The prenatal identification of fetal compatibility in neonatal alloimmune thrombocytopenia using amniotic fluid and variable number of tandem repeat (VNTR) analysis

Abstract

Most severe episodes of neonatal alloimmune thrombocytopenic purpura (NATP) are caused by antiplatelet alloantibodies against the HPA-1a (PlA1) antigen. However, half of subsequent fetuses produced from a HPA-1a/b father (genotypic frequency 28%) will result in a child who is not affected. Some investigators manage NATP by confirming the fetal platelet phenotype using percutaneous umbilical cord sampling, a procedure that carries a low but real risk of fetal morbidity and mortality. More recently, physicians determine the fetal platelet antigen genotype using DNA derived from amniotic fluid or chorionic villus samples. All therapy is withdrawn for a fetus who genotypes as HPA-1b/b. However, since the fetus is the same genotype as the mother, there can be uncertainty about the origin of the genetic material and thus the validity of the fetal genotype. The inappropriate withdrawal of therapy for a erroneously genotyped fetus could be fatal, and consequently many physicians advocate fetal HPA-1 phenotyping with confirmation using percutaneous umbilical blood sampling. In this report we describe the management of two pregnancies with previously affected infants due to anti-HPA-1a alloantibodies. Both husbands were HPA-1a/b. For the current pregnancies, amniotic fluid was collected at 20 or 29 weeks of gestation, and the platelet genotype indicated that the fetuses were HPA-1b/b. The fetal origin of the amniotic fluid derived DNA was confirmed by the forensic technique of DNA profiling using variable number of tandem repeat (VNTR) analysis. All therapy was withdrawn, percutaneous umbilical blood sampling was not performed, and both women vaginally delivered healthy non-thrombocytopenic infants. The application of platelet alloantigen genotyping using DNA from amniotic fluid cells identified the HPA-1b/b fetus, and VNTR analysis confirmed that the tissue was fetal derived, thus avoiding the necessity for percutaneous umbilical blood sampling. The use of this approach in patients at risk will avoid additional investigation and treatment in approximately one-seventh of all NATP pregnancies involving the HPA-1a antigen.

RhD status of a fetus at risk for haemolytic disease with a discrepant maternal DNA-based RhD genotype

“…DNA was isolated from EDTA whole blood or amniotic fluid (Denomme et al, 1995) and various segments of the RHD gene were PCR-amplified using two previously described methods (Bennett et al, 1993;Simsek et al, 1995). These methods rely on the presence of either exon 10 or exons 4-5 of the RHD gene to infer the expressed RhD status.…”

Prenatal genotyping of Jka and Jkb of the human Kidd blood group system by allele-specific polymerase chain reaction

“…Contamination of fetal samples with maternal cells is a well-acknowledged problem which can influence the results of prenatal testing by molecular diagnostic methodologies (Denomme et al, 1995;Colucci et al, 1993;Rebello et al, 1994). However, the extreme sensitivity of PCR-based assays is beneficial when attempting to identify antigen incompatibilities between a pregnant woman and her fetus (Hessner et al, 1997).…”

“…In order to define the sensitivity of the Kidd ASPCR assay, reconstitution experiments were conducted that demonstrated paternally inherited alleles can be detected even in cases of high levels (>90 per cent) of maternal contamination. However, it is always advisable to confirm the fetal origin of DNA used for genotyping by variable number of tandem repeat (VNTR) analysis before withdrawing further monitoring or therapy in fetuses identified as being not at risk (Denomme et al, 1995).…”

Fetal blood group genotyping

“…Cultured amniocytes are established routinely to avoid repeat amniocentesis should blood group genotyping be inconclusive using DNA extracted directly from cells in the fluid. 21 The most striking advancement in fetal blood group genotyping is the ability to detect RHD in DNA extracted from Rh-maternal plasma. The technique was first described by Lo et al 22 and several groups have evaluated its efficacy in predicting fetal inheritance of RHD (see Table 1).…”