ABSTRACT
INTRODUCTION: The traditional cytogenetic analysis requires relatively long cell culture time, intensive labour and trained personnel. But, in clinical situations, rapid diagnosis of genetic disease is very important for urgent decision for future management. So we need more rapid and precise diagnostic tools for clinical genetic counselling. The fluorescence in situ hybridization (FISH) has been studied for detecting chromosomal aneuploidies because this method can get rapid and precise results of cytogenetic studies. OBJECTIVE: To evaluate the clinical utility of fluorescence in situ hybridization technique as a diagnostic tool of chromosomal anomaly. METHODS: Peripheral blood or gonadal tissue were obtained from the patients (n=63) clinically suspicious of genetic disease. Chorionic villi (n=6), amniotic fluid (n=9), and fetal cord blood (n=2) were obtained from 15 pregnancies undergoing fetal karyotyping at 9 to 30 weeks of gestation for prenatal genetic counselling. Karyotyping was performed by both traditional cytogenetics and FISH, using commercially available kits. After the procedures, the results of FISH were compared with the results of traditional cytogenetic studies. RESULTS: In a blind series of 17 samples all, including trisomy 21 (1 case), trisomy 18 (1 case), monosomyX (1 case), 47,XYY (1 case), and 47,XXY (1 case), were correctly identified. FISH results were correspondent with conventional karyotyping results in 7 patients with intersex except one case of suspicious of mosaicism. In nine children of Turner syndrome, the results of two methods were correspondent too. There was a fluorescent signal defect in band 15 q11-q13 in one of chromosome 15 in 18 children of 29 patients, clinically suspicious of Prader-Willi syndrome, with FISH method and only four patients were diagnosed as Prader-Willi syndrome with G-banding microscope. It was impossible to identify the defect in chromosome 15 q11-q13 in 10 (34%) children by both methods. Two children of 11 patients, clinically suspicious of Angelman syndrome, were diagnosed as Angelman syndrome with both method respectively. And four children were diagnosed as Angelman syndrome only with FISH method. In 5 cases, we cannot detect the defect in chromosome 15 q11-q13 with both methods. In four cases of Williams syndrome, the results of both methods were as follows; 1 case (25%): diagnosed as Williams syndrome by both methods; 2 cases (50%): diagnosed
Subject(s)
Child , Female , Humans , Pregnancy , Amniotic Fluid , Aneuploidy , Angelman Syndrome , Cell Culture Techniques , Chorionic Villi , Chromosomes, Human, Pair 15 , Cytogenetic Analysis , Cytogenetics , Diagnosis , Down Syndrome , Fetal Blood , Fluorescence , Gonads , In Situ Hybridization , Karyotyping , Mosaicism , Prader-Willi Syndrome , Trisomy , Turner Syndrome , Williams SyndromeABSTRACT
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.