Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Performance of Streck cfDNA Blood Collection Tubes for Liquid Biopsy Testing.

Author(s): Medina Diaz I, Nocon A, Mehnert DH, Fredebohm J, Diehl F, Holtrup F

Publication: PLoS One, 2016, Vol. 11, Page e0166354

PubMed ID: 27832189 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of storing blood in Streck blood collection tubes (BCT) at room temperature on cell-free DNA (cfDNA) yield, genomic DNA contamination, and mutation detection in plasma from healthy volunteers and patients with colorectal cancer. Venous blood was collected into K2EDTA (10 mL) and BCT tubes (10 mL) and inverted 10 times before storage. Specimens from 60 healthy donors were stored in K2EDTA tubes for 2 h at room temperature and in BCT tubes for 2 h, 3 days (with or without shaking), and 5 days at room temperature before plasma isolation and DNA quantification and mutation analysis with BEAMing and Safe-SeqS. Specimens from 21 patients with CRC and specimens from eight healthy donors spiked with DNA carrying specific mutations were stored in K2EDTA tubes for 2 h at room temperature and in BCT tubes for 2 h and 3 days at room temperature before plasma isolation and DNA quantification and mutation analysis with BEAMing. Plasma was obtained by centrifugation for 10 min at 1600 x g followed by centrifugation of the supernatant at 6000 x g for 10 min, aliquoted, and frozen at -80˚C. Plasma was thawed at room temperature and DNA was extracted using the QIAamp Circulating Nucleic Acid Kit with an extension of the Proteinase K digestion step to 1 h. DNA was quantified by real-time PCR amplification of 96 bp (cfDNA and gDNA) and 402 bp (gDNA only) of LINE-1. Mutation analysis was conducted using the flow cytometry-based BEAMing method and the next-generation sequencing based SafeSeqS.

   Summary of Findings:

   The real-time PCR assay revealed approximately 20% of the DNA in plasma was ≥ 402 bp (a size likely to represent genomic DNA) when blood was stored in K2EDTA or BCT tubes for 2 h at room temperature. Comparable plasma yields of cfDNA, ratios of long to short LINE-1 PCR products, mutation rates by BEAMing, and number of each type of base changes by SafeSeq were obtained from blood stored in BCT or EDTA tubes for 2 h at room temperature and from blood stored in a BCT at room temperature for 3 days with or without shaking or stored for 5 days. The authors state that the electrophoretic migration was also comparable for plasma obtained from blood stored in BCT and K2EDTA tubes. A slight increase in the average mutant fraction of EGFR (0.6% versus 0.4%) and a decrease in the mutant fraction of KRAS (0.6% versus 0.7%) occurred when blood spiked with DNA carrying mutations was stored for 5 days in BCT tubes rather than 2h in K2EDTA or BCT tubes, but no change in the average mutant fraction of PIK3CA was observed and it was unclear if the effect was statistically significant or was within the expected variability. Further, analysis showed than when individual values were normalized to their respective K2EDTA control there was no difference in fold change. Plasma DNA yields were comparable and mutation rates were strongly to very strongly correlated (R²=0.876 to 0.973) between plasma from blood stored for 2h in K2EDTA tubes and that stored for 2 h or 3 days in BCT tubes in patients with colorectal cancer. 

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)
   • Streck/BCT

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   DNA	Electrophoresis
   DNA	Flow cytometry
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	Streck BCT K2EDTA tube
   Biospecimen Preservation	Type of fixation/preservation	Blood collection tube additive EDTA
   Storage	Storage conditions	With agitation Without agitation
   Storage	Time at room temperature	2 h 3 days 5 days
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated

   View more

2. Study Purpose

   This study investigated the effects of storing blood at various temperatures (4˚C, 6˚C, 10˚C, 20˚C, room temperature 30˚C, 37˚C, or 40˚C) on plasma volume and genomic DNA contamination was also investigated in specimens from healthy volunteers. Venous blood was collected into K2EDTA (10 mL) and BCT (10 mL) tubes and inverted 10 times before storage. Specimens from six healthy donors were stored in K2EDTA tubes for 2 h at room temperature and in BCT tubes for 2 h at room temperature or for 3 and 5 days at 4˚C, room temperature, and 40˚C before plasma isolation and DNA quantification.  Specimens from eight healthy donors were stored in BCT tubes for 3 days at 6, 10, 20, 30, and 37˚C before plasma isolation and DNA quantification.  Plasma was obtained by centrifugation for 10 min at 1600 x g followed by centrifugation of the supernatant at 6000 x g for 10 min, aliquoted, and frozen at -80˚C. Plasma was thawed at room temperature and DNA was extracted using the QIAamp Circulating Nucleic Acid Kit with an extension of the Proteinase K digestion step to 1 h. DNA was quantified by real-time PCR amplification of 96 bp (cfDNA and gDNA) and 402 bp (gDNA only) of LINE-1.

   Summary of Findings:

   Plasma volume decreased non-significantly when blood was stored in a BCT for 3 days or more regardless of temperature, but the magnitude of the decrease was generally larger at extreme temperatures (4, 6, or 10 or 40˚C) than at room temperature. Storage of blood for 5 days in BCT tubes at 4˚C resulted in a larger cellular interface area and storage at 40˚C resulted in darker plasma, indicating hemolysis. As expected based on the changes in the visual appearance of the plasma, an increase in the ratios of long to short LINE-1 PCR products indicative of genomic DNA contamination also occurred when blood was stored for 5 days in BCT tubes at 4˚C or 40˚C (P<0.01, both), but there was no effect of storing blood for 5 days at room temperature in a BCT rather than 2 h in a K2EDTA tube at room temperature.  Further experimentation revealed a significant increase in the ratios of long to short LINE-1 PCR products in specimens stored for 3 days in BCT at 6˚C compared to those stored at 20˚C (P<0.05), but those stored at 10˚C or 37˚C only showed non-significant increases in the LINE-1 ratio.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • Streck/BCT

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Macroscopic observation
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	K2EDTA Vacutainer Streck BCT
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Storage	Storage temperature	4˚C Room temperature 40˚C 6˚C 10˚C 20˚C 30˚C 37˚C
   Storage	Storage duration	2 h 3 days 5 days
   Biospecimen Preservation	Type of fixation/preservation	Blood collection tube additive EDTA Refrigeration

   View more