Frequency and characterization of known and novel RHD variant alleles in 37 782 Dutch D-negative pregnant women.

To guide anti-D prophylaxis, Dutch D- pregnant women are offered a quantitative fetal-RHD-genotyping assay to determine the RHD status of their fetus. This allowed us to determine the frequency of different maternal RHD variants in 37 782 serologically D- pregnant women. A variant allele is present in at least 0·96% of Dutch D- pregnant women The D- serology could be confirmed after further serological testing in only 54% of these women, which emphasizes the potential relevance of genotyping of blood donors. 43 different RHD variant alleles were detected, including 15 novel alleles (11 null-, 2 partial D- and 2 DEL-alleles). Of those novel null alleles, one allele contained a single missense mutation (RHD*443C>G) and one allele had a single amino acid deletion (RHD*424_426del). The D- phenotype was confirmed by transduction of human D- erythroblasts, consolidating that, for the first time, a single amino acid change or deletion causes the D- phenotype. Transduction also confirmed the phenotypes for the two new variant DEL-alleles (RHD*721A>C and RHD*884T>C) and the novel partial RHD*492C>A allele. Notably, in three additional cases the DEL phenotype was observed but sequencing of the coding sequence, flanking introns and promoter region revealed an apparently wild-type RHD allele without mutations.

Fetal RHD genotyping after bone marrow transplantation.

BACKGROUND: Fetal RHD genotyping allows targeted diagnostic testing, fetal surveillance, and eventually intrauterine treatment to D-alloimmunized pregnant women who carry an RHD+ fetus. However, false-positive and false-negative results of noninvasive prenatal fetal RHD genotyping have been described due to a variety of causes. In this case report we present two cases where noninvasive fetal RHD typing was complicated by a previous bone marrow transplantation (BMT). CASE REPORT: We describe two women with a history of allogeneic BMT in early childhood. Both were born D+ and received a transplant of their D- male sibling. Anti-D were detected during pregnancy in one of them. The biologic father of this pregnancy was D+. In both cases polymerase chain reaction procedures specific for RHD on maternal plasma DNA were positive whereas a D- neonate was born in one case (Case 1). CONCLUSION: False-positive results of noninvasive fetal RHD genotyping occur in D+ women transplanted with marrow of a D- donor, due to circulating cell-free DNA originating from nonhematopoietic tissue. The cases highlight that health care professionals and laboratories should be aware that allogeneic BMT can be a cause for false-positive results in fetal RHD genotyping with cell-free DNA in maternal plasma, and likewise the wrong fetal sex can be reported in the case of a male donor and a female fetus. Based on one of the cases we also recommend giving D- blood products to young female patients who receive a BMT of D- donors.

Use of bi-allelic insertion/deletion polymorphisms as a positive control for fetal genotyping in maternal blood: first clinical experience.

Amplification of fetal DNA in maternal plasma is a new way for non-invasive fetal genotyping in pregnancies at risk for disorders where the presence of a paternal DNA sequence contributes to the risk status of the fetus. We describe the use of a panel of 10 bi-allelic highly polymorphic markers to ascertain the presence and amplification of fetal DNA in case the fetus is negative for the targeted paternal "disease" sequence.

Clinical applications of cell-free fetal DNA from maternal plasma.

OBJECTIVE: To describe our clinical experience with detection and analysis of cell-free fetal DNA derived from maternal plasma for prenatal sexing and fetal rhesus-D typing. METHODS: Real-time quantitative polymerase chain reactions (PCRs) of rhesus-D sequences and the SRY gene were validated and offered to patients with an enhanced risk for sex-linked fetal pathology and patients with rhesus-D antibodies. RESULTS: In the validation group, 72 samples were analyzed. Sensitivity of the rhesus-D real-time quantitative PCR in maternal plasma was 100% (95% confidence interval [CI]91.8%, 100%) and specificity was 96.6% (95% CI 82.2%, 99.9%). Sensitivity of the SRY real-time quantitative PCR was 97.2% (95% CI 85.5%, 99.9%), and specificity was 100% (95% CI 88.1%, 100%). The technique was used successfully in a clinical setting in 24 women. Overall, invasive tests were avoided in 41.7% of these patients. CONCLUSION: Detection of cell-free fetal DNA from maternal plasma is a reliable technique that can substantially reduce invasive prenatal tests.

Reliability of fetal sex determination using maternal plasma.

OBJECTIVE: To determine the diagnostic accuracy of noninvasive fetal sex determination in maternal plasma. METHODS: All consecutive patients for whom fetal sex determination in maternal plasma was performed in our laboratory from 2003 up to 2009 were included in the study. Real-time polymerase chain reaction was performed for the SRY gene and multicopy DYS14 marker sequence. A stringent diagnostic algorithm was applied. In the case of a positive result for both Y chromosome-specific assays, a male-bearing pregnancy was reported. In the case of a negative result, the presence of fetal DNA was ascertained through the use of 24 biallelic insertion/deletion polymorphisms or paternally inherited blood group antigens. Only if the presence of fetal DNA was confirmed was a female-bearing pregnancy reported. Results were compared with the pregnancy outcomes. RESULTS: A total of 201 women were tested. The median gestational age was 9 0/7 weeks (interquartile range 8 0/7 to 10 0/7 weeks). In 189 of 201 cases (94%), a test result was issued; in 10 cases, the presence of fetal DNA could not be confirmed; in two cases, an early miscarriage was observed. Pregnancy outcome was obtained in 197 cases, including 105 male-bearing and 81 female-bearing pregnancies and 11 miscarriages. Sensitivity and specificity of the test were 100% (95% confidence intervals 96.6-100% and 95.6-100%, respectively). In all 10 cases in which the presence of fetal DNA could not be confirmed, a female was born. CONCLUSION: Noninvasive fetal sex determination in maternal plasma is highly reliable and clinically applicable. LEVEL OF EVIDENCE: III.