American Journal of Medical Genetics 85:66–75 (1999)
Investigation of Maternal Blood Enriched for Fetal
Cells: Role in Screening and Diagnosis of Fetal Trisomies
Raghad Al-Mufti, Henry Hambley, Farzin Farzaneh, and Kypros H. Nicolaides*
Harris Birthright Research Center for Fetal Medicine, Departments of Haematology and Molecular Medicine, Kings
College Hospital School of Medicine and Dentistry, London, England
Prenatal diagnosis of chromosomal abnor-
malities relies on assessment of risk fol-
lowed by invasive testing in the group with
highest risk. Assessment of risk by a combi-
nation of maternal age and fetal nuchal
translucency and invasive testing in the 5%
of the population with the highest risk
would identify about 80% of trisomy 21 preg-
nancies. Preliminary reports suggest that
chromosomal abnormalities can also be di-
agnosed by fluorescent in situ hybridization
(FISH) in maternal blood enriched for fetal
cells. This study examines the potential role
of this method on the prenatal diagnosis of
fetal trisomies. Maternal blood was ob-
tained before invasive testing in 230 preg-
nancies at 10–14 weeks of gestation. After
enrichment for fetal cells, by triple density
centrifugation and anti-CD71 magnetic cell
sorting, FISH was performed and the pro-
portion of cells with positive signals in the
chromosomally normal and abnormal
groups was determined. Fetal karyotype
was normal in 150 cases and abnormal in 80
cases, including 36 with trisomy 21. Using a
21 chromosome-specific probe, three-signal
nuclei were present in at least 5% of the en-
riched cells from 61% of the trisomy 21 preg-
nancies and in none of the normal pregnan-
cies. For a cut-off of 3% of three-signal nu-
clei the sensitivity for trisomy 21 was 97%
for a false positive rate of 13%. Similar val-
ues were obtained in trisomies 18 and 13 us-
ing the appropriate chromosome-specific
probe. Examination of fetal cells from ma-
ternal blood may provide a noninvasive pre-
natal diagnostic test for trisomy 21 with the
potential of identifying about 60% of af-
fected pregnancies. Alternatively, this tech-
Contract grant sponsor: Fetal Medicine Foundation.
*Correspondence to: Professor K.H. Nicolaides, Harris Birth-
right Research Centre of Fetal Medicine, Kings College Hospital
School of Medicine and Dentistry, Denmark Hill, London SE5
8RX, England.
Received 17 July 1998; Accepted 14 January 1999
© 1999 Wiley-Liss, Inc.
nique can be combined with maternal age
and fetal nuchal translucency as a method
of selecting the high-risk group for invasive
testing. Potentially, 80% of trisomy 21 preg-
nancies could be identified after invasive
testing in less than 1% of the pregnant popu-
lation. Am. J. Med. Genet. 85:66–75, 1999.
© 1999 Wiley-Liss, Inc.
KEY WORDS: prenatal diagnosis; fetal tri-
somy; magnetic cell sorting;
fetal cells in maternal blood;
fluorescent in situ hybridiza-
tion; nuchal translucency
thickness; assessment of risk
INTRODUCTION
Prenatal diagnosis of chromosomal abnormalities
currently relies on invasive testing, by chorionic villus
sampling or amniocentesis, in pregnancies considered
to be at high risk for such abnormalities. Amniocente-
sis and chorion villus sampling provide accurate diag-
nosis of the fetal karyotype but they can cause a mis-
carriage in up to a 1% of pregnancies. The risk for
chromosomal abnormalities increases with maternal
age and invasive testing in the 5% of the population
with the highest age risk identifies about 30% of tri-
somy 21 pregnancies. An improved method of assessing
risks is by a combination of maternal age and second
trimester maternal serum biochemistry (alpha-fetopro-
tein, human chorionic gonadotrophin, and estriol), be-
cause for a 5% invasive testing rate about 60% of tri-
somy 21 pregnancies are identified. A more recent
method for assessment of risks is the combination of
maternal age and measurement of subcutaneous
edema in the fetal neck, visualized by ultrasonography
as nuchal translucency, in the first trimester of preg-
nancy [Nicolaides et al., 1992; Taipale et al., 1997;
Snijders et al., 1998]. This method identifies about 80%
of trisomy 21 fetuses for an invasive testing rate of 5%
[Snijders et al., 1998]. Preliminary data also suggest
that the risk can be estimated by a combination of ma-
ternal age, fetal nuchal translucency, and maternal se-
rum biochemistry (free ␤-hCG and PAPP-A) at 10–14
weeks of gestation to identify the 5% highest risk group
 Fetal Cells in Maternal Blood 67

Fig. 1. Nuchal translucency thickness in the 230 chromosomally normal (left) and abnormal (right; trisomy 21, other) groups plotted on the normal
range for crown-rump length (median 5th and 95th centiles).

