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.

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.