Objective
The objective of the study was the evaluation of a novel multiplex assay to detect fetal Rh blood group D-antigen gene ( RHD) loci in maternal plasma from RhD-negative, pregnant women.
Study Design
An RHD genotyping assay was designed to detect exons 4, 5, 7, and 10 and RHDΨ (pseudogene) of the RHD gene along with a Y chromosome–specific assay and a generic polymerase chain reaction amplification control. Plasma samples from 150 RhD-negative pregnant women were assayed for fetal RHD genotype using the MassARRAY system.
Results
The fetal RHD status of 148 of 150 samples (98.7%) was correctly classified; 86 (57.3%) and 62 (41.3%) were positive and negative, respectively.
Conclusion
This study demonstrates that noninvasive prenatal diagnostics with a single-reaction multiplexed assay is a viable path toward routine characterization of fetal RHD genotypes using circulating cell-free fetal DNA in maternal plasma on the MassARRAY system and is perhaps preferable to serologic testing as currently used clinically.
The presence of circulating cell-free fetal (ccff) deoxyribonucleic acid (DNA) in maternal plasma of pregnant woman was demonstrated nearly a decade ago and has expanded the repertoire of methods for prenatal diagnosis by noninvasive methods without risk for the fetus. Although significant efforts are required to develop more challenging tests for the detection of fetal chromosomal aneuploidies, the detection of unique fetal genetic targets such as the Y chromosome from a male fetus or the Rh blood group D-antigen gene ( RHD ) of an RhD-positive fetus is more readily achievable.
The determination of fetal RHD status is a significant aid to the management of pregnancies at risk for hemolytic disease of the fetus and newborn (HDN) as a result of maternal anti-D antibody. For example, if an immunized mother is known to be carrying an RHD negative fetus, there is no risk of HDN. In contrast, if the fetus tests RHD positive, then appropriate monitoring and follow-up, including therapy can proceed. Recently, ccff DNA in maternal plasma has led to noninvasive procedures to predict fetal RHD sequences and is already offered as a routine service in several European countries to women at risk for HDN.
The availability of noninvasive assays for prenatal RHD typing also makes it possible to restrict prenatal prophylactic treatment to only those women at risk for immunization. A practice that delivers prophylactic treatment only to pregnant women with RhD-positive fetuses would reduce unnecessary injections of anti-D therapy by about 40% in Western societies.
In this study, we evaluated a novel noninvasive assay for fetal RHD genotyping using the MassARRAY system (Sequenom, Inc, San Diego, CA) that analyzes RHD exons 4, 5, 7, and 10 and includes analysis of RHDΨ , the most common cause of an RhD-negative phenotype in Africans ( Table 1 ). It had been reported previously that the fetal RHD status can be accurately detected using polymerase chain reaction (PCR) amplification and detection by mass spectrometry. The aim of this study was to develop a multiplexed RHD assay that combines the detection of multiple RHD exons along with a Y-chromosomal marker and an amplification control into a single reaction. To measure the performance of this multiplexed assay, we analyzed 150 plasma samples from pregnant woman who had previously been characterized fetal RhD status serologically and molecularly by real-time PCR.
| Assay | Designation | Description |
|---|---|---|
| Rhesus D gene exon 4 | RHD_exon4 | Detection of RHD exon 4 loci; distinguishes RHD from RHCE by extend product mass |
| Rhesus D Ψ-pseudogene | Detection of RHD exon 4 loci containing the RHDΨ 37 bp insertion; distinguishes RHD from RHCE by extend product mass | |
| Rhesus D Ψ-pseudogene zygosity | psi | Detection of polymorphism linked to RHDΨ insertion; wild-type RHD and RHCE are not distinguishable |
| Rhesus D gene exon 5 | RHD_exon5 | Detection of RHD exon 5 loci; distinguishes RHD from RHCE by extend product mass |
| Rhesus D gene exon 7 | RHD_exon7 | Detection of RHD exon 7 loci; distinguishes RHD from RHCE by extend product mass |
| Rhesus D gene exon 10 | RHD_exon10 | Detection of RHD exon 10 loci |
| Amelogenin gene-XY chromosomes | AMG | Assay control for amplifiable X and Y chromosomal DNA in sample; estimation of male fetal DNA in maternal plasma |
| Human serum albumin | ALB | Assay control for amplifiable DNA in sample and estimation of total DNA copy when synthetic ALB standard included |
| Sex-determining region Y | SRY | Fetal sex; verifies presence of amplifiable male fetal DNA |
Materials and Methods
Details of sample collection, preparation, storage, and DNA extraction for real-time PCR have been published recently.
