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Sinuhe Hahn

Publications and source records attributed to Sinuhe Hahn.

At least 55 records · Page 3Linked to original sources
PubMed2005

Numerous erythroblasts in maternal blood are impervious to fluorescent in situ hybridization analysis, a feature related to a dense compact nucleus with apoptotic character.

BACKGROUND AND OBJECTIVES: The analysis by fluorescence in situ hybridization (FISH) of fetal erythroblasts enriched from maternal blood remains an attractive alternative for risk-free prenatal diagnosis of aneuploidies. However, current results are discouraging because of the low levels of sensitivity or the inability to detect fetal erythroblasts by FISH. DESIGN AND METHODS: Erythroblasts were enriched from 35 maternal blood samples by magnetic cell sorting (MACS), identified morphologically following May-Grünwald Giemsa staining and examined by FISH for chromosomes X, Y and 18. RESULTS: We observed that circulating erythroblasts comprised two distinct groups: one was clearly of maternal origin and could be reliably analyzed by FISH, whereas the other, which appeared to be of fetal origin, was largely impervious to FISH analysis. This latter feature seemed to be related to an abnormally dense nucleus with an apoptotic character. Since the oxygen tension in the maternal circulation is higher than that in the fetus, we cultured fetal cord blood erythroblasts in conditions mimicking this difference in oxygen concentrations and found that high oxygen concentrations rapidly induced shrinkage of the erythroblast nucleus, rendering it impervious to FISH analysis. INTERPRETATION AND CONCLUSIONS: Our data show that circulating erythroblasts of presumed fetal origin cannot be reliably analyzed by FISH because of an abnormally dense nucleus. This nuclear phenotype appears to be induced by the higher oxygen tension present in the maternal circulation than in fetal blood.

Aneuploidy↗
PubMed2004

Size separation of circulatory DNA in maternal plasma permits ready detection of fetal DNA polymorphisms.

BACKGROUND: Analysis of fetal DNA in maternal plasma has recently been introduced as a new method for noninvasive prenatal diagnosis, particularly for the analysis of fetal genetic traits, which are absent from the maternal genome, e.g., RHD or Y-chromosome-specific sequences. To date, the analysis of other fetal genetic traits has been more problematic because of the overwhelming presence of maternal DNA sequences in the circulation. We examined whether different biochemical properties can be discerned between fetal and maternal circulatory DNA. METHODS: Plasma DNA was examined by agarose gel electrophoresis. The fractions of fetal and maternal DNA in size-fractionated fragments were assayed by real-time PCR. The determination of paternally and maternally inherited fetal genetic traits was examined by use of highly polymorphic chromosome-21-specific microsatellite markers. RESULTS: Size fractionation of circulatory DNA indicated that the major portion of cell-free fetal DNA had an approximate molecular size of <0.3 kb, whereas maternally derived sequences were, on average, considerably larger than 1 kb. Analysis of size-fractionated DNA (</=0.3 kb) from maternal plasma samples facilitated the ready detection of paternally and maternally inherited microsatellite markers. CONCLUSIONS: Circulatory fetal DNA can be enriched by size selection of fragment sizes less than approximately 0.3 kb. Such selection permits easier analysis of both paternally and maternally inherited DNA polymorphisms.

DNA↗
PubMed2004

Interlaboratory comparison of fetal male DNA detection from common maternal plasma samples by real-time PCR.

BACKGROUND: Analysis of fetal DNA from maternal plasma by PCR offers great potential for noninvasive prenatal genetic diagnosis. To further evaluate this potential, we developed and validated a standard protocol to determine whether fetal DNA sequences could be reproducibly amplified and measured across multiple laboratories in a common set of specimens. METHODS: Each of five participating centers in a National Institute of Child Health and Human Development consortium collected 20 mL of peripheral blood from 20 pregnant women between 10 and 20 weeks of gestation. The plasma fraction was separated according to a common protocol, divided, and frozen in five aliquots. One aliquot was shipped to each participating laboratory, where DNA was extracted according to a standard protocol. All plasma samples (n = 100) were then analyzed blindly for the presence and quantity of total DNA (GAPDH) and male fetal DNA (SRY) by real-time PCR. Genomic DNA was isolated from female and male cells at one center, quantified, and shipped to the others to serve as calibrators for GAPDH and SRY, respectively. RESULTS: The amplification of known quantities of DNA was consistent among all centers. The mean quantity of male DNA amplified from maternal plasma when the fetus was male ranged from 51 to 228 genome equivalents (GE)/mL. Qualitative concordance was found overall among centers. The sensitivity of the assay for detection of male DNA when the fetus was male varied from 31% to 97% among centers. Specificity was more consistent (93-100%) with only four false-positive results obtained across the entire study. CONCLUSIONS: All centers were able to consistently amplify frozen and shipped DNA. The PCR procedure used here is reliable and reproducible. Centers that extracted and amplified more DNA per milliliter of maternal plasma had superior sensitivities of Y chromosome sequence detection. The specificity of the assay was more consistent among centers. A robust and thoroughly optimized protocol for the extraction of DNA from maternal plasma is needed to make testing of fetal DNA in maternal plasma a clinically relevant analytical tool.

