Effectiveness and costs of non-invasive foetal RHD genotyping in rhesus-D negative mothers: a French multicentric two-arm study of 850 women.

BACKGROUND: The determination of foetal Rhesus D (RHD) status allows appropriate use of IgRh prophylaxis by restricting its use to cases of RHD feto-maternal incompatibilities. There is a degree of uncertainty about the cost-effectiveness of foetal RHD determination, yet screening programs are being introduced into clinical practice in many countries. This paper evaluates the impact of non-invasive foetal Rhesus D (RHD) status determination on the costs of managing RHD-negative pregnant women and on the appropriate use of anti-D prophylaxis in a large sample of RHD-negative pregnant women using individual prospectively collected clinical and economic data. METHODS: A prospective two-armed trial of RHD negative pregnant women was performed in 11 French Obstetric Departments. Non-invasive foetal RHD genotyping was performed before 26 weeks' gestation in the experimental arm whereas the control arm participants received usual care. The costs associated with patient management in relation to their RHD negative status (biological tests, anti-D prophylaxis and visits) were calculated from inclusion to the end of the postpartum period. The costs of hospital admissions during pregnancy and delivery were also determined. RESULTS: A total of 949 patients were included by 11 centres between 2009 and 2012, and 850 completed follow-up, including medical and biological monitoring. Patients were separated into two groups: the genotyping group (n=515) and the control group (n=335). The cost of the genotyping was estimated at 140 euros per test. The total mean cost per patient was estimated at €3,259 (SD ± 1,120) and €3,004 (SD ± 1,004) in the genotyping and control groups respectively. The cost of delivery represented three quarters of the total cost in both groups. The performance of managing appropriately RHD negative anti-D prophylaxis was 88% in the genotyping group, versus 65% in the control group. Using the costs related to RHD status (biological tests, anti-D immunoglobulin injections and visits) the incremental cost-effectiveness ratio (ICER) was calculated to be €578 for each percentage gain in women receiving appropriate management. CONCLUSION: Early knowledge of the RHD status of the foetus using non-invasive foetal RHD genotyping significantly improved the management of RHD negative pregnancies with a small increase in cost. TRIAL REGISTRATION: Clinical trials registry- NCT00832962 -13 January 2009 - retrospectively registered.

Prenatal diagnosis of anoxic cerebral lesions caused by profound fetal anemia secondary to maternal red blood cell alloimmunization.

BACKGROUND: The long-term neurological prognosis of severe fetal anemia is usually considered favorable, especially when fetal hydrops regresses after successful in utero transfusion. CASES: We report two cases of prenatally diagnosed fetal cerebral anoxic lesions associated with severe fetal anemia despite appropriate and successful treatment by in utero transfusion. The two pregnancies were terminated. CONCLUSION: Profound fetal anemia may cause anoxic lesions of the fetal brain that may be diagnosed prenatally. If new onset ventriculomegaly is observed on ultrasonography after in utero transfusion for severe fetal anemia, anoxic lesions could be suspected.

Fatal hemolytic disease of the fetus and newborn associated with anti-Jr.

BACKGROUND: Jr(a) is a high-prevalence red cell (RBC) antigen. The clinical significance of anti-Jr(a) is controversial. When hemolytic disease of the fetus and newborn (HDFN) occurred, most reported cases were clinically mild. We report the first case of fatal HDFN due to anti-Jr(a). CASE REPORT: A 28-year-old Caucasian woman with transfusion history was monitored at the 29th week of pregnancy (G4P1). An ultrasound scan showed fetal cardiomegaly and hepatomegaly. An antibody directed against a high-prevalence antigen was detected, but without conclusive identification. An emergency cesarean section was performed at the 36th week. The newborn was hydropic and showed severe anemia. Death occurred 30 hours after birth. MATERIALS AND METHODS: Serologic methods were performed to investigate the mother's RBCs and serum. An in vitro functional cellular assay and semiquantitative measurement of anti-Jr(a) were used to determine the clinical significance of the antibody. RESULTS: Anti-Jr(a) was identified in the serum and Jr(a-) phenotype was confirmed. The anti-Jr(a) titer was 1024, with predominant immunoglobulin (Ig)G1 and minor IgG4 subclasses. The functional cellular assay was consistent with an antibody unlikely to cause HDFN. Semiquantitative measurement of anti-Jr(a) showed a reactivity equivalent to a 25 IU per mL (5 microg/mL) concentration of anti-D, a value associated with a significant risk of HDFN. CONCLUSION: This is the first documented case of fatal HDFN due to anti-Jr(a). Therefore, we recommend close monitoring of pregnant women with a high-titer anti-Jr(a), especially those with an incompatible transfusion history and/or multiple pregnancies. This case report provides new arguments about the clinical significance of anti-Jr(a) in the transfusion setting.

