Nondestructive Identifcation Of Rare Trophoblastic Cells By ER-LDA For Noninvasive Prenatal Testing Of Thalassemia

Background&ObjectiveFetal cells carrying the entire fetal genome in maternal reproductive tract have been demonstrated to be a promising cell source for noninvasive detection of many congenital defects,including chromosomal aneuploidies,microdeletions,monogenic diseases,and hemolytic disease of the newborn.However,an easy-to-operate assay with high sensitivity,high specificity and a simple process is not available for the detection of these fetal trophoblastic cells.Currently,few studies have closely investigated the fetal origin of trophoblastic cells by sequencing a large panel of genetic markers.Furthermore,whether these cells are adapted to test for single-nucleotide variations or micro-deletions in monogenic diseases is not well investigated.Here,we demonstrate a novel cell-based noninvasive prenatal testing(NIPT)system,in which rare trophoblastic cells in Papanicolaou smears(Pap)can be rapidly screened and isolated for fetal genotyping.Additionally,we also characterized the origin of candidate cells by sequencing of genetic markers.Importantly,the feasibility of using fetal trophoblastic cells for prenatal diagnosis of thalassemia is also substantiated.Methods1.The number of fetal cells in samples from pregnancies with a male fetus was calculated by ddPCR and qPCR.The fluorescence intensity of ER-Tracker in trophoblastic cells and maternal cells were determined by fluorescence imaging and flow cytometry.Moreover,a novel assay based on endoplasmic reticulum staining and linear discriminant analysis(ER-LDA)was developed and evaluated in clinical samples.2.Candidate trophoblastic cells were isolated by single-cell picking.Then,immunofluorescent staining was applied to single trophoblastic cells to characterize the expression of β-hCG and HLA-G,trophoblastic cell markers.To confirm the fetal origin of candidate cells,short tandem repeat(STR)profiling,single nucleotide polymorphism(SNP)genotyping and fluorescence in situ hybridization(FISH)analysis,were performed on rare cells.3.To investigate whether the rare trophoblastic cells can be applied to noninvasive prenatal testing for thalassemia,pregnancies whose fetuses were at risk for thalassemia were recruited.Moreover,our cell-based test was also applied to fetal RHD(Rh blood group D antigen)genotyping by qPCR.We also performed whole exome sequencing(WES)on rare fetal cells to test its feasibility in the detection of other monogenic diseases.Results1.About 72 to 5150 fetal cells could be detected in Pap samples from pregnant women.We found that the fluorescence intensity of ER-Tracker in trophoblastic cells was about 3.13±1.13 and 2.64±0.73 fold higher than that in squamous epithelial cells and WBCs,respectively.The LDA showed a sensitivity of 99%,a specificity of 98.75%and an accuracy of 98.83%for discrimination of trophoblastic cells versus non-trophoblastic cells.We also found that all the 22 genetic loci in single cells showed successful amplifications,which supports their high genomic coverage.2.As expected,putative trophoblastic cells showed a much brighter ER fluorescence than that of background cells and trophoblast-like morphology.We observed that most of these cells were β-hCG and HLA-G positive.ERhigh cells separated from pregnancies with a male fetus showed positive probe binding for the Y chromosome,suggesting their fetal origin.These samples were found to be harboring two to eleven informative STR and SNP markers that distinguish the fetus from the mother.These results indicated that the ERhigh single cells we isolated were indeed fetal cells.3.We found that rare trophoblastic cells were successful used for the detection of__SEA/thalassemia,-α4.2/thalassemia and β thalassemia.Results of all cases were confirmed by invasive or postnatal tests and the genotype of fetuses,including abnormal fetuses and normal fetuses,was accurately detected by our cell-based test.Moreover,the RHD genotype could also be evaluated by our cell-based test.A lot of SNVs and InDels could be detected in rare fetal cells by WES,which indicated that rare fetal cells may also be suitable for use in identifying inheritable mutations in a variety of monogenic diseases by high throughput sequencing.ConclusionOverall,the ER-LDA single cell platform presented here offers a noninvasive method for acquiring valuable molecular information from fetuses that are at risk for monogenic disorders.The findings of our study imply that the detection of rare fetal cells in Pap samples by ER-LDA holds great promise for identifying fetuses with thalassemia,and other congenital defects.