Severity of haemolytic disease of the fetus and newborn in patients with a history of intrauterine transfusions in a previous pregnancy: A nationwide retrospective cohort study.

OBJECTIVE: Pregnant women who received at least one intrauterine transfusion (IUT) for haemolytic disease of the fetus and newborn (HDFN) in the preceding pregnancy are presumed to have a high likelihood of requiring IUTs again, often starting at an earlier gestational age. Our aim was to quantify these risks in a large national cohort. DESIGN: Retrospective cohort study of a nationwide Dutch database. SETTING: The Netherlands. POPULATION: All women treated in The Netherlands with IUTs for Rhesus D (RhD)- or Kell-mediated HDFN between 1999 and 2017 and their follow-up pregnancies were included. Pregnancies with an antigen-negative fetus were excluded. METHODS: Electronic patient files were searched for the number and gestational age of each IUT, and analysed using descriptive statistics and linear regression. MAIN OUTCOME MEASURES: Percentage of women requiring one or more IUTs again in the subsequent pregnancy, and gestational age at first IUT in both pregnancies. RESULTS: Of the 321 women in our study population, 21% (69) had a subsequent ongoing pregnancy at risk. IUTs were administered in 86% (59/69) of cases. In subsequent pregnancies, the median gestational age at first IUT was 3 weeks earlier (interquartile range -6.8 to 0.4) than in the preceding pregnancy. CONCLUSIONS: Our study shows that pregnant women with a history of IUTs in the previous pregnancy are highly likely to require IUTs again, and on average 3 weeks earlier. Clinicians need to be aware of these risks and ensure timely referral, and close surveillance from early pregnancy onwards. Additionally, for women with a history of IUT and their caregivers, this information is essential to enable adequate preconception counselling.

A case of haemolytic disease of the fetus and newborn attributed to a novel antigen in the RHAG blood group system.

BACKGROUND AND OBJECTIVES: A newborn presented with jaundice in Thailand. The cord red cells tested positive by direct antiglobulin test (DAT) for an unknown maternal red cell antibody. Initial blood group sequencing suggested that the infant carried a novel variant RHAG c.140T>C, responsible for a low-prevalence antigen in the RHAG blood group system (ISBT 030). We report here on testing of samples from the infant's parents and older sibling to define a new antigen in the RHAG system. MATERIALS AND METHODS: Massive parallel sequencing (MPS) using a custom-designed panel was performed on all four family members. Extended serological testing was also performed to determine whether family members with the same variant as the infant showed reactivity with the antibody in the maternal plasma. RESULTS: We identified a novel single nucleotide variant (SNV) (RHAG c.140T>C, p.[Phe47Ser]) in samples from three of the four family members tested (the infant, the older sibling and the father). The variant was not detected in the mother's sample. Maternal plasma showed positive agglutination with all family members tested; however, when tested with routine panel cells, no reactivity was observed. CONCLUSION: This case study showed that the presence of the novel variant (RHAG c.140T>C), encoding a p.(Phe47Ser) change in the RhAG glycoprotein, was the apparent cause of incompatibility between maternal plasma and that of red cells from the proband, father and older sibling of the proband. We propose this variant to be a new low-prevalence antigen in the RHAG blood group system.

Hemolytic disease of the fetus and newborn caused by anti-sD antibody in a GP.Mur/Mur Thai mother and review of the prevalence of sD in Thai blood donors.

