Circulating cell free DNA: Preanalytical considerations.
Author(s): El Messaoudi S, Rolet F, Mouliere F, Thierry AR
Publication: Clin Chim Acta, 2013, Vol. 424C, Page 222-230
PubMed ID: 23727028 PubMed Review Paper? Yes
Purpose of Paper
The purpose of this paper was to determine the effects of whole blood and plasma storage duration and temperature and freeze-thaw cycling of plasma on levels of circulating cell-free DNA (ccfDNA) in plasma.
Conclusion of Paper
A slight increase in ccfDNA concentration in EDTA-plasma was noted with longer storage of whole blood prior to centrifugation or storage of plasma, but significant effects were only observed after 6 and 4 h at room temperature, respectively. When whole blood was agitated during storage, the increase in ccfDNA concentration was slightly greater and the ccfDNA was more degraded than when specimens were not agitated. When plasma was stored frozen for 3 h there was more ccfDNA extracted and less fragmentation of the ccfDNA than when the specimens were stored at 4 degrees C or room temperature. When plasma was subjected to multiple freeze-thaw cycles, DNA fragmentation increased after the third cycle. Storage of plasma at -80 degrees C for 3-9 months did not affect the ccfDNA concentration compared to plasma stored 0-3 months, but storage at -20 degrees C for 3-5 months led to a slight decrease in ccfDNA concentration from that found in plasma stored at -20 degrees C for 0-3 months. When plasma was stored frozen, there was greater variability in concentration between replicates than when extracted DNA was stored frozen.
Studies
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Study Purpose
The purpose of this study was to determine the effects of whole blood storage duration and temperature and agitation during storage on levels of ccfDNA in EDTA plasma. Plasma was frozen at -80 degrees C until analysis. ccfDNA integrity was determined by the ratio of a 288 bp amplicon to a 105 bp amplicon of the proto-oncogene B-Raf (BRAF).
Summary of Findings:
A slight increase in ccfDNA concentration in EDTA-plasma was noted with increasing whole blood storage time prior to centrifugation; however, a significant increase was only observed after 6 h at room temperature and not at 4 degrees C. When whole blood was agitated during storage, the increase in ccfDNA concentration was slightly greater than when specimens were not agitated. Further, when whole blood was stored at room temperature for 6 h or agitated during storage, there was slightly more degradation of the ccfDNA in the plasma that that observed after shorter storage durations or when specimens were not agitated.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 15 min
30 min
40 min
45 min
1 h
2 h
3 h
4 h
6 h
Storage Storage temperature 4 degrees C
Room temperature
Storage Storage conditions Agitated
Not agitated
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
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Study Purpose
The purpose of this study was to investigate the effects of freeze-thaw cycling and plasma storage temperature and duration on levels of extracted ccfDNA. Freeze-thaw cycling was at -80 degrees C. ccfDNA integrity was determined by the ratio of a 288 bp amplicon to a 105 bp amplicon of BRAF.
Summary of Findings:
While the amount of ccfDNA extracted from plasma increased with increasing durations of room temperature storage, storing plasma for up to 4 h had no effect on ccfDNA fragmentation. When plasma was stored frozen for 3 h, there was more ccfDNA extracted and less fragmentation of the ccfDNA than when the specimens were stored at 4 degrees C or room temperature. When plasma was subjected to multiple freeze-thaw cycles, after the third cycle the ccfDNA was more fragmented. Storage of plasma at -80 degrees C for 3-9 months did not affect the ccfDNA concentration compared to plasma stored for 0-3 months, but storage at -20 degrees C for 3-5 months led to a slight decrease in ccfDNA concentration compared to that found in plasma stored at -20 degrees C for 0-3 months. When plasma was stored frozen, there was greater variability in ccfDNA concentration between replicates than when extracted DNA was stored frozen.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Freeze/thaw cycling 1 cycle
2 cycles
3 cycles
Storage Storage duration 0-3 months
0 h
1 h
2 h
3 h
4 h
3-5 months
3-9 months
Storage Storage temperature -80 degrees C
-20 degrees C
4 degrees C
Room temperature