Minimal impact of maternal intravenous immunoglobulin infusion on cell-free DNA sequencing for fetal aneuploidy

A range of maternal factors are known to influence the performance of cell-free DNA (cfDNA) screening for aneuploidy, including autoimmune disease1, treatment with low-molecular-weight heparin2, maternal weight3 and maternal malignancy4. We reported previously our experience of repeated failed cfDNA screening in a woman with severe autoimmune disease and low fetal fraction5. In that report, our serial samples suggested that intravenous immunoglobulin (IVIG) therapy caused increased variance in cfDNA counts, leading to a failed cfDNA result. We now report our subsequent experience of cfDNA screening in a healthy pregnant woman receiving weekly IVIG therapy for a fetal indication of prior history of a newborn affected by alloimmune thrombocytopenia. A 36-year-old pregnant woman, gravida 6 para 2, had previously undergone cfDNA screening at 10 weeks' gestation with a whole-genome massively parallel sequencing assay (PerceptTM prenatal test, Victorian Clinical Genetics Services, Royal Children's Hospital Melbourne, Parkville, VIC, Australia) as part of her routine obstetric care. She received a low-risk result for trisomies 21, 18 and 13 in a male fetus. The fetal fraction of cfDNA in the maternal plasma was 12.4% and maternal body mass index was 27.6 kg/m2. Clinically indicated weekly IVIG infusions were administered from 20 weeks' gestation for prevention of neonatal alloimmune thrombocytopenia. We obtained ethical approval and patient consent to perform repeat blood sampling before and after IVIG infusion to investigate its effect on cfDNA testing and quality control metrics. At 29 weeks' gestation, 10 mL of maternal peripheral venous blood was collected in a StreckTM DNA blood collection tube just prior to a 75-g IVIG infusion. A repeat sample was taken at the end of the infusion, 6 h later. Both samples were sent to the clinical laboratory that performed the 10-week cfDNA test. The laboratory staff were not blinded to the research samples but adhered to the routine clinical workflow for prenatal screening samples. Plasma isolation, cfDNA extraction, library preparation and sequencing batch run were performed on pre- and post-IVIG samples simultaneously. Both samples returned low-risk results for trisomies 21, 18 and 13, and identified a male fetus. Fetal fractions in the pre- and post-IVIG samples were 17.0% and 20.9%, respectively. Aside from an observed three-fold increase in library concentration after IVIG, there was little difference in the quality control metrics between the two samples. The chromosome-wide variation in read counts (i.e. a measure of the sample- or library-level noise) showed no discernible difference between any of the samples tested. This case demonstrates that IVIG in itself does not appear to adversely affect the performance of cfDNA screening in healthy women with a satisfactory fetal fraction. The adverse effect of IVIG noted in our prior case5 may have been due the borderline acceptable fetal fraction (4.6%) prior to IVIG infusion, differences in the type of cfDNA assay (chromosome selective vs whole genome) and the presence of severe autoimmune disease. Our negative finding adds to the accumulating literature on the influence of maternal factors on cfDNA screening and may assist clinicians in pretest counseling of pregnant women receiving IVIG. L. Hui*†‡§, M. Pertile¶**, M. Tassone†† and D. Bruno¶‡‡ †Perinatal Medicine, Mercy Hospital for Women, Heidelberg, VIC, Australia; ‡Obstetrics and Gynaecology, University of Melbourne, Parkville, VIC, Australia; §Public Health Genetics, Murdoch Childrens Research Institute, Parkville, VIC, Australia; ¶Cytogenetics, Victorian Clinical Genetics Services, Parkville, VIC, Australia; **Paediatrics, University of Melbourne, Parkville, VIC, Australia; ††Department of Obstetrics and Gynaecology, Mercy Hospital for Women, Heidelberg, VIC, Australia; ‡‡Translational Genomics Unit, Murdoch Childrens Research Institute, Parkville, VIC, Australia *Correspondence. (e-mail: lisahui77@gmail.com)