Kirstin Finning

Prediction of fetal D status from maternal plasma: introduction of a new noninvasive fetal RHD genotyping service.

BACKGROUND : Invasive procedures to obtain fetal DNA for prenatal blood grouping present a risk to the fetus. During pregnancy, cell‐free fetal DNA is present in maternal blood. The detection of RHD sequences in maternal plasma has been used to predict fetal D status, based on the assumption that RHD is absent in D– genomes.

Effect of high throughput RHD typing of fetal DNA in maternal plasma on use of anti-RhD immunoglobulin in RhD negative pregnant women: prospective feasibility study.

Objectives To assess the feasibility of applying a high throughput method, with an automated robotic technique, for predicting fetal RhD phenotype from fetal DNA in the plasma of RhD negative pregnant women to avoid unnecessary treatment with anti-RhD immunoglobulin. Design Prospective comparison of fetal RHD genotype determined from fetal DNA in maternal plasma with the serologically determined fetal RhD phenotype from cord blood. Setting Antenatal clinics and antenatal testing laboratories in the Midlands and north of England and an international blood group reference laboratory. Participants Pregnant women of known gestation identified as RhD negative by an antenatal testing laboratory. Samples from 1997 women were taken at or before the 28 week antenatal visit. Main outcome measures Detection rate of fetal RhD from maternal plasma, error rate, false positive rate, and the odds of being affected given a positive result. Results Serologically determined RhD phenotypes were obtained from 1869 cord blood samples. In 95.7% (n=1788) the correct fetal RhD phenotype was predicted by the genotyping tests. In 3.4% (n=64) results were either unobtainable or inconclusive. A false positive result was obtained in 0.8% (14 samples), probably because of unexpressed or weakly expressed fetal RHD genes. In only three samples (0.2%) were false negative results obtained. If these results had been applied as a guide to treatment, only 2% of the women would have received anti-RhD unnecessarily, compared with 38% without the genotyping. Conclusions High throughput RHD genotyping of fetuses in all RhD negative women is feasible and would substantially reduce unnecessary administration of anti-RhD immunoglobulin to RhD negative pregnant women with an RhD negative fetus.

Noninvasive prenatal diagnosis of fetal blood group phenotypes: current practice and future prospects

Fetuses of women with alloantibodies to RhD (D) are at risk from hemolytic disease of the fetus and newborn, but only if the fetal red cells are D‐positive. In such pregnancies, it is beneficial to determine fetal D type, as this will affect the management of the pregnancy. It is possible to predict, with a high level of accuracy, fetal blood group phenotypes from genotyping tests on fetal DNA. The best source is the small quantity of fetal DNA in the blood of pregnant women, as this avoids the requirement for invasive procedures of amniocentesis or chorionic villus sampling (CVS). Many laboratories worldwide now provide noninvasive fetal D genotyping as a routine service for alloimmunized women, and some also test for c, E, C and K.

Non‐invasive prenatal determination of fetal sex: translating research into clinical practice

Hill M, Finning K, Martin P, Hogg J, Meaney C, Norbury G, Daniels G, Chitty LS. Non‐invasive prenatal determination of fetal sex: translating research into clinical practice.

Fetal blood group genotyping from DNA from maternal plasma: an important advance in the management and prevention of haemolytic disease of the fetus and newborn

The cloning of blood group genes and subsequent identification of the molecular bases of blood group polymorphisms has made it possible to predict blood group phenotypes from DNA with a reasonable degree of accuracy. The major application of this technology, which has now become the standard of care, is the determination of a fetal RHD genotype in women with anti‐D, to assess whether the fetus is at risk of haemolytic disease of the fetus and newborn (HDFN). Initially, the procurement of fetal DNA required the invasive procedures of amniocentesis or chorionic villus sampling. Since the discovery of fetal DNA in maternal plasma in 1997, the technology of detecting an RHD gene in this very small quantity of fetal DNA has developed rapidly, so that non‐invasive fetal D typing can now be provided as a diagnostic service for D‐negative pregnant women with anti‐D. Within a few years, it is probable that fetuses of all D‐negative pregnant women will be tested for RHD, to establish whether the mother requires antenatal anti‐D immunoglobulin prophylaxis.

Fetal genotyping for the K (Kell) and Rh C, c, and E blood groups on cell‐free fetal DNA in maternal plasma

BACKGROUND: When a pregnant woman has an antibody with the potential to cause hemolytic disease of the fetus and newborn, it is beneficial to determine whether her fetus has the corresponding antigen to assess risk. In many countries this is now done routinely for RhD, by testing cell‐free fetal DNA in the maternal plasma. Similar tests for K, C, c, and E are reported.

