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Optimization of Preanalytical Variables for cfDNA Processing and Detection of ctDNA in Archival Plasma Samples.

Author(s): Nesic M, Bødker JS, Terp SK, Dybkær K

Publication: Biomed Res Int, 2021, Vol. 2021, Page 5585148

PubMed ID: 34307658 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare cfDNA yield between plasma from blood subjected to 30 min-24 h processing delays in Streck BCT or EDTA tubes, between EDTA plasma obtained by centrifugation at different speeds, and among different extraction kits. The potential to use archival plasma to identify mutations present in matched biopsy specimens in patients with DLBCL was also investigated. Peripheral blood from eight healthy donors was collected into EDTA tubes and Streck BCTs.  Blood was stored in each tube type for <30 min, 1 h, and 4 h at room temperature; in Streck tubes 24 h at room temperature; and in EDTA tubes for 24 h at 4°C before plasma separation. Plasma was obtained from Streck blood by centrifugation at 1600 x g for 10 min followed by 16,000 x g for 10 min and from EDTA blood by centrifugation at 2000 or 3000 x g for 10 min. Plasma was stored at -80°C. cfDNA was extracted from thawed plasma using the QIAamp Circulating Nucleic Acid Kit and quantified by Qubit. cfDNA size was confirmed by bioanalyzer. To investigate the effect of cfDNA extraction method, cfDNA was extracted from the plasma (tube type and centrifugation conditions not specified) of three healthy volunteers using the QIAamp Circulating Nucleic Acid, Quick-cfDNA Serum & Plasma, and DNeasy Blood & Tissue kits. To investigate if the workflow could be used to identify mutations in archival plasma, cfDNA was extracted using the QIAamp Circulating Nucleic Acid Kit from EDTA plasma (obtained by centrifugation at 2000 x g) specimens collected from three patients with DLBCL and known mutations in tumor (whole exome sequencing) and stored in liquid nitrogen for a median of 9 years. Genotypes were confirmed in cfDNA using mutation specific ddPCR (EZH2, CD58, and TNFRSF14) and frequency was compared to mutations detected in matched tumor specimens by whole exome sequencing and ddPCR.

    

   Summary of Findings:

   The cfDNA yield was comparable between tube types when plasma was isolated ≤4 h after blood collection but, when blood was stored for 24 h at 4°C in EDTA tubes, the cfDNA yield was higher than when stored in Streck tubes for 24 h at room temperature (P=0.03125). The authors note less individual variation in cfDNA yield when Streck tubes were used rather than EDTA tubes. For EDTA tubes, the cfDNA yield was higher when plasma was obtained by centrifugation at 2000 x g rather than 3000 x g, regardless of centrifugation delay. All three kits isolated DNA of 150-180 bp, indicating isolation of cfDNA. The yield of cfDNA was highest when extraction was with the QIAamp Circulating Nucleic Acid Kit. The expected mutations were found in archival cfDNA but, as expected, the fractional abundance was lower than found in the matched tumor biopsy.

   Biospecimens

   • Bodily Fluid - Blood
   • Tissue - Lymph Node
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Lymphoma
   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Digital PCR
   DNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated Multiple speeds compared
   Biospecimen Acquisition	Type of collection container/solution	Streck BCT EDTA tube
   Biospecimen Acquisition	Biospecimen location	Biopsy Plasma
   Analyte Extraction and Purification	Analyte isolation method	QIAamp Circulating Nucleic Acid Kit Quick-cfDNA Serum & Plasma Kit DNeasy Blood & Tissue Kit
   Storage	Storage duration	30 min 1 h 4 h 24 h

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