EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples.
OBJECTIVE
The extracellular DNA occurring in plasma-EDTA and serum is a biomarker of growing interest, especially in prenatal diagnosis and oncology. The objectives of the present study were to compare the DNase activity in these specimens and to investigate its ex-vivo impact over the circulating cell-free DNA yield (ccfDNA), using the circulating cell-free fetal DNA (ccffDNA) as a tool.


METHODS
EDTA-plasma and serum from women bearing male fetus were submitted to an endogenous DNase activity assay based on qPCR hydrolysis probe degradation, they were treated with DNAse I to investigate the action of an exogenous nuclease and also submitted to different temperature conditions to investigate the temperature-dependent degradation of the ccffDNA. In all instances, all male ccffDNA were quantified by qPCR targeting the Y chromosome-specific sequence DYS-14. Moreover, a serial dilution of EDTA was added to nonanticoagulated plasma and serum before the endogenous DNAse activity assay, to investigate the EDTA-mediated inhibition of the blood's DNase.


RESULTS
The endogenous nuclease activity was 14.9-fold higher in serum compared to EDTA-plasma. The DNAse I treatment did not alter the ccffDNA yields in EDTA-plasma, but completely degraded it in serum. The addition of increasing doses of EDTA to nonanticoagulated plasma and serum resulted in a stepwise inhibition of their nucleases activity. Finally, we observed a much more pronounced temperature-mediated decrease on the ccffDNA amount in serum compared to EDTA-plasma.


CONCLUSIONS
The exogenous and endogenous DNases are more active in serum, the anticoagulant EDTA indirectly inhibits blood DNases, and consequently ccfDNA is protected from the blood's DNase preanalytical impact in EDTA-plasma.
Sabin Laboratory of Clinical Analysis, Brasilia, Federal District, Brazil; Post-Graduation in Health Sciences, University of Brasilia, Brasilia, Federal District, Brazil. Electronic address: gbbarra@gmail.com.
Gustavo Barcelos
GB
Barra
Sabin Laboratory of Clinical Analysis, Brasilia, Federal District, Brazil; Post-Graduation in Health Sciences, University of Brasilia, Brasilia, Federal District, Brazil.
Ticiane Henriques
TH
Santa Rita
Sabin Laboratory of Clinical Analysis, Brasilia, Federal District, Brazil; Post-Graduation in Health Sciences, University of Brasilia, Brasilia, Federal District, Brazil.
Júlia
J
de Almeida Vasques
Post-Graduation in Health Sciences, University of Brasilia, Brasilia, Federal District, Brazil.
Camilla Figueiredo
CF
Chianca
Sabin Laboratory of Clinical Analysis, Brasilia, Federal District, Brazil.
Lídia Freire Abdalla
LF
Nery
Sabin Laboratory of Clinical Analysis, Brasilia, Federal District, Brazil.
Sandra
S
Santana Soares Costa
D000925:Anticoagulants / Q000494:pharmacology*; D015415:Biomarkers / Q000097:blood / Q000378:metabolism; D065096:Calcium Chelating Agents / Q000494:pharmacology*; D041322:Chromosomes, Human, Y / Q000378:metabolism; D004247:DNA / Q000097:blood* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000037:antagonists & inhibitors / Q000097:blood / Q000378:metabolism; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors* / Q000097:blood / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D008297:Male; D025202:Molecular Diagnostic Techniques / Q000379:methods; D010949:Plasma / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods; D044967:Serum / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology; D013696:Temperature
D000925:Anticoagulants; D015415:Biomarkers; D065096:Calcium Chelating Agents; D004791:Enzyme Inhibitors; D004247:DNA; D004492:Edetic Acid; D003851:Deoxyribonucleases; D003850:Deoxyribonuclease I
Cell-free DNA; Cell-free fetal DNA; DNase activity; Maternal plasma; Maternal serum; Nuclease activity