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Keywords:

  • fetal DNA;
  • maternal plasma;
  • SAFE;
  • targeted Rh-prophylaxis

Abstract

  1. Top of page
  2. Abstract
  3. Management of pregnancies complicated by anti-Rh immunization
  4. Blood sampling and transportation
  5. Cff DNA extraction methods
  6. Prenatal RhD testing in anti-D alloimmunized pregnancies
  7. Prenatal C, c and E testing in anti-C, c and E alloimmunized pregnancies
  8. Fetal-specific controls
  9. Targeted administration of anti-D immunoglobulin
  10. Final political statement
  11. Acknowledgements
  12. Disclosures
  13. References

Background  Prenatal rhesus testing is currently not well standardized. The clinical guidelines and technologies differ between countries and regions.

Aims  To facilitate implementation of non-invasive prenatal diagnosis of the foetal Rh type (NIPD of Rh) from cell free foetal DNA in maternal plasma. To propel the implementation of targeted administration of anti-D immunoglobulin only to those women who carry a D-positive foetus.

Materials and Methods  A review of the English literature was performed.

Results  For foetal DNA extraction a variety of detailed protocols have been published which allow the prediction of the foetal Rh phenotype with an accuracy of above 99%. There are manual as well as automated systems on the market. Several thousand D-negative women have been tested in clinical trials for the foetal D status with real-time PCR. Primers and probes have been designed to be applicable in multi-ethnic populations despite the complex genetic background of the Rh blood group system. A control for foetal DNA has been published in some papers in order to provide an in-process control for cases where the foetus is D (C, c or E) negative.

Discussion  The scientific community reached a high level of accuracy which allows NIPD of Rh on a large scale.

Conclusion  NIPD of Rh is now available for all alloimmunized women. In most countries health politicians need to change guidelines now before targeted administration of anti-D immunoglobulin can be offered by obstetricians.


Management of pregnancies complicated by anti-Rh immunization

  1. Top of page
  2. Abstract
  3. Management of pregnancies complicated by anti-Rh immunization
  4. Blood sampling and transportation
  5. Cff DNA extraction methods
  6. Prenatal RhD testing in anti-D alloimmunized pregnancies
  7. Prenatal C, c and E testing in anti-C, c and E alloimmunized pregnancies
  8. Fetal-specific controls
  9. Targeted administration of anti-D immunoglobulin
  10. Final political statement
  11. Acknowledgements
  12. Disclosures
  13. References

Children of women immunized by Rhesus (Rh) antigens are at high risk to develop the haemolytic disease of the fetus and newborn (HDFN). In the 1990s, cells cultured from amniotic fluid were used to predict the fetal Rh D, C, c, E or e status in pregnancies complicated by anti-Rh immunization [1]. This specimen was easily available in those days because the degree of fetal haemolysis has usually been estimated by measuring the change of optical density at 450 nm in amniotic fluid in many prenatal care centres [2]. Given the fact that invasive procedures such as amniocentesis always bear a considerable risk of boosting antibodies [3] or even fetal loss [4], fetal anaemia is estimated in most prenatal units by the measurement of the peak systolic velocity of the fetal middle cerebral artery using today’s improved ultrasound technologies. Additionally, the antibody titre is determined and repeatedly tested every 1–4 weeks depending on the week of gestation, changes in the titre and ultrasound findings [5]. Since the beginning of this millennium, we have been offering a service to determine the paternal D zygosity for women with anti-D alloimmunization [6]. Surprisingly, this new tool for decision making was only rarely requested by clinicians, possibly because of the fear that paternity is equivocal.

According to an international survey, about 50% of the centres involved in monitoring and treatment of anti-D in pregnancy determine the fetal genotype with molecular methods from cell-free fetal (cff) DNA sequences extracted form maternal plasma. These centres predict the fetal Rh type according to the presence or absence of characteristic blood group polymorphisms [5]. One aim of this article is to increase this figure up to 100% with a subsequent reduction of costs and psychological pressure on women who carry an Rh D (C, c, or E)-negative fetus. A second aim is to propel the implementation of national guidelines that allow clinicians to restrict the prophylactic antenatal administration of anti-D immunoglobulin only to those women who carry a D-positive fetus.

