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Technical Briefs |
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Departments of Clinical Chemistry,
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Epidemiology and Biostatistics, and
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Obstetrics and Gynaecology, University Medical Centre, Nijmegen, PO Box 9101, 6500 HB, Nijmegen, The Netherlands
aaddress correspondence to this author at: Department of Clinical Chemistry/564, University Medical Centre Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands; fax 31-243541743, e-mail D.Swinkels{at}CKCL.azn.nl
Fetal DNA in plasma and serum of pregnant women has been reported to be significantly increased in preeclampsia (1)(2). This increase may even precede clinical diagnosis (3). We hypothesized that subsequent development of hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome will further increase fetal DNA in maternal plasma and serum. In addition, total cell-free DNA may be increased in plasma as a result of tissue destruction, which generally occurs in HELLP syndrome.
To confirm our hypothesis, we recruited three groups of pregnant women with informed consent and matched for maternal age [(MA), in years] and gestational age [(GA), in weeks+days] at the time of blood drawing; the median ages (and ranges) were as follows: normotensive women (n = 10), MA, 30 (24–37) and GA, 32+6 (27+0-34+3); preeclamptic women without HELLP syndrome (n = 7), MA, 28 (24–36) and GA, 33+0 (27+6-34+3); and preeclamptic women with HELLP syndrome (n = 10), MA, 31 (26–38) and GA, 26+6-33+6. All women in the latter group had HELLP syndrome at the time of the blood drawing, except two who developed HELLP only after delivery. All participants were carrying single male fetuses. Preeclampsia was defined as a diastolic blood pressure >90 mmHg on two or more consecutive occasions after 20 weeks of pregnancy in previously normotensive women, with proteinuria >0.3 g/L in a 24-h collection period. HELLP syndrome was defined as the simultaneous occurrence of hemolysis (lactate dehydrogenase, >600 U/L), increased liver enzymes (serum aspartate aminotransferase and alanine aminotransferase >70 U/L), and low platelets (<100 x 109/L) (4). Diastolic blood pressure [median (ranges) in mmHg] was 70 (60–80), 120 (110–130), and 118 (110–140) for women with normotensive and preeclamptic pregnancies without and with HELLP syndrome, respectively.
Blood was collected in a plain and an EDTA-containing Vacutainer Tube from all pregnant women (n = 27). Serum and plasma were obtained by centrifugation for 10 min at 1200g and stored at -30 °C. We isolated DNA from 500 µL of thawed plasma or serum using the PureGene and QIAquick protocols and eluted the DNA in 50 µL of 10 mmol/L Tris-HCl (pH 8.5), as described previously (5). Real-time quantitative PCR (ABI Prism 7700 Sequence Detection System; Applied Biosystems) was used for gene quantification. Copy numbers of the SRY gene (GenBank Accession No. X53772) and albumin gene (GenBank Accession No. M12523) were quantified separately in split samples to determine the amount of fetal DNA and total DNA, respectively. PCR primers and probe for SRY were as follows: SRY-106F, 5'-AATTGGCGATTAAGTCAAATTCG-3'; SRY-192R, 5'-TAACGTTGACTACTTGCCCTG CT-3'; and SRY-168VIC, 5'-VIC-ATCTGCCTCCCTGACTGCTCTACTGCTGT-TAMRA-3'. For each amplification reaction (50 µL), we used 300 nM each primer, 160 nM probe, 25 µL of TaqMan Universal Master Mix (Applied Biosystems), and 5 µL of DNA sample. After 10 min at 95 °C, 40 two-step cycles were performed (30 s at 95 °C and 1 min at 60 °C). Primer and probe sequences for albumin were as follows: ALB-15659F, 5'-TGAAACATACGTTCCCAAAGAGTTT-3'; ALB-15739R, 5'-CTCTCCTTCTCAGAAAGTGTGCATAT-3'; and ALB-TET, 5'-TET-TGCTGAAACATTCACCTTCCATGCAGA-TAMRA-3'. Reaction conditions were identical to SRY amplification, except that 120 nM of the ALB-TET probe was used. Cycle threshold values for SRY and ALB were presented by the computer and were transformed to copy numbers with calibration curves of known concentrations of male genomic DNA (Sequence Detection Systems 1.7.1; 6 pg of DNA = 1 SRY copy = 2 ALB copies = 1 cell-equivalent). Finally, copy numbers of SRY and ALB were expressed in cell-equivalents/mL of maternal plasma or serum.
