EDTA Is a Better Anticoagulant than Heparin or Citrate for Delayed Blood Processing for Plasma DNA Analysis

Abstract

Plasma is usually chosen for cell-free circulating DNA studies because ex vivo release of DNA from hematopoietic cells was demonstrated in serum during the clotting process from whole blood (1)(2)(3). Among the most commonly used anticoagulants, EDTA, heparin, and citrate, heparin was suggested by some investigators to be an unsuitable anticoagulant because it could inhibit PCR (4)(5)(6). However, the quantitative effects of the choice of anticoagulants for plasma DNA analysis have not been studied systematically. We therefore conducted an experiment to investigate the effect of three commonly used anticoagulants on plasma DNA quantification. Ten healthy individuals were recruited, and peripheral blood was collected into four EDTA, four heparin, and four citrate tubes (Vacuette; Greiner) for each individual. Blood samples were kept at room temperature (24°C). Plasma was obtained by centrifugation from the EDTA, heparin, and citrate tubes at four different time points, 0, 2, 6, and 24 h after venesection, respectively, with one tube used for each time point. The plasma samples were stored at −80°C until analysis. DNA was extracted from 200 μL of plasma according to the “blood and body fluid protocol” of the QIAamp Blood Kit (Qiagen), with a final elution volume of 50 μL. We then subjected 5 μL of the DNA to real-time quantitative PCR for the β-globin gene as described previously (7) on a 7700 Sequence Detector (Applied Biosystems). DNA concentration is expressed as genome-equivalents/mL.