Noninvasive Prenatal Diagnosis Of Chromosome Aneuploidies Using Fetal Nucleated Erythrocytes Circulating In Maternal Peripheral Blood

PART ISorting of Fetal Nucleated Erythrocytes in Maternal Peripheral Circulation with Immunochemistry Staining of Gamma Heamoglobinand MicromanipulationObjective To establish an effective seperating method for fetal nucleated red blood cells (FNRBC) from maternal peripheral blood to obtain highly concentrated and purified target cells and explore the optimal time for FNRBCs sorting. Methods 188 cases of pregnant women were taken as the study group. After preliminary enrichment using density gradient centrifugation, FNRBCs were identified by immunohistochemistry stain with anti- γ -globin monoclonal antibody and collected by micromanipulation. Primer extension preamplification (PEP) and ploymerase chain reaction (PCR) based on single cell were adopted to detect SRY gene. Results Fetal NRBCs displayed unique morphological characteristics and haemoglobin stain. The number of FNRBCs among 0.6 ×10~8 nucleated cells in each case was 0 ~89. In 188 cases, 160 gravid women had normal courses of pregnancy, who are further grouped according to first, second and third trimester with a mean number of FNRBCs of 10.44 ±3.19, 29.96 ± 4.10, 22.00±3.55 respectively. Very significant difference existed between the FNRBCs amounts of the above three groups (P<0.01), and the group of second trimester had the greatest number of FNRBCs. FNRBCs sorted in 18 pregnancies affected by fetal growth restriction were remarkably more than that of common pregnancies (P<0. 01). There were one pregnant woman bearing Down syndrome fetus and one bearing Turner syndrome fetus, from whom FNRBCs in peripheral blood were 89 and 43 respectively, much higher than the mean number of normal pregnancies. SRY gene was detected with PEP-PCR with a sensitivity of 95. 74% and a specificity of 100%. The accurate rate of determination of fetal sex using this method was 97.85%. Conclusions The sorting method associated γ-haemoglobin immunohistochemistry stain and micromanipulation can guarantee the quantity and quality of research template, which make accurate genetic analysis using FNRBC in maternal blood more accessible. The number of FNRBCs sorted from maternal blood is highest in the second trimester and elevates in pregnancies affected with aneuploidy, which offers a guidance for application of the noninvasive prenatal diagnosis. PART II Identification of Fetal Nucleated Erythrocytes Sorted from MaternalPeripheral Blood with Short Tandem RepeatsObjective To evaluate the applicability of X-specific short tandem repeats (X-STRs) to identification of fetal cells and detection of fetal sex in noninvasive prenatal diagnosis. Methods Peripheral blood samples were obtained from 47 pregnant women and their husbands. Fetal nucleated erythrocytes in maternal blood were discerned by heamoglobin γ chain staining and micromanipulated separately. After primer extension preamplification (PEP), single candidate cells were subjected to amplification of two X-STRs (DXS101, DXS6797) and AMXY 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 were realized by comparing the genotype of fetus with that of its parents. Results 3 cases out of the total 47 samples failed to yeild PCR products. In 40 cases NRBCs could be proved as fetal original and fetal sex can be inferred by analysis of DXS101, DXS6797 with AMXY gene in each fetus-parents group. The sensitivity of diagnosis of female fetuses or male fetuses is 90%, 91. 67% respectively, with a concordance rate of 90. 91% in the determination of fetal sex. Conclusions Amplification of X-STRs and AMXY gene can accurately determine the origin of NRBCs and detect fetal sex. This procedure is rapid, cheap and able to discern male fetus and female fetus as well. STRs may gain wider use in noninvasive prenatal diagnosis in the near future. part IIIPrenatal Diagnosis of Common Aneuploidies Using Fetal Nucleated Red Blood Cells Sorted from Maternal Peripheral CirculationTEST I Prenatal Diagnosis of Aneuploidies by Fluorescence in SituHybridization on Fetal Nucleated Erythrocytes from Maternal BloodObjective To explore the feasibility of noninvasive prenatal diagnosis of commom chromosome aneuploidies by FISH on fetal NRBCs separated from maternal peripheral blood. Methods High-risk pregnant women were picked out with type B ultrasonic and triplex serum screening test of AFP, uE3 and β -HCG. A puerperal woman was also recruited for her delivery of a Down syndrome (DS) neonatus and the maternal blood sample was collected six days after delivery. Fetal NRBCs were identified with immunochemistry stain of γ heamoglobin and separated by micromanipulation. Fluorescence in situ hybridization using CEP X/Y or LSI 21 probes was applied on the sorted cells in order to evaluate the number and structure of the correspond choromsomes. Results 34 high-risk mothers were screened out from 114 women taking genetic counseling. 16 cases including the woman who had born a DS baby accepted FISH on fetal NRBCs from maternal peripheral blood. The result of FISH suggested that four babies affected by aneuploidies, two with Down syndrome and two with Turner syndrome. It also showed that there were 8 male and 8 female babies. Except one fetus that was diagnosed as Turner syndrome by FISH but was acturally normal, the results of the other babies were in coincidence to their real karyotypes. The concordance rate was 93.75%. Conclusions Fetal aneuploidies can gain intrauterine determination by FISH on NRBCs of fetal origin in maternal blood. This is the first report about successful diagnosis of fetal aneuploidies using the noninvasive technique in our country.TEST II Application of STRs to Noninvasive Prenatal Diagnosis ofChromosome AneuploidiesObjective The purpose of the study is to develop a Polymerase chain reaction assay employing STRs for the noninvasive prenatal diagnosis of aneuploidies. Methods Blood samples were collected from 50 pairs of pregnant or puerperal women and their partners who came to take genetic counseling. Determined by heamoglobin γ chain staining, fetal nucleated erythrocytes in maternal blood were picked out by micromanipulation. After primer extension preamplification (PEP), single candidate cells undertook amplification of three 21 chromosome specific STR loci (D21S11, D21S1411, D21S1412), two X chromosome specific STR loci (DXS101, DXS6797) and AMXY gene. The above gene loci were also evaluated in each pair of parents. The results of a fetus was compared with that of its parents, by which the information about correspond chromosomes of the fetus could be obtained. Results 3 cases among the 50 samples of FNRBCs failed to produce PCR results. In the other 47 cases, the rates of imformativeness of D21S11, D21S1411, D21S1412 were 85.11 %,78. 72%,76.60% respectively. By combination of the three 21-STRs, the fetal origin of NRBCs could be confirmed in 45 samples and the number of 21 chromosome in fetal cells were also determined with a concordance rate of 95.74% (45/47) . Two babies affected by Down syndrome were discerned using this technique, which were verified by fetal real karyotypes. Fetal sexes were detected by gene analysis associating DXS101, DXS6797 with AMXY gene, the sensitivity of this method in diagnosing female babies was 90.48%, and that of male babies was 92.31%. The concordance rate of fetal sex detection using this procedure was 91.49 % . A fetus suffering Turner' s syndrome was also gain successful diagnosis using this noninvasive technique. Conclusion Multiple STR loci analysis based on single cell PEP-PCR can be applied on prenatal diagnosis of fetal aneuploidies, which is possible to be used as the alternative or supplementary technique to FISH. Besides, it is indicated that autosomal polymorphic loci are also capable of determining the origin of cells, which free the identification of fetal cells from male specific markers.