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Updated: Sep 30, 2025
Detection of Rare Mutations in CtDNA Using Next Generation Sequencing
Published on: August 24, 2017
Effects of Different Centrifugation Protocols on the Detection of EGFR Mutations in Plasma Cell-Free DNA
Kyung-Hwa Shin1,2,3, Sun Min Lee3,4,5, Kongkyoung Park4
1Department of Laboratory Medicine, Busan, South Korea.
Objectives:
Various preanalytical factors, including the collection tube, storage conditions, and centrifugation, affect the detection results of plasma cell-free DNA (cfDNA). We compared the effect of different centrifugation protocols on the detection of EGFR mutations in cfDNA.
Methods:
We analyzed 117 plasma specimens from 110 patients with non-small cell lung cancer using the cobas EGFR Mutation Test v2 (Roche Diagnostics). We compared the identified EGFR mutations and semiquantitative index values from the 1- and 2-step centrifugation groups and confirmed the clinical impact of differences in the results after further high-speed centrifugation.
Results:
We detected EGFR mutations in 44 (37.6%) and 47 (40.2%) samples that were centrifuged once and twice, respectively; the 2 groups showed an 89.7% (105/117) concordance and a strong correlation in their semiquantitative index values (r = 0.929). Among the 12 inconsistent result pairs, 9 samples of 2-step centrifugation (75%) were consistent with the results of a recent tissue biopsy.
Conclusions:
Additional high-speed centrifugation has been shown to increase the sensitivity of EGFR mutation detection in a commercial in vitro diagnostic real-time polymerase chain reaction device and is an optimal preanalytical factor for detecting low-allele frequency gene mutations using low concentrations of cfDNA.
