DNA is increased in patients with neoplastic diseases (1), pregnancy-related complications (2, 3), trauma (4), and certain autoimmune diseases (5). In addition to quantitative analysis, qualitative changes in plasma DNA have also been investigated in different physiologic and pathologic conditions. In this regard, our group has shown that fetal DNA is shorter than the maternal counterpart in the plasma of pregnant women (6), and this has allowed the enrichment of fetal DNA from maternal plasma by size fractionation (7). It has also been shown that plasma DNA fragments are longer in patients suffering from a variety of neoplastic diseases (8). In addition, circulating Epstein–Barr virus DNA molecules in patients with nasopharyngeal carcinoma have been found to consist mainly of short DNA fragments 180 bp in size (9). Because the samples collected from patients and healthy controls in these studies were usually collected at different time points and under different conditions, it is important to ensure that the observed variations in the sizes of plasma DNA are the result of biological changes rather than artifacts attributable to differences in sample handling. Therefore, in this study, we investigated the effects of several preanalytical factors, including clotting, delayed separation of blood cells from plasma, freezing–thawing, and storage, on the integrity of circulating cell-free DNA. We recruited 27 healthy volunteers for modules 1 to 4 of the study. Venous blood (30 mL) was collected from each volunteer into eight tubes containing EDTA and one plain tube. The samples serving as references were processed immediately after collection. Plasma was separated from the blood cells by centrifugation at 1600g for 10 min, and the plasma was then microcentrifuged at 16 000g for 10 min to ensure the complete removal of cells. DNA was extracted from the plasma samples with use of the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer’s recommendations and was stored at 80 C until analysis.

Module 1. To evaluate the effect of clotting, blood collected in the plain tubes was allowed to stand at room temperature for 30 min to allow clotting. Serum was then separated from the blood cells by centrifugation and microcentrifugation as described above. DNA was extracted from the serum samples immediately and stored at 80 C until analysis.