Rarity of fetal cells in exocervical samples for noninvasive prenatal diagnosis

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Laura Bourlard ◽  
Yannick Manigart ◽  
Catherine Donner ◽  
Guillaume Smits ◽  
Julie Désir ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Control Cell ◽  
Genetic Diagnosis ◽  
Positive Control ◽  
Specific Marker ◽  
Cell Detection ◽  
Fetal Cells ◽  
Rare Cells ◽  
Endocervical Canal ◽  
Extravillous Trophoblasts

Abstract Objectives The possibility to isolate fetal cells from pregnant women cervical samples has been discussed for five decades but is not currently applied in clinical practice. This study aimed at offering prenatal genetic diagnosis from fetal cells obtained through noninvasive exocervical sampling and immuno-sorted based on expression of HLA-G. Methods We first developed and validated robust protocols for cell detection and isolation on control cell lines expressing (JEG-3) or not (JAR) the HLA-G antigen, a specific marker for extravillous trophoblasts. We then applied these protocols to noninvasive exocervical samples collected from pregnant women between 6 and 14 weeks of gestational age. Sampling was performed through insertion and rotation of a brush at the ectocervix close to the external os of the endocervical canal. Finally, we attempted to detect and quantify trophoblasts in exocervical samples from pregnant women by ddPCR targeting the male SRY locus. Results For immunohistochemistry, a strong specific signal for HLA-G was observed in the positive control cell line and for rare cells in exocervical samples, but only in non-fixative conditions. HLA-G positive cells diluted in HLA-G negative cells were isolated by flow cytometry or magnetic cell sorting. However, no HLA-G positive cells could be recovered from exocervical samples. SRY gene was detected by ddPCR in exocervical samples from male (50%) but also female (27%) pregnancies. Conclusions Our data suggest that trophoblasts are too rarely and inconstantly present in noninvasive exocervical samples to be reliably retrieved by standard immunoisolation techniques and therefore cannot replace the current practice for prenatal screening and diagnosis.

scholarly journals Factors determining microbial colonization of liquid nitrogen storage tanks used for archiving biological samples

2019 ◽  
Vol 104 (1) ◽  
pp. 131-144 ◽  
Author(s):  
F. Bajerski ◽  
A. Bürger ◽  
B. Glasmacher ◽  
E. R. J. Keller ◽  
K. Müller ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Epifluorescence Microscopy ◽  
Gene Copy ◽  
Marker Genes ◽  
Rrna Gene ◽  
Specific Marker ◽  
Cell Detection ◽  
Storage Tanks ◽  
Gene Copies ◽  
Ice Phase

AbstractThe availability of bioresources is a precondition for life science research, medical applications, and diagnostics, but requires a dedicated quality management to guarantee reliable and safe storage. Anecdotal reports of bacterial isolates and sample contamination indicate that organisms may persist in liquid nitrogen (LN) storage tanks. To evaluate the safety status of cryocollections, we systematically screened organisms in the LN phase and in ice layers covering inner surfaces of storage tanks maintained in different biobanking facilities. We applied a culture-independent approach combining cell detection by epifluorescence microscopy with the amplification of group-specific marker genes and high-throughput sequencing of bacterial ribosomal genes. In the LN phase, neither cells nor bacterial 16S rRNA gene copy numbers were detectable (detection limit, 102 cells per ml, 103 gene copies per ml). In several cases, small numbers of bacteria of up to 104 cells per ml and up to 106 gene copies per ml, as well as Mycoplasma, or fungi were detected in the ice phase formed underneath the lids or accumulated at the bottom. The bacteria most likely originated from the stored materials themselves (Elizabethingia, Janthibacterium), the technical environment (Pseudomonas, Acinetobacter, Methylobacterium), or the human microbiome (Bacteroides, Streptococcus, Staphylococcus). In single cases, bacteria, Mycoplasma, fungi, and human cells were detected in the debris at the bottom of the storage tanks. In conclusion, the limited microbial load of the ice phase and in the debris of storage tanks can be effectively avoided by minimizing ice formation and by employing hermetically sealed sample containers.

scholarly journals Fragile X syndrome carrier screening in pregnant women in Chinese Han population

