Empowering Fertility - Rh Factor (part 2): New Strategies to Manage Rh(-) Pregnancies

Rh Factor (part 2): New Strategies to Manage Rh(-) Pregnancies

By Paul Bergh, MD

A great deal of heartache has been averted and countless young lives have been saved since it was discovered that the timely administration of anti-Rh immunoglobulin (RhIg / Rhogam) to Rh(-) women, could prevent hemolytic disease of the fetus and newborn (HDFN).  Prior to the introduction of this practice in the 1960’s, 14% of Rh(-) pregnancies were affected by this terrible disease. Of these affected pregnancies, 50% resulted in stillbirths.   Of the other 50% of children that survived to birth, half would either die in the neonatal period or develop cerebral injury.

RhIg is a human blood product that is procured from paid serum donors. Thus, its administration carries a small, but real risk of transmission of a disease-causing virus or prion.   Currently, there are four RhIg products available in the United States and they all undergo a micropore filtration to eliminate viral transmission.  While there was an outbreak of hepatitis C related to tainted RhIg in Ireland in the 1970’s, there has never been a reported case of viral infection related to RhIg in the United States.  The micropore filtration is effective in eliminating viral risk. However, it is not known if it can effectively clear plasma of prions which can cause spongiform encephalopathies including “mad cow disease” and Creutzfeldt-Jakob Disease.

It is always desirable, when possible, to avoid the  use of human blood products. One strategy is the use of completely synthetic RhIg.   A new recombinant RhIg is under development and may ultimately replace the current human product.  Rozrolimupab is a recombinant mixture of 25 fully human RhD-specific monoclonal antibodies. It is a member of a new class of recombinant human antibody mixtures that has been used without any serious adverse side effects in both phase I and phase II clinical trials.  Compared to human plasma derived RhIg, Rozrolimupab use results in a similar dose dependent rate of Rh(+) red cell clearance from the circulation.

A second strategy to avoid the administration of human blood products is to identify those pregnant Rh(-) women  who are in fact carrying a Rh(-) fetus and thus do not require RhIg.  Depending on the population and ethnicity, up to  40% of Rh(-) pregnant women unknowingly carry a Rh(-) fetus and thus unnecessarily receive this human blood product – RhIg.   We now have to ability to determine as early as 8wks of pregnancy, with a high degree of confidence, the Rh status of a fetus.

In 1997, it was first reported that cell-free fetal DNA could be detected in the maternal circulation. Of the cell-free DNA found in the plasma of pregnant women, 3% to 6% is of fetal origin.  Unlike fetal cells that have been known to persist for years, the fetal cell-free DNA is cleared from the maternal circulation within hours after delivery.  The very first assay developed to detect cell-free DNA was based on the presence of the Y chromosome.  Shortly afterward, in 1999, the detection of fetal Rh status in Rh(-) women was reported.

It goes without saying, that fetal testing is only necessary if the father is Rh(+). The first step in this new paradigm is to determine the paternal Rh zygosity or genotype; i.e. if the father contains one (heterozygous) or two copies (homozygous) of the RhD antigen gene.  If he is homozygous for RhD, then there is no reason to test for the fetal status as the fetus will always be Rh(+), and thus the mother will require treatment with RhIg.  If the father is heterozygous for RhD, then the fetus has a 50% chance of being Rh(-) in which case the mother would not require treatment with RhIg.  Serologic testing methods are typically sufficient to determine the genotype of most blood alleles because both a major and minor allele is expressed.   However, for RhD, there is no minor alleles, so serologic testing, especially in low prevalence populations can predict heterozygosity with only a 50% confidence.  Fortunately, there are now labs that offer extremely accurate tests for paternal Rh zygosity using quantitative polymerase chain reaction.  The typical turn-around for this test is 2-7 days.

For those Rh(-) pregnant women with a heterozygous Rh(+) mate, free-fetal DNA in maternal blood can be analyzed as early as 38 days post conception.  Due to apoptosis of the placental cytotrophoblastic cells, fetal DNA will compromise up to 6% of the total circulating DNA pool.  Given its short half-life, there should not be any residual DNA from the previous pregnancy. There is has been a rapid accumulation of experience in managing patients with this new technology such that, cell-free fetal DNA testing to determine fetal RhD status in women at risk for HDNF, has now become the standard practice in many European countries.  In the event of a result suggesting a Rh(-) fetus, the presence of a Y chromosome for a male fetus or the presence of informative SNPs are used to confirm the presence of fetal DNA.

Although several European countries have replaced the routine administration of RhIg with paternal zygosity typing and cell-free DNA testing, there is considerable controversy regarding the large scale adaptation of this practice.  It is now available in the United States but has not been widely implemented.  The international community has made great strides in standardizing this testing and has demonstrated it to be very accurate.  However, there is still a very small possibility of a false negative result in which case, the  routine cord blood phenotyping at birth will correctly identify a RhD positive fetus.  In these cases, postnatal RhIg can be administered to the mother within the 72-hour window.  There still exists a small risk, estimated to be 1 out of 86,000,  in this situation of sensitization in these women that could affect a future pregnancy.