Blood sampling and transportation

  1. Top of page
  2. Abstract
  3. Management of pregnancies complicated by anti-Rh immunization
  4. Blood sampling and transportation
  5. Cff DNA extraction methods
  6. Prenatal RhD testing in anti-D alloimmunized pregnancies
  7. Prenatal C, c and E testing in anti-C, c and E alloimmunized pregnancies
  8. Fetal-specific controls
  9. Targeted administration of anti-D immunoglobulin
  10. Final political statement
  11. Acknowledgements
  12. Disclosures
  13. References

Time of blood sampling and transport conditions are crucial for accurate fetal genotyping. The quantity of cff DNA in maternal plasma is not only at the limit of detection at the beginning of the pregnancy and increases during gestation [7] but also significantly fluctuates when blood is taken on different days within short intervals [8]. To prevent false-negative results, I recommend performing the first test not before week 9 of gestation. If the first test reveals an Rh-negative result, the result should be confirmed from a second sample which has been taken not before week 12 of gestation [5]. After sample collection, cff DNA concentration in maternal blood is stable for at least 24 hours [9].

In our large-scale validation trial, the noninvasive prenatal diagnosis of the fetal RhD type (NIPD of RhD) failed in the first test in one of 662 (0·15%) pregnancies with a D-positive fetus and only was positive in a repeat test [7]. Detailed investigation of the transport condition revealed that transport at room temperature in that case lasted 6 days, resulting in a maternal DNA concentration high above average. Similarly, Finning et al. [10] observed in samples older than 14 days an increase of false-negative and inconclusive results. Based on these observations, I suggest separating plasma from cells within 2–3 days after phlebotomy as a reasonable compromise between laboratory and logistic requirements. If the separation is delayed, the amount of maternal DNA increases dramatically because of the release of DNA from maternal leucocytes.

Cff DNA extraction methods

  1. Top of page
  2. Abstract
  3. Management of pregnancies complicated by anti-Rh immunization
  4. Blood sampling and transportation
  5. Cff DNA extraction methods
  6. Prenatal RhD testing in anti-D alloimmunized pregnancies
  7. Prenatal C, c and E testing in anti-C, c and E alloimmunized pregnancies
  8. Fetal-specific controls
  9. Targeted administration of anti-D immunoglobulin
  10. Final political statement
  11. Acknowledgements
  12. Disclosures
  13. References

For fetal DNA extraction, in principle, any DNA extraction method can be used; however, because most methods have been optimized for genomic DNA from leucocytes, every new method has to be validated. We published a manual protocol, a slight modification of the original protocol used for the extraction of viral DNA and RNA with the QIAamp DSP Virus kit (QIAGEN, Hilden, Germany) [11], which seemed to be very promising in an international workshop [12] and which can be used in any study as a reference method. In a large-scale trial on this nucleic acid extraction method combined with a real-time PCR specific for RHD, sensitivity and specificity turned out to be as high as 99·7 and 99·2 per cent, respectively [7]. Several robotic systems are available to perform automated cff DNA extraction from low to high throughput. Our centre was involved in the evaluation of some robotic systems [11–13] and decided to evaluate the Chemagen Magnetic Separation Module 1 (Chemagen, Baesweiler, Germany) in a large-scale trial [7]. In this study, sensitivity and specificity of prenatal RHD genotyping were 99·8 per cent and 98·1 per cent, respectively. In the meantime, new robotic systems have been developed. To my knowledge, however, none of these has obtained CE approval for NIPD of RhD or other fetal markers as intended use so far.

Prenatal RhD testing in anti-D alloimmunized pregnancies

  1. Top of page
  2. Abstract
  3. Management of pregnancies complicated by anti-Rh immunization
  4. Blood sampling and transportation
  5. Cff DNA extraction methods
  6. Prenatal RhD testing in anti-D alloimmunized pregnancies
  7. Prenatal C, c and E testing in anti-C, c and E alloimmunized pregnancies
  8. Fetal-specific controls
  9. Targeted administration of anti-D immunoglobulin
  10. Final political statement
  11. Acknowledgements
  12. Disclosures
  13. References

In many countries, a service for NIPD of RhD is offered to anti-D alloimmunized pregnant women [5]. Target sequences have to be amplified, and amplification products can be analysed with either real-time PCR or matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF) [2,7,10,14–20]. Both technologies are patented. In most large-scale studies (n > 1000) revealing a > 99 per cent accuracy, real-time PCR has been applied and other technologies need validation in large cohorts first before they can be recommended for diagnostic laboratories [2]. To ensure the accurate detection of the most frequent D variants, at least two exons of the RHD gene should be analysed. An assay reacting negative with the RHD pseudogene (RHDψ) should be included if the patient population does not consist exclusively of Whites or Asians [2]. Primers specific for RHD exon 10 may lead to many false positive results in Asians and Blacks because of the relatively high frequency of RHD-CE-D hybrid alleles in these ethnic groups [21].

