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DOI: 10.1369/jhc.4A6400.2005
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Journal of Histochemistry and Cytochemistry
Volume 53 (3): 307-314, 2005
Copyright ©The Histochemical Society, Inc.

Application of Fetal DNA Detection in Maternal Plasma : A Prenatal Diagnosis Unit Experience

Cristina González-González, Maria Garcia-Hoyos, M. Jose Trujillo-Tiebas, Isabel Lorda-Sanchez, Marta Rodríguez de Alba, Fernando Infantes, Jesus Gallego, Joaquín Diaz-Recasens, Carmen Ayuso and Carmen Ramos

Department of Genetics (CGG,MGH,MJTT,ILS,MRdA,FI,JG,CA,CR) and Department of Gynecology and Obstetrics (JDR), Fundación Jiménez Díaz, Madrid, Spain

Correspondence to: Cristina González-González, Fundacion Jimenez Diaz, Genetica, Avda Reyes Catolicos 2, Madrid, 28040, Spain. E-mail: cgonzalezg{at}megalab.es


   Summary
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 Summary
Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Non-invasive prenatal diagnosis tests based on the analysis of fetal DNA in maternal plasma have potential to be a safer alternative to invasive methods. So far, different studies have shown mainly fetal sex, fetal RhD, and quantitative variations of fetal DNA during gestation with fetal chromosomal anomalies or gestations at risk for preeclampsia. The objective of our research was to evaluate the use of fetal DNA in maternal plasma for clinical application. In our study, we have established the methodology needed for the analysis of fetal DNA. Different methods were used, according to the requirements of the assay. We have used quantitative fluorescent polymerase chain reaction (QF-PCR) to perform fetal sex detection with 90% sensitivity. The same technique permitted the detection of fetal DNA from the 10th week of gestation to hours after delivery. We have successfully carried out the diagnosis of two inherited disorders, cystic fibrosis (conventional PCR and restriction analysis) and Huntington disease (QF-PCR). Ninety percent of the cases studied for fetal RhD by real-time PCR were correctly diagnosed. The detection of fetal DNA sequences is a reality and could reduce the risk of invasive techniques for certain fetal disorders in the near future. (J Histochem Cytochem 53:307–314, 2005)

Key Words: fetal DNA • maternal plasma • non-invasive diagnosis • QF-PCR • real-time PCR • cystic fibrosis • Huntington disease • fetal RhD


   Introduction
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 Summary
 Introduction
Materials and Methods
 Results
 Discussion
 Literature Cited
 
THE DISCOVERY of extracellular fetal DNA in maternal blood has exciting possibilities for non-invasive prenatal diagnosis (Lo et al. 1997Go). These findings have suggested that this fetal material would be very valuable for performing accurate diagnosis of certain fetal conditions. Due to the coexistence of fetal and maternal DNA, these analyses would be applicable only when the involved target sequence is in the fetus but absent from the mother, such as paternally inherited disorders or "de novo" mutations. Earlier analyses have mainly shown fetal sex detection and fetal rhesus D status (Lo et al. 1997Go,1998aGo). Other authors (Saito et al. 2000Go) have also detected a fetus affected with achondroplasia.

In the last few years, some characteristics have been described regarding the behavior of this fetal DNA. Quantitative variations have been observed in gestations at risk for preeclampsia or fetal chromosomal anomalies (Lo et al. 1999Go; Zhong et al. 2001Go).

Advances in laboratory technology have helped the development of this non-invasive fetal diagnosis with the use of real-time PCR (Lo et al. 1998bGo). Because prenatal diagnosis is an established part of routine obstetrical care in most countries, these non-invasive procedures are very interesting for both laboratory personnel and for parents, because of the low risk for the fetus.