The cost of non-invasive testing (~$450) is similar to the cost of the route  of  RhIg (~$400) administration.  However, since half of women with heterozygous Rh(+) partners will still require RhIg in addition to the non-invasive testing, the cost profile becomes less favorable.  It is  estimated that if the cost per non-invasive evaluation dropped by $140, then the non-invasive testing would become more cost-effective.   Many would argue that the cost is irrelevant if it prevents one from practicing ethically; i.e.  exposing pregnant women to a blood product that is of no benefit.  While the practice may not be widespread in the United States, shouldn’t providers be discussing the risks and benefits of this option with  their Rh(-) patients?  Shouldn’t patients  at least have this information and the option to have paternal  zygosity  and cell-free DNA testing?

Labs for paternal Rh zygosity & cell free DNA testing:




Lo, Y. M., Corbetta, N., Chamberlain, P. F., Rai, V., Sargent, I. L., Redman, C. W., and Wainscoat, J. S. Presence of fetal DNA in maternal plasma and serum. Lancet 350(9076), 485-487. 8-16-1997.

Lo, Y. M. Fetal RhD genotyping from maternal plasma. Ann.Med. 31(5), 308-312. 1999.

Pirelli, K. J., Pietz, B. C., Johnson, S. T., Pinder, H. L., and Bellissimo, D. B. Molecular determination of RHD zygosity: predicting risk of hemolytic disease of the fetus and newborn related to anti-D. Prenat.Diagn. 30(12-13), 1207-1212. 2010.

Moise, K. J., Jr. and Argoti, P. S. Management and prevention of red cell alloimmunization in pregnancy: a systematic review. Obstet.Gynecol. 120(5), 1132-1139. 2012.

Mackenzie, I. Z., Roseman, F., Findlay, J., Thompson, K., and McPherson, K. Clinical validation of routine antenatal anti-D prophylaxis questions the modelling predictions adopted by NICE for Rhesus D sensitisation rates: results of a longitudinal study. Eur J Obstet Gynecol Reprod Biol 139(1), 38-42. 2008.

Clausen, F. B., Damkjaer, M. B., and Dziegiel, M. H. Noninvasive fetal RhD genotyping. Transfus.Apher.Sci. 50(2), 154-162. 2014.

Hyland, C. A., Gardener, G. J., O’Brien, H., Millard, G., Gibbons, K., Tremellen, A., Ochoa-Garay, G., Flower, R. L., and Hyett, J. A. Strategy for managing maternal variant RHD alleles in Rhesus D negative obstetric populations during fetal RHD genotyping. Prenat.Diagn. 34(1), 56-62. 2014.

Robak, T., Windyga, J., Trelinski, J., von Depka, Prondzinski M., Giagounidis, A., Doyen, C., Janssens, A., Alvarez-Roman, M. T., Jarque, I., Loscertales, J., Rus, G. P., Hellmann, A., Jedrzejczak, W. W., Kuliczkowski, K., Golubovic, L. M., Celeketic, D., Cucuianu, A., Gheorghita, E., Lazaroiu, M., Shpilberg, O., Attias, D., Karyagina, E., Svetlana, K., Vilchevska, K., Cooper, N., Talks, K., Prabhu, M., Sripada, P., Bharadwaj, T. P., Naested, H., Skartved, N. J., Frandsen, T. P., Flensburg, M. F., Andersen, P. S., and Petersen, J. Rozrolimupab, a mixture of 25 recombinant human monoclonal RhD antibodies, in the treatment of primary immune thrombocytopenia. Blood 120(18), 3670-3676. 11-1-2012.

Zhu, Y. J., Zheng, Y. R., Li, L., Zhou, H., Liao, X., Guo, J. X., and Yi, P. Diagnostic accuracy of non-invasive fetal RhD genotyping using cell-free fetal DNA: a meta analysis. J Matern.Fetal Neonatal Med. 27(18), 1839-1844. 2014.

Hawk, A. F., Chang, E. Y., Shields, S. M., and Simpson, K. N. Costs and clinical outcomes of noninvasive fetal RhD typing for targeted prophylaxis. Obstet.Gynecol. 122(3), 579-585. 2013

Duplantie, J., Martinez, Gonzales O., Bois, A., Nshimyumukiza, L., Gekas, J., Bujold, E., Morin, V., Vallee, M., Giguere, Y., Gagne, C., Rousseau, F., and Reinharz, D. Cost-effectiveness of the management of rh-negative pregnant women. J.Obstet.Gynaecol.Can. 35(8), 730-740. 2013

Kent, J., Farrell, A. M., and Soothill, P. Routine administration of Anti-D: the ethical case for offering pregnant women fetal RHD genotyping and a review of policy and practice. BMC.Pregnancy.Childbirth. 14, 87. 2014.

Empowering Fertility: An educational blog for patients & healthcare professionals that empowers individuals to take charge of their fertility. Visit us at http://empoweringfertility.com.

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