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Performance comparison of blood collection tubes as liquid biopsy storage system for minimizing cfDNA contamination from genomic DNA.

Author(s): Zhao Y, Li Y, Chen P, Li S, Luo J, Xia H

Publication: J Clin Lab Anal, 2018, Vol. , Page e22670

PubMed ID: 30191594 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared genomic DNA and hemoglobin levels in plasma from blood stored for up to 14 days pre-centrifugation in K₂EDTA, Streck, and Roche tubes. The effect of storing blood for three days in Roche or Streck tubes on NGS library yield and complexity was also examined. Blood was collected from six healthy donors into K₂EDTA tubes, Streck BCT, and Roche tubes and stored at room temperature for 0, 3, 7, or 14 days before centrifugation. At the allotted time, specimens were mixed gently and aliquots removed before centrifugation at 2000 x g for 10 min at 4 ˚C followed by centrifugation of the supernatant at 16000 x g for 10 min at 4˚C. The resultant plasma was stored at -80˚C. Hemolysis was determined using a spectrophotometer. Total genomic DNA in plasma was quantified by real-time PCR of L1PA2. cfDNA was extracted from plasma using the Circulating Nucleic Acid Kit, sheared using a Covaris M220, end-repaired, adapter-ligated, selected using Agencourt beads, and amplified. The library was quantified by Qubit and LabChip GXII Touch. cfDNA was sequenced using a NextSeq500 sequencer.

   Summary of Findings:

   As expected, genomic DNA contamination of plasma increased linearly with pre-centrifugation storage of blood in K₂EDTA tubes (5.35, 118.26, and 449.85-fold after 3, 7, and 14 days, respectively). In contrast, much smaller increases were noted when blood was stored for 3, 7, or 14 days in Streck (2.18, 2.87 and 11.49-fold, respectively) or Roche tubes (1.30, 2.50, and 2.73-fold, respectively). Importantly, the Roche tubes were more effective than the Streck tubes at preventing the release of gDNA for storage durations of 14 days. Further, plasma hemoglobin levels were significantly lower than in K₂EDTA blood when blood was stored for up to 7 days in Streck (P=3.55 x 10^-6) or Roche (P=8.80 x 10^-8) tubes. Hemolysis increased when blood was stored for 14 days in either Streck or Roche tubes but the magnitude of the hemolysis was less in Roche than Streck Tubes. Importantly, the authors state that the rupture of WBCs, as indicated by genomic DNA contamination, and the rupture of RBCs, as indicated by hemolysis, were not synchronized.  NGS library yield before and after capture and library complexity were comparable when blood in K₂EDTA was processed within 2 h and when blood was stored in Streck or Roche tubes for 3 days before processing.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

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

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   Protein	Spectrophotometry
   DNA	Real-time qPCR
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	K2EDTA tube Streck tube Roche tube
   Storage	Storage duration	0 days 3 days 7 days 14 days
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated

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