The role of preimplantation genetic diagnosis in the management of severe rhesus alloimmunization: first unaffected pregnancy: case report

Rhesus (Rh) D alloimmunization may cause haemolytic disease of the fetus and newborn if the fetal Rh blood type is positive. Although the incidence of severe RhD alloimmunization has decreased with prophylactic anti-D immunoglobulin administration during and after pregnancy, sensitization still occurs in a small group of women. In such women, Rh disease will continue to be significant problem and for their babies who may be affected. Preimplantation genetic diagnosis (PGD) may be utilized to avoid materno-fetal blood group incompatibility in an RhD-sensitized woman. Biopsy of a single cell from early cleavage-stage embryos screening for RhD-negative embryos allows the transfer of only RhD-negative embryo(s) into the uterus. This avoids any complications related to haemolytic disease of the fetus and newborn. This article describes the first reported case of an unaffected pregnancy using PGD for Rh disease. IVF and embryo transfer resulted in a clinical pregnancy and the birth of a healthy girl confirmed to be blood type RhD negative. PGD in couples with a heterozygous RhD-positive male partner provides an option for avoiding haemolytic disease of the newborn in RhD alloimmunized mothers.     

Prenatal typing of fetal DNA in cases of potential alloimmune haemolytic disease of the newborn: clinical benefit outweighs disadvantage.

Haemolytic disease of the newborn (HDN) is caused by the action of maternal antibodies on paternally derived antigens present on fetal erythrocytes. In its most severe form it may lead to death in utero. Although the introduction of anti-D prophylaxis has greatly reduced the incidence of the disease, a significant number of cases appear each year. Current antenatal management aims to predict whether the fetus will be severely affected, correct the fetal anaemia if present and deliver the baby at the optimal time. Indirect methods include antibody quantitation performed on maternal blood samples, and amniocentesis to determine bilirubin levels in the amniotic fluid. Fetal blood sampling offers a direct method to determine the antigen status of the fetus, and enables measurement of fetal haemoglobin and haematocrit if appropriate. Both fetal blood sampling and amniocentesis are invasive procedures and have the potential to produce further antibody stimulation which can compound existing problems and compromise the pregnancy. The ultimate aim is to develop an accurate, non-invasive technique for fetal DNA typing which can be carried out in the first trimester. Research methods outlined here suggest that this is feasible. Pinpointing those pregnancies where further intervention is not required will reduce the demand on NHS resources.     

Anti-M antibodies in the pathogenesis of hemolytic disease of the newborn

Two cases are presented of serological fetomaternal incompatibility in the MNSs group system diagnosed for the first time in Poland. Anti-M antibodies of the mother caused in one newborn haemolytic disease with acute anaemia requiring blood transfusions. In the other newborn clinical signs of the disease failed to appear despite a positive direct antiglobulin test. The results are presented of immunohematological studies calling attention to difficulties connected the diagnosis of haemolytic disease of the newborn caused by anti-M antibodies. This problem is discussed more extensively in the light of available literature.     

新生儿ABO溶血病相关母亲因素分析

目的观察分析孕妇血清IgG抗A（B）效价、妊娠次数及年龄因素与新生儿溶血病的相关性。方法检测母婴血型不合孕妇血清IgG抗A（B）效价,分别统计不同IgG抗A（B）效价的孕产妇中发生因ABO血型不合所致的新生儿溶血病的比率,并比较不同妊娠次数、不同年龄段的孕妇发生新生儿溶血的比率。结果孕妇不同血清IgG抗A（B）效价时、不同妊娠次数时及孕妇的不同年龄段时发生新生儿ABO溶血病的比率之间均有统计学差异（P〈0．05）,随着孕妇血清IgG抗A（B）效价的增高、妊娠次数的增加及孕妇年龄的增加,新生儿ABO溶血病的发病也增多。结论为保障母婴安全,减少新生儿溶血病的发生率,有条件时检测孕妇血清IgG抗A（B）效价,并尽量减少妊娠次数及避免高龄怀孕。     

The fetal and neonatal outcomes of Rhesus D antibody affected pregnancies in Northern Ireland

