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.     

Noninvasive fetal RHD genotyping from maternal plasma: Use of a new developed Free DNA Fetal Kit RhD

Fetal RHD genotyping from maternal plasma was performed by real-time PCR amplification of exons 7 and 10 of the RHD gene and the amplified products were detected either with SYBR Green I dye according to our previously published method [Mol Diagn 8 (2004) 23–31] or with hydrolysis probes in a new Free DNA Fetal Kit RhD^®. Plasma specimen from 300 RhD-negative pregnant women (between 10 to 34 weeks of gestation) were analysed and validation of the results was ascertained either by RHD genotyping on amniotic cells or by blood typing of the neonate at birth. We found 100% concordant results when comparing the two methods. Two false-positive but no false-negative results were found. Thus, the sensitivity of the assay was 100% and the specificity superior than 99%. These data confirm the accuracy of fetal RHD genotyping on maternal plasma using the Free DNA Fetal Kit RhD^®, thus allowing to propose non invasive PCR-based fetal RHD genotyping for all RhD-negative pregnant women and to restrict the use of anti-D immunoglobulins only to those bearing an RhD-positive fetus.     

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.     

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.     

The clearance kinetics of autologous RhD-positive erythrocytes coated ex vivo with novel recombinant and monoclonal anti-D antibodies

Anti-D is given routinely to pregnant RhD-negative women to prevent haemolytic disease of the fetus and newborn. To overcome the potential drawbacks associated with plasma-derived products, monoclonal and recombinant forms of anti-D have been developed. The ability of two such antibodies, BRAD-3/5 monoclonal anti-D IgG (MAD) and rBRAD-3/5 recombinant anti-D IgG (RAD), to clear RhD-positive erythrocytes from the circulation was compared using a dual radiolabelling technique. Six RhD-positive males received autologous erythrocytes radiolabelled with (99m)Tc and (51)Cr and coated ex vivo with MAD and RAD. Blood samples were collected up to 1 h following intravenous injection, and percentage dose of radioactivity in the samples determined. Three different levels of coating were used on three separate occasions. No significant differences between MAD and RAD were observed in the initial clearance rate constant at any dose level. The log[activity]-time clearance plots were curved, showing a reduction in the clearance rate constant with time. This reduction was more marked for RAD than for MAD. The results support a dynamic model for the clearance of antibody-coated erythrocytes that may have wider relevance for the therapeutic use of antibodies.     

Pregnancy immunology and maternal alloimmune responses

Pregnancy is an “immunological Paradox”. This is because pregnant women are tolerant of their semi-allogeneic fetus but are not immunosuppressed and indeed make strong antibody responses. The immunological changes required are driven mainly by the placenta, an organ derived from cells of the blastocyst surrounding the embryo. Trophoblast cells on the surface of the placenta form the interface between the fetus and maternal tissues and blood. They differentiate into (a) villous syncytiotrophoblast (ST) that are in contact with maternal blood and transfer gases and nutrients between maternal and fetal blood and (b) extravillous trophoblast cells that invade into the maternal decidua lining the uterus. Trophoblast cells escape allorecognition because they lack classical HLA class I and II molecules. Local immunoregulation, or tolerance, in the decidua is mediated mainly by HLA-G+ extravillous trophoblasts that prevent killing of fetal and placental cells by maternal natural killer cells, cytotoxic T cells and macrophages, thus ensuring the fetus is not rejected. Placental hormones orchestrate the composition and regulatory function of maternal immune cells in the decidua. ST at the surface of chorionic villi, together with ST debris that is shed into maternal blood, maintain a state of mild maternal systemic inflammation by enhancing innate immunity and skewing the immune response towards humoral (antibody) immunity. This enables a healthy immune system to be maintained in pregnant women, with robust protective antibody responses to pathogens whilst enabling survival of the fetus. However, this immunological skewing ensures that pregnant women readily form alloantibodies to incompatible fetal alloantigens. If fetomaternal haemorrhage (FMH) occurs, they can make antibodies to fetal red cells, platelets and leucocytes that are in her blood. The alloantibodies are initially of low affinity but after re-immunisation of the woman with further FMH (most commonly at delivery), they become functionally effective, high titre IgG. Maternal IgG is transferred by the ST to the fetus, to give the baby protective immunity to survive infancy. This has the unfortunate consequence that alloantibodies to blood cells are also transported, leading to haemolytic disease of the fetus and newborn (HDFN) and fetal and neonatal alloimmune thrombocytopenia (FNAIT). Both HDFN and FNAIT may result in death or serious neurological damage to the baby. HDFN due to anti-D can be prevented by administration of prophylactic anti-D to the mother, destroying or clearing D-positive fetal red cells in FMH from her circulation before they can become immunogenic.     

