Guidance
3 The diagnostic tests
The assessment compared 1 intervention test with 1 comparator test.
The intervention
3.1 High-throughput non‑invasive prenatal testing (NIPT) for fetal RHD genotype is a laboratory developed test offered by the International Blood Group Reference Laboratory, Bristol (NHS Blood and Transplant). The test uses a real-time quantitative polymerase chain reaction (PCR) method for identifying fetal RHD genotype from fetal DNA in the plasma of rhesus‑D (D) negative women. The test analyses cell-free fetal DNA, in the form of small fragments of fetal extracellular DNA shed from the placenta and circulating freely in the maternal plasma. The level of cell-free fetal DNA in maternal blood increases throughout the pregnancy and rapidly falls after delivery. Most women who are D negative do not have copies of the RHD gene; therefore, the presence of the RHD gene in a D‑negative pregnant woman suggests a D‑positive fetus.
3.2 High-throughput NIPT is carried out using 4 ml to 6 ml of maternal anti-coagulated blood. DNA extraction is done using an automated robotic platform (MDx BioRobot, Qiagen), which can rapidly process samples. The robotic platform is also used as a liquid handler to dispense samples and reagents. PCR is then done on an ABI Prism 7900HT analyser (Applied Biosystems). Primers and probes for exons 5 and 7 of the RHD gene are used, and the following controls are tested alongside the samples: RHD positive DNA; RHD negative DNA; RHD pseudogene positive DNA; and no DNA. The samples can be tested in batches of between 32 and 88 samples. The time to complete the test from sample receipt to report generation is 5 to 6 hours.
3.3 The exon 5 assay amplifies the RHD gene, whereas the exon 7 assay amplifies both the RHD gene and the RHD pseudogene. A threshold value of less than 42 cycles is interpreted as a positive signal and an algorithm is used to determine the fetal RHD genotype. Results are reported as 'D‑positive', 'D‑negative' or 'indeterminate – treat as D‑positive'. The result would influence whether to offer routine antenatal anti‑D prophylaxis and anti‑D immunoglobulin to D‑negative women, who are not sensitised to D antigen, after potentially sensitising events.