|Year : 2016 | Volume
| Issue : 1 | Page : 21-28
Noninvasive fetal RHD genotyping from maternal plasma
Disha S Parchure, Swati S Kulkarni
From the Department of Transfusion Medicine, National Institute of Immunohaematology, Mumbai, Maharashtra, India
|Date of Web Publication||3-Mar-2016|
Swati S Kulkarni
From the Department of Transfusion Medicine, National Institute of Immunohaematology, Mumbai, Maharashtra
Source of Support: None, Conflict of Interest: None
Alloimmunization to antigens of Rh blood group system is of clinical relevance during pregnancy. Despite the use of antenatal anti-D immunoglobulin prophylaxis, some proportion of RhD-negative pregnant women still become immunized. RhD-negative pregnant women with a heterozygous partner can be reassured and managed less intensively if RhD-negative status of the fetus was confirmed. The conventional techniques used for prenatal testing of fetal RhD status are mainly invasive such as chorionic villus sampling, amniocentesis and cordocentesis, and carry risk of transplacental hemorrhage and pregnancy loss. The discovery of cell-free fetal DNA (cffDNA) in maternal plasma in 1997 has opened up new possibilities for noninvasive prenatal diagnosis. With the use of real time polymerase chain reaction technology, circulating fetal DNA has been detected robustly in the plasma of pregnant women, even in the first trimester of pregnancy. Various assays have been developed to confirm the fetal RHD status by targeting the cffDNA using a combination of multiple RHD exons. The commonly used exons are 4, 5, 7, and 10. Common causes leading to discordant results in fetal RHD typing are low fetal DNA concentration due to extraction inefficiency, lack of fetal DNA control, silent maternal RHD alleles, and manual error. False negative results can prove critical in case of alloimmunized pregnancies and hence use of appropriate controls and strict reporting criteria is important to increase the sensitivity of the assay. Owing to the wide genetic variation of the Rh blood group system, the development of fetal genotyping strategies according to ethnic origin of the patients would further increase the sensitivity. Fetal blood group genotyping by noninvasive method is safe, and numerous groups have reported fetal RHD genotyping in D-negative mothers with close to 100% accuracy. Noninvasive fetal RHD typing can be performed in RhD-negative alloimmunized and nonimmunized pregnancies to decide on clinical management and to restrict the anti-D immunoglobulin prophylaxis, respectively. Thus, the introduction of this test for screening all RHD-negative pregnant women is highly desirable.
Keywords: Antenatal prophylaxis, cell-free fetal DNA, maternal plasma, noninvasive fetal RHD genotyping
|How to cite this article:|
Parchure DS, Kulkarni SS. Noninvasive fetal RHD genotyping from maternal plasma. Glob J Transfus Med 2016;1:21-8
| Introduction|| |
Hemolytic disease of fetus and newborn (HDFN) is caused by maternal alloantibodies directed against the fetal red cell surface antigens that the mother lacks. The D antigen of the Rh blood group system is most frequently involved in HDFN. The RhD antigen of the fetus is well-developed by 30–40 days of gestation and hence there is a risk of fetomaternal hemorrhage (FMH) from the 6th week of gestation. The risk of FMH increases as the pregnancy progresses (7% in the first trimester, 16% in the second trimester, and 29% in the third trimester) with greatest risk occurring at birth (15–50%). After the initial sensitization events, a secondary exposure of fetal D antigen leads to the production of maternal immunoglobulin G (IgG) anti-D antibodies that can cross the placenta and bring about destruction of fetal red cells. The clinical severity of HDFN ranges from mild to severe hemolytic anemia leading to jaundice, kernicterus, hydrops fetalis, and intrauterine or neonatal death.
