RESUMEN
Non-invasive prenatal testing (NIPT) to detect fetal aneuploidy by sequencing the cell-free DNA (cfDNA) in maternal plasma is being broadly adopted. To detect fetal aneuploidies from maternal plasma, where fetal DNA is mixed with far-larger amounts of maternal DNA, NIPT requires a minimum fraction of the circulating cfDNA to be of placental origin, a level which is usually attained beginning at 10 weeks gestational age. We present an approach that leverages the arrangement of alleles along homologous chromosomes-also known as chromosomal phase-to make NIPT analyses more conclusive. We validate our approach with in silico simulations, then re-analyze data from a pregnant mother who, due to a fetal DNA fraction of 3.4%, received an inconclusive aneuploidy determination through NIPT. We find that the presence of a trisomy 18 fetus can be conclusively inferred from the patient's same molecular data when chromosomal phase is incorporated into the analysis. Key to the effectiveness of our approach is the ability of homologous chromosomes to act as natural controls for each other and the ability of chromosomal phase to integrate subtle quantitative signals across very many sequence variants. These results show that chromosomal phase increases the sensitivity of a common laboratory test, an idea that could also advance cfDNA analyses for cancer detection.
Asunto(s)
Ácidos Nucleicos Libres de Células , Diagnóstico Prenatal , Aneuploidia , Ácidos Nucleicos Libres de Células/genética , Cromosomas , ADN/genética , Femenino , Feto , Humanos , Placenta , Embarazo , Diagnóstico Prenatal/métodos , Trisomía/diagnóstico , Trisomía/genéticaRESUMEN
PURPOSE: Because of the 4 to 6-month interval between a diagnostic amniocentesis and birth, clinical application of amniotic mesenchymal stem cell (AMSC)-based therapies demands a 3-stage cell manufacturing process, including isolation/primary expansion, cryopreservation, and thawing/secondary expansion. We sought to determine the feasibility and cell yield of such a staged cell manufacturing process, within regulatory guidelines. METHODS: Human AMSCs isolated from diagnostic amniocentesis samples (n = 11) were processed under Food and Drug Administration-accredited good manufacturing practice. Expanded cells were characterized by flow cytometry and cryopreserved for 3 to 5 months. Cell release criteria included more than 90% CD29+, CD73+, and CD44+; less than 5% CD34+ and CD45+; negative mycoplasma quantitative polymerase chain reaction (QPCR) and endotoxin assay; and at least 70% viability. RESULTS: Isolation and ample expansion of AMSCs was achieved in 54.5% (6/11) of the samples. Early processing and at least a 2-mL sample were necessary for reliable cell manufacturing. Cell yield before cryopreservation was 223.2 +/- 65.4 x 10(6) cells (44.6-fold expansion), plus a 14.7 x 10(6)-cell backup, after 36.3 +/- 7.8 days. Cell viability postthaw was 88%. Expanded cells maintained a multipotent mesenchymal progenitor profile. CONCLUSIONS: Human amniotic mesenchymal stem cells can be manufactured in large numbers from diagnostic amniocentesis, by an accredited staged processing, under definite procurement guidelines. These data further support the viability of clinical trials of amniotic mesenchymal stem cell-based therapies.
Asunto(s)
Líquido Amniótico/citología , Trasplante de Células/normas , Células Madre Mesenquimatosas , Ingeniería de Tejidos/normas , Amniocentesis , Separación Celular/métodos , Separación Celular/normas , Supervivencia Celular , Criopreservación/métodos , Femenino , Citometría de Flujo , Edad Gestacional , Guías como Asunto , Humanos , Embarazo , Sensibilidad y Especificidad , Manejo de Especímenes/normasRESUMEN
BACKGROUND: Dermatomyositis is rare during pregnancy and, if untreated, is associated with poor fetal outcome. Corticosteroids are a standard treatment for dermatomyositis in pregnancy, but they have adverse effects. Intravenous immune globulin is an effective therapy for this condition and may have few adverse effects. CASE: A young, white primigravida presented with dermatomyositis at 4 5/7 weeks of gestation (creatine kinase 2,762 units/L). Intravenous immune globulin was administered monthly at a dose of 1 g/(kg.d) for 2 consecutive days. The patient's symptoms resolved and no complications were experienced during therapy. At term, creatine kinase was 29 units/L and a healthy 3,657.5-g (8-lb, 1-oz) neonate was born. CONCLUSION: Pregnant patients with dermatomyositis can be treated with intravenous immune globulin, resulting in good fetal outcome, thus avoiding the deleterious effects of corticosteroid therapy on pregnancy.
