Molecular testing for high-risk anti-D HDFN screening of RH negative expectant mothers

Nov. 20, 2014

Advances in molecular testing make it possible to determine a fetus’s gender, Rh status, and other genetic chromosomal anomalies as early as 10 weeks of maternal gestation. In the past, chromosome analysis, fetus gender, and Rh status could only be performed using specimens collected through invasive procedures such as amniotic fluid or chorionic villus sampling (CVS). Now the same can be accomplished with a routine blood draw. This represents a possible benefit in the early monitoring of Rh negative mothers who are expecting Rh positive babies.

Approximately 16% of Rh negative mothers who carry an Rh positive fetus may produce anti-D,1 a potent antibody that can cross the placenta and cause massive hemolysis of the baby’s red blood cells. By finding out the fetus’s Rh status during the first trimester of pregnancy, physicians and healthcare professionals can better care for their patients and plan for early interventions. This can result in better outcomes for both mother and baby. 

Molecular testing to determine the Rh status of the fetus can also prevent unnecessary administration of antenatal RhIg (Rh immunoglobulin) at 28 weeks of gestation. This practice will prevent unneeded exposure of the mother to a biological product. It will also reduce healthcare costs.

Some questions may be legitimately raised, however:

How reliable are these new molecular tests? How are these tests performed? Are the tests sufficiently sensitive and specific to justify not prescribing antenatal RhIg, with possible jeopardy to a later pregnancy? 

Current RhIgG guidelines in the United States

RhIgG was introduced approximately four decades ago, when the incidence of stillborn babies from Rh negative mothers was high. Treatment options for the baby after birth were slim, and the mortality rate was high.1 The administration of RhIg is now well established, and physicians may be skeptical about switching to a different protocol. 

Currently in the United States, Rh negative mothers receive one antenatal intramuscular dose of 300ug RhIg (1500 UI) at 28 weeks, and one dose up to 72h post-delivery if indicated. This practice decreases the risk of the sensitization of Rh negative mothers to produce anti-D when pregnant with an Rh positive fetus from 16% to only 0.14%.1 The risk of sensitization is lower, 1% to 2%, if an Rh negative mother carries an ABO incompatible fetus, which carries an overall risk for sensitization of 13.2%.1

 RhIg is manufactured by sensitizing Rh negative donors with Rh positive blood and fractionating the plasma via a modified cold ethanol process. This product is still considered biological, and the risk of a transmission of infectious disease cannot be totally eliminated.1,2 Many Rh negative expectant mothers have been refusing the injection. But the information gathered by this type of test would benefit expectant mothers and their physicians.

Molecular testing to determine the fetus’s Rh status (RHD gene) 

First trimester molecular testing using maternal peripheral blood is accomplished by analysis of circulating cell free DNA (ccffDNA) in maternal plasma. EDTA samples are collected and centrifuged, the plasma is separated, and fetal DNA is extracted. Studies have shown that maternal plasma carries an approximately 10% concentration of circulating fetal DNA.3

In 2005, a study was performed by testing the cell-free plasma DNA (ccff DNA) of 98 RHD negative mothers for the presence of exons 4, 5, and 10 of RHD (D gene). This was done by confirming the presence of fetal DNA by detecting the marker SRY (the presence of Y chromosome in maternal blood) and biallelic insertion/deletion polymorphisms in the maternal plasma and buffy coat. The molecular RHD test results obtained were then compared with serologic testing performed on the cord blood of the respective babies after delivery, with a correct result in 94% of the samples tested.2 

In the same year, a study performed in the Netherlands used a noninvasive PCR test to screen the plasma of 2,415 Rh negative pregnant patients, with gestational age of 28 to 30 weeks. The goal was to identify mothers carrying an Rh negative fetus and not administer an antenatal RhIG to them. This test found 1,465 (61.59%) of mothers to be carrying an Rh positive fetus, and 915 (37.88%) were found to be carrying an Rh negative fetus. Antenatal RhIg was then administered only to the mothers who had an Rh positive result through the screening test. 

