Noninvasive Fetal Blood Group Typing

Treatment of HDFN

If alloimmunization is detected in time, the disease can be prevented in most cases. Treatment of HDFN depends on the severity of fetal anemia. In the past, bilirubin levels in amniotic fluid were used to assess the anemia. Today, advances in Doppler ultrasonography have made noninvasive detection of fetal anemia possible. Mari et al. showed that ultrasonic Doppler determination of the middle cerebral artery peak systolic velocity (MCA-PSV) can be used as a surrogate measurement of fetal anemia . In an international multicenter study it was demonstrated that MCA-PSV is superior to amniocentesis, with a sensitivity of 88% and a specificity of 82% . In case of severe fetal anemia, the anemia can be corrected by intrauterine blood transfusions (IUT) and/or preterm labor might be induced followed by neonatal treatment. The first IUT is on average given around 26 weeks, range 16–35 weeks, the earlier transfusions are especially given in pregnancies complicated with anti-K antibodies. The perinatal survival rate of cases treated with IUT is around 95% with a normal neurodevelopmental outcome in > 95% . The development of kernicterus after birth can be prevented by exchange transfusion after birth. In mild cases of HDFN phototherapy is sufficient. For timely treatment, all developed countries have screening programs in place to identify alloimmunized pregnancies at risk. Without a screening program, HDFN during pregnancy can only be suspected by decreased fetal movements or sudden fetal death, or by (early) neonatal jaundice after birth.

Anti-D Immunoprophylaxis to Prevent Immunization

Before the introduction of postnatal anti-D immunoprophylaxis HDFN was one of the major causes of perinatal death. In the Netherlands postnatal immunoprophylaxis was introduced in 1969 and resulted in a drastic decrease of the prevalence of anti-D immunization from 3.5% to 0.5% in the 1990s . Postnatal prophylaxis has to be given within 72 h after delivery and traditionally on the basis of postnatal RhD typing of cord blood from the newborn . Already in 1978, Bowman and colleagues demonstrated that the use of antenatal anti-D prophylaxis can further reduce immunization incidents . The effects of combined antenatal and postnatal prophylaxis have been substantiated by more recent meta-analyses . Combined prophylaxis reduces the immunization risk by half as compared to postnatal prophylaxis only, with a parallel reduction by half in severe HDFN cases . Thus in several countries, antenatal prophylaxis is currently combined with postnatal prophylaxis to further minimize immunization risk. Antenatal prophylaxis is routinely given either as a single dose of 250–300 μg of immunoglobulin at gestational weeks 28–31, or as two 150-μg doses, respectively, at gestational weeks 29 and 34 . In addition, it is given after invasive procedures during pregnancy, abdominal trauma, late miscarriage, or termination of pregnancy .

We recently obtained strong evidence that anti-D immunoprophylaxis is also preventing immunization toward other RBC antigens. Only 1 out of 99 pregnant women who became alloimmunized against non-Rh antigens during their first pregnancy had received anti-D immunoprophylaxis in this first pregnancy, which was significantly less often than the 10 women expected based on calculations for the general population . Also ABO incompatibility can protect against immunization against other blood group antigens . Theoretically, also immunoprophylaxis against Rhc or K, or by giving antibodies against a universal fetal antigen could have the same “wider” preventive effect, but so far, only anti-D immunoprophylaxis is available.

HDFN Mediated by RhD and Non-RhD Alloantibodies

Despite adequate antenatal and postnatal anti-D immunoprophylaxis still 1–3 in 1000 D-negative women develop anti-RhD antibodies, and in about 30% of these cases severe HDFN develops . The immunization against RBC antigens other than RhD became relatively more important when the incidence of anti-D immunization decreased. The prevalence of non-D immunization as determined in large-scale (> 70,000 women) studies of unselected pregnant women is calculated to be about 3 in 1000 women ( Table 1 ). The specificities of non-D alloantibodies detected in a predominantly European population of pregnant women are, in order of frequency, anti-E, anti-K, anti-c, anti-C ^w , anti-Fy ^a , anti-S, anti-Jk ^a , and anti-e . Occasionally antibodies against Kp ^a , anti-f, anti-Jk ^b , and anti-s are detected. Antibodies against other blood group antigens are extremely rare. Anti-M antibodies are frequently found, but almost always of the IgM subclass and therefore harmless.

