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Letters |
1 A&E Medicine Academic Unit and
2 Department of Chemical Pathology The Chinese University of Hong Kong Prince of Wales Hospital Shatin, New Territories, Hong Kong SAR
aAuthor for correspondence. Fax 852-2194-6171; e-mail loym{at}cuhk.edu.hk.
To the Editor:
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 ß-globin gene as described previously (7) on a 7700 Sequence Detector (Applied Biosystems). DNA concentration is expressed as genome-equivalents/mL.
The results are summarized in Fig. 1
. We found no statistically significant differences (P >0.05, Friedman test) among the median plasma DNA concentrations in samples collected into different anticoagulants at 0, 2, and 6 h after venesection. After 24 h of whole blood incubation at room temperature, median plasma DNA concentrations were significantly increased in EDTA (940 vs 590 genome-equivalents/mL; 1.6-fold; P = 0.013, Wilcoxon test), heparin (4100 vs 540 genome-equivalents/mL; 7.6-fold; P = 0.005, Wilcoxon test), and citrate (4300 vs 540 genome-equivalents/mL; 8-fold; P = 0.005, Wilcoxon test) compared with their respective concentrations at time 0 h. We also found a statistically significant difference among the median plasma DNA concentrations for the three different anticoagulant types at 24 h (P = 0.0005, Friedman test). In addition, the differences in median plasma DNA concentrations between EDTA/heparin (P = 0.005, Wilcoxon test) and EDTA/citrate (P = 0.005, Wilcoxon test) were both highly statistically significant, whereas the difference in plasma DNA concentrations between heparin and citrate was not statistically significant (P = 0.96, Wilcoxon test).
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The results from our study show that EDTA, heparin, and citrate produced similar quantitative plasma DNA results within 6 h of venesection. At 24 h after sampling, however, DNA concentrations were higher with all three anticoagulants than at 0 or 6 h. Therefore, high false-positive results are likely to occur if samples are stored for 24 h before being analyzed. Plasma DNA originates at least partly from hematopoietic cells (1) but does not come from red blood cells and platelets because they are predominantly anucleate cell populations. Increases in plasma DNA concentrations after storage for 24 h are likely a result of leukocyte necrosis or apoptosis. The underlying mechanism leading to the much higher plasma DNA concentrations from heparin and citrate tubes are not clearly known. Further investigations may be needed to explore on this area.
The efficiencies of the real-time PCR assays in the present study were similar irrespective of the choice of anticoagulant. This is evident from the lack of difference in the plasma DNA concentrations among samples collected into tubes containing the three anticoagulants at time 0 h. This finding is different from previous observations, where heparin was reported to have an inhibitory effect on PCR (4)(5)(6).
It is therefore acceptable to use EDTA, heparin, or citrate as the anticoagulant for quantitative plasma DNA analysis provided that plasma is collected within 6 h after venesection. However, EDTA is the anticoagulant of choice if delayed blood processing is anticipated.
References
The following articles in journals at HighWire Press have cited this article:
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  E C W Hung, R W K Chiu, and Y M D Lo Detection of circulating fetal nucleic acids: a review of methods and applications J. Clin. Pathol., April 1, 2009; 62(4): 308 - 313. [Abstract] [Full Text] [PDF]
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 K. Saukkonen, P. Lakkisto, V. Pettila, M. Varpula, S. Karlsson, E. Ruokonen, K. Pulkki, and for the Finnsepsis Study Group Cell-Free Plasma DNA as a Predictor of Outcome in Severe Sepsis and Septic Shock Clin. Chem., June 1, 2008; 54(6): 1000 - 1007. [Abstract] [Full Text] [PDF]
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 G. Borthakur, C. Butryee, M. Stacewicz-Sapuntzakis, and P. E. Bowen Exfoliated Buccal Mucosa Cells as a Source of DNA to Study Oxidative Stress Cancer Epidemiol. Biomarkers Prev., January 1, 2008; 17(1): 212 - 219. [Abstract] [Full Text] [PDF]
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G. T.Y. Chung, R. W.K. Chiu, K.C. A. Chan, T. K. Lau, T. N. Leung, and Y.M. D. Lo Lack of Dramatic Enrichment of Fetal DNA in Maternal Plasma by Formaldehyde Treatment Clin. Chem., March 1, 2005; 51(3): 655 - 658. [Full Text] [PDF]
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 F. Z. Bischoff, D. E. Lewis, and J. L. Simpson Cell-free fetal DNA in maternal blood: kinetics, source and structure Hum. Reprod. Update, January 1, 2005; 11(1): 59 - 67. [Abstract] [Full Text] [PDF]
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, heparin; 
, citrate. Plasma ß-globin DNA concentrations (genome-equivalents/mL) are plotted on the y axis, and the hours after venesection are indicated on the x axis. The upper and lower limits of the boxes and the lines across the boxes indicate the 75th/25th percentiles and the medians, respectively. The upper and lower error bars indicate the 90th and 10th percentiles, respectively. The symbols indicate outliers.