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Chia-Cheng Hung ◽  
Chien-Nan Lee ◽  
Yu-Chu Wang ◽  
Chih-Ling Chen ◽  
Tze-Kang Lin ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Fragile X Syndrome ◽  
Chinese Women ◽  
Fragile X ◽  
Genetic Diagnosis ◽  
Cost Effective ◽  
Carrier Screening ◽  
Chinese Han ◽  
Cgg Repeat ◽  
Taiwanese Women

Abstract Fragile X syndrome (FXS) is the most frequent genetic cause of intellectual disability (ID). It was previously believed that the FXS prevalence was low in Chinese population, and the cost-efficiency of FXS carrier screening was questioned. This retrospective observational study was conducted between September 2014 and May 2017 to determine the prevalence of FXS carriers in a large Chinese cohort of pregnant women. The FMR1 CGG repeat status was determined in 20,188 pregnant Taiwanese women and we identified 26 women with premutation (PM). The PM allele was transmitted to the fetus in 17 pregnancies (56.6%), and six of 17 expanded to full mutation (FM). One asymptomatic woman had a FM allele with 280 CGG repeats. Prenatal genetic diagnosis of her first fetus revealed a male carrying a FMR1 gene deletion of 5′ UTR and exon 1. Her second fetus was a female carrying a FM allele as well. This is so far the largest study of the FXS carrier screening in Chinese women. The prevalence of premutation allele for FXS in normal asymptomatic Taiwanese women was found to be as high as 0.13% (1 in 777) in this study. The empirical evidence suggests that reproductive FXS carrier screening in Taiwan might be cost-effective.

scholarly journals A Silicon-based Coral-like Nanostructured Microfluidics to Isolate Rare Cells in Human Circulation: Validation by SK-BR-3 Cancer Cell Line and Its Utility in Circulating Fetal Nucleated Red Blood Cells

Micromachines ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 132 ◽  
Author(s):  
Gwo-Chin Ma ◽  
Wen-Hsiang Lin ◽  
Chung-Er Huang ◽  
Ting-Yu Chang ◽  
Jia-Yun Liu ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Pregnant Women ◽  
Blood Cells ◽  
De Novo ◽  
Capture Rate ◽  
Genetic Diagnosis ◽  
Maternal Blood ◽  
Capture Efficiency ◽  
Nucleated Red Blood Cells ◽  
Silicon Based

Circulating fetal cells (CFCs) in maternal blood are rare but have a strong potential to be the target for noninvasive prenatal diagnosis (NIPD). “Cell RevealTM system” is a silicon-based microfluidic platform capable to capture rare cell populations in human circulation. The platform is recently optimized to enhance the capture efficiency and system automation. In this study, spiking tests of SK-BR-3 breast cancer cells were used for the evaluation of capture efficiency. Then, peripheral bloods from 14 pregnant women whose fetuses have evidenced non-maternal genomic markers (e.g., de novo pathogenic copy number changes) were tested for the capture of circulating fetal nucleated red blood cells (fnRBCs). Captured cells were subjected to fluorescent in situ hybridization (FISH) on chip or recovered by an automated cell picker for molecular genetic analyses. The capture rate for the spiking tests is estimated as 88.1%. For the prenatal study, 2–71 fnRBCs were successfully captured from 2 mL of maternal blood in all pregnant women. The captured fnRBCs were verified to be from fetal origin. Our results demonstrated that the Cell RevealTM system has a high capture efficiency and can be used for fnRBC capture that is feasible for the genetic diagnosis of fetuses without invasive procedures.

scholarly journals Detection of Apoptotic Fetal Cells in Plasma of Pregnant Women

2000 ◽  
Vol 46 (5) ◽  
pp. 729-731 ◽  
Author(s):  
Inge J van Wijk ◽  
Anneke C de Hoon ◽  
Rishma Jurhawan ◽  
May Lee Tjoa ◽  
Sandra Griffioen ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Fetal Cells

The Number of Endovascular Trophoblasts in Maternal Blood Increases Overnight and after Physical Activity: An Experimental Study

2015 ◽  
Vol 40 (1) ◽  
pp. 54-58 ◽  
Author(s):  
Jacob Mørup Schlütter ◽  
Ida Kirkegaard ◽  
Anne Sigaard Ferreira ◽  
Lotte Hatt ◽  
Britta Christensen ◽  
...  
Keyword(s):  
Physical Activity ◽  
Experimental Study ◽  
Pregnant Women ◽  
Transvaginal Ultrasound ◽  
Maternal Blood ◽  
Interindividual Variation ◽  
Intraindividual Variation ◽  
Fetal Cells ◽  
Repeated Sampling ◽  
Endovascular Trophoblast