Incidence of hereditary spherocytosis in a population of jaundiced neonates.

As most of hereditary spherocytosis-affected individuals experience jaundice at birth, it seemed of interest to evaluate the proportion of hereditary spherocytosis in 402 severely jaundiced neonates with a bilirubinemia level prompting phototherapy. Red cell dehydration, a hallmark of spherocytosis whether constitutional or acquired, was demonstrated in 74 of them, among whom 23 disclosed a typical pattern of spherocytosis upon red cell deformability studies. Acquired spherocytosis of immune origin was diagnosed in 19/23 and hereditary spherocytosis in 4, making the proportion of hereditary spherocytosis-affected individuals among a severely jaundiced population of neonates amount to 1%, an incidence at least 30-fold that of the overall population.

Large-scale pre-diagnosis study of fetal RHD genotyping by PCR on plasma DNA from RhD-negative pregnant women.

BACKGROUND: The routine prenatal determination of fetal RhD blood group would be very useful in the management of pregnancies in RhD-negative women, as up to 40% of these pregnancies bear a RhD-negative fetus. The fetal DNA present in maternal plasma offers an opportunity for risk-free prenatal diagnosis. AIM: This study focused on the feasibility and accuracy of large-scale RhD fetal diagnosis in non-immunized and anti-D immunized RhD-negative women. METHODS: Plasma DNA was extracted from 893 RhD-negative pregnant women and amplified in exons 7 and 10 of the RHD gene using conventional and real-time PCR. The results were then compared with the RHD fetal genotype determined on amniotic cells and/or the RhD phenotype of the red blood cells of the infants at birth. RESULTS: After exclusion of 42 samples from women exhibiting a nonfunctional or rearranged RHD gene, fetal RhD status was predicted with a 99.5% accuracy. A strategy is also proposed to avoid the small number of false-positive and -negative results. CONCLUSION: Fetal RHD genotyping from maternal plasma DNA in different clinical situations may be used with confidence.

Prospective flow cytometric evaluation of nucleated red blood cells in cord blood units and relationship with nucleated and CD34(+) cell quantification.

BACKGROUND: Cord blood (CB) represents an alternate source of stem cells in transplantation. Nucleated red blood cells (NRBCs) are a physiological subset of CB population. Although it is important to have an accurate estimate of CD34(+) cell number, NRBCs could compromise white blood cell count and interfere with CD34(+) cell quantification. STUDY DESIGN AND METHODS: A total of 826 CB units were analyzed for total nucleated cells (TNCs), NRBCs, and CD34(+) cells by flow cytometry. NRBCs were also counted conventionally by manual microscopy. Percentages of CD34(+) cells corrected by NRBC count (CD34+c) were determined as follows: %CD34+c = CD34(+)/CD45(+) (x10(6))/(TNCs (x10(8)) - NRBCs (x10(8))). RESULTS: The mean percentages of CD34+ cells and NRBCs were 0.27 percent (range, 0.01%-1.25%) and 7.64 percent (range, 0.13%-84%), respectively. Comparison between flow cytometric and microscopic NRBC count showed a regression of y = 0.685 + 0.719x and a coefficient of determination of r(2) = 0.721. When corrected with NRBC count, the mean percentage of CD34(+) c cells was 0.295 percent (p = 0.0008 compared with CD34(+)%) and mean TNCc count was 14.8 x 10(8) (p < 10(-4) compared to TNC count). CONCLUSION: The determination of NRBCs with a flow cytometric method might represent a new strategy for providing satisfactory quality assurance controls of CB products.

Measurement of the affinity of anti-D in the serum of immunized mothers and in immunoglobulin preparations with unlabeled antibodies.

