Validation study for noninvasive single-cell-based prenatal genetic testing

ObjectiveTo clinically validate a cell-based noninvasive prenatal genetic test using sequence-based copy number analysis of single trophoblasts from maternal blood. MethodsBlood was obtained from 401 individuals (8-22 weeks) and shipped overnight. Red blood cells were lysed, and nucleated cells stained for cytokeratin (CK) and CD45 using fluorescent antibodies and enriched for positive CK staining. Automated microscopic scanning was used to identify and pick single CK+/CD45- trophoblasts which were subjected to whole genome amplification and next-generation sequencing. ResultsBlood was obtained from 243 pregnancies scheduled for CVS or amniocentesis. Luna results were normal for 160 singletons while 15 cases were abnormal (14 aneuploidy and one monozygotic twin case with Williams syndrome deletion). These Luna results agreed with CVS/amniocentesis. Placental mosaicism occurred in 7 of 236 (3.0%) Luna cases and in 3 of 188 (1.6%) CVS cases (total 4.6%). No scorable trophoblasts were recovered in 32 of 236 (13.6%) usable samples. Additionally, 158 low-risk pregnancies not undergoing CVS/amniocentesis showed normal results for 133 cases. Seven had aneuploidy results, and there were 3 likely pathogenic deletions or duplications including one15q11-q13 deletion. ConclusionThis noninvasive cell-based prenatal genetic test detected aneuploidy and deletions/duplications with high sensitivity and specificity based on concordance with CVS/amniocentesis. Key pointsO_ST_ABSWhats already known about this topic?C_ST_ABSO_LIAs a proof of principle for noninvasive genetic prenatal diagnosis, circulating fetal trophoblasts have been isolated from maternal blood and analyzed for detection of aneuploidy and genomic deletions and duplications. C_LIO_LIThese trophoblasts reflect the genotype of the current placenta(s) but not necessarily the genotype of the fetus because of placental mosaicism. C_LI What does this study add?O_LIThis study demonstrates the advantages of single cell analysis and the feasibility of launching a test for reliable detection of cytogenetic aneuploidy, deletions, and duplications. C_LIO_LIThis test has improved detection of deletions and duplications compared to cell-free NIPT, but widespread adoption will require improved recovery of fetal cells from maternal blood and reduced cost through automation and high-throughput. C_LI