Rh Immune Globulin (RhIG) has been highly successful in reducing the incidence of HDFN since its introduction in 1970s. Initially, prophylaxis was given postnatally to RhD-negative women after birth of RhD-positive fetus and to women having abortion/miscarriage or after invasive procedure. This reduced maternal immunization risk from 16% to 2%. Antenatal prophylaxis given at 28–34 weeks gestation along with postnatal prophylaxis has successfully further reduced the incidence of Rh immunization from 1.8% to 0.1%. This treatment is offered to all RhD-negative pregnant women irrespective of the fetal RhD status. Despite the widespread use of antenatal and postnatal RhIG prophylaxis, RhD alloimmunization is still a significant cause of fetal and neonatal morbidity and mortality. The prophylaxis failure is mainly due to Rh immunization prior to 28 weeks of gestation, late administration, and quality of RhIG used. In India, about 5% of the population is RhD-negative and around 1.7% of RhD-negative women develop anti-D after receiving postnatal prophylaxis. The incidence has further reduced to 1% with the use of antenatal prophylaxis in Mumbai, India.
RhD-negative pregnant women at high risk of HDFN are monitored by serial assessment of maternal antibody levels, Doppler ultrasound of the middle cerebral artery, and intrauterine cord blood sampling. In alloimmunized pregnant women, knowledge of fetal antigen status is useful to tailor pregnancy management. If the fetus inherits the D antigen, then careful monitoring for fetal anemia is required. If the father is heterozygous for D antigen, there is a 50% chance that the fetus does not carry the D antigen and thus has no risk of HDFN. The RhD-negative pregnant women with a heterozygous partner can thus be reassured and managed less intensively if RhD-negative status of the fetus was confirmed early in pregnancy. As 40% of European and around 25% of RhD-negative Indian pregnant women bear an RhD-negative fetus and are not at risk of immunization, knowing the fetal RhD status would enable selective antenatal RhIG administration. The prophylactic RhIG is prepared from pooled plasma of immunized donors and hence it carries the risk associated with human blood products and the possibility of transmission of unknown infections. The supply of RhIG is not unlimited and therefore should only be given when necessary.
Molecular bases of most of the blood group system have been determined and various molecular techniques have been established for blood group genotyping. To assess the risk of HDFN in RhD alloimmunized women, earlier the fetal RhD status was assessed by invasive means such as amniocentesis and chorionic villus sampling. However, these methods have a risk of transplacental hemorrhage and pregnancy loss. Hence, an alternative to these methods was required for prenatal diagnosis and management of RhD-negative pregnancies. Noninvasive analysis of cell-free fetal DNA (cffDNA) is currently recognized as a safe and reliable technique for fetal RhD genotyping and is being offered as a clinical service.
| Cell-Free Fetal Dna|| |
Mandel and Metais in 1948 first observed the presence of circulating nucleic acids in the plasma/serum of healthy donors, pregnant women, and clinical patients. Later, the rise in DNA levels in the blood of patients of systemic lupus erythematosus and rheumatoid arthritis were observed and similarly, high amounts of circulating cell-free tumor DNA were found in the plasma and serum of cancer patients. Thus, cell-free DNA as a source of nucleic acids in plasma and serum was established. This led Lo et al. to look for the presence of cffDNA in plasma and serum of healthy pregnant females and it was first demonstrated in 1997, but the presence of Y chromosome signals in pregnant women carrying a male fetus was the first evidence to prove that this technique could be used for prenatal diagnosis. In 1998, noninvasive fetal RHD gene detection was demonstrated in D-negative pregnant women and cffDNA has now been used clinically for some inherited diseases and genetic disorders.
Various sources of circulating fetal nucleic acids in maternal blood are (1) direct release from circulating fetal cells,, (2) trafficking from other fetal organs or from the amniotic fluid, and (3) from the placenta. Many investigators have tried to isolate fetal cells from maternal blood using various cell separation techniques and have found about one fetal cell per milliliter of maternal blood. Due to the scarcity of fetal cells in maternal circulation, they show no significant correlation for both the volume and turnover of cffDNA. Nucleic acid trafficking is not the main contributor of cffDNA as the presence of circulating fetal nucleic acids has been demonstrated well before the establishment of fetal circulatory system.