BACKGROUND: Low-prevalence antigen sD (MNS23) is encoded by GYPB c.173C > G. Hemolytic disease of the fetus and newborn (HDFN) due to anti-sD is rare. A mother delivered a newborn whose red blood cells (RBCs) were DAT-positive and was later diagnosed with HDFN. Serum from the mother was incompatible with the father's RBCs and was used to screen 184 Thai blood donors. This study aimed to investigate the cause of HDFN in a Thai family and determine the prevalence of sD in Thai blood donors. MATERIALS AND METHODS: Three family members and four blood donors were investigated in the study. Massively Parallel Sequencing (MPS) was used for genotyping. Standard hemagglutination techniques were used in titration studies, phenotyping, and enzyme/chemical studies. Anti-s, anti-Mia , anti-JENU, and anti-sD reagents were used in serological investigations. RESULTS: The mother was GYP*Mur/Mur. The father and the four donors were GYPB*s/sD predicting S - s + sD +. The baby was GYP*Mur/sD and his RBCs were Mia +, s + w with anti-s (P3BER) and JENU+w . RBCs from two GYPB*sD -positive blood donors reacted with anti-sD (Dreyer). Proteolytic enzyme α-chymotrypsin-treated sD + cells did not react with anti-sD (Wat) produced by the GP.Mur/Mur mother but reacted with the original anti-sD (Dreyer). DISCUSSION: This is the first report of HDFN due to anti-sD in the Asian population. The genotype frequency for GYPB*sD in a selected Thai blood donor population is 2.2% (4/184). Anti-sD should be considered in mothers with Southeast Asian or East Asian background when antibody identification is unresolved in pregnancies affected by HDFN.

The spectrum of ABO haemolytic disease of the fetus and newborn in neonates born to group O mothers.

BACKGROUND AND OBJECTIVES: ABO haemolytic disease of the fetus and newborn (HDFN) is a lesser recognized entity; however, the severity may vary in neonates. This prospective observational study was performed to determine the severity and risk of ABO-HDFN in neonates born to O group mothers. MATERIALS AND METHODS: A total of 260 neonates born to non-alloimmunized blood group O mothers were recruited. Blood group O neonates were excluded from the study. Neonatal direct antiglobulin test (DAT) was performed using the column agglutination technique. They were monitored for clinical and laboratory parameters and followed up at 6-8 weeks. The maternal anti-A and anti-B titres (IgM and IgG) were also done. RESULTS: A total of 176 neonates with blood group A (77/260; 29.6%) and B (99/260; 38.1%) were finally included in the study, and 15 (8.5%) of them were DAT positive. Overall, 26.7% (47/176) neonates received phototherapy, 172 (97.7%) survived and none required readmission. The median (inter-quartile range [IQR]) maternal IgG anti-B titre (32 [32-64]) was significantly higher (p < 0.001) than the IgG anti-A titre (16 [8-64]). The maximum total serum bilirubin in neonates had a significant positive association with neonatal birth weight (p = 0.045), positive DAT (p = 0.006) and requirement of phototherapy (p < 0.001). The relative risk (95% CI) of a DAT-positive neonate requiring phototherapy was 4.55 (3.12-6.33). CONCLUSION: The frequency of ABO incompatibility in neonates born to group O mothers was 67.69% (176/260). The maternal IgG titre of ≥64 could be a good predictor for identifying the neonates at risk of developing hyperbilirubinaemia requiring phototherapy.

The role of non-invasive prenatal testing (NIPT) for fetal blood group typing in Australia.

Maternal alloimmunisation against red blood cell antigens can cause haemolytic disease of the fetus and newborn (HDFN). Although most frequently caused by anti-D, since the implementation of rhesus D (RhD) immunoglobulin prophylaxis, other alloantibodies have become more prevalent in HDFN. Recent advances in non-invasive prenatal testing (NIPT) have allowed early prediction of HDFN risk in alloimmunised pregnancies and allow clinicians to focus health resources on those pregnancies that require intervention. This article aims to provide updates on the current status of NIPT in Australia as both a diagnostic and screening tool in pregnancy.

Optimization of diagnostic strategy for non-invasive cell-free foetal RHD determination from maternal plasma.

