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Technical Briefs |
1 Department of Chemical Pathology and2 Accident and Emergency Medicine Academic Unit, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong Special Administrative Region, China
aaddress correspondence to this author at: Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong Special Administrative Region, China; fax 852-2194-6171, e-mail loym{at}cuhk.edu.hk
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.
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.
Module 2.
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.
Module 3.
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.
Module 4.
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.
Module 5.
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 concentrations measured by the 105-bp assay are shown in Fig. 1A
. The median DNA concentrations for the serum samples and the fresh plasma samples were 975 and 600 copies/mL, respectively. The difference between these concentrations was statistically significant (P <0.05). The increased serum DNA concentration suggests that additional DNA may be released from leukocytes into the serum during clotting. The plasma DNA concentrations were also significantly increased when the whole-blood samples were stored for 24 h at room temperature or at 4 °C. The median concentrations for the two groups were 1000 and 750 copies/mL, respectively. On the other hand, delayed separation of blood cells for up to 6 h, freezing and thawing of plasma samples up to three times, and storage of plasma at –80 °C for 2 weeks did not significantly affect the plasma DNA concentrations.
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The 201/105 ratios of plasma/serum DNA for different preanalytical conditions are shown in Fig. 1B
. The principle of the determination of the size of plasma DNA has been described previously (9). The observation that assays with longer amplicons would give lower concentrations is unlikely to be attributable to the discrepancy in PCR efficiencies (9). The median 201/105 ratios for the serum samples and the immediately handled plasma samples were 50% and 33%, respectively. The difference between these median ratios was statistically significant (P <0.05). The median 201/105 ratios of samples stored for 24 h at room temperature and at 4 °C were 48% and 41%, respectively. The 201/105 ratio for samples stored at room temperature was significantly increased. A possible explanation for the increase in DNA size in serum and plasma samples processed after 24 h would be the release of high-molecular-weight DNA from leukocytes during clotting and prolonged storage. On the other hand, when the plasma samples were frozen and thawed three times, the 201/105 ratio was significantly decreased to 24%. This decrease in the 201/105 ratio indicates that the plasma DNA may become fragmented during repeated freezing and thawing of plasma. In contrast, delayed separation of plasma from blood cells for up to 6 h, freezing and thawing of plasma samples once, and storage of plasma at –80 °C for 2 weeks did not significantly affect the fractional concentration.
The 356/105 ratios for serum/plasma DNA are shown in Fig. 1C
. The median 356/105 ratio for freshly prepared plasma samples was 12%, and that of the serum samples was 26%. The difference between these ratios was statistically significant (P <0.05). Although the median 356/105 ratios for samples stored for 24 h at room temperature and at 4 °C increased to 21% and 20%, respectively, the differences did not reach statistical significance (P >0.05). There was also no significant difference in the 356/105 ratios for samples subjected to other preanalytical conditions.
Because we have shown that repeated freezing and thawing of plasma samples would affect the integrity of plasma DNA, it is logical to investigate whether freezing and thawing of extracted DNA would also lead to fragmentation of DNA. The 201/105 and 356/105 ratios for plasma DNA subjected to one and three cycles of additional freezing and thawing of extracted DNA are shown in Fig. 1D
. There was no significant change in DNA concentration when the extracted DNA was frozen and thawed up to three times.
In this study, we have shown that clotting and delayed separation of plasma from blood cells for 24 h significantly increases the concentration and observed size of cell-free DNA in blood samples. Moreover, we have also shown that repeated freezing and thawing of plasma samples, but not extracted DNA, leads to fragmentation of DNA. Therefore, blood samples collected for investigation of the integrity of circulating DNA should be handled within 6 h after collection. The harvested plasma should be aliquoted into smaller portions to avoid repeated freezing and thawing of samples. Alternatively, DNA can be extracted from the plasma samples for storage because DNA appears to be more resistant to fragmentation when stored in DNA extraction solution than in plasma.
Acknowledgments
This project is supported by a Central Allocation Grant (CUHK1/03C) from the Hong Kong Research Grants Council.
References
The following articles in journals at HighWire Press have cited this article:
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  M. L. Gulley and W. Tang Laboratory Assays for Epstein-Barr Virus-Related Disease J. Mol. Diagn., July 1, 2008; 10(4): 279 - 292. [Abstract] [Full Text] [PDF]
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 K.C. A. Chan, S.-F. Leung, S.-W. Yeung, A. T.C. Chan, and Y.M. D. Lo Persistent Aberrations in Circulating DNA Integrity after Radiotherapy Are Associated with Poor Prognosis in Nasopharyngeal Carcinoma Patients Clin. Cancer Res., July 1, 2008; 14(13): 4141 - 4145. [Abstract] [Full Text] [PDF]
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 N. UMETANI, S. HIRAMATSU, and D. S.B HOON Higher Amount of Free Circulating DNA in Serum than in Plasma Is Not Mainly Caused by Contaminated Extraneous DNA during Separation. Ann. N.Y. Acad. Sci., September 1, 2006; 1075: 299 - 307. [Abstract] [Full Text] [PDF]
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