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Technical Briefs |
1
Molecular Biology Laboratory, Department of Clinical Chemistry and
2
Department of Obstetrics and Gynecology, University Hospital ‘Vrije Universiteit’, 1007 MB Amsterdam, The Netherlands;
a address correspondence to this author at: Molecular Biology Laboratory, Department of Clinical Chemistry, University Hospital ‘Vrije Universiteit’, PO Box 7057, 1007 MB Amsterdam, The Netherlands
Fetal DNA is present in plasma of pregnant women in amounts almost 1000-fold higher than the DNA present in intact fetal cells circulating in maternal peripheral blood (1)(2). The clinical usefulness of fetal DNA from plasma for noninvasive prenatal diagnosis has been demonstrated recently through analysis of the fetal RhD status by PCR-based approaches (3)(4)(5). We explored the possibility that not all of this fetal DNA is soluble and cell-free, but that part of it is still cell-associated.
Heparin blood samples of 25–30 mL were obtained after informed consent from 38 pregnant women attending the Prenatal Diagnostic Center at week 7–16 of gestation. Several weeks after blood withdrawal, chorionic villus sampling or amniocentesis was performed as planned at each patient’s first visit. The protocol was approved by the Committee of Medical Ethics of the University Hospital ‘Vrije Universiteit’. Blood samples were processed on the day of withdrawal and were centrifuged on a discontinuous Percoll gradient as described previously (6)(7). In brief, after dilution of the heparin blood in Hanks’ balanced salt solution; 1:2 dilution (Life Technologies), blood was layered on a 5-step Percoll density gradient consisting of 15 mL of 600 mL/L and 5 mL each of 550, 500, 450, and 400 mL/L Percoll in Hanks’ balanced salt solution. After centrifugation for 25 min at 1000g at room temperature, plasma samples were removed from the upper part of the gradient, and cells with density 1.053–1.060 kg/L were removed for our study on circulating fetal trophoblast cells (7). The plasma samples were centrifuged twice for 10 min at 500g in a microcentrifuge, and pellets were washed in phosphate-buffered saline. After the cells were stained with 4',6-diamidine-2'-phenylindole dihydrochloride, we noticed the presence of intact nuclei by microscopic analysis of these samples (300–400 cells/mL blood). Interestingly, the majority of these nuclei showed morphological features suggestive of apoptosis (a lobular appearance). When we used the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL) with the In Situ Cell Death Detection Kit (Roche Diagnostics), the majority of these cells (>95%) were labeled. In contrast, in control TUNEL assays using samples consisting of cells removed from the same gradient with density 1.053–1.060 kg/L, <1% of the cells were found to be in apoptosis.
Cells isolated from plasma were pretreated by proteinase K incubation
(final concentration, 0.5 g/L) for 60 min at 55 °C in PCR buffer and
subjected to a nested X/Y-chromosome-specific PCR assay followed by
Southern blotting to assess the presence of fetal DNA. The PCR was
performed as described previously (6). All samples were
positive for the X chromosome, indicating efficient isolation of DNA in
all samples. The Y-chromosome-specific 254-bp end-product of the PCR
assay was labeled with digoxigenin 11-dUTP and used as a probe for
Southern blotting. Results of PCRs followed by blotting (Table 1
) were compared with results of conventional karyotyping after
chorionic villus sampling or amniocentesis in the same women (gold
standard). PCR results of cells isolated from plasma of 31 pregnant
women of 38 patients in total correlated with the gold standard (i.e.,
81.6% correct). In two patients, PCR results were not interpretable.
Three of 16 female samples were false positives, presumably because of
contamination; at least one of these samples was negative in a
subsequent assay. Two of 22 male samples were false negatives, probably
because of the low amount of fetal DNA present in these very early
pregnancy samples (all false-negative samples were obtained before 11
weeks of gestation). Lo et al. (3) described their RhD-PCR
test as reliable at the beginning of the second trimester. When only
samples taken at >11 weeks of gestation were included in our study
(n = 10 male fetuses), no false-negative results were observed.