BACKGROUND AND OBJECTIVES: The aim of the study was to optimize routine non-invasive prenatal detection of fetal RHD gene from plasma of RhD-negative pregnant women (the median of gestational age was 25 weeks, range 10-38) to detect RhD materno-fetal incompatibility and to avoid the redundant immunoprophylaxis. MATERIALS AND METHODS: Initially only one exon of RHD gene (exon 10) was investigated in 281 plasma samples (144 verified after delivery), in the second phase three RHD exons (5, 7, 10) were analyzed in 246 samples of plasma and maternal genomic DNA (204 verified) by real-time PCR method. Detection of Y-chromosomal sequence DYS-14 and five X-chromosomal insertion/deletion polymorphisms was used to confirm the fetal cfDNA detectability in plasma. Specific polymorphisms in RHD gene were detected by sequence-specific primer PCR in nine samples. RESULTS: When only the RHD exon 10 was tested, 2·8% of verified samples were false positive and 3·5% false negative. With three RHD exons (5, 7, 10) and maternal genomic DNA testing, only one case was false negative (0·5%). Nine samples were inconclusive due to RHD-positive results in maternal genomic DNA. These samples were analyzed for specific mutations in RHD gene. Combination of both methods for fetal cfDNA verification succeeded in 75% of tested group. CONCLUSION: Implementation of analysis of three RHD exons and maternal genomic DNA to routine practice lowers dramatically the ratio of false positive and negative results. This method enables more accurate determination of fetal RHD status with the reduction of unnecessary medical care and RhD immunoprophylaxis.

The risk to future pregnancies of transfusing Rh(D)-negative females of childbearing potential with Rh(D)-positive red blood cells during trauma resuscitation is dependent on their age at transfusion.

BACKGROUND: A risk assessment model for predicting the risk of haemolytic disease of the fetus and newborn (HDFN) in future pregnancies following the transfusion of Rh(D)-positive red blood cell (RBC)-containing products to females of childbearing potential (FCP) was developed, accounting for the age that the FCP is transfused in various countries. METHODS: The HDFN risk prediction model included the following inputs: risk of FCP death in trauma, Rh(D) alloimmunization rate following Rh(D)-positive RBC transfusion, expected number of live births following resuscitation, probability of carrying an Rh(D)-positive fetus, the probability of HDFN in an Rh(D)-positive fetus carried by an alloimmunized mother. The model was implemented in Microsoft R Open, and one million FCPs of each age between 18 and 49 years old were simulated. Published data from eight countries, including the United States, were utilized to generate country-specific HDFN risk estimates. RESULTS: The risk predictions showed similar characteristics for each country in that the overall risk of having a pregnancy affected by HDFN was higher if the FCP was younger when she received her Rh(D)-positive transfusion than if she was older. In the United States, the overall risk of HDFN if the FCP was transfused at age 18 was 3·4% (mild: 1·20%, moderate: 0·45%; severe: 1·15%; IUFD: 0·57%); the risk was approximately 0% if the FCP was 43 years or older at the time of transfusion. CONCLUSION: This model can be used to predict HDFN outcomes when establishing transfusion policies as it relates to the administration of Rh(D)-positive products for massively bleeding FCPs.

Clinical consequences of the extremely rare anti-PP1Pk isoantibodies in pregnancy: a case series and review of the literature.

BACKGROUND: The absence of the red cell antigens P, P1 and Pk , known as 'p', represents an extremely rare red cell phenotype. Individuals with this phenotype spontaneously form anti-PP1Pk isoantibodies, associated with severe haemolytic transfusion reactions, recurrent spontaneous abortion and haemolytic disease of the fetus and newborn (HDFN). METHODS: We report a series of four successful pregnancies in three women with anti-PP1Pk isoantibodies, one complicated by HDFN, another by intrauterine growth restriction, all managed supportively. We also review the literature regarding the management of pregnancy involving anti-PP1Pk isoimmunization. RESULTS: The literature surrounding anti-PP1Pk in pregnancy is limited to a very small number of case reports. The majority report management with therapeutic plasma exchange (TPE) with or without intravenous immunoglobulin. The relationship between titre and risk of pregnancy loss remains unclear, though a history of recurrent pregnancy loss appears important. Although a positive cord blood direct antiglobulin test is frequently noted, clinically significant HDFN appears uncommon, though possible. CONCLUSION: Early initiation of TPE in high risk patients should be strongly considered. If possible, pregnancies should be managed in a high-risk obstetric or maternal fetal medicine service. The fetus should be monitored closely with interval fetal ultrasound and middle cerebral artery peak systolic volume Doppler to screen for fetal anaemia. Timely sourcing of compatible blood products is likely to be highly challenging, and both directed and autologous donation should be contemplated where appropriate. The International Red Cell Donor Panel may also provide access to compatible products.

Non-invasive prenatal testing for management of haemolytic disease of the fetus and newborn induced by maternal alloimmunisation.