BACKGROUND: Few data are available on the affinity of maternal anti-D responsible for hemolytic disease of the fetus and the newborn (HDN) and of anti-D used for the prophylaxis of that disease. A method was recently described to measure the affinity (K(a)) of untagged anti-D monoclonal antibodies (MoAbs). In this work, the same method was applied to determine the K(a) of polyclonal anti-D. STUDY DESIGN AND METHODS: O R(1)r red blood cells (RBCs) were sensitized with increasing concentrations of native anti-D in serum samples from immunized mothers and donors and in RhIG preparations. At equilibrium, the amount of anti-D bound to RBCs was measured by enzyme-linked immunosorbent assay. Scatchard and Langmuir equations were used to determine Ka. RESULTS: The experimental data fitted well with the Scatchard equation (mean r2=0.95) but a better correlation was observed with the Langmuir equation (mean r2=0.99). The mean Ka of anti-D in 11 maternal serum samples, in 6 immunized donors, and in 5 lots of RhIG were 5.6x10(8) per M (from 2.8x10(8) to 12x10(8)/M), 3.9x10(8) per M (from 1.5x10(8) to 6.8x10(8)/M), and 3.4x10(8) per M (from 3.1x10(8) to 4.2x10(8)/M), respectively. The comparison of anti-D affinity in 5 cases of HDN with fetal anemia and in 6 cases of HDN with postnatal anemia showed no significant difference. CONCLUSION: The method previously described for anti-D MoAbs was applied to polyclonal anti-D present in the serum of immunized subjects and in immunoglobulin preparations. The experimental data fitted well with the Langmuir equation, and the affinity of polyclonal of anti-D was measured with accuracy.

Intravenous fetal exchange transfusion before 22 weeks of gestation in early and severe red-cell fetomaternal alloimmunization.

OBJECTIVES: To determine the perinatal outcome in severe red-cell fetomaternal alloimmunization. METHODS: Retrospective series of 32 affected fetuses treated with intravenous fetal exchange transfusion (IFET) before 22 weeks of gestation. The main outcome measures were the degree of fetal anemia, fetal transfusions and perinatal outcome. RESULTS: The first IFET was performed at 19.8 +/- 1.8 weeks of gestation. All fetuses were severely anemic and hemoglobin levels were not different between 20 hydropic and 12 nonhydropic fetuses (4.1 +/- 2.5 vs. 5.6 +/- 2.8 g/dl, p=0.33). The initial maternal anti-D level ranged from 4 to 76 microg/l and was not correlated to fetal anemia (r=-0.07). CONCLUSION: The overall perinatal survival rate was 78% compared to a previous perinatal loss rate excluding first pregnancies of 55.5%.

IgG1 and IgG3 anti-D in maternal serum and on the RBCs of infants suffering from HDN: relationship with the severity of the disease.

BACKGROUND: Anti-D IgG antibodies that are responsible for severe cases of HDN belong chiefly to IgG1 and IgG3 subclasses. The relationship between the concentrations of IgG1 anti-D and IgG3 anti-D in maternal serum and the amount bound to the surface of infants' RBCs is not known. In addition, the contribution of the two subclasses to the severity of HDN is not well established. STUDY DESIGN AND METHODS: Blood samples from 40 infants suffering from severe forms of HDN due to anti-D were collected before transfusion together with sera from their respective mother. The amount of total anti-D IgG as well as IgG1 anti-D and IgG3 anti-D on infants' RBCs and the concentration in maternal sera were determined by ELISA. RESULTS: The median percentages of IgG1 anti-D and of IgG3 anti-D in maternal sera were 90 and 10 percent, respectively, whereas on infants' RBCs they were 97 and 3 percent, respectively. The differences between maternal and infantile percentages were significant (p < 0.001). IgG1 and IgG3 anti-D bound to infants' RBCs increased concomitantly with the concentration of IgG1 and IgG3 anti-D in maternal sera. The severity of HDN correlated positively with the concentration of IgG1 anti-D in maternal sera, but negatively with the amount of IgG3 anti-D bound to infants' RBCs. In addition, the existence of a high proportion of IgG3 anti-D in maternal serum was associated with a delayed risk of fetal anemia. CONCLUSION: The proportion of IgG3 anti-D relative to the total anti-D IgG on infants' RBCs is only one- third of the proportion present in maternal serum. The study of the correlations between the amount of IgG1 anti-D and IgG3 anti-D and the severity of HDN suggests that IgG1 anti-D are more important than IgG3 anti-D in the pathogenesis of fetal anemia.

Sub-lethal hydrops as a manifestation of dehydrated hereditary stomatocytosis in two consecutive pregnancies.

Dehydrated hereditary stomatocytosis (DHS) is a rare congenital hemolytic anemia mapping to 16q23-q24. We showed recently that it is part of a pleiotropic syndrome likely to display pseudohyperkalemia and/or different forms of fetal and placental fluid collections. Here, we report a woman with DHS. She had two consecutive pregnancies associated with severe fetal hydrops. Hydrops would probably have been lethal in the absence of appropriate removal of ascites and excess amniotic fluid. In utero exchange transfusion, performed once, was useless, because anemia was not pronounced enough to be the cause of the hydrops. In both newborns, ascites resolved within a week following birth and never recurred. The association of hydrops and hemolytic anemia suggests the possibility of DHS. Symptomatic treatment of the hydrops assists survival until spontaneous resorption occurs.