Prenatal C, c and E testing in anti-C, c and E alloimmunized pregnancies

  1. Top of page
  2. Abstract
  3. Management of pregnancies complicated by anti-Rh immunization
  4. Blood sampling and transportation
  5. Cff DNA extraction methods
  6. Prenatal RhD testing in anti-D alloimmunized pregnancies
  7. Prenatal C, c and E testing in anti-C, c and E alloimmunized pregnancies
  8. Fetal-specific controls
  9. Targeted administration of anti-D immunoglobulin
  10. Final political statement
  11. Acknowledgements
  12. Disclosures
  13. References

In addition to anti-D, anti-c, anti-E and anti-C alloantibodies are also known to lead to severe HDFN occasionally (Table 1) [22]. Therefore, we developed a real-time PCR protocol for the noninvasive determination of the fetal rhesus C, c and E status [23]. Because of the fragmentation of fetal DNA in maternal plasma, shorter amplicons have to be amplified to obtain a sensitivity and specificity of more than 99 per cent when samples are drawn during the early second trimester [24,25].

Table 1.   NIPD Rh cases analysed from August 2008 until March 2010 at the University Medical Centre Göttingen, Germany
Antibody specificityn
anti-D24
anti-D plus C10
anti-D plus E4
anti-c2
anti-c plus E1
anti-E2
No alloimmunization6
Total49

Fetal-specific controls

  1. Top of page
  2. Abstract
  3. Management of pregnancies complicated by anti-Rh immunization
  4. Blood sampling and transportation
  5. Cff DNA extraction methods
  6. Prenatal RhD testing in anti-D alloimmunized pregnancies
  7. Prenatal C, c and E testing in anti-C, c and E alloimmunized pregnancies
  8. Fetal-specific controls
  9. Targeted administration of anti-D immunoglobulin
  10. Final political statement
  11. Acknowledgements
  12. Disclosures
  13. References

Ideally, NIPD of Rh assays should include a control to detect fetal DNA in all samples, especially in those samples obtained from women with a D (C, c, E)-negative fetus [2]. Recently, test methods using methylation-sensitive restriction enzymes have been proposed to confirm the presence of fetal DNA irrespective of the target sequence [26,27]. As an alternative, multiple polymorphisms can be investigated and compared with maternal polymorphisms [28,29]. Hyland and colleagues amplified SRY-specific sequences, which are located on the Y-chromosome, in 140 pregnant women [19] and performed a supplemental test in all D-negative female fetuses (n = 16), detecting the hypermethylated (fetal-specific) promoter of the tumour suppressor gene RASSF1A. In all these 16 cases, they confirmed the presence of fetal DNA and, thus, avoided false-negative results.

Targeted administration of anti-D immunoglobulin

  1. Top of page
  2. Abstract
  3. Management of pregnancies complicated by anti-Rh immunization
  4. Blood sampling and transportation
  5. Cff DNA extraction methods
  6. Prenatal RhD testing in anti-D alloimmunized pregnancies
  7. Prenatal C, c and E testing in anti-C, c and E alloimmunized pregnancies
  8. Fetal-specific controls
  9. Targeted administration of anti-D immunoglobulin
  10. Final political statement
  11. Acknowledgements
  12. Disclosures
  13. References

Routine Rh prophylaxis is nowadays an established procedure in all D-negative women. However, in about 40 per cent of all pregnancies in D-negative women, the fetus is D-negative and Rh prophylaxis is not indicated. That means these women are unnecessarily exposed to a potentially infectious human immunoglobulin derived from plasma of volunteer donors who participated in an anti-D immunization programme.