Anti-D immunoglobulin prophylaxis reduces the risk of RhD negative women becoming alloimmunised to the RhD antigen and is a major preventative strategy in reducing the burden of haemolytic disease of the fetus and newborn (HDFN). HDFN also arises from other maternal red cell antibodies, with the most clinically significant, after anti-D, being anti-K, anti-c and anti-E. Among the 39 human blood group systems advanced genomic technologies are still revealing novel or rare antigens involved in maternal alloimmunisation. Where clinically significant maternal antibodies are detected in pregnancy, non-invasive prenatal testing (NIPT) of cell-free fetal DNA provides a safe way to assess the fetal blood group antigen status. This provides information as to the risk for HDFN and thus guides management strategies. In many countries, NIPT fetal RHD genotyping as a diagnostic test using real-time PCR has already been integrated into routine clinical care for the management of women with allo-anti-D to assess the risk for HDFN. In addition, screening programs have been established to provide antenatal assessment of the fetal RHD genotype in non-alloimmunised RhD negative pregnant women to target anti-D prophylaxis to those predicted to be carrying an RhD positive baby. Both diagnostic and screening assays exhibit high accuracy (over 99 %). NIPT fetal genotyping for atypical (other than RhD) blood group antigens presents more challenges as most arise from a single nucleotide variant. Recent studies show potential for genomic and digital technologies to provide a personalised medicine approach with NIPT to assess fetal blood group status for women with other (non-D) red cell antibodies to manage the risk for HDFN.

First report of the rare RhCE-depleted D--phenotype in sixteen people of Iranian origin.

BACKGROUND AND OBJECTIVES: In transfusion medicine, it may be a challenge to acquire compatible blood for patients who have clinically important alloantibodies to high-prevalence antigens. The aim of this study was to study prevalence of rare D-- phenotype in samples from patients and their relatives referred to the Immunohematology reference laboratory of the Iranian Blood Transfusion Organization and the detection and identification of the phenotype and associated antibodies, particularly in an antenatal setting. This is the first report of the cases evaluated by the IBTO and family studies of the D-- proposita in Iran and possibly the first attempted comprehensive study in the current transfusion-related literatures. MATERIALS AND METHODS: This retrospective cross-sectional study was carried out on 6720 pregnant women and individuals with difficult positive pretransfusion testing referred for ABO/Rh(D) typing and antibody screening during a period of 8 years from 2008 to December 2016 in the Immunohematology Reference Laboratory of the Iranian Blood Transfusion Organization, Tehran, Iran. RESULTS: During 2008 to December 2016, 16 persons from ten families were detected to have rare D-- phenotype. Anti-Rh17 and anti-c were identified in plasma of the 11 persons, including 10 females with a history of multiple unsuccessful pregnancy and the total number of 24 abortions and one male with history of blood transfusion vs. 5 individuals, including an unmarried single woman, 1 person with a history of first-time pregnancy and 3 persons with a history of multiple pregnancy, who showed no alloimmunization. Based on these collective findings, we interpreted these results as being confirmed as D-- phenotype (0.23%). CONCLUSION: Irrespective of Rh (D) group a serological antibody screening test is recommended to be required in a National prenatal testing guideline.

Factors associated with persistence of red blood cell antibodies in woman after pregnancies complicated by fetal alloimmune haemolytic disease treated with intrauterine transfusions.

Red blood cell (RBC) antibodies can persist for decades or decrease quickly to undetectable levels. Antibody persistence has not been systematically studied. Women whose children are treated with intrauterine transfusions (IUT) for haemolytic disease of the fetus (HDFN) often produce additional antibodies, which can be evoked by the intrauterine transfusion or by fetomaternal haemorrhage during the procedure. Factors associated with persistence of both the antibodies responsible for HDFN and additional antibodies were studied in 260 women whose children were treated with IUT between 1988 and 2008. They possessed 499 (205 anti-D and 294 non-D) antibodies after the last IUT. After a median follow-up of 8·7 years, all 260 antibodies primarily responsible for HDFN had persisted. Additional antibodies directed against antigens of the children persisted in 70·6%, and in 32·3% if they were not child-specific (P < 0·001). Antibodies induced by irradiated IUT persisted in only 7·1%. Multivariate analyses showed that non-HDFN antibody persistence was dependent on the antibody titre and specificity. In conclusion, persistence of antibodies mainly depends on antibody strength and specificity. Difference between fetal or non-fetal immunogens suggests maintenance of antigenic stimulation possibly by long-term fetomaternal chimerism.