Majority of the cffDNA originates from the placenta due to its size, close proximity to maternal circulation, and abundant cellular activity. It reflects placental mosaicism and epigenetic modifications similar to trophoblast tissue. During the continuous development of the placenta, the cffDNA is released mainly from the syncytiotrophoblast cells of the chorionic villi through apoptosis. Increase in the release of cffDNA is observed on damage and destruction of the placental cells in clinical conditions such as hyperemesis gravidarum, preeclampsia, and invasive placenta.
cffDNA is highly fragmented in nature (fragments of length <300 bp) and smaller than maternally derived sequences., It forms 3–6% of total cell-free DNA in maternal circulation and the levels increase as the gestation progresses. Lo et al. reported approximately 25.4 GEq/ml cffDNA at 11–17 weeks and 292.2 GEq/ml at gestational weeks 37–43 with a sharp increase of fetal DNA during the last 8 weeks of pregnancy. The quantitation of cffDNA is subjected to variation as it depends on the gestational age, extraction method used, and sensitivity of the detection technique.
In pregnancies, as a result of in vitro fertilization, cffDNA can be detected in maternal blood as early as 18 days following embryo transfer. It has been detected around 4–5 weeks of gestation, but reliable detection is possible from 10 to 11 weeks., Kinetic studies have shown that unlike fetal cells in maternal blood which can persist for many years, cffDNA is cleared from maternal plasma after delivery with a half-life of approximately 15 min and thus has no contamination from pervious pregnancies.
| Factors Affecting Cell-Free Fetal Dna Detection|| |
cffDNA has been detected both in serum and plasma., Plasma seems to be the choice for extraction of cffDNA as the process of clotting leads to release of excess maternal DNA in serum. Fetal DNA levels can be affected by transport, processing, centrifugation, and storage. EDTA is the choice of anticoagulant as cffDNA remains stable even during prolonged transportation. The amount of maternal DNA increases with delay in processing time due to lysis of maternal cells leading to decreased cffDNA fraction. The maternal plasma separated can be stored for several weeks below −70°C and tested later. cffDNA is stable and maternal DNA release is inhibited if blood is collected in specialized Streck ® cell-free DNA ™ BCT.
Both manual and automated kits are used for cffDNA extraction. Automation leads to higher yields of DNA, reproducibility of results, and decreased manual error. The participating laboratories for fetal RHD detection at the International Society of Blood Transfusion's Fourth International Workshop on molecular blood group genotyping used both manual kits such as Qiagen MinElute Virus kit, Qiagen Blood Mini kit, and automated kits such as Roche MagnaPure, Biomerieux EasyMag ®. Sufficient amount of cffDNA can be obtained by increasing the amount of maternal plasma.
| Fetal Rhd Typing|| |
Molecular basis of Rh blood group system
The Rh blood group system consists of two highly homologous genes: RHD producing the D antigen and RHCE producing C, c, E, and e antigens. Both RHD and RHCE genes have 10 exons each [Figure 1]a, containing 417 aminoacids and have 97% identity. RhD-positive phenotype individual has one or two functional copies of RHD gene. More than 50 antigens have been recognized in Rh blood group system and 251 RHD alleles and 123 RHCE alleles have been described. The frequency of D-negative phenotype is 15–17% in Europeans, 7% in Africans, 5% in Indians, and <1% in East Asians. In Caucasians, the deletion of the RHD gene is primarily responsible for the D-negativity [Figure 1]b. Sixty-six percent of the D-negative Africans carry an inactive RHD gene called the pseudogene [Figure 1]c and 15% carry the RHD-CE-D s hybrid allele [Figure 1]d. The high degree of homology between the RHD and RHCE genes and their opposite orientation favors the production of numerous RHD hybrid alleles. These different RHD alleles give rise to D variants with either absent, weakly, or partially expressed D antigen. The frequency of these D variants varies in different populations. Missense mutations, deletions, and nonsense mutations are also responsible for the D variants. These molecular events produce variants such as weak D, partial D, and the DEL phenotype.
