THE LANCET
`
`Presence of fetal DNA in maternal plasma and serum
`
`Y M Dennis Lo, Noemi Corbetta, Paul F Chamberlain, Vik Rai, Ian L'Sargent, Christopher W G Redman,
`James S Wainscoat
`
`Summary
`
`Background The potential use of plasma. and serum for
`molecular diagnosis has generated interest. Tumour DNA
`has been found in the plasma and serum of cancer patients,
`and molecular analysis has been done on this material. We
`investigated the equivalent condition in pregnancy-that is,
`whether fetal DNA is present in maternal plasma and serum.
`
`Methods We used a rapid-boiling method to extract DNA
`from plasma and serum. DNA from plasma, serum, and
`nucleated blood cells from 43 pregnant women underwent a
`sensitive V-peR assay to detect circulating male fetal DNA
`from women bearing male fetuses.
`
`Findings Fetus-derived Y sequences were detected in 24
`(80%) of the 30 maternal plasma samples, and in 21 (70%)
`of the 30 maternal serum samples, from women bearing
`male fetuses. These results were obtained with only 10 f-LL
`of the samples. When DNA from nucleated blood cells
`extracted from a similar volume of blood was used, only five
`(17%) of the 30 samples gave a positive Y signal. None of
`the 13 women bearing female fetuses, and none of the ten
`non-pregnant
`control women,
`had
`positive
`results
`for
`plasma, serum or nucleated blood cells.
`
`of circulating fetal DNA in
`finding
`Interpretation Our
`maternal plasma may have implications for non-invasive
`prenatal diagnosis, and for improving our understanding of
`the fetomaternal relationship.
`
`Lancet 1997; 350: 485-87
`
`Nuffield Department of Clinical Biochemistry (Y M D La MRCP),
`Nuffield Department of Obstetrics and Gynaecology
`.(P F Chamberlain MO, Vik Rai MRCOG, I L Sargent PhD,
`Prof C W G Redman FRCP), and Department of Haematology, John
`Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK
`(J W Wainscoat FRCPath); and Istituto dl Mediclna Interna e
`F1siopatologia Medica, IRCCS, Ospedale Maggiore, Milan, Italy
`(N Corbetta)
`Correspondence to: Dr Y M Dennis La, Department of Chemical
`Pathology, Chinese University of Hong Kong, Prince of Wales
`Hospital, Shatin, New Territories, Hong Kong
`
`Introduction
`The passage of nucleated cells between mother and fetus is
`well recognised.'·2 One important clinical application is the
`use of fetal cells in maternal blood for non-invasive prenatal
`diagnosis.' This approach avoids the risks associated with
`conventional
`invasive techniques, such as amniocentesis
`and chorionic-villus sampling. Substantial advances have
`been made in the enrichment and isolation of fetal cells for
`analysis.'" However, most techniques are time-consuming
`or require expensive equipment.
`There has becn much interest in the use of DNA derived
`from plasma or
`serum for molecular diagnosis,s
`In
`particular, there have been reports that tumour DNA can
`be detected by molecular
`techniques in the plasma or
`serum of cancer patients.'o-R Such reports prompted us to
`investigate whether fetal DNA can be detected in maternal
`plasma and serum.
`
`Methods
`Patients
`Pregnant women attending the John Radcliffe Hospital (Oxford,
`UK) were recruited before amniocentesis or delivery. We obtained
`approval of the ptoject ftom the Central Oxfordshire Research
`Ethics Committee. Informed consent was obtained in each case.
`5-10 mL maternal peripheral blood was collected into one tube
`containing edetic acid and one plain tube. For women undergoing
`amniocentesis, maternal blood was always
`taken before the
`procedure, and 10 mL amniotic fluid was also collected for fetal
`sex determination. For women recruited just before delivery, fetal
`sex was noted at the time of delivery. Control blood samples were
`also taken from ten non-pregnant women, and the samples were
`processed in the same way as those obtained from the pregnant
`women.
`
`Sample preparation
`Maternal blood samples were processed 1-3 h after venesection.
