Collection of cell-free DNA for genomic analysis of solid tumors in a clinical laboratory setting.
Author(s): Raymond CK, Hernandez J, Karr R, Hill K, Li M
Publication: PLoS One, 2017, Vol. 12, Page e0176241
PubMed ID: 28448587 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of room temperature storage of K2EDTA blood prior to centrifugation and K2EDTA plasma after centrifugation and obtaining plasma by plasmapheresis rather than centrifugation on the yield and integrity of cell-free DNA (cfDNA). The authors also compared the size distribution and sequence of cfDNA between healthy patients and those with lung cancer and compared the sequence of cfDNA with sonicated DNA.
Conclusion of Paper
There was an increase in gDNA contamination when blood was stored at room temperature for 24 h before centrifugation, but there was no effect on cfDNA yield or integrity due to storing blood for 24 h prior to centrifugation or plasma for 172 h after centrifugation. Further, the detection of mutations was not hindered by storage of plasma containing mutated DNA for up to 4 days at room temperature. The authors state that cfDNA obtained from plasmapheresis specimens appeared to be intact nucleosomal fragments such as found in plasma obtained by centrifugation. Similar cfDNA size distributions were found for tumor-derived EML4-ALK fusion fragments and germline DNA fragments. Sequencing of cfDNA from healthy patients and those with cancer showed that unlike sonicated genomic DNA there was a bias toward more cytosine in the first few nucleotides (35% versus 21%) and decreased thymidine in the second position and guanosine in the third position, indicating that the fragmentation of cfDNA is not random.
Studies
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Study Purpose
This study investigated the effects of room temperature storage of K2EDTA blood prior to centrifugation and K2EDTA plasma after centrifugation and obtaining plasma by plasmapheresis rather than centrifugation on the yield and integrity of cfDNA. Blood was collected from 84 healthy individuals and at least three patients with non-small cell lung cancer into K2EDTA tubes. Plasma was obtained by double centrifugation of the blood at 2000 x g for 10 min. An additional 40 plasma specimens were obtained by autopheresis using sodium citrate as the anticoagulant. cfDNA was isolated from plasma using the Qiagen Circulating Nucleic acids kit and quantified by Qubit. The size of isolated DNA was determined by electrophoresis. Cloning and next generation sequencing analysis was performed with the Illumina NextSeq NGS system using paired-end sequencing. The effects of delayed centrifugation were investigated by storing blood from a healthy volunteer at room temperature for 0, 1, 2, 3, 4, and 24 h before centrifugation and the effects of delayed extraction were investigated by storing plasma at room temperature for 30, 52, 77, 100, and 172 h before overnight shipping and DNA extraction.
Summary of Findings:
Analysis of cfDNA from 84 healthy patients showed that nucleosomal-sized fragments were the dominant DNA form. There was an increase in gDNA contamination when blood was stored at room temperature for 24 h before centrifugation, but it did not affect cFDNA yield or integrity nor did it interfere with the hybrid capture required for NGS. cfDNA yields and integrity were comparable in specimens from plasma held for 30, 53, 77, 100, or 172 h before overnight shipping and DNA extraction. Further, there was no change in the detection of the mutations when DNA fragments containing mutations were added to plasma from healthy individuals and stored for up to 4 days at room temperature. Plasmapheresis specimens yielded 4.4 ng of cfDNA per mL, which the authors state all appeared to be intact nucleosomal fragments such as found in plasma obtained by centrifugation.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Fluorometry DNA Electrophoresis DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Method of fluid acquisition Apheresis
Venipuncture
Storage Time at room temperature 0 h
1 h
2 h
3 h
4 h
24 h
30 h
52 h
77 h
100 h
172 h
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Biospecimen Aliquots and Components Blood and blood products Plasma
Whole blood
-
Study Purpose
This study compared the size distribution and sequence of cfDNA between healthy patients and those with lung cancer and compared the sequence characteristics of cfDNA and sonicated genomic DNA. Blood was collected from 84 healthy individuals and at least three patients with non-small cell lung cancer into K2EDTA tubes. Plasma was obtained by double centrifugation of the blood at 2000 x g for 10 min. cfDNA was isolated from plasma using the Qiagen Circulating Nucleic acids kit and quantified by Qubit. The size of isolated DNA was determined by electrophoresis. Cloning and next generation sequencing analysis were performed with the Illumina NextSeq NGS system using paired end sequencing. The effects of delayed centrifugation were investigated by storing blood from a healthy volunteer at room temperature for 0, 1, 2, 3, 4, and 24 h before centrifugation and the effects of delayed extraction were investigated by storing plasma at room temperature for 30, 52, 77, 100, and 172 h before overnight shipping and DNA extraction.
Summary of Findings:
cfDNA was predominantly nucleosome-sized monomers and dimers. Similar size distributions were found for tumor- derived EML4-ALK fusion fragments and germline DNA fragments. Sequencing of cfDNA from healthy patients and those with cancer showed that there was a bias toward more cytosine in the first few nucleotides in sonicated genomic DNA (35% versus 21%) and decreased thymidine in the second position and decreased guanosine in the third position, indicating that the fragmentation was not random.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Neoplastic - Carcinoma
- Normal
Platform:
Analyte Technology Platform DNA Electrophoresis DNA Next generation sequencing DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Next generation sequencing Specific Targeted nucleic acid Sonicated gDNA
cfDNA
Preaquisition Diagnosis/ patient condition Healthy
NSCLC