Development of a flow cytometric test for the detection of D-positive fetal cells after fetomaternal hemorrhage and a survey of the prevalence in D-negative women

A sensitive test for the presence of D-positive fetal red blood cells (RBCs) in the maternal circulation of D-negative women has been developed. It was used to investigate the possibility that the occasional failure in preventing alloimmunization might be due to the administration of inadequate amounts of prophylactic anti-D Rh immune globulin. The standard dose in Australia contains 125 microg of antibody, and can suppress immunization by an estimated 6 mL of packed D-positive RBCs. A fetomaternal hemorrhage (FMH) of this volume is detectable in the maternal circulation as approximately 0.25 percent of the total RBCs. Our test utilizes a commercially available human monoclonal IgG anti-D that has been biotinylated and used with a dye-conjugated streptavidin. Flow cytometry is used to quantitate fluorescing D-positive RBCs. To date, 2,288 tests have been performed on blood samples from D-negative women attending local antenatal clinics or at the time of delivery. Evidence for an FMH has been obtained in six cases (0.26%). In one case, the FMH was only 0.1 percent, and in another (confirmed by the Kleihauer-Betke method), fetal cells constituted only 0.2 percent. Additional Rh immune globulin was not given to these patients. In the other four cases, the D-positive fetal cells were estimated to be 0.7,0.5,0.5, and 0.4 percent, and additional prophylactic Rh immune globulin was administered. Although the prevalence of FMH is low, screening D- negative women at risk of alloimmunization has proved to be simple, fast, and inexpensive.     

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.     

Hemolytic disease of the newborn due to anti-C and anti-G masquerading as anti-D

After several transfusions with D-negative blood, an O Rh-negative women was apparently sensitized to the C and D antigens. In her prenatal workup, it became evident that she had in fact not been sensitized to D but to G, which initially appeared as anti-D plus anti-C. This sensitization pattern is an unusual occurrence in itself. Moreover, the fetus was affected significantly and was delivered at 32 1/2 weeks with moderate hemolytic disease of the newborn. Consideration is given to points regarding current methods of screening Rh-negative women for transfusion, the use of anti-Rh immune globulin in patients sensitized to anti-C and anti-G, medicolegal implications, and continuous attention to the risk-benefit ratio in decisions to use transfusions.     

Clinical Practice Guidelines for Prenatal Aneuploidy Screening and Diagnostic Testing from Korean Society of Maternal-Fetal Medicine: (2) Invasive Diagnostic Testing for Fetal Chromosomal Abnormalities

The Korean Society of Maternal Fetal Medicine proposed the first Korean guideline on prenatal aneuploidy screening and diagnostic testing, in April 2019. The clinical practice guideline (CPG) was developed for Korean women using an adaptation process based on good-quality practice guidelines, previously developed in other countries, on prenatal screening and invasive diagnostic testing for fetal chromosome abnormalities. We reviewed current guidelines and developed a Korean CPG on invasive diagnostic testing for fetal chromosome abnormalities according to the adaptation process. Recommendations for selected 11 key questions are: 1) Considering the increased risk of fetal loss in invasive prenatal diagnostic testing for fetal genetic disorders, it is not recommended for all pregnant women aged over 35 years. 2) Because early amniocentesis performed before 14 weeks of pregnancy increases the risk of fetal loss and malformation, chorionic villus sampling (CVS) is recommended for pregnant women who will undergo invasive prenatal diagnostic testing for fetal genetic disorders in the first trimester of pregnancy. However, CVS before 9 weeks of pregnancy also increases the risk of fetal loss and deformity. Thus, CVS is recommended after 9 weeks of pregnancy. 3) Amniocentesis is recommended to distinguish true fetal mosaicism from confined placental mosaicism. 4) Anti-immunoglobulin should be administered within 72 hours after the invasive diagnostic testing. 5) Since there is a high risk of vertical transmission, an invasive prenatal diagnostic testing is recommended according to the clinician''s discretion with consideration of the condition of the pregnant woman. 6) The use of antibiotics is not recommended before or after an invasive diagnostic testing. 7) The chromosomal microarray test as an alternative to the conventional cytogenetic test is not recommended for all pregnant women who will undergo an invasive diagnostic testing. 8) Amniocentesis before 14 weeks of gestation is not recommended because it increases the risk of fetal loss and malformation. 9) CVS before 9 weeks of gestation is not recommended because it increases the risk of fetal loss and malformation. 10) Although the risk of fetal loss associated with invasive prenatal diagnostic testing (amniocentesis and CVS) may vary based on the proficiency of the operator, the risk of fetal loss due to invasive prenatal diagnostic testing is higher in twin pregnancies than in singleton pregnancies. 11) When a monochorionic twin is identified in early pregnancy and the growth and structure of both fetuses are consistent, an invasive prenatal diagnostic testing can be performed on one fetus alone. However, an invasive prenatal diagnostic testing is recommended for each fetus in cases of pregnancy conceived via in vitro fertilization, or in cases in which the growth of both fetuses differs, or in those in which at least one fetus has a structural abnormality. The guidelines were established and approved by the Korean Academy of Medical Sciences. This guideline is revised and presented every 5 years.     