Introduction: Fetal cells in maternal blood may be used for noninvasive prenatal diagnostics, although their low number is a challenge. This study's objectives were to evaluate whether physical activity, transabdominal and transvaginal ultrasound scans of the uterus, as well as overnight or day-to-day variation affect the number of isolated fetal cells, more specifically the presumed endovascular trophoblast (pEVT). Material and Methods: In each of 3 different experiments, 10 normal singleton pregnant women (gestational age 10+4-14+4 weeks) participated. The number of pEVTs was assessed in 30-36 ml blood using specific markers for enrichment and identification. Results: The number of pEVTs increased overnight (p = 0.001) from a median of 1.5 to 3.5 and even further to a median of 6.0 after 30 min of physical activity (p = 0.04) but was not affected by transabdominal and transvaginal ultrasound scans. Repeated sampling showed that the interindividual variation of pEVTs was higher than the intraindividual variation (p < 0.001). However, even in pregnant women with a consistently low number of pEVTs, isolation of the pEVTs for prenatal diagnoses was possible in all cases by doing 2 separate blood samplings a few days apart. Discussion: The number of pEVTs identified in maternal blood can be increased by presampling conditions or repeated sampling.

scholarly journals Modulation of phospholipase A2 activity by epidermal growth factor (EGF) in CHO cells transfected with human EGF receptor. Role of receptor cytoplasmic subdomain

1991 ◽  
Vol 274 (3) ◽  
pp. 715-721 ◽  
Author(s):  
S Clark ◽  
M Dunlop
Keyword(s):  
Arachidonic Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase A2 ◽  
Egf Receptor ◽  
Control Cell ◽  
Cell Types ◽  
Positive Control ◽  
Prostaglandin Production ◽  
Epidermal Growth

Activation of phospholipase A2 (PLA2) in response to external stimuli may play a pivotal role in signal-transduction pathways via the generation of important cellular intermediates, including prostaglandins. Epidermal growth factor (EGF) has been shown to modulate prostaglandin production, possibly via direct activation of PLA2 or indirectly via interaction with a PLA2-modifying protein such as lipocortin I. We have investigated these pathways with two CHO cell-lines, one (CHOwt) transfected with the full-length human EGF receptor and the second (CHO 11) with a deletion mutant, delta 990, that has lost the autophosphorylation sites and part of the internalization domain. CHOwt cells responded to EGF with a rapid rise in lysophosphatidylcholine and arachidonic acid release concomitant with an increase in prostaglandin production. However, in the non-internalizing CHO 11 cells no such activation of PLA2 was observed. This was not due to an intrinsic lack of PLA2 in these cells, as PLA2 activation was shown on melittin addition, nor was this difference due to a defect in intracellular pathways, as arachidonic acid was released from both cell types by Ca2+ and protein kinase C modulators. However, only in CHOwt cells were these responses potentiated by concomitant addition of EGF. Thus the cytoplasmic subdomain of the EGF receptor, containing the major sites of autophosphorylation and the internalization domain, seems to be involved in the activation of PLA2 by EGF. In addition, we have shown that phosphorylation of lipocortin I is unlikely to play a role in PLA2 activation. In CHOwt cells and a positive control cell line, A431, activation of PLA2 was complete by 10 min, at which time there was no evidence of lipocortin I phosphorylation.

scholarly journals Failure to Find DUP25 in Patients with Anxiety Disorders, in Control Individuals, or in Previously Reported Positive Control Cell Lines

2003 ◽  
Vol 72 (3) ◽  
pp. 535-538 ◽  
Author(s):  
Melody Tabiner ◽  
Sheila Youings ◽  
Nicholas Dennis ◽  
David Baldwin ◽  
Christel Buis ◽  
...  
Keyword(s):  
Anxiety Disorders ◽  
Cell Lines ◽  
Control Cell ◽  
Positive Control

scholarly journals Development of an immunofluorescent AR-V7 circulating tumor cell assay – A blood-based test for men with metastatic prostate cancer

2020 ◽  
