Non-invasive fetal Rhesus D blood genotyping

June 14, 2021

Reducing the need for prophylactic anti-D treatment for pregnant women.

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Non-invasive prenatal testing (NIPT) using cell-free fetal DNA in maternal plasma is being used to determine fetal RhD blood group antigen status so that RhD-negative pregnant women can avoid receiving antenatal anti-D if they are carrying a RhD-negative baby.

The Rhesus D factor

The Rhesus (Rh) system is one of the most important blood group systems, particularly in pregnant women, because of the large number of antigens and their role in determining maternal and fetal blood compatibility. There are two Rhesus proteins, RhD and RhCE, found on the surface of red blood cells (RBCs). Of these, the D antigen is considered the most important. Whilst most people have RBCs that express active RhD antigen, around 1 in 6 people, do not and are observed to be RhD-negative. (Zipursky & Paul, 2011; Westhoff, 2007).

The Rhesus D factor in pregnancy

Being RhD-negative per se is not problematic; the issue arises when an RhD-negative woman becomes pregnant with an RhD-positive fetus. If RhD-positive fetal blood is mixed with the RhD-negative blood of the mother, the immune system of the mother will develop antibodies against RhD antigens in an event called 'sensitisation'. As a consequence, should the mother fall pregnant with another RhD-positive fetus, the now-present anti-RhD antibodies can cross the placenta and enter fetal circulation. There, they will encounter and bind to RhD-positive fetal RBCs and mark them for elimination. Once this occurs, fetal RBCs will undergo haemolysis; this is called Haemolytic Disease of the Fetus and Newborn (HDFN). Mild rates of haemolysis may be tolerated by the fetus and resolve after birth but severe cases will likely be fatal (Dean, 2005).

Treatment of RhD-negative women in pregnancy

To prevent HDFN, routine antenatal care in many countries consists of the administration of anti-RhD immunoglobulin (anti-D) to all RhD-negative pregnant women. Currently, anti-D prophylaxis is given to all RhD-negative pregnant women, irrespective of fetal RhD status. When no maternal-fetal incompatibility exists, as in 30-40% of pregnancies for RhD-negative mothers, this approach leads to unnecessary costs to national healthcare systems and increased risk of side-effects such as anaphylaxis associated with anti-D therapy (O'Brien et al., 2009). It is therefore beneficial to know the RhD phenotype of a fetus.

Rhesus D genotypes

Understanding the genetics of Rhesus D has meant that RhD phenotypes can be predicted with a high degree of accuracy by real-time PCR which determines the presence or absence of RHD-specific sequences. However for an rtPCR assay to be suitable for testing African population or any populations containing a substantial proportion of people with African ethnicity multiplex PCR test that includes primers to detect the presence of RhD-Psi is required.

Predicting RhD phenotype from fetal DNA

The first methods for fetal RHD genotyping used cell free fetal DNA isolated from fetal tissue by amniocentesis or chorionic villus sampling. Both techniques are associated with an increased risk of miscarriage and/or transplacental haemorrhage and the latter event could also result in a pre-natal sensitisation event, which would further increase the risk of severe HDFN (Urbaniak, 1998). The lack of safe, non-invasive methods to assess the fetal RhD status has meant that standard recommendations have been to give all RhD-negative pregnant women anti-D immunoglobulin.

Non-invasive fetal RhD blood group genotyping

The discovery of fetal cell-free DNA in maternal blood from as early as the 7th week of gestation has offered an alternative non-invasive approach to fetal RHD genotyping.  During early pregnancy, around 3% of the total cell-free DNA in maternal blood is of fetal origin, with this value increasing to 6-7% by late pregnancy (Lo et al., 1998). As the RhD status is genetically determined, cell-free DNA presents itself as a readily available source of fetal DNA that can be sampled non-invasively, bypassing the risks of amniocentesis or chorionic villus sampling (Cardo et al., 2010). Studies have shown that cell-free DNA in maternal blood can be genotyped using real time PCR to determine the RhD status of the fetus, guiding anti-D prophylaxis treatment with up to 100% sensitivity and over 90% specificity.

