Maldi-tof mass spectrometry compared with real-time PCR for detection of fetal cell-free DNA in maternal plasma.
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DNA↗
Biomedical subjects
Sinuhe Hahn
Publications and source records attributed to Sinuhe Hahn.
Use of real-time polymerase chain reaction for the detection of fetal aneuploidies.
With the advent of real-time polymerase chain reaction (PCR), it is now possible to measure nucleic acid concentrations with an accuracy that was not possible only a few years ago. Examples are the analysis of gene expression or gene duplications/losses, where twofold differences in nucleic acid concentration have routinely been determined with almost 100% accuracy. As our primary interest is in prenatal diagnosis, we have investigated whether real-time PCR could be used for the diagnosis of chromosomal anomalies, in particular the aneuploidies such as trisomy 21, where the difference in copy number is only 50%. The feasibility of such an approach was first tested in a pilot study, in which we were able to demonstrate that trisomy 21 samples could be detected with 100% specificity. We have recently modified this test in order to permit the simultaneous analysis of trisomies 18 and 21, and have in a large scale analysis demonstrated that our approach can be used for the highly reproducible and robust detection of only 1.5-fold differences in gene copy number.
Amniocentesis↗
Macrophage migration inhibition factor is elevated in pregnancy, but not to a greater extent in preeclampsia.
BACKGROUND: Maternal serum concentrations of macrophage migration inhibitory factor (MIF) have recently been reported to be elevated in cases with preeclampsia. These findings may be important in increasing our understanding of the underlying events leading to the development of preeclampsia, as this cytokine is also expressed in the placenta, where it has been shown to possess immunemodulatory activities. For this reason we attempted to independently verify this report. METHODS: Plasma levels of MIF were assessed by ELISA in plasma samples collected from normal healthy male and female blood donors (n=20 per group), as well as healthy normal pregnant women in all three trimesters of pregnancy (n=60). In addition, MIF levels were examined from cases with mild and severe preeclampsia (n=20 per study cohort) and matched normotensive pregnancies (n=20). RESULTS: MIF levels were found to be elevated in pregnancy (median=10.1 ng/ml) when compared to non-pregnant controls (median=1.7 ng/ml). A moderate, but not significant, elevation was found to occur from the first to the third trimester of pregnancy. No significant difference was found to occur between the two preeclampsia study groups when compared to the normotensive control group. CONCLUSIONS: Our data suggest that circulatory MIF concentrations are elevated throughout pregnancy, but are not further increased in preeclampsia.
Blood Donors↗
Analysis of plasma elastase levels in early and late onset preeclampsia.
BACKGROUND: Circulatory neutrophils have been reported to be activated in preeclampsia. It has been suggested that maternal plasma levels of elastase may serve as a possible cell-free marker to quantify such activation. Although plasma elastase levels have been found to be elevated in cases with manifest preeclampsia and eclampsia, this has not yet been examined in cases with early and late onset preeclampsia. We have now examined this aspect. METHODS: In this retrospective study, maternal plasma samples were examined from eight cases with early onset preeclampsia (<34 weeks of gestation), eight cases with late onset preeclampsia (>34 weeks of gestation) and an equal number of gestational age matched normotensive term controls. Plasma concentrations of elastase were measured by ELISA using a commercially available assay. RESULTS: Plasma elastase concentrations were significantly elevated the preeclampsia study group when compared to the normotensive control group (median=139.2 ng/ml versus median=72.1 ng/ml; P=0.0025). These elevations remained significant when the preeclampsia study group was stratified into case with early onset preeclampsia (median=118.8 ng/ml versus median=62.2 ng/ml; P=0.03), but jailed failed to attain significance for those cases with late onset preeclampsia (median=181.3 ng/ml versus median=86.3 ng/ml; P=0.061). CONCLUSIONS: Our data indicate that elastase levels are elevated in both early and late onset forms of preeclampsia, and imply that the activation of neutrophils may be more acute in the former than in the latter (238 words).
Case-Control Studies↗
Syncytiotrophoblast micro-particles do not induce apoptosis in peripheral T lymphocytes, but differ in their activity depending on the mode of preparation.
Recent studies have suggested that pre-eclampsia may result from endothelial cell damage and overt immune activity triggered by the elevated release of syncytiotrophoblast-derived micro-particles (STBM). In this context, STBM have been reported to inhibit lymphocyte proliferation and induce Jurkat T cell apoptosis. In this study, STBM were prepared by three different in vitro methods (mechanical dissection, villous explant culture, and placental perfusion) and their functional properties were tested on T lymphocytes enriched from peripheral blood samples. Mechanically- and villous explant-derived STBM significantly inhibited activation, proliferation and cytokine release by T lymphocytes, while placental perfusion-derived STBM significantly induced T cell proliferation and a slight increase in IFNgamma release. None of the STBM preparations caused T cell apoptosis. Therefore, STBM prepared by different methods in vitro exhibit different effects on circulating T cells, a feature that will have to be taken into account when considering their potential role in normal pregnancy and pre-eclampsia.
