The Research Of Application Of Nucleated Red Blood Cells Captured By Nanostructure Microchips In Prenatal Diagnosis

Part 1:Sorting of Fetal Nucleated Red Blood Cells from Maternal Peripheral Blood Using Ppy-Biotin Nanostructure Microchips and Identification by "Two Positive Indicators"Fluorescence StainingObjective To isolation,capture and identification fetal nucleated red blood cells(fNRBCs)from maternal peripheral blood by Ppy-biotin nanostructure microchips,confirmation the optimal pregnancy week to capture of fNRBCs,and exploration the relation between abnormal pregnancy about birth defects and the number of fNRBCs.Methods 48 cases of normal pregnancy women(pregnancy week range from 10 weeks to 30 weeks)without any complications of pregnancy and 12 cases of abnormal pregnancy women with a fetal birth defect were enrolled into my study group.Drawing their peripheral bloods and obtaining the monocyte layers by Ficoll density gradient centrifugation,cells were captured by antibody CD 147 modified Ppy-biotin nano-microchips,and identification by immunofluorescence(IF)and fluorescence in situ hybridization(FISH)as "two positive indicators"fluorescence staining.Results Antibody CD 147 had a significant specificity to fNRBCs.The accuracy rate of "two positive indicators" fluorescence staining in identification of fNRBCs is 100%.The number of fNRBCs in maternal peripheral blood in 48 cases of normal pregnancy women had an elevating trend.The number of fNRBCs in first trimester was less than it in second and third trimester and in the 18th pregnancy week it climbed to the peak.The number of fNRBCs in the 12 cases of abnormal pregnancy women was significant more than it in the normal pregnancy women in the same pregnancy week.Conclusion Antibody CD 147 modified Ppy-biotin nanostructure microchip should be regarded as one of the best methods to capture fNRBCs because of its easy fabrication,simple operation and economic application.The 18th week of gestation is determined to be the optimum time point of fNRBCs capture.fNRBCs in abnormal pregnancy women with fetal chromosomal aneuploidies are more than normal pregnancy women,which could be considered as a guidance of non-invasive prenatal diagnosis.Part 2:Captured Fetal Nucleated Red Blood Cells in Situ Released in Ppy-Biotin Nanostructure Microchips and Fetal Origin IdentificationObjective To evaluate the applicability of in situ release of captured fetal nucleated red blood cells(fNRBCs)and detection it in non-invasive prenatal diagnosis.Methods Release of captured fNRBCs in nanostructure microchips using electric’stimulation under different times and voltages,detection cell release efficiency and cell loss ratio,obtaining the optimum employed time and voltage.26 cases of umbilical cord blood was called for testing,separately.After cells released from the microchips,candidate cells were recycled and subjected to Polymerase Chain Reaction(PCR)by amplification of two X-STRs(DXS10101,DXS10103)and SRY gene.Meanwhile,the above gene loci were detected in DNA from each pair of parents.Identification of NRBCs of fetal origin and judgment of fetal sex was realized by comparing the genotype of fetus with that of their parents.Results The cell release efficiency by 0.3 V,0.5 V and 0.8 V in microchips were 68.3%,76.2%and 94.6%respectively.The cell release efficiency by 0-8 V on 5 s,15 s and 20 s in microchips were 54.7%,70.3%and 91.2%.In 26 cases of released fNRBCs could be proved as fetal original and fetal sex be inferred by analysis of DXS10101 DXS10103 with SRY gene in each fetus-parents group.The fetal sex identification efficiency of combination of the genes is high than each individual gene.Conclusion Using 0.8 V with 15 s of electric stimulation should be the optimum release parameter.After release and recycle procedure,cells could be used for further testing.Part 3:Application of Fetal Nucleated Red Blood Cells from Maternal Peripheral Blood for Non-Invasive Prenatal DiagnosisTest 1 Non-Invasive Prenatal Diagnosis of Chromosomal Aneuploidies by Fluorescence in Situ Hybridization on Fetal Nucleated Red Blood Cells from Maternal Peripheral BloodObjective To explore the feasibility of non-invasive prenatal diagnosis of fetal chromosomal aneuploidies on fNRBCs separated from maternal peripheral blood.Methods 18 cases of suspected chromosomal aneuploidies screened by cell-free fetal DNA(cffDNA)based non-invasive prenatal testing(NIPT)or/and imageological examination pregnancy women were enrolled into the test.fNRBCs were isolated and captured by microchips.Fluorescence in situ hybridization using GLP21/13,CSP18/X/Y probes was applied on the fNRBCs in order to evaluate the structure of the correspond choromsomes,and then compared with amniocentesis and G-banding karyotyping analysis.Results 13 cases were diagnosed fetal chromosomal aneuploidies by amniocentesis and G-banding karyotyping analysis in 18 suspected cases,fNRBCs-based non-invasive prenatal diagnosis confirmed 12 cases of chromosomal aneuploidies.5 cases of trisomy 21,3 cases of trisomy 18,2 cases of trisomy 13 and 2 cases of Klinefelter’s syndrome were identified.Only 1 cases of Klinefelter’s syndrome was undetected.The agreement rate between fNRBCs-based non-invasive prenatal diagnosis and invasive prenatal diagnosis was 92.3%.Conclusion fNRBCs sorted from maternal peripheral blood successfully diagnosed chromosomal aneuploidies.Test 2 Non-Invasive Prenatal Diagnosis of Microdeletion Syndrome by Whole Exome Sequencing on Fetal Nucleated Red Blood Cells from Maternal Peripheral BloodObjective To explore the feasibility of non-invasive prenatal diagnosis of fetal microdeletion syndrome on fNRBCs separated from maternal peripheral blood.Methods A 18q21 microdeletion syndrome was detected by amniocentesis and chromosomal microarray(CMA),whole exome sequencing(WES)was applied on the fNRBCs which was isolated and sorted from maternal peripheral blood,comparation with the gravida exome message from mouth mucosa exfoliative cells.Results Amplification of 30 genes and deletion of 35 genes in chromosome 18 were detected by comparation whole exome sequencing of fNRBCs captured from maternal peripheral blood with maternal whole exome message,including CYM gene which was detected from amniocentesis and CMA.Conclusion Whole exome sequencing,using in the fNRBCs isolated and captured from maternal peripheral blood,provides effective technological support in diagnosis of microdeletion/microduplication syndrome.