Targeted Rh prophylaxis based on NIPD of RhD is offered as an option to pregnant women in some countries (e.g. Switzerland), and it is about to become mandatory in others (e.g. the Netherlands). As an RHD-specific international reference material has been established and regular workshops have been implemented by members of the ISBT (International Society of Blood Transfusion), appropriate external quality control is already available for such a new large-scale RhD screening [30].

In cost-efficiency studies, the number of false-negative cases per year and population is the most emphasized figure because each HDFN case that would have been avoided by the current general antenatal Rh-prophylaxis programme is associated with high costs for the health care system [31]. As false-negative NIPD of RhD results has been shown to be rare events in recent studies, appropriate trials have to include a large number of cases (>1000) and are, thus, expensive. The observation that D-variants in newborns are detected more reliably with NIPD of RhD than with serology underlines that the assessment of the cost efficiency is rather complex [7]. Another advantage of the screening of D-negative pregnant women with NIPD of RhD is that inaccurate determination of the maternal D-status with serology owing to weak D phenotype will sometimes lead to wrong decisions. In 1998, we published the first RHD gene variation in the weak D phenotype [32] and there is an agreement in most countries today that the majority of pregnant women with weak D do not require Rh prophylaxis because they are not subject to anti-D immunization. A molecular investigation of leucocyte DNA in women who are apparently D-negative with serology and identified as carriers of a variant RHD gene by NIPD of RhD will allow more accurate decision making on provision of Rh prophylaxis [33–35].

Final political statement

  1. Top of page
  2. Abstract
  3. Management of pregnancies complicated by anti-Rh immunization
  4. Blood sampling and transportation
  5. Cff DNA extraction methods
  6. Prenatal RhD testing in anti-D alloimmunized pregnancies
  7. Prenatal C, c and E testing in anti-C, c and E alloimmunized pregnancies
  8. Fetal-specific controls
  9. Targeted administration of anti-D immunoglobulin
  10. Final political statement
  11. Acknowledgements
  12. Disclosures
  13. References

It is very unusual in medical sciences to administer any kind of therapy to a patient who does not need it, although a diagnostic tool such as NIPD of RhD is available to identify patients at risk. To overcome this dilemma, our society has to reconcile the conflicting interests of two groups:

On the one hand, there is the interest of patent holders whose intellectual property rights are protected by law and they take their time to develop a CE-approved NIPD of RhD, which includes a control for fetal DNA for the future. This interest is supported by health politicians who fear the increase in costs through a new test-based algorithm in pregnancy and the manufacturers of anti-D immunoglobulin who fear decreasing sales of their valuable product. On the other hand, there is the interest of several thousand D-negative women who are pregnant now, read about NIPD of RhD for alloimmunized women in the media and notice that appropriate high throughput tests are already available in clinical trials. Their interest unfortunately is not supported by a comparable lobby.

In my opinion, the D-negative pregnant women should be moved into the centre stage and should not be withhold from feasible state-of-the-art diagnostic tools. Politicians should pave the way for cost-effective, standardized, high-throughput NIPD of RhD screening assays (including a control for fetal DNA) by convincing patent holders to license their technology to others at a reasonable price.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Management of pregnancies complicated by anti-Rh immunization
  4. Blood sampling and transportation
  5. Cff DNA extraction methods
  6. Prenatal RhD testing in anti-D alloimmunized pregnancies
  7. Prenatal C, c and E testing in anti-C, c and E alloimmunized pregnancies
  8. Fetal-specific controls
  9. Targeted administration of anti-D immunoglobulin
  10. Final political statement
  11. Acknowledgements
  12. Disclosures
  13. References

The author is grateful to Dr. Alexander Strate who carefully read and improved the style of the manuscript. He acknowledges the skills and support of the technical and medical staff of the department of Transfusion Medicine in Göttingen, the cooperation and support of clinical and research group colleagues around the globe who contributed to the implementation of prenatal Rh testing in clinical settings.

References

  1. Top of page
  2. Abstract
  3. Management of pregnancies complicated by anti-Rh immunization
  4. Blood sampling and transportation
  5. Cff DNA extraction methods
  6. Prenatal RhD testing in anti-D alloimmunized pregnancies
  7. Prenatal C, c and E testing in anti-C, c and E alloimmunized pregnancies
  8. Fetal-specific controls
  9. Targeted administration of anti-D immunoglobulin
  10. Final political statement
  11. Acknowledgements
  12. Disclosures
  13. References