The Preliminary Studies On Non-invasive Prenatal Diagnosis Utilizing Fetal Nucleated Red Cell Blood Cell From Maternal Blood

Objective:To explore the feasibility of isolating and enriching these fetal erythroid cells from maternal blood for prenatal genetic analysis we employed a two-phase liquid culture that supports the growth and differentiation of erythroid progenitor cells..Methods:(1) Using a Percoll discontinuous gradient, the fetal nucleated erythrocytes were enriched from the Cell suspension of 12 villi. the half of enriched cells were stained with theε-chain immunofluorescence antibody, Wright's staining method was used to mark the other half of isolated special cells previously spread on the slides. (2)Mononuclear cells were separated from ten maternal blood samples at 7-9weeks of gestation and cultured in the first phase. After 4-5 days, the non-adherent cells were harvested and recultured with erythropoietin for another 3-4days.Results:(1) The green dyed FNRBC were observed under fluorescence microscope. Red blood cells in various stages of division were found with Wright's staining, and there were 30% of the red blood cells having the ability to be amplified. (2) Most of the mononuclear cells were died. in the first phase after3-4 days,no living cells survived in the next 10-14 days.Conclusion:(1) There are Erythropoiesis having the Proliferative capacity in villi,which can be amplified by cell culture. Erythropoiesis is expected to be amplified by cell culture, but the experiments need further validation (2) According to experimental results the FNRBC from peripheral blood of pregnant women still can not be effectively amplified under the present culture conditions.It is possible that a clinically satisfactory, acceptable non-invasive prenatal diagnosis could be achieved through improving the culture conditions or searching for new types of fetal cells to amplify. Object:The purpose of this paper is to establish a effective method using fetal nucleated red blood cells form peripheral maternal blood for non-invasive prenatal diagnosis.Methods:1:villous and 20mL peripheral maternal blood were collected from 10 cases of pregnant women in 7-9 weeks.2:Detection of sry/zfx gene fragments to determine fetal sex by PCR of villous after abortion.3:Enrichment of nucleated red blood cells (nRBCs) from 20 mL maternal blood was done using a Percoll discontinuous density gradient and isolation by flow sorting using a combination of three monoclonal antibodies:Anti-CD71 (A group), anti-CD45dim (B group) or hemoglobinε-chain immunofluorescence antibody (C group) labeled before Flow cytometry sorting in 25 cases between 7 and 9 weeks' gestation.Selected cells will be thrown on the slide.4:Anti-CD45, and hemoglobin s-chain antibody immunofluorescence double-labeled mononuclear cells of 5 cases between 7 and 9 weeks'gestation before sorting by flow cytometry with CD45dim 5:single out Anti-ε-chain positive NRBCs by Micromanipulation technique.6:Selected cells were amplified by multiple displacement amplification (MDA) for whole genome amplification.Fetal gender, Confirmed by conventional PCR of Alphoid fragments with whole genome amplification product as a template, compared with the sex identification by PCR of the villous.Results:1:The percentage of positive signals after flow sorting,the number of signal and the cells thrown on the slide A group were 0.305±0.051%,6500±2000/10ml,4±3/10ml; B group,0.781±0.051%,10000±3000/10ml,20±8/10ml; C group 0.007±0.002%,-2500±1500/10ml,0/10ml. There are significant differences among the three groups, of which B the highest rate of cell harvest, C group the lowest..2:Double labeling mononuclear cells of the 5 cases with Hemoglobinε-chain immunofluorescence antibody and CD45 antibody, after sorting by flow cytometry with CD45dim, all specimens were observed inε-chain of hemoglobin-positive cells, which were fetal nucleated red blood cells(fetal nucleated red blood cell, FNRBC) 3:There were two men in these five cases, by conventional PCR detection of sry/ zfx fragments of villous to determine fetal gender.These sub-positive cells of two male cases and one female case were selected for the whole genome amplification and subsequent detection of gender by conventional Alphoid gene fragment amplification.The results showed that one cases of whole genome amplification failed; 1 case diagnosed as male fetuses, and 1 female, which were in full compliance with the gender results of PCR with villous..Conclusion:1:Enrichment of nucleated red blood cells (nRBCs) from maternal blood was done using a Percoll discontinuous density gradient and isolation by flow sorting using a combination of three monoclonal antibodies:Anti-CD71, anti-CD45 or hemoglobinε-chain immunofluorescence antibody labeled before Flow cytometry sorting, of which anti-CD45dim group has the highest rate of cell harvest, hemoglobinε-chain immunofluorescence antibody group has the lowest.2:Double labeling with Hemoglobin s-chain immunofluorescence antibody and CD45 antibody,after sorting by flow cytometry with CD45dim,s-chain of hemoglobin-positive cells were singled out for whole genome amplification,this technique can effectively obtain an adequate amount of template and ensure the accuracy of the follow-up analysis for clinical sex-linked genetic diseases, non-invasive prenatal diagnosis.