Effects of Different Centrifugation Protocols on the Detection of EGFR Mutations in Plasma Cell-Free DNA

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.