A novel approach to stabilize fetal cell-free DNA fraction in maternal blood samples for extended period of time.
Author(s): Fernando MR, Jiang C, Krzyzanowski GD, Somer-Shely T, Ryan WL
Publication: PLoS One, 2018, Vol. 13, Page e0208508
PubMed ID: 30521613 PubMed Review Paper? No
Purpose of Paper
This paper investigated if Blood Exo DNA ProTeck (ProTeck) tubes stabilize plasma cell-free DNA (cfDNA) and percentage of cell-free fetal DNA (cffDNA) and reduces genomic DNA contamination in blood from pregnant women stored at room temperature for up to 28 days, at 4˚C for up to 21 days, and at 30˚C for up to 14 days. The effect of ProTeck agent on cell metabolism was also investigated by measuring glucose concentration.
Conclusion of Paper
Glucose levels, an indicator of cell metabolism, remained at approximately 95% of initial levels when blood was stored in ProTeck tubes at room temperature (22˚C) for up to 28 days but declined rapidly with storage in K3EDTA tubes. Storage of blood in K3EDTA tubes resulted in significant increases in plasma DNA levels and decreased in percentage of cffDNA at the shortest durations examined (1 day at room temperature, 3 days at 4˚C, and 2 days at 30˚C). When blood was stored in ProTeck tubes, the levels of cfDNA were stable for 14 days at room temperature, 21 days at 4˚C, and 7 days at 30˚C and the percentage of cffDNA was stable for 28 days at room temperature, 21 days at 4˚C, and 7 days at 30˚C. Bioanalyzer results showed an increase in high molecular weight DNA with increased storage in K3EDTA tubes at room temperature, 4˚C, and 30˚C but not in ProTeck tubes; indicating the increased plasma DNA was due to genomic contamination.
Studies
-
Study Purpose
This study investigated if ProTeck tubes stabilize plasma cfDNA and percentage of cffDNA and reduce genomic DNA contamination in blood from pregnant women stored at room temperature for up to 28 days, at 4˚C for up to 21 days, and at 30˚C for up to 14 days. The effect of ProTeck agent on cell metabolism was also investigated by measuring glucose concentration. Blood was collected from women who were 8-9 weeks pregnant into matched K3EDTA and ProTeck tubes. Blood was aliquoted for within 2 h of blood draw and stored for 0, 1, 2, 3, and 4 days (30 specimens) or 0, 3, 7, 14, and 28 days (28 specimens) at room temperature; 0, 3, 7, 14, and 21 days at 4˚C (7 specimens); or 0 ,2, 3, 7, and 14 days at 30˚C (7 specimens). After storage, plasma was obtained by centrifugation at 1600 x g for 10 min followed by 16000 x g for 10 min. cfDNA was extracted using the QIAamp Circulation Nucleic Acid kit and stored at -80˚C. Concentration and size of cfDNA in specimens stored for 0, 3, 7, 14, and 28 days was determined by a bioanalyzer. cfDNA and cffDNA were further quantified by droplet digital PCR amplification of a 140 bp region of the RASSF1A gene promoter in BstU1 digested and undigested DNA. Blood glucose levels were determined in blood stored at room temperature for 0, 3, 7, 14, and 28 days using a blood glucose meter.
Summary of Findings:
After 3 days at room temperature, glucose was undetectable in blood in K3EDTA tubes but glucose levels were approximately 95% of those at day 0 in blood stored in ProTeck reagent. No further declines in glucose levels were noted after storage at room temperature in ProTeck tubes for up to 28 days. Plasma DNA levels rose and the percentage of cffDNA declined only non-significantly with storage of blood in ProTeck tubes at room temperature for up to 14 days, but there was a 1.4% increase in the cfDNA concentration over baseline after 28 days (P<0.0004). In contrast, plasma DNA levels rose rapidly with storage in K3EDTA tubes and were already significantly different from baseline in specimens stored for 1 day at room temperature before centrifugation (P<0.05). As the plasma DNA levels rose, the percentage of cffDNA in plasma declined such that there was a 48.5% decrease noted by Day 2 in K3EDTA tubes (P<0.01). Similar to storage at room temperature, increased plasma DNA concentration and decreased percentage of fetal DNA were observed after storage of blood in K3EDTA tubes for the shortest durations examined: 3 days at 4˚C (P<0.02 and P<0.01, respectively) and 2 days at 30˚C (P<0.0006, P<0.03). Increased plasma DNA and decreased percentage of cffDNA was observed in specimens stored in ProTeck tubes after 14 days at 30˚C (P<0.03, both) and were not observed even after 21 days at 4˚C in ProTeck tubes. Bioanalyzer results showed an increase in high molecular weight DNA in plasma with increased storage of blood in K3EDTA tubes at room temperature, 4˚C, and 30˚C but not ProTeck tubes. NMR analysis of the Proteck agent demonstrated that it is formaldehyde free.
Biospecimens
Preservative Types
- Other Preservative
- None (Fresh)
Diagnoses:
- Pregnant
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer Carbohydrate Clinical chemistry/auto analyzer DNA Digital PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution K3EDTA tube
Blood Exo DNA ProTeck tube
Storage Storage duration 0 days
1 day
2 days
3 days
4 days
7 days
14 days
21 days
28 days
Storage Storage temperature 4˚C
22˚C
30˚C