Sample collection
One hundred fifty samples from RhD-negative women in the second trimester of pregnancy were collected in Germany through a network of 173 practicing gynecologists. No participants over week 32 of gestation were included in the study. All women provided informed consent and had been typed as serologically D negative using two monoclonal immunoglobulin (M) anti-D antibodies. If both tests were clearly negative, the patient was typed as RHD negative. A weak result was interpreted as RHD positive. After informed consent, 15 mL of EDTA anticoagulated blood was obtained from each participant.
Blood samples were shipped at room temperature to the University Medical Center, Goettingen, Germany. Transport time varied from a few hours to a maximum of 8 days (median, 2 days). Upon receipt, plasma was separated from cells by centrifugation for 10 minutes at 2700 × g and transferred to fresh tubes without disturbing the buffy coat before being recentrifuged for 45 minutes at 12,500 × g . Plasma supernatants were aliquoted into 1 mL portions in individual polypropylene tubes and stored at –80°C until analysis.
DNA extraction for real-time PCR
DNA was extracted from maternal plasma using a modified spin-column method (QIAamp DSP virus kit, catalog no. 60704; Qiagen, Hilden, Germany). Briefly, 500 μL of plasma was mixed with lysis buffer and 100 μL of protease before being incubated at 56°C for 20 minutes. After ethanol (96-100%) was added and the mixture was incubated at room temperature for 10 minutes, it was applied to the QIAamp column and centrifuged at 6000 × g for 1 minute. The columns were washed 3 times and prewarmed at 56°C for 3 minutes before elution of DNA with 40 μL elution buffer.
Real-time PCR testing
DNA was amplified immediately after extraction using a real-time PCR detection system (ABI 7300; Applied Biosystems, Foster City, CA). A duplex exon 5/exon 7 PCR was set up for the detection of fetal RHD. All PCR primers were purchased from Purimex (Grebenstein, Germany). An oligonucleotide probe, labeled with a fluorescent reporter and quencher dye, accompanied each primer pair and was used to monitor amplification of the DNA target. An external control PCR detecting a β-globin sequence in fetus and mother was included in an identical run and performed from the same DNA eluate as used for the detection of RHD. Further details of the real-time PCR protocols have been published previously. Duplicate RHD PCR was set up per individual and extraction method, and β-globin PCR was performed only once. If results in duplicate were inconclusive, DNA extraction and PCR setup were repeated, resulting in a maximum number of 4 RHD PCR procedures and 2 β-globin PCR procedures per individual sample and extraction method.
Sample processing on the MassARRAY system
For the 150 samples processed on the MassARRAY system, maternal plasma DNA was prepared using a modification of the Qiagen MinElute protocol. Plasma was thawed and centrifuged to remove cryoprecipitate. One milliliter of plasma supernatant was combined with 150 μL Qiagen protease and 1 mL of buffer AL containing 5.6 μg carrier RNA. After incubation at 56°C for 15 minutes, 100 μL of buffer SB1 containing a proprietary buffer composition was added, and the plasma lysate was vortexed and then incubated for 10 minutes at room temperature prior to addition to a Qiagen MinElute column. After centrifugation, plasma lysate flow-through was collected and an additional 1200 μL buffer SB1 was added to the plasma lysate flow-through. The sample was vortexed and incubated for 10 minutes at room temperature prior to the addition to a second viral MinElute column. From this final column, plasma DNA was eluted with 55 μL water and collected by centrifugation. Twenty microliters of eluate were used for PCR amplification.
MassARRAY technology for RHD and sex determination
Details of the MassARRAY methods have been described in detail elsewhere. Briefly, the method involves a multiplex PCR followed by a multiplexed single-base primer extension reaction that targets specific positions within the amplified sequences. As a first step after the PCR, nucleotides are deactivated by shrimp alkaline phosphatase treatment. A reaction mix containing 4 terminator nucleotides and a DNA polymerase is added to perform the single-base primer extension reaction. The extension products were then analyzed using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry, and the sequence identity of the amplification products was deduced from the molecular masses of each of the extension products. An overview of primers used in this study is provided in Table 2 .