Asunto(s)
Dermatomiositis/diagnóstico , Inmunoglobulinas Intravenosas/uso terapéutico , Complicaciones del Embarazo/diagnóstico , Diagnóstico Prenatal , Adulto , Creatina Quinasa/sangre , Dermatomiositis/sangre , Dermatomiositis/tratamiento farmacológico , Dermatomiositis/patología , Diagnóstico Diferencial , Esquema de Medicación , Femenino , Humanos , Inmunoglobulinas Intravenosas/administración & dosificación , Recién Nacido , Embarazo , Complicaciones del Embarazo/sangre , Complicaciones del Embarazo/tratamiento farmacológico , Complicaciones del Embarazo/patología , Primer Trimestre del EmbarazoRESUMEN
Recovery of nucleated cord blood cells after storage in liquid nitrogen was evaluated. Red cells were depleted using Ficoll-Paque or Puregene red cell lyses. Freeze Medium contained 10% dimethylsulfoxide and 20% serum for cryoprotection. Recovery of the original cell population remaining serviceable for fluorescence activated cell sorting (FACS) was 12 +/- 10% (average +/- standard deviation), with a range of 1% to 55%. Viability measured by FACS analysis after freezing was significantly lower than that of the same specimens prior to freezing, 62 +/- 20% compared to 91+/-11% (p<0.001). Percentage CD45+34+ cells were the same for fresh and frozen cells. Gestational age at which specimens were collected had no effect on the percent cells carrying the CD45+34+ markers. We conclude that better cryoprotective supplements are needed to insure consistent high recovery of viable nucleated umbilical cord blood cells after preservation in liquid nitrogen.
Asunto(s)
Conservación de la Sangre/métodos , Criopreservación/métodos , Sangre Fetal/citología , Leucocitos Mononucleares/citología , Antígenos CD34 , Supervivencia Celular , Crioprotectores , Dimetilsulfóxido , Femenino , Edad Gestacional , Humanos , Recién Nacido , Antígenos Comunes de Leucocito , EmbarazoRESUMEN
OBJECTIVE: This study was undertaken to determine whether induction of labor with oral misoprostol will result in fewer cesarean deliveries than intravenous oxytocin in nulliparous women with premature rupture of membranes at term. STUDY DESIGN: Three hundred five women at 10 centers were randomly assigned to receive oral misoprostol, 100 microg every 6 hours to a maximum of two doses or intravenous oxytocin. The primary outcome measure was cesarean deliveries. Secondary outcomes were time from induction to vaginal delivery and measures of maternal and neonatal safety. RESULTS: The study was stopped prematurely because of recruitment difficulties. We present the results for the 305 enrolled women. There was no difference in the proportion of women who underwent cesarean delivery (20.1% in the misoprostol group, 19.9% in the oxytocin group). The time interval from induction to vaginal delivery was also similar (11.9 hours for the misoprostol group, and 11.8 hours for the oxytocin group). Maternal and neonatal safety outcomes were similar for the two treatments. More infants born to women in the misoprostol group received intravenous antibiotics in the neonatal period (16.4% vs 6.9%, P=.01), although there were no differences in chorioamnionitis or in proven neonatal infections. Women receiving misoprostol were less likely to have postpartum hemorrhage than those receiving oxytocin (1.9% vs 6.2%, P=.05). CONCLUSION: Oral misoprostol does not offer any advantage in time from induction to vaginal delivery or risk of cesarean section.