EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples.
Author(s): Barra GB, Santa Rita TH, Vasques JA, Chianca CF, Nery LF, Costa SS
Publication: Clin Biochem, 2015, Vol. 48(15), Page 976-81
PubMed ID: 25746148 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of EDTA on endogenous and exogenous DNAse activity in serum and plasma, and to determine the effects of storage temperature and duration on circulating cell-free DNA (ccfDNA) levels in serum and plasma from pregnant women.
Conclusion of Paper
EDTA plasma had less endogenous and exogenous DNAse activity than serum, and the addition of EDTA to serum or non-anticoagulated plasma decreased the endogenous DNAse activity. EDTA plasma had 1.4-fold more ccfDNA than serum. When stored at 37°C, serum ccfDNA levels declined significantly after 24 h, and after 48 h, EDTA plasma ccfDNA levels had declined. EDTA plasma ccfDNA levels were comparable when stored for 24 h at -20°C, 4°C or 22°C, but ccfDNA levels in serum were lower when serum was stored for 24 h at 22°C than when stored at 4°C or -20°C.
Studies
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Study Purpose
The purpose of this study was to compare DNAse activity in EDTA plasma and serum, determine if EDTA can inhibit DNAse activity in non-anticoagulated plasma and serum, and to determine the effects of storage temperature and duration on ccfDNA levels in serum and plasma from pregnant women. K2EDTA plasma, non-anticoagulated plasma and serum were collected from 34 pregnant women carrying male fetuses. K2EDTA plasma and serum tubes were centrifuged, transported for an unspecified duration and then frozen at -20°C. Non-anticoagulated plasma was obtained through immediate centrifugation of blood, transfer of the supernatant to a new tube and a second centrifugation. After thawing, 1 mL of the supernatants were transferred to a polypropylene tube and recentrifuged before addition of EDTA to supernatant. DNA was extracted using the NucliSens easyMAG system and quantified by amplification of the Y-chromosome specific sequence DYS-14 (DYS14).
Summary of Findings:
When incubated at 37°C, degradation of the hydrolysis probe in EDTA plasma plateaued after 1-3 h, but degradation of the hydrolysis probe in serum continued for the entire 24 h. After 24 h there was 14.9-fold more degradation in serum than EDTA plasma (p=0.0156). Further, the degradation of the hydrolysis probe in non-anticoagulated plasma or serum was inhibited in a dose-dependent manner by the addition of EDTA. When specimens were incubated at 37°C for 1 h with 1 unit of DNAse, serum ccfDNA levels declined (p=0.0039), but EDTA plasma levels remained unchanged. EDTA plasma had 1.4-fold more ccfDNA than serum. When stored at 37°C for 24 h, serum ccfDNA levels declined by 3.52-fold (p=0.001), and after 48 h, EDTA plasma and serum levels of ccfDNA had declined by 1.10-fold (p=0.001) and 5.9-fold (p=0.001), respectively. EDTA plasma ccfDNA levels were comparable when stored for 24 h at -20°C, 4°C or 22°C, but ccfDNA levels were 1.6-fold and 1.54-fold lower when serum was stored for 24 h at 22°C than when stored at 4°C (p=0.0039) or -20°C (p=0.0039), respectively.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Pregnant
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant EDTA
None
Multiple concentrations evaluated
Biospecimen Aliquots and Components Biospecimen components 5 x 10^-9 M EDTA
5 x 10^-8 M EDTA
5 x 10^-7 M EDTA
5 x 10^-6 M EDTA
5 x 10^-5 M EDTA
5 x 10^-4 M EDTA
5 x 10^-3 M EDTA
5 x 10^-2 M EDTA
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Real-time qPCR Specific Targeted nucleic acid DYS14
Storage Storage temperature -20°C
4°C
22°C
37°C
Analyte Extraction and Purification Nucleic acid digestion 25 U DNase I
No DNase added
Storage Storage duration 0 h
24 h
48 h