Fig. 2. FISH-analyzed cells, in maternal blood enriched for fetal cells, from trisomy 21 (A), trisomy 18 (B), and trisomy 13 (C) pregnancies demon-
strating three-signal nuclei with the appropriate chromosome-specific probe. In each case, cells were hybridized on two slides; the first slide with
combination of chromosomes 18 (aqua blue), X (green), and Y (red) and the second slide with combination of chromosomes 21 (red) and 13 (green).
68 Al-Mufti et al.
Fig. 3. Percentage of cells with three-signal nuclei using the 21 chromosome–specific probe in maternal blood enriched for fetal cells from chromo-
somally normal pregnancies and those with trisomy 21 and triploidy.
that would contain about 90% of trisomy 21 pregnan-
cies [Spencer et al., 199x].
During the last 30 years extensive research has
aimed to develop a noninvasive method for prenatal
diagnosis based on the isolation and examination of
fetal cells found in the maternal circulation [Walknow-
ska et al., 1969]. It is now believed that about 1 in 107
nucleated cells in maternal blood are fetal [Bianchi et
al., 1990; Price et al., 1991; Hamada et al., 1993]. Al-
though complete separation of fetal from maternal cells
is not yet possible, the proportion of fetal cells can be
enriched by such techniques as magnetic cell sorting
(MACS) or fluorescence-activated cell sorting (FACS)
after attachment of magnetically labeled or fluorescent
antibodies onto specific fetal cell surface markers [Bi-
anchi et al., 1990; Ganshirt-Ahlert et al., 1992; Wachtel
et al., 1991].
The resulting sample is unsuitable for traditional cy-
togenetic analysis because it is still highly contami-
nated with maternal cells. However, with the use of
chromosome-specific DNA probes and fluorescent in
situ hybridization (FISH) it is possible to suspect fetal
trisomy by the presence of three-signal nuclei in some
of the cells of the maternal blood enriched for fetal
cells. Thus, Bianchi et al. detected three-signal nuclei
from a trisomy 21 pregnancy after enrichment for fetal
cells in maternal blood by FACS [Bianchi et al., 1992].
Ganshirt-Ahlert et al. found three-signal nuclei in
9–17% of cells from ten trisomy 21 and six trisomy 18
pregnancies after sorting by magnetic cell sorting
(MACS); in 10 chromosomally normal pregnancies
0–7% of cells had three-signal nuclei [Ganshirt-Ahlert
et al., 1993]. Simpson and Elias reported the presence
of three-signal nuclei, after sorting by FACS, in 2.8–
74% of cells from five trisomy 21 and two trisomy 18
pregnancies, but in none of 61 chromosomally normal
pregnancies [Simpson and Elias, 1994].
This study examines the potential role of FISH in
maternal blood enriched for fetal cells by triple density
centrifugation and MACS in screening and the prena-
tal diagnosis of fetal chromosomal abnormalities.
METHODS
FISH using multicolor probes for chromosomes Y, X,
21, 18, and 13 was carried out in samples of maternal
blood enriched for fetal cells from 222 singleton preg-
nancies at 10–14 weeks of gestation. The patients were
referred to our center for fetal karyotyping following
assessment of risk for trisomy 21 by maternal age and

Fig. 4. Percentage of cells with three-signal nuclei using the 18 chromosome–specific probe in maternal blood enriched for fetal cells from chromo-
somally normal pregnancies and those with trisomy 18 and triploidy.
fetal nuchal translucency thickness. Written informed
consent was obtained from all patients.
Immediately before chorionic villus sampling, mater-
nal blood (20 ml) was obtained from the antecubital
vein into lithium heparinized bottles and stored at 4°C.
The samples were processed within 24 hr of collection
and before the result of the fetal karyotype became
available.
