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Diagnostic accuracy of fetal rhesus D genotyping using
cell-free fetal DNA during the first trimester of pregnancy
Alexandre Vivanti, MD; Alexandra Benachi, MD, PhD; Franois-Xavier Huchet, MD; Yves Ville, MD, PhD;
Henri Cohen, MD; Jean-Marc Costa, PharmD
BACKGROUND: Rhesus D genotyping with cell-free fetal DNA reaction, with amplification of exon 10. Results were compared with
currently is used throughout the world. Although this technique has spread RhD phenotype data that were obtained by cord blood sampling of
rapidly, its optimal use is still a matter of debate. This screening test has neonates.
been introduced mainly for the treatment of RhD-negative pregnant RESULTS: In total, 416 serum samples from RhD-negative pregnant
women during the third trimester of pregnancy, thereby avoiding sys- women were collected during the first trimester of pregnancy. The tests
tematic anti-D prophylaxis, yet such a strategy has proved cost-ineffective. overall sensitivity and specificity were 100% (95% confidence interval,
Publications reporting on fetal RHD genotyping with cell-free DNA in 96.9e100.0) and 95.2% (95% confidence interval, 90.5e97.6),
maternal plasma, specifically during the first trimester of pregnancy, are respectively. The negative and positive predictive values were 99.8%
scarce in the scientific literature. (95% confidence interval, 94.9e100.0) and 97.1% (95% confidence
OBJECTIVE: This study sought to assess the performance of nonin- interval, 94.2e98.6), respectively. Fetal RHD status was inconclusive in 9
vasive fetal Rhesus D genotyping in the first trimester of pregnancy with a cases (2.2%).
single-exon real-time polymerase chain reaction assay. CONCLUSION: Noninvasive fetal RHD determination by single-exon
STUDY DESIGN: This was a retrospective observational multicenter quantitative polymerase chain reaction during the first trimester of preg-
study. Cell-free fetal DNA was extracted from maternal blood of both nancy exhibits high accuracy.
nonimmunized and immunized women at 10e14 weeks of gestation.
RHD sequence was determined by quantitative polymerase chain Key words: cell-free fetal DNA, maternal serum, RHD genotyping
sample was kept at room temperature that was observed for the RHD reaction procedures (6.0%) were performed at
for a maximum of 72 hours before being appeared much earlier than for the 10e11 weeks of gestation. Most women
processed. Anti-D immunoglobulins positive control. In that specic case, it (90.6%) were tested as part of systematic
were not administered to patients whose means that the amount of RHD se- screening; 22 women (5.3%) were tested
fetus was found to be RhD-negative. quences that are detected in maternal before an invasive procedure, and 17
serum is too large to be of fetal origin. women (4.1%) were tested because of
Real-time PCR for RHD gene Based on these ndings, it can be existing RhD immunization. Among
Detection of sequences derived from the concluded that RHD gene sequences are women who were tested for fetal RHD
human RHD gene was performed, as present in the maternal genome, which status, 48 women (11.5%) were of Afri-
described elsewhere8; the mouse GALT suggests the presence of a mother variant can or Caribbean origin. Neonatal RhD
gene was used as a positive control of of RHD gene. Denitive RhD-negative status was negative in 162 cases (38.9%)
DNA extraction. Briey, as a tracer for genotype was considered established and positive in 254 cases (61.1%).
DNA extraction and amplication steps, when all RHD PCR reactions were In this study cohort, fetal RHD status
a low amount (250 pg) of mouse DNA negative, and RHD-positive genotype was inconclusive in 9 cases (2.2%;
(Sigma, Grenoble, France) was added to was considered established when at least Figure), with 6 of them being of African
each patients sample (1 mL of serum) 3 of 4 PCR reactions were positive. or Caribbean origin. Concerning these
immediately before DNA extraction. The results were compared with inconclusive results, 2 newborn infants
Total DNA was then extracted with the those obtained by RhD serology of the were determined to be RhD-positive, and
use of the PCR Template Preparation Kit neonate cord blood sample. 7 infants were determined to be RhD-
(Roche Diagnostics, Meylan, France), negative (based on cord blood sera).
