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Technical Briefs |
1 Division of Genetics, Departments of Pediatrics, Obstetrics and Gynecology, Tufts-New England Medical Center and Tufts University School of Medicine, Boston, MA 02111
2 Department of Chemical Pathology and Institute of Molecular Oncology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong Special Administrative Region, China
3 Women’s Health Service, Chestnut Hill, MA 02467
aaddress correspondence to this author at: New England Medical Center, 750 Washington St., Box 394, Boston, MA 02111; fax 617-636-1469, e-mail DBianchi{at}lifespan.org
The discovery of intact fetal cells in maternal blood has led to the possibility of noninvasive prenatal genetic testing. Although extensive work has been performed on maternal blood in the hope of developing a reliable method for the recovery of intact fetal cells, the total number of recoverable cells is quite small, averaging 19 nucleated fetal cells in 16 mL of blood (1)(2). This is contrasted by a relatively large amount of cell-free fetal DNA detectable in maternal plasma (3). Recent studies suggest that cell-free fetal DNA in maternal plasma can be used as a diagnostic tool for diseases of pregnancy, such as preeclampsia or preterm labor, or for fetal anomalies, such as Down syndrome (4)(5)(6)(7). Because of these potential applications, it is of great importance to determine whether cell-free fetal DNA concentrations remain stable in a test tube after phlebotomy.
In this report we evaluate the effects of time after phlebotomy on the quantity of cell-free fetal DNA in blood samples from a pregnant woman and a woman who has just undergone termination of pregnancy. The post-termination samples were used because in the first trimester, there are relatively few fetal cells present in maternal blood. The termination procedure increases fetomaternal transfusion (8). We hypothesized that after phlebotomy, apoptotic fetal cells that die in the venipuncture tube would release their DNA. We also asked whether more fetal cells undergo apoptosis as a result of exposure to the maternal bloodstream and cytokines. In both cases, we hypothesized that cell-free fetal DNA concentrations would increase in the blood samples over time.
This study was performed with Institutional Review Board approval and with informed consent from participants at Tufts-New England Medical Center. For early-gestation samples, 29 women who were 6–17 weeks pregnant were recruited from a private women’s health clinic. Blood was drawn 5–15 min after the termination procedure. For late-gestation samples, five pregnant women at gestational ages of 25–33 weeks with known male fetuses were recruited.
Blood was collected in 10-mL EDTA tubes and placed on a gentle agitator. An initial 1.8 mL of blood was aliquoted into a microcentrifuge tube (t0) and centrifuged at 1600g. The plasma layer was then removed without disturbing the underlying cellular layer, placed on dry ice, and stored at -80 °C.
While the original blood sample remained on the agitator, the aliquoting, centrifugation, and freezing procedures were repeated at the following sequential time points: 15 and 30 min and 1, 2, 4, 6, and 20–24 h. The plasma was later thawed, centrifuged at 13 500g, and transferred into a sterile microcentrifuge tube, leaving any pellet at the bottom of the tube undisturbed.
Placental tissue from the termination cases was collected and refrigerated at 4 °C for the purpose of determining fetal gender.
We used 400–800 µL of plasma for DNA extraction with the QIAamp Mini Blood Kit (Qiagen) as described by the manufacturer. The DNA was eluted into a final volume of 25–50 µL and stored at 4 °C until analysis.
DNA was extracted from 25 mg of placental tissue, by the method recommended by Qiagen. The DNA was eluted in a final volume of 100 µL. Standard precautions to prevent DNA contamination were followed.
DNA was quantified by TaqMan PCR on a Perkin-Elmer Applied Biosystems (PE-ABI) 7700 Sequence Detector. The early-gestation samples were analyzed in Boston with DYS1 and ß-globin probes and their primer sets as described previously (9). The late-gestation samples were shipped to Hong Kong on dry ice. DNA was detected with the following DYS14 and ß-globin primers and probes (see below) as described by Lo et al. (3):
where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine. DYS14, although once thought to be a single-copy gene, is a multicopy locus with variability among individuals but is consistent within an individual.
Each sample was run in triplicate, and the mean was used for further calculations. An amplification calibration curve was created using titrated, purified male DNA.
Initially, 29 women were enrolled in the early-gestation portion of the experiment. Gender determination was performed on the placental tissue samples collected. The copy numbers of DYS1 and ß-globin were not always in a 1:1 ratio as expected. We identified a potential source of DNA contamination in the way placental samples were handled in the termination clinic. For this reason, nine samples at 7–10 weeks of gestational age with the strongest and most unequivocal Y DNA values [>20 000 genome-equivalents (GE)/25 mg] were selected for further analysis (see Table 1 in the Data Supplement, available with the online version of this Technical Brief at http://www.clinchem.org/content/vol49/issue1/). Three samples from confirmed female fetuses were chosen as negative controls.
In the early-gestation samples, the initial (t0) concentration of total DNA (ß-globin) varied between 638 and 3812 GE/mL. An ANOVA on ranked averages in these specimens showed no difference between samples from women carrying male and female fetuses. The copy number stayed relatively constant through the 4-h mark, when there was a significant increase in the overall number at 6 h and an even sharper increase overnight (P <0.001, Student–Newman–Keuls test; see Table 2 in the Data Supplement and Fig. 1A
).
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In contrast, there was no change in the copy number of fetal cell-free DNA (DYS1; Fig. 1B
). The initial DYS1 copy number ranged from 9 to 142 GE/mL. The samples from women carrying female fetuses had no evidence of DYS1 signal. ANOVA of the DYS1 data showed no differences over time (P = 0.30).
