Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy.
Author(s): Sato A, Nakashima C, Abe T, Kato J, Hirai M, Nakamura T, Komiya K, Kimura S, Sueoka E, Sueoka-Aragane N
Publication: Oncotarget, 2018, Vol. 9, Page 31904-31914
PubMed ID: 30159131 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of precentrifugation storage of blood in K2EDTA, sodium citrate, Streck BCT, and PAXgene tubes on cell-free DNA (cfDNA) yields and fragment size. The effects of delayed processing of sodium citrate tubes, long-term frozen storage of cfDNA or plasma, and cfDNA extraction method on EGFR mutation quantification were also examined.
Conclusion of Paper
cfDNA concentrations and concentration of large DNA fragments increased in sodium citrate and K2EDTA plasma obtained by either centrifugation protocol with pre-centrifugation storage of blood at 4˚C and room temperature. The increases in cfDNA yield were only significant for sodium citrate plasma after 72 h at room temperature for 72 h or 4˚C for 1 week and in K2EDTA plasma after 72 h at either temperature, regardless of centrifugation protocol used. Importantly, sodium citrate plasma obtained after 72 h at 4˚C had comparable cfDNA levels and fragment size distribution to plasma from specimens stored for 72 h pre-centrifugation at room temperature in Streck BCT or PAXgene tubes.
The area under mutation peak for L858R was lower when blood was stored for 72 h preprocessing in sodium citrate tubes at room temperature than when blood was processed immediately or stored at 4˚C for 72 h. The area under mutation peak decreased when cfDNA was stored for 7 years compared to initial levels but the magnitude of the decrease was weakly correlated with mutation frequency. The magnitude of the decrease over the 7 years of storage was even larger when plasma was stored instead of extracted cfDNA. Importantly, the decrease was smaller when extraction was with cellulose magnetic beads than spin columns.
Studies
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Study Purpose
This study compared the effects of precentrifugation storage at room temperature and 4˚C on cfDNA yield and fragment size in K2EDTA and sodium citrate plasma. A total of 32 mL of blood was collected from each of ten healthy volunteers and divided among eight sodium citrate tubes and eight K2EDTA tubes. Blood was stored at room temperature for 0, 4, 24, and 72 h and at 4˚C for 4 h, 24 h, 72 h, and 1 week before plasma isolation. Plasma was obtained by centrifugation at 3000 rpm for 20 min. To investigate if these effects could be mitigated by centrifugation protocol, blood from five healthy volunteers was divided among four sodium citrate and four K2EDTA tubes. Sodium citrate plasma was obtained by centrifugation at 3000 x g for 20 min and K2EDTA plasma was obtained by centrifugation 1600 × g for 10 min followed by 16000 × g for 10 min. cfDNA was extracted from all plasma specimens using the Maxwell RSC kit and quantified by Quantus. cfDNA size was determined using a bioanalyzer.
Summary of Findings:
The median cfDNA concentration in sodium citrate plasma increased progressively with pre-centrifugation storage of blood at 4˚C room temperature, but the increase was only significant when stored at room temperature for 72 h (P<0.001) or 4˚C for 1 week (P=0.019). Similarly, median cfDNA concentrations increased with increasing processing delay of K2EDTA plasma, but the changes were only significant when stored for 72 h or 1 week at 4˚C (P=0.021 and P<0.001, respectively) or for 72 h at room temperature (P<0.001). While a single 170 bp peak was observed in sodium citrate plasma from blood stored at 4˚C for up to 72 h, peaks of larger DNA (1-9 kb) were observed when blood was stored in either tube type at room temperature and when EDTA plasma was stored for 72 h or more at 4˚C. The concentration of DNA fragments of 1000-9000 bp in K2EDTA plasma was significantly higher when blood was stored at 4˚C for 72 h or longer (P<0.001, both), but the concentration of fragments of 100-250 bp was unaffected. Plasma from blood stored in K2EDTA tubes for 72 h and centrifuged twice also had higher cfDNA concentrations and a higher concentration of large fragments (>1000 bp) than that processed immediately (P=0.043, both), but there was no change in the concentration of short fragments.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Fluorometry DNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Potassium EDTA
Sodium citrate
Storage Storage temperature Room temperature
4˚C
Storage Storage duration 0 h
4 h
24 h
72 h
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
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Study Purpose
This study investigated the effects of precentrifugation storage at room temperature or 4 ˚C on cfDNA yield, fragment size, and detection of the L858R mutation in sodium citrate plasma. Blood was drawn from a single patient with non-small cell lung cancer (NSCLC) and a L858R mutation into sodium citrate tubes and then divided among three sodium citrate tubes. One tube was immediately processed for cfDNA extraction, while the others were stored for 72 h at room temperature or 4˚C. Plasma was obtained by centrifugation at 3000 rpm for 20 min. cfDNA was extracted from all plasma specimens using the Maxwell RSC kit and quantified by Quantus. cfDNA size was determined using a bioanalyzer. The L858R EGFR mutation was detected using the PCR based i-densy IS 5320.
