Noninvasive Single-Exon Fetal RHD Determination in a Routine Screening Program in Early Pregnancy LEVEL OF EVIDENCE: I

OBJECTIVE: To develop a simple and robust assay suitable for fetal RHD screening in first-trimester pregnancy and to estimate the sensitivity and specificity of the test after its implementation in an unselected pregnant population. METHODS: Pregnant women attending their first antenatal visit were included, and fetal RHD determination was performed for all women who typed RhD-negative by routine serology. DNA was extracted by an automated system and quantitative polymerase chain reaction was done by an assay based on exon 4. Reporting criteria were simple and strict. RESULTS: Four thousand one hundred eighteen pregnancies, with a median gestational age of 10 weeks, were included. After 211 (5.1%) reanalyses, fetal RHD was reported positive in 2,401 (58.3%), negative in 1,552 (37.7%), and inconclusive in 165 (4.0%) based on the first sample. After a second sample in 147 of 165, only 14 remained inconclusive, all resulting from a weak or silent maternal RHD gene. Using blood group serology of the newborns as the gold standard, the false-negative rate was 55 of 2,297 (2.4%) and the false-positive rate was 15 of 1,355 (1.1%). After exclusion of samples obtained before gestational week 8, the false-negative rate was 23 of 2,073 (1.1%) and the false-positive rate was 14 of 1,218 (1.1%). Both sensitivity and specificity were close to 99% provided samples were not collected before gestational week 8. From gestational week 22, sensitivity was 100%. CONCLUSION: Fetal RHD detection in early pregnancy using a single-exon assay in a routine clinical setting is feasible and accurate. (Obstet Gynecol 2012;120:227-34 LEVEL OF EVIDENCE: I I n 1997, Lo and coworkers 1 reported that approximately 3-6% of cell-free DNA in the plasma of pregnant women is of fetal origin. Unlike fetal cells, which can persist for many years, cell-free fetal DNA is rapidly cleared from the maternal circulation and undetectable within hours of delivery. 2 The first assay of cell-free fetal DNA was based on the detection of the Y chromosome in a female background. Shortly thereafter, it was demonstrated that fetal RHD genes could be detected in RHD-negative women. 3 Many studies have been published since then that confirm the safety aspects of this technology. 4 -6 Initially the focus was on the diagnosis of RHD status of the fetus in a small number of immunized women, but recent screening studies have included several hundred to several thousand nonimmunized pregnant women

Noninvasive Single-Exon Fetal RHD Determination in a Routine Screening Program in Early Pregnancy.

OBJECTIVE: To develop a simple and robust assay suitable for fetal RHD screening in first-trimester pregnancy and to estimate the sensitivity and specificity of the test after its implementation in an unselected pregnant population. METHODS: Pregnant women attending their first antenatal visit were included, and fetal RHD determination was performed for all women who typed RhD-negative by routine serology. DNA was extracted by an automated system and quantitative polymerase chain reaction was done by an assay based on exon 4. Reporting criteria were simple and strict. RESULTS: Four thousand one hundred eighteen pregnancies, with a median gestational age of 10 weeks, were included. After 211 (5.1%) reanalyses, fetal RHD was reported positive in 2,401 (58.3%), negative in 1,552 (37.7%), and inconclusive in 165 (4.0%) based on the first sample. After a second sample in 147 of 165, only 14 remained inconclusive, all resulting from a weak or silent maternal RHD gene. Using blood group serology of the newborns as the gold standard, the false-negative rate was 55 of 2,297 (2.4%) and the false-positive rate was 15 of 1,355 (1.1%). After exclusion of samples obtained before gestational week 8, the false-negative rate was 23 of 2,073 (1.1%) and the false-positive rate was 14 of 1,218 (1.1%). Both sensitivity and specificity were close to 99% provided samples were not collected before gestational week 8. From gestational week 22, sensitivity was 100%. CONCLUSION: : Fetal RHD detection in early pregnancy using a single-exon assay in a routine clinical setting is feasible and accurate. LEVEL OF EVIDENCE: : I

Targeted routine antenatal anti-D prophylaxis in the prevention of RhD immunisation--outcome of a new antenatal screening and prevention program.

OBJECTIVE: To estimate the incidence of RhD immunisation after implementation of first trimester non-invasive fetal RHD screening to select only RhD negative women carrying RHD positive fetuses for routine antenatal anti-D prophylaxis (RAADP). MATERIALS AND METHODS: We present a population-based prospective observational cohort study with historic controls including all maternity care centres and delivery hospitals in the Stockholm region, Sweden. All RhD negative pregnant women were screened for fetal RHD genotype in the first trimester of pregnancy. Anti-D immunoglobulin (250-300 µg) was administered intramuscularly in gestational week 28-30 to participants with RHD positive fetuses. Main outcome measure was the incidence of RhD immunisation developing during or after pregnancy. RESULTS: During the study period 9380 RhD negative women gave birth in Stockholm. Non-invasive fetal RHD genotyping using cell-free fetal DNA in maternal plasma was performed in 8374 pregnancies of which 5104 (61%) were RHD positive and 3270 (39%) RHD negative. In 4590 pregnancies with an RHD positive test the women received antenatal anti-D prophylaxis. The incidence of RhD immunisation in the study cohort was 0.26 percent (24/9380) (95% CI 0.15-0.36%) compared to 0.46 percent (86/18546) (95% CI 0.37 to 0.56%) in the reference cohort. The risk ratio (RR) for sensitisation was 0.55 (95% CI 0.35 to 0.87) and the risk reduction was statistically significant (p = 0.009). The absolute risk difference was 0.20 percent, corresponding to a number needed to treat (NNT) of 500. CONCLUSIONS: Using first trimester non-invasive antenatal screening for fetal RHD to target routine antenatal anti-D prophylaxis selectively to RhD negative women with RHD positive fetuses significantly reduces the incidence of new RhD immunisation. The risk reduction is comparable to that reported in studies evaluating the outcome of non selective RAADP to all RhD negative women. The cost-effectiveness of this targeted approach remains to be studied

Study participants and outcomes.

<p>Study participants and outcomes.</p

Details on women RhD immunised during study period.

*<p>GA = gestational age.</p>#<p>RAADP = routine antenatal anti-D prophylaxis.</p>¶<p>HDFN = haemolytic disease of the fetus and newborn.</p