Antenatal RHD screening to guide antenatal anti-D immunoprophylaxis in non-immunized D- pregnant women.

In pregnancy, D- pregnant women may be at risk of becoming immunized against D when carrying a D+ fetus, which may eventually lead to hemolytic disease of the fetus and newborn. Administrating antenatal and postnatal anti-D immunoglobulin prophylaxis decreases the risk of immunization substantially. Noninvasive fetal RHD genotyping, based on testing cell-free DNA extracted from maternal plasma, offers a reliable tool to predict the fetal RhD phenotype during pregnancy. Used as a screening program, antenatal RHD screening can guide the administration of antenatal prophylaxis in non-immunized D- pregnant women so that unnecessary prophylaxis is avoided in those women who carry a D- fetus. In Europe, antenatal RHD screening programs have been running since 2009, demonstrating high test accuracies and program feasibility. In this review, an overview is provided of current state-of-the-art antenatal RHD screening, which includes discussions on the rationale for its implementation, methodology, detection strategies, and test performance. The performance of antenatal RHD screening in a routine setting is characterized by high accuracy, with a high diagnostic sensitivity of ≥99.9 percent. The result of using antenatal RHD screening is that 97-99 percent of the women who carry a D- fetus avoid unnecessary prophylaxis. As such, this activity contributes to avoiding unnecessary treatment and saves valuable anti-D immunoglobulin, which has a shortage worldwide. The main challenges for a reliable noninvasive fetal RHD genotyping assay are low cell-free DNA levels, the genetics of the Rh blood group system, and choosing an appropriate detection strategy for an admixed population. In many parts of the world, however, the main challenge is to improve the basic care for D- pregnant women.

Noninvasive fetal blood group antigen genotyping.

Noninvasive fetal blood group antigen genotyping serves as a diagnostic tool to predict the risk of hemolytic disease of the fetus and newborn in pregnancies of immunized women. In addition, fetal RHD genotyping is used as an antenatal screening to guide targeted use of immunoglobulin prophylaxis in non-immunized RhD negative, pregnant women. Based on testing of cell-free DNA extracted from maternal plasma, these noninvasive assays demonstrate high performance accuracies. Consequently, noninvasive fetal blood group antigen genotyping has become standard care in transfusion medicine.

Prediction of ABO hemolytic disease of the newborn using pre- and perinatal quantification of maternal anti-A/anti-B IgG titer.

BACKGROUND: Hemolysis in fetus/newborns is often caused by maternal antibodies. There are currently no established screening procedures for maternal ABO antibodies harmful to fetus/newborn. We investigated the clinical significance, and predictive value of maternal anti-A/B titer for hyperbilirubinemia in ABO-incompatible newborns. METHODS: We conducted a case-control study of blood group O mothers and their ABO-compatible (O) vs. -incompatible (A/B) newborns receiving phototherapy, and of ABO-incompatible newborns receiving phototherapy vs. no phototherapy. Newborn data and treatment modalities were recorded, and total serum bilirubin and hemoglobin were measured. Maternal anti-A/B immunoglobulin-γ (IgG) titers were measured prenatally and perinatally, and negative and positive predictive values (NPV, PPV) were calculated to assess the risk of developing hyperbilirubinemia requiring phototherapy. RESULTS: We found a significantly higher maternal IgG antibody titer in the case group (p < 0.001). Maternal anti-A/B titers at first trimester had modest predictive values: NPV = 0.82 and PPV = 0.65 for neonatal hyperbilirubinemia; titers at birth improved the predictive values: NPV = 0.93 and PPV = 0.73. Newborn hemoglobin was significantly lower in incompatibles compared to compatibles (p = 0.034). Furthermore, increased anti-A/B IgG production during pregnancy was associated with hyperbilirubinemia and hemolysis in incompatible newborns. CONCLUSIONS: There was a significant association between maternal anti-A/B IgG titer and hyperbilirubinemia requiring treatment. IMPACT: Maternal anti-A/B IgG titer in the first trimester and at birth is predictive of hemolytic disease of the ABO-incompatible newborn. Increased IgG anti-A/B production throughout pregnancy in mothers to ABO-incompatible newborns developing hyperbilirubinemia contrasts a constant or reduced production in mothers to newborns not developing hyperbilirubinemia. Screening tools available in most immunohematology laboratories can identify clinically important IgG anti-A/B. Use of maternal samples taken at birth yielded NPV = 0.93 and PPV = 0.73.

Targeted Rhesus immunoglobulin for RhD-negative women undergoing an induced abortion: A clinical pilot study.

