| IntroductionIn 1969 Walknowska et al. identified cell of karyotype 46, XY in the peripheral blood of pregnant woman. This was the first to demonstrate that fetal cells enter the maternal circulation in normal pregnancy and suggested that these cells had potential for use in non - invasive prenatal diagnosis. According to the difference of the dignosis method, Prenatal diagnosis can be divided into two kinds; invasive and non - invasive. Invasive prenatal diagnosis include Chorionic villus sampling^ Amniocentesis et al. Though having a high accuracy , these methods have significant risk of fetal loss. While enrichment and purification fetal cell or free DNA from maternal blood belongs to non - invaisve prenatal diagnosis. Because of many benefits,it has become the major field of prenatal diagnosis. This method takes the fetal cells from peripheral blood of pregnant women as materials, using these cells' DNA as sample to diagnose the genetic disease prenatally. With the progress of the investigation,nucleated red blood cell (NR-BC) was regard as the candidate for non - invasive prenatal diagnosis. However , two main problems need to be overcome. First, the low frequency of the target cells i. e. 1 in 104 ~ 106nucleated blood cells, depending upon detection method and gestational age , and second, the need for an unequivocal marker for the fetal origin of the cells detected. Several seperated method, such as flow - sorting and magnetic sorting et al, had been used to enrich for NRBCs, However, enrichment procedures inevitably lead to cell loss. So how to get fetal NRBC accurately , simplify the experiment procedure and decrease the loss of fetal cells becomes the most important thing.Duchenne muscular dystrophy is a common motal X - Linked recessive disease, whose incidence of male birth is 1/3500. It characterized by progressivelymuscular atrophy and weakness. The patient commonly appear symptom at 3 ~ 5 years old and die before 20. Dystrophin gene located in Xp21.2 , about 2.4Mb long, the biggest disease - related gene as well - known at present. The main gene disorder of dystrophin is deletion of different regions, account for 55 ~65% or so, repetition about 6%. There are two deletions hot spot regions, with one was exon 4 ~21 in 5'end,and the other was exon 44 ~52. Using multiplex PCR could detected 98% of the detection, other non - deletions have to depend on STR linkage analysis. At present,this disease can not be cured. The only measure is to prevent patient child born by prenatal diagnosis. Therefore non - invasive prenatal diagnosis of DMD is beneficial for society and econimy.Materials and Methods:We obtained maternal blood samples from 40 pregnancies, at 7 - 26 weeks' gestation, 15 of these pregnancies were at high risk for DMD and were followed up with molecular genetic analysis of chorionic villus or amniotic fluids. Blood from 3 normal non - pregnant women and 3 normal men was used as negative control, and 2 fetal umbilical cord blood was used as positive control.NRBCs were separated with percoll using a discontinuous density gradient method ,and then smeared on microscope slides using cytocentifugation. Slides were stained with antibodies against the - chain of fetal hemoglobin (HbF). NRBC were detected,counted and localized. After this, all positive NRBCs were collected by micromanipulator under microscopic observation, and then amplified them by improved primer extension preamplification(PEP). Sex and DMD genetic diagnosis were determind from a small aliquot of the PEP reaction. This can further verificated the origin of NRBCs and make the prenatal diagnosis of DMD at the same time.Results:NRBCs stained with HbF were found earliest at 8 week of gestation. And the optimum week in which to perform a reliable non - invasive prenatal diagno-sis was around 14 ~ 18 week. The number of detected NRBCs from 10ml of maternal blood ranged from 2. 5 to 17. 14 in each case and average was 9. 15 in 10ml. No HbF positive were found in peripheral blood of normal non - pregnant wo...
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