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Technical Briefs |
1 Maternité, Hôpital Necker-Enfants Malades, AP-HP-Université Paris, Paris, France 2 Centre de Diagnostic Prénatal, American Hospital of Paris, Neuilly, France
aaddress correspondence to this author at: M. Dassault Molecular Biology Laboratory, Centre de Diagnostic Prénatal, American Hospital of Paris, 63 bd Victor Hugo, 92200 Neuilly-sur-Seine, France; e-mail jean-marc.costa{at}ahparis.org
Analysis of cell-free fetal DNA circulating in maternal blood is now well recognized as a useful tool for noninvasive prenatal diagnosis. Determination of fetal sex has modified prenatal diagnosis for women at risk of transmitting X-linked disorders (1), as well as management of pregnancies at risk for congenital adrenal hyperplasia (2). Routine determination of fetal rhesus D status has been evaluated by several groups (3)(4)(5). Despite the increasing number of potential applications, the low proportion of fetal DNA in a high background of maternal DNA dramatically limits its study, although some groups have already reported such analyses (6)(7)(8).
Recently, Dhallan et al. (9) reported that the addition of formaldehyde to maternal blood increased the percentage of fetal DNA recovered. Because of its potential implications, this requires confirmation. We conducted a study similar to that of Dhallan et al. (9) but used an automated procedure for the extraction of nucleic acids and real-time quantitative PCR to provide more precise measurements of DNA.
We recruited 30 pregnant women carrying a male fetus for this study. The mean (range) gestational age was 29 (15–39) weeks. After receipt of informed consent, blood was collected in 5-mL Vacutainer® Tubes with or without (EDTA tube) clot activator, with or without a neutral solution of formaldehyde (1 mL/L final concentration).
Less than 24 h after blood sampling, each EDTA blood was centrifuged at 1600g for 10 min at room temperature, and the plasma was carefully transferred to a new tube, avoiding disruption of the buffy coat. The plasma was again centrifuged in a similar manner and stored at –80 °C until further processing. For tubes containing clot activator, serum was obtained after a single centrifugation at 3000g for 10 min at room temperature and then immediately stored at –80 °C.
DNA was extracted from 1 mL of plasma or serum by the Total Nucleic Acid LV extraction procedure on the MagNaPure Compact instrument (Roche Diagnostics). DNA was finally eluted in 50 µL of elution buffer, and 5 or 10 µL (see above) of eluate was used per PCR reaction.
The SRY (fetal DNA) and ß-globin (total DNA) genes were quantified by real-time quantitative PCR in a LightCycler 2.0® instrument (Roche Diagnostics). PCR reactions were set up in a final volume of 20 µL and included the reagents from the Fast DNA Master Hybridization Probes Kit (Roche Diagnostics), 0.5 µM each primer, 0.25 µM each probe (Proligo), 1.25 U of uracil DNA glycosylase (Biolabs), and 4.75 mM magnesium chloride. After an initial 1-min incubation at 50 °C, a first denaturation step of 8 min at 95 °C was followed by 50 cycles of amplification [denaturation at 95 °C for 10 s (ramp rate, 20 °C/s), annealing at 56 °C for 10 s (ramp rate, 20 °C/s), and extension at 72 °C for 15 s (ramp rate, 2 °C/s)]. The primer and probe (Proligo) sequences are described in Table 1 of the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue1/.
The SRY and ß-globin assays were performed in triplicate for each sample, using 10 and 5 µL of the DNA extract, respectively. Calibration curves were generated from successive dilutions of a male DNA; these curves were used to quantify the samples by interpolation for both the SRY and the ß-globin genes. Results are expressed in kilogenome-equivalents/L (kgeq/L) and are summarized in Table 1
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No significant difference in fetal DNA concentration was observed in the four sample groups (Fig. 1 in the online Data Supplement). The mean (SD) SRY concentrations were 204 (257), 169 (204), 163 (180), and 144 (162) kgeq/L for samples with and without EDTA and formaldehyde. Fetal DNA was 6.8 (8.7)% [mean (SD)] of total DNA in plasma vs 0.06 (0.09)% in serum. These proportions increased to 36.8 (26.5)% and 6.5 (9.4)% when maternal blood was collected in tubes containing the neutral solution of formaldehyde. The percentage of fetal DNA in these maternal formaldehyde-treated plasmas was correlated with gestational age (Fig. 1
). The increase in the percentage of fetal DNA was related to the dramatic decrease in total DNA concentration when the sample was treated with formaldehyde (P <0.0001). The mean (SD) ß-globin concentrations were 3.5 (0.36) x 103, 0.45 (0.05) x 103, 431.8 (461) x 103, and 5.8 (4.4) x 103 kgeq/L, respectively (Fig. 2 in the online Data Supplement). However, the enrichments for each patient were not similar in plasma and serum.