Such reports have prompted us to evaluate the analysis of fetal DNA in maternal plasma as a possible alternative tool in routine laboratory prenatal diagnosis. In our assay, we have used maternal plasma to determine fetal sex, to study the behavior of fetal DNA throughout gestation, to detect two Mendelian inherited disorders: cystic fibrosis (CF) (Gonzalez-Gonzalez et al. 2002Go) and Huntington disease (HD) (Gonzalez-Gonzalez et al. 2003aGo,bGo), and to determine fetal RhD status. Different methods were used according to assay requirements.


   Materials and Methods
Top
 Summary
 Introduction
 Materials and Methods
Results
 Discussion
 Literature Cited
 
Patients
For fetal sex detection, 81 pregnant women from the 10th to the 20th weeks of gestation participated in the study. One pregnant woman and her husband were selected to assay the behavior of fetal DNA in maternal plasma throughout gestation. This pregnant woman donated blood from the 8th week of gestation to 24 hr after delivery (13 samples). Another pregnant woman in the 13th week of gestation, and her relatives, participated in the study of CF. We studied two pregnant women in the 13th week of gestation whose husbands were affected with HD. The husbands and relatives also participated in the study. In the last study, 20 RhD-negative pregnant women from the 11th to the 16th week of gestation participated in the fetal RhD detection. All samples were taken after obtaining informed consent and before the invasive procedure was carried out [amniocentesis or chorionic villus sample (CVS)]. Blood was collected in 10-ml EDTA tubes.

DNA Extraction
DNA was extracted from 0.8 to 2 ml of maternal plasma with the QIAamp DNA Blood Mini Kit (QIAGEN; Hilden, Germany). We also extracted DNA from amniotic fluid and CVS to confirm our results.

All procedures were performed avoiding exogenous DNA contamination and in a sterile room with sterile pipettes and separate areas for DNA extraction and post-PCR handling.

PCR
Fetal DNA Detection
Ten µl of the extracted DNA was used as a template. PCR was performed in a volume of 50 µl, with 1.5 mM MgCl2, 10x PCR buffer (Roche; Mannheim, Germany), 0.2 mM of dNTP (Amersham Pharmacia Biotech, Inc.; Piscataway, NJ) and 1 U of Fast Start DNA polymerase (Roche). We used 10 pmol of each specific primer for DYS390 short tandem repeat (STR) located at the Y chromosome, F: 5'-TAT ATT TTA CAC ATT TTT GGG CC-3' and R: 5'-TGA CAG TAA AAT GAA CAC ATT GC-3'. The forward primer was labeled at the 5' end with the fluorescent dye 6-FAM (Applied Biosystems; Foster City, CA). Amplification conditions were 55 cycles at 94C, 30 sec; 55C, 30 sec; and 72C, 30 sec. DNA fragments obtained were diluted 10 times and mixed with deoinized formamide and 0.4 µl of GS-400HD ROX standard size (Applied Biosystems). The mix was heated at 95C for 5 min and resolved on a DNA sequencer (ABI PRISM 310 Genetic Analyzer) using GeneScan analysis software (Applied Biosystems).

Fetal DNA throughout Gestation
Ten µl of the DNA extracted from maternal plasma was used as a template. DNA from pregnant women and their husbands were also amplified by PCR in a previous analysis to determine an informative STR. We selected primers for the HPTR gene (located on the X chromosome) due to the different sizes of mothers' and fathers' alleles. DNA PCR was performed in a volume of 50 µl, with 1.5 mM MgCl2, 10x PCR buffer (Roche), 0.2 mM of dNTP (Amersham Pharmacia Biotech), and 1 U of Fast Start DNA polymerase (Roche). We used 10 pmol of each specific primer, F: 5'-CTC TCC AGA ATA GTT AGA TGT AGG TAT-3' and R: 5'-ATG CCA CAG ATA ATA CAC ATC CCC-3'. The forward primer was labeled at the 5' end with the fluorescent dye 6-FAM (Applied Biosystems). Amplification conditions were 55 cycles at 94C, 30 sec; 55C, 30 sec; and 72C, 30 sec. DNA fragments were resolved on a DNA sequencer (ABI PRISM 310 Genetic Analyzer; Applied Biosystems).

