Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Liquid biopsy - Performance of the PAXgene® Blood ccfDNA Tubes for the isolation and characterization of cell-free plasma DNA from tumor patients.

Author(s): Schmidt B, Reinicke D, Reindl I, Bork I, Wollschläger B, Lambrecht N, Fleischhacker M

Publication: Clin Chim Acta, 2017, Vol. 469, Page 94-98

PubMed ID: 28373054 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of storing uncentrifuged blood for 7 days in K3EDTA or PAXgene tubes and DNA extraction method used on levels of ERV-3, beta-actin, and methylated SHOX-2 in plasma from 29 lung cancer patients. Blood was collected into two S-Monovette K3-EDTA tubes and two PAXgene blood ccfDNA tubes. One tube of each type from each patient was stored at room temperature for 2-3 h before plasma isolation by centrifugation at 550 x g for 15 min followed by 3000 x g for 15 min. The other tube of each type was stored for 7 days at room temperature before plasma isolation by centrifugation at 550 x g (PAXgene tube) or 1000x g (K3EDTA tube) for 15 min followed by 3000 x g for 15 min. The resultant plasma was stored at -80˚C until DNA extraction. DNA was extracted from the plasma of 10 patients using the QIAamp Circulating Nucleic Acid Kit and from the remaining 19 patients using the automated QIAsymphony PAXgene Blood ccfDNA Kit after correcting input volume for the preservative agent. DNA was stored at -80˚C. ERV-3 was quantified by real-time PCR amplification. DNA was bisulfite converted using a magnetic bead-based method and mSHOX2 DNA was quantified by real-time PCR with Beta-actin as a standard.

   Summary of Findings:

   ERV-3 (79 bp) levels were comparable in blood stored for 7 days in PAXgene tubes before plasma isolation to specimens stored for 2-3 h, regardless of extraction method, but were significantly lower when blood was stored in K3EDTA tubes for 7 days rather than 2-3 h before centrifugation when DNA was extracted manually using the QIAamp Circulating Nucleic Acid Kit (P<0.01) and by the automated method (P<0.01). Interestingly, one of the ten specimens stored in PAXgene tubes for 7 days that was extracted manually did not show a difference in ERV-3 (79 bp) after storage and one of the specimens extracted by the automated method showed a decline in ERV-3 (79 bp). Similarly, levels of the 297 bp fragment of ERV-3 and Beta-actin were lower in plasma from blood stored for 7 days in K3EDTA tubes than in plasma from blood processed within 2-3 h when extracted manually (P<0.01, both) or by an automated method (P<0.01, both) but were comparable in plasma from blood stored in PAXgene tubes for 2-3 h and 7 days. In contrast, levels of mSHOX2 DNA were unaffected by storage of blood for 7 days in either tube type, regardless of DNA extraction method.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • PAXgene
   • None (Fresh)

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Bisulfite conversion assay
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	S-Monovette K3-EDTA tube PAXgene ccfDNA tube
   Storage	Storage duration	0 days 7 days
   Biospecimen Preservation	Type of fixation/preservation	EDTA PAXgene
   Bisulfite conversion assay Specific	Targeted nucleic acid	SHOX2
   Real-time qPCR Specific	Targeted nucleic acid	ERV-3 Beta-actin mSHOX2
   Real-time qPCR Specific	Length of gene fragment	79 bp 297 bp
   Analyte Extraction and Purification	Analyte isolation method	QIAsymphony PAXgene Blood ccfDNA Kit QIAamp Circulating Nucleic Acid Kit

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