Apoptosis↗
Cell-free foetal DNA in maternal plasma does not appear to be derived from the rich pool of cell-free foetal DNA in amniotic fluid.
BACKGROUND: Large quantities of cell-free foetal DNA have been detected in amniotic fluid, and it has been proposed that this material may contribute to the pool of cell-free foetal DNA in maternal plasma. METHODS: Twelve maternal blood samples were obtained from pregnant women about to undergo an amniocentesis. Cell-free DNA was extracted from the maternal plasma samples and the matched amniotic fluid samples. The amount of cell-free foetal DNA was quantified by real-time PCR assays for the SRY and RHD genes. RESULTS: Amniotic fluid was found to contain vast quantities of cell-free DNA (median concentration = 3,978 copies/ml amniotic fluid). The concentration of cell-free foetal DNA in maternal plasma was much lower (median concentration = 96.6 copies/ml maternal plasma). No significant correlation could, however, be determined between these two pools of cell-free foetal DNA. CONCLUSIONS: Our data confirm that amniotic fluid contains prodigious quantities of cell-free foetal DNA, but as no relationship exists between this material and that in the maternal circulation, it is unlikely that the amnion contributes to the presence of cell-free foetal DNA in maternal plasma.
Amniotic Fluid↗
Microparticle-free placentally derived soluble factors downmodulate the response of activated T cells.
Soluble placental factors may have immunoregulatory properties and have been demonstrated to inhibit T-lymphocyte proliferation in vitro. On the other hand, placentally derived syncytiotrophoblast microparticles and crude placental homogenates have been demonstrated to inhibit proliferation of mixed lymphocytes in vitro. Because previous studies on placentally derived soluble factors may have been contaminated by the presence of trophoblast-derived microparticles, we prepared microparticle-free placental supernatants. Such supernatants reduced the activation response of T cells, as well as their proliferation and the production of cytokines such as interleukin-2 and interferon gamma, in a dose-dependent manner. This reduction in T-cell proliferation does not appear to be caused by indoleamine 2,3-dioxygenase (IDO) because it was not reversed by the addition of L-tryptophan or an inhibitor of IDO (1-methyl-DL-tryptophan). No evidence was found for the presence of IDO in these supernatants when we used a biochemical assay measuring tryptophan catabolism. We conclude that the placenta produces currently unknown soluble factors that reduce T-cell activation, proliferation, and cytokine production.
Antigens, CD↗
Direct detection of fetal cells in maternal blood: a reappraisal using a combination of two different Y chromosome-specific FISH probes and a single X chromosome-specific probe.
BACKGROUND: We have recently explored the detection of circulatory male fetal cells directly in maternal whole blood samples by fluorescence in-situ hybridization (FISH). In order to improve the efficacy of fetal cell detection, we have now examined whether this could be enhanced by the use of two different Y chromosome-specific FISH probes (alpha-satellite and classical satellite III regions) in combination with an X chromosome-specific FISH probe. METHODS: Nineteen maternal blood samples (median gestational age = 28 weeks, range = 12-37 weeks) were examined in a blinded manner. No enrichment procedure was performed. Following hypotonic treatment and Carnoy's fixation, total nucleated cells were examined by two color FISH with a single X and two Y chromosome-specific probes. Nine cases were examined in parallel by conventional XY-FISH. RESULTS: Fetal cell detection was superior when using two Y chromosome-specific probes (specificity = 75%; sensitivity = 91%) when compared to the conventional XY-FISH approach (specificity = 50%; sensitivity = 60%). CONCLUSIONS: Male fetal cells can be detected in most maternal blood samples examined. Specificity and sensitivity is improved when using a combination of single X and two Y chromosome-specific probes when compared to a conventional XY-FISH protocol.
Chromosomes, Human, X↗
Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma.
