A. Kolialexi

Clinical Manifestations and Molecular Investigation of 50 Patients with Williams Syndrome in the Greek Population

Williams syndrome (WS) is a well-recognized neurodevelopmental disorder manifested by both connective tissue and CNS abnormalities. The study depicts the 8-y experience and follow-up of 50 Greek children with the clinical diagnosis of WS. Clinical data on the facial features and cardiovascular, endocrinologic, and neurodevelopmental evaluation are presented. The most consistent findings were dysmorphic features (100%), followed by dental anomalies (90%) and hyperacousis (90%). Only eight of 50 children had severe cardiovascular defects that required surgical intervention during the first year of life. Supravalvular aortic stenosis was less frequent (28%) than shown in the literature. Severe hypertension was noticed in 22% of our patients, and infantile hypercalcemia was noticed in 6%. Twelve percent of our patients showed an elevation of CPK. Most children presented with moderate to severe mental retardation with IQ ranging from 20 to 85. Elastin hemizygosity was detected by fluorescence in situ hybridization. Dinucleotide repeat polymorphism analysis was performed in an attempt to correlate phenotype with genotype. The origin of deletions was more frequently maternal (59%), and a more severe phenotype seemed to be associated with those deletions. This is the first report on WS patients in the Greek population.

Apoptosis in Maternal Peripheral Blood during Pregnancy

Objective: To investigate the mononuclear cell apoptosis rate during pregnancy. Materials and Methods: Apoptosis was quantitated by EtBr staining in whole peripheral blood samples of 135 women in different gestational weeks and 85 nonpregnant women used as controls. Apoptosis was also qualitated by TUNEL assay. Results: The apoptosis rate increased during pregnancy according to gestational age. In chromosomally abnormal fetuses apoptosis was 2.5-fold higher than that found in pregnancies with normal embryos matched for gestational age. FISH in TUNEL-positive cells using X, Y and 21 chromosome probes verified the fetal origin of part of the apoptotic population. Conclusion: Apoptosis is stimulated in maternal peripheral blood during pregnancy, possibly accounting partly for the presence of free fetal DNA in maternal serum. The increased apoptosis rate in pregnancies with chromosomally abnormal fetuses may have additional clinical importance.

Improved Specificity of NRBC Detection in Chorionic Villus Sample Supernatant Fluids Using Anti-Zeta and Anti-Epsilon Monoclonal Antibodies

Objective: Fetal erythrocytes leak from fetal capillaries at the time of chorionic villus sampling (CVS). It has been reported that in approximately 60% of CVS cases fetal nucleated red blood cells (NRBC) can be isolated from the supernatant fluid by immunophenotyping with monoclonal antibody (Ab) against the γ-chain of fetal hemoglobin and used as an additional source for confirmation of the fetal karyotype. However, the increased prevalence of β-thalassemia mutations in countries such as Greece results in many pregnant women who produce γ-positive cells. This makes it difficult to distinguish between the fetal and maternal origin of the NRBC. Use of Abs against embryonic hemoglobin chains ζ and ε may increase specificity for fetal NRBC detection. Methods: Mouse monoclonal Abs against Hb-ζ and Hb-ε were used in order to examine if specificity for fetal NRBC detection in CVS supernatant fluids could be improved. 41 samples were studied using anti-ζ and 20 using anti-ε monoclonal Abs. Results: Anti-ζ or anti-ε positive erythrocytes were, respectively, identified in 52 of 61 CVS samples and anti-ζ or anti-ε positive NRBC were present in all cases. The mean number of Hb-positive erythrocytes identified with the anti-ζ Ab was 58 and the mean number of NRBC 29. The mean number of anti-ε positive erythrocytes was 30 and of NRBC 23. FISH with X and Y chromosome specific probes was performed in 26 cases and the results were concordant with the CVS karyotype. Statistical analysis using the correlation test showed that anti-ζ and anti-ε were more specific for the detection of embryonic NRBCs. Conclusions: Since embryonic monoclonal Abs show increased specificity, they should be preferentially used for NRBC detection in CVS supernatant fluids. Furthermore, the increased specificity of anti-ζ and anti-ε Abs may considerably improve prenatal diagnosis from fetal cells isolated from maternal circulation.