Rh阴性孕妇44例临床分析

目的Rh血型不合是我国新生儿溶血病的主要原因之一,其病情往往较重甚至出现胎儿水肿,胎死宫内。本研究主要探讨Rh阴性孕妇孕期处理及妊娠结局。方法回顾分析1999年1月至2004年12月我院发现的Rh阴性孕妇44例,观察其临床特点,孕期处理及妊娠结局。随机抽取同期分娩的无合并症并发症妇女44例作为对照组。结果Rh阴性孕妇的羊水粪染,新生儿窒息,低体重儿,新生儿转入重症监护室及产后出血的发生率分别为11.36%,4.55%,11.36%,9.09%,13.6%与对照组比较无显著差异。早产及新生儿高胆红素血症的发生率均为15.9%,与对照组比较有显著差异。经多元回归分析发现发生血型不合的危险因素为死胎史及抗-D抗体效价大于1∶16。结论对Rh阴性孕妇进行系统管理(包括产检常规查Rh血型,定期检测抗-D抗体效价,系列B超检查,羊水检查及脐带穿刺)及时治疗(包括抗-D免疫球蛋白,宫内输血,及时终止妊娠)可明显改善围生儿预后,对提高产科质量有重要意义。     

Rh阴性孕妇外周血基因鉴定胎儿RhD血型的方法

目的建立可靠有效的检测孕妇外周血中胎儿游离DNA的方法,探讨其在非创伤性产前诊断中的价值。结论采用PCR技术对5例RhD阴性孕妇外周血血浆DNA进行分析,然后对出生后婴儿脐带血进行Rh血清学表型分析,回顾性评价无创性预测胎儿RhD基因分析的准确性。结果 5例RhD阴性血浆标本扩增出180bp与156bp两条特异性片段,产后新生儿脐血血清学检测结果为RhD阳性,与PCR检测结果完全吻合。结论孕妇外周血浆中胎儿游离DNA的测定能实现对胎儿RhD基因型的预测判定。孕妇外周血检测胎儿DNA可应用于非创伤性产前诊断。     

黏多糖病Ⅱ型的产前诊断

目的 应用胎儿羊水细胞艾杜糖-2-硫酸酯酶(iduronate-2-sulfatase,IDS)活性测定和基因突变检测的方法,对2例黏多糖病Ⅱ型(mucopolysaccharidosis typeⅡ,MPSⅡ)高危妊娠孕妇进行产前诊断.方法 胎儿羊水细胞培养,测定其IDS活性,并抽取羊水细胞基因组DNA,做胎儿性别鉴定和IDS基因突变检测.结果 2例胎儿羊水细胞IDS活性均明显下降,基因测序发现1例男性胎儿为IDS突变半合子,另1例女性胎儿为IDS基因突变携带者.结论 胎儿羊水细胞酶活性测定结合基因突变分析是一种准确、可靠、灵敏的MPSⅡ产前诊断方法,可对高危妊娠孕妇作出快速的产前诊断.     

Implementation of Low Titer Whole Blood for French overseas operations: O positive or negative products in massive hemorrhage?

Massive hemorrhage is the leading preventable cause of death during military operations. During these operations, the delay between initial treatment and arrival at a surgical facility is considerably longer than in Metropolitan settings. This increased prehospital period requires the availability of blood products during the prehospital stage and justifies the availability of Low Titer Whole Blood (LTOWB) for surgical facilities. This product is a fully authorized labile blood product processed by the French Military Blood Institute according to French regulations. Its shelf life is 21 days when stored between 2 and 6 °C. It provides the three products necessary for the transfusion management of war injuries in a single product and in physiological proportions. The low anti-A and anti-B titers (<1/64) make LTOWB compatible with any recipient. However, the RhD antigen remains an issue due to its potential harm in cases of transfusion to a D-negative childbearing-age woman due to the potential risk of fetal–maternal incompatibility. This risk must be balanced with the availability of D-negative blood products. Considering the epidemiology of war injuries and LTOWB use guidelines, in addition to the current knowledge on anti-RhD fetal–maternal alloimmunization, the harm–benefit assessment favors the use of RhD-positive LTOWB during overseas operations. Follow-up of childbearing recipients and setup of countermeasures to prevent alloimmunization in those cases remain key points of transfusion safety.     