INTRODUCTION: The aim of this clinical pilot study was to examine the accuracy of noninvasive fetal RHD genotyping in early pregnancy (8+0  to 11+6  weeks) and to clarify whether targeted administration of Rhesus immunoglobulin (RhIg) is possible for women undergoing an induced abortion such that unnecessary injections can be avoided. The study examines the correlation between gestational age and the amount of cell-free fetal DNA in maternal plasma, the fetal fraction of DNA and whether transportation time or body mass index affects these parameters. MATERIAL AND METHODS: Fifty-two RhD-negative women undergoing a surgically induced abortion were included. A maternal blood sample was collected prior to the abortion and a tissue sample was collected from the placental part of the abortion material after the intervention. Fetal RhD type was determined by PCR analysis of cell-free fetal DNA extracted from maternal plasma and on DNA from the tissue sample, with the latter providing a reference standard. Copies of RHD/mL were determined on RHD-positive samples and the fetal fraction of DNA was calculated. RESULTS: We demonstrated complete concordance between results from plasma and tissue, with 31 RhD-positive and 21 RhD-negative samples, corresponding to 40% being RhD-negative, specificity 100% [95% confidence interval (CI) 88.8-100] and sensitivity 100% (95% CI 83.9-100). We found no significant correlation between gestational age and the amount or the fraction of cell-free fetal DNA in maternal plasma, nor did we find that transportation time or BMI significantly affected these factors in this setup. CONCLUSIONS: Fetal RHD genotyping can be accurately performed from the 8th week of gestation and unnecessary injections of RhIg can be avoided for women undergoing an induced abortion. A larger study is needed to determine a more accurate sensitivity for the analysis early in pregnancy.

High levels of fetal DNA are associated with increased risk of spontaneous preterm delivery.

OBJECTIVE: To assess whether spontaneous preterm delivery can be predicted from the amount of cell free fetal DNA (cffDNA) as determined by routine fetal RHD genotyping at 25 weeks' gestation. STUDY DESIGN: Cohort study including RhD negative women participating in a routine RHD screening programme. A standard dilution curve was used to quantify the amounts of cffDNA. Values above the 95(th) centile for the study population defined high levels of cffDNA. RESULTS: We found a highly significant association between preterm delivery and cffDNA levels above the 95(th) centile (p = 0.002). Using logistic regression, women with high levels of cffDNA had an odds ratio of 6.3 (95% confidence interval: 1.9-20.9) for preterm delivery before 37 weeks and an odds ratio for delivery before 34 weeks of 16.6 (95% confidence interval: 3.2-84.7) when adjusting for gestational age at sampling, body mass index and previous miscarriages/terminations of pregnancy. CONCLUSION: High levels of cffDNA at 25 weeks are associated with increased risk of spontaneous preterm delivery.

Massive fetomaternal hemorrhage: clearance of fetal red blood cells after intravenous anti-D prophylaxis monitored by flow cytometry.

BACKGROUND: The clearance of D+ red blood cells (RBCs) from the circulation in D- individuals mediated by passively administered anti-D occurs by opsonization with the antibody and subsequent removal in the spleen. Few data exist on the kinetics of clearance of large volumes of D+ RBCs from the maternal circulation by anti-D in clinical cases of massive fetomaternal hemorrhage (FMH). CASE REPORT: A 33-year-old D- woman delivered a D+ female infant by emergency cesarean section for suspected fetal anemia. A massive FMH, initially estimated to be approximately 142 mL of RBCs, was found. In addition to the standard dose of intramuscular (IM) anti-D (300 microg) given immediately after delivery, 2700 microg of anti-D was administered intravenously (IV). The clearance of D+ fetal cells from the maternal circulation was monitored by flow cytometry in samples obtained on a daily basis using anti-D. The mother had no detectable anti-D 6 months after delivery. RESULTS: No clearance of fetal cells was apparent after the insufficient dose of IM anti-D. The IV administration of anti-D caused accelerated clearance of D+ fetal RBCs with a t1/2 of 24.5 hours. D+ reticulocytes comprised 4.2 percent of all D+ cells in the maternal circulation at delivery suggesting acute fetal blood loss. CONCLUSIONS: The approach used in this report allowed a detailed analysis of the kinetics related to the clearance of fetal D+ RBCs. Simultaneous measurements of fetal reticulocytes and fetal RBCs in maternal blood may establish the timing of an FMH.

Mosaicism due to myeloid lineage restricted loss of heterozygosity as cause of spontaneous Rh phenotype splitting.

Spontaneous Rh phenotype alteration interferes with pretransfusion and prenatal blood group examinations and may potentially indicate hematologic disease. In this study, the molecular background of this biologic phenomenon was investigated. In 9 patients (3 with hematologic disease), routine RhD typing showed a mixture of D-positive and D-negative red cells not attributable to transfusion or hematopoietic stem-cell transplantation. In all patients, congenital and acquired chimerism was excluded by microsatellite analysis. In contrast to D-positive red cells, D-negative subpopulations were also negative for C or E in patients genotyped CcDdee or ccDdEe, respectively, which suggested the presence of erythrocyte precursors with an apparent homozygous cde/cde or hemizygous cde/- genotype. Except for one patient with additional Fy(b) antigen anomaly, no other blood group systems were affected. RH genotyping of single erythropoietic burst-forming units, combined with microsatellite analysis of blood, different tissues, sorted blood cell subsets, and erythropoietic burst-forming units, indicated myeloid lineage-restricted loss of heterozygosity (LOH) of variable chromosome 1 stretches encompassing the RHD/RHCE gene loci. Fluorescent in situ hybridization studies indicated that LOH was caused by either somatic recombination or deletion. Therefore, most cases of spontaneous Rh phenotype splitting appear to be due to hematopoietic mosaicism based on LOH on chromosome 1.