Routine use of a spike-in DNA in-process control for foetal RHD genotyping: Testing the real-world effectiveness of this 'canary'.

BACKGROUND AND

OBJECTIVES

Non-invasive foetal RHD (fRHD) genotyping is widely implemented to prevent unnecessary administration of antenatal anti-D prophylaxis. Reliable assay performance is critical. In line with expert recommendations, we validated and implemented an artificial spike-in extraction control in our previously published assay. In this study, we report on assay verification and its performance in a 2-year cohort.

STUDY DESIGN AND

METHODS

fRHD typing was performed with cell-free DNA isolated from maternal plasma from gestational age week 27 or later. A circular plasmid with a fragment of glycoprotein B gene of the Phocid herpes virus type 1 (PhHV1-gB) (spike-in control) was added to the plasma before DNA extraction. Assay accuracy was verified with gestational week-27 plasma samples and corresponding cord blood samples from D-negative pregnant women. In addition, assay performance over time was evaluated in a 2-year cohort.

RESULTS

The performance verification of our modified assay showed no false negative and one false positive test result in a small clinical cohort (n = 191). In a further 47,391 samples across 1111 runs, we observed eight false negative results due to technical failures that were prevented by the addition of the spike-in control. In this larger series, the spike-in control was the sole detector of a technical problem most likely related to different batches of the DNA extraction kit.

CONCLUSION

This study demonstrates the prevention of false negative fRHD typing results by the addition of an artificial extraction control. This control allows improved monitoring of assay performance, thereby ensuring assay consistency. Findings underscore the importance of thorough quality assurance measures in fRHD genotyping.