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Finally, using fluorescent DNA in situ hybridization (FISH) with
directly labeled X- and Y-chromosome-specific probes (CEPX-SA,
CEPY-SATIII-SO; Vysis, Downers Grove, IL), we found X/Y-positive cells,
i.e., fetal cells, on microscope slides made from samples obtained
identically from pregnant women carrying a male fetus. Cells isolated
from plasma were layered on Superfrost Plus microscope slides
(Mentzel-Gläser), air dried, fixed in acetone for 10 min, and
stored at -20 °C. Before FISH, slides were rehydrated, postfixed in
10 g/L paraformaldehyde in phosphate-buffered saline (pH 7.0) for 10
min at room temperature, washed in phosphate-buffered saline, and
dehydrated. The probe mixture, containing 1 µL of each probe, 1 µL
of H2O, and 7 µL of CEP hybridization buffer
(Vysis), and the target were co-denatured on a hot plate at 75 °C
for 2.5 min. Hybridizations were performed overnight in a moist chamber
at 42 °C. Slides were washed three times in 2x standard saline
citrate (SSC) containing 500 mL/L formamide (pH 7–8) and once
in 2x SSC (pH 7–7.5) for 10 min each at 45 °C and once in 2x SSC
containing 1 g/L NP-40 for 5 min at 45 °C. Following
dehydration, cells were embedded in Vectashield (Vector), and
photographs were made using a Fuji film Sensia II 400 on a Zeiss
Axiophot microscope equipped with filters for fluorescence and a 100 W
mercury arc lamp. In a pilot experiment, samples from nine pregnant
women (gestational age, 7–13 weeks) were analyzed using this X/Y-FISH
assay: one false-positive and no false-negative results were obtained
(88% correct). A representative FISH result is shown in Fig. 1
. In five samples from women with male fetuses, X/Y cells were
found in all slides, varying from 1 to 5 cells per slide (~2000 cells
per slide). In four samples from women with female fetuses, in only one
sample, one single positive X/Y cell was found. The relative proportion
of apoptotic fetal and maternal cells isolated from plasma were between
1:500 and 1:2000.
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These data indicate that part of the fetal DNA present in plasma of pregnant women circulates in the form of intact cells and can easily be obtained by centrifugation of the plasma samples. Moreover, despite being in the process of apoptosis, these cells are amenable to both FISH and PCR analysis (at least X- and Y-chromosome identification). If confirmed for other chromosomes, this approach will allow not only rapid noninvasive analysis of fetal DNA by PCR, but also cytogenetic diagnosis of fetal chromosomal aneuploidies by FISH.
Acknowledgments
This study was supported in part by the Dutch ‘Praeventiefonds/ZON' (Grant 28-3022). We thank Drs. A.W.M. Nieuwint, Y.M. Heins, and K. Madan from the Department of Clinical Genetics and the physicians of the Division of Prenatal Diagnosis of the Department of Obstetrics and Gynecology for their support. We thank Vysis Inc. for their collaboration and for providing the chromosome-specific probes used in this work.
Footnotes
fax 31-20-4443895, e-mail ij.vanwijk{at}azvu.nl
References
The following articles in journals at HighWire Press have cited this article:
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  T.V. Zolotukhina, N.V. Shilova, and E. Y. Voskoboeva Analysis of Cell-free Fetal DNA in Plasma and Serum of Pregnant Women J. Histochem. Cytochem., March 1, 2005; 53(3): 297 - 299. [Abstract] [Full Text] [PDF]
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 T. H. Rainer, N. Y.L. Lam, N. B.Y. Tsui, E. K.O. Ng, R. W.K. Chiu, G. M. Joynt, and Y.M. D. Lo Effects of Filtration on Glyceraldehyde-3-Phosphate Dehydrogenase mRNA in the Plasma of Trauma Patients and Healthy Individuals Clin. Chem., January 1, 2004; 50(1): 206 - 208. [Full Text] [PDF]
