The use of maternal plasma for prenatal RhD blood group genotyping.

Alloimmunization to the blood group antibody anti-RhD (anti-D) is the most common cause of hemolytic disease of the fetus and newborn. Knowledge of fetal D type in women with anti-D makes management of the pregnancy much easier and avoids unnecessary procedures in those women with a D-negative fetus. Fetal D typing can be performed by detection of an RHD gene in cell-free DNA in the plasma of D-negative pregnant women. The technology involves real-time quantitative polymerase chain reactions targeting exons 4, 5, and 10 of RHD, with the exons 4 and 10 tests performed as a multiplex. Testing for SRY in multiplex with the RHD exon 5 test provides an internal control for the presence of fetal DNA when the fetus is male. Fetal D typing has become the standard of care in England in pregnant women with a significant level of anti-D.

Fetal blood group genotyping: present and future.

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 detect RHD exons 4, 5, and 10, which also reveals RHDpsi. SRY and, 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 the KEL and RHCE genes. We have begun trials on an automated method for fetal RhD typing from DNA in maternal plasma. This is designed to test fetal RhD in all pregnant RhD- women, to identify the 40% with an RhD- fetus so that antenatal RhD immunoglobulin (Ig) prophylaxis can be avoided. Similar trials have already been reported by Sanquin Research Laboratories in Amsterdam.

Prevalence of RhD status and clinical application of non-invasive prenatal determination of fetal RHD in maternal plasma: a 5 year experience in Cyprus.

BACKGROUND: After the discovery that cell-free fetal DNA (cffDNA) is circulating in the maternal plasma of pregnant women, non-invasive prenatal diagnosis for fetal RhD in maternal plasma in RhD negative women at risk for haemolytic disease of the newborn (HDN) was clinically established and used by many laboratories. The objectives of this study are: (a) to assess the feasibility and report our experiences of the routine implementation of fetal RHD genotyping by analysis of cffDNA extracted from maternal plasma of RhD negative women at risk of HDN, and (b) to estimate the RhD phenotype frequencies, the RHD genotype frequencies and the RhD zygosity in the Cypriot population. METHODS: cffDNA was extracted from maternal plasma of 73 RhD negative pregnant women. Real-Time Multiplex-PCR was used to amplify regions of RHD gene in exons 4, 5 and 10. RhD phenotypes were determined on 445 random samples using conventional agglutination slide test. RESULTS: The fetus was predicted to be positive in 53 cases and negative in 18 cases. Two of cases were identified as D-variants, weak D type-1 and 11. The frequency of RhD negative homozygosity in the Cypriot population was estimated to be 7.2%, while the frequencies of RHD hemizygosity and RhD positive homozygosity was calculated to be 39.2 and 53.6%, respectively. CONCLUSION: Fetal RHD genotyping can be accurately determined using cffDNA from maternal plasma. The implementation of the test has eliminated all use of unnecessary anti-D and reduced the total use of anti-D by 25.3% while achieving appropriate management of the RhD negative pregnancies.

Noninvasive fetal blood grouping: present and future.

Identification of the molecular basis of the D polymorphism of the Rh blood group system in the 1990s made it possible to predict D phenotype from DNA. The most valuable application of this has been the determination of fetal D type in pregnant D-negative women with anti-D. Knowledge of fetal D type reveals whether the fetus is at risk of hemolytic disease of the fetus and newborn so that the pregnancy can be managed appropriately. Noninvasive fetal D typing for D-negative pregnant women with anti-D, performed on the small quantity of fetal DNA present in the blood of pregnant women, is now routine practice in several European countries. Noninvasive fetal blood grouping for C, c, E, and K also may be provided as a routine service for alloimmunized pregnant women. In many countries, all D-negative pregnant women are offered anti-D prophylaxis antenatally, yet in a predominantly Caucasian population, about 38% will be carrying a D-negative fetus and will receive the treatment unnecessarily. Large-scale trials to ascertain the accuracy of high-throughput, automated methods suggest that fetal D screening of all D-negative pregnant women is feasible, and it is likely that fetal D screening in D-negative pregnant women will be policy in some European countries within the next few years.

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.In many countries, anti-D immunoglobulin injections are offered to D-negative pregnant women, to reduce the chances of prenatal immunization, even though up to 40% of these women will have a D-negative fetus. High-throughput, noninvasive fetal D genotyping technologies are being developed so that unnecessary treatment of pregnant women can be avoided. Trials suggest that fetal D typing of all D-negative pregnant women is feasible and should become common practice in the near future.

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.

Use of maternal plasma for noninvasive determination of fetal RhD status.

Determination of the fetal RhD typing using free fetal DNA in maternal plasma is beginning to enjoy widespread acceptance in Europe. Case 1, the partner of an RhD-sensitized patient, was identified with a heterozygous paternal phenotype by serologic testing. Maternal plasma was drawn at 18 weeks' gestation to determine the fetal RhD status. The result was unable to be reported as RhD negative; the patient subsequently underwent amniocentesis to confirm an RhD-negative fetus. Case 2, a partner of another RhD-sensitized patient, was similarly identified with a heterozygous paternal phenotype by serologic testing. Maternal plasma was also drawn at 18 weeks' gestation to determine the fetal RhD status. It returned RhD negative and allowed for the avoidance of invasive testing for the remainder of the pregnancy. Therefore, maternal plasma testing for fetal RhD status represents a new tool in the management of the cases of RhD alloimmunization in pregnancy.