Study On A Method Using Fetal Nucleated Erythrocytes Screened From Maternal Peripheral Blood By Biological Nanotechnology For Diagnosis Of Down Syndrome

In China, there are as many as 800,000 to 1,200,000 newborns with birth defects every year and incidence rate with birth defects arrive at 4%-6%. Down syndrome is most common chromosome abnormalities. Incidence rate of Down syndrome in newborns is 1/600-1/800, and it's one main reason of congenital mental retardation. Without valid therapy for Down syndrome, prenatal screening and diagnosis as early as possible and then termination of pregnancy after confirmation is an effective way to cut down the birth rate. Prenatal diagnosis is an important measure to control the occurrence of genetic disease and improve the quality of the population. Now the rountine methods include amniocentesis or chorionicvillus sampling (CVS) and fetus cord blood sampling. These methods are accurate but they are invasive, with inherent risk to the fetus and the mother, such as uterus infection, abortion and fetal death. Non-invasive prenatal diagnosis methods include ultrasound and serum screening, but these methods have limited sensitivity and accuracy. Non-invasive prenatal diagnosis methods include ultrasound and serum screening, but these methods have limited sensitivity and accuracy. Therefore exploring non-invasive prenatal method on diagnosis of down syndrome is important for prevention of birth defects and elevation of population quality.The development of biological nanotechnology brings first light of morning for non-invasive prenatal diagnosis. This technology makes use of the special physical, chemical characterization of the nanoparticles and interaction between nanoparticles and cells or molecules, to explore high efficient and hypersensitized technology for detection, diagnosis, and therapy of disease. Magnetic screening of magnetic nanoparticles and labeling of fluorescent quantum dots is applied widespreadly in the field of biomedicine.Because there exists rare fetal nucleated red blood cells (FNRBC) in maternal peripheral blood, the isolation fetal cells from maternal circulation represent a promising challenge for investigators seeking a new approach to noninvasive prenatal diagnosis. Density gradient centrifugation, magnetic activated cell sorting (MACS), flow sorting cell sorting (FACS), micromanipulation of individual cells and culture and enrichment of fetal cells have been used to enrich target cells by reducing the ratio of maternal cells to FNRBC. Combination density gradient centrifugation with MACS is the most common method. Compared with FACS, MACS is cheaper, faster and needs less professional technology, in addition, it allows a large volume of blood to be separated in a short period of time. Anti-CD71 mAb, which specifically recognizes the transferrin receptor, is the immunoreagent most commonly used in fetal NRBC isolation protocols. Most investigators are prone to use two major magnetic beads for separation of FNRBC:big magnetic bead (1200-4500 nm in diameter) produced by Dynal company and small microbeads(≈50 nm in diameter) produced by Miltenyi company that are coupled directly or indirectly with anti-CD71 monoclonal antibody. But these magnetic beads have different defects and are expensive because they are made by foreign company.Because anti-CD71 antibody binds not only FNRBC, but also some activated maternal lymphocytes and reticulocytes. In order to make sure that the enriched cells are of fetal origin, different strategies to identify FNRBC were utilized. One strategy is subjecting the cells to PCR with Y chromosome-specific sequence or FISH analysis with X and Y chromosome -specific probes. But these two methods identify only the FNRBC from male fetus. Another strategy is immunocytochemistry or immunofluorescence to identify the hemoglobin specific to fetus. But the results are not satisfactory because of lower sensitivity and specificity.In this study, FNRBC in maternal blood were enriched by magnetic nanoparticles coupling with anti-CD71 antibody and identified by quantum dots coupling with anti-y-hemoglobin antibody. Finally, Down syndrome was detected by real-time quantity PCR. The progress of this experiment is as follows:1. In the optimal condition, by comparation of different methods of density gradient centrifugation, we found that Percoll 1.090 single density gradient centrifugation can enrich more NRBC in umbilical cord blood (170.58±5.67×104 NRBC/ml) and more purity (129 NRBC/2000 total cells). Moreover, the FNRBC in maternal blood were acquired by Percoll 1.090 single density gradient centrifugation. This method is simple and quick. After enrichment, FNRBC have intact shape and are qualified for next experiment.2. Anti-CD71 monoclonal antibody was conjugated with magnetic nanoparticles (MNPs) of medium size by EDC coupling reagent. The conjugates distributed evenly by TEM, the coupling rate of anti-CD71 antibody with nanoparticles arrived at 44.28±1.74%; the antibody after conjugation has good activity by FITC-Ab2; NRBC were enriched by cells bonding experiments; separation efficiency of the conjugates arrived 25.19±10.15% in umbilical cord blood model. The method of preparing anti-CD71-MNP conjugates was developped. It lays on the foundation for the enrichment of the scarce FNRBC in maternal blood.3. Aqueous soluble CdTe quantum dots were prepared by using thioglycolic acid as the stabilizer in aqueous solution and then characterized by absorption and fluorescence emission spectrum. The results show that these quantum dots had good optical properties. EDC-mediated condensation method was used to couple anti-y-hemoglobin antibody to CdTe quantum dots. The conjugates can label specifically the NRBC in umbilical cord blood and have better stability. It's the first time to bind magnetic enrichment with quantum dot labelling for screening FNRBC in maternal blood.4. The trace amount genome DNA from FNRBC that were enriched magnetically and identified by QDs, were extracted by specialised kit; relative quantitation in Down syndrome critical region gene 3 of genome DNA from samples were detected by real-time quantitive PCR, and the results showed the value of seven samples distributed within normal limits, in coincidence with those results karyotyping analysis from CVS. Down syndrome is successfully diagnosed by real-time quantitive PCR using screened FNRBC from maternal blood.In this study, a method using fetal nucleated erythrocytes screened from maternal Blood by biological nanotechnology for diagnosis of Down syndrome was developed. If it was feasible and can be applied to the clinic in the future, prenatal diagnosis would be more safe,simple,quick and popular. It will have an active and practical effect on decreasing incidence rate of the birth defects and improving population quality of newborns.