Any antibody that can pass the placenta and react with an antigen expressed by fetal RBCs can theoretically result in HDFN. However, only in a minority of immunized pregnancies, the presence of the antibody results in clinical disease. In 2008 we performed a Dutch nationwide prospective index-cohort study on 305,000 consecutive pregnancies screened in the 12th week of pregnancy, and determined the risk that the presence of non-RhD antibodies will lead to clinical signs of HDFN . In Table 2 the results of this study are summarized. In total non-RhD antibodies were detected in 1279 pregnancies and in 403 cases the fetus was positive for the antigen. Severe HDFN, defined as need for intrauterine treatment and/or blood transfusion after birth, was almost exclusively seen in pregnancies with anti-c and anti-K antibodies, and rarely in pregnancies with other (non-D/c) Rh-antibodies. The risk of severe HDFN in case of anti-K antibodies has probably been underestimated in Table 2 , because at present we observe that 50% of cases with anti-K antibodies lead to severe HDFN. Others have reported similar data . All other antibodies (observed in this single center study) did not cause severe HDFN. In a cohort of 426 IUT-treated fetuses from the reference center for fetal treatment in the Netherlands after the introduction of antenatal prophylaxis, the vast majority of IUTs were still given because of alloimmunization against D (78%), compared to K in 15%, c in 3.5% and occasionally, antibodies against other antigens. Similar observations were done in Denmark and Germany.

In literature case reports of severe HDFN caused by a wide variety of other specificities have been described, but in population studies these specificities are seldom found as cause of severe HDFN. Anti-Duffy, anti-Kidd, and anti-MNS antibodies are relatively frequently found, but rarely result in severe HDFN , although they may lead to slightly decreased hemoglobin levels at birth and need for phototherapy. For antibodies against I, Le, P1, Lu, and Yt there is no risk for developing disease, because of very low expression of these antigens by fetal cells. Albeit the high rate of ABO incompatibility between the mother and her fetus, clinically significant hemolysis in neonates due to anti-A or anti-B is relatively low. And, if it occurs, it is usually mild . For these reasons in Western countries pregnant women are not screened for the presence of anti-A or anti-B of IgG class. Because placental tissue expresses A and B antigens the maternal alloantibodies may be absorbed to some extent by the placenta. Furthermore, fetal RBCs show weak expression of A and B blood group antigens. There is a striking, and unexplained, difference in the incidence of ABO-mediated HDFN between populations. The incidence is around 0.3%–0.8% in the Caucasian population, vs 3%–5% in Black or Asian populations, also with a more severe clinical course . Other alloantibodies detected in East Asian populations from China and Taiwan are antibodies specific for hybrid glycophorins of the MNS system, especially antibodies reactive with RBC expressing GP.Mur. This phenotype is virtually absent in European populations, whereas it occurs in 6%–8% of Chinese and Thai individuals, and 0.13% of pregnant Chinese women have anti-Mur antibodies . The majority of anti-MUR antibodies are IgM, but IgG anti-MUR antibodies can result in very severe HDFN and might cause, in the absence of antibody screening, fatal hydrops fetalis .

In conclusion, in the Western world especially pregnancies complicated with anti-D, anti-c, anti-K, and possibly also anti-E should be carefully monitored. If fetal blood group typing demonstrates that the fetus is antigen positive, the titer of the antibody has to be investigated at regular intervals to check whether a critical titer is exceeded . In that case the woman can be referred to a special care center for further clinical examinations like Doppler ultrasonography to determine the severity of anemia, and to start IUT in time or to induce labor, if needed.

Rh System

The Rh blood group system consists of two genes RHD and RHCE on chromosome 1 (1p36.11), positioned in opposite directions and separated by 31.8 kb, in which the TMEM50A gene (previously SMP1) is located . The RHD gene arose as a duplication of the RHCE gene in the common ancestors of humans, chimpanzees, and gorillas . Both genes have 10 exons and share an overall 93.8% gene sequence identity and 96.4% exon sequence identity . The RHD gene is flanked by two 9 kb regions of 98.6% homology, the so-called Rh boxes . The function of the RhD and RhCE polypeptides is unknown. They belong to the Rh family, but in contrast to three other members of this family (RhAG, RhBG, and RhCG) they most likely do not transport ammonia . It has been suggested that they might play a role in CO ₂ transport in RBC but are functionally redundant. The RHD gene ( NG_007494 ) encodes the RhD protein (CD240D), carrying the D antigen (RH1). The RHCE gene ( NG_009208 ) encodes the RhCE protein (CD240CE), carrying the C (RH2) or c (RH4) and E (RH3) or e (RH5) antigens. Both proteins are red cell specific, consist of 417 amino acids, are not glycosylated, and have 12 membrane spanning domains. As shown in Fig. 1 A , 37 nucleotides are specific for the coding sequence of RHD and not present in any of the RHCE alleles. Five nucleotides (in exon 2) are specific for RHc , of which the 307C encoding 103Pro is best correlated with c expression . Exon 2 of RHC is identical to exon 2 of RHD , therefore only 48C (in exon 1) (16Cys) is specific for RHC . However, 16Cys is not correlated to C-expression and 74% of African blacks have 48C in a ce-allele with normal expression of Rhc . Therefore RHC genotyping assays have to be based on a 109-bp insert in intron 2, that is, only present in RHC . The E/e polymorphism is caused by a single nucleotide variation 676C > G (Pro226Ala) in exon 5 of RHCE . The 676G is also present in the RHD allele, but is in RHD surrounded by seven D specific nucleotides . The frequency of the different alleles in the different populations is indicated in Fig. 1 A.