In the late-gestation samples, the initial (t0) male DNA (DYS14) copy number ranged from 235 to 549 copies/mL. There was no discernable increase in the quantity of cell-free fetal DNA related to time (ANOVA on ranks, P = 0.36). Similar to the first trimester data, a difference in ß-globin DNA concentrations was observed among the samples (ANOVA on ranks, P = 0.022). Pairwise comparison using the Student–Newman–Keuls test indicated that the difference was attributable to the increase in ß-globin DNA concentrations in the 24-h samples (P <0.05; see Table 3 in the Data Supplement and Fig. 1
, C and D).
This study analyzed two types of maternal blood samples from two different gestational age ranges: first trimester (7–10 weeks) post-termination, and third trimester (25–33 weeks) in an ongoing pregnancy. The early-gestation samples had lower DNA concentrations, consistent with earlier findings at 7–10 weeks of gestational age (3)(10). The results were the same for both types of samples, i.e., cell-free fetal DNA remained stable up to 24 h after phlebotomy. This demonstrates that cell-free fetal DNA is a stable analyte that can be measured in referral laboratories after the sample has been shipped overnight.
Previous studies indicate that the amount of maternal and fetal DNA fluctuates in maternal plasma (11). The method of blood processing, specifically the force of the centrifugation procedure, can account for some fluctuation of total DNA but not cell-free fetal DNA (12). These data, in conjunction with our own, indicate that the amount of time a sample spends in a tube before processing affects the amount of total but not cell-free fetal DNA. The total DNA, which increases in the test tube over time, is most likely attributable to apoptosis, cell death, and lysis.
Apoptosis is a rapid process that occurs within hours of being triggered (13). Large numbers of apoptotic fetal cells have been found in the plasma of pregnant women (14) and in the cord blood of newborn infants (15). Early-gestation samples were collected after surgical termination of the pregnancy. This procedure provides a transfusion of fetal cells into the maternal bloodstream, which are then exposed to the maternal circulation and immune system. If these or other cells were apoptotic or were triggered to undergo apoptosis, they would release their DNA over time in a postphlebotomy sample, making intact cell recovery more difficult. Our data indicate that this is not the case, as cell-free fetal DNA concentrations remained constant.
The source of the cell-free fetal DNA remains unknown, but our data show that whatever amount is in the plasma is not augmented by the ongoing release of DNA by fetal cells that may exist in a maternal blood sample. Whether the cell-free fetal DNA is derived from the placenta, the fetal hematopoietic system, or the amniotic fluid, it appears to be transferred as a naked molecule or very quickly assumes that form. Experiments investigating the size of the molecules, examining specifically any patterns suggestive of apoptosis, may yield clues to the origin of this DNA.
The stable concentrations of cell-free fetal DNA may be attributable to another mechanism. Fetal DNA may be simultaneously released from cell nuclei and metabolized in the plasma. Plasma deoxyribonucleases are known to exist, but their activity in vitro at room temperature is not well characterized. The increasing concentrations of total DNA with concurrent stable concentrations of cell-free fetal DNA argue against this theory, although what happens after 24 h remains unknown.
Intact fetal cells in the cellular fraction of the maternal blood do not measurably contribute to the amount of cell-free DNA after blood sampling. Cell-free fetal DNA concentrations are stable up to 24 h after phlebotomy and therefore are useful as an additional analyte for the noninvasive screening for complications of pregnancy.
Acknowledgments
This work was supported by NIH Contracts HD 43204 (to Dr. Bianchi) and T32 HD 07492 (to Dr. Angert), and by Innovation and Technology Fund Grant AF/90/99 (to Dr. Lo).
References
The following articles in journals at HighWire Press have cited this article:
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  C. Gonzalez-Gonzalez, M. Garcia-Hoyos, M. J. Trujillo-Tiebas, I. Lorda-Sanchez, M. R. de Alba, F. Infantes, J. Gallego, J. Diaz-Recasens, C. Ayuso, and C. Ramos Application of Fetal DNA Detection in Maternal Plasma: A Prenatal Diagnosis Unit Experience J. Histochem. Cytochem., March 1, 2005; 53(3): 307 - 314. [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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H. M. Muller, L. Ivarsson, H. Schrocksnadel, H. Fiegl, A. Widschwendter, G. Goebel, S. Kilga-Nogler, H. Philadelphy, W. Gutter, C. Marth, et al. DNA Methylation Changes in Sera of Women in Early Pregnancy Are Similar to Those in Advanced Breast Cancer Patients Clin. Chem., June 1, 2004; 50(6): 1065 - 1068. [Full Text] [PDF]
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 H Masuzaki, K Miura, K-i Yoshiura, S Yoshimura, N Niikawa, and T Ishimaru Detection of cell free placental DNA in maternal plasma: direct evidence from three cases of confined placental mosaicism J. Med. Genet., April 1, 2004; 41(4): 289 - 292. [Full Text] [PDF]
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K. L. Johnson, K. A. Dukes, J. Vidaver, E. S. LeShane, I. Ramirez, W. D. Weber, F. Z. Bischoff, S. Hahn, A. Sharma, D. X. Dang, et al. Interlaboratory Comparison of Fetal Male DNA Detection from Common Maternal Plasma Samples by Real-Time PCR Clin. Chem., March 1, 2004; 50(3): 516 - 521. [Abstract] [Full Text] [PDF]
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