|Figure 1: RHD alleles. The black boxes and the gray boxes represent RHD and RHCE exons 1–10, respectively. The arrows represent mutations and the red triangle, a 37 bp insert. (a) RHD-positive (b) RHD-negative (due to deletion of RHD gene) (c) RHD-negative (due to the presence of pseudogene) (d) RHD negative (due to hybrid RHD-CE-Ds) (e) partial D (DV1 Type III) (f) partial D (DBT)|
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| Techniques Used|| |
Prenatal noninvasive fetal RHD genotyping in alloimmunized women from maternal plasma has been implemented in many laboratories in the Western countries. cffDNA detection is mostly performed by real time polymerase chain reaction (PCR) technique, but matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), digital PCR, multiplex ligation-dependent probe amplification, and next generation sequencing have also been used recently.,, All the 24 laboratories that participated in the Fourth International Workshop on molecular blood group genotyping used real time PCR for fetal RHD detection  and it seems to be the technique of choice because of its ease of use, sensitivity, and ability to automate and avoid contamination., Many studies performed with this technique have shown high degree of sensitivity [Table 1]. Mass spectrometry  and digital PCR  provide an alternative approach to prenatal RHD genotyping. Mass spectrometry was used to detect multiple RHD exons and the fetal identifier assay was used to screen 92 single nucleotide polymorphisms in case of RhD-negative female fetus. Thus, these assays will be used in future as they are of high throughput, sensitive to detect small amounts of fetal DNA, and also can be multiplexed to detect multiple RHD exons.
|Table 1: Noninvasive fetal RhD typing studies from different countries showing 100% sensitivity|
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| Selection of Rhd Exons|| |
The fetal RHD gene detection is facilitated by the absence of the maternal RHD gene, i.e., the deletion of it. This forms the basic premise for its detection. Thus, selection of a single region of the RHD gene will give a concordant result in majority of the cases. However, owing to the large genetic variation of the Rh system, this approach is not feasible and selection of two to three regions of RHD gene is recommended. Exons 4, 5, 7, and 10 are the common RHD targets used. Exon 4–7 sequence encodes the all-known exofacial D epitope sites of the RhD protein. The special noninvasive advances in fetal and neonatal evaluation network was established under the European Commission Sixth Framework Program with an aim to develop the technology required for robust and cost-effective noninvasive prenatal diagnosis and recommends the use of exon 5 and 7 for fetal RHD detection. In case of an RHD pseudogene, exon 5 detection is negative and exon 7 is positive whereas in case of hybrid RHD-CE-D s, both are not detected. Thus, it avoids false positive results. Some RHD variants will be missed by these approaches such as DVI Type III and DBT [Figure 1]e and [Figure 1]F. Inclusion of exon 10 assay would help in the identification of these variants, but may generate a lot of false positive like in the case of RHD-CE(4-7)-D or RHD-CE(2-9)-D partial D variants. Methods employing exon 7 and exon 10 assays will not be suitable for testing people of African origin. Real time PCR assays using exons 4, 5, 6, and 10 by Finning et al. in UK differentiated RHD positive, RHD negative from RHD pseudogene and RHD-CE-D s variants with 100% concordance. Thus, the detection of multiple RHD exons will increase the sensitivity of the assay, but it is difficult to avoid false positive results in case of rare D-negative alleles. Ethnic variations also play an important role in accurate fetal RHD detection. Knowledge of the paternal and maternal ethnicity would help in designing of supplemental assays to target population-specific variants.