`Blood samples were centrifuged at 3000 g, and plasma and serum
`were carefully removed from the edetic-acid-containing and plain
`tubes, respectively, and transferred into plain polypropylene tubes.
`Great care was taken to ensure that the buffy coat or the blood clot
`was undisturbed when plasma or serum samples, respectively, were
`removed. Mter removal of the plasma samples, the red-cell pellet
`and buflY coat were saved for DNA extraction with a Nucleon
`DNA extraction kit (Scotlabs, Strathclyde, Scotland, UK). The
`plasma
`and
`serum samples
`then
`underwent
`a
`second
`centrifugation at 3000 g, and the recentrifuged plasma and serum
`samples were collected into fresh polypropylene tubes. The
`samples were stored at -20°C until further processing.
`
`DNA extraction from plasma and serum samples
`Plasma and serum samples were processed for PCR by a modified
`version of Emanuel and Peska's method.' 200 f-LL plasma or serum
`in a 0·5 mL eppendorf tube was heated at 99°C for 5 min on a
`heat block. The heated sample was then centrifuged at maximum
`speed in a microcentrifuge, after which the clear supernatant was
`collected and 10 f.LL used for peR.
`
`Vol 350 • August 16, 1997
`
`485
`
`Ariosa Exhibit 1035, pg. 1
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`

`M 1 2 3 4 567
`
`M 8 9 10 1112 13 14
`
`15 16
`
`.
`
`I"
`
`'
`
`[2529432834"
`,1722 5 393236 38,
`Case
`Controls
`Maternal plasma
`Maternal serum
`number
`Amplification of fetal Y-ehromosomal sequences from maternal
`plasma and serum
`Lanes 13 and 14~Y·PCR on male genomic DNA (positive controls); lane
`13 DNA equivalent to ten male cells; lane 14 DNA equivalent of one
`male cell. Land 15. 1 fLg female genomic DNA (negative control). Lane
`16, water (negative control). Arrow marks position of 198 bp Y·PCR
`product. M~molecular weight marker (<\>X174 DNA digested with
`Hincll).
`
`neural-tube defects. An approach by which DNA-based
`diagnosis is done on serum samples could be incorporated
`into existing screening programmes.
`Our data show that fetal DNA can be detected in as little
`as 10 iLL maternal plasma and serum. The detection rate is
`much higher than that for DNA from nucleated blood cells
`extracted from a similar volume of whole blood. This
`fmding suggests a relative enrichment of fetal DNA in
`maternal plasma and serum, a phenomenon analogous to
`the relative enrichment of tumour DNA in the plasma and
`serum of cancer patients.'·s The low detection rate of fetal
`DNA sequences for DNA from nucleated blood cells
`
`Case number
`
`Male fetuses
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`24
`25
`26
`27
`28
`29
`30
`
`Female fetu59S
`31
`32
`33
`34
`35
`36
`37
`38
`39
`40
`41
`42
`43
`
`Gestation
`(weeks)
`
`y·peR
`
`Plasma
`
`Serum
`
`Blood
`
`+
`+
`+
`+
`
`+
`t
`+
`+
`
`+
`+
`+
`+
`+
`
`+
`+
`
`+
`
`+
`
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`
`12
`14
`15
`15
`15
`15
`15
`15
`15
`15
`15
`15
`16
`16
`16
`16
`16
`16
`16
`17
`17
`22
`40
`40
`40
`40
`40
`40
`40
`40
`
`15
`16
`16
`16
`16
`16
`17
`17
`17
`18
`40
`40
`40
`
`Amplification of fetal Y-ehromosomal sequences from maternal
`plasma, serum, and blood
`
`THEU\NCET
`
`DNA extraction from amniotic fluid
`The amniotic-fluid samples were processed for PCR by the
`method of Rebello and colleagues." 100 floL amniotic fluid was
`transferred into a 0·5 mL eppendorf rube, and mixed with an
`equal volume of 10% Chelex-100 (Bio-Rad). After addition of 20
`floL mineral oil to prevent evaporation, the tube was incubated at
`56°C for 30 min on a heat block. The tube was then vortexed
`briefly and incubated at 99°C for 20 min. The treated amniotic
`fluid was stored at 4°C until PCR, and 10 floL was used in a
`100 floL reaction.