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The effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma have been emphasized by Chiu et al. (10). These authors showed that different protocols significantly affect the quantification and the day-to-day fluctuation of total (ß-globin sequences) but not fetal DNA (SRY sequences). They suggested that most of the fetal DNA circulates in an extracellular form; therefore, adding a cell stabilizer to the medium will not modify the absolute amount of free DNA because intact fetal cells contribute a very small proportion of the fetal DNA. The absolute amount of fetal DNA does not significantly change in plasma and serum regardless of whether the blood is pretreated with formaldehyde. The present study confirms this hypothesis.
Dhallan et al. (9) suggested that the increase in relative percentage of free fetal DNA likely results from a combination of factors in which inhibition of maternal cells lysis seems predominant. By stabilizing the maternal cells, the formaldehyde reduces the in vitro release of cell-free DNA and thus decreases the amount of maternal free DNA. In the present study, this phenomenon is striking in the serum groups. The higher total DNA concentration in serum (compared with plasma) was attributable to the release of DNA by hematopoietic cells during the clotting process (11). This concentration decreased by 74-fold in formaldehyde-treated serum, highlighting its cell-stabilizing effect.
We observed no differences in fetal DNA concentrations in the samples treated or not treated with EDTA and formaldehyde. These results support the notion that use of formaldehyde to inhibit enzymes that destroy DNA, such as deoxyribonucleases, can be eliminated. Moreover, Lo et al. (12) previously demonstrated the limited effect of such in vitro DNA degradation. In their study, Dhallan et al. (9) had no control group and compared their values with those of Lo et al. (12), obtained not only with a different methodology but also at different gestational ages, despite the fact that the absolute amount of fetal DNA increases with gestational age. It is most likely that formaldehyde does not affect DNA stability.
Another possible factor is the use of gentle centrifugation. Chiu et al. (10) have shown that centrifugation alone is not enough to obtain maternal cell-free plasma and that the number of cells left in plasma after centrifugation is variable. Therefore, in the study of Dhallan et al. (9), the role of centrifugation is certainly low when compared with the role of formaldehyde. In our study, although we used similar shipping, handling, and processing conditions, we measured a higher absolute concentration of free fetal DNA (169 vs 66.1 kgeq/L), even taking into account the mean gestational age at sampling.
Dhallan et al. (9) also addressed the need for a very short time delay between the venipuncture procedure and the addition of formaldehyde. To circumvent this problem, we added the formaldehyde to the sampling tube before venipuncture. As a result, the formaldehyde enrichment effect was shown for all patients in our study, whereas Dhallan et al. (9) reported a lack of effect in three cases. We observed, however, a high variability for this enrichment (2- to 27-fold for plasma and 21- to 138-fold for serum). This variability seems to be unrelated to gestational age (P >0.05) and to the amount of fetal DNA (P >0.05). We speculate that it reflects a variable effect of the formaldehyde or the natural fluctuation in the amount of cell-free maternal DNA present before processing.
We conclude that formaldehyde increases the percentage of free fetal DNA in maternal plasma or serum by inhibiting maternal cell lysis. This new simple procedure facilitates study of fetal genetic markers such as single nucleotide mutations, but some issues still need to be addressed, especially the reason for the variability in results. Other strategies are actually under evaluation to achieve this final goal (13)(14) before use of this technique for noninvasive prenatal diagnosis.
Acknowledgments
We are grateful to Annie Gougelet and Michèle Thomas for technical assistance. We thank Dr. Lavergne for critical review of the manuscript.
References
The following articles in journals at HighWire Press have cited this article:
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  Y. M. D. Lo, F. M. F. Lun, K. C. A. Chan, N. B. Y. Tsui, K. C. Chong, T. K. Lau, T. Y. Leung, B. C. Y. Zee, C. R. Cantor, and R. W. K. Chiu From the Cover: Digital PCR for the molecular detection of fetal chromosomal aneuploidy PNAS, August 7, 2007; 104(32): 13116 - 13121. [Abstract] [Full Text] [PDF]
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