In the RBC membrane the Rh proteins are complexed with RhAG, another member of the Rh family. The most likely in vivo subunit configuration of the Rh core complex is RhD-(RhAG) ₂ and RhCcEe-(RhAG) ₂ . RhAG is essential for the assembly of the Rh complex in the erythrocyte membrane . Mutations in the RHAG gene can result in the complete absence (regulator Rh _null ) or severe reduction (Rh _mod ) of both RhD and RhCE antigens . The Rh complex is directly anchored to the cytoskeleton via Ankyrin R, and also mutations in ANK1 can result in weakened expression of RhD and RhCE antigens . The RH locus is highly polymorphic, and many hybrid RHD-RHCE gene variants have been described. The Rh antigens and RH alleles are numbered by the Working Party on Red Cell Immunogenetics and Blood Group Terminology of the International Society of Blood Transfusion and published on their website ( http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/ ). A complete database for RHD variants has been established by Wagner and Flegel and is continuously updated ( http://www.rhesusbase.info /) . At present (December 2017), 54 antigens, 378 RHD alleles, and 116 RHCE alleles have been recognized in the Rh blood group system. The main mechanism responsible for the generation of hybrid RH genes is thought to be gene conversion, explained by the opposite orientation of the highly homologous RHD and RHCE genes. Multiple exons can be converted, but often microconversion events lead to single amino acid changes. In addition, gene conversion events can be associated with untemplated mutations, in which the mutated nucleotides are not derived from either gene .

RhD Negativity

The frequency of the D negative phenotype is 15%–17% in populations of European descent, 5% in Africa, and very rare in Asian populations. Because D negativity will reduce reproductive fitness due to HDFN, it is surprising that D negativity has become so frequent in Europe, suggesting a positive selection for an as-of-yet unknown fitness benefit of the RHD deletion. Perry et al. found, however, no evidence that positive natural selection affected the frequency of the RHD deletion . Thus the emergence of the RHD deletion in European populations may simply be explained by genetic drift/founder effect. Mourant indeed proposed already in 1954 a mixing of two populations, one essentially D − (Paleolithic peoples from the Basque region, which survived the last ice age) and the other D + (Neolithic migrants) as cause for the high frequency of D negativity in Europeans . Genetic analysis using mtDNA and Y chromosome markers has now provided strong evidence for this hypothesis . In European and Asian people the D negative phenotype is usually caused by deletion of RHD , which has occurred between the Rh boxes, flanking the RHD gene . Although many different nonsense mutations or splice site mutations have been described, each of them is rare. In contrast, in the African population deletion of RHD gene is not the only mechanism responsible for D negativity. Approximately 66% carry the RHDΨ gene and 15% carry a RHD-CE-Ds (r’s) hybrid allele ( Fig. 1 B) . The RHDΨ gene (RHD⁎08N.01) contains a 37 base pair duplication causing a frameshift, consisting of the last nucleotides of intron 3 and the first nucleotides of exon 4, and a nonsense mutation in exon 6 (807T > G, Tyr269stop) . The African RHD-CE-Ds allele consists of exon 1, 2, and 3 of RHD⁎DIIIa ; exons 4–7 of RHCE ; and exons 8–10 of RHD (type 1 = RHD⁎03N.01) , but occasionally it is a hybrid RHD (exon1–2)- RHCE (exon3–7)- RHD (exon8–10) allele (type 2 = RHD⁎01N.06) . These two genes produce no D, but do produce an abnormal C antigen. Because the RHD-CE-Ds alleles miss intron 2 on which RHC genotyping assays are based, reliable RhC prediction is also complicated in Africans. The observation that in Africa different mechanisms resulting in D negativity have emerged might indicate that there has been an ancient (unknown) selective pressure in Africa .