Triple-density gradient centrifugation was carried
out as previously described and the middle layer, con-
taining nucleated erythrocytes and neutrophilic granu-
locytes, was separated [Ganshirt-Ahlert, 1993]. These
cells were then isolated and incubated with magneti-
cally labeled CD71 antibody to the transferrin receptor
antigen (Miltenyi Biotech, Bergisch Gladbach, Ger-
many) for 15 min at 4°C. Enrichment of the magneti-
cally labeled cells was performed by MACS as previ-
ously described [Ganshirt-Ahlert, 1993]. An aliquot
of the positive cell fraction was cytocentrifuged at 14.3
g for 10 min (Shandon, Frankfurt, Germany), the
cells were cytospinned onto slides, stained with the
Kleihauer-Betke method, and then counterstained
with methylene blue stain (Gurr-Giemsa, BDH Merck
Ltd., Poole, England). The slides were examined by
light microscopy (Nikon Corporation, Tokyo, Japan)
and in 222 (97%) of cases there were fetal hemoglobin-
Fetal Cells in Maternal Blood 69
positive cells accounting for 1–9% (median 4%) of the
nucleated cells; in eight (3%) cases no fetal hemoglobin-
positive cells were observed.
The cells in the positive fraction were centrifuged at
400 g for 5 min and the pellets were suspended in 5 ml
of 75 mM KCl. After incubation at 37°C for 15 min, the
suspension of cells was recentrifuged at 400 g for 5
min, resuspended in 5 ml of Methanol/Glacial acetic
acid (3:1, vol/vol) for 10 min and then kept frozen
at −20°C. For FISH, the cells were transferred to
glass slides and hybridization was carried out with
multicolor chromosome-specific DNA probes as recom-
mended by the manufacturers (Aneuscreen Vysis Inc.,
Downers Grove, Illinois, USA). Two slides were used in
each case; one was hybridized with the multicolor
probes for chromosomes 18 (aqua blue), X (green), and
Y (red) and the other slide was hybridized with probes
for chromosomes 21 (red) and 13 (green).
The slides were examined under a fluorescence mi-
croscope (Nikon Corporation), using a DAPI/FITC/
TRITC triple band pass filter set (Vaysis Inc.). Image
capture and processing was by the Microsoft comput-
erized system Cytovision (Applied Imaging Corpora-
tion, California, USA). In each case 100–300 cells were
examined on each slide and the percentage of cells with
one signal for the Y chromosome probe, one and three
70 Al-Mufti et al.
Fig. 5. Percentage of cells with three-signal nuclei using the 13 chromosome–specific probe in maternal blood enriched for fetal cells from chromo-
somally normal pregnancies and those with trisomy 13 and triploidy.
signals for the X chromosome probe, and three signals
with the 21, 18, and 13 chromosome-specific probes
were calculated. Only intact cells that were not over-
lapping were chosen for the analysis. The enrichment
for fetal cells, FISH, and examination of slides were
carried out without knowledge of clinical details or fe-
tal karyotype.
The distribution of cells with positive signals in the
chromosomally normal and abnormal groups was com-
pared and the sensitivity and false positive rates for
different cut-off percentages of positive cells were cal-
culated. The significance of the possible association be-
tween the percentage of positive cells with fetal nuchal
translucency thickness in the chromosomally normal
and abnormal groups was determined by regression
analysis.
RESULTS
The study consisted of 230 cases with median mater-
nal age of 34 years (range 16–46 years) and median
gestation at the time of sampling of 12 weeks (10–
14 weeks). Examination of the enriched cell fraction
after staining with the Kleihauer-Betke method dem-
onstrated that in 222 (97%) cases there were fetal he-
moglobin-positive cells accounting for 1–9% (median
4%) of the nucleated cells. In eight (3%) cases, all from
the chromosomally normal group, no fetal hemoglobin-
positive cells were observed. The fetal nuchal translu-
cency thickness was above the 95th centile of the nor-
mal range for crown-rump length in 97 (68%) of the
chromosomally normal and in 79 (99%) of the abnormal
group (Fig. 1) [Snijders et al., 1998].