Identification of Fetal Nucleated Red Blood Cells in the Maternal Circulation during Pregnancy Using Anti-Hemoglobin-ε Antibody

Aim: To investigate the use of anti-hemoglobin-Ε antibody in order to identify fetal cells in the maternal circulation during pregnancy. Materials and Methods: 48 blood samples were obtained from pregnant women, 26 in the 1st trimester and 22 in the 2nd trimester. Magnetic activated cell sorting was used for fetal cell enrichment followed by immunophenotyping with a monoclonal antibody against hemoglobin-Ε. FISH with X, Y and 21 chromosome-specific probes was performed in 29 cases. Results: The mean number of Ε-positive cells was 9.2 (range 2–23) in the 1st trimester, 4.8 (range 3–13) in the 2nd trimester and 22 (range 15–28) in pregnancies with Down syndrome. No significant difference was noted in the number of Ε-positive nucleated red blood cells (NRBCs) isolated from carriers and noncarriers of β-thalassemia. FISH analysis was successful in 24 cases. In 4 cases with known male fetuses, an average of 4.7 Ε-positive cells showed a Y signal. In 4 cases with Down syndrome, all Ε-positive cells showed 3 signals for chromosome 21. Conclusion: Anti-hemoglobin-Ε antibody has increased specificity for fetal NRBCs and should be preferentially used to improve noninvasive prenatal diagnosis of chromosome abnormalities from fetal cells in maternal blood.

Fetal nucleated erythrocytes (NRBCS) in chorionic villus sample supernatant fluids: an additional source of fetal material for karyotype confirmation

Fetal erythrocytes leak from the fetal capillaries at the time of chorionic villus removal. The purpose of this study was to determine if fetal nucleated erythrocytes (NRBCs) could be isolated from the chorionic villus sampling (CVS) supernatant fluid and used as an additional source of fetal material in order to confirm the fetal karyotype in cases of CVS mosaicism. One hundred CVS supernatant fluids were studied by simultaneous immunophenotyping, using a mouse antifetal haemoglobin antibody, UCHγ, combined with fluorescent in situ hybridization (FISH) analysis using X‐ and Y‐specific DNA probes. A chromosome 18 probe was also used in the case of a known male fetus with trisomy 18. Fetal haemoglobin (HbF)‐positive cells were identified in 73 supernatant fluids and HbF‐positive nucleated cells were present in 60 samples. The number of cells detected per sample showed great variation among the individual samples. FISH analysis was performed in 41 cases. FISH prediction of the fetal gender was concordant with the CVS karyotype in all cases, and the fetal trisomy 18 was correctly verified. In five cases in which Y sequences were detected, a small number of HbF‐positive cells with two X signals were also identified; interestingly, in three of the five cases, the mother was a β‐thalassaemia carrier. This technique can be used as a quick and accurate method for the immediate verification of CVS results in cases of mosaicism, thus avoiding second‐trimester amniocentesis.

13th International Congress The Fetus as a Patient

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Apoptosis in maternal peripheral blood during pregnancy

OBJECTIVE To investigate the mononuclear cell apoptosis rate during pregnancy. MATERIALS AND METHODS Apoptosis was quantitated by EtBr staining in whole peripheral blood samples of 135 women in different gestational weeks and 85 nonpregnant women used as controls. Apoptosis was also qualitated by TUNEL assay. RESULTS The apoptosis rate increased during pregnancy according to gestational age. In chromosomally abnormal fetuses apoptosis was 2.5-fold higher than that found in pregnancies with normal embryos matched for gestational age. FISH in TUNEL-positive cells using X, Y and 21 chromosome probes verified the fetal origin of part of the apoptotic population. CONCLUSION Apoptosis is stimulated in maternal peripheral blood during pregnancy, possibly accounting partly for the presence of free fetal DNA in maternal serum. The increased apoptosis rate in pregnancies with chromosomally abnormal fetuses may have additional clinical importance.

Improved Specificity of Nrbc Detection in Cvs Supernatant Fluids Using An Anti-ζ Monoclonal Antibody 70

Improved Specificity of Nrbc Detection in Cvs Supernatant Fluids Using An Anti-ζ Monoclonal Antibody 70