| Controls|| |
Appropriate controls are required for correct prediction of the fetal genotype. Mainly controls for total DNA and those specific for fetal DNA are used. Controls for total DNA extraction include housekeeping genes such as CCR5, β-actin, β-globin, GAPDH, and albumin. These controls amplify both the maternal and fetal DNA and thus indicate that amplification has occurred and are used in quantification of total DNA. They can also work as a quality check for the plasma sample when excessive maternal DNA is detected by quantitative PCR, which may interfere with the detection of cffDNA sequences. Comparison of cycle threshold (Ct) values of total DNA and the fetal DNA can signify the presence of silent maternal gene, and thus also act as a check for false positive reporting. Spiking of plasma with exogenous DNA such as maize, mouse, Escherichia coli plasmid, and amplification of the same act as a control for both adequate DNA extraction and PCR amplification.
Since the mother is RhD-negative, an RHD-negative fetal genotyping result needs to be confirmed by a fetal-specific marker. Inadequate amounts of starting cffDNA, due to loss during storage, extraction, or any other reason can give rise to a false negative result. The most common fetal-specific control used is the Y-linked gene SRY. SRY being a single copy gene as opposed to DYS14 (a multicopy gene) is the preferred choice as a control in case of RHD detection. SRY is informative in only 50% of the pregnancies where the fetus is male. Paternally inherited polymorphisms absent from the maternal genome can be exploited for the detection of cffDNA. Short tandem repeats  and sets of biallelic polymorphisms have been used. The disadvantages associated with them are low sensitivity; require testing of large number of different markers, and maternal and paternal DNA genotyping for the assessment of polymorphic status. SensiGENE ® RHD genotyping laboratory detection test kit using MALDI-TOF mass spectrometry utilizes 92 single nucleotide polymorphisms and a minimum of six informative paternal alleles can be used to establish the presence of fetal DNA. In 2006, Chan et al. described the promoter of the tumor suppressor gene Ras-association domain family 1 isoform A (RASSF1A) as a universal fetal marker. The promoter of the gene is hypermethylated in the fetal (placental) DNA and hypomethylated in the maternal DNA. Exploitation of this epigenetic difference with the use of restriction enzyme-mediated digestion using methylation sensitive enzyme such as BstU1 can leave the fetal RASSF1A sequence intact, cleaving the maternal sequence. An extra digestion control having the same efficiency as the fetal marker can avoid the risk of incomplete digestion and has been used as a fetal-specific marker.
PCR controls such as a no template control containing water, an RhD positive and negative control are highly recommended for interpretation of results. Along with it, the replicate testing of multiple RHD exons and strict criteria for reporting of the fetal genotype on the basis of number of positive replicates obtained are required.
| Discordant Results|| |
In case of alloimmunized women, fetal RHD status should be known early in pregnancy to assess the risk of HDFN. False negative results can prove critical for further management of pregnancy and hence high levels of sensitivity and specificity are required. In the case of nonimmunized antenatal women, the main objective is the decision making on Rh prophylaxis and hence false negative results would lead to missing of the RhIG dose. False positive results in this case are not critical although would lead to unnecessary wastage of RhIG. The genotyping results are compared to cord blood serology report, which is the gold standard for diagnostic accuracy.
Most false positive results are due to silent nonfunctional maternal D-variants. To prevent false positives due to the presence of RHD pseudogene and hybrid RHD-CE-D s, fetus was considered negative when there was a lack of amplification of exon 4 and exon 5 irrespective of the result of exon 10. In case of fetal amplification leading to low Ct values for RHD exons, the maternal buffy coat is tested for maternal RHD variants [Figure 2]. Another reason for false positive reporting could be the previous history of the antenatal woman receiving a solid organ transplant. Thus, a thorough clinical history of the D-negative woman is recommended. In few studies, a positive real time result was obtained, but cord blood serology was found to be negative. In such cases, an indirect anti-globulin test will reveal the presence of weak or partial D allele. This can be confirmed by a second sample either using venipuncture or a buccal swab of the newborn.
