Prenatal Diagnosis of Fetal Trisomy 21 from Maternal Peripheral Blood

This study was undertaken to establish a noninvasive prenatal genetic diagnostic method for trisomy 21 using the fetal nRBCs that is rarely present in maternal circulation. Peripheral venous blood samples were collected from 30 women with an advanced maternal age, abnormal triple marker test results, or abnormal ultrasound findings such as an increased nuchal translucency. The blood samples were treated with heparin. The triple density gradient centrifugation, and MACS using CD45 and CD71 were used to isolate the fetal cells. FISH analysis using probe 21 was performed with GPA-immunostaining. The study population consisted of 30 patients from 13 to 25 weeks of gestation, and nRBCs were separated in all cases. In GPA-immuno FISH analysis using probe 21, 3 cases of trisomy 21 were diagnosed and these results were confirmed by the amniocentesis. In conclusion, a prenatal diagnosis of trisomy 21 through GPA- immuno fluorescence in situ hybridization (FISH) analysis using separated fetal nRBCs is a useful, innovative, accurate, rapid and non-invasive diagnostic method.

Isolation of Fetal Cells in Maternal Peripheral Blood and Genetic Analysis of Trisomy 21 by GPA-immuno FISH(Fluorescence in situ hybridization / 대한산부인과학회잡지

INTRODUCTION: Down's syndrome is the most common congenital chromosomal anomalies which occurs 1 out of 700-1000 births. Until now, for prenatal diagnosis of Trisomy 21, invasive techniques such as amniocentesis, chorionic villus sampling(CVS) and cordocentesis were used, but they encompass the rare possibility of morbidity to the mother and fetus. Triple marker using maternal serum is a currently used noninvasive method, but it only shows the accuracy of 60%. Accordingly, a noninvasive method, using fetal cells from maternal blood is under extensive investigation. This study was undertaken to establish a noninvasive prenatal genetic diagnostic method of trisomy 21 using fetal nRBCs rarely present in maternal circulation. MATERIALS AND METHODS: Peripheral venous blood samples were collected from 76 women and treated by heparin. For the isolation of fetal cells, we used a triple density gradient centrifugation, and Vario-MACS and Mini-MACS using CD45 and CD71, and then, the morphological differentiation of the fetal nRBC was performed by Kleihaur-Betke stain. With GPA immunostain, nRBCs were identified by cytoplasm and GPA attatchment, and after marking the site, a FISH was performed. RESULTS: This study population included 76 patients from 8 to 41 weeks of gestation, and nRBC was separated from all cases. The morphological differentiation was achieved by K-B stain. The mean number of nRBC collected from 20 ml of maternal peripheral blood was 15. The number of nRBCs retrieved reached its peak in 12-18 gestational weeks(18.9 6.0) which decreased from 20 gestational weeks and thereafter. Fetal sex was determined by FISH analysis using probe X, Y with GPA-immunostained cells. GPA-immuno FISH analysis using probe 21 in 30 cases of advanced maternal age or positive triple markers, we confirmed 3 cases of Down's syndrome. These results were also confirmed using the CVS or amniocentesis. CONCLUSIONS: Fetal nRBCs were separated from all cases after 8 gestational weeks. Prenatal diagnosis of trisomy 21 through GPA-immuno FISH analysis of chromosome 21 using separated fetal nRBCs is an useful, innovative, accurate, rapid and non-invasive diagnostic method. But for clinical use, more cases of experiments will be needed.

Prenatal genetic diagnosis from maternal blood: simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative and positive magnetic activated cell sorting

Fetal nucleated red blood cells (nRBCs) are rare in maternal circulation, but their presence constitutes a potential source of non-invasive prenatal genetic diagnosis. This study was undertaken to establish a non-invasive prenatal genetic diagnosis method using isolated fetal nRBCs. A multi-step method including triple density gradient and magnetic activated cell sorting (MACS) using CD45 and CD71, cytospin centrifugation, K-B staining, and glycophorin A-immuno fluorescence in situ hybridization (GPA-immuno FISH) was performed. The study population included 65 patients from 8 to 41 weeks of gestation, and fetal nRBC was separated from all cases. The number of fetal nRBCs retrieved was 12.8 +/- 2.7 in 8 to 11 gestational weeks, 15.2 +/- 6.5 in 12 to 18 gestational weeks, 16.4 +/- 6.5 in 19 to 23 gestational weeks, 10.6 +/- 3.2 in 24 to 28 gestational weeks, and 5.5 +/- 1.9 in 35 to 41 gestational weeks: the mean number of nRBCs collected from 20 ml of maternal peripheral blood was 13.7 +/- 6.2. The highest value of yield was 45.6% from 12 to 18 weeks gestation. The fetal sex determination confirmed by amniocentesis or chorionic villus sampling showed 100% sensitivity and 91.7% specificity for males; 91.7% sensitivity and 100% specificity for females. We showed that fetal cells can be reliably enriched from maternal blood and that they can be used for detecting specific chromosomes by FISH with a specificity superior to current non-invasive methods.