	48 hr	6 hr	24 hr	48 hr
HC11	0.193	0.185	0.172	0.178	0.227	0.222
HC12	0.166	0.176	0.168	0.176	0.201	0.257
HC13	0.270	0.271	0.207	0.253	0.314	0.413
HC14	0.339	0.287	0.325	0.305	0.315	0.333
HC15	0.428	0.417	0.451	0.366	0.485	0.511
Mean	0.2792	0.2672	0.2646	0.2556	0.3084	0.3472
SD	0.1073	0.0974	0.1221	0.0821	0.1112	0.1174
Shapiro-Wilk test for Normal distribution	W=0.9520	W=0.9006	W=0.8493	W=0.9124	W=0.8923	W=0.9566
	accept normality (P =0.7516)	accept normality (P =0.4130)	accept normality (P =0.1924)	accept normality (P =0.4821)	accept normality (P =0.3686)	accept normality (P =0.7840)

Abbreviation: SD, standard deviation.

Table 4.

Detection of EGFR gene mutation in plasma samples from patients with lung cancer

Sample collection and storage conditions	Input plasma volume (mL)	cfDNA extraction kit manufacturer	Case No. LC06 (L858R)	Case No. LC08 (Exon 19 deletion)	Case No. LC10 (Exon 19 deletion)
Sample collection and storage conditions	Input plasma volume (mL)	cfDNA extraction kit manufacturer	EGFR mutation analysis (semi-quantitative index)∗
K2-EDTA, _<1 hr	2	Roche	L858R (5.70)	Exon 19 deletion (10.97)	Exon 19 deletion (9.42)
K2-EDTA, <1 hr	1	Thermo Fisher	L858R (5.12)	Exon 19 deletion (8.41)	Exon 19 deletion (8.71)
K2-EDTA, 6 hr	1	Thermo Fisher	L858R (5.79)	Mutation not detected	Exon 19 deletion (8.23)
K2-EDTA, 24 hr	1	Thermo Fisher	L858R (5.03)	Exon 19 deletion (10.17)	Exon 19 deletion (8.97)
K2-EDTA, 48 hr	1	Thermo Fisher	L858R (4.00)	Exon 19 deletion (8.40)	Exon 19 deletion (8.69)
BCT, 1 day	1	Thermo Fisher	L858R (6.04)	Exon 19 deletion (9.32)	Exon 19 deletion (6.98)
BCT, 3 days	1	Thermo Fisher	L858R (6.98)	Exon 19 deletion (9.01)	Exon 19 deletion (8.26)
BCT, 7 days	1	Thermo Fisher	L858R (6.00)	Exon 19 deletion (9.22)	Exon 19 deletion (8.71)
BCT, 14 days	1	Thermo Fisher	L858R (4.92)	Exon 19 deletion (9.12)	Exon 19 deletion (8.66)

^∗ EGFR mutation was determined using cobas® EGFR Mutation Test v2 (Roche Diagnostics, Indianapolis, IN, USA). Abbreviations: BCT, cell-free DNA BCT container; SD, standard deviation.

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