The Role of Immunoglobulins in Neonatal Rhesus Haemolytic Disease

Rhesus (Rh) isoimmunisation is the most common form of severe haemolytic disease of the newborn (HDN). The introduction of prophylaxis with anti-D Rh0 immunoglobulin (anti-D) has resulted in a marked reduction in the sensitisation of Rh-negative women and deaths attributable to Rh HDN. The sensitisation rate could be further decreased if there was strict adherence to the guidelines for administration of anti-D prophylaxis. Whether additional prophylaxis at 28 and 34 weeks of gestation would be cost effective is controversial. Intrauterine transfusions to treat fetal anaemia, postnatal exchange transfusions and phototherapy are all part of the standard management of affected individuals. Intravenous immunoglobulin given to pregnant women can reduce fetal haemolysis, and when administered to neonates with Rh isoimmunisation has been associated with a reduction in the requirement for exchange transfusion. There are, however, potential risks of immunoglobulin administration, including haemolysis due to the presence of anti-A or anti-B antibodies, allergy and the transmission of disease.     

唐氏综合征高危孕妇血浆游离胎儿DNA的检测

目的探讨实时荧光定量PCR(RQ-PCR)检测孕妇血浆游离胎儿DNA;在筛查唐氏综合征高危孕妇中的应用。方法采用RQ-PCR检测22例唐氏综合征高危孕妇及20例低危孕妇血浆中GA;PDH及SRY水平,2-△△Ct法分析两组间的差异。结果 22例孕男胎均检出SRY基因,20例孕女胎中出现2例假阳性,高危组游离胎儿DNA;水平明显高于低危组(P=0.006,<0.05),比值为2.79。结论孕妇血浆游离胎儿DNA;的定量检测在唐氏综合征筛查中有重要价值。     

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.     

荧光定量PCR检测孕妇外周血中游离胎儿DNA在子痫前期中的价值

目的探讨孕妇外周血中的游离胎儿DNA水平作为子痫前期预测指标的可能。方法普通PCR检测子痫前期孕妇22例和正常孕妇25例的血浆中Y染色体性别决定基因（SRY）,然后应用荧光定量PCR技术对有SRY基因表达者的胎儿DNA水平进行定量分析并比较子痫前期孕妇组和正常孕妇组胎儿DNA水平的差异。结果22例子痫前期患者及25例对照组中各有12例出现SRY阳性信号。子痫前期组的胎儿DNA水平明显高于正常对照组（P〈0.01）。结论孕妇外周血中的游离胎儿DNA可望作为预测子痫前期的指标之一。     

Parvovirus B-19 infections

Although HPV infection is usually benign in healthy people, in patients with hereditary anemias and in patients genetically prone toward arthritis, infection with HPV can be of significant consequence. In pregnant females, HPV infection represents a serious health risk to the fetus. Infection with HPV may replace rubella as the chief threat in pregnant women. The increasing frequency of nonimmune hydrops fetalis over the previous decade coupled with the potential for parvovirus induced fetal abnormalities has prompted medical personnel to address this issue. It has been proposed that HPV testing be elevated to routine screening status and, therefore, to include this assay in the TORCH series. Development of a vaccine appears warranted. Efforts to find a suitable cell system for propagating human parvoviruses have been unsuccessful. The virus does infect erythroid progenitor cells, producing a conspicuous cytotoxic effect, but recovery of infectious virus has not yet been achieved. Continued research along these lines and further clarification of the relationship of this agent with human disease is necessary.     

Evaluation of the enzyme test for the detection of clinically significant red blood cell antibodies during pregnancy

In the Croatian transfusion medicine, no general agreement has yet been achieved whether red blood cell (RBC) Rhesus (Rh) antibodies detected during pregnancy only by enzyme tests can cause hemolytic disease of the newborn (HDN). Results of the detection of clinically significant RBC antibodies by low-ionic-strength additive solution antiglobulin test (LISS-IAT) and trypsin enzyme test in 22,947 pregnant women are presented. All pregnant women in whom clinically significant RBC antibodies (RBC-CSA) were detected by LISS-IAT and/or enzyme tests were followed and observed during pregnancy. The women who had enzyme-only anti-D antibodies in their serum were followed up during subsequent pregnancies. Out of 302 positive results obtained by both techniques, irregular clinically significant enzyme-only antibodies (anti-RhD and anti-RhE specificity) were detected in 14 (4.6%) pregnant women. None of 11 RhD positive newborns whose mothers had enzyme-only anti-D antibodies, had signs of HDN after delivery. In these 11 women, anti-D antibodies were detected by LISS-IAT in the first trimenon of subsequent pregnancy. Nine infants born from subsequent pregnancies to women who had previously had enzyme-only anti-D, had clinical signs of HDN. The authors concluded that there is no need for enzyme tests in prenatal testing because enzyme tests are not reliable in the prediction of HDN.