Effects of Collection and Processing Procedures on Plasma Circulating Cell-Free DNA from Cancer Patients.
Author(s): Risberg B, Tsui DWY, Biggs H, Ruiz-Valdepenas Martin de Almagro A, Dawson SJ, Hodgkin C, Jones L, Parkinson C, Piskorz A, Marass F, Chandrananda D, Moore E, Morris J, Plagnol V, Rosenfeld N, Caldas C, Brenton JD, Gale D
Publication: J Mol Diagn, 2018, Vol. , Page
PubMed ID: 30165204 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of processing delays, tube type, centrifugation protocol, and shipment in Streck Cell-Free DNA BCT blood collection tubes (cfDNA BCTs) on cell-free DNA (cfDNA) levels, mutant allele fractions, and circulating tumor DNA (ctDNA) yield. The effects of tube type and processing delay on the distribution of next generation sequencing (NGS) non-reference calls and the effect of shipment on copy number profile were also investigated.
Conclusion of Paper
The levels of cfDNA increased and mutant allele frequency declined when blood was stored in K3EDTA tubes at room temperature or at 4˚C but the yield increases were smaller than when stored at 4˚C instead of room temperature. In contrast, cfDNA levels and mutant allele frequency were not significantly different when blood was processed immediately, stored for a week in BCTs before processing, or shipped in a BCT. Importantly, the ratio of non-reference to reference calls in targeted sequencing for each mutation type was unaffected by tube type or processing delay. No significant differences in the copy number distributions were identified in specimens shipped in BCTs compared to those processed immediately or stored at room temperature before processing. Centrifugation speed had no effect on the yield of cfDNA or the mutant allele frequency.
Studies
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Study Purpose
This study investigated the effects of processing delays, tube type, and shipment in BCT on cfDNA levels, mutant allele fractions, and ctDNA yield. The effect of tube type and processing delay on the distribution of NGS non-reference calls and the effect of shipment on copy number profile were also investigated. Peripheral blood was collected from 47 patients with serous ovarian cancer and 15 patients with metastatic breast cancer into tubes for each experiment. The effects of delayed processing at room temperature were investigated by storing EDTA blood from 5-26 patients for 0 h, 6 h, 24 h, 48 h, 96 h, and 1 week before centrifugation. The effects of storage temperature before centrifugation were investigated by storing EDTA blood from 5-26 patients at 4˚C and room temperature (19-25˚C) for 0, 24, 48, and 96 h before centrifugation. The effects of collection tube type on levels of cfDNA was investigated by collecting blood into K3EDTA and BCT tubes and processing immediately (20 and 10 specimens, respectively), after 96 h at room temperature (5 and 10 specimens, respectively), and after 1 week at room temperature (5 and 15 specimens, respectively). To investigate the effects of shipping blood in BCT tubes, one specimen from each of thirteen patients was collected in K3EDTA tubes and processed immediately, another was stored in a BCT at room temperature, and the third was shipped in a BCT back to the same laboratory and processed (48 h delay for 10 specimens, 96 h delay for 2 specimens, and 5-day delay for 1 specimen). Plasma was obtained by centrifugation at 820 x g for 10 min, followed by recentrifugation of the supernatant at 14000 x g for 10 min. Cell-free DNA was extracted from 1.4 mL plasma using the QIAamp Circulating Nucleic Acid kit with the addition of carrier RNA and a spike-in extraction efficiency control and then stored at -20˚C. cfDNA was quantified by digital PCR amplification of a 65-bp fragment of RPP30 and ctDNA was quantified using Taqman assays for wildtype and mutant TP53 or PIK3CA.
Summary of Findings:
The levels of cfDNA increased significantly when blood was stored in K3EDTA tubes at room temperature for 48 h or longer (P<0.05) and the mutant allele fraction declined significantly when blood was stored in K3EDTA tubes at room temperature for 96 h or more (P=0.005) but the copies of ctDNA per mL of plasma was unaffected by room temperature processing delay of up to 1 week. Storage of K3EDTA at 4˚C for 48 h or more also resulted in significant increases in cfDNA yield (P<0.05) and decreased mutant allele fraction (insufficient data for statistical analysis) but the yield increases were smaller than when stored at room temperature and this difference was significant only for specimens stored for 96 h (P<0.05). cfDNA levels were not significantly different when blood was processed immediately or stored for a week in BCT tubes before processing but there was a trend toward higher levels in specimens stored in BCT for 1 week rather than 96 h. Similarly, the mutant allele fraction appeared to be minimally affected by storage for up to 1 week in BCT, but significance was not tested. Importantly, the ratio of non-reference to reference calls in targeted sequencing for each mutation type was unaffected by tube type and processing delay. Specimens shipped in BCTs had comparable levels of ctDNA and mutant allele fraction as those stored in BCT for an equivalent time at room temperature or in K3EDTA tubes processed immediately. The copy number profiles were modestly to very strongly correlated among shipped specimens, specimens stored in BCT, and specimens in K3EDTA tubes processed immediately (average R=0.76, range R=0.44-0.98) and no significant differences in the copy number distributions were identified.
Biospecimens
Preservative Types
- Streck/BCT
- None (Fresh)
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Digital PCR DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution K3EDTA
BCT
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Biospecimen Preservation Type of fixation/preservation EDTA
Blood collection tube additive
Storage Storage duration 0 h
6 h
24 h
48 h
96 h
1 week
Storage Storage temperature Room Temperature
4˚C
Storage Between site transportation method Mailed
Not transported
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Study Purpose
This study investigated the effects of centrifugation speed on cell-free DNA (cfDNA) levels and mutant allele fractions. Peripheral blood was collected into K3EDTA tubes from 13 patients with either serous ovarian cancer or metastatic breast cancer. Plasma was obtained by centrifugation at 820 x g for 10 min followed by recentrifugation of the supernatant at 14000 x g for 10 min, centrifugation at 1600 x g for 10 min followed by recentrifugation of the supernatant at 14,000 x g for 10 min, or centrifugation at 1600 x g for 10 min followed by recentrifugation of the supernatant at 3,000 x g for 10 min. Cell-free DNA was extracted from 1.4 mL of plasma using the QIAamp Circulating Nucleic Acid kit with the addition of carrier RNA and a spike-in extraction efficiency control and then stored at -20˚C. cfDNA was quantified by digital PCR amplification of a 65-bp fragment of RPP30 and ctDNA was quantified using Taqman assays for wildtype and mutant TP53 or PIK3CA.
Summary of Findings:
cfDNA yields and mutant allele frequencies were not significantly affected by centrifugation protocol.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Digital PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Multiple speeds compared