Detection of a CF Mutation
We studied a family whose first-born son was diagnosed with CF. A molecular study of the son confirmed two different mutations of CF: G542x and Q890x. Molecular analysis of the parents was performed, and we discovered that the mother was a heterozygous carrier of G542x mutation and the father was a heterozygous carrier of Q890x. From the plasma-extracted DNA, 20 µl and 30 µl were used as a template for PCR. We also used DNA from peripheral whole blood from relatives, and water was used as a negative PCR control. For the Q890x locus, 15 pmol of each primer, F: 5'-GGT GCA TGC TCT TCT AAT G-3' and R: 5'-AAG GCA CAT GCC TCT GTG C-3' was used in a 50-µl reaction along with 1.5 mM MgCl2, 10x PCR buffer (Roche), 0.2 mM of dNTP (Amersham Pharmacia Biotech), and 1.5 U of Fast Start DNA polymerase (Roche). Cycling conditions were 94C, 5 min; and 48 cycles of 94C, 1 min; 56C, 1 min; and 72C, 1.5 min. The Q890x mutation consists of an A to G substitution that creates an MseI restriction site that is used to detect the mutation. The PCR product was incubated at 65C for 3 hr with 20 U of MseI and 3.5 µl of buffer R (MBE) (MBI Fermentas; Quimigranel, Madrid) and water up to a total volume of 35 µl. Obtained fragments were visualized after 2 hr of electrophoresis in a 3% agarose gel and in a 6% polyacrylamide gel stained with ethidium bromide.

HD Diagnosis
We used the quantitative fluorescent polymerase chain reaction (QF-PCR) technology to diagnose paternally inherited HD in maternal plasma. We studied two pregnant women, both at 13 weeks of gestation and married to HD patients, attending our unit for prenatal diagnosis of HD. The prenatal diagnosis is comprised of the extraction of DNA from CVS at 10–13 weeks of gestation to detect the number of expansions of the polymorphic (CAG)n repeats in the Huntington gene that causes the disease (9 to 36 in normal individuals and 37 to 86 in HD patients). PCR was performed in a volume of 50 µl with 10 pmol of each primer (F: 5'-ATG GCG ACC CTG GAA AGC TGA TGA A-3' and R: 5'-GGC GGC TGA GGA AGC TGA GGA-3') along with 1.5 mM MgCl2, 10x PCR buffer (Roche), 0.2 mM dNTP (Amersham Pharmacia Biotech), 1 U of FastStart Taq DNA polymerase (Roche) per reaction. Cycling conditions were 94C, 5 min; 55C, 1 min; 72C, 1 min; 50 cycles of 94C, 45 sec; 58C, 40 sec; and a final extension of 72C, 10 min. The forward primer was labeled at the 5' end with the fluorescent dye 6-FAM (Applied Biosystems). DNA fragments were diluted 10 times and mixed together with deionized formamide and 0.4 µl of GS-400HD ROX standard size (Applied Biosystems). The mix was then heated at 95C for 5 min and resolved on a DNA sequencer (ABI PRISM 310 Genetic Analyzer) using GeneScan analysis software (Applied Biosystems).

Fetal RhD Detection
Ten µl of DNA extracted from maternal plasma was used to perform a real-time multiplex PCR. Primers located in exon 7 of the RhD gene were used (F: 5'-CCT CTC ACT GTT GCC TGC ATT-3'and R: 5'-TTA CAA GCG CGT CCG TGA-3') and a TaqMan probe (5'-TAC GTG AGA AAC GCT CA-3') labeled with 6-FAM (Applied Biosystems). As amplification control, primers that amplify the beta-globin gene were used (F: 5'-GTG CAC CTG ACT TCC TGA GGA GA-3'and R: 5'-GAC CCG TCC AAC CAT AGT TCC-3') and TaqMan probe labeled with VIC (5'-TCT GCC GTT ACT GCC CT-3'). PCR conditions were 50 nM of each probe, 50 nM of each primer for beta-globin, and RhD gene with 25 µl of TaqMan Universal Mastermix (all products from Applied Biosystems) in a final volume of 50 µl with 40 cycles of 94C, 1 min; 62C, 1.30 min; and 72C, 1 min.


