Quantification of Cell-Free DNA: A Comparative Study of Three Different Methods

Kibum Jeon; Jiwon Lee; Jee-Soo Lee; Miyoung Kim; Han-Sung Kim; Hee Jung Kang; Young Kyung Lee

doi:10.15263/jlmqa.2019.41.4.214

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Original Article

Lab Med Qual Assur 2019; 41(4): 214-219

Published online December 31, 2019

https://doi.org/10.15263/jlmqa.2019.41.4.214

Quantification of Cell-Free DNA: A Comparative Study of Three Different Methods

Kibum Jeon, Jiwon Lee, Jee-Soo Lee, Miyoung Kim, Han-Sung Kim, Hee Jung Kang, and Young Kyung Lee

Department of Laboratory Medicine, Hallym University Sacred Heart Hospital, Anyang, Korea

Correspondence to:Jee-Soo Lee
Department of Laboratory Medicine, Hallym University Sacred Heart Hospital, 22 Gwanpyeong-ro 170beon-gil, Dongan-gu, Anyang 14068, Korea
Tel: +82-31-380-4729 Fax: +82-31-380-1798 E-mail: jsleemd85@gmail.com

Received: July 2, 2019; Revised: August 8, 2019; Accepted: August 22, 2019

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

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Abstract

Background:

Cell-free DNA (cfDNA) provides unique potential as a biomarker for cancer patients or in the field of prenatal care. Following extraction from plasma, cfDNA should be quantified before downstream analysis. The standardization of the quantification methods is essential for application of these techniques to laboratory practice. We aimed to compare three different quantification methods (spectrophotometry, fluorometry, and the electrophoresis-based method) and to determine the contributor(s) to the differences, if any.

Methods:

A total of 135 plasma samples obtained from cancer patients (n=71) and normal individuals (n=15) and cfDNA were extracted from the samples. The extracted cfDNA was quantified using three different methods: NanoDrop spectrophotometry, Quantus fluorometry, and the electrophoresis-based 4200 TapeStation method.

Results:

NanoDrop exhibited the highest estimates (median, 12.3 ng/uL). The estimates of Quantus (median, 0.33 ng/uL) were higher than those of 4200 TapeStation (median, 0.22 ng/ uL). Quantus and TapeStation were significantly correlated (r=0.802, P<0.001). The Bland-Altman plot showed a positive bias of 0.24 ng/uL for Quantus compared with TapeStation. The difference between Quantus and TapeStation values demonstrated a significantly moderate correlation with the amount of high molecular weight DNA (r=0.357, P<0.001).

Conclusions:

Nanodrop is considered as a non-specific method of cfDNA quantification. Quantus depicted more specific quantification compared to Nanodrop; however, TapeStation may be used in parallel or as a complementary method as it enables to determine the size distribution of DNA fragments.

Keywords: Cell-free nucleic acids, Spectrophotometry, Fluorometry