Haemolytic disease of the fetus and newborn.

Haemolytic Disease of the Fetus and Newborn (HDFN) is caused by maternal alloimmunization against red blood cell antigens. In severe cases, HDFN may lead to fetal anaemia with a risk for fetal death and to severe forms of neonatal hyperbilirubinaemia with a risk for kernicterus. Most severe cases are caused by anti-D, despite the introduction of antental and postnatal anti-D immunoglobulin prophylaxis. In general, red blood cell antibody screening programmes are aimed to detect maternal alloimmunization early in pregnancy to facilitate the identification of high-risk cases to timely start antenatal and postnatal treatment. In this review, an overview of the clinical relevance of red cell alloantibodies in relation to occurrence of HDFN and recent views on prevention, screening and treatment options of HDFN are provided.

Low anti-RhD IgG-Fc-fucosylation in pregnancy: a new variable predicting severity in haemolytic disease of the fetus and newborn.

Haemolytic disease of the fetus and newborn (HDFN) may occur when maternal IgG antibodies against red blood cells (RBCs), often anti-RhD (anti-D) antibodies, cross the placenta and mediate the destruction of RBCs via phagocytic IgG-Fc-receptors (FcγR). Clinical severity is not strictly related to titre and is more accurately predicted by the diagnostically-applied monocyte-based antibody-dependent cellular cytotoxicity (ADCC), a sensitive test with relatively low specificity. This suggests that other factors are involved in the pathogenesis of HDFN. Binding of IgG to FcγR requires the N-linked glycan at position 297 in the IgG-Fc-region, consisting of several different glycoforms. We therefore systematically analysed IgG-derived glycopeptides by mass spectrometry from 70 anti-D IgG1 antibodies purified from the plasma of alloimmunized pregnant women. This revealed a variable decrease in Fc-fucosylation in the majority of anti-D IgG1 (even down to 12%), whereas the total IgG of these patients remained highly fucosylated, like in healthy individuals (>90%). The degree of anti-D fucosylation correlated significantly with CD16 (FcγRIIIa)-mediated ADCC, in agreement with increased affinity of defucosylated IgG to human FcγRIIIa. Additionally, low anti-D fucosylation correlated significantly with low fetal-neonatal haemoglobin levels, thus with increased haemolysis, suggesting IgG-fucosylation to be an important pathological feature in HDFN with diagnostic potential.

Iron status in infants with alloimmune haemolytic disease in the first three months of life.

BACKGROUND AND OBJECTIVES: Ferritin levels are often highly elevated at birth in neonates with alloimmune haemolytic disease of the fetus and newborn (HDFN). Data on ferritin levels in these infants in the first 3 months of life are lacking. Objective of this study was to examine the course of iron status and incidence of iron deficiency and overload in neonates with alloimmune HDFN up to 3 months of age. Secondary objective was to analyse bilirubin levels, liver enzymes and red-blood-cell indices in the same time period and the association with intrauterine transfusion (IUT). MATERIALS AND METHODS: Observational study of neonates with alloimmune HDFN admitted to our centre between November 2010 and March 2012. Data on iron status, bilirubin levels, liver enzymes and red-blood-cell indices up to 3 months of age were routinely collected and compared between neonates treated with and without IUT. RESULTS: Thirty-five infants with alloimmune HDFN were included. Iron overload occurred in 70% of neonates at birth and in 50% and 18% at the age of 1 and 3 months, respectively. No cases of iron deficiency at birth and only one case of iron deficiency at 3 months of age were found. No infants received iron therapy. Infants who received IUT had a significantly lower haemoglobin level and reticulocyte count and higher ferritin level at birth. CONCLUSION: The vast majority of neonates with alloimmune HDFN have iron overload at birth. Incidence of iron overload gradually decreases within the first 3 months without iron supplementation.