   Results
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 Summary
 Introduction
 Materials and Methods
 Results
Discussion
 Literature Cited
 
We have correctly diagnosed fetal sex in 92% of the cases. Sensitivity was 0.935 and reached 100% in the samples obtained in the second trimester (Figure 1).



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  		Figure 1

Number of samples per week of gestation. Black bars represent successful detection and white bars represent failed detection.

 
The fetal DNA follow-up during gestation assay showed that DNA could be detected from the 10th week of gestation and no longer than 24 hr after delivery (Table 1). We could also observe that the fetal DNA peak area seen in QF-PCR was greater as gestation advanced, confirming that fetal DNA amount increases as gestation advances (Figures 2 and 3). In 2 out of 13 samples, one from the 13th week and another from the 27th week, we were not able to detect fetal DNA. A second analysis was performed, but only positive results were obtained from the sample of the 27th week.


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  		Table 1

Fetal DNA during gestation

 


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  		Figures 2 and 3

Figure 2 Results obtained from parental DNA amplification of the HPTR gene and maternal plasma at the 10th week of gestation.

Figure 3 Results obtained from maternal plasma at 32 + 4 weeks of gestation.

 
In the detection of the Q890x mutation of CF, a 485 band is expected in a normal homozygote patient in an acrylamide/agarose gel. The heterozygote carrier pattern is expected to show three fragments of 485, 350, and 135 bp. Figure 4 shows the obtained results. Lane 1 (20 µl of DNA from maternal plasma) shows 485 bp and 350 bp bands (the 135 bp band is absent because of long electrophoresis), corresponding to the paternally inherited mutation Q890x. In lane 2 (30 µl of DNA from maternal plasma), we did not amplify either maternal DNA or fetal DNA, possibly because of a PCR failure. The presence of the mutation in the fetus was confirmed by molecular analysis of the chorion biopsy. The results in agarose gel showed extremely faint bands.



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  		Figure 4

Results obtained in CF study. Lanes 1 and 2: amplification of maternal plasma (20 µl and 30 µl, respectively). Lane 3, mother; lane 4, father; lane 5, affected son; lane 6, brother of the father; lane 7, healthy son; lane 8, negative PCR control. (From González-González et al. Prenat Diagn 2002;22:946–948. Copyright John Wiley & Sons, Ltd. Reproduced with permission.)

 
Concerning the HD assay, a healthy fetus and an affected fetus were correctly diagnosed. In the first family, results from the PCR of paternal DNA showed an allele of 17 CAG repeats and an expanded one of 37 repeats. PCR from the mother's DNA showed two alleles of 20 and 24 CAG repeats. PCR from maternal plasma showed three normal alleles of 17, 20, and 24. Because the allele of 17 repeats was not found in the analysis of the maternal whole blood DNA, this allele corresponded to the paternally inherited allele of the fetus. Thus, the fetus was unaffected (Figure 5). In the second family, PCR analysis of the maternal plasma allowed the detection of three alleles, two normal of 15 and 17 CAG repeats and an expanded one of 40 repeats. The expanded one was not found in the analysis of the maternal whole blood DNA; therefore, the fetus would be affected with HD (Figure 6). All results were confirmed with the analysis of DNA isolated from CVS.



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  		Figures 5 and 6

Figure 5 HD study of a healthy fetus in maternal plasma. (From González-González et al. Prenat Diagn 2003;23:232–234. Copyright John Wiley & Sons, Ltd. Reproduced with permission.)