|Figure 2: General flow chart depicting the common steps involved in fetal RHD detection|
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False negatives can result due to lack of a fetal-specific marker. Scheffer et al. reviewed that false negative results occur at a low frequency of 0.1–0.2% in studies lacking a fetal DNA-specific marker. They mainly result due to low amount of fetal DNA. False negative results can be avoided by the screening of two or more RHD regions. False negative results occur due to manual handling errors, misreporting, and improper labeling of samples. Avent has recommended that all RhD-negative women should be genotyped for potential expression of partial D and weak D alleles known to be implicated in HDFN, as certain positive signals for the fetal RHD exons will be explained by the maternal variant genes. However, this would lead to increased workload. Chitty et al. recommend fetal RHD genotyping on a mass scale to be performed only after 11 weeks of gestation to reduce the number of false negatives.
Excessive maternal DNA interferes with of fetal DNA identification and results in inconclusive reporting. This can happen due to long transport time of the maternal sample. In such cases, a repeat sample can clear the discrepancy [Figure 2]. Clausen et al. however do not support this finding and reveal that fetal DNA was detectable even with very high levels of mean total DNA and after 8 days of transport. Inconclusive results are usually obtained with maternal variant D genes such as weak D, DVI, pseudogene, and RHD-RHCE hybrid alleles., Routine antenatal anti-D prophylaxis is recommended for D-variant women with inconclusive results, thus covering the theoretical risk of being immunized by their D-positive fetus. Samples are also termed inconclusive when sufficient numbers of positive and negative replicates are not obtained and the test is then repeated on a second sample. Hyland et al. reported a threshold of 9/12 replicates to be positive for stating that a fetus is RhD positive when using exons 4, 5, and 10 in quadruplicate. [Figure 2] depicts the common steps involved and the general interpretation of results in fetal RHD genotyping.
| Diagnostic Accuracy of Fetal Rhd Genotyping in Published Studies|| |
Lo et al. in 1998 demonstrated a sensitivity of 78% for the first trimester and 100% for the second and the third trimester thus showing the promise and paving the way for further testing of RHD gene from the fetal fraction. The accuracy and robustness of cffDNA assays was reviewed from 1998 to 2005 by Geifmann-Holtzmann describing 44 different protocols. The analysis showed varied accuracy ranging from 32% to 100% depending on protocol and study design with overall accuracy of 94.8%. Legler et al. reviewed eight studies from 2006 to 2008 for prenatal RHD genotyping comprising 14 different protocols and found that sensitivity of large-scale studies ranged from 99.5% to 99.8% and specificity ranged from 94% to 99.5%.
Studies since 2009 reported decrease in false negatives results leading to higher sensitivity. In large-scale studies performed, Clausen et al. and Wikman et al. reported sensitivity of 99.9% (25 weeks median gestation) and 99.3% (10 to 40 weeks gestation), respectively. [Table 1] shows some of the recent studies from different countries with 100% sensitivity using multiple RHD exons and different controls. A recent meta-analysis covering 41 publications including 11,129 samples shows an overall diagnostic accuracy of 98.5% and sensitivity and specificity of 99% and 98%, respectively.
Thus, noninvasive prenatal RHD typing is now increasingly being studied and will be used diagnostically by many more countries in future. This would enable to target RHD-negative alloimmunized pregnancies that need close monitoring. Routine early noninvasive fetal RHD typing will reduce unnecessary administration of antenatal prophylaxis to women carrying RHD-negative fetus. Further large-scale studies in different ethnic populations have to be planned by taking into consideration early detection, high accuracy, and cost-effectiveness for it to be implemented diagnostically.
In India, at present, the standard care for RhD-negative pregnant women is antenatal antibody screening and postnatal anti-D administration. Antenatal prophylaxis is mostly given to women in metro cities. Hence, it is the time we update antenatal screening program and include noninvasive prenatal RhD typing routinely for the management of RHD-negative pregnancies.
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Conflicts of Interest
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[Figure 1], [Figure 2]
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