The battle against Rh disease is about to get a lot easier

April 15, 2006

With the help of PCR analysis, cell-free fetal RhD antigen can be detected in maternal serum, offering the promise of detecting mother/child incompatibilities long before any clinical damage can occur. Here's a brief overview of the technology and why it's still not ready for prime time in the U.S.

While modern medicine is sometimes accused of providing band-aid treatments for complex disorders, the history of Rh disease represents one of medicine's genuine success stories. When the erythrocyte's rhesus D antigen was first discovered in the 1950s, about one out of every two Rh-negative women who became sensitized to the antigen lost her baby. But by 1986, with the help of amniocentesis, amniotic fluid analysis, and intrauterine transfusions, the odds of a child surviving such an immunological nightmare had vastly improved, with mortality dropping to 2% to 3%.1

The real victory, however, will come when all these mother/child immunological incompatibilities are detected long before they become nightmares. Middle cerebral artery Doppler is helping physicians achieve that goal, but even more impressive is the technology that allows clinicians to noninvasively determine the presence of fetal Rh antigen in maternal blood.

With the assistance of polymerase chain reaction analysis, noninvasive cell-free fetal RhD genotyping has become a routine part of prenatal care in several European countries. Our purpose here is to briefly describe the technology, discuss its advantages and disadvantages, and answer the question on the minds of many clinicians: Why isn't it part of the prenatal workup in the United States?



Impressive technology

There are two clinical situations in which cell-free fetal DNA testing of an Rh-negative mother holds the potential for being a major breakthrough. John T. Queenan, MD, a well-respected expert in Rh disease, points out that the Rh-negative immunized mother with a heterozygous Rh-positive father has a 50-50 chance of having an Rh-negative fetus.2 If the fetus turns out to be Rh negative, there's no risk of hemolytic disease and testing can be stopped. If, on the other hand, the fetus tests Rh positive, then appropriate diagnostic tests, and therapy, if needed, can proceed.

The second situation in which fetal genotyping holds promise is in the Rh-negative, nonimmunized mother who is a candidate for 28-week Rh immunization (Rh immunoglobulin). By testing the mother for fetal Rh antigen, the clinician can determine if the fetus is Rh positive, which would mean immunoglobulin is indicated. Of course, if the fetus is Rh negative, the clinician "can eliminate the expensive and wasteful practice of administering Rh immune globulin at 28 weeks to those who subsequently deliver Rh-negative babies."1

The test procedure starts with a maternal blood sample, which is then subjected to DNA amplification and gel electrophoresis to pinpoint the presence of the specific DNA sequence for the RhD protein. More specifically, short priming DNA sequences are designed that are complementary to the ends of each of the Rh antigen's single DNA strands. DNA is extracted from the maternal blood sample and heated to "unzip" the double helix chains of DNA, which yields long single chains; then the primers are added to the solution. The mixture is then cooled to allow the primer DNA sequences to bind to the ends of the single DNA strands of the Rh antigen, and reheated in the presence of a polymerase, which triggers the synthesis of additional copies of each strand.

Eventually enough DNA is synthesized-the amplification process. Because the chain reaction has generated enough RhD antigen molecules, a discrete pattern emerges, which can then be displayed graphically to signal the presence of the DNA fingerprint for the antigen.



Appreciating the procedure's shortcomings

Like any diagnostic test, noninvasive cell-free fetal RhD genotyping yields some false-negative and false-positive results. As Diana Bianchi, MD, a prominent researcher in the field, points out, "false-negative cases are mainly due to a lack of fetal DNA in the maternal sample due to early gestation or insensitive methods."2 False negatives pose a greater threat than false positives. In scenario one mentioned above, in which an Rh-negative mother is immunized, a false-negative finding means telling the patient that her unborn doesn't have the antigen when it does, which of course means you run the risk of not treating a fetus with hemolytic anemia. A false-positive test in this setting only results in wasted resources as additional unnecessary procedures are ordered.

In a woman who is not immunized and being considered for Rh-immunoglobulin prophylaxis, false-negative results would deprive her of valuable protection. A false-positive test, on the other hand, has the potential to subject a woman to RhD immunoglobulin when her baby is Rh negative and not likely to be subjected to any threat.

In one study, four out of 197 women who had the blood test performed were falsely negative. But a repeat test correctly confirmed the fetal genotype.3 In a separate investigation, more than 1,200 maternal blood samples were directly compared to cord blood serology; only seven false negatives were reported.4 A third study of 283 RhD-negative women found no false-negative results-or false positives for that matter.5 In the other two studies just mentioned, there were three confirmed false positives among 654 women and five in the larger trial involving over 1,200 women, which translates into a diagnostic accuracy rate of 99.1%.

Getting a handle on quality control

Unfortunately, while all this research is impressive, applying it to the real world can be daunting. As one report in Clinical Chemistry pointed out, much of the research on PCR analysis of maternal blood has only been done in individual laboratories under very specific conditions.6 Establishing a test protocol that can be used across the country in a variety of commercial labs is quite another issue.

When investigators did an interlaboratory comparison to determine the value of cell-free fetal DNA genotyping in maternal blood, they found considerable variations. Testing 100 blood samples from 20 pregnant women at five different labs, they attempted to compare their ability to detect male gender (by the presence of Y-chromosome DNA strands) in women carrying fetuses of known gender.

Even after establishing a common protocol for preparing the blood samples, doing DNA extraction, and PCR analysis across all five labs, there were still disappointing results. The ability of the labs to identify known fetal gender by correctly detecting the presence of Y-chromosome DNA varied from 31% to 94%.

In this particular study, differences in the labs' DNA extraction procedure seemed to be responsible for the variations. Variations in the amounts of PCR primer, the amount of time between drawing samples and DNA analysis, the type of PCR instrumentation, and reagents are among the many other possible confounding factors that could affect test results in laboratories that have not been subjected to the vigorous quality control safeguards required for the above study.




1. Queenan JT. Noninvasive fetal Rh genotyping. Obstet Gynecol. 2005;106:682-683.

2. Bianchi DW, Avent ND, Costa JM, et al. Noninvasive prenatal diagnosis of fetal Rhesus D. Obstet Gynecol. 2005;106:841-844.

3. Rouillac-Le Sciellour C, Puillandre P, Gillot R, et al. Large-scale pre-diagnosis study of fetal RHD genotyping by PCR on plasma DNA from RhD-negative pregnant women. Mol Diagn. 2004;8:23-31.

4. van der Schoot CE, Ait Soussan A, Dee R, et al. Screening for foetal RhD-genotype by plasma PCR in all D-negative pregnant women is feasible. Vox Sang. 2004;87(suppl):9.

5. Gautier E, Benachi A, Giovangrandi Y, et al. Fetal RhD genotyping by maternal serum analysis: a two-year experience. Am J Obstet Gynecol. 2005;192:666-669.

6. Johnson KL, Dukes KA, Vidaver J, et al. Interlaboratory comparison of fetal male DNA detection from common maternal plasma samples by real-time PCR. Clin Chem. 2004;50:516-521.