Cell-free DNA in serum and plasma has been suggested to have diagnostic potential because associations between DNA concentrations and several disorders have been described (1). The concentration of cell-free DNA circulating in plasma and serum has been analyzed in several studies and used as an interchangeable index of the quantity of circulating DNA in blood (2–4). However, it is known that the DNA concentration in serum is 3-to 24-fold higher than in plasma (3–6). Recently published articles in this journal showed that various preanalytical factors of blood sampling and processing can affect the DNA concentration in plasma (5, 7, 8), but these findings do not explain the difference between plasma and serum concentrations of DNA mentioned above. Comparative investigations of the preanalytical conditions influencing the DNA concentration in serum and plasma are lacking. In addition, reference intervals for the concentration of cell-free DNA in serum were established without considering these factors (9). Thus, to complement the data of Lui et al.(5), we analyzed the influence of time delay in blood processing for plasma and serum at room temperature and at 4 C. Venous blood samples from 10 healthy volunteers (5 females and 5 males; mean age, 42 years) were simultaneously collected in Monovette plastic tubes without any additive for native serum (cat. no. 02.1726. 001; Sarstedt), in plastic tubes with kaolin-coated plastic granulate coagulation accelerator (cat. no. 04.1904. 001; Sarstedt) for preparation of serum samples, and in Monovette plastic tubes coated with potassium EDTA (cat. no. 05.1167. 001; Sarstedt) for preparation of plasma samples. The tubes were either centrifuged at 2000g for 10 min at 4 C immediately after venipuncture or were stored for 2, 4, or 8 h at room temperature (25 C) and at 4 C for 8 and 24 h, respectively, before being centrifuged under similar conditions. The supernatants were carefully removed and centrifuged again at 16 000g for 10 min at 4 C and stored at 80 C until analysis. Using a NucleoSpin Blood Kit (Macherey-Nagel), we extracted DNA from 400 L of sample per column, eluted it in 100 L of buffer according to the manufacturer’s instructions, and stored the eluted DNA at 20 C until use. DNA equivalents were quantified by amplifying the-globin gene by realtime PCR (LightCyclerTM; Roche), using the QuantiTect SYBR Green PCR Kit (Qiagen). A 110-bp fragment of the-globin gene was generated with the forward primer 5-ACACAACTGTGTTCACTAGC-3 and the reverse primer 5-CAACTTCATCCACGTTCACC-3 (TIB Mol-Biol) in a final concentration of 0.5 M each. The cycle conditions were as follows: initial activation step at 95 C for 15 min, followed by 45 cycles of denaturation at 95 C for 15 s, annealing at 56 C for 20 s, and extension at 72 C for 15 s. The reaction volume was 20 L, including 2 L of DNA eluate. The product-specific melting temperature was 82 C. A calibration curve obtained with serial dilutions of a control

DNA template (LightCycler Control Kit DNA; Roche) was linear at least up to 4546 genome-equivalents of-globin [genome-equivalents/mL were calculated using a conversion factor of 6.6 pg of DNA per single diploid human cell (5)]. The within-run (n 12) and between-run (n 10) imprecisions (CVs) for a DNA template (807.6 genome-equivalents/mL) were 3.1% and 7.2%, respectively. Statistical analyses were made with GraphPad Prism 3.03 for Windows (GraphPad Software). Parametric statistical tests were used because the DNA concentrations in our 10 samples had a gaussian distribution (Kolmogorov–Smirnov test).