It has been suggested that routine antenatal prophylactic anti-D should be introduced for prevention of Rhesus D (RhD) haemolytic disease. Before making changes to the current prevention program it is important, therefore, to have up-to-date data on affected infants. Pregnant women with anti-D antibodies between September 1994 and February 1997 were identified by the Northern Ireland Blood Transfusion Service. The records of 124 women and 130 babies were examined. 26% of planned deliveries occurred in hospitals without paediatric cover. Rhesus isoimmunisation affected 78 babies. Fifty-nine infants were admitted to one of seven neonatal units. There were 2 stillbirths and 1 neonatal death. Two infants have severe neurodevelopmental delay. There is still significant morbidity associated with RhD haemolytic disease. Care for RhD affected pregnancies should be centralised to guarantee training opportunities and maintenance of expertise. The current management of these pregnancies should be re-examined before changing the Rhesus prevention program.     

Immunoglobulin G-mediated regulation of the murine immune response to transfused red blood cells occurs in the absence of active immune suppression: implications for the mechanism of action of anti-D in the prevention of haemolytic disease of the fetus and newborn?

Anti-D has been widely and effectively used in Rhesus blood group D negative mothers for the prevention of haemolytic disease of the fetus and newborn; its mechanism of action however, often referred to as antibody-mediated immune suppression (AMIS), remains largely unresolved. We investigated, in a murine model, whether active immune suppression or clonal deletion mediated by anti-red blood cell (RBC) immunoglobulin G (IgG) could explain the phenomenon of AMIS. Transfusion of IgG-opsonized foreign RBCs (i.e. AMIS) strongly attenuated antibody responses compared to transfusion of untreated foreign RBCs. When the AMIS-mice were subsequently transfused with untreated RBCs, no immune suppression was observed at 5 and 35 days after AMIS induction; in fact, the mice responded to retransfusion with untreated RBCs in a manner that was characteristic of a secondary immune response. When IgG-opsonized RBCs were transfused concurrently with untreated RBCs, a dose-dependent reduction of the antibody response was observed. This work suggests that the attenuation of the antibody responsiveness by anti-RBC IgG is not associated with active immune suppression or clonal deletion at either the T-cell or B-cell level; rather, the effect appears more characteristic of B-cell unresponsiveness to IgG-opsonized RBCs. These results may have implications for the understanding of the mechanism of action of anti-D in haemolytic disease of the fetus and newborn.     

Follow-up of the feto-maternal allo-immunization]

Immunohaematological tests used in antenatal patients have come a long way. However, despite a great deal of progress, we should not loose sight of the fact that these tests give only an indirect measurement and will only help the obstetrician, in conjunction with other fetal parameters, to assess the severity of the haemolytic disease (HD) of the fetus and newborn. The best method to assess the severity is the direct determination of foetal blood group hemoglobin after foetal blood sampling but this procedure is not without risk. Since 10 years ago, it is possible to determine the RHD genotype of the fetus using amniocytes and, today, maternal plasma directly. All pregnant women should be grouped for ABO-RH-KEL1 and the sera tested for clinically irregular antibodies (anti-RH are still the most frequent). The trend in anti-RH levels is more important than the level itself. The perfect technique for anti-RH quantitation has not been developed. Manual titration is simple but only provides rough, semiquantitatives estimates of anti-RH concentration. Quantitative haemagglutination methods, using auto-analyzers and appropriate anti-RH1 standards, measure in microg/ml, are sensitive, rapid and have acceptable intra-laboratory reproductibility.     

ABH secretion and natural selection

The secretor gene, which determines secretion of the ABH blood group antigens into saliva and other fluids, has widely varying frequencies in different healthy populations. Its frequency in patients suffering from certain diseases also differs from that in healthy persons. These observations suggest that frequencies of the gene are to some extent the results of natural selection. One means of selection is through haemolytic disease of the newborn. Women, mostly of group O, become immunized to the A or B antigen of a fetus, and this or subsequent fetuses are at risk from haemolytic disease of the newborn. Affected fetuses are preponderantly secretors; this is probably a genetic consequence of all or most immunizing fetuses being secretors. Selection against the secretor factor as well as against blood groups A and B is thus liable to occur. Similar processes of selection seem to operate through fetal loss by early abortion. It is suggested that counter-selection favouring secretors may result from certain epidemic infectious diseases.     

