Comparative analysis of circulating tumor DNA stability In K<sub>3</sub>EDTA, Streck, and CellSave blood collection tubes.
Author(s): Kang Q, Henry NL, Paoletti C, Jiang H, Vats P, Chinnaiyan AM, Hayes DF, Merajver SD, Rae JM, Tewari M
Publication: Clin Biochem, 2016, Vol. 49, Page 1354-1360
PubMed ID: 27129799 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of blood collection tube, delayed centrifugation duration, and temperature on plasma levels of circulating tumor DNA (ctDNA) and wildtype DNA (wtDNA) in patients with metastatic breast cancer.
Conclusion of Paper
Duration of pre-centrifugation storage significantly affected the quantity of wtDNA in plasma for three of six patients when blood was stored in EDTA or CellSave tubes, but there was no effect of storage duration on specimens stored in Streck tubes. Similarly, pre-centrifugation storage in EDTA tubes led to increased plasma ctDNA in one of four patients. When stored for 48 h, ctDNA levels in one patient were lower in CellSave tubes and higher in Streck tubes compared to EDTA tubes. Importantly, the tumor allele fraction was significantly lower when blood was stored for 48 h rather than 2 h in EDTA tubes for two of four patients and when stored for 48 h in CellSave or Streck rather than EDTA tubes (one patient each). Although storage temperature had no effect on levels of wtDNA or ctDNA in EDTA plasma, levels of wtDNA but not ctDNA were increased in two of six patients when blood was stored in Streck tubes at 4˚C rather than room temperature.
Studies
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Study Purpose
This study investigated the effects of blood collection tube, delayed centrifugation duration, and temperature on plasma levels of ctDNA and wtDNA in six patients with metastatic breast cancer. Blood was collected from a single venipuncture into two Streck BCT tubes, two Monoject glass K3EDTA tubes, and one CellSave tube. Within 30 min of collection, one tube of each of the specimens in Streck BCT and EDTA tubes were placed on ice while the other specimens (one of each tube type) remained at room temperature. Each specimen was then divided into three aliquots within 1 h and returned to storage on ice or at room temperature for 2, 6, or 48 h. Specimens were centrifuged at 2500 x g for 10 min and the resultant supernatant was centrifuged at 16,000 x g to obtain cell-free plasma. Cell-free plasma was stored at -80˚C until DNA extraction. Plasma was thawed at room temperature and centrifuged at 16000 x g for 5 min before DNA extraction with a modification of the QIAamp Circulating Nucleic Acid Kit protocol (no carrier RNA used and extension of digestion for Streck and CellSave specimens). DNA was quantified using the Qubit dsDNA HS Assay kit and real-time PCR amplification of LINE-1. Mutant and wildtype copies of PIK3CA and TP53 DNA were quantified by droplet digital PCR.
Summary of Findings:
Regardless of storage temperature, significantly higher amount of wtDNA in plasma were found when blood was stored in EDTA tubes for 48 h rather than 2 h before centrifugation in three of six patients (P<0.001, P=0.048, and P<0.001). Qubit confirmed an increase in total cfDNA after 48 h at room temperature in two of three patients and levels were at the limit of detection by Qubit but were confirmed to be increased after 48 h by LINE-1 PCR for the third patient. Similarly, significantly lower levels of wtDNA in plasma were found when blood was stored in CellSave tubes for 6 h rather than 2 h in one patient (P=0.008) and for 48 h rather than 2 h in two additional patients (P=0.001, P=0.035). No effect was found on plasma levels of wtDNA due to duration of blood storage in Streck tubes, but significantly higher wtDNA levels were found when blood was stored in Streck tubes for 48 h at 4˚C rather than room temperature in two of six patients (P=0.035 and P=0.005).
Four of the six patients had detectable ctDNA in plasma and there was no effect of tube type or delayed centrifugation for three of these patients. For the fourth patient, a significantly lower level of ctDNA was found when blood was stored in EDTA tubes for 48 h rather than 2 h at 4˚C or room temperature (P<0.0005, both). In one patient, there was a marginal decrease in stability after 48 h pre-centrifugation storage in the CellSave rather than EDTA tube (P=0.041) and an in increase in stability when stored for 48 h in Streck rather than EDTA tubes (P=0.01). Importantly, storage on ice versus room temperature did not impact the stability of ctDNA.
The tumor allele frequencies were lower after storage of blood in EDTA tubes for 48 h in two of four patients (P= 0.009 and P= 0.019). This decrease in tumor allele fraction was attributable to the decrease in ctDNA in one patient and to the increase in wtDNA in another. Similarly, the tumor allele fraction in plasma was found to be significantly higher in specimens when blood was stored for 48 h in CellSave than EDTA tubes in one patient (P=0.012) and Streck tubes than EDTA tubes in another (P=0.012).
Biospecimens
Preservative Types
- Other Preservative
- None (Fresh)
- Streck/BCT
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Fluorometry DNA Digital PCR DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Streck BCT
EDTA tube
CellSave tube
Biospecimen Preservation Type of fixation/preservation EDTA
Blood collection tube additive
Real-time qPCR Specific Targeted nucleic acid LINE-1
Storage Storage temperature On ice
Room temperature
Digital PCR Specific Targeted nucleic acid Mutant and wildtype copies of PIK3CA
Mutant and wildtype copies of TP53
Storage Storage duration 2 h
6 h
48 h