Implementation of NIPT for fetal RHD genotyping

From 2016, NICE has recommended non-invasive prenatal testing (NIPT) for fetal RHD genotyping for pregnant women who are RhD negative as a cost-effective option to guide antenatal prophylaxis with anti-D within the UK (For further information see https://www.nice.org.uk/ guidance/dg25). National fetal RhD testing programmes for all D-negative pregnant women have subsequently been introduced successfully in other countries (Denmark, Netherlands, France, Finland and parts of Sweden) and is scheduled to become part of reimbursement programmes within the public health system in Germany and Australia (Gordon et al., 2017; Haimila et al., 2017; Neovius et al., 2016; Szczepura et al., 2011).

The implementation of this genotyping test has allowed anti-D Ig to be used in a more precise and indicated way not only improving care for RhD-negative women by reducing the number of unnecessary treatments when the fetus and mother are not RhD-incompatible but reducing the cost to national healthcare systems by conserving the use of an expensive product that is often in short supply.

Direct from plasma fetal RHD genotyping

Despite the significant advantages of NIPT for fetal RHD genotyping, the low abundance of cffDNA in maternal blood presents a challenge. Most methods require the purification of cffDNA prior to rtPCR so its extraction is a critical step in ensuring enough cffDNA for reliable detection. In order to make the process more reliable and efficient, Nonacus developed the first, direct from plasma, fetal RhD genotyping kit, Cell3™ Direct. With no cffDNA extraction required, Cell3 Direct not only improves reliability of results, it takes under 3 hours from sample receipt to result, reducing technician time and processing costs.

References

  1. Cardo et al. Non-invasive fetal RHD genotyping in the first trimester of pregnancy. Clin Chem Lab Med. 2010 Aug; 48(8):1121-1126.
  2. Chitty et al. Diagnostic accuracy of routine antenatal determination of fetal RHD status across gestation: population based cohort study. BMJ. 2014 Sep 4; 349:g5243.
  3. Dean L. Blood Groups and Red Cell Antigens [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2005.
  4. Gordon et al. Noninvasive fetal RHD genotyping of RhD negative pregnant women for targeted anti-D therapy in Australia: A cost-effectiveness analysis. Prenat Diagn. 2017 Dec; 37(12):1245-1253.
  5. Haimila et al. Targeted antenatal anti-D prophylaxis program for RhD-negative pregnant women - outcome of the first two years of a national program in Finland. Acta Obstet Gynecol Scand. 2017 Oct; 96(10):1228-1233.
  6. Lo et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet. 1998 Apr; 62(4):768-75.
  7. Manfroi et al. Prenatal non-invasive foetal RHD genotyping: diagnostic accuracy of a test as a guide for appropriate administration of antenatal anti-D immunoprophylaxis. Blood Transfus. 2018 Nov; 16(6):514-524.
  8. Mitra et al. Blood groups systems. Indian J Anaesth. 2014 Sep; 58(5):524-8.
  9. Neovius et al. Cost-effectiveness of first trimester non-invasive fetal RHD screening for targeted antenatal anti-D prophylaxis in RhD-negative pregnant women: a model-based analysis. BJOG. 2016 Jul; 123(8):1337-46.
  10. O'Brien et al. Reaction to anti-D immunoglobulin - can we manage it? Obstet Med. 2009 Mar; 2(1):38-9.
  11. Szczepura et al. A new fetal RHD genotyping test: Costs and benefits of mass testing to target antenatal anti-D prophylaxis in England and Wales. BMC Pregnancy Childbirth. 2011; 11(5).
  12. Singleton et al. The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in africans with the Rh D-negative blood group phenotype. Blood. 2000 Jan 1; 95(1):12-8.
  13. Urbaniak SJ. The scientific basis of antenatal prophylaxis. Br J Obstet Gynaecol. 1998 Nov; 105 Suppl 18:11-8.
  14. Westhoff CM. The structure and function of the Rh antigen complex. Semin Hematol. 2007 Jan;44(1):42-50.
  15. Zipursky A, Paul VK. The global burden of Rh disease. Arch Dis Child Fetal Neonatal Ed. 2011 Mar; 96(2):F84-5.
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