DNA from each plasma sample was isolated and quantified on two different occasions: day 1 and day 2. DNA from serum was analyzed once on day 2. Therefore, SRY and albumin gene copy numbers could be quantified in 27 x 3 (x 2 plasma and x 1 serum) = 81 samples. Because of insufficient material, however, only 73 samples were available, and in 1 sample, quantification of the SRY gene failed because of an erroneous laboratory procedure. As a result, 27 pregnant women provided 73 DNA samples, leading to 73 albumin and 72 SRY measurements, respectively.
Both fetal and total DNA in plasma increased markedly with the severity of the hypertensive disorder of pregnancy (Fig. 1
). Only plasma DNA values of day 1 were presented because these were quantified in all 27 individuals. Note that the two preeclamptic women who developed HELLP syndrome after delivery had fetal cell-free DNA concentrations that were higher than those of women who developed HELLP syndrome before delivery.
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The linear mixed model (6) [dependent variables: SRY, albumin, SRY/albumin ratio; random independent variable: sample; fixed independent variables: pregnancy groups (n = 3) and the determinations (n = 3)] indicated that median DNA concentrations differed significantly between pregnancy groups [P <0.001 for all of them (SRY/albumin); Table 1
]. For circulating fetal DNA, we found an overall 6.1-fold [95% confidence interval (CI), 3.6–10.3] increase in women with preeclampsia compared with normotensive pregnancies. Previously, a median increase of 5.5-fold was detected in sera from preeclamptic women (1) and a 2-fold increase in plasma (2). With the onset of HELLP, circulating fetal DNA concentrations increased another 4.3-fold (95% CI, 1.1–7.0) in our study. Zhong et al. (2) also detected such an increase in plasma from 12 women who developed various severe forms of preeclampsia, among them 3 women with HELLP syndrome. Similarly, the median concentration of total (fetal + maternal) free circulating DNA increased 5.5-fold (95% CI, 1.1–13.6) from normal to preeclamptic pregnancies. For total DNA, Zhong et al. (2) found a threefold increase in plasma, using the GAPDH gene for total DNA quantification. The simultaneous increase in both fetal DNA and total DNA from normotensive to preeclamptic pregnancies led to a relatively constant fetal DNA concentration (4–5% of total DNA) in both pregnancy groups. Typically, with the onset of HELLP syndrome, another 17.9-fold increase (95% CI, 7.5–42.7) in total DNA was observed. This increase in total DNA (17.9-fold) was much higher than that in fetal DNA (4.3-fold). Consequently, the median percentage of cell-free fetal DNA in the total free circulating DNA (SRY/albumin) differed significantly between preeclamptic women with and without HELLP syndrome (1.2% vs 4.9%; P <0.001; linear mixed model, contrast test according to Tukey; Table 1
).
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Fetal and total DNA increased similarly with increasing severity of the hypertensive disease, as indicated by a significant correlation between the concentrations of fetal and total DNA (Spearman rank correlation test, 
= 0.83, P <0.001; n = 72). A similar high correlation was found when the data were restricted to any of the following: plasma day 1, plasma day 2, and serum day 2. This relationship became evident only with data of all three pregnancy groups: normotensive women and preeclamptic women with and without HELLP syndrome. When these three groups were analyzed separately, this correlation was not significant within any one of them, probably because of the small DNA concentration ranges of each of these groups. This might explain the results of Zhong et al. (2), which did not indicate a correlation between fetal and total DNA in normotensive pregnancies. In that study, only in the very heterogeneous group of mild to severe preeclamptic pregnancies, with a much wider range of both fetal and total free circulating DNA concentrations, was a significant correlation found (7).
