Enrichment of Fetal Nucleated Red Blood Cells by Multi-core Magnetic Composite Particles for Non-invasive Prenatal Diagnosis

A novel kind of multi-core magnetic composite particles, the surfaces of which were respectively mo- dified with goat-anti-mouse IgG and antitransferrin receptor（anti-CD71）, was prepared. The fetal nucleated red blood cells（FNRBCs） in the peripheral blood of a gravida were rapidly and effectively enriched and separated by the mo- dified multi-core magnetic composite particles in an external magnetic field. The obtained FNRBCs were used for the identification of the fetal sex by means of fluorescence in situ hybridization（FISH） technique. The results demonstrate that the multi-core magnetic composite particles meet the requirements for the enrichment and speration of FNRBCs with a low concentration and the accuracy of detetion for the diagnosis of fetal sex reached to 95%. Moreover, the obtained FNRBCs were applied to the non-invasive diagnosis of Down syndrome and chromosome 3p21 was de- tected. The above facts indicate that the novel multi-core magnetic composite particles-based method is simple, relia- ble and cost-effective and has opened up vast vistas for the potential application in clinic non-invasive prenatal diag- nosis.

Isolation of Fetal Nucleated Red Blood Cells from Maternal Blood

To find a simple, effective method of isolating fetal cells from maternal peripheral blood for prenatal diagnosis, 45 women were studied with their gestation being 6-14 weeks and age 21- 30 years. The fetal cells were isolated from maternal blood by using discontinuous density gradient centrifugation. Some of the isolated cells were made smear and counted under the microscope; others were used for predicting fetal sex by PCR amplification of Y chromosome specific DYZ1 gene. The major cells in the upper separation interface were lymphocytes and monocytes, with occasionally seen nucleated red blood cells (NRBC); while those in the middle separation interface were neutrocytes, with NRBC scattering. The ratio of NRBC/nucleated cells was 1. 98±0. 28× 10-5. There was no significant difference between the first and second trimester (P>0. 05). The amount of isolated fetal cells was sufficient for prenatal genetic diagnosis. Male pregnancy was correctly predicted in 10 out of 13 cases. It is concluded that the method of discontinuous density gradient centrifugation was of considerable importance in the development of non-invasive prenatal genetic diagnosis.

Detection of Fetal Nucleated Erythrocytes and Fetal DNA from Peripheral Blood of Pregnant Women

In order to search for a more reliable method of sorting fetal nucleated red blood cells (NRBCs) and DNA from maternal peripheral blood and to identify origin of NRBCs and DNA, NRBCs were isolated from peripheral blood of 88 pregnant women by density gradient centrifugation and fluorescence activated cell sorter (FACS) respectively. Nested polymerase chain reaction was used to detect normal male SRY gene from blood plasma DNA of 65 pregnant women. The results revealed that fetal NRBCs were found in 14 of 27 maternal samples by density gradient centrifugation. The number of cells was from 1 to 10. Using FACS, CD71 + cells were identified among all 61 samples. The frequency was (0.35±0.25)×10 -2; The detectable rate of the SRY gene of blood plasma DNA from 46 women carrying male fetuses was 65.22 % (30/46). Non-detectable rate for 19 women carrying female fetuses was 94.74 % (18/19). It was concluded that the methods of sorting fetal NRBCs and DNA have already made great progress. The methods for fetal NRBCs and plasma DNA from maternal peripheral blood to diagnose genetic diseases seem to be the best methods of noninvasive prenatal diagnosis.

Investigation on the Maternal-Infantile Infection with Human Parvovirus B19

To investigate the maternal-infantile infection with human parvovirus B19, the IgG and IgM antibodies against human parvovirus and the B19-DNA in serum and peripheral blood mononuclear cells (PBMC) of pregnant women as well as the serum IgM antibody against B19 and the B19-DNA in serum and cord blood nucleated cells (CBNC) of newborns were determined by ELISA and nested PCR respectively. It was found that the positive rate of the IgG antibody against human parvovirus B19 in sera of 92 pregnant women was 38.04% (35/92), and that of the IgM antibody in 720 pregnant women was 9.03% (65/720). However, the IgM antibody against human parvovirus B19 was negative in the cord blood sera of 95 newborns. As to the human parvovirus B19 DNA, none of 720 pregnant women and 95 newborns was proved to be positive in their sera. Nevertheless, the positive rate of the parvovirus B19 DNA in PBMC was 3.06% (3/98) in 98 pregnant women and 1.12% (1/89) in CBNC of 89 newborns. It is concluded that the history of infection with human parvovirus B19 exists in certain pregnant women with a small percentage of pregnant women infected with recent or acute infections of B19 virus. The detection rates of the B19 viral DNA in PBMC of pregnant women and CBNC of newborns were higher than those in sera, indicating that the risk for vertical transmission is very low.