Summary of Findings:
The cfDNA concentration was 7.9 ng/mL in the specimen processed immediately and 6.9 ng/mL and 140.5 ng/mL in the specimens processed after 72 h at 4˚C and room temperature, respectively. Further, the specimen processed after room temperature storage had more cfDNA >1000 bp than the other specimens. The area under mutation peak for L858R was lower when blood was stored for 72 h preprocessing at room temperature than when blood was processed immediately or stored at 4˚C for 72 h.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Storage temperature Room temperature
4˚C
Storage Storage duration 0 h
72 h
-
Study Purpose
This study compared cfDNA yield and fragment size in specimens processed immediately and after 72 h in sodium citrate, Streck BCT, and PAXgene ccfDNA tubes. Blood was collected from five healthy volunteers and divided among two sodium citrate tubes, two Streck BCT tubes, and two PAXgene ccfDNA tubes. Specimens were stored for 0 and 72 h at 4˚C (sodium citrate tubes) or room temperature (stabilizer tubes) before processing to plasma. Plasma was obtained by centrifugation of sodium citrate tubes at 3000 rpm for 20 min, Streck BCT tubes at 1600 x g for 10 min followed by 160000 x g for 10 min, and PAXgene tubes by centrifugation at 1900 x g for 10 min and 19000 x g for 10 min. DNA was extracted from plasma using the MAXwell RSC kit and quantified by Quantus. cfDNA size was determined using a bioanalyzer.
Summary of Findings:
After 72 h, there were no differences in cfDNA levels or fragment size distribution between specimens stored in sodium citrate, Streck BCT, or PAXgene tubes.
Biospecimens
Preservative Types
- None (Fresh)
- PAXgene
- Streck/BCT
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Streck BCT
Sodium citrate tube
PAXgene ccfDNA tube
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Storage duration 0 h
72 h
Biospecimen Preservation Type of fixation/preservation PAXgene
Blood collection tube additive
None (fresh)
-
Study Purpose
This study compared detection of the EGFR mutation in fresh specimens, specimens stored as extracted cfDNA, and specimens stored as plasma and subjected to extraction using two different kits. Plasma and cfDNA was obtained from 22 patients with advanced NCSLC and the EGFR T790M mutation. Plasma was obtained immediately after blood collection and, subsequently, cfDNA was isolated using a silica membrane spin column. Plasma was stored at -80˚C and cfDNA was stored in LoBind tubes at -20˚C for 7 years before the study. cfDNA was re-extracted from plasma for this study using silica membrane spin columns (QIAamp DNA Mini Kit) and cellulose magnetic beads (Maxwell RSC ccfDNA plasma cartridge). The EGFR T790M mutation was detected using the PCR-based i-densy IS 5320.
Summary of Findings:
The area under mutation peak decreased when cfDNA was stored for 7 years compared to initial levels but the magnitude of the decrease was significantly larger when the initial area under mutation peak was ≤93.6 (approximately 50% reduction) compared to >93.6 (P=0.014). Further, the decrease was weakly correlated with the initial area under mutation peak (R2=0.1542). The magnitude of the decrease over the 7 years of storage was even larger when plasma was stored instead of extracted cfDNA. Importantly, the decrease was smaller when extraction was with cellulose magnetic beads (Maxwell RSC ccfDNA plasma cartridge) than QIAamp spin columns.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage conditions Plasma
Extracted cfDNA
Analyte Extraction and Purification Analyte isolation method Maxwell RSC ccfDNA plasma cartridge
QIAamp DNA Mini Kit
Storage Storage duration 0 days
7 years