`
`peR
`The PCR was carried out broadly as described elsewhere" with
`reagents from a GeneAmp DNA Amplification Kit (Perkin Elmer,
`Foster City, CA, USA). The detection ofY-specific fetal sequence
`from maternal plasma, serum, and cellular DNA was done as
`described with primers Y1·7 and Y1·S, designed to amplifY a
`single-copy sequence (DYS14)." The V-specific product was 19S
`bp. 60 cycles of Hot Start PCR with Ampliwax technology were
`used on 10 floL maternal plasma or serum, or on 100 ng maternal
`nucleated blood-cell DNA; each cycle consists of a denaturation
`step at 94°C for 1 min, and a combined reannealinglextension step
`at 57°C for 1 min. 40 cycles were used for amplification of
`amniotic fluid. PCR products were analysed by agarose-gel
`electrophoresis and ethidium-bromide staining. PCR results were
`scored before fetal sex was revealed to the investigator.
`
`Results
`Serial dilutions of male genomic DNA in 1 iLg female
`genomic DNA were carried out and amplified by the Y(cid:173)
`PCR system with 60 cycles of amplification. Positive
`signals were detected up to the 1/100000 dilution-ie, the
`approximate equivalent of a single male cell.
`The optimum volume of heated plasma and serum
`samples for PCR amplification was assessed by subjecting
`1 iLL, 2 iLL, 10 iLL, 30 iLL, and 50 iLL heated plasma or
`serum samples from male individuals to Y-PCR. The best
`serum.
`signal was obtained from 10 iLL plasma or
`Complete inhibition of amplification was reached with 50
`iLL. Thus, 10 iLL heated plasma or serum samples were
`used for subsequent experiments.
`Maternal plasma and serum samples were collected from
`43 women who were between 12 and 40 weeks pregnant.
`There were 30 male and 13 female fetuses. Among the 30
`women bearing male fetuses, Y-positive
`signals were
`detected in 24 plasma samples and 21 serum samples
`when 10 iLL of the samples was used for PCR (figure and
`table). When DNA from nucleated blood-eells was used for
`Y-PCR, positive signals were detected in only five of the 30
`cases (table). None of the 13 women bearing female
`fetuses, and none of the ten non-pregnant control women,
`had a positive Y signal when plasma, serum, or cellular
`DNA was amplified.
`
`Discussion
`Our results show that fetal DNA is present in maternal
`plasma and serum. Use of maternal plasma or serum for
`the detection of fetal DNA for non-invasive prenatal
`diagnosis may therefore be possible. Ironically, plasma is
`the material routinely discarded in the early stages of many
`DNA-extraction protocols, and also after
`the density(cid:173)
`centifugaton step used by many investigators for non(cid:173)
`invasive prenatal diagnosis. This is probably one of the
`reasons why the presence of fetal DNA in maternal plasma
`has not been explored previously.
`Maternal serum is being used by many centres for
`biochemical screening of chromosomal aneuploidies and
`
`486
`
`Vol 350 • August 16, 1997
`
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`IPR2013-00276
`
`

`

`resulted from the use of only 100 ng DNA, compared with
`1 j.Lg in our previous study. 12 We chose 100 ng because this
`was the average quantity of DNA extracted from 10 j.LL
`whole blood with our genomic DNA-extraction method.
`This
`approach
`allows
`us
`to
`compare
`the
`relative
`detectability of fetal DNA in 10 j.LL plasma or serum and
`the cellular component of 10 j.LL whole blood.
`The detection rate of fetal DNA in 10 j.LL plasma and
`serum is already high at 80% and 70%, respectively, but
`these rates can probably be improved. 1 mL maternal blood
`or serum, for example, will result in a one hundred-fold
`increase in the absolute amount of fetal genetic material
`available for analysis. Such a magnitude of increase should
`lead to a robust and non-invasive system for detection of
`paternally inherited fetal DNA sequences. However, more
`work is needed before this can be achieved, since the
`boiling method we used resulted in a relatively impure
`DNA extract, which inhibited PCR when volumes much
`larger than 10 j.LL were used. We are investigating other
`DNA-extraction methods for plasma and serum. We believe
`that concentration methods for plasma and serum DNA
`could potentially be easier than many fetal-cell
`isolation
`methods, such as cell sorting and micromanipulation.
