DETERMINATION OF FETAL RHESUS D STATUS
BY MATERNAL PLASMA DNA ANALYSIS Aykut A1,*, Onay H1, Sagol S2, Gunduz C3, Ozkinay F1, Cogulu O1 *Corresponding Author: Ayça Aykut, M.D., Ph.D., Department of Medical Genetics, Ege University Faculty
of Medicine, 35100, Bornova, Izmir, Turkey; Tel.: +90-232-390-3961; Fax: +90-232-390-3971; E-mail:
aycaaykut@hotmail.com page: 33
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MATERIALS AND METHODS
Blood samples (9 mL), collected in EDTA vacutainers,
from 30 RhD-negative Turkish women between
9 and 39 weeks of gestation, who were referred
to us for invasive testing because of advanced
maternal age, increased maternal serum screening
test, fetal sonographic abnormality and previous history
of chromosomal or single gene disorder. Routine
assay for ABO and RhD typing and testing for
unexpected antibodies were performed to include
RhD negative women in the study. The positive
control for the RHD and SRY genes was a heterozygous
RHD-positive man, while the negative control
for both genes was an RhD-negative non pregnant
woman. The SRY gene served as an internal control
marker to confirm the presence of male fetal DNA.
All analyses were performed blind, that is, the fetal
RHD genotyping was performed without knowing
the fetus RhD status, which was confirmed by serological
methods postpartum. Nine mL of maternal
blood was collected in EDTA vacutainers and sent to
the laboratory at room temperature. The blood was
centrifuged at 2840 rpm for 10 min., the plasma was
transferred without disturbing the buffy coat and recentrifuged
again at 3600 rpm for 20 min. and the
supernatants were collected and stored at −80 °C before
DNA extraction.
Written informed consent was obtained from
all the families. The study was approved by the Faculty
Ethics Committee of Ege University Faculty of
Medicine, Izmir, Turkey.
DNA Extraction from Plasma Samples and
Fetal Samples. DNA was extracted from 500 mL
plasma using QIAamp DSP Virus Kit (Qiagen,
Hilden, Germany) according to the manufacturer’s
instructions. DNA was eluted in 20 Elution buffer
(AVE) and 4.0 μL was used as a template for the
polymerase chain reaction (PCR).
DNA from amniocentesis or CVS specimens
was isolated using Chelex (InstaGene Matrix™,
Bio-Rad Laboratories, Mississauga, Ontario, Canada)
in a rapid isolation technique according to the
manufacturer’s instructions. The specimens were
stored at −20 °C before being studied.
Real-time Polymerase Chain Reaction Analysis.
The TaqMan real-time PCR assay protocol
(LightCycler 1.5, Roche Diagnostics, Mannheim,
Germany) was performed. The primers and probes
used for RHD genotyping were targeted towards exons
7 and 10. For the detection of chromosome Y,
primers and probes were targeted for the SRY gene
on chromosome Y (Table 1). Amplicon lengths for
exons 7, 10 and SRY were 82, 122 and 137 bp, respectively.
All primers and probes were synthesized
by TIB MOLBIOL (Berlin, Germany). At least two
regions of the RHD gene were used for the complex
genetic variant forms of RHD. The primers and
probes designed for exons 7 and 10 did not permit
amplification of a non functional rearranged RHD gene, which has been demonstrated in this and in
other studies generating positive results in exon 10.
The RHD/SRY amplification reactions were set
up in a volume of 20 mL. Each reaction contained
4 mL of Light Cycler DNA Master Hybridization
Probes (Roche Diagnostics, Basel, Switzerland; 10×
concentrated), 100 nM of each probe, and 200 nM
of each amplification primer. A 4 mL volume of the
extracted DNA was used for amplification. Thermal
cycling was initiated by a denaturation step of 10
min. at 95 °C, followed by 50 cycles at 95 °C for 15
seconds and at 60 °C for 60 seconds. Amplification
data were analyzed using the Light-Cycler software
(Roche Diagnostics).
A duplicated RHD/SRY PCR was set up for
each individual sample. If results in the duplicate
tests were inconclusive or all data for the RHD and
SRY genes were negative, the PCR set up was repeated,
resulting in a maximum number of four
RHD/SRY PCR procedures.
The presence or absence of the fetal RHD gene
was determined as follows: first, the samples were
examined in duplicate. If both reactions were positive,
we considered the fetus was carrying the paternal
RHD gene. If both reactions were negative or
one of two was positive, the sample was repeated in
another duplicate. In total, if the fetal RHD sequence
was detectable in two or more of four reactions, the
result was scored as positive. Otherwise, it was considered
to be negative. Confirmation of our results
was performed by the analysis of the serological test
on cord blood after delivery or by genotyping the fetal
DNA obtained from amniocentesis or CVS. Only
samples with cycle threshold (Ct) values of less than
40 were considered positive. The results were finally
interpreted if all performed PCR procedures for
RHD and SRY were consistent. If the PCR replicates
for RHD or SRY were discrepant, the plasma was
retested from DNA isolated from the second tube.
For gender detection, the SRY sequence was
used to identify male fetal DNA that was present in
each sample. The experiment was repeated in three
separate reactions. If at least two of the three amplification
reactions were positive for the SRY sequence,
the fetus was considered to be male. If there was no
SRY sequence amplification in all separate reactions,
the fetus was considered to be female. The results
were confirmed by karyotype, examination after birth
or Doppler ultrasound after 20 weeks of gestation.
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