In the 222 pregnancies with fetal hemoglobin-
positive cells, the fetal karyotype was normal in 142
cases (46,XY, n ⳱ 88; 46,XX, n ⳱ 54) and abnormal in
80 cases (47,XY + 21, n ⳱ 17; 47,XX + 21, n ⳱ 19;
47,XY + 18, n ⳱ 12; 47,XX + 18, n ⳱ 12; 47,XY + 13, n
⳱ 7; 47,XX + 13, n ⳱ 3; 45,XO, n ⳱ 6; 47XXY, n ⳱ 1;
69,XXX, n ⳱ 3).
The results of FISH using the 21, 18, and 13 chro-
mosome-specific probes on the samples obtained after
enrichment of the maternal blood for fetal cells in the
chromosomally normal and abnormal groups are
shown in Figures 3–5.
Using the Y probe, there were no pregnancies with
female fetuses, either chromosomally normal (n ⳱ 54)
or abnormal (n ⳱ 43), that demonstrated any signals.
In the 125 pregnancies with male fetuses there were 71

Fig. 6. Percentage of cells with zero-, one-, three-, and four-signal nuclei using the X chromosome–specific probe in maternal blood enriched for fetal
cells from chromosomally normal female pregnancies.
(57%) with positive signals in 1–10% (median 3%) of
the cells and 54 (43%) with no signals. The median
percentage of cells with positive signals in the chromo-
somally normal pregnancies (n ⳱ 88) was 2% and in
the abnormal pregnancies (n ⳱ 37) was 3%.
Using the X probe, in the pregnancies with chromo-
somally normal and abnormal (other than Turner syn-
drome and triploidy) female fetuses 0–5% (median 1%)
of cells demonstrated one-signal nuclei, and 0–3% (me-
dian 1%) demonstrated three-signal nuclei (Figs. 6 and
7). In the three pregnancies with triploidies 4–5% of
cells had three-signal nuclei and in the six pregnancies
with Turner syndrome 3–6% of cells had one-signal nu-
clei (Fig. 7). In the pregnancies with male fetuses,
0–3% (median 1%) of cells had three-signal nuclei, 85–
100% (median 95%) had two-signal nuclei, and 0–11%
(median 3%) had one-signal nuclei.
Using chromosome 21-, 18-, and 13-specific probes,
the percentage of cells demonstrating three-signal nu-
clei was higher in the respective trisomies as well as
the triploides than the normal group (Figs. 3–5). There
was no significant association between fetal nuchal
translucency thickness and the percentage of cells with
three signals in either the chromosomally abnormal
group (Fig. 8; r ⳱ 0.088, P ⳱ 0.468), or the normal
group (chromosome 21 probe, r ⳱ 0.004, P ⳱ 0.958;
chromosome 18 probe, r ⳱ 0.025, P ⳱ 0.763; chromo-
some 13 probe, r ⳱ 0.053, P ⳱ 0.534). In 68 (97%) of the
70 trisomic pregnancies there were three signals with
Fetal Cells in Maternal Blood 71
the respective specific probe in at least 3% of the cells,
whereas in the normal group, the median percentage of
cells with three signals was 1–2%. In the 36 trisomic
male pregnancies the relation of the percentage of cells
with positive signals for the Y-specific probe and the
percentage of cells with three-signal nuclei for chromo-
some-specific probes 21, 18 or 13 is shown in Figure 9
(r ⳱ 0.312, P ⳱ 0.064).
The percentage of chromosomally abnormal and nor-
mal pregnancies with at least 3%, 4%, and 5% of cells
with three-signal nuclei are shown in Table I. Using all
three probes (for chromosomes 21, 18, and 13), three-
signal nuclei were present in at least 3% of cells in 68
of the 70 trisomic pregnancies (sensitivity of 97%) and
in 74 of the 142 normal pregnancies (false positive rate
of 52%); for a cut-off of 5% of three-signal cells the
sensitivity was 57% (40 of 70) and the false positive
rate was 0%.
In all eight fetal hemoglobin-negative cases, the fetal
karyotype was normal (three male and five female).
Using the Y probe, there were no pregnancies that
demonstrated any signals.