BACKGROUND: Circulating fetal DNA (cfDNA) in maternal plasma has been measured to investigate its possible relationship with pregnancy-related disorders, including fetal trisomy 21 and preeclampsia. The circulating concentrations of single-copy fetal genes, however, are close to the detection limits of PCR methods. METHODS: We optimized a protocol for the real-time quantitative PCR amplification of the multicopy sequence DYS14 on the Y-chromosome. This was compared with an established real-time PCR assay for the single-copy SRY gene. RESULTS: By probit regression analysis, the measurements of male DNA by the DYS14 assay had a 10-fold lower detection limit (0.4 genome equivalents) than did measurements of SRY. For plasma samples from women in the first trimester of pregnancy, imprecision (CV) was 2%-22% when amplifying DYS14 compared with 26%-140% for SRY. CONCLUSIONS: The low copy numbers of fetal DNA in plasma of women in the first trimester of pregnancy cannot be measured precisely when targeting single-copy sequences. Better results are obtained by amplifying a sequence that is present in multiple copies per male genome.
Chromosomes, Human, Y↗
Detection of paternally inherited fetal point mutations for beta-thalassemia using size-fractionated cell-free DNA in maternal plasma.
CONTEXT: Currently, fetal point mutations cannot be reliably analyzed from circulatory fetal DNA in maternal plasma, due to the predominance of maternal DNA sequences. However, analysis of circulatory fetal DNA sequences in maternal plasma have been shown to selectively enrich for fetal DNA molecules on the basis of a smaller molecular size than maternal DNA. OBJECTIVE: To examine the prenatal analysis of 4 common beta-thalassemia point mutations: IVSI-1, IVSI-6, IVSI-110, and codon 39. DESIGN, SETTING, AND PATIENTS: A total of 32 maternal blood samples were collected at 10 to 12 weeks of gestation (mean, 10.7 weeks) between February 15, 2003, and February 25, 2004, in Bari, Italy, from women with risk for beta-thalassemia in their newborns immediately prior to chorionic villous sampling. Samples in which the father and mother did not carry the same mutation were examined. Circulatory DNA was size-fractionated by gel electrophoresis and polymerase chain reaction (PCR) amplified with a peptide-nucleic-acid clamp, which suppresses amplification of the normal maternal allele. Presence of the paternal mutant allele was detected by allele-specific real-time PCR. MAIN OUTCOME MEASURE: Detection of paternally inherited beta-globin gene point mutations. RESULTS: Presence or absence of the paternal mutant allele was correctly determined in 6 (86%) of 7 cases with the IVSI-1 mutation, 4 (100%) of 4 with the IVSI-6 mutation, 5 (100%) of 5 with the IVSI-110 mutation, and 13 (81%) of 16 with the codon 39 mutation. One false-positive test result was scored for the IVSI-1 mutation. Two cases with the codon 39 mutation were classified as uncertain and 1 case was excluded due to lack of a diagnostic test result at the time of analysis. These results yielded an overall sensitivity of 100% and specificity of 93.8%, with classified cases removed. CONCLUSION: Our recently described technique of the size-fractionation of circulatory DNA in maternal plasma may be potentially useful for the noninvasive prenatal determination of fetal point mutations.
Chorionic Villi Sampling↗
Circulatory nucleosome levels are significantly increased in early and late-onset preeclampsia.
INTRODUCTION: Elevations in circulatory DNA, as measured by real-time PCR, have been observed in pregnancies with manifest preeclampsia. Recent reports have indicated that circulatory nucleosome levels are elevated in the periphery of cancer patients. We have now examined whether circulatory nucleosome levels are similarly elevated in cases with preeclampsia. METHODS: Maternal plasma samples were prepared from 17 cases with early onset preeclampsia (<34 weeks gestation) with 14 matched normotensive controls, as well as 15 cases late-onset preeclampsia (>34 weeks gestation) with 10 matched normotensive controls. Levels of circulatory nucleosomes were quantified by commercial ELISA (enzyme-linked immunosorbant assay). RESULTS: The level of circulatory nucleosomes was significantly elevated in both study preeclampsia groups, compared to the matched normotensive control group (p = 0.000 and p = 0.001, respectively). CONCLUSIONS: Our data suggests that preeclampsia is associated with the elevated presence of circulatory nucleosomes, and that this phenomenon occurs in both early- and late-onset forms of the disorder.
Case-Control Studies↗
Soluble factors released by placental villous tissue: Interleukin-1 is a potential mediator of endothelial dysfunction.
OBJECTIVE: The purpose of this study was to analyze the potential of placental-conditioned medium to activate endothelial cells in vitro and to identify the placental factors that mediate this effect. STUDY DESIGN: Placental-conditioned medium was generated by the culturing of normal term placental villous explants for up to 48 hours. Human umbilical vein endothelial cells were exposed to the conditioned media, and cellular proliferation, viability, and activation were investigated. RESULTS: The proliferation of endothelial cells that were exposed to 20% placental-conditioned medium was reduced by 25%, but their survival was not compromised. Conditioned medium also up-regulated the expression of E-selectin and stimulated the release of soluble intercellular adhesion molecule-1 and the secretion of interleukin-6. Treatment with interleukin-1 receptor antagonist, but not with an anti-tumor necrosis factor-alpha neutralizing antibody, blocked the release of soluble intercellular adhesion molecule-1 and interleukin-6. CONCLUSION: Placentally derived interleukin-1 may be 1 of the potential mediators of the maternal inflammatory response that is observed in late pregnancy.