and the adsorbed DNA was eluted with Data and statistical analyses Fetal RHD status was conclusive for
50 mL of elution buffer, 10 mL of which For each woman who was included in the 407 maternal sera (97.8%): 259 nonin-
was used per PCR reaction. Amplica- study, the following data were collected: vasive assays (63.6%) were positive, and
tion was carried out in a Light-Cycler gestational age at noninvasive RHD 148 assays (36.4%) were negative. When
V2.0 instrument (Roche Diagnostics). determination, indication for RHD conclusive, all sera from women carrying
PCR reactions were performed with the determination, results of real-time PCR an RhD-positive fetus (n252) provided
use of the FastDNAMaster Hybridiza- for RHD gene, neonatal RhD determi- positive results for RHD gene detection
tion Probes Kit (Roche Diagnostics) in a nation (cord blood sampling) as gold vs 7 sera from women carrying an RhD-
nal volume of 20 mL, with 0.5 mmol/L standard, antenatal and postnatal negative fetus. None of the RhD-positive
of each primer (Table), 0.25 mmol/L screenings for irregular antibodies, fetuses provided negative results for
of each probe (Sigma), 1.25 units of antenatal or postnatal anti-D adminis- RHD gene detection. The tests overall
uracil-DNA glycosylase (Biolabs, Saint- tration, and geographic origin of both sensitivity and specicity were 100%
Quentin en Yvelines, France), and 4.75 parents. (95% CI, 96.9e100.0) and 95.2% (95%
mmol/L of magnesium chloride. After Data were analyzed with JMP 10 Sta- CI, 90.5e97.6), respectively. In our
an initial 1-minute incubation at 50 C, a tistical Discovery software (SAS Insti- cohort, the negative and positive pre-
rst denaturation step of 8 minutes at tute, Cary, NC). All estimates were dictive values were 99.8% (95% CI,
95 C was followed by amplication presented with 95% condence intervals 94.9e100.0) and 97.1% (95% CI,
performed for 50 cycles of denaturation (CI). 94.2e98.6), respectively.
(95 C, 10 seconds, ramping rate 20 C/ According to antenatal and postnatal
sec), annealing (56 C, 10 seconds, Details of ethics approval screenings for irregular antibodies,
ramping rate 20 C/sec), and extension This observational retrospective study none of the nonimmunized women had
(72 C, 20 seconds, ramping rate 2 C/ was approved by the institutional review been sensitized during their pregnancies.
sec). Each sample was treated twice for board of the French college of obstetri- Among the 17 immunized women, all
DNA extraction, and the RHD assay was cians and gynecologists (CEROG OBS RHD statuses were determined correctly,
performed in duplicate on each DNA 2013-02-04 R1). All data were deidenti- with 7 fetuses who tested RhD-negative.
extract. During each run, sera that were ed to ensure patient privacy and Of the 7 women with false-positive RHD
obtained from women carrying an RhD- condentiality. According to the French antenatal noninvasive genotyping tests,
positive or RhD-negative fetus were used regulation regarding prenatal diagnosis, 4 had been administered anti-D immu-
as positive and negative controls. RHD written informed consent was obtained noglobulins during pregnancy.
reaction was considered to be positive from all patients.
when a uorescent signal was detected Comment
for the RHD gene and to be negative Results Our study results revealed that fetal RHD
when a signal was detected for the Among the 416 RhD-negative women status can be determined with very high
mouse GALT gene only. The results who were tested for fetal RHD status, the accuracy with the use of a single-exon
were considered inconclusive when the mean gestational age at blood sampling assay, even during the rst pregnancy
crossing point value (uorescent signal) was 13.1 1.0 weeks. Overall 25 trimester and in a mixed population.
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Daniels G, Nicolaides KH. Fetal RHD genotyping 20. Hawk AF, Chang EY, Shields SM, cine de la Reproduction, Hopital Antoine Beclere,
in maternal plasma at 11-13 weeks of gestation. Simpson KN. Costs and clinical outcomes of Universite Paris Sud, Clamart, France (Drs Vivanti and
Fetal Diagn Ther 2011;29:301-6. noninvasive fetal RhD typing for targeted pro- Benachi); the Laboratoire de Biologie Medicale
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occurred in the Rhesus box. Blood 2000;95: 21. Teitelbaum L, Metcalfe A, Clarke G, Montsouris (Dr Cohen), Paris, France; the Service de
3662-8. Parboosingh JS, Wilson RD, Johnson JM. Costs Gynecologie-Obstetrique, Hopital Necker-Enfants Mal-
14. Singleton BK, Green CA, Avent ND, et al. and benets of non-invasive fetal RhD determi- ades, Paris, France (Dr Ville); the Departement de Biologie
The presence of an RHD pseudogene contain- nation. Ultrasound Obstet Gynecol 2015;45: Specialisee et de Genetique, Laboratoire CERBA, Saint-
ing a 37 base pair duplication and a nonsense 84-8. Ouen lAumone, France (Dr Costa).
mutation in Africans with the Rh D-negative 22. Neovius M, Tiblad E, Westgren M, Received March 31, 2016; revised June 8, 2016;
blood group phenotype. Blood 2000;95:12-8. Kublickas M, Neovius K, Wikman A. Cost-effec- accepted June 28, 2016.
15. Chitty LS, Finning K, Wade A, et al. Diagnostic tiveness of rst trimester non-invasive fetal RHD J.-M.C. is an employee and shareholder of CERBA.
accuracy of routine antenatal determination of screening for targeted antenatal anti-D prophy- The other authors report no conflict of interest.
fetal RHD status across gestation: population laxis in RhD-negative pregnant women: a model- Corresponding author: Jean-Marc Costa, PharmD.
based cohort study. BMJ 2014;349:g5243. based analysis (2015). BJOG 2016;123:1337-46. jmcosta@lab-cerba.com