DISCUSSION
This study provides further evidence on the feasibil-
ity of diagnosing fetal chromosomal abnormalities by
the application of FISH in maternal blood enriched for
fetal cells. The results confirm those of previous studies
72 Al-Mufti et al.
Fig. 7. Percentage of cells with zero-, one-, three-, and four-signal nuclei using the X chromosome–specific probe in maternal blood enriched for fetal
cells from chromosomally abnormal female pregnancies (䊉, trisomies; 䊊, triploidy, 䊐, Turner’s).
that in trisomic pregnancies the percentage of three-
signal nuclei in maternal blood enriched for fetal cells,
either by MACS or FACS, is higher than in chromo-
somally normal pregnancies [Bianchi et al., 1992; Gan-
shirt-Ahlert et al., 1993; Simpson and Elias, 1994]. The
presence of three-signal nuclei in a small percentage of
cells from the chromosomally normal group and varia-
tions in the hybridization efficiency of the different
probes are well-recognized phenomena with the use of
FISH [Ward et al., 1993; Bryndorf et al., 1997].
Concerning fetal sexing with the Y chromosome–
specific probe, in our study positive signals were de-
tected in 57% of the pregnancies with male fetuses and
in none of those with female fetuses. In two previous
studies on a total of 39 male and 15 female pregnan-
cies, positive signals with the Y chromosome–specific
probe were detected in 74% of the pregnancies with
male fetuses and 7% of those with female fetuses [Gan-
shirt-Ahlert et al., 1993; Simpson et al., 1995]. In the
pregnancies with chromosomally abnormal male fe-
tuses there was a higher percentage of cases with
three-signal nuclei using the 21, 18, or 13 probes than
positive cells for the Y probe. The Y, X, and 18 chromo-
some–specific probes are hybridized simultaneously
onto one slide and therefore differences in hybridiza-
tion between the Y and other probes can not be ex-
plained by differences in the handling of the slides.
However, the signals for the Y probe are smaller than
those with the 18 and X chromosome probes and there-
fore more likely to be washed away with post-hybrid-
ization washings.
Our population was preselected because the patients
had undergone assessment of risk by maternal age and
fetal nuchal translucency thickness and consequently
the majority of chromosomally abnormal fetuses had
increased nuchal translucency. However, our data may
be applicable to chromosomal abnormalities irrespec-
tive of nuchal translucency thickness because there
was no correlation between this measurement and the
percentage of cells with three-signal nuclei in the ma-
ternal blood enriched for fetal cells. Furthermore, this
finding suggests that the data from nuchal translu-
cency thickness and percentage of cells with three-
signal nuclei can be combined to improve the effective-
ness of screening.
Using a 21 chromosome–specific probe, three-signal
nuclei were present in at least 5% of the enriched cells
from about 60% of trisomy 21 pregnancies and in none
of the normal pregnancies. These preliminary results
suggest that this method could be associated with the
same rate of detection of trisomy 21 as second trimester
serum biochemistry but with the advantage that the
invasive testing rate may be as low as 0% rather than
5%. However, unlike serum biochemistry testing,
which is relatively easy to apply for mass population
screening, enrichment of fetal cells by triple-density
 Fetal Cells in Maternal Blood 73

Fig. 8. Association between the percentage of cells with three-signal nuclei using the 21, 18, or 13 chromosome–specific probes in maternal blood
enriched for fetal cells from trisomic pregnancies and fetal nuchal translucency thickness (䊉, trisomy 21; 䊐, trisomy 18; 䉭, trisomy 13).

gradient centrifugation and MACS followed by FISH,
is both labor intensive and requires highly skilled op-
erators. In the case of FISH there are promising devel-
opments for automated computerized analysis of cells
that are likely to simplify processing of the slides. The
extent to which the techniques for enrichment of fetal
cells could be improved, to achieve a higher yield of the
necessary cells, as well as become automated, to allow
simultaneous analysis of a large number of samples,
remains to be seen.