`The relative merit of the use of plasma or serum samples
`requires further investigation. In seven cases-2, 7,8,9,10,
`22, and 29-there was discordance in the PCR results
`obtained
`from the
`plasma
`and
`serum. A possible
`explanation for the discrepancies is that the quantity of fetal
`DNA in these cases may have a limiting effect, and thus
`sampling errors could be contributing to the observed
`results. Future research with quantitative PCR assays may
`elucidate this effect.
`In four cases with male fetuses, both plasma and serum
`were negative for fetal DNA (cases 1, 11, 12, and 30).
`Three of
`these women were tested at 15 weeks of
`pregnancy or earlier (cases 1, 11, and 12). Furthermore, of
`the seven cases in which there was discordance between
`plasma and serum samples, all but one were tested before
`23 weeks. Taken together,
`these results suggest
`that
`the
`concentration
`of
`fetal DNA increases
`as
`gestation
`progresses, possibly owing to the increase in fetal size. This
`result is analogous to that of N awroz and colleagues,' who
`detected in head and neck cancer patients mutant plasma
`DNA predominantly in those with high tumour
`load.
`Future studies should investigate the temporal
`relation
`between gestation and the appearance and concentration of
`fetal DNA in maternal plasma.
`for
`teclmiques
`disorders,
`as
`sex-linked
`As well
`fetal-DNA detection in maternal plasma or serum can also
`be used to detect many paternally inherited DNA
`sequences that differ from their maternal counterparts.
`Clinical examples include fetal rhesus D status assessment"
`and .detection of certain haemoglobinopathies. I
`' The
`plasma or serum-based approach might also be applicable
`to screening for chromosomal
`aneuploidies
`(such as
`Down's syndrome) if there is a quantitative difference in
`the concentration of fetal DNA in maternal plasma and
`serum between affected and normal pregnancies; this is a
`situation analogous to the high concentration of fetal cells
`detectable in pregnancies that involve aneuploid fetuses. I
`Fetal-cell isolation, however, will nevertheless be necessary
`for
`the
`definitive
`cytogenetic
`diagnosis
`of
`fetal
`chromosomal aneuploidies, and for the direct mutational
`analysis of autosomal
`recessive disorders caused by a
`single mutation.
`The presence of
`
`fetal DNA in maternal plasma
`
`'
`
`THE LANCET
`
`and serum has not been previously described. The
`underlying processes
`that cause free fetal DNA to be
`released into the maternal circulation have yet
`to be
`explained. Possible mechanisms include cell lysis resulting
`from physical
`and
`immunological
`damage,
`and
`developmentally regulated apoptosis of certain fetal tissues.
`There are interesting similarities between a growing fetus
`and a neoplasm: both are immunologically foreign to--and
`have an extensive vascular
`interface with-their hosts.
`Investigation of the variations in fetal-DNA concentrations
`in conditions in which the placental interface is damaged
`(eg, pre-eclampsia) would be of clinical and biological
`interest.
`
`Contributors
`Y M Dennis La and James S Wainsco3r initiated the project. Y M Dennis La
`reviewed literature, supervised the daily practical work and analysis of
`laboratory results. Noemi Corbetta designed the experimental methods and
`was the main laboratory worker. Pa ul F Chamberlain, Vik Rai,
`Ian L Sargem and ChristopherW G Redman designed the clinical module
`of the project. Paul F Chamberlain and Vik Rai were responsible for the
`clinical correlation of laboratory results. Ian L Sargent,
`ChriSlopherW G Redman and James SWainscoat analysed laborarory
`results. James S Wainscoat designed the DNA-extraction methods, and
`supervised the writing of the manuscript. All the authors read and
`contributed to the writing of the paper.
`
`Acknowledgments
`YMDL is supported by the Wellcome Trust. We thank N Manning for
`assistance in sample collection and S L Wong for helpful discussion.
`
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