The PCR results of the fetus Rh status were then compared with cord blood serology after the babies were born. The cord blood serology results were obtained by sending a questionnaire to the participants in the study; 55% were compliant in responding to the questionnaire. Discrepant results between the PCR test at 28-30 weeks of gestational samples and the cord blood serology were found in 21 of 1,257 candidates. The 21 discrepant results were further investigated by also testing the mothers for the presence of RHD variant type or weak D, giving an overall agreement between the molecular testing and the cord serology of 99.4%.6

In 2011, the results of a study performed in London were published. Cell-free fetal DNA (ccff DNA) from 591 maternal samples of Rh negative women of 11-13 weeks gestational age was tested for the presence of exons 5 and 7 of the RHD gene to determine the RHD genotype. In this study, 85.7% of the samples had a conclusive result for the fetus D genotype, with 14.3% yielding an inconclusive profile. The test had a predictive value of 100% if the fetus was found to be Rh positive; the predictive value was 96.5% if the fetus was found to the Rh negative.3

In 2012, a new study was conducted not only to evaluate the accuracy of determining the RHD status of a fetus using ccff DNA, but the gender as well. The study enrolled 120 Rh negative mothers, with samples drawn in three different intervals of maternal gestational age: 120 samples at 12.4 weeks, 118 samples at 17.6 weeks, and 113 samples at 28.7 weeks. The RHD determination was found to be accurate in all the intervals: 99.1% in the first trimester, 99.1% in the second trimester, and 98.1% in the third trimester. However for gender determination, the accuracy increased from 99.1% in the first and second trimester to 100% in the third trimester.5 

A commercial laboratory test has been available since 2012. The laboratory utilizes ccff DNA to determine the RHD status (D genotype) of a fetus as early as 10 weeks of maternal gestational age. The test is based on identifying fetus markers on maternal plasma and triggers three RHD exon targets on chromosome 1, as well as other markers that increase the sensitivity of the test and assure that the DNA extracted is from the fetus and not maternal contamination. If the fetus is found to be Rh negative due to absence of or insufficient fetus markers on maternal plasma, a second sample is retested to confirm that the fetus Rh status is not due to maternal interference.7

Non-invasive molecular tests offer promising advances and valuable information on the fetus Rh status of Rh negative mothers early in their pregnancy. This information especially offers an advantage for multigravida patients who have been previously sensitized with anti-D. If a previously sensitized mother is known to be carrying an Rh positive fetus early in a pregnancy, she can be more carefully monitored. An antibody titer can be performed early, possibly minimizing problems later in the pregnancy. Recommendations and changes in the current RhIg administration may require further study, and education of healthcare professional and patients.

Cristine Fior Clemente dos Santos, PhD, MLS (ASCP)CM SBB, is an assistant professor in the School of Allied Health and Communicative Disorders, Northern Illinois University.


  1. Bowman J. Thirty-five years of Rh prophylaxis. Transfusion. 2003;43(12):1661-1666.
  2. Zhou L, Thorson JA, Nugent C, Davenport R D, Butch SH, Judd WJ. Noninvasive prenatal RHD genotyping by real-time polymerase chain reaction using plasma from D-negative pregnant women. American Journal of Obstetrics and Gynecology. 2005;193(6):1966-1971.
  3. Akokelar R, Finning K, Kuppusamy R, et al. Fetal RHD genotyping at 11-13 weeks of gestation. Fetal Diagnosis and Therapy. 2011;29(4):301-306.
  4. Bombard A T, Akokelar R, Farkas D H, et al. Fetal RHD genotype detection from circulating cell-free fetal DNA in maternal plasma in non-sensitized RhD negative women. Prenatal Diagnosis. 2011;31(8):802-808.
  5. Moise KJ, Boring NH, Simpson LL, et al. Circulating cell-free fetal DNA for the detection of RHD status and sex using reflex fetal identifiers. Prenatal Diagnosis. 2013;33(1):95-101.
  6. Van der Schoot E, Soussan A A , Bonsel GJ, et al. Non invasive screening for fetal RHD-genotype in ALL D-Negative women is reliable and cost-effective. Blood. 2005;106: Abstract 556. 
  7. Sensigene Fetal RHD Genotyping. Accessed October 1, 2014.