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EDTA is a better anticoagulant than heparin or citrate for delayed blood processing for plasma DNA analysis.

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 (1-3). 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 (4-6). 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[degrees]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[degrees]C until analysis. DNA was extracted from 200 [micro]L of plasma according to the "blood and body fluid protocol" of the QIAamp Blood Kit (Qiagen), with a final elution volume of 50 [micro]L. We then subjected 5 [micro]L of the DNA to real-time quantitative PCR for the [beta]-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).

[FIGURE 1 OMITTED]

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 (4-6).

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

(1.) Lui YYN, Chik KW, Chiu RWK, Ho CY, Lam CWK, Lo YMD. Predominant hematopoietic origin of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation. Clin Chem 2002;48:421-7.

(2.) Lee TH, Montalvo L, Chrebtow V, Busch MP. Quantitation of genomic DNA in plasma and serum samples: higher concentrations of genomic DNA found in serum than in plasma. Transfusion 2001;41:276-82.

(3.) Jung M, LKlotzek S, Lewandowski M, Fleischhacker M, Jung K. Changes in concentration of DNA in serum and plasma during storage of blood samples. Clin Chem 2003;49:1028-9.

(4.) Beutler E, Gelbart T, Kuhl W. Interference of heparin with the polymerase chain reaction. Biotechniques 1990;9:166.

(5.) Jung R, Lubcke C, Wagener C, Neumaier M. Reversal of RT-PCR inhibition observed in heparinized clinical specimens. Biotechniques 1997; 23:24-8.

(6.) Famert A, Arez AP, Correia AT, Bjorkman A, Snounou G, do Rosario V. Sampling and storage of blood and the detection of malaria parasites by polymerase chain reaction. Trans R Soc Trop Med Hyg 1999;93:50-3.

(7.) Lo YMD, Tein MSC, Lau TK, Haines CJ, Leung TN, Poon PMK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768-75.

Nicole Y.L. Lam [1]

Timothy H. Rainer [1]

Rossa W.K. Chiu [2]

Y.M. Dennis Lot [2] *

[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

* Author for correspondence. Fax 852 2194-6171; e-mail loym@cuhk.edu.hk.

DOI: 10.1373/clinchem.2003.026013
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Title Annotation:Letters
Author:Lam, Nicole Y.L.; Rainer, Timothy H.; Chiu, Rossa W.K.; Lo, Y.M. Dennis
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Jan 1, 2004
Words:945
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