Noninvasive Prenatal Diagnosis Of Congenital Toxoplasma Infection Using Fetal Nucleated Erythrocytes Circulating In Maternal Peripheral Blood

【Objective】Using micromanipulation techniques to isolate the fetal nucleated red blood cells (FNRBC) from maternal peripheral blood, and explore the feasibility of noninvasive prenatal diagnosis of congenital Toxoplasma infection by PEP-PCR on fetal NRBCs.【Methods】58 samples of 5ml maternal peripheral blood with positive Toxoplasma IgM,the gestational age 6-27 weeks were collected. Mononuclear cells were preliminary enriched by density gradient centrifugation. FNRBCs were observed,located,counted and isolated by micromanipulation technique under 400×inverted microscope after Wright's staining. Single cell DNA was obtained by alkaline denaturation, then primer extension preamplification (PEP) and polymerase chain reaction (PCR) were adopted to detect SRY and TOX B1 gene. Congenital Toxoplasma infections will final diagnosis if TOX B1 gene was positive in FNRBC.【Results】Fetal NRBCs displayed unique morphological characteristics under Wright's staining, and easy to discern. The number of FNRBCs in each case was 2～12. 58 samples of maternal blood were all obtained form the first and second trimester which had the mean number of FNRBCs of 3.20±1.15,7. 86±2.42 respectively. Very significant difference existed between the two groups (P<0.01), and the group of second trimester had the greatest number of FNRBCs. FNRBCs were not found in peripheral blood of unpregnancies. SRY gene was detected by PEP-PCR with a sensitivity of 93.75% (30/32) and a specificity of 100%. The accurate rate of determination of fetal sex using this method was 96.55% (56/58). 5 fetuses had final diagnosis of congenital TOX infection in 58 pregnant women with positive TOX IgM. The vertical infection rate was 8.6%(5/58). The PEP-PCR result of TOX B1 gene showed that there was one case of congenital TOX infection had been prenatal diagnosed by this method, the accurate rate was 20%(1/5)and the detection rate was 1.72%(1/58).【Conclusions】single fetal NRBC can be isolated by Micromanipulative technique associated Wright's staining method in a convenient,quick and accurate way. Fetal NRBCs with high quantity and quality can be obtained by this method effectively and then used for further research, which make genetic analysis using FNRBC in maternal blood more reliable. The FNRBCs sorted form maternal blood are more in the second trimester than the first, what offers a guidance for the best time to get mothers'blood for prenatal diagnosis. Fetal congenital TOX infection can gain intrauterine determination by PEP-PCR on NRBCs of fetal origin in maternal blood. This is the first report about successful diagnosis of fetal intrauterine infection of TOX using a new technique in our country, in a earlier,noninvasive way.