Algorithm development and diagnostic accuracy testing for non-invasive foetal RHD genotyping: an Indian experience.

BACKGROUND

The discovery of the cell-free foetal DNA (cffDNA) circulating in the maternal plasma enabled prediction of foetal RHD thus eliminating the risks associated with invasive procedures. Non-invasive foetal RHD genotyping has now become the standard approach in developed countries for management of alloimmunised women and is also used for targeted antenatal prophylaxis in non-alloimmunised women.

MATERIALS AND METHODS

cffDNA was extracted from the plasma of 217 RhD negative pregnant women at a gestational age of 10-34 weeks. The foetal RHD genotype was determined by real-time polymerase chain reaction (real-time PCR) amplification of exons 4, 5 and 10 in duplicates. After an initial 54 samples, foetal typing was carried out with RHD exons 5 and 10 for the remaining samples. CCR5, SRY and RASSF1A genes were used as controls. Results were compared with cord blood serological typing at birth.

RESULTS

Out of the 217 women, 193 were non-immunised and 24 were alloimmunised. A conclusive diagnosis was obtained in 203 samples. Diagnosis was inconclusive in 14 samples; of these, foetal RHD genotype could be resolved in six samples after maternal and paternal RHD genotyping. A 100% diagnostic accuracy, sensitivity and specificity were demonstrated in 209 women who had had a conclusive result. When the inconclusive samples were included, diagnostic accuracy and sensitivity were more than 95% and specificity was 78.95%.

DISCUSSION

Anti-D is still the leading cause of haemolytic disease of the foetus and the newborn in India. There is, therefore, a need to establish and develop an algorithm for antenatal RhD negative women in India. The positive results of non-invasive foetal RHD genotyping, from the start of the 10 week of gestation using two RHD exons giving 100% diagnostic accuracy, show promise for routine diagnostic use to the benefit of the antenatal RhD negative Indian population.