The Effect of Preservative and Temperature on the Analysis of Circulating Tumor DNA.
Author(s): Parpart-Li S, Bartlett B, Popoli M, Adleff V, Tucker L, Steinberg R, Georgiadis A, Phallen J, Brahmer J, Azad N, Browner I, Laheru D, Velculescu VE, Sausen M, Diaz LA Jr
Publication: Clin Cancer Res, 2017, Vol. 23, Page 2471-2477
PubMed ID: 27827317 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of delayed centrifugation, storage temperature, and collection tube type on levels of cell-free DNA (cfDNA) and mutant allele frequency in plasma. Additionally, cfDNA yield, fragment size, and mutant allele frequency were compared in plasma and serum specimens.
Conclusion of Paper
Serum had a much higher number of genome equivalents cfDNA but a much lower mutant allele fraction and a larger fragment distribution. The volume of plasma obtained decreased with increasing room temperature storage, regardless of tube type. The genome equivalents of cfDNA increased and the mutant allele frequency decreased when blood was stored in K2EDTA tubes at room temperature but were stable when blood was stored in BCT. Storage of blood in K2EDTA tubes at 4˚C mitigated most of the effects of delayed processing, but specimens in K2EDTA tubes after three days had a noticeable increase in genomic DNA and a larger percent change in mutant allele fraction than those stored in BCT.
Studies
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Study Purpose
This study compared cfDNA yield, fragment size, and mutant allele frequency in serum and plasma specimens from patients with pancreatic cancer. Blood was collected from 15 pancreatic cancer patients with mutations in KRAS, EGFR, or BRAF into serum, K2EDTA, and BCT tubes. Serum was obtained 15-30 min after blood collection by centrifugation of tubes without anticoagulant at 1000-2000 x g for 10 min followed by recentrifugation of the supernatant in 1 mL aliquots at 18,400 x g for 10 min. Plasma was obtained by centrifugation of blood at 375 x g for 10 min and recentrifugation of the supernatant in 1 mL aliquots at 18,400 x g for 10 min. Plasma and serum aliquots were frozen at -80˚C until use. cfDNA was extracted using the QIAamp Circulating Nucleic Acid Kit and quantified using the Qubit dsDNA HS Assay Kit. Genomic equivalents of wild-type and mutant DNA were determined by droplet digital PCR and fragment size was determined by bioanalyzer.
Summary of Findings:
Serum yielded more than 10-fold more genome equivalents of cfDNA than plasma (P<0.001), but the mutant allele fraction in serum was approximately 10% of that observed in plasma (P<0.001). The largest peaks of cfDNA in plasma were 155 bp but there were multiple large fragments ranging from 150 to 2000 bp in serum.
Biospecimens
Preservative Types
- Streck/BCT
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer DNA Digital PCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
-
Study Purpose
This study investigated the effects of storing blood for up to 7 days before centrifugation in K2EDTA and BCT tubes on plasma volume, the yield of cfDNA, and the mutant allele fraction. Blood was collected from 9 patients with KRAS mutations into five K2EDTA and five BCT tubes which were inverted 10 times and then stored at room temperature. Plasma was obtained 0, 24, 72, 120, and 168 h after collection of blood by centrifugation at 375 x g for 10 min. The supernatant was aliquoted and recentrifuged at 18,400 x g for 10 min. Plasma aliquots were frozen at -80˚C until use. cfDNA was extracted using the QIAamp Circulating Nucleic Acid Kit and quantified using the Qubit dsDNA HS Assay Kit. Genomic equivalents of wildtype and mutant DNA were determined by droplet digital PCR.
Summary of Findings:
The volume of plasma obtained decreased with increasing room temperature storage, regardless of tube type. Storage of BCT at room temperature for 168 h (7 days) before processing did not affect the genome equivalents of cfDNA or the mutant allele fraction of the resultant plasma. In contrast, storage of K2EDTA tubes at room temperature for 24 h resulted in a 12-66% increase in the genome equivalents of cfDNA (P<0.05) and a 47-93% decline in the mutant allele frequency. Importantly, the increase in genomic equivalents and decrease in mutant allele frequency increased with increasing storage duration at room temperature.
Biospecimens
Preservative Types
- Streck/BCT
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Digital PCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Streck BCT
K2EDTA tube
Storage Storage duration 0 h
24 h
72 h
120 h
168 h
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
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Study Purpose
This study investigated the effects of storing blood for up to 72 h in K2EDTA tubes at 4˚C or in BCT at room temperature on the yield of cfDNA and the mutant allele fraction. Blood was collected from six colorectal cancer patients with KRAS mutations into three K2EDTA tubes and then stored at 4 ˚C and into three BCT tubes stored at room temperature. Plasma was obtained from blood <4, 24, and 72 h after collection by centrifugation at 375 x g for 10 min and recentrifugation of the supernatant in 1 mL aliquots at 18,400 x g for 10 min. Plasma aliquots were frozen at -80˚C until use. cfDNA was extracted using the QIAamp Circulating Nucleic Acid Kit and quantified using the Qubit dsDNA HS Assay Kit. Genomic equivalents of wild-type and mutant DNA were determined by droplet digital PCR and fragment size was determined by a bioanalyzer. Sequence mutations were detected in specimens from three patients by next generation sequencing using the Agilent SureSelect Target Enrichment System.
Summary of Findings:
Total genome equivalents of cfDNA remained constant when blood was stored in K2EDTA tubes at 4˚C or BCT tubes at room temperature for up to 3 days before processing, but storage in K2EDTA tubes at 4˚C for 72 h resulted in an increase of genomic DNA that did not occur when storage was in BCT at room temperature. Additionally, while changes in the mutant allele fraction remained insignificant over the 3 days of storage under either condition, the percent change in mutant allele fraction after 72 h was significantly greater in specimens stored for 3 days in K2EDTA tubes at 4˚C than in BCT tubes at room temperature (P<0.05). The base error rate was comparable in K2EDTA and BCT plasma and did not seem to be affected by the processing delay.
Biospecimens
Preservative Types
- Streck/BCT
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer DNA Next generation sequencing DNA Fluorometry DNA Digital PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Streck BCT
K2EDTA tube
Storage Storage temperature 4˚C
Room temperature
Storage Storage duration <4 h
24 h
72 h
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated