Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics.

Author(s): van Ginkel JH, van den Broek DA, van Kuik J, Linders D, de Weger R, Willems SM, Huibers MMH

Publication: Cancer Med, 2017, Vol. 6, Page 2297-2307

PubMed ID: 28940814 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of blood collection tube type, delayed centrifugation, centrifugation protocol, extraction method, and quantification method on the yield of cell-free DNA from blood specimens. The effect of delayed processing on the mutant allele fraction was also investigated. Blood specimens were obtained from 46 healthy individuals and 18 non-small cell lung cancer patients with progressive disease. Additionally, three pools of blood composed of blood from 60 cancer patients, were used. To investigate the effect of blood collection tube, 8-25 blood specimens from healthy donors were collected into K2/K3 EDTA, heparin, silicone-coated and citrate tubes. To investigate the effects of delayed processing, blood from six patients was stored in CellSave, Streck BCT, or EDTA tubes for 0, 3, 6, or 24 h at room temperature and in CellSave and Streck BCT tubes for 2 or 5 days before centrifugation. Using EDTA blood from six healthy patients, three centrifugation protocols were compared: a) centrifugation for 10 min at 800 x g, b) centrifugation for 10 min at 800 x g followed by centrifugation of the supernatant for 1 min at 11,000 x g, and c) centrifugation for 10 min at 800 x g followed by post-thaw centrifugation of the supernatant for 1 min at 11,000 x g. Using EDTA blood from five cancer patients, two centrifugation protocols were compared: d) centrifugation for 20 min at 380 x g followed by centrifugation of the supernatant for 10 min at 20,000 x g, and e) centrifugation for 20 min at 380 x g followed by post-thaw centrifugation of the supernatant for 1 min at 20,000 x g.  Following centrifugation, plasma was stored frozen at -20˚C. The effect of DNA isolation method was investigated using the blood from four healthy patients and six cancer patients, DNA was isolated using the Jena PME free-circulating DNA extraction kit, QIAsymphony Circulating NA kit, QIAamp Circulating NA kit, MagNAPure LC Total Nucleic Acid Isolation Large Volume kit, and Zymo Quick cfDNA serum & plasma kit. Extracted DNA was quantified by spectrophotometry, Qubit Fluorometry, and droplet digital PCR of BRAF, RPP30, EIF2C1, a single-nucleotide polymorphism (SNP) variant of LEPREL2, 6 mutant EGFR, and 8 mutant KRAS multiplex assays.

   Summary of Findings:

   cfDNA levels were significantly higher in serum than EDTA (P<0.001), citrate (P<0.05), or heparin (P<0.05) plasma and higher in EDTA than heparin plasma (P<0.05). Mean concentrations cfDNA were unaffected by storage in EDTA, Streck BCT, or CellSave tubes for up to 24 h, but the mutant fraction cfDNA appeared to decrease with storage in Streck BCT or CellSave tubes (EDTA not investigated) for 2 days as compared to 1 day (2.1% and 2.6% versus 18.0% and 14.8%) or 5 days (22.5% and 22.3%). The mean cfDNA levels were higher when isolated by a single centrifugation at 800 x g for 10 min than when the supernatant was centrifuged for 1 min at 11,000 x g immediately (72 versus 27.7 copies/µL) or after thawing (72 versus 36.2 copies/µL).  The mean copies of cfDNA/mL were comparable when the supernatant from the specimens centrifuged for 20 min at 380 x g was centrifuged for 10 min at 20,000 x g before or after freezing (31.5 versus 39.8 copies/µL). The average percentage of positive droplets was highest using the Zymo Quick cfDNA kit (0.54) followed by the QIAamp Circulating NA Kit (0.43). In cancer patients, cfDNA extracted using the QIAamp Circulating NA Kit had higher mean positive droplets than when extracted using the MagNAPure LC Total Nucleic Acid Isolation Large Volume kit (P<0.05) but comparable mean positive droplets to specimens extracted using QIAsymphony Circulating NA kit, Jena PME free-circulating DNA extraction kit, or Zymo Quick cfDNA serum & plasma kit. The QIAsymphony kit yielded a higher mean number of droplets than the QIAamp kit (P<0.05) or MagNA Pure kit (P<0.05). Regression analysis showed cfDNA concentrations as determined by ddPCR were very strongly correlated with those by Qubit (R2=0.96, P<0.0001) but not those measured by NanoDrop (R2=0.002, P=0.81).

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)
   • Other Preservative
   • Streck/BCT

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Digital PCR
   DNA	Spectrophotometry
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Healthy NSCLC
   Biospecimen Acquisition	Anticoagulant	Citrate EDTA Heparin
   Biospecimen Acquisition	Type of collection container/solution	CellSave Streck BCT EDTA
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated Different number of centrifugation steps compared Multiple durations compared Multiple speeds compared
   Biospecimen Preservation	Type of fixation/preservation	Blood collection tube additive EDTA
   Storage	Time at room temperature	0 h 6 h 24 h 2 days 5 days
   Analyte Extraction and Purification	Analyte isolation method	Jena PME free-circulating DNA extraction kit QIAsymphony Circulating NA kit QIAamp Circulating NA kit MagNAPure LC Total Nucleic Acid Isolation Large Volume kit Zymo Quick cfDNA serum & plasma kit
   Digital PCR Specific	Targeted nucleic acid	BRAF EGFR
   Digital PCR Specific	Technology platform	NanoDrop Qubit

   View more