Preparation of test cells for the antiglobulin test

Erythrocytes may be coated with blood group antibodies with or without reacting complement or sometimes apparently with complement alone. This may occur in vivo in such conditions as autoimmune acquired haemolytic anaemia, haemolytic disease of the newborn, or after transfusions of incompatible blood. It may occur in vitro also by the deliberate sensitization of erythrocytes during laboratory serological investigations.Blood group antibodies may be of immunoglobulin types gammaM, gammaA, or gammaG; we have never seen gammaD antibodies. The presence of these antibodies on the erythrocyte surface, together with complement components or the presence of complement components alone, may be detected by the direct antiglobulin test where sensitization occurs in vivo or by the indirect antiglobulin test where there is sensitization in vitro.     

Lessons learnt from many years of experience using anti-D in humans for prevention of RhD immunization and haemolytic disease of the fetus and newborn

For 40 years prophylactic anti-D has been given to D-negative women after parturition to prevent haemolytic disease of the fetus and newborn. Monoclonal or recombinant anti-D may provide alternatives to the current plasma-derived polyclonal IgG anti-D, although none of them have yet proved as effective in phase 1 clinical trials. The variation in efficacy of the antibodies may have been influenced by heterogeneity in glycosylation of anti-D produced from different cell lines. Some aspects of the conduct of the human studies, most notably the use of low doses of anti-D and target D positive red cells in vivo, may aid the design of the clinical development of other immunomodulatory drugs in order to minimize adverse effects.     

Mechanism of anti-D-mediated immune suppression--a paradox awaiting resolution?

During pregnancy, women can be immunized by fetal red blood cells (RBCs) of an incompatible blood group. Subsequent transplacental passage of the antibodies can result in fetal morbidity or mortality due to RBC destruction. The administration of anti-D antibodies to D(-) women after delivery of a D(+) infant, and subsequent prevention of Rhesus (Rh) D haemolytic disease of the fetus and newborn, is the most successful clinical use of antibody-mediated immune suppression. The passive IgG anti-D might prevent immunization to D(+) RBCs by an IgG Fcgamma receptor (Fcgamma R)-dependent mechanism such as crosslinking the D-specific B-cell receptor and inhibitory FcgammaRIIb. However, recent murine studies demonstrate that the suppressive effects of antibodies to heterologous RBCs can be Fcgamma R-independent, suggesting other mechanisms might contribute.     

Blood group molecular genotyping

DNA-based typing methods of red cell antigens are applied in several fields. Genotyping is used to clarify problems in patient serology. It is also increasingly applied for routine mass-scale typing of blood donors for minor red cell antigens and screening for donors with rare blood types. An advance in this field is the non-invasive fetal RHD diagnostics in pregnancies of D-negative women to determine the fetal RHD. Reference centres for immunohaematology commonly use molecular methods to clarify problems, discrepancies or unusual results in patient serology. It is often applied in individuals with variant RhD expression to detect the contributing weak D or partial D types. Genotyping is also used to identify the individual ABO and RhD blood group in patients after mismatched transfusion. In some patients with positive direct antiglobulin test (DAT) it is necessary to type for antigens by DNA techniques. Genotyping is also helpful when reagents are not available or only weakly reactive, or to confirm weakly expressed antigens. Blood establishments are constantly challenged with the blood supply for patients carrying irregular red cell antibodies. A high number of donors have to be typed to find compatible blood units. Increasingly, DNA-based methods are used alongside standard serological typing. The advantages of molecular methods are the wider range of different antigens available for typing and that some DNA methods are less expensive than phenotyping. Extensive donor antigen typing leads to short response times, from request to issuing blood units. Genotyping is also used for identifying donors with extremely weak RhD expression (DEL) to prevent mistyping as RhD-negative. Non-invasive fetal typing of RHD from maternal blood in pregnancies of D-negative women is implemented in many Caucasian blood establishments. It allows that D-negative fetuses can be identified at early stage. In these cases, no anti-D immunoglobulin is required, which provides a more efficient use of this human blood derivate. Furthermore, in anti-D alloimmunized women, the assay allows to accurately identify fetuses at risk for haemolytic disease.     