Clinical Laboratory Techniques↗
PubMed2004

In utero transplantation of autologous and allogeneic fetal liver stem cells in ovine fetuses.

OBJECTIVE: The purpose of this study was to assess the feasibility of autologous stem cell transplantation in fetal sheep and to compare short-term engraftment of allogeneic and autologous fetal liver stem cells in an immunocompetent large animal model. STUDY DESIGN: Fetal liver stem cells were collected from preimmune sheep fetuses with an open or ultrasound-guided technique. After being labeled with PKH26, the cells were transplanted intraperitoneally into allogeneic and autologous fetal recipients at 48 to 64 days of gestation. Engraftment was determined by flow cytometry and real-time polymerase chain reaction 1 to 2 weeks after transplantation. RESULTS: Fetal loss rate was 29% (allogeneic transplantation) and 73% (autologous transplantation). Engraftment of donor cells was found in all fetuses, with a level of < or =4.7% in fetal liver, spleen, bone marrow, blood and thymus. Overall, there was no difference between allogeneic and autologous grafts. CONCLUSION: Autologous in utero transplantation of fetal liver stem cells in fetal sheep is feasible, but yields a high loss rate. Differences in the major histocompatibility complex between donor and recipient seems not to have a major impact on stem cell engraftment early in gestation; major histocompatibility complex-independent donor/host competition might be responsible for low engraftment in immunocompetent recipients.

Animals↗
PubMed2004

Anti-phospholipid and anti-DNA antibodies are not associated with the elevated release of circulatory fetal DNA in pregnancies affected by preeclampsia.

OBJECTIVES: We have previously shown that the levels of circulatory fetal DNA are elevated in preeclampsia and that these increases correspond to disease severity. Several reports have indicated that increased levels of antiphospholipid (anti-PL) and anti-DNA antibodies may be associated with preeclampsia, in particular with the severe forms of the disorder. Since the release of cell-free DNA by the placenta is attributed to some form of cell death or damage and as anti-PL and anti-double-stranded DNA (dsDNA) antibodies have been proposed to lead to placental damage, we have studied the relationship between these parameters in preeclampsia. METHODS: Circulating fetal DNA levels in samples taken from pregnant women with mild (n = 12) or severe (n = 12) preeclampsia and from normal pregnant controls (n = 35) were quantified using a Taqman real-time Polymerase Chain Reaction (PCR) assay. The Anti-PL antibodies (IgG and IgM) were assayed by anticardiolipin ELISA and by commercial anti-beta2-Glycoprotein I (GPI) ELISA kits. Anti-dsDNA antibodies (IgG and IgM) were analyzed by a commercially available anti-dsDNA ELISA kit. RESULTS: No correlation could be drawn with the quantity of circulatory fetal DNA in the samples analyzed and corresponding anti-PL or anti-dsDNA antibody levels. Furthermore, no significant difference existed between the levels of these antibodies in the two study groups and the control cohort. CONCLUSION: Our data suggest that the mechanism leading to the increased release of cell-free circulatory DNA from the placenta does not involve trophoblast damage mediated by these agents. Our analysis also questions the reported involvement of anti-PL and anti-DNA antibodies in preeclampsia.

Antibodies↗
PubMed2004

Engraftment kinetics of human cord blood and murine fetal liver stem cells following in utero transplantation into immunodeficient mice.

This study was undertaken to evaluate the kinetics of engraftment after in utero transplantation of murine fetal liver and human cord blood stem cells in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. NOD/SCID fetuses were injected with murine fetal liver or human cord blood CD34+ cells at day 13.5 of gestation. Frequencies of donor cells were analyzed by flow cytometry up to 48 h post transplantation and 4-16 weeks postnatally. Hematopoietic multilineage reconstitution capacity was assessed. Both types of donor cells home rapidly. However, the frequency of human cord blood stem cells rapidly diminished while the murine fetal liver stem cells expanded over time, resulting in multilineage hematopoietic reconstitution. Differences in long-term reconstitution of allogeneic versus xenogeneic donor cells were ascribed to the inability of the human cells to self-renew and differentiate in the fetal mouse environment, demonstrating the limitations of this commonly used xenograph.

