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.

Allelotyping and Comparative Genomin Hybridization Studies in Papillary Thyroid Carcinomas and Follicular Adenomas / 대한내분비학회지

BACKGROUND: In our previous study, the prevalence of the known causes of thyroid tumorigenesis was relatively rare in Korean population, suggesting genetic and environmental differences exist. Screening of genetic alteration in papillary thyroid carcinoma(PTC) and follicular adenoma(FA) in whole genomic scale was needed prior to search on individual genes of possible causes. METHODS: Ten cases of PTC without ret/PTC-I, -2, -3 rearrangement and 5 cases of follicular adenoma were included in the study of microsatellite marker allelotyping. Sixty two microsatellite markers available, were chosen to cover the known sites of loss of heterozygosity(LOH) involved in thyroid tumors, tumor suppressor genes and terminal portion of each chromosomes. PCR was performed on tumor DNA and leukocytes DNA from each patient with MDE gel electrophoresis to detect LOH. Same specitnens as above, 3 case of normal thyroid tissues and NPA, ARO cell lines were included in the study of comparative genomic hybridization(CGH). Tumor and control DNAs were hybridized to metaphase chromosome with differential stainings with fluorescein and rhoda-mine-dUTP. Obtained results were analyzed by multicolor fluorescence computer assisted image analyzer. RESULTS: In allelotyping, LOH were detected in 5 cases of PTC, 2 cases on D10S1435, 1 case each on D2S1780, DSS1099, D11S1986, D16S539, 1 case of PTC revealed LOH on DSS1099, D11S1986. In FA, LOH were detected in 3 cases on D1S534, D1S226, Dl 1S907, D22S683, DXS9807. In CGH, Xp addition was noticed in 1 case of PTC, 12q and 10p addition was noticed in 1 case each, 16q deletion and 17q addition in 1 case of FA. CONCLUSION: No hot spot of LOH was noticed in microsatellite marker allelotyping, neither of common chromosomal change in CGH study suggesting unbalanced translocation or gene amplification more than 5-10 Mb may be involved in the genetic alteration of PTC and FA.

Cytogenetic analysis of meningiomas

Cytogenetic analysis of 4 cases of meningiomas from 3 male and 1 female patients is reported. One of male patients suffered from neurofibromatosis type 2. Histologically, the meningiomas were meningotheliomatous (1), transitional (2), and psammomatous (1). Chromosomal abnormalities were found in all cases with a karyotype 45,XY,-22, 45,XY,-16, 45,XX,-2, and 45,XY,t (15p;22q), respectively. Monosomy of chromosome 22 was detected only in the patient with neurofibromatosis type 2. These cytogenetic analysis demonstrates that variable clonal karyotype aberrations exist in meningiomas.