In Asian populations (China, Korea, Japan), 15%–30% of serologically typed D negative individuals carry the RHD⁎01EL.01 ( RHD⁎1227A ) gene, harboring a single mutation (rs549616139) causing a splice site defect resulting in the DEL phenotype . In DEL individuals D expression on RBCs is extremely weak and can only be recognized by sensitive serological techniques such as the adsorption-elution test. These individuals are not at risk for RhD immunization . Between 3% and 8% of truly D negative Asian individuals carry the D-negative hybrid RHD⁎D-CE(2–9)-D allele (RHD⁎01N.03) ( Fig. 1 B). In addition, the silent RHD⁎711delC (RHD⁎01N.16) allele is relatively frequent (> 1%) in Chinese D negative individuals .

The presence of silent RHD genes in D negative individuals, shown in Fig. 1 B, might hamper noninvasive fetal RHD genotyping assays. If the mother carries a silent RHD allele that is detected in the fetal genotyping assay, the genotype of the fetus cannot be reliably predicted. If the fetus inherited a silent RHD gene from the father, this will result in false-positive results. For that reason, it is important to take these variant RHD genes into account when designing fetal RHD genotyping assays, or at least in the interpretation of the assay results. This might also be true for the Asian silent alleles. In Asia, routine fetal RHD genotyping for D negative pregnant women seems to be not that relevant, because < 5% of fathers are hemizygous for RHD . However, in a multiethnic society fetal genotyping assays should also be reliable for Asian D negative pregnant women, thus assays based on RHD exon 10 should be preferably avoided.

RHD Variant Genes With D Expression

As described previously the RH locus is highly polymorphic and numerous RHD variants have been described (see http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/ ). RHD variants were originally subdivided into so-called DEL, weak D, and partial D. The most frequently occurring variant alleles are shown in Fig. 1 C.

The Del phenotype is characterized by a very weak expression of the complete RhD protein, which expression is only detectable with the very sensitive absorption-elution technique . The Del phenotype is most often caused by mutations that disturb splice sites or mutations in the C-terminal region of the RhD protein. DEL types are common in Asia (see earlier) and less common in Europe. The DEL frequency in the European population is 1:350 to 1:2000 .

Individuals with weak D expression express the complete RhD protein, however, in low quantities . 0.2%–1% of populations of European descent express weak D antigens. Weak D expression is caused mostly by single mutations in RHD that cause amino acid change in the transmembrane or intracellular parts of the protein . At present (December 2017), 139 different weak D antigens have been described. The most frequent weak D types are weak D type 1 (rs121912763; allele frequency 0.3% in ExaC database), weak D type 2 (rs71652374; allele frequency 0.1%), and weak D type 3 (rs144969459; allele frequency 0.3%).

RBCs of individuals carrying a partial D variant express an RhD protein that lacks 1 or several of the 30 D-epitopes and/or express new RhD epitopes, due to amino acid changes in (an) extracellular loop(s) of the RhD protein. Most partial variants arise from hybrid alleles in which parts of RHD are exchanged with the very homologous RHCE gene. In many of the partial D variants the expression is also weakened compared to normal RhD expression .

Fetal RBC expressing any of the gene products of these RHD variant genes can theoretically induce alloimmunization in the mother. DEL individuals cannot make anti-D, since they express the complete protein, so pregnant women do not need immunoprophylaxis. Furthermore, it was supposed that also weak D individuals cannot make anti-D, in contrast to individuals carrying partial D variants that miss some D epitopes. However, since the serological recognition of partial D variants can be difficult and it became clear that some weak D individuals, for example, with weak D type 4.2 (DAR), type 11, 15, 21, or 57 (reviewed in ) and also DEL individuals carrying the RHD⁎DEL8 allele or the RHD⁎DEL5 can become immunized against D, the DEL/weak D/partial D nomenclature is misleading and nowadays the term “D variant” is applied for all RHD alleles that result in qualitatively and/or quantitatively aberrant expression . The general consensus is that pregnant women or transfusion recipients with weak D type 1, 2, and 3 and Asian type DEL should be considered as RhD positive and not at risk for alloimmunization . Pregnant women carrying any of the other RHD variants should be categorized as D negative and included in prophylaxis programs . As outlined previously, the presence of these variant RHD genes will mask the fetal RHD , thereby hindering a straightforward prediction of the fetal D type. Furthermore, fetuses carrying a D-expressing- RHD variant might be missed by fetal genotyping and result in false-negative results.