Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Pre-Analytical Evaluation of Streck Cell-Free DNA Blood Collection Tubes for Liquid Profiling in Oncology.

Author(s): Diaz IM, Nocon A, Held SAE, Kobilay M, Skowasch D, Bronkhorst AJ, Ungerer V, Fredebohm J, Diehl F, Holdenrieder S, Holtrup F

Publication: Diagnostics (Basel), 2023, Vol. 13, Page

PubMed ID: 37046506 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare cfDNA yield, the level of genomic DNA contamination, and MAF in plasma isolated from blood that was stored in K₂EDTA tubes for 2 or 6 h and in Streck Cell-Free DNA blood collection tubes (BCTs) for 2 h or 3 days. The study used specimens from patients with colorectal cancer, pancreatic cancer and NSCLC. Case-matched blood was collected from 21 patients with colorectal cancer, 11 with pancreatic cancer, and 21 with non-small cell lung cancer (NSCLC) into K₂EDTA tubes and Streck Cell-Free DNA BCTs. Plasma was separated from both tube types after 2 h, from K₂EDTA tubes after 6 h at room temperature and from Streck Cell-Free DNA BCTs after 3 days by centrifugation at 1600 g for 10 min followed by 6000 g for 10 min. Plasma was aliquoted and stored at -80°C until DNA isolation using the QIAamp Circulating Nucleic Acid Kit and a modified protocol (extended Proteinase K digestion). DNA was quantified by real-time PCR of 96 and 402 bp fragments of LINE-1 and the ratio was used as a marker of genomic DNA contamination. The mutational load of isolated cfDNA was analyzed by BEAMing of common KRAS, NRAS, and EGFR mutations.

   Summary of Findings:

   cfDNA yields were comparable in plasma isolated from blood collected from patients with colorectal cancer, pancreatic cancer and NSCLC in to K₂EDTA tubes and Streck Cell-Free DNA BCTs that were analyzed after 2 h (both tube types), 6 h (K₂EDTA tube only), and 3 days (Streck BCT only) at room temperature.  Further, the ratio of long (402 bp) to short (96 bp) LINE-1 fragments was comparable when blood was stored in Streck BCTs for 2 h versus 3 days and when blood was stored in K₂EDTA tubes for 2 versus 6 h, indicating blood storage for these durations did not increase the level of genomic DNA contamination or decrease cfDNA levels. A total of 16 mutations were identified in 15 specimens by BEAMing analysis (one specimen had two mutations). MAFs were similar between the tube types evaluated and the processing delays investigated.  Further, MAF was very strongly correlated between specimens stored in K₂EDTA tubes for 2 h and those stored in Streck BCTs for 2 h (R²=0.9768) or 3 days (R²=0.929) or in K₂EDTA tubes for 6 h (R²=0.99923 in pancreatic cancer patients and R²=0.9957 in NSCLC patients).

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Flow cytometry
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Colorectal cancer Pancreatic cancer Non-small cell lung cancer
   Biospecimen Acquisition	Type of collection container/solution	K2EDTA tube Streck Cell-Free DNA blood collection tube
   Real-time qPCR Specific	Length of gene fragment	96 bp LINE-1 402 bp LINE-1
   Storage	Time at room temperature	2 h 6 h 3 days
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated

   View more