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S. Hristoskova, W. Holzgreve, and S. Hahn Fetal Nucleated Erythrocytes in Maternal Circulation Do Not Display a Classic Membrane-associated Apoptotic Characteristic (Phosphatidylserine Exposure) Despite Being Positive by Terminal dUTP Nuclear End Labeling Clin. Chem., November 1, 2003; 49(11): 1934 - 1937. [Full Text] [PDF]
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 J. Guibert, A. Benachi, A.-G. Grebille, P. Ernault, J.-R. Zorn, and J.-M. Costa Kinetics of SRY gene appearance in maternal serum: detection by real time PCR in early pregnancy after assisted reproductive technique Hum. Reprod., August 1, 2003; 18(8): 1733 - 1736. [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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R. M. Angert, E. S. LeShane, Y.M. D. Lo, L. Y.S. Chan, L. C. Delli-Bovi, and D. W. Bianchi Fetal Cell-free Plasma DNA Concentrations in Maternal Blood Are Stable 24 Hours after Collection: Analysis of First- and Third-Trimester Samples Clin. Chem., January 1, 2003; 49(1): 195 - 198. [Full Text] [PDF]
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 N. C. Lambert, Y. M. D. Lo, T. D. Erickson, T. S. Tylee, K. A. Guthrie, D. E. Furst, and J. L. Nelson Male microchimerism in healthy women and women with scleroderma: cells or circulating DNA? A quantitative answer Blood, September 26, 2002; 100(8): 2845 - 2851. [Abstract] [Full Text] [PDF]
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 X. Y. Zhong, W. Holzgreve, and S. Hahn Cell-free fetal DNA in the maternal circulation does not stem from the transplacental passage of fetal erythroblasts Mol. Hum. Reprod., September 1, 2002; 8(9): 864 - 870. [Abstract] [Full Text] [PDF]
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E. K.O. Ng, N. B.Y. Tsui, N. Y.L. Lam, R. W.K. Chiu, S. C.H. Yu, S.C. C. Wong, E. S.F. Lo, T. H. Rainer, P. J. Johnson, and Y.M. D. Lo Presence of Filterable and Nonfilterable mRNA in the Plasma of Cancer Patients and Healthy Individuals Clin. Chem., August 1, 2002; 48(8): 1212 - 1217. [Abstract] [Full Text] [PDF]
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 G. Vona, C. Beroud, A. Benachi, A. Quenette, J. P. Bonnefont, S. Romana, Y. Dumez, B. Lacour, and P. Paterlini-Brechot Enrichment, Immunomorphological, and Genetic Characterization of Fetal Cells Circulating in Maternal Blood Am. J. Pathol., January 1, 2002; 160(1): 51 - 58. [Abstract] [Full Text] [PDF]
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D. W. Bianchi, E. S. LeShane, and J. M. Cowan Large Amounts of Cell-free Fetal DNA Are Present in Amniotic Fluid Clin. Chem., October 1, 2001; 47(10): 1867 - 1869. [Full Text] [PDF]
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S. Hristoskova, W. Holzgreve, S. Hahn, I. J. van Wijk, and C. B.M. Oudejans More Than One-Half of the Erythroblasts in the Fetal Circulation and Cord Blood Are TUNEL Positive Drs. van Wijk and Oudejans respond: Clin. Chem., October 1, 2001; 47(10): 1870 - 1871. [Full Text] [PDF]
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R. W. K. Chiu, L. L. M. Poon, T. K. Lau, T. N. Leung, E. M. C. Wong, and Y. M. D. Lo Effects of Blood-Processing Protocols on Fetal and Total DNA Quantification in Maternal Plasma Clin. Chem., September 1, 2001; 47(9): 1607 - 1613. [Abstract] [Full Text] [PDF]
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Y.M. D. Lo Fetal DNA in Maternal Plasma: Biology and Diagnostic Applications Clin. Chem., December 1, 2000; 46(12): 1903 - 1906. [Abstract] [Full Text] [PDF]
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L. L.M. Poon, T. N. Leung, T. K. Lau, and Y.M. D. Lo Presence of Fetal RNA in Maternal Plasma Clin. Chem., November 1, 2000; 46(11): 1832 - 1834. [Full Text] [PDF]
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Y.M. D. Lo, T. K. Lau, L. Y.S. Chan, T. N. Leung, and A. M.Z. Chang Quantitative Analysis of the Bidirectional Fetomaternal Transfer of Nucleated Cells and Plasma DNA Clin. Chem., September 1, 2000; 46(9): 1301 - 1309. [Abstract] [Full Text] [PDF]
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