Non-invasive foetal RhD determination

Non-invasive fetal RHD determination is clinically indicated in RhD-negative pregnant women in whom fetal RHD status is required to guide clinical management. In clinical practice, this may include:

• RhD-negative pregnant women in whom the need for anti-D prophylaxis requires assessment
• RhD-negative pregnant women with evidence of anti-D antibodies (RhD sensitisation), in whom fetal RHD status may help assess whether the fetus is at risk and guide further obstetric monitoring

RhD-negative pregnancies carry a risk of RhD sensitisation when maternal exposure to RhD-positive fetal erythrocytes occurs. This can lead to severe fetal hemolytic anemia and hydrops fetalis. Determining fetal RHD status has direct implications for clinical management, enabling appropriate use of anti-D prophylaxis and reducing unnecessary treatment.

Non-invasive fetal RHD testing provides clinically actionable information by identifying pregnancies in which the fetus is predicted to be RhD-negative. This supports evidence-based decision-making, can avoid unnecessary administration of antenatal anti-D prophylaxis in approximately 35–40% of cases of RhD-negative pregnancies, and reduces exposure to a human plasma-derived product where it is not medically indicated.

Positive result: RHD sequences detected and the fetus is predicted to be RhD-positive. The result should be managed as RhD-positive for clinical purposes, while recognising that fetal RhD phenotype is predicted from the RHD genotype rather than measured directly. Anti-D prophylaxis should be administered in the 28th-30th week of pregnancy.

Negative result: No RHD sequences are detected. A valid negative result indicates that the fetus is predicted to be RhD-negative; however, a low amount of cell-free fetal DNA can contribute to an inconclusive or false-negative result. Under this test’s confirmation protocol, a negative result before week 19 is considered provisional and should be confirmed at least two weeks later and after week 17. Anti-D prophylaxis is not necessary when the fetus is confirmed RhD-negative.

Regardless of the prenatal non-invasive fetal RHD result, the baby’s RhD status should be determined immediately after birth, preferably from umbilical cord blood, for every baby born to an RhD-negative mother to exclude rare false-negative results. If the baby is RhD-positive and the mother is not already sensitised, the mother should receive a standard dose of anti-D immunoglobulin (300 µg) within 72 hours of birth, in accordance with current guidance.

Test results should always be interpreted in the context of clinical presentation, gestational age, maternal antibody status and sample quality. An unclear or non-evaluable result does not necessarily exclude the possibility of determining fetal RHD status on a repeat sample, as testing can be limited by the amount of cell-free fetal DNA, RHD variants, available methodologies and pre-analytical sample factors.

Depending on the findings, the following next steps may be considered:

• Positive result: If the mother is not already sensitised, anti-D prophylaxis should be administered in the 28th-30th week of pregnancy according to current guidelines.
• Negative result before week 19: Repeat testing after week 17 to confirm the result and minimise false-negative risk.
• Negative result after week 19: Anti-D prophylaxis is not required if the fetus is confirmed RhD-negative.
• Unclear result: Repeat sampling may be considered where clinically appropriate. If no reliable fetal RHD result is available by the time prophylaxis is due, anti-D prophylaxis should be given as a precaution in a non-sensitised RhD-negative pregnancy.
• Post-birth confirmation: RhD status should be determined, preferably from umbilical cord blood, for every baby born to an RhD-negative mother, regardless of the prenatal result.
• Genetic counselling: The responsible clinician should provide appropriate pre-test information, explain the possible results and implications for pregnancy management, and obtain informed consent in accordance with local requirements.

Clinical consultation is an important part of the testing pathway. Before testing, the treating obstetrician/gynaecologist or another appropriately qualified clinician should explain the purpose and timing of fetal RHD determination, its benefits and limitations, and how the result may affect anti-D prophylaxis. After testing, the result should be interpreted alongside gestational age, maternal antibody status and the wider obstetric picture, with further monitoring or referral arranged where indicated. If anti-D antibodies are already present, anti-D prophylaxis does not treat established sensitisation and specialist obstetric or fetal-medicine input may be required. Counselling and consent requirements vary by country; where prenatal genetic testing is subject to specific legal requirements, local regulations must also be followed.

In our laboratory, we use advanced techniques designed to provide accurate, clinically useful diagnostic results.

• Detection of cell-free fetal DNA from maternal plasma
• Analysis of RHD sequences using real-time PCR with three target regions (exons) to detect as many variants of the RHD gene as possible
• CE-IVD certified test with high diagnostic sensitivity (>99%) and specificity (>98%)