Noninvasive Prenatal Diagnosis By Utilizing Fetal Nucleated Red Blood Cells In Maternal Blood

Part I Enrichment of Fetal Nucleated Red Blood Cells in the Peripheral Blood of Pregnant WomenExperiment I Minor Enrichment of Fetal Nucleated Red Blood Cells in Maternal Blood by Density Gradient Centrifugation Combined with Micromanipulation and Fatal Origin Identification by PEP-PCRObjective To explore the feasibility of enriching fetal nucleated red blood cells (FNRBCs) in maternal blood by density gradient centrifugation combined with micromanipulation.Methods Peripheral blood samples were obtained from 40 pregnant women. Minorenrichment of fetal nucleated red blood cells was done by density gradient centrifugation. After smearing and Wright's staining, positive cells were observed and counted. FNRBCs were collected by micromanipulation and then primer extension pream-plification (PEP) and polymerase chain reaction (PCR) were adopted to detect the sex-determining region Y chromosome (SRY) gene.Results Typical morphological features of FNRBCs were observed, which were characterized by larger diameter than the negative cells, round nucleus without nucleolus, massive chromatin and abundant cytoplasm. Positive cells can be found in 31 cases and the detection rate was 77.5 %( 31/40). One to eight cells can be found in 6 ml maternal blood samples and the average was 3.55±1.73. The positive coincidence of SRY detection by PEP-PCR base on FNRBCs was 76.5% (13/17) and the negative coincidence was100% (14/14).The total gender coincidence was 87.1%.Conclusion Fetal nucleated red blood cells exist in maternal blood with special morphological features which were benefit for identification. All these proved that noninvasive technique of getting fetal cells was soundly feasible, providing the morphological reference for following-up enrichment. According to the existence of unsatisfied identification, conclusion was made that the method of density gradient centrifugation combined with micromanipulation is very subjective, which requires high technically skills and can't achieve the ideal degree of generalization.ExperimentⅡEnrichment of Fetal Nucleated Red Blood Cells in Maternal Blood by Flow Cytometry and Fatal Origin Identification by PEP-PCRObjective To explore the feasibility of enriching fetal nucleated red blood cells (FNRBCs) in maternal blood by flow cytometry and find the optimal method of FNRBCs enrichmentMethods Peripheral blood samples were obtained from 15 normal pregnant women. After density gradient centrifugation , FITC-CD71 and PE-GPA monoclonal antibodies were used to do cell-sorting by flow cytometry. PEP-PCR was adopted to detect SRY gene of the obtained cells.Results The suspension of positive cells after sorting was dropped into chips and the fluorescent cells were observed using fluorescence microscope. After Wright's stained, the positive cells can be seen with morphological characteristics of FNRBCs. The cell detection rate was 100%. Three to nine cells were found in 6ml maternal blood samples and the average was 5.13±1.68. SRY gene detection was done by PEP-PCR based on the obtained cells and the gender coincidence rate was 100%.Conclusion The combination of density gradient centrifugation and flow cytometry can improve the isolating rate of fetal nucleated red blood cells in maternal blood and the achievement ratio of micromanipulation, which could be considered as an effective method to enrich FNRBCs for doing noninvasive prenatal diagnosis.PartⅡChromosomal Detection of Fetal Nucleated Red Blood Cells by Multi-PRINSObjective To evaluate the applicability of utilizing PRINS as a fast way to perform chromosomal detection of FNRBCsMethod FITC-12-dUTP, Texas red-12-dUTP were used to do multiple in situ hybridization (Primed in situ labeling, PRINS) to detect chromosomes of FNRBCs: 1. PRINS was used to detect the X, Y chromosome at the same time; 2. PRINS was used to detect the 21, Y chromosome at the same time.Results 1. Two red fluorescent signals (on behalf of two X chromosomes) were observed in fetal nucleated red blood cells of female fetuses. One red and one green fluorescent signal (on behalf of one X chromosome and one Y chromosome) was observed in fetal nucleated red blood cells of male fetuses. 2. Two green fluorescent signals (on behalf of two 21 chromosomes) were observed and there was no red signal in fetal nucleated red blood cells of female fetuses. Two green fluorescent signals(on behalf of two 21 chromosomes) and one red signal(on behalf of one Y chromosome) was observed in fetal nucleated red blood cells of male fetuses. The testing results were consistent with the actual fetal karyotypes.Conclusion PRINS was found to be an effective and accurate method to perform chromosomal detection. It is economic, fast, and the results were direct-viewing. Multi-PRINS can be applied to detect different chromosomes at the same time, which may serve as an ideal method to do prenatal diagnosis of chromosomal abnormalities. PartⅢPreliminary Establishment and Verification of Rapid Noninvasive Prenatal Diagnostic Model of Down's SyndromeObjective To establish a rapid and effective noninvasive prenatal diagnostic model of Down's syndrome.Methods The model was initially proposed and put into practice specifically: 8 women with high-risk of Down's syndrome pregnancy were chosen by triple serum screening, ultrasonic inspection and case history. Blood samples were collected and FNRBCs were enriched by density gradient centrifugation combined with flow cytometry. Multi-PRINS was applied to detect the Y chromosome and 21 chromosomes.Results Diagnostic conclusions can be made in two days: there were 7 cases of normal fetuses, including two cases of male and five cases of female fetuses; one male fetus with Down's syndrome was diagnosed. Three 21 chromosomes and one Y chromosome can be observed in the cells isolated from the positive case. All the results were consistent with the actual fetal karyotypes.Conclusion The model was found to be effective to do prenatal diagnosis of fetal Down's syndrome and may provide reference for prenatal diagnosis of other fetal chromosomal or gene abnormalities and intrauterine infection.