Animals↗
PubMed2004

Can circulatory fetal DNA be used to study placentation at high altitude?

Pregnancy at high altitude (>2700 m) is associated with higher rates of fetal growth restriction and preeclampsia as well as alterations in placentation, including increased villous vascularization and reduced remodeling of maternal spiral arteries. Because circulatory fetal DNA concentrations were shown to be enhanced in pregnancies affected by preeclampsia, we investigated whether similar elevations are also apparent in pregnancies at high altitude by examining ethnic Tibetans and recent migrant Han Chinese residents in Lhasa (altitude 3650 m) as well as Han Chinese residents in Guangzhou (altitude 7 m). Our data from this preliminary study (n = 10/study group) indicate that circulatory fetal DNA levels were significantly higher in the unremarkable pregnancies of Han women who moved to Tibet when compared to Han women at sea level. No significant difference could be discerned between migrant Han Chinese and ethnic Tibetans living in Lhasa. Our data, therefore, suggest that pregnancy at high altitude is associated with an increased liberation of cell-free fetal DNA and that no major ethnic differences are apparent.

Altitude↗
PubMed2003

Intact fetal cells in maternal plasma: are they really there?

Rare fetal cells can be recovered from maternal blood, which suggests that non-invasive prenatal diagnosis is possible. However, recovery and analysis of fetal cells from blood is complex, and sensitivity is low because of the rarity of these cells in the maternal circulation. An alternative strategy, which suggested that intact fetal cells can be found in maternal plasma by use of simple enrichment methods, has been reported. We aimed to replicate this technique. However, five independent laboratories were unable to identify any intact male cells from the plasma of 38 women known to be carrying male fetuses. Although apoptotic intact fetal cells could contribute to the detection of fetal DNA in maternal plasma, we believe that recovery of these cells is difficult and not clinically practical.

Aneuploidy↗
PubMed2003

Inability to detect cell free fetal DNA in the urine of normal pregnant women nor in those affected by preeclampsia associated HELLP syndrome.

OBJECTIVE: Recent reports have indicated that cell-free fetal DNA can be detected in the urine of pregnant women. We attempted to reproduce those data. METHODS: Urine samples were collected from 18 normal pregnant women (11 with a male fetus). Urinary DNA was examined by Y-chromosome-specific nested polymerase chain reaction (PCR) or real-time PCR. Samples were also examined from two pregnancies complicated by HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, which had very high levels of cell-free fetal DNA in the maternal plasma. To validate our data, a quantitative comparison of different DNA extraction procedures used in the previous reports was performed. RESULTS: In no instance were we able to detect any fetal DNA in maternal urine, although copious quantities of cell-free fetal DNA were present in the maternal plasma of those pregnancies affected by HELLP syndrome. Our quantitative analysis of the various extraction procedures used indicated that the commercial column elution method we used was comparable, if not superior, to the noncommercial methods used in previous reports. CONCLUSION: Our data strongly suggest that cell-free fetal DNA is not readily detectable in maternal urine, even under conditions known to increase kidney permeability.

Chromosomes, Human, Y↗
PubMed2002

Circulating erythroblasts in maternal blood are not elevated before onset of preterm labor.

OBJECTIVE: Preterm labor has recently been reported to be associated with an increased release of cell free fetal deoxyribonucleic acid (DNA) into the maternal circulation. We have previously observed increases in both fetal cell traffic and cell free fetal DNA in preeclamptic pregnancies. In this study, we investigated whether fetal cell traffic is also disturbed in pregnancies with preterm labor. METHODS: In a case-control study, we examined 47 pregnancies complicated by preterm contractions that occurred between 20 and 34 weeks' gestation and an equal number of matched controls. Erythroblasts were enriched for by magnetic cell sorting and enumerated. These values were then correlated with subsequent pregnancy outcome. RESULTS: In the study group 16 patients delivered prematurely (subgroup A). The other 31 (subgroup B) delivered at term, as did all those in the control group. No significant difference was noted in erythroblast numbers between either one of the subgroups and the controls. CONCLUSION: Contrary to the reported increased levels of free fetal DNA in maternal serum, erythroblasts in maternal blood are not elevated significantly in pregnancies with threatened premature labor or in those that deliver preterm.

Abortion, Threatened↗