Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy
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Akemi Sato1, Chiho Nakashima2, Tomonori Abe2, Junichi Kato3, Mitsuharu Hirai3, Tomomi Nakamura2, Kazutoshi Komiya2, Shinya Kimura2, Eisaburo Sueoka1 and Naoko Sueoka-Aragane2
1Department of Clinical Laboratory Medicine, Faculty of Medicine, Saga University, Saga, Japan
2Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Saga, Japan
3ARKRAY Inc., Kyoto, Japan
Naoko Sueoka-Aragane, email: [email protected]
Keywords: liquid biopsy; pre-analytical procedure; circulating free DNA; EGFR; lung cancer
Received: March 06, 2018 Accepted: July 21, 2018 Published: August 07, 2018
Liquid biopsy with circulating free DNA (cfDNA) is a recommended alternative method of re-biopsy. Quality control with cfDNA is indispensable for precise examinations, and it is desirable to achieve high-quality cfDNA separation. We investigated two issues: the influence of pre-analytical procedures on cfDNA analysis performed as a routine procedure in a standard clinical laboratory, and the extent of deterioration of cfDNA quality due to long-term storage. Comparisons among blood collection tube types, storage temperatures, and periods of blood separation were performed in terms of cfDNA quantification, cfDNA size distribution, and detection of EGFR mutations. Quality of cfDNA was better with collection tubes containing 3.2% sodium citrate than with those containing EDTA 2K, and was maintained with storage at 4° C for up to 72 h after blood collection, equivalent to results with cell-stabilizing blood collection tubes. Analysis of cfDNA stored for 7 years showed that samples with low allele frequency (AF) deteriorated more readily than samples with high AF. Despite the same storage period and extraction method, AF of plasma stored for 7 years was remarkably lower than that of cfDNA. However, deterioration due to long-term plasma storage was overcome by changing the DNA extraction method from a silica membrane spin column to a cellulose magnetic beads system. These results can guide the establishment of standardized pre-analytical procedures for liquid biopsy with cfDNA.
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