On the basis of our results and the currently avail-
able technology, examination of fetal cells from the ma-
ternal blood is more likely to find an application as a
method for assessment of risk, rather than the nonin-
vasive prenatal diagnosis of chromosomal defects. With
this method in combination with maternal age and fe-
tal nuchal translucency the detection of trisomy 21
pregnancies could remain at 80% but the need for in-
vasive testing could potentially be reduced from 5% to
less than 1%. In the USA and most western European
countries the median maternal age is about 27 years
and the prevalence of trisomy 21 at 12 weeks of gesta-
tion is about one in 400 [Snijders et al., 1995]. In a
representative sample of 10,000 pregnancies, assess-
ment of risk by a combination of maternal age and fetal
nuchal translucency thickness would classify 500 such
pregnancies as being at high risk and this group would
contain 20 (80%) of the estimated 25 cases of trisomy
21 (Fig. 10). One option in the management of this
high-risk group of 500 pregnancies is to carry out an
invasive test, such as chorionic villus sampling, which
would diagnose all 20 cases of trisomy 21 but such

TABLE I. Cut-off Percentages of Cells With Three-Signal Nuclei in the Chromosomally Abnormal and Normal Group Using
Fluorescent Probes for Chromosomes 21, 18, and 13
Chromosome 21 probe Chromosome 18 probe Chromosome 13 probe
Trisomy 21 Normal Trisomy 18 Normal Trisomy 13 Normal
Cut-off (N ⳱ 36) (N ⳱ 142) (N ⳱ 24) (N ⳱ 142) (N ⳱ 10) (N ⳱ 142)
ⱖ3 35 (97.22%) 19 (13.38%) 23 (95.83%) 23 (16.2%) 10 (100%) 32 (22.5%)
ⱖ4 30 (83.33%) 4 (2.8%) 20 (83.30%) 9 (6.34%) 8 (80%) 7 (4.93%)
ⱖ5 22 (61.11%) 0 13 (54.16%) 0 5 (50%) 0
74 Al-Mufti et al.

Fig. 9. Association between the percentage of cells with three-signal nuclei for chromosome-specific probes 21, 18, or 13 and the percentage of cells
with positive signals for the Y-specific probe in the 36 pregnancies with trisomic male fetuses (䊉, trisomy 21; 䊐, trisomy 18; 䉭, trisomy 13).

policy would be associated with a procedure-related cency, in trisomies 18 and 13 there are multiple other
miscarriage of five pregnancies. An alternative policy findings that help establish prenatal diagnosis; thus, in
would be to carry out FISH on maternal blood enriched trisomy 18 there is often early-onset growth retarda-
for fetal cells and reserve chorionic villus sampling only tion, exomphalos, and bradycardia, whereas trisomy 13
for those pregnancies where no fetal hemoglobin- is associated with holopresencephaly and tachycardia
positive cells are recovered and those where at least 3% [Sherod et al., 1997; Snijders et al., 1997]. Therefore,
of the cells demonstrate three signals with the 21 chro- the use of multiple chromosome-specific probes could
mosome–specific probe. According to our preliminary be reserved for the few cases of increased nuchal trans-
results, such a policy could reduce the need for invasive lucency with additional sonographic indicators of a
testing to less than 1% of the whole population (or 16% chromosomal abnormality.
of the 500 pregnancies considered to be at high risk by
maternal age and nuchal translucency) with a minor
loss (about 3%) in the sensitivity for detection of tri-
somy 21 (see Table I).
In this study FISH was carried out with multiple
chromosome–specific DNA probes. In simultaneous ex-
amination for trisomies 21, 18, and 13, three-signal
nuclei were present in at least 3% of cells in 97% of the
trisomic pregnancies but also in 52% of the normal
pregnancies. Assessment of risk by a combination of
maternal age and fetal nuchal translucency identifies
within the top 5% group with the highest risk 80% of
those with trisomy 21 or 18 or 13 [Snijders et al., 1998].
The application of FISH in maternal blood enriched for
fetal cells could be used to reduce the need for invasive
testing from 5% to about 2.5%. Alternatively, FISH can
be carried out with the 21 chromosome–specific probe
alone to reduce the invasive testing rate to less than
Fig. 10. Summary of screening for trisomy 21 using the combination of
1%. Unlike trisomy 21, where the only sonographic maternal age and fetal nuchal translucency followed by maternal blood
marker at 10–14 weeks is increased nuchal translu- enrichment for fetal cells.

The application of FISH in maternal blood enriched
for fetal cells can potentially diagnose noninvasively
about 60% of trisomy 21 pregnancies. Alternatively,
this technique can be combined with maternal age and
fetal nuchal translucency as a method of selecting the
high-risk group for invasive testing. Potentially, 80% of
trisomy 21 pregnancies could be identified after inva-
sive testing in less than 1% of the pregnant population.
ACKNOWLEDGMENT
The study was supported by a grant from the Fetal
Medicine Foundation (Registered Charity no. 1037116).
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