New insights in fetal and neonatal alloimmune thrombocytopenia

During the recent years considerable advances in the clinical and laboratory diagnosis of alloimmune thrombocytopenia have been done. Feto-maternal alloimmunization is the commonest cause of severe isolated fetal and neonatal thrombocytopenia. The condition results from maternal immunization against specific fetal platelet antigens (HPA). Unlike the haemolytic disease of the fetus and newborn the first newborn was found to be affected. The diagnosis is usually made at birth when an otherwise ‘well’ term infant exhibits bleeding at delivery or few hours afterwards. The most feared complication of this disorder is the occurrence of intracranial haemorrhage (ICH) as a result of severe thrombocytopenia leading to death or neurological sequelae. The diagnosis of alloimmune thrombocytopenia enables appropriate management of the index case and future pregnancies. The diagnosis is confirmed by laboratory testing with the identification of the maternal alloantibody and the offending antigen present in the fetus or neonate that is absent in the mother. In a recent retrospective study concerning 75 HPA-1b1b women, we have shown that the diagnosis of maternal alloimmunization was mostly established at delivery and the women were primigravida in 51% of the cases. The deleterious consequence of this severity was evidenced by in utero or postnatal ICH. Subsequent pregnancies were managed according to three different protocols, steroids only, intravenous immunoglobulin (IVIG) or IVIG and steroids. We found that the most efficacious antenatal therapy for fetal alloimmune thrombocytopenia is maternal treatment with IVIG and steroids. In summary, this study shows (1) that the morbidity linked with this condition is important to be considered for further antenatal screening (2) that antenatal management in referral centers should be suggested to high-risk pregnant women.     

Update on HDFN: new information on long-standing controversies

Hemolytic disease of the fetus and newborn (HDFN) results from maternal IgG antibodies that cross the placenta to the fetal circulation during gestation and cause RBC destruction and complications before birth (HDF), or anemia and hyperbilirubinemia after birth (HDN), or both. In its most severe form,HDF produces hydrops fetalis, which is characterized by total body edema, hepatosplenomegaly, and heart failure and can lead to intrauterine death. Before discovery of Rh immunoglobulin (RhIG), HDFN from anti-D was a significant cause of perinatal mortality or long-term disability. Routine administration of RhIG to D- women during pregnancy and shortly after the birth of D+ infants effectively reduced the incidence of HDFN caused by anti-D. Maternal alloimmunization to other RBC antigens in the Rh, Kell, and other blood group systems can not be routinely prevented and these antibodies can also cause HDFN. Advances in prenatal care, noninvasive monitoring, and intrauterine transfusion have improved the outlook for affected pregnancies to the extent that even hydrops fetalis can be reversed and effectively treated in many cases. This review will provide an update on the current issues in prevention and treatment of HDFN, emphasizing recent insights into long-standing controversies regarding maternal weak D phenotypes and D alloimmunization, noninvasive fetal diagnosis and monitoring of affected pregnancies, and recent treatment guidelines.     

Studies on the blood group antigen Ina

Anti-Ina antibodies have been observed in 30 of the 41 anti-Rh donors hyperimmunised with group O Ror In(a+) blood. They have also been found in four of 60 Rh immunised women. In three of these the husbands and previous children were In(a+). However, there was no evidence of haemolytic disease of the newborn due to anti-Ina antibodies alone. The Ina antigen is developed at birth though it is weaker than in adults. The strength of the Ina antigen decreases during pregnancy and returns to the previous level three months afer termination of pregnancy. The Ina antigen is affected by papain, trypsin, bromelin and neuraminidase. The antibodies in all the donors, even though their sera were preserved for more than 10 years at -20 degrees C, reacted in saline and albumin milieux and in the indirect antiglobulin procedure using anti-IgG reagents. These antibodies are complement binding as shown by the two-stage complement binding test. Treatment with 2-mercaptoethanol and DEAE-column chromatography suggested the anti-Ina antibodies were of the IgG class. Anti-Ina antibodies could possibly cause a transfusion reaction since In(a+) red cells when transfused into individuals possessing anti-Ina antibodies are eliminated from the circulation within 20 minutes.     

Fetal RhD genotyping: A more efficient use of anti-D immunoglobulin

The most important application of blood group genotyping by molecular genetics is the prediction of fetal RhD phenotype in pregnant women with anti-D, in order to assess the risk of haemolytic disease of the fetus and newborn. This diagnostic test performed on cell-free fetal DNA in the maternal plasma, is now a routine procedure in some countries. High-throughput modifications of this form of fetal D-typing would be valuable for testing fetuses of all D-negative pregnant women to avoid unnecessary antenatal treatment with anti-D immunoglobulin in the 40% of D-negative pregnant women with a D-negative fetus. The results of trials in Bristol and Amsterdam suggest that such routine testing is feasible and accurate.     

母儿ABO血型不合孕妇血清IgG抗体效价与新生儿溶血的相关性分析

目的探讨母亲孕期血型血清学的IgG抗体效价与新生儿ABO溶血病(HDN)的关系。方法选取2005年1月～2009年12月在我院分娩的2168例ABO血型不合孕妇及其新生儿为研究对象。以分娩前孕妇最后一次IgG抗A(B)效价值为标准,效价≥1∶64者列为研究范围。新生儿HDN则观察溶血3项筛查指标及其红细胞、血红蛋白、网织红细胞等。结果 2168例ABO血型不合孕妇中血清IgG抗体效价1∶64有710例,1∶128有800例,1∶256有519例,效价≥1∶512有171例。HDN发病患儿共529例。2次及以上妊娠孕妇的抗A、抗B抗体效价值大于1次妊娠者,差异有显著性,P<0.05;年龄30-40岁组孕妇的抗A和抗B抗体效价高于21-29岁组,差异有显著性,P<0.05;母亲IgG抗A或抗B抗体效价与新生儿ABO溶血的发生率相关,r=0.8119,P<0.05。结论孕妇血清中血型免疫性抗体IgG是引起HDN的主要原因,且随着IgG抗体效价的增高,HDN的发病率也增高,但不是HDN发病的唯一因素,妊娠次数的增多、年龄增大、不良生育史等因素可加重IgG抗体效价对HDN的发生。     

19S γM, followed by 7S γG anti-Jka antibodies associated with pregnancy

Mrs H., a white woman, with three previous pregnancies, developed anti-Jk^a blood group antibodies in her serum, but her twins, both the Jk(a+), did not develop haemolytic disease of the newborn. It was shown that her antibodies were 19S γM anti-Jk^a antibodies, that they did not cross the placental barrier, and were absent from the serum of the children. One month after delivery, 7S γG antibodies appeared in her serum. Five months later, the 19S antibodies faded leaving 7S anti-Jk^a antibodies behind.

This change is considered to be as a result of immunization of pregnancy due to the influx of Jk(a +) foetal cells into the maternal circulation.

Various aspects of the investigations on the maternal serum are discussed.

     

The impact of intrauterine transfusions on morphological picture of red cells in Rh incompatibility

The purpose of the study was to analyse the changing picture of red cells in fetuses treated with intrauterine transfusions for Rh incompatibility. The study included 28 live-born infants who were given 48 intrauterine transfusions. The reference group consisted of 232 newborns with haemolytic disease of the newborn (HDN), untreated in the fetal period. The number of erythroblasts and reticulocytes in the red cells along with fetal haemoglobin (HbF), in relation to the number of the own erythrocytes of the fetus and the total number of red cells in cord blood were examined. The suppressing influence of the transfused blood on haematopoiesis and a significant stimulation of erythrocyte renowal, in cases of smaller number of HbF erythrocytes in cord blood were observed.     

Prenatal screening

In most countries, pregnant women are early in pregnancy typed for ABO and RhD and screened for the presence of red cell antibodies. Maternal red cell alloantibodies of IgG class are actively transported by the placenta to the fetus and destruct the fetal red cells, if these carry the involved antigen leading to haemolytic disease of the fetus and newborn, HDFN. HDFN is a disease which –if untreated– can cause perinatal mortality and morbidity, with a substantial risk for long-term sequelae. In most cases of HDFN, alloimmunization against the RhD antigen is involved, although introduction of anti-D immunoglobulin (Ig) prophylaxis drastically decreased anti-D-mediated HDFN. In the last decade, non-invasive fetal genotyping, with cell-free fetal DNA present in maternal plasma, for RhD, RhC, Rhc, RhE and K became a diagnostic reality. In a few countries non-invasive fetal RhD genotyping has already been implemented to target antenatal and postnatal anti-D Ig prophylaxis to RhD-negative women carrying RhD-positive fetuses. Both applications of non-invasive fetal genotyping are summarized in this article.