Diagnostic accuracy of routine antenatal determination of fetal RHD status across gestation: population based cohort study

Objectives To assess the accuracy of fetal RHD genotyping using cell-free fetal DNA in maternal plasma at different gestational ages. Design A prospective multicentre cohort study. Setting Seven maternity units in England. Participants RhD negative pregnant women who booked for antenatal care before 24 weeks’ gestation. Interventions Women who gave consent for fetal RHD genotyping had blood taken at the time of booking for antenatal care and, when possible, at other routine visits such as for Down’s syndrome screening between 11 and 21 weeks’ gestation, at the anomaly scan at 18-21 weeks, and in the third trimester when blood was taken for the routine antibody check. The results of cord blood analysis, done routinely in RhD negative pregnancies, were also obtained to confirm the fetal RHD genotyping. Main outcome measures The accuracy of fetal RHD genotyping compared with RhD status predicted by cord blood serology. Results Up to four analyses per woman were performed in 2288 women, generating 4913 assessable fetal results. Sensitivity for detection of fetal RHD positivity was 96.85% (94.95% to 98.05%), 99.83% (99.06% to 99.97%), 99.67% (98.17% to 99.94%), 99.82% (98.96% to 99.97%), and 100% (99.59% to 100%) at <11, 11-13, 14-17, 18-23, and >23 completed weeks’ gestation, respectively. Before 11 weeks’ gestation 16/865 (1.85%) babies tested were falsely predicted as RHD negative. Conclusions Mass throughput fetal RHD genotyping is sufficiently accurate for the prediction of RhD type if it is performed from 11 weeks’ gestation. Testing before this time could result in a small but significant number of babies being incorrectly classified as RHD negative. These mothers would not receive anti-RhD immunoglobulin, and there would be a risk of haemolytic disease of the newborn in subsequent pregnancies.

Noninvasive genotyping fetal Kell blood group (KEL1) using cell‐free fetal DNA in maternal plasma by MALDI‐TOF mass spectrometry

Alloimmunization against the fetal Kell (KEL1) blood group antigen is gaining importance relative to the Rhesus problem and is the second most important cause of hemolytic disease of the fetus and newborn. Molecular diagnosis for fetal KEL1, which currently involves invasive procedures, is routinely done for accessing whether a fetus is at risk. Here we developed a matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS)‐based single allele‐based extension reaction (SABER) to examine the fetal KEL1 gene from KEL1‐negative pregnant women using cell‐free fetal DNA in maternal plasma.

Fetal Blood Group Genotyping

Abstract:  Prediction of fetal blood group from DNA is usually performed when the mother has antibodies to RhD, to assess whether the fetus is at risk from hemolytic disease of the fetus and newborn (HDFN). Over the last five years RhD testing on fetal DNA in maternal plasma has been introduced. At the International Blood Group Reference Laboratory (IBGRL) we employ real‐time quantitative polymerase chain reaction (RQ‐PCR) to detectRHDexons 4, 5, and 10, which also revealsRHDψ. SRYand, in RhD‐negative (RhD–) females, eight biallelic polymorphisms are incorporated in an attempt to provide an internal positive control. Since 2000 we have tested 533 pregnancies for RhD. In 327 pregnancies where the RhD of the infant is known, we had one false‐positive and one false‐negative result. In 2004 we introduced fetal typing from DNA in maternal plasma for K, Rhc, and RhE, which represent single nucleotide polymorphisms (SNPs) on theKELandRHCEgenes.

Fetal RHD Genotyping in Maternal Plasma at 11–13 Weeks of Gestation

Objective: To examine the feasibility of fetal RHD genotyping at 11–13 weeks’ gestation from analysis of circulating cell-free fetal DNA (ccffDNA) in the plasma of RhD negative pregnant women using a high-throughput robotic technique. Methods: Stored plasma (0.5 ml) from 591 RhD negative women was used for extraction of ccffDNA by a robotic technique. Real-time quantitative polymerase chain reaction (PCR) with probes for exons 5 and 7 of the RHD gene was then used to determine the fetal RHD genotype, which was compared to the neonatal RhD phenotype. Results: In total there were 502 (85.7%) cases with a conclusive result and 84 (14.3%) with an inconclusive result. The prenatal test predicted that the fetus was RhD positive in 332 cases and in all of these the prediction was correct, giving a positive predictive value of 100% (95% CI 96.8–100). The test predicted that the fetus was RhD negative in 170 cases and in 164 of these the prediction was correct, giving a negative predictive value for RhD positive fetuses of 96.5% (95% CI 93.7–99.2). Conclusion: The findings demonstrate the feasibility and accuracy of non-invasive fetal RHD genotyping at 11–13 weeks with a high-throughput technique.