Figure 6 HD study of affected fetus in maternal plasma. (From González-González et al. Neurology 2003;60:1214–1215. Copyright 2003 by AAN Enterprise. Reproduced with permission.)

 
Results from Fetal RhD Detection
Ninety percent of the cases studied for fetal RhD detection were correctly diagnosed. All results were confirmed with the analysis of DNA isolated from CVS and amniotic fluid (Table 2).


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  		Table 2

Obtained results of the fetal RHD study in maternal plasma

 

   Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
Literature Cited
 
Our first purpose was to detect fetal DNA to develop a non-invasive prenatal diagnosis technique, and fetal sex detection was considered a successful assay. This study allowed us to introduce an adequate methodology of DNA extraction and techniques of PCR amplification and analysis of the obtained fragments. It was also useful to establish some important rules for sample handling, such as very strict precautions against contamination that are necessary to avoid false-positive results. We confirmed that fetal DNA is detected from late first trimester (10th week of gestation) by QF-PCR. Follow-up throughout gestation confirmed that it is easier to detect fetal DNA as pregnancy advances. This study shows the diagnosis of female fetuses and demonstrates that it is possible to detect any sequence absent in the mother but present in the fetus. In these cases, it is essential to have a previous analysis of parental DNA. This technique for fetal sex detection has an important clinical application as a non-invasive tool in those cases of X-linked disorders. The QF-PCR technique, with a similar design, has been successfully used by other groups to detect a paternally inherited X-chromosome polymorphism (Tang et al. 1999Go) and a paternally inherited translocation (Chen et al. 2000Go).

Presently, the main purpose of prenatal diagnosis is the detection of fetal aneuploidies. However, due to the overriding presence of maternal DNA, we were able to detect only fetal sequences of paternal origin in maternal plasma, and most of the aneuploides, such as trisomy 21 (Down Syndrome), are maternally inherited. Previous research from our group has been concerned with the analysis of fetal cells in maternal blood (Rodríguez de Alba et al. 2001Go). The high degree of maternal-cell contamination makes the identification and analysis of single fetal cells difficult and time consuming, but currently it appears to be the only strategy for the non-invasive detection of fetal aneuploidies.

There are many Mendelian disorders in which affected families require prenatal diagnosis. We consider that fetal DNA detection in maternal plasma could be very useful in those cases. The prevalence of CF is very high, depending on the population, and many studies focus on the need for prenatal/neonatal screening of CF (Richards and Grody 2004Go). CF is a disorder in which the exocrine glands of the epithelia produce abnormally thick secretions of mucus and elevated sweat electrolytes. It is caused by many different mutations (~1000) and is characterized by progressive respiratory and gastrointestinal problems and serious impairments of the pancreas, intestine, and liver. In couples at risk of having a fetus affected by CF, the analysis of fetal DNA in maternal plasma could be an alternative to an invasive procedure. This technique is performed routinely in the laboratory but does not have high sensitivity. Nasis et al. (2004)Go have developed an allele-specific real-time PCR to improve the sensitivity and specificity to detect a fetal CF mutation in maternal plasma.

We used QF-PCR for the study of HD as a more sensitive method to detect fetal DNA than conventional acrylamide. HD is a dominantly inherited disease with a variable age of onset and causes motor abnormalities, gradual loss of cognition, and ultimately, death. A parental study was always performed to determine the size of the alleles for the CAG repeats. In both studies, we correctly diagnosed the fetal status at an early gestational age. An important aim of our study was to perform a fetal diagnostic study early in pregnancy, when CVS or amniocentesis might be done.

More experience in this field is needed to confirm the results obtained in our study. At present, our group is working with more patients. It is likely that the analysis of fetal DNA by QF-PCR could be very useful as a pretest suitable for monitoring paternally inherited expanded alleles in HD. The low quantity of fetal DNA in the maternal circulation and interference from an excessive amount of maternal DNA make it necessary to use very sensitive methods such as QF-PCR to analyze fetal mutations. This method would also permit the diagnosis of other paternally inherited fetal disorders, but different approaches are needed for each specific situation. Additional advantages of this technique are its feasibility and the rapidity of carrying it out.

To improve the sensitivity of fetal DNA detection, we decided to introduce the technology of real-time PCR. Many authors have described a sensitivity and specificity close to 100% for fetal DNA detection using this technique (Lo et al. 1998bGo). We considered fetal RhD status detection to be a useful tool in cases of sensitized pregnancies of RhD-negative women with partners heterozygous for the RhD gene. These pregnancies continue to present a considerable obstetric problem of hemolytic disease of the newborn, requiring transfusions in utero and having a mortality risk. Our study demonstrates promising results with 90% correct diagnosis. To determine the sensitivity and usefulness of this technique, an assay with a larger number of samples is being performed in our laboratory. It is also important to consider the RhD polymorphism prevalent in many ethnic populations (deletions, pseudogenes) to avoid incorrect results (Wagner and Flegel 2000Go).

Our experience with the analysis of fetal DNA in maternal plasma demonstrates the reliability of these approaches. As a prenatal diagnosis unit, we focus on the clinical application of fetal DNA detection, mainly in the prenatal diagnosis of Mendelian disorders. These procedures could be used as an alternative method prior to CVS or amniocentesis, for those parents discouraged from having an invasive procedure.

Understanding the technical parameters affecting the reliability of the detection of fetal DNA in maternal plasma is very important for its use as a routine prenatal diagnosis procedure. We have observed that after phlebotomy of the mother, it is necessary to obtain the plasma as soon as possible and then aliquot and freeze it until time of DNA extraction. We collected maternal blood and processed it for no longer than 24 hr. A few samples were processed after this time (48 and 72 h), and using these samples we were able to detect maternal DNA but not fetal DNA. Previous studies indicate that fetal DNA in maternal plasma is stable even 24 hr after collection (Angert et al. 2003Go). The time a sample spends in the tube before processing could not only affect the amount of total DNA by releasing DNA due to apoptosis of maternal cells, but also affect the stability of free-cell fetal DNA. The overriding maternal DNA interferes with fetal DNA amplification, and the degradation of fetal DNA is obviously inconvenient for PCR; therefore, it is important to consider the processing time as an important factor. These data need to be studied at length to establish protocols for the transmission of samples from distant places.

Another limitation of the use of fetal DNA PCR for diagnosis is the possibility of false-positive results. These are attributed mainly to PCR product carry-over, but we believe that sample handling is also an important factor. It is essential to avoid any risk of exogenous DNA contamination; this mandates the use of sterile pipettes and filter tips, and separate sterile areas for DNA extraction and PCR handling. Real-time PCR offers, to date, the highest level of safety and represents the most secure amplification procedure because of its closed-tube systems and the multiplex PCR design that includes an internal PCR amplification control.

Johnson et al. (2004)Go have evaluated a standard protocol to determine whether fetal DNA detection could be reproducible in multiple laboratories. They reported that their PCR procedure is reliable but suggested that an optimized protocol for DNA extraction would be useful to improve the results. We conclude that fetal detection from maternal plasma is a promising technique but that more experience in this field is needed. Further studies are essential to avoid the interference of maternal DNA to obtain a non-invasive diagnosis with results as precise as those obtained by conventional procedures. Poon et al. (2002)Go have focused on the biology of epigenetic phenomena, reasoning that using DNA methylation differences between the mother and fetus, it may be possible to overcome this limitation. It is highly likely that advances in technology will allow the analysis of fetal DNA in maternal plasma to soon be routine in prenatal diagnosis.


   Acknowledgments
 
Comunidad de Madrid (08.6/0028/2000 3) supported this project. We thank Diego Cantalapiedra for scripting assistance and Foundation Conchita Rábago for their support to CGG.


   Footnotes
 
Presented in part at the 14th Workshop on Fetal Cells and Fetal DNA: Recent Progress in Molecular Genetic and Cytogenetic Investigations for Early Prenatal and Postnatal Diagnosis, Friedrich-Schiller-University, Jena, Germany, April 17–18, 2004.

Received for publication May 17, 2004; accepted September 23, 2004


   Literature Cited
Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

Angert RM, LeShane ES, Lo YM, Chan LY, Delli-Bovi LC, Bianchi DW (2003) Fetal cell-free plasma DNA concentrations in maternal blood are stable 24 hours after collection: analysis of first- and third-trimester samples. Clin Chem 49:195–198[Free Full Text]

Chen CP, Chern SR, Wang W (2000) Fetal DNA in maternal plasma: the prenatal detection of a paternally inherited fetal aneuploidy. Prenat Diagn 20:20355–20357

Gonzalez-Gonzalez MC, Garcia-Hoyos M, Trujillo MJ, Rodríguez de Alba M, Lorda-Sanchez I, Diaz-Recasens J, Gallardo E, et al. (2002) Prenatal detection of a cystic fibrosis mutation in fetal DNA from maternal plasma. Prenat Diagn. 22:946–948[CrossRef][Medline]

Gonzalez-Gonzalez MC, Trujillo MJ, Rodríguez de Alba M, Garcia-Hoyos M, Lorda-Sanchez I, Diaz-Recasens J, Ayuso C, et al. (2003a) Huntington disease-unaffected fetus diagnosed from maternal plasma using QF-PCR. Prenat Diagn 23:232–234[CrossRef][Medline]

Gonzalez-Gonzalez MC, Trujillo MJ, Rodríguez de Alba M, Ramos C (2003b) Early Huntington disease prenatal diagnosis by maternal semiquantitative fluorescent-PCR. Neurology 60:1214–1215[Free Full Text]

Johnson KL, Dukes KA, Vidaver J, LeShane ES, Ramirez I, Weber WD, Bischoff FZ, et al. (2004) Interlaboratory comparison of fetal male DNA detection from common maternal plasma samples by real-time PCR. Clin Chem 50:516–521[Abstract/Free Full Text]

Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, Wainscoat JS (1997) Presence of fetal DNA in maternal plasma and serum. Lancet 16:485–487

Lo YM, Hjelm NM, Fidler C, Sargent IL, Murphy MF, Chamberlain PF, Poon PM, et al. (1998a) Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma. N Engl J Med. 339:1734–1738[Abstract/Free Full Text]

Lo YM, Lau TK, Zhang J, Leung TN, Chang AM, Hjelm NM, Elmes RS, et al. (1999) Increased fetal DNA concentrations in the plasma of pregnant women carrying fetuses with trisomy 21. Clin Chem 45:1747–1751[Abstract/Free Full Text]

Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, Wainscoat JS, et al. (1998b) Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet. 62:768–775[CrossRef][Medline]

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Poon LL, Leung TN, Lau TK, Chow KC, Lo YM (2002) Differential DNA methylation between fetus and mother as a strategy for detecting fetal DNA in maternal plasma. Clin Chem 48:35–41[Abstract/Free Full Text]

Richards CS, Grody WW (2004) Prenatal screening for cystic fibrosis: past, present and future. Expert Rev Mol Diagn 4:49–62[CrossRef][Medline]

Rodríguez de Alba M, Palomino P, Gonzalez-Gonzalez C, Lorda-Sanchez I, Ibanez MA, Sanz R, Fernandez-Moya JM, et al. (2001) Prenatal diagnosis on fetal cells from maternal blood: practical comparative evaluation of the first and second trimesters. Prenat Diagn 21:165–170[CrossRef][Medline]

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