Cell Proliferation↗
Tissue-specific engraftment after in utero transplantation of allogeneic mesenchymal stem cells into sheep fetuses.
OBJECTIVE: Mesenchymal stem cells (MSC) have multiorgan differentiation capacity, providing the potential for prenatal treatment of genetic disorders. We address the question if in utero transplantation of MSC results in short-term organ-specific engraftment in the fetal sheep. STUDY DESIGN: Sheep fetal liver-derived MSC selected by adherence culture (passage 1) were transplantated into the fetal peritoneal cavity with ultrasound-guidance (mean gestational age, 59 days). After 14 days recipient fetuses were analyzed by fluorescence-activated cell sorting (FACS), real-time polymerase chain reaction (PCR), and immunohistochemistry. RESULTS: Fetuses (n = 11) were transplanted with 7.7 x 10(6) MSCs (mean). All surviving fetuses (n = 5) showed engraftment with mean levels of 3.2% (lung), 0.8% (spleen), 0.6% (liver, brain), 0.4% (bone marrow), 0.1% (blood, thymus), and <0.1% (kidneys) by flow cytometry. Immunohistochemistry showed organ-specific distribution. CONCLUSION: In utero transplantation of allogeneic MSC results in low level, multiorgan engraftment at 14 days post transplant. This supports the potential of in utero MSC transplantation for the treatment of nonhematopoietic genetic disorders of the fetus.
Animals↗
Nucleic acid based biosensors: the desires of the user.
The need for nucleic acid based diagnostic tests has increased enormously in the last few years. On the one hand, this has been stimulated by the discovery of new hereditary genetic disease loci following the completion of the Human Genome Project, but also by the presence of new rapidly spreading viral threats, such as that of the SARS epidemic, or even micro-organisms released for the purpose of biological warfare. As in many instances rapid diagnoses of specific target genetic loci is required, new strategies have to be developed, which will allow this to be achieved directly at the point-of-care setting. One of these avenues being explored is that of biosensors. In this review, we provide an overview of the current state of the art concerning the high-throughput analysis of nucleic acids, and address future requirements, which will hopefully be met by new biosensor-based developments.
Biosensing Techniques↗
FISH analysis of all fetal nucleated cells in maternal whole blood: improved specificity by the use of two Y-chromosome probes.
Current cytogenetic approaches in noninvasive prenatal diagnosis focus on fetal nucleated red blood cells in maternal blood. This practice may be too restrictive because a vast proportion of other fetal cells is ignored. Recent studies have indicated that fetal cells can be directly detected, without prior enrichment, in maternal blood samples by fluorescence in situ hybridization (FISH) analysis for chromosomes X and Y (XY-FISH). In our blinded analysis of 40 maternal blood samples, we therefore examined all fetal cells without any enrichment. Initial examinations using conventional XY-FISH indicated a low specificity of 69.4%, which could be improved to 89.5% by the use of two different Y-chromosome-specific probes (YY-FISH) with only a slight concomitant decrease in sensitivity (52.4% vs 42.9%). On average, 12-20 male fetal cells/ml of maternal blood were identified by XY- and YY-FISH, respectively.
Cell Count↗
Evaluation of a soybean lectin-based method for the enrichment of erythroblasts.
We performed a comparative study of the enrichment of erythroblasts by a soybean agglutinin galactose-specific lectin method and a standardized magnetic cell-sorting (MACS) protocol. Blood samples, obtained from 11 pregnant women at between 11 and 40 weeks of gestation, were split and examined by each method in parallel. The number of erythroblasts recovered by the lectin method was approximately eightfold higher than the number obtained by MACS. Our data suggest that the lectin-based method may provide a better approach for the enrichment of rare fetal erythroblasts from maternal blood.
Cell Separation↗
Treatment of maternal blood samples with formaldehyde does not alter the proportion of circulatory fetal nucleic acids (DNA and mRNA) in maternal plasma.
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Blood Specimen Collection↗
Circulatory corticotropin-releasing hormone mRNA concentrations are increased in women with preterm delivery but not in those who respond to tocolytic treatment.
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Corticotropin-Releasing Hormone↗