No significant differences were detected between duplicate plasma (day 1 and day 2) and single serum (day 2) measurements in the median concentrations of fetal DNA, total DNA, nor their ratio (linear mixed model albumin, P = 0.11; SRY, P = 0.48, SRY/albumin, P = 0.19). For fetal DNA, Lo et al. (7) obtained similar data in their study on cell-free fetal DNA in normotensive early and late pregnancies. However, results from other studies (8)(9)(10) contrast with our findings and the findings of Lo et al. For total DNA, our similar quantification results for plasma and serum are in conflict with previous reports showing that serum contains substantially more free DNA than plasma (7). Discrepancies were possibly caused by differences between blood-processing and DNA-extraction protocols (11).
Furthermore, the precision of the quantification assay for cell-free DNA (consisting of the sum of the variances of the isolation and the quantification) was calculated from the three repetitive determinations for the same pregnant women. This led to total CVs for the concentrations of SRY DNA and the albumin gene of 34% and 26%, respectively. These CVs were similar to the CV of 27% we found previously in a methodologic study in plasma, demonstrating the good DNA isolation efficiency and reproducibility of our method (5).
In conclusion, we have shown potential possibilities for the clinical diagnosis of preeclampsia and HELLP with fetal and total circulating DNA. The increased concentrations of DNA in two preeclamptic patients that preceded the development of HELLP syndrome make us especially optimistic about the use of cell-free fetal or total DNA as markers to predict the onset of HELLP syndrome. Future prospective studies are needed to determine whether abnormal patterns of free circulating fetal DNA concentrations can be used as a predictive screening test. Moreover, autosomal genetic markers outside the Y chromosome may be used to extend the application to women carrying female fetuses (12).
References
The following articles in journals at HighWire Press have cited this article:
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  O. Lapaire, H. Stroh, I. Peter, J. M. Cowan, U. Tantravahi, B. O'Brien, D. W. Bianchi, and K. L. Johnson Larger Columns and Change of Lysis Buffer Increase the Yield of Cell-Free DNA Extracted from Amniotic Fluid Clin. Chem., January 1, 2006; 52(1): 156 - 157. [Full Text] [PDF]
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 Y.M. D. Lo Recent Advances in Fetal Nucleic Acids in Maternal Plasma J. Histochem. Cytochem., March 1, 2005; 53(3): 293 - 296. [Abstract] [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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 Y. Li, X. Y. Zhong, A. Kang, C. Troeger, W. Holzgreve, and S. Hahn Inability to Detect Cell Free Fetal DNA in the Urine of Normal Pregnant Women nor in Those Affected by Preeclampsia Associated HELLP Syndrome Reproductive Sciences, December 1, 2003; 10(8): 503 - 508. [Abstract] [PDF]
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E. K.O. Ng, T. N. Leung, N. B.Y. Tsui, T. K. Lau, N. S. Panesar, R. W.K. Chiu, and Y.M. D. Lo The Concentration of Circulating Corticotropin-releasing Hormone mRNA in Maternal Plasma Is Increased in Preeclampsia Clin. Chem., May 1, 2003; 49(5): 727 - 731. [Abstract] [Full Text] [PDF]
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 E. K. O. Ng, N. B. Y. Tsui, T. K. Lau, T. N. Leung, R. W. K. Chiu, N. S. Panesar, L. C. W. Lit, K.-W. Chan, and Y. M. D. Lo From the Cover: mRNA of placental origin is readily detectable in maternal plasma PNAS, April 15, 2003; 100(8): 4748 - 4753. [Abstract] [Full Text] [PDF]
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D. W. Swinkels, E. Wiegerinck, E. A.P. Steegers, and J. B. de Kok Effects of Blood-Processing Protocols on Cell-free DNA Quantification in Plasma Clin. Chem., March 1, 2003; 49(3): 525 - 526. [Full Text] [PDF]
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T.-W. Lau, T. N. Leung, L. Y.S. Chan, T. K. Lau, K.C. A. Chan, W. H. Tam, and Y.M. D. Lo Fetal DNA Clearance from Maternal Plasma Is Impaired in Preeclampsia Clin. Chem., December 1, 2002; 48(12): 2141 - 2146. [Abstract] [Full Text] [PDF]
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) and the 95% CIs (bars) are indicated. Concentrations of circulating male fetal DNA are expressed in SRY cell-equivalents/mL. Concentrations of total cell-free DNA are expressed in albumin cell-equivalents/mL. 
and • represent the results from two preeclamptic women who developed HELLP syndrome only after delivery.