Haemolytic disease of the fetus and newborn: advancements in precision and prevention

In pregnant patients, the impact of blood type and the presence of red blood cell antibodies influence the course of the pregnancy and the health of the fetus and newborn infant. Throughout history, haemolytic disease of the fetus and newborn has played a fundamental role in the discovery of the blood group antibodies and their cognate antigens. The mid‐1900s were noted for the advent of Rh immunoglobulin. Now, the technological advancements in diagnosis and treatment of haemolytic disease of the fetus and newborn have provided the critical tools needed to support mothers with affected pregnancies. The knowledge of blood typing has been further refined with the explosion of understanding about blood group genes, particularly in the RH blood group. Genomic blood group typing, improvements in ultrasound technology and transfusion medicine progress have advanced the field. The care of women with potentially affected pregnancies has never been more robust. Despite this, the risk to the fetus is significant, and prevention strategies for maternal alloimmunization deserve continued attention.     

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.     

Rh血型系统的分子遗传学及其医学应用

Rh血型系统是人类较为复杂和重要的血型系统。它有两个同源结构基因串联排列在1p34.3-36.1，编码的Rh蛋白为有2个跨膜域的红细胞膜蛋白。Rh抗原有很多变异体；RhD阴性个体存在3种遗传多态性。Rh血型系统在临床输血及新生儿溶血病（hemolytic disease of the newborn,HDN)中意义重大，可利用PCR进行Rh基因分型方法对胎儿进行产前诊断，但此方法仍有一定缺陷。需要对Rh血型系统进行更深入的认识，以解决这一问题。     

Early Lyme disease: Humoral immune status and treatment

Objective: To investigate the humoral immune status and the effect of antibiotic treatment in Bulgarian patients with early Lyme disease.
Method: A total of 34 early Lyme disease patients was examined, 16 with erythema migrans and 18 with non-specific systemic symptoms. Serum samples from all patients and from 12 healthy controls were tested for total immunoglobulins (IgG, IgA and IgM), hemolytic activity of complement (CH₅₀) and immune complexes (ICs). The patients were treated with doxycycline (100 mg orally, twice daily for 10 to 15 days, in one or two courses).
Results: The patients showed significantly increased IC levels ( P <0.01 for patients with erythema migrans and P <0.001 for patients with non-specific symptoms). There were no significant changes in the levels of total hemolytic complement and total immunoglobulins (IgG, IgA and IgM). The clinical outcome was satisfactory in 21 of the 34 patients (61.8%) after treatment with doxycycline for 10 to 15 days. The rest of the patients (38.2%) failed to respond to the therapy, and continued to report various complaints, such as arthralgia, myalgia, paresthesia, headache, fatigue or recurrent rash. All of these had elevated levels of IC. After a second course of treatment with the same antibiotic regimen these patients had resolution of symptoms (12 patients) or improvement (1 patient).
Conclusions: Immunologic investigation may be useful in determining treatment strategy in Lyme disease. Elevated IC levels may indicate a need for more prolonged antibiotic therapy.     

新生儿Rh溶血病的检查分析及预防

目的检查分析15例新生儿Rh溶血病及发生原因，预防新生儿Rh溶血病的发生。方法采用盐水法检测患儿和其父母Rh血型，用新生儿溶血病血型血清学检查检测新生儿Rh溶血病，用Rh抗原谱细胞鉴定孕妇血清、患儿血清和红细胞抗体放散液中的Rh系统抗体。结果15例Rh溶血病患儿中由抗D引起的溶血病有8例，由抗D和Rh其他系统抗体联合引起的有2例，共10例，占66．7％；由抗E引起的有3例，由抗E和抗C联合引起的1例，占26．7％；由抗C引起的1例，占6．7％。15例患儿母亲都曾有生产或流产或输血史。结论为预防新生儿Rh溶血病的发生．对产前尤其是对曾有过生产史、流产史或输血史的孕妇作产前夫妇Rh血型和孕妇Rh免疫性抗体筛查极有必要。     

Placental Transport of Maternal Immunoglobulin G in Pregnancies at Risk of Rh (D) Hemolytic Disease of the Newborn

PROBLEM: The following questions were addressed: Is the placental transport of immunoglobulin (Ig)G, IgG1, and IgG3 diminished in pregnancies at risk of hemolytic disease of the newborn? Is the placental transport of IgG, IgG1, and IgG3 correlated with the hemoglobin concentration in the fetus and AutoAnalyzer (AA) quantitations of maternal anti-D? METHOD OF STUDY: IgG concentrations were determined retrospectively in 41 paired fetal/maternal (f/m) samples in 31 Rh (D) alloimmunized pregnancies. IgG1 and IgG3 concentrations were determined in those 23 cases in which the results of fetal hemoglobin concentration and quantitations of maternal anti-D were available. The results were compared with values found in normal pregnancy and correlated with maternal anti-D AA quantitations and fetal hemoglobin concentrations. RESULTS: Fetal IgG, IgG1, and IgG3 concentrations, and the corresponding fetomaternal ratios in Rh (D) alloimmunized pregnancies, increased with gestational age according to the following formulas (obtained by simple regression): Fetal IgG = ?8.846 + 0.491.gestational age (GA), (R² = 0.544); fetal IgG1 = ?10.021 + 0.46GA, (R² = 0.463); fetal IgG3 = ?0.865 + 0.039GA, (R² = 0.327); f/m IgG = ?1.006 + 0.054?GA, (R² = 0.557); f/m IgG1 = ?1.876 + 0.085GA, (R² = 0.654); f/m IgG3 = ?0.199 + 0.026GA, (R² = 0.146). CONCLUSIONS: The placental transport of IgG, IgG1, and IgG3 in women with Rh (D) immunizations is not diminished compared with normal pregnancy. However, AA quantitations of anti-D are inversely correlated with f/m IgG ratio, f/m IgG1 ratio, and fetal IgG and IgG1 concentrations (P = 0.002, P = 0.004, P = 0.02, and P = 0.02 respectively). The placental transport of IgG3 is significantly higher in pregnancies at risk of hemolytic disease of the newborn compared with IgG3 concentrations in normal pregnancy.     

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.     

An insight into neonatal unconjugated hyperbilirubinaemia from the transfusion medicine perspective

Unconjugated hyperbilirubinaemia is a common cause of morbidity and mortality among neonates in Asian and African countries. The aetiology is multifactorial and has wide range of presentation ranging from simple physiological jaundice to severe HDN requiring prompt intervention to prevent long‐term neurological sequelae. Extensive detection of borderline raised bilirubin level in newborn is debatable as it may lead to inappropriately increased phototherapy and unnecessary increased hospital stay. However, those at risk like early presentation of hyperbilirubinaemia within 48 h, prolonged hyperbilirubinaemia, and family history in siblings or having history of HDN must be screened for immune as well as non‐immune causes to prevent the neonate from further severe form of complications. Along with direct antiglobulin test, elution, antibody screening/identification is performed to evaluate the immune causes. Improvement in the molecular technology leads to prompt diagnosis of non‐immune causes which were previously remained as idiopathic. This will aid in the early management like phototherapy or exchange transfusion as per the indication. This review will basically focus on the aetiopathogenesis of neonatal unconjugated hyperbilirubinaemia and approach to immune causes from transfusion medicine prospective.     

微柱凝胶技术应用于新生儿溶血病的实验室诊断

目的采用微柱凝胶技术进行新生儿溶血病（HDN）的实验室诊断分析。方法对1627例新生儿进行血型血清学分析,包括母婴血型、直接抗人球蛋白试验、血清游离抗体试验、热放散试验、ABO血型系统以外的抗体鉴定试验。结果 1627例病例中确诊为HDN的有163例,其中母-婴血型为O-A及O-B的最多,共占97.55%。确诊为HDN血清学结果中放散试验和游离抗体试验同时阳性最多,占76.07%。诊断为HDN可疑病例血清学结果中,直接抗人球蛋白试验阳性率最多,占79.55%。在出生后3～7d内确诊HND的患儿数最多,占66.26%。ABO血型系统以外的抗体鉴定试验阳性有6例,其中5例诊断为RhHDN。结论红细胞抗体释放试验、直接抗人球蛋白试验和血清游离抗体试验是早期诊断HDN的有效方法。HDN检出率与所采用的实验方法和采血时机有很大关系。     

Rh阳性个体RHD杂合性分析

目的 分析中国汉族Rh(D)阳性个体的RHD杂合性,讨论Rh阴性妇女产前Rh同种免疫预防策略.方法 血清学检测31 115名汉族捐血者的Rh(D)表型,分析Rh(D)阳性个体的RHD杂合率;对其中3628名随机Rh阳性个体采用PCR方法直接测定RHD合子型,计算杂合率与前者比较.结果 31 115名捐血者中采用间接抗人球蛋白试验(indirect antiglobulin test,IAT)确认99名个体为Rh(D)阴性(0.318%),d基因频率0.056 41,D基因频率0.943 59,Dd杂合型0.106 45(10.6%),考虑IAT检测D放散型为Rh(D)阴性,计算后实际Dd杂合型为0.090 32(约9.0%);PCR测定3628名Rh阳性个体RHD合子型测定显示DD纯合型3383人(93.2%),Dd杂合体245名(6.8%),由于无效RHD等位基因的PCR结果为阳性(D),重新分析后实际携带1条功能性RHD基因的杂合性个体约7.4%.提示中国汉族Rh(D)阴性妇女当配偶为Rh(D)阳性时,子女Rh(D)阴性的比率约3.7%～4.5%.结论 中国新生儿Rh同种免疫预防进行侵入性胎儿Rh(D)血型预测意义不大,或可直接假定新生儿为Rh(D)阳性进行产前检查和同种免疫防护.

Abstract：

Objective To investigate the RHD zygosity of Rh(D)-positive Chinese Hans in order to study the mother-fetus Rh isoimmunization prophylaxis. Methods Rh(D) blood group of 31 115 donors were serotyped, and the RHD zygosities were analyzed, or determined through a PCR method for 3628 donors of Rh(D)-positive individuals. Results Among the 31 115 donors, 99 were tested Rh(D)-negative by indirect antiglobulin test (IAT) (0. 318%). The d frequency was 0. 056 41, D was 0. 943 59, and Dd heterozygosity was 0. 106 45 (10.6%). However the rate was 0.090 32 (about 9.0%) after excluding DEL (IAT-negative). For the 3628 PCR tested donors, 3383 were DD (93. 2%), 245 were Dd (6.8%). After excluding nonfunctional RHD alleles, 7. 4% of the donors were carrying one functional RHD. It showed that an Rh(D)-negative Chinese Hah woman gives an Rh(D)-negative child at a rate of 3.7%-4. 5% when her husband is Rh (D)-positive. Conclusion Fetus Rh (D)-genotyping may be unnecessary for Chinese Hans if invasive operation was needed for prenatal diagnosis. The Rh prophylaxis could be chosen assuming an Rh(D)-positive fetus.     

Gestational trophoblastic disease: histopathological diagnosis in the molecular era

Gestational trophoblastic disease is a heterogeneous group of proliferative disorders of the placental trophoblasts, often with challenging morphology, posing diagnostic problems for the general, even specialty pathologists. In this review we discuss characteristic histological features of complete- and partial hydatidiform moles, with special focus on the diagnosis of hydatidiform moles evacuated at early stage and their mimics. The diagnostic criteria and potential pitfalls in the diagnosis of gestational choriocarcinoma and two rare, unique neoplasms of the intermediate trophoblast – placental site trophoblastic tumour and epithelioid trophoblastic tumour – are presented, along with their differential diagnoses from two tumour-like conditions including exaggerated placental site and placental site nodule. We also review the current practice of immunohistochemistry, ploidy analysis and molecular genotyping in the diagnosis of gestational trophoblastic disease. Finally, how molecular testing may change the future diagnostic algorithm of gestational trophoblastic diseases is discussed.     

Early detection of RhD status in pregnancies at risk of hemolytic disease of the newborn

The aim of this work was to investigate the presence of the RHD gene in fetal cells obtained from amniotic fluid. We studied 65 samples of amniotic fluid, 11 from RhD-negative mothers sensitized with anit-D alloantibodies. The fetal origin of the DNA was confirmed with the analysis of 1 VNTR locus and 3 STR loci in DNA samples from amniotic fluid and maternal blood. The RHD genotyping was performed in non-contaminated samples (n=62) using a multiplex polymerase chain reaction strategy that yields three amplification products from RhD-positive phenotypes (intron 4 of both RHCE and RHD genes and exon 10 of the RHD gene) and 1 DNA fragment from RhD-negative phenotypes (intron 4 of the RHCE gene). We genotyped 54 RhD-positive fetuses (8 from RhD-negative sensitized mothers) and 8 RhD-negative fetuses (3 from RhD-negative sensitized mothers). The fetal DNA genotyping allows the diagnosis, from a single amniocentesis, of fetuses at real risk of hemolytic disease of the newborn. When the fetus is determined to be RhD-negative invasive procedures can be avoided. Received: 17 October 2001 / Accepted: 26 May 2002     

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。     

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。     

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.     

Cell‐free fetal DNA and fetal blood group genotyping: non‐invasive prenatal testing

In transfusion medicine and clinical immunology, cell‐free fetal DNA (cffDNA) is analysed from maternal plasma of pregnant women to predict fetal blood groups with the purpose of (1) assessing the risk of haemolytic disease of the fetus and newborn (HDFN) in immunized women and (2) guiding targeted Rh prophylaxis in non‐immunized RhD‐negative women. National programmes for guiding prophylaxis are now implemented in around 6–7 European countries; assay accuracy is very high, with sensitivities of 99·9%. Sensitivity is challenged by low quantities of cffDNA, especially in early pregnancy. Specificity is challenged by the polymorphic Rh blood group system, where careful attention is needed to navigate among the many RHD variants that may complicate cffDNA analysis and interpretation of results, especially in populations with mixed ethnicities. However, fetal RHD testing is feasible when implemented with careful attention to these issues. The success of predicting fetal RhD and its successful clinical implementation should encourage widespread implementation. For blood groups that are determined by SNPs, such as KEL or Rhc, novel techniques such as next‐generation sequencing and droplet digital PCR are now providing accurate non‐invasive prediction of these fetal blood groups. Future work on non‐invasive prenatal testing of fetal blood groups determined by SNPs may consolidate the application for cell‐free DNA testing for such targets. At ISBT, the newly formed cfDNA subgroup of the Red Cell Immunogenetics and Blood Group Terminology Working Party will work to facilitate clinical applications, implementation and evaluation of cell‐free DNA testing.     

Is intravenous immunoglobulin effective to reduce exchange transfusion in Rhesus haemolytic disease of the newborn?

Background Haemolytic disease of the newborn (HDN) is a persistent public health problem in Brazil due to the lack of efficient prophylactic measures. The standard therapeutic interventions for the newborn are phototherapy and exchange transfusion (ET). Intravenous immunoglobulin has been used for several years in some neonatal centres to reduce the need for ET among newborns with jaundice, but the scientific evidence on the efficacy of this treatment is still not definitive. Objective To assess the evidence of efficacy of non-specific immunoglobulin in decreasing the need for ET in newborns with haemolytic disease of the newborn. Methods All randomized controlled trials on the use of intravenous immunoglobulin (IVIG) to reduce the need of ET in the treatment of Rhesus haemolytic disease were included by searching MEDLINE, Cochrane Library, EMBASE, Scopus, Lilacs, Scielo, http://www.clinicaltrial.gov and abstracts from SPR. Results Six trials were found including 281 neonates: Rubo et al. (1992) (32); Alpay et al. (1999) (23); Dagoglu et al. (1995) (41); Nasseri et al. (2006) (13); Santos et al. (2010) (92); Smits-Wintjens et al. (2011) (80). Most of the trials used the immunoglobulin in a prophylactic approach and only one used it when the jaundice was already established. Only two studies used placebo in the control group and described a method of blinding the intervention after allocation. The phototherapy protocols were not the same neither the efficacy of the units used. There was difference on the doses of the IVIG and on the number of infusions. Conclusions Although current guidelines support the therapeutic effect of the intravenous immunoglobulin effect on decreasing the need for ET in newborns with Rhesus haemolytic disease it shall not yet be routinely recommended.