Comparison of Blood Collection Tubes from Three Different Manufacturers for the Collection of Cell-Free DNA for Liquid Biopsy Mutation Testing.
Author(s): Alidousty C, Brandes D, Heydt C, Wagener S, Wittersheim M, Schäfer SC, Holz B, Merkelbach-Bruse S, Büttner R, Fassunke J, Schultheis AM
Publication: J Mol Diagn, 2017, Vol. , Page
PubMed ID: 28732213 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of blood collection tube type and delayed centrifugation on mutation detection in plasma from blood spiked with epidermal growth factor receptor gene (EGFR) T790M mutated DNA fragments.
Conclusion of Paper
The EGFR T790M mutation was detected in plasma specimens from blood spiked with ≥0.5 ng DNA, regardless of blood collection tube type or storage duration. The EGFR T790M mutation was detectable when blood in Streck BCT was spiked with 0.05 ng DNA but not when spiked with 0.1 ng DNA. Conversely, EGFR T790M mutation was detected when blood in Cell-Free or PAXgene tubes was spiked with 0.1 ng DNA but not when spiked with 0.05 ng.
Studies
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Study Purpose
This study investigated the effects of blood collection tube type and delayed centrifugation on mutation detection in plasma from blood spiked with epidermal growth factor receptor gene (EGFR) T790M mutated DNA fragments. Venous blood was collected from an unspecified number of healthy patients using a 21-gauge needle into Streck cfDNA BCT, Roche Cell-Free DNA collection tubes, and Qiagen PAXgene Blood ccfDNA tubes; inverted ten times; and stored at room temperature. Before storage blood was spiked with 0.01-3 ng of EGFR T790M mutated double-stranded DNA that had been fragmented by ultrasonication to a median size of 160 bp. Blood was transferred to Falcon tubes 0, 1, 2, 3, 4, or 7 days after collection and centrifuged at 1600 x g for 10 min at 4˚C. Plasma was transferred to fresh LoBind tubes and then aliquoted into cryogenic vials for storage at -80˚C. DNA was extracted using the Maxwell RSC LV ccf DNA Kit and stored at 4˚C. The mutated DNA was detected by real-time PCR using the AmoyDx EGFR T790M Mutation Test Kit.
Summary of Findings:
The EGFR T790M mutation was detected in plasma specimens regardless of blood collection tube type or storage duration when blood was spiked with 1 or 3 ng of DNA. Although EGFR T790M mutation detection was comparable between tube types when blood was spiked with ≥ 0.5 ng DNA, it was not detectable when blood in Streck BCT was spiked with 0.1 ng DNA but was detected in the other tube types. Conversely, it was detected when blood in Streck BCT was spiked with 0. 05 ng DNA but was not in Cell-Free DNA or PAXgene tubes spiked with ≤0.05 ng DNA. The EGFR T790M mutation was not detectable when blood was spiked with 0.01 ng, regardless of tube type.
Biospecimens
Preservative Types
- PAXgene
- Other Preservative
- Streck/BCT
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Streck BCT
Roche Cell-Free DNA collection tube
Qiagen PAXgene blood ccfDNA tube
Biospecimen Aliquots and Components Biospecimen components 0.01 ng EGFR T790M mutated DNA added
0.05 ng EGFR T790M mutated DNA added
0.1 ng EGFR T790M mutated DNA added
0.5 ng EGFR T790M mutated DNA added
1 ng EGFR T790M mutated DNA added
3 ng EGFR T790M mutated DNA added
Real-time qPCR Specific Targeted nucleic acid EGFR
Biospecimen Preservation Type of fixation/preservation Blood collection tube additive
PAXgene
Storage Time at room temperature 0 days
1 days
2 days
3 days
4 days
7 days
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated