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`Human Reproduction Update, Vol.11, No.1 pp. 59–67, 2005
`
`Advance Access publication November 29, 2004.
`
`doi:10.1093/humupd/dmh053
`
`Cell-free fetal DNA in maternal blood: kinetics, source
`and structure
`
`Farideh Z.Bischoff1,4, Dorothy E.Lewis2 and Joe Leigh Simpson1,3
`
`Departments of 1Obstetrics and Gynecology, 2Immunology and 3Human and Molecular Genetics, Baylor College of Medicine,
`Houston, TX 77030, USA
`
`4To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Baylor College of Medicine,
`6550 Fannin Street, Suite 885, Houston, TX 77030, USA. E-mail: bischoff@bcm.tmc.edu
`
`The kinetics and structure of cell-free fetal DNA in maternal plasma is currently under investigation. Plasma fetal
`DNA seems quite stable albeit cleared rapidly following birth, suggesting continuous fetal DNA release into the
`maternal circulation during pregnancy. However, to understand better the kinetics of circulating DNA, studies to
`determine the biological (structural) form in which fetal and maternal DNA exist and the mechanisms underlying
`variation in plasma are warranted to ensure quantitative diagnostic reliability. It is likely that circulating fetal
`DNA is released from fetal and/or placental cells undergoing apoptosis. Thus, the majority of fetal DNA is
`proposed to circulate in membrane-bound vesicles (apoptotic bodies). This review summarizes the latest reports in
`this field.
`
`Key words: apoptosis/apoptotic bodies/fetal DNA in maternal plasma/non-invasive prenatal diagnosis/quantitative PCR
`
`Introduction
`
`Prenatal genetic diagnosis has traditionally required invasive
`procedures such as amniocentesis or chorionic villous sampling
`(CVS); however, each carries a small but finite risk of fetal loss
`and injury. Maternal serum analyte screening and ultrasound can
`identify individuals at risk for fetal aneuploidy (predominantly
`trisomy 21), but like other non-invasive screening methods, they
`are hampered by non-optimal sensitivities and high false-positive
`(procedure) rates. For several years, we and others have focused
`on the isolation of intact fetal cells from maternal blood, a non-
`invasive method that can yield definitive results. Universal pre-
`sence of fetal cells in maternal blood is now accepted, but their
`occurrence is rare and requires complex enrichment and identifi-
`cation strategies. There exist one to six fetal cells per millilitre
`of blood from normal pregnant women (Hamada et al., 1993;
`Krabchi et al., 2001). The largest study concerning efficacy is
`the multicentre National Institute of Child Health and Develop-
`ment (NICHD) fetal cell study group in which we and other
`groups collaborated. Of 2744 maternal samples (Bianchi et al.,
`2002), fetal male cells were correctly identified in 41.4% when
`the fetus was euploid (n ¼ 1292). Among confirmed aneuploid
`cases, the detection rate was higher: 74.4%. Although further
`improvement of existing enrichment and isolation protocols is
`warranted, progress remains hampered by both rarity of fetal
`cells and the lack of fetal-specific cell markers. More recently,
`we and other researchers have also focused on non-cellular fetal
`
`DNA in the plasma fraction of maternal blood for quantification
`and analysis of locus-specific sequences. In this review we
`emphasize advancements related to evaluation of the dynamic
`changes and biological nature of circulating nucleic acids in
`maternal plasma.
`
`Existence of circulating nucleic acids in plasma
`of cancer patients
`
`Nucleic acids (DNA and RNA) in plasma were first observed
`. 50 years ago. In the early 1970s increased quantities of DNA
`were verified in the plasma of cancer patients (Leon et al.,
`1977). In the late 1980s and 1990s several groups demonstrated
`that plasma DNA derived from cancer patients displayed
`tumour-specific characteristics, including decreased strand stab-
`ility, Ras and p53 mutations, mircrosatellite alterations, abnor-
`mal promoter hypermethylation of selected genes, mitochondrial
`DNA mutations and tumour-related viral DNA (Stroun et al.,
`1989; Sorenson et al., 1994; Vasioukhin et al., 1994; Chen
`et al., 1996; Nawroz et al., 1996; Anker et al., 1999; Chan et al.,
`2002). Tumour-specific DNA for a wide range of malignancies
`has been found: haematological, colorectal, pancreatic, skin,
`head-and-neck,
`lung, breast, kidney, ovarian, nasopharyngeal,
`liver, bladder, gastric, prostate and cervix. In aggregate,
`the
`above data show that tumour-derived DNA in plasma is ubiqui-
`tous in affected patients, and likely the result of a common bio-
`logical process such as apoptosis. Investigations into the size of
`
`Human Reproduction Update vol. 11 no. 1 q European Society of Human Reproduction and Embryology 2004; all rights reserved
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`compared with normal gestational age-matched controls (n ¼ 10;
`102 copies/ml) (Ng et al., 2003).
`
`Temporal changes in circulating cell-free DNA
`
`Total DNA
`
`Given establishment of fetal DNA in maternal plasma, attention
`turned to kinetics of the phenomenon. The concentration of total
`DNA in plasma of healthy (non-pregnant) adults is in the range
`of 10 – 100 ng or 103 – 104 GEq/ml Wu et al. (2002). Total plasma
`DNA (fetal and maternal) levels are significantly higher during
`pregnancy (Lo et al., 1998b; Lo, 2000) as well as cancer (Wu
`et al., 2002; Taback et al., 2004). The explanation of increased
`concentration of total DNA during pregnancy is unclear. DNA
`from fetal sources comprises only a small portion (5 – 7%) of
`total circulating DNA. Moreover, fetal DNA in maternal plasma
`seems quite stable (Angert et al., 2003) albeit cleared at an
`extremely rapid rate following birth (Lo et al., 1999d). Thus, a
`continuous supply of fetal DNA is emitted into the maternal cir-
`culation. Such findings suggest that during pregnancy, cellular
`turnover of maternal cells is enhanced. Although such quantita-
`tive changes may serve as a means for distinguishing between
`euploid and abnormal pregnancies, clinical application will
`require a better understanding of the cause associated with varia-
`bility among normal specimens.
`
`Fetal DNA
`
`As expected, detection of relatively low levels of fetal DNA
`sequences (as compared to maternal DNA levels) is dependent
`on the sensitivity of the assay as well as the amount of target
`fetal sequences. Several reports have confirmed that gestational
`age correlates positively with amount of fetal DNA in plasma;
`thus, higher detection rates are reported with increased gestation.
`Lo et al. (1998b) reported fetal concentrations to be low in the
`first trimester, rising in the second and third trimester. Ariga
`et al. (2001) combined real-time kinetic PCR with liquid oligo-
`mer hybridization with 32P-labelled probes
`to quantify Y
`chromosome-specific sequences throughout pregnancy. In 20
`women confirmed to have a male fetus and followed from the
`first to third trimester, fetal DNA concentrations increased from
`10.1 to 130.5 copies per 0.5 ml maternal plasma. Rijnders et al.
`(2003) studied pregnant women after assisted reproduction and
`reported detection of fetal DNA as early as 5 weeks and 2 days
`gestation in one of two patients; however, detection reached
`100% by 9 weeks gestation. It is likely that earlier studies (i.e.
`Thomas et al., 1995) demonstrating 100% Y-sequence detection
`using maternal whole blood between 4 and 7 weeks were actu-
`ally measuring cell-free fetal DNA and not intact cells. Although
`there appears to be considerable variability among subjects in
`the quantity and timing of fetal DNA’s initial presence in
`maternal circulation, the overall trend of increased quantity of
`this DNA with increasing gestational age is consistent.
`During the last 8 weeks of pregnancy there is a sharp increase
`of fetal DNA in maternal plasma (Lo et al., 1998). This might
`be related to gradual breakdown of the maternal – fetal interface/
`placental barrier (Bianchi, 2000). To address this, Chan et al.
`(2003) performed serial analysis of fetal DNA concentrations in
`late pregnancy, showing a positive correlation with gestational
`
`F.Z.Bischoff, D.E.Lewis and J.L.Simpson
`
`these plasma DNA fragments from cancer patients has revealed
`that
`the majority show lengths in multiples of nucleosomal
`DNA, a characteristic of apoptotic DNA fragmentation (Giacona
`et al., 1998; Jahr et al., 2001).
`If a cancer shows specific viral DNA sequences or tumour
`suppressor and/or oncogene mutant sequences, PCR-specific
`strategies can be developed. However, for most cancers (and
`most Mendelian disorders), clinical application awaits optimiz-
`ation of methods to isolate, quantify and characterize the
`tumour-specific DNA compared to the patient’s normal DNA,
`which is also present in plasma. Therefore, understanding the
`molecular structure and dynamics of DNA in plasma of normal
`individuals will be necessary to achieve further advancement in
`this field.
`
`Presence of cell-free fetal nuceic acids in maternal blood
`
`Circulating cell-free fetal DNA in maternal plasma and serum
`
`As studies of tumour-derived DNA detection in plasma of cancer
`patients were being pursued, Lo et al. (1997) demonstrated fetal
`DNA in plasma and serum from healthy pregnant women. Using
`quantitative real-time PCR, surprisingly high mean concen-
`trations (6.2% of total plasma DNA) of fetal DNA were found in
`maternal plasma in early and late pregnancy (Lo et al., 1998b).
`In plasma, fetal DNA reached a mean of 25.4 genome equi-
`valents (GEq)/ml
`(range 3.3 – 69.4)
`in early pregnancy and
`292.2 GEq/ml (range 76.9 – 769) in late pregnancy. Mean con-
`centration was less (3.4% of total serum DNA) but still substan-
`tive in maternal serum. As assessed by the numbers of copies of
`SRY (a single-copy Y chromosome-specific sequence), the ratio
`of fetal
`to maternal DNA was 775 – 970-fold greater in the
`plasma than amount of DNA derived from intact fetal cells
`would indicate.
`Interestingly, Jimenez et al. (2003) demonstrated detection of
`fetal DNA in the maternal serum of rhesus monkeys with appar-
`ently similar kinetics related to gestational age and postnatal
`clearance characteristics observed in humans. The availability of
`such an animal model system should prove very useful
`in
`addressing further questions relating to origin, mechanism and
`nature of fetal DNA in maternal circulation.
`
`Fetal/placental-derived RNA in maternal plasma
`
`In plasma from cancer patients, RNA is also shown to be present
`(Kopreski et al., 1999; Lo et al., 1999a; Chen et al., 2000; Silva
`et al., 2001). These molecules are likely packaged in apoptotic
`bodies and, hence, rendered more stable compared to ‘free
`RNA’ (Hasselmann et al., 2001; Anker et al., 2002; Tsui et al.,
`2002; Ng et al., 2003). Thus, it is not surprising that stable fetal
`RNA is also present in maternal plasma (Poon et al., 2000b). Ng
`et al. (2003a) detected maternal plasma mRNA transcripts exclu-
`sively expressed from the placenta. Two placenta-expressed
`genes (human placental lactogen, hPL; b subunit of hCG) were
`detected in each of 10 maternal samples. This study provides
`direct evidence that RNA is stable in whole blood prior to pro-
`cessing and clearance following delivery. Investigations invol-
`ving another plasma-expressed gene, corticotrophin-releasing
`hormone (CRH), have shown increased CRH mRNA in plasma
`of women with pre-eclampsia (n ¼ 12; mean 1070 copies/ml)
`
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`Cell-free fetal DNA in maternal blood
`
`Indeed, a recent report by Chan et al. (2004) investigated the
`size distribution of plasma DNA among pregnant and non-
`pregnant women. They have shown that plasma DNA (based on
`amplicon sizes of the leptin gene) in maternal samples is signifi-
`cantly longer than DNA in plasma of non-pregnant women. In
`addition, among maternal
`samples, maternal-derived DNA
`(based on leptin gene sequences) was longer than fetal-derived
`(based on SRY) DNA. In their study of 31 pregnant women,
`median percentage of plasma DNA with size . 201 bp was 57%
`compared to 14% among non-pregnant women (n ¼ 34). Of the
`fetal-derived DNA, 20% displayed sizes . 193 bp but not
`exceeding 313 bp. These studies support the hypothesis that fetal
`DNA may be distinguishable from maternal DNA, enabling
`development of fetal DNA enrichment strategies.
`
`Fetal DNA likely exists in the form of apoptotic bodies
`
`Irrespective of the source, various forms of circulating DNA in
`plasma can be suggested: shed cells, apoptotic bodies, nucleo-
`somes, other nucleoproteins, and free DNA. Halicka et al.
`(2000) described formation of apoptotic bodies during apoptosis
`of different cell
`types induced by various treatments. These
`authors reported that apoptosis is not a random or chaotic pro-
`cess but under regulatory control. Thus, formation of apoptotic
`bodies is a specific stage of programmed cell death. Apoptotic
`bodies usually contain either DNA or RNA, but not both
`(Halicka et al., 2000). Evidence that apoptotic bodies are the
`vehicle for cell-free DNA in plasma has been provided by detec-
`tion of stabilized circulating RNA in patients with malignant
`melanoma (Kopreski et al., 1999) and breast cancer (Chen et al.,
`2000). The same holds for fetal DNA in normal pregnant
`women (Tsui et al., 2002). Given that plasma is rich in RNase
`activity and that RNA is highly sensitive to nuclease attack,
`detection of stable RNA further suggests that this nucleic acid is
`protected likely in vesicles or apoptotic bodies.
`
`Evidence that apoptotic bodies contain fetal DNA: Baylor results
`
`Although nucleosomes have been observed and quantified in
`plasma of cancer patients, little is known of the basic properties
`of apoptotic bodies which are known to contain nucleosomes. It
`is unclear how apoptotic bodies are distributed during blood
`fractionation into cellular and plasma fractions by centrifugation
`or ultrafiltration. Because we have such compelling evidence for
`the presence and utility of fetal DNA in maternal circulation for
`clinical diagnostic application, a better understanding of the
`nature of this potential source of fetal genetic material is war-
`ranted. To address some of these issues, we performed studies
`that enable more efficient recovery and concentration of circulat-
`ing DNA from maternal plasma.
`
`Transmission electron microscopy
`
`Most isolation protocols target a particular form of DNA; thus,
`standard approaches will inevitably result in loss of non-targeted
`DNA (fetal and/or maternal). Thus, to maximize recovery of all
`possible forms of fetal DNA, we have used the Microcon cen-
`trifugal filter device (Millipore, USA). These filter units employ
`a low-binding, anisotropic, hydrophilic regenerated cellulose
`membrane that allows high yield recovery rates (. 95% of the
`sample) with concentration factors as high as 100-fold. Using
`
`61
`
`age in the third trimester. During the late third trimester, they
`observed a mean increase of 29.3% of fetal DNA each week.
`Thus,
`they provide normative values for comparative studies
`involving pregnancy related pathological conditions such as pre-
`term labour and pre-eclampsia.
`Ohashi et al. (2002) report a correlation of fetal DNA and
`hCG concentrations using second trimester maternal serum
`samples. Farina et al. (2002) demonstrated a significant corre-
`lation between early gestational age (10 – 12 weeks) and total
`fetal DNA concentrations among 63 euploid pregnancies that
`was normally distributed. In addition, several studies to assess
`the sensitivity and specificity of fetal DNA in first and second
`trimester maternal plasma have also been reported, with rela-
`tively large sample sizes (Costa et al., 2001; Sekizawa et al.,
`2001; Zhong et al., 2001a; Honda et al., 2002). Real-time quan-
`titative PCR was employed based on Y-specific sequences in
`pregnancies carrying male fetuses. Overall, 95 – 100% sensitivity
`with 100% specificity was observed. Sensitivity of detection in
`first trimester (7 – 12 weeks) euploid samples was less efficient
`(70 – 95%), likely reflecting lower amounts of fetal DNA.
`
`In what form does fetal DNA exist? Apoptotic bodies
`as likely vehicle
`
`Apoptosis as ubiquitous source of circulating DNA
`
`Three sources of circulating DNA can be plausibly hypoth-
`esized: (i) dying cells (necrotic or apoptotic); (ii) active secretion
`of DNA; (iii) terminal differentiation. Apoptosis (programmed
`cell death) is the most common form of cell death, continuing
`through life from early stages of embryogenesis to death.
`Because 1011 – 1012 cells divide daily and the same amount
`should be lost to maintain tissue homeostasis, , 1 – 10 g of DNA
`can be expected to be degraded each day in the human (Rudin
`et al., 1997). Given the high turnover, it is not surprising that
`some DNA escapes final cleavage/degradation and thus appears
`in the plasma. The concentration of DNA in plasma is in the
`range of 10 – 100 ng or 103 – 104 GE/ml (Jen et al., 2000a; Wu
`et al., 2002). During pregnancy this DNA could be fetal and/or
`placental in origin.
`
`Apoptosis giving rise to fetal DNA: distinguishable size fragments
`
`The biochemical hallmark of apoptosis is fragmentation of geno-
`mic DNA, an irreversible event that commits the cell to die.
`This occurs before changes in plasma membrane permeability
`(prelytic DNA fragmentation) (Barrett et al., 2001; Martelli
`et al., 2001). In many systems, this DNA fragmentation has
`been shown to result from activation of endogenous Ca2þ
`- and
`Mg2þ
`-dependent nuclear endonuclease. This enzyme selectively
`cleaves DNA at sites located between nucleosomal units (linker
`DNA) generating mono- and oligonucleosomal DNA fragments.
`The process of DNA cleavage is not random but likely under
`regulatory control. Thus, certain DNA sequences are relatively
`more susceptible to cleavage, generating predictable size frag-
`ments. Given that the normal mechanisms of apoptosis may be
`saturated during pregnancy or that an entirely different mechan-
`ism of apoptosis is involved, fetal and/or placental cells under-
`going apoptosis may contain DNA that is distinguishable from
`maternal DNA in plasma based on molecular characteristics.
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`plasma was analysed by real-time PCR to quantify DYS1 (fetal)
`and GAPDH sequences. Microscopic analysis revealed the pre-
`sence of apoptotic bodies and nucleosomes in plasma following
`separation. Real-time PCR quantification demonstrated signifi-
`cant enrichment with mean detection of 28% fetal sequences in
`sorted compared to 2.8% in non-sorted specimens. Thus, based
`on these observations, fetal DNA enrichment is feasible and can
`enhance fetal sequence detection.
`
`Technical advances in assaying cell-free fetal DNA
`
`Recovery of plasma DNA
`
`More efficient or selective methods of plasma fetal DNA
`isolation should improve fetal DNA sequence detection in first
`trimester cases. The amount of DNA isolated from plasma is
`dependent on the specific method of DNA isolation employed
`(Houfflin-Debarge et al., 2000; Jen et al., 2000b). Centrifugation
`speed is critical for blood separation and recovery of fetal DNA
`from plasma (Lui et al., 2002). Chiu et al. (2001) showed that
`inefficient processing could lead to residual cells in the plasma
`and interfere with accuracy in quantification of fetal sequences.
`This likely explains the discrepancies between groups reporting
`on whether intact fetal cells are actually present in maternal
`plasma (Poon et al., 2000a; Bayrak-Toydemir et al., 2003;
`Bischoff et al., 2003b). Because intact fetal cells in maternal
`plasma are likely apoptotic (Kolialexi et al., 2001), their locali-
`zation to the plasma fraction after centrifugation is likely the
`indirect result of these cells floating from the mononuclear cell
`layer due to reduced cellular density.
`To address some of the obvious variables associated with
`plasma DNA isolation and utilization,
`the five participating
`centres of the NICHD fetal cell study group developed a stan-
`dard protocol for inter-laboratory comparison of fetal DNA
`detection (Johnson et al., 2004). This collaborative effort exam-
`ined variables impacting data quality, and determined sensitivity
`and specificity for detection of the Y chromosome. These studies
`conclusively showed not only that fetal DNA, as judged by the
`Y chromosome, is consistently present in maternal plasma, but
`defined the range of fetal DNA during the weeks of gestation
`relevant for prenatal diagnosis. Overall results among the five
`centres were based on matched maternal plasma specimens
`(total of 35 known male and 28 known female fetuses). Effi-
`ciency of amplification of known quantities of standard DNA
`was consistent between all centers. Surprisingly, we found that
`detecting the fetal allele, and thus sensitivity and negative pre-
`dictive power, was disproportionately impacted by total DNA
`recovery. The quantity of male DNA (SRY sequences) amplified
`from maternal plasma when the fetus was male ranged from 51
`to 228 GEq/ml, whereas control DNA (GAPDH sequences) ran-
`ged from 5939 to 12 397 GEq/ml
`in these cases. Sensitivity
`among centres varied from 31.4 to 97.1% with specificity of
`92.8 to 100%. Given that the amount of DNA recovered corre-
`lates positively with sensitivity, variable sensitivities are predict-
`able. This result puts a premium on efficient DNA purification.
`
`Sampling of isolated plasma DNA
`
`The quantity of background (circulating maternal) DNA can
`influence the detection rate of low copy number sequences
`
`F.Z.Bischoff, D.E.Lewis and J.L.Simpson
`
`Figure 1. Transmission electron microscopic analysis of maternal plasma
`pellets. Images display electron micrographs of maternal plasma pellets at
`two magnifications. (A) Arrows identify the presence of nucleosomes among
`various structures that are likely ruptured vesicles (apoptotic bodies).
`(B) Higher magnification illustrates presence of chromatin (arrow).
`
`these filter units enables collection of all nucleic acids . 10 bp
`in size in a plasma sample. By concentrating the plasma DNA
`content, suitable size plasma pellets were prepared and subjected
`to electron microscopic analysis that demonstrated for the first
`time presence of substantial quantities of nucleosomes in plasma
`of pregnant women. In Figure 1, the images display transmission
`electron micrographs of maternal plasma pellets at two magnifi-
`cations. In Figure 1A, arrows identify the presence of small
`spherical bodies that appear quite symmetrical among various
`other structures that are likely ruptured vesicles, such as apopto-
`tic bodies. These structures are nucleosomes (DNA bound to
`histones), which had likely been engulfed by apoptotic bodies.
`Because the initial step in our plasma concentration protocol
`involves a 16 000 g centrifugation to force the suspension
`through the filter, it is not surprising that such membrane-bound
`components would rupture and release their components in the
`plasma pellet. At higher magnification (Figure 1B), these struc-
`tures clearly contain and are attached to chromatin (arrow). This
`chromatin is abundant, with varying size and level of conden-
`sation, consistent with an apoptotic pathway. This finding sup-
`ports the hypothesis that fetal DNA may exist in distinct forms
`and thus be subject to enrichment.
`
`Real-time quantitative PCR
`
`In a preliminary study, maternal plasma (n ¼ 28; 15 male, 13
`female) was separated by centrifugation (800 g) from 12 – 16
`week gestations. Acridine Orange (AO), a nucleic acid stain,
`was used to label recovered plasma, followed by flow cytometric
`separation of
`the AO-positive non-cellular
`fraction. Sorted
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`Cell-free fetal DNA in maternal blood
`
`different fetal growth and placental pathologies may result in
`different levels of fetal DNA.
`Interestingly, Hromadnikova et al. (2002) were not able to
`detect increased fetal DNA levels or differences in the fetal:
`maternal DNA ratio in maternal plasma of patients with affected
`trisomy 21 fetuses compared to normal controls. Although
`Spencer et al. (2003) could not detect increased levels of fetal
`cell-free DNA in serum of ten trisomy 21-affected women, total
`DNA (fetal and maternal) was observed to be increased. Based
`on
`real-time
`quantification
`of
`ubiquitous
`albumin
`gene
`sequences,
`the median level of total DNA was significantly
`greater in women with trisomy 21 (36 152 GEq/ml) compared to
`controls (5832 GEq/ml). Given that maternal cell lysis increases
`the overall quantity of DNA in serum, analysis using maternal
`serum rather than plasma in this study likely explains the
`inability to detect increased fetal DNA levels in trisomy 21.
`Wataganara et al. (2003) found maternal serum fetal DNA
`levels
`to be
`elevated in cases of
`trisomy 13 (29.2 –
`187.0 GEq/ml) but not
`trisomy 18 (18.6 – 77.6 GEq/ml). The
`level of fetal DNA detected was quite variable in the two groups
`as well as in controls (3.7 – 127.4 GEq/ml). Archival maternal
`serum samples displayed a 1.8-fold increase in the amount of
`fetal DNA levels in trisomy positive pregnancies compared to
`gestational age-matched controls.
`Although methods to improve consistency of fetal DNA quan-
`tities are likely to be achieved with more efficient plasma/serum
`DNA isolation, these findings point to the possible use of either
`fetal DNA or total DNA (fetal and maternal) as an additional
`aneuploidy screening analyte. Farina et al. (2003) evaluated the
`use of circulating fetal DNA as a second trimester maternal
`serum marker of Down’s syndrome. The median fetal DNA con-
`centration was 1.7-fold greater in Down’s syndrome cases than
`in controls. Used singularly as a non-invasive marker
`for
`Down’s syndrome, fetal DNA gives a 21% detection rate with a
`set 5% false-positive rate. If combined with the quadruple mar-
`ker screen, non-invasive aneuploidy detection would improve to
`86% at a fixed 5% false-positive rate.
`
`Cell-free fetal DNA to identify pregnancy complications
`
`Increased fetal DNA concentrations
`
`Circulating fetal DNA has also been targeted as a marker for
`assessing feto-maternal well-being. Increased fetal DNA concen-
`trations have been reported for
`several pregnancy-related
`complications (Table I). The best studied is pre-eclampsia,
`
`Table I. Pregnancy-related disorders shown to be associated with increased
`fetal DNA concentrations in maternal plasma
`
`Pregnancy-related disorders
`
`References
`
`Pre-eclampsia
`Preterm labour
`Invasive placentation
`Hyperemesis gravidarum
`Intrauterine growth restriction
`(IUGR)
`Feto-maternal haemorrhage
`Polyhydramnious
`
`Lo et al., 1999; Zhong et al., 2001
`Leung et al., 1998
`Sekizawa et al., 2002
`Sekizawa et al., 2001; Sugito et al., 2003
`Caramelli et al., 2003
`
`Lau et al., 2000
`Zhong et al., 2000
`
`63
`
`(Stenman et al., 2001). When fetal DNA is present in very low
`copy numbers (e.g. 1 – 3 GEq/ml), real-time PCR is likely at the
`limit of its assay sensitivity. Thus, vicissitudes of sampling
`affect detection rate and quantitative measurements. Detection of
`the target DNA may therefore be influenced not only by the
`amount of material used per test but by the number of fractions
`tested. As a result, an increased number of replicates is likely
`necessary to ensure reliable results (Hromadnikova et al., 2003).
`
`Stabilization and transportability of specimens prior to
`DNA isolation
`
`It is not surprising that intact cell lysis due to time delay prior to
`blood processing or treatment conditions (i.e. centrifugation
`speed) would result
`in increased concentrations of maternal
`background DNA, thus causing dilution of relatively low levels
`of circulating fetal DNA (Dukes et al., 2004). Dhallan et al.
`(2004) propose treatment of blood samples with formaldehyde.
`This seems plausible in preventing lysis of intact cells (maternal
`and fetal). Data support this hypothesis with increased percen-
`tage of fetal DNA detected in seven of 10 matched formal-
`dehyde-treated (mean 20.2% fetal) versus untreated (mean 7.7%
`fetal) cases. If fetal DNA exists in the form of apoptotic bodies,
`then treatment of maternal blood samples with formaldehyde is
`also likely to assist in the stabilization of this DNA. Formal-
`dehyde-treated plasma DNA may be more resilient
`to the
`adverse affects caused by delay in processing time, centrifu-
`gation speeds, and storage conditions (i.e. 2 80 versus 2 208C
`temperature) prior to DNA extraction. Lam et al. (2004) also
`report improved recovery of plasma DNA and analysis when
`blood is collected in EDTA rather than heparin or citrate if
`processing is to be delayed by . 6 h after blood draw.
`Transportability of specimens soon after venipuncture has
`been considered. This potential problem is likely to be obviated
`by technical advances as well. For example, our group reported
`success in detection of fetal sequences using dried maternal
`blood spots and real-time PCR (Bischoff et al., 2003a). Fetal
`Y-specific (DYS1) sequences were detected in all 19 (100%;
`4.20 – 24.68 GEq/ml of blood) maternal blood specimens from
`women carrying male fetuses. Feasibility of detecting fetal
`sequences in maternal blood spots dried onto filter paper allows
`for more efficient collection and transport of specimens, thus
`enabling cell-free DNA to be incorporated into non-invasive
`screening regimes on a wide scale.
`
`Cell-free fetal DNA for detecting aneuploidy
`
`Trisomies 13, 18 and 21
`
`Cell-free fetal DNA can play a role in non-invasive prenatal gen-
`etic diagnosis of aneuploidy by helping to identify pregnancies
`at sufficient risk for trisomy 21 or 18 such that an invasive diag-
`nostic procedure should be offered for definitive diagnosis. The
`fetal DNA is likely derived from genes located throughout the
`fetal genome, not just fetal chromosome 21. Using real-time
`PCR to quantify Y-specific sequences, Lo et al. (1999b) demon-
`strated a 2-fold increase in fetal DNA levels for trisomy 21,
`compared to euploid cases. Subsequent studies have supported
`these observations on trisomy 21, although increase is not
`observed in trisomy 18 (Zhong et al., 2000a). This suggests that
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`(background) DNA, approaches have
`amounts of maternal
`focused on cases where the allele of interest would not be
`present in the maternal genome (e.g. paternal inheritance of a
`dominant disease). PCR-based assays for fetal DNA have been
`developed for a number of single gene disorders. In particular,
`Rh determination during pregnancies in which a rhesus-negative
`mother is at risk for a rhesus-positive child has been validated
`(Faas et al., 1998; Bischoff et al., 1999). In our retrospective
`study of 20 frozen serum samples from sensitized RhD-negative
`pregnant women confirmed to have RhD-positive fetuses, we
`demonstrated positive fetal RhD detection in 70% of cases using
`conventional fluorescent PCR for simultaneous amplification of
`the RhD and RhCE (control) genes (Bischoff et al., 1999). Fail-
`ure to detect RhD sequences in 100% of cases was most likely
`due to either analysis using serum samples (lysis of maternal
`cells further dilutes fetal DNA), DNA degradation as a result of
`freezing and thawing serum specimens or inefficient DNA iso-
`lation. Using improved methods of plasma DNA isolation and
`more sensitive sequence detection assays, namely real-time
`PCR, highly accurate (98 – 100%) fetal RhD genotyping can be
`achieved (Lo et al., 1998a; Zhong et al., 2000b; Finning et al.,
`2002).
`Indeed,
`the high detection and accuracy rates have
`resulted in the implementation of this non-invasive test at the
`International Blood Group Reference Laboratory (IBGRL)
`(Finning et al., 2002). Several other single gene disorders have
`also been tested for feasibility in detection or exclusion of an
`affected fetus based on DNA sequences isolated from maternal
`plasma. Table II summarizes these disorders; however, each
`reflects analysis of only one or very few cases.
`
`Epigenetic modifications
`
`Inability of PCR to distinguish readily between maternally inher-
`ited fetal DNA and native maternal DNA is clearly a diagnostic
`impediment. A comparable equivalent to Y-specific DNA that
`could serve as a facile internal control to verify presence of fetal
`DNA in the sample being assessed is lacking. However, studies
`of epigenetic changes resulting in distinguishable patterns of
`DNA methylation between fetal and maternal DNA provide
`promise in the development of unique gene or global fetal-
`specific sequence detection assays (Poon et al., 2002). Similarly,
`placental-specific transcripts in maternal plas