Fetal RhD genotyping by maternal serum analysis

American Journal of Obstetrics and Gynecology (2005) 192, 666–9
www.ajog.org
EDITORS’ CHOICE
Fetal RhD genotyping by maternal serum analysis:
A two-year experience
Evelyne Gautier, MD,a Alexandra Benachi, MD,b Yves Giovangrandi, MD,c
Pauline Ernault,a Martine Olivi,a Thierry Gaillon,a Jean-Marc Costaa,*
Centre de Diagnostic Pre´natal, American Hospital of Paris, Neuilly, France,a Maternite´, Hoˆpital Necker-Enfants
Malades, Paris, France,b and Maternite´, Hoˆpital Notre-Dame de Bon Secours, Paris, Francec
Received for publication June 29, 2004; revised October 1, 2004; accepted October 28, 2004
KEY WORDS
Fetal DNA
Maternal serum
RHD genotype
Objective: The purpose of this study was to determine the accuracy of the none-invasive prenatal
determination of polymerase chain reaction (PCR)-based fetal RhD genotyping.
Study design: A prospective case series was undertaken on all RhD-negative pregnant women
presenting for genetic counseling in our prenatal diagnosis center from January 2001 until
December 2002. Results were compared with serologic RhD typing of the newborns.
Results: Among the 285 pregnant women who participated in the study, fetal RhD status could
be determined for 283 patients. In 2 cases, the RhD-negative phenotype of the mother was not the
result of a complete RHD gene deletion, and therefore, the status of the fetus could not be
determined. Neither false-negative nor false-positive results were observed.
Conclusion: The present report demonstrates that a reliable fetal RHD genotype determination
can be achieved with 100% accuracy. It is therefore possible to consider that such an assay could
be systematically proposed to all RhD-negative pregnant women in order to more effectively
utilize RhD prophylaxis.
Ó 2005 Elsevier Inc. All rights reserved.
The incidence of hemolytic disease of the fetus/
newborn has dropped dramatically (1 to 6 per 1000
live births) since 1968, when eﬀective prophylaxis by
anti-D immunoglobulin injection to the mother became
available. However, administration of blood derivatives
is not devoid of risk, and every eﬀort should be made to
improve techniques that could reduce the number of
injections.1 Therefore, the new possibility of fetal RhD
genotyping using polymerase chain reaction (PCR) is
* Reprint requests: Jean-Marc Costa, Centre de Diagnostic Pre´natal, American Hospital of Paris, 63 bd Victor Hugo, 92200 Neuillysur-Seine, France.
E-mail: [email protected]
0002-9378/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ajog.2004.10.632
a signiﬁcant advance for clinical purposes. Fetal RhD
genotype determination may also be useful in the
management of RhD-negative sensitized women, but
genotyping is also useful in RhD-negative pregnant
women at risk for RhD immunization2 in order to
avoid unnecessary administration of anti-D immunoglobulin in the case of an RhD-negative fetus.
Although anecdotal diagnosis has been made using
circulating fetal DNA (ie, myotonic dystrophy,3 achondroplasia4), most of the studies on the accuracy of fetal
circulating DNA analysis have focused on both Y
chromosome and RHD gene. In the past few years,
some studies have reported a 100% accuracy in fetal sex
determination in the ﬁrst trimester.5,6 This approach has
Gautier et al
667
Real-time PCR for the RHD gene
in maternal serum
Figure Detection of the RHD gene in maternal serum using
real-time PCR.Typical results observed from patient samples:
one giving a positive result for fetal DNA (1), the second
a negative one (2), and the third one corresponding to an RhDnegative woman but carrying the RHD gene in her genome (3).
now modiﬁed the strategy of prenatal diagnosis in Xlinked disorders,7 as well as the management of pregnancies at risk for congenital adrenal hyperplasia.8 The
use of cell-free DNA in maternal plasma or serum has
also been achieved for noninvasive fetal RhD genotyping by several groups,9-13 but most often on a limited
number of cases.
More recently, 2 large studies14,15 have been conducted on 137 and 893 patients, respectively (most of
them being alloimmunized), before implementation of
routine clinical practice.14 In the present publication, we
report the results of our 2-year experience in the noninvasive prenatal determination of fetal RhD genotyping routinely proposed in a prenatal diagnosis center.
Material and methods
Patients and samples
Noninvasive prenatal determination by maternal serum
analysis was systematically proposed to every RhDnegative pregnant woman presenting for genetic counseling in our prenatal diagnosis center from January
2001 to December 2002. Two hundred and eighty-ﬁve
women elected to participate; all were of Caucasian
origin but none were alloimmunized. After informed
consent, 5 mL of blood were collected into Vacutainer
SSTÒ tubes (Becton Dickinson, Meylan, France) before
amniocentesis or CVS if indicated. Anti-D immunoglobulin was not provided to patients whose fetus tested to be
RhD-negative. Immediately after clotting, serum was
obtained by centrifugation for 10 minutes at 3000g,
aliquoted and stored at 80(C until further processing if
the assay was not performed on the same day. The mean
gestational age was 15.2 weeks, ranging from 8 to 35.
The procedure was similar to that previously described.5,13 Brieﬂy, as tracer for the DNA extraction
and ampliﬁcation steps, a low amount (250 pg) of mouse
DNA (Sigma, Grenoble, France) was added to each
patient’s sample (400 mL of serum) immediately before
DNA extraction. Total DNA was then extracted by the
PCR Template Preparation Kit (Roche Biochemicals,
Meylan, France), and the adsorbed DNA eluted with
50 mL of elution buﬀer, 10 mL of which was used per
PCR reaction. Ampliﬁcation was carried out in a LightCyclerÒ instrument (Roche Biochemicals). PCR reactions were set up in a ﬁnal volume of 20 mL using the
Fast DNA Master Hybridization Probes Kit (Roche Biochemicals), with 0.5 mmol/L of each primer, 0.25 mmol/L
of each probe (Proligo, France) (Table), 1.25 units of
uracil DNA glycosylase (UDG) (Biolabs, Saint-Quentin
en Yvelines, France), 4.75 mmol/L of magnesium chloride. After an initial 1-minute incubation at 50(C, a ﬁrst
denaturation step of 8 minutes at 95(C was followed by
an ampliﬁcation performed for 50 cycles of denaturation
(95(C, 10 seconds, ramping rate 20(C/second), annealing
(56(C, 10 seconds, ramping rate 20(C/second), and
extension (72(C, 20 seconds, ramping rate 2(C/second).
Each sample was treated twice for DNA extraction,
and the RHD assay was performed in duplicate on each
DNA extract. Deﬁnitive results were considered only
when the 4 PCR reactions were concordant. During
each run, sera obtained from patients carrying an RhDpositive or an RhD-negative fetus were used as a positive
and a negative control.
The results were compared with those obtained later
in pregnancy on amniotic ﬂuid cells and by RHD
serology of the newborn.
Results
Among the 285 pregnant women tested for RHD gene
presence in their serum, the status of the fetus could not
be determined in 2 cases because the RhD-negative
phenotype of the mother was not in relation with
a complete RHD gene deletion. These 2 particular cases
were easily identiﬁed using real-time PCR because the
amount of RHD sequences detected in maternal serum
was abnormally high (expressed by an unusual crossing
point) to be from fetal origin (Figure). Because the
maternal DNA represents the major part of DNA
isolated from serum, it can be concluded that RHD
sequences were present in the maternal genome. This
hypothesis was conﬁrmed by analysis of DNA extracted
from the maternal leukocytes.
Fetal RhD genotype could be determined from all
other 283 maternal sera. In 179 cases, RHD sequences
had been detected in maternal serum, and the fetuses
668
Table
Gautier et al
Characteristics and sequences of primers and probes used in the PCR assay
Gene target
Primers sequences
Probes sequences
Human RHD exon 10
50 -GCCTGCATTTGTACGTGAGA-30
50 -CAAAGAGTGGCAGAGAAAGGA-30
30 FITC-TGACAGCAAAGTCTCCAATGTTCG
50 LCRed640-GCAGGCACTGGAGTCAGAGAAAA-30 Ph
could therefore be considered as RhD-positive, while the
others (n = 104) were RhD negative.
For 11 patients, the result obtained by maternal
serum analysis could not be conﬁrmed due to an early
pregnancy termination in relation to fetal chromosomal
abnormality (3 cases), or because the patients were lost
to follow-up (8 cases). The results of the fetal RHD
genotyping on maternal serum could be controlled for
the other 272 patients either by analysis of amniotic
ﬂuids for the presence of RHD gene (n = 209) and/or
by serologic study of the newborn (n = 232). Results
were in complete concordance, and neither false-negative
nor false-positive results were observed; all sera from
women carrying an RhD-positive fetus (n = 170) gave
positive results for RHD gene detection, while all sera
from women carrying an RhD-negative fetus (n = 102)
gave negative results. Speciﬁcity and sensitivity of the
assay were 100% (95% CI: 98-100).
Comment
Fetal RHD genotype can be determined with a high
level of accuracy by analysis of fetal DNA circulating in
maternal plasma and serum. It is therefore possible to
consider that such an assay could be included in
prenatal care of RhD-negative women.16
The results of a 2-year experience of such a clinical
practice are reported herein. In 2 cases, the pregnant
women did not carry a complete deletion of the RHD
gene and a ﬁnal answer regarding the fetal RhD status
could not be provided. Although a thorough analysis
was not undertaken, theses patients were highly suspected of carrying a RHD pseudogene or variant.
Except for these 2 pregnant women, the fetal RhD
status could be addressed for the other 283 patients who
had accepted the test (overall success rate 99.3%). The
result was in complete concordance with that obtained
either by analysis of amniotic ﬂuid for the presence of
RHD gene (n = 209) and/or by serologic study of the
newborn (n = 232). Therefore, the present report demonstrates that a reliable fetal RHD genotype determination can be achieved with 100% accuracy. Although
the presence of fetal DNA cannot be formally proven in
maternal serum in case of RhD-negative fetus, a falsenegative result was never observed in our experience.
This was the result of the use of a similar procedure
successfully used for many years for fetal sex determination7 that includes a careful ultrasonographic control
of the date of the pregnancy, the assay never being
realized before 8 weeks. On the other hand, a false
positive due to the transmission by the father of a silent
RHD gene (women carrying such genes being easily
detected by our assay) could not be excluded in the
future. The patients were systematically informed about
this without any medical risk possibility during genetic
counseling.
Among the studied women, 104 (36.8%) carried an
RhD-negative fetus. Injection of anti-D immune globin
was thus avoided, and pregnancy follow-up was therefore made easier, decreasing the patient’s anxiety.
Determination of fetal RHD genotype using a noninvasive approach is an important challenge because it
has crucial implication in the clinical management of
sensitized RhD-negative pregnant women. Fetal DNA
analysis in maternal serum oﬀers the opportunity to
achieve this challenge.
Attempts to identify the RhD-negative women who
are at risk of sensitization contribute to a more eﬃcient
prophylaxis of alloimmunization in obstetrics being
targeted speciﬁcally at these women. Our data could
be considered as part of a novel strategy in order to
maintain and increase anti-D supplies, as well as (1) the
use of minidose of anti-D in some circumstances (ie, ﬁrst
trimester indications), (2) increased and accurate use of
tests to assess feto-maternal hemorrhage, and (3) increase compliance with guidelines on the use of anti-D.
Moreover, while eﬀorts are made to ensure that the
blood used for the anti-D preparation is safe and free of
human pathogens, it cannot be formally excluded that
an infectious agent is present because of lack of
availability or sensitivity of tests at present.17
The high accuracy rate achieved in the prediction of
fetal D status has resulted in the implementation of
a noninvasive fetal RHD genotyping service. Although
this test is actually performed by a limited number of
specialized laboratories in the world, increasing demand
is anticipated in the near future. As a consequence, the
actual ‘‘made-by-hand’’ assays must move towards more
automated ones (until the availability of commercial
tests) in order to ensure robustness and reliability. This
major point was recently highlighted by an interlaboratory study.18 Therefore, time has come for an external
quality control scheme.
Finally, large-scale studies regarding the economic
aspect of such routine oﬀer of fetal RhD genotyping are
necessary. A consideration of cost-eﬀectiveness is particularly important because of the limited supply of anti-D
Gautier et al
available. These economic evaluations must be countrydependent because guidelines for anti-D use, if existing,
may vary from one country to the other and from
centers where no recommendations exist. Such a study is
actually in progress in France.
Attempts to predict other fetal blood groups (ie, Kell,
Duﬀy) by maternal blood analysis is currently under
investigation by some groups. However, the accurate,
sensitive, and reliable determination of such ‘‘single
base’’ modiﬁcations are diﬃcult to achieve using the
actual methods caused by the presence of a high
background of negative maternal DNA.
Acknowledgments
We are indebted to Dr Jocelyn McGinnis for reviewing
this manuscript.
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