Effects of Preanalytical Factors on the Molecular Size of Cell-Free DNA in Blood

Abstract

Recently, plasma DNA analysis has been increasingly explored for different clinical diagnostic purposes. Previous studies have shown that the concentration of plasma 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. 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. To evaluate the effect of delayed separation of plasma from blood cells, we stored the blood samples in the EDTA tubes at either 4 °C or room temperature for 6 or 24 h after collection. Plasma was then isolated from the blood samples, and DNA was extracted as described above. To evaluate the effect of freezing and thawing of plasma samples, we stored the plasma samples at −80 °C for 24 h and then allowed them to thaw at room temperature for 30 min. For evaluation of the effect of one freeze–thaw cycle, after the samples had thawed, we extracted the DNA and stored it at −80 °C until analysis. For evaluation of the effect of three freeze–thaw cycles, we repeated the freeze-thaw cycle two more times before DNA extraction. To evaluate the effect of storage of plasma samples, we separated plasma from the blood cells immediately after blood collection and stored it at −80 °C for 2 weeks before DNA extraction. To evaluate the effect of freezing and thawing of extracted DNA, we took 10 mL of venous blood from each of nine healthy volunteers, extracted the DNA from the plasma, and aliquoted it into three portions. One portion was stored at −80 °C until analysis. The other portions underwent either one or three additional freeze–thaw cycles as described above. The plasma/serum DNA concentration in each sample was determined by three real-time quantitative PCR assays targeting the leptin gene. The amplicon sizes of the three real-time PCR assays were 105, 201, and 356 bp, respectively. The sequences of the primers and the probe and the set-up of the reactions have been described previously (6). For modules 1 to 4, samples from the same individual but subjected to different preanalytical conditions were analyzed in the same batch to avoid interanalysis variation. For module 4, all samples were analyzed in the same batch. The concentrations measured in the 201- or 356-bp assays were compared with those measured by the 105-bp assay and expressed as the 201/105 and 356/105 ratios. An increase in these ratios indicates an increase in the observed size of plasma DNA, whereas a decrease in these ratios indicates the reverse. The plasma/serum DNA concentrations for different handling protocols were compared by the Friedman test, followed by post hoc comparison with the control group with the Dunn method if the P value for the Friedman analysis was <0.05. The analyses were performed with SigmaStat Ver. 3.0 software. The plasma/serum DNA