Isolating The Fetal Nucleated Red Blood From Maternal Blood And Non-invasive Prenatal Diagnosis Of Fetal Aeuploid Using Fetal Nucleated Red Blood

Part I Isolating Fetal Nuleated Red Blood Cells from MaternalBloodObjective To investigate the best method of isolating the fetal nucleated red blood cells (NRBCs) through the comparison isolating methods using micromanipulation techniques and flow cytometry.Method 120 samples of maternal blood with the gestational age 10-32 weeks were collected. NRBCs are isolated by micromanipulation technique and flow cytometry after density gradient centrifugation respectively. When flow cytometry was used, 5 groups were divided in each sample {Group I: Anti-CD71-FITC McAb was added; Group II: Anti-GPA-PE McAb was added; Group III: Anti- anti-e—Hb McAb was added; Group IV: Anti-CD71-FITC McAb + Anti-GPA-PE McAb were added jointly; Group V: Anti-CD71-FITC McAb + Anti-GPA-PE McAb+ Anti- anti-e-Hb McAb were added jointly }. PCR were used to identify the SRY gene from isolated FNRBCs finally.Result FNRBCs were found in 97 of 120 (80.83%) samples by micromanipulation after density gradient centrifugation and the concordance rate was 61.7% (74/120). The gain rates and the concordance rates of the five groups were: 2.8%, 70.8%;5.74%, 65.5%; 0.25%, 95.8%; 0.74%, 95.8%; 0.02%,43.3%.3-15 NRBCs were foundin every samples combined flow cytometry with micromanipulation and theconcordant rate was 82.5%.Conclusion: high purity NRBCs could be received by flow cytometry combined withmicromanipulation, which might be served as foundation for next non-invasiveprenatal diagnosis.Part II Identifying Fetal Nucleated Red Blood Cells and theCell-free Fetal DNAObjective To compare some techniques of identifying single fetal cells and cell-freefetal DNA which existed in maternal blood, and to investigate the feasibility of thesemethods.Method 8ml maternal peripheral blood were collected in 175 pregnancy womenwhose gestation age were 10-33weeks. Flow cytometry combined withmicromanipulation was used for isolating sing fetal cells and cell-free fetal DNA wastaken. Every sample was divided into 3 groups: Group 1 SRY gene in single FNRBCwas amplification by PEP-PCR; Group 2 Y chromosomal was detected by PRINS;Group 3 SRY gene in cell-free fetal DNA from maternal blood was detected usingQF-PCR.Result: 1. 82 SRY gene in single FNRBC were detected by PEP-PCR, and thepositive rate was 91.1% (82/90) ; 2. 90 SRY gene from cell-free fetal DNA inmaternal blood were detected through QF-PCR. The positive rate was 100% (90/90);3. 89 Y chromosomal in single FNRBC were detected by PRINS, and the positive rate was 98.9% (89/90) .Conclusion: PRINS and QF-PCR were the method of identifying fetal cells and fetalcell-free DNA with high accuracy and specificity.Part III Non-invasive Prenatal Diagnosis of Fetal Aneuploid UsingMateral Peripheral BloodExperiment 1 Screening the Fetal Down's Sydrome through Detecting the AFP, β-HCG, and μE3 from Materal SerumAbstract: Objective A combined test in pregnancy women had been introduced in the detection of fetal Down's syndrome ,which involves serum markers. The three hormones tested are AFP, β-HCG, and μE3. Inadditional, the abnormal reading of the three hormones during the screening tests had been developed to try to identify pregnancies that were at "high risk". These pregnancies were then candidates for further diagnostic testing. Method: Using enzyme-linked immunosorbent assay to measure the three hormones that had been applied to 278 pregnant women for August 2003 to August 2005, who had volunteered to had a Down's syndrome serum markers screening in our hospital, during which their pregnancies were monitored till the birth of their children. The screening results combined with other data collected from the tests about the pregnant women had been analyzed by using Down's syndrome's risky software to identify higher risked pregnancies.Result: Among 278 screened pregnant women, 25 cases had been identified as high risk, which was 8.9% of the total screened pregnancies. 1 fetus had been discovered to have DS, and one was still birth. Also. in the 10 pregnancy cases that had been screened to have high Down's syndrome risk had also exhibit complications duringpregnancies.Conclusion Prenatal testing can give information about a pregnant woman's risk ofhaving a baby with certain birth defects, such as fetal aneuploid. Prenatal testing canalso reduced both death rate and defect rate of new born babies, which had a benefitfor the society.Experiment 2 Diagnosing Fetal Aneuploid Using Fetal Nuleated Red Blood from Materal Peripheral BloodObjective: To investigate a new technique of non-invasive prenatal diagnosing fetalaneuploid using maternal peripheral blood.Method: peripheral blood in 25 high risk pregnant women in part 1 and 87 pregnantwomen who came forward to access our experiment were collected. Flow cytometrywas used to isolate the fetal erythroblast from 112 maternal peripheral blood, thenPRINS was applied to detect the X, Y and 21 chromosome in single fetal cells.Result: X chromosome was detected in every samples. The sensitivity and specificitywere both 100% . 69 Y chromosome were detected. The sensitivity and specificitywere 92% and 100%, respectivity. In additional, 2 XXY syndrome and 2 Down'ssyndrome were detected.Conclusion: PRINS is a reliable new technique for the gene analysis of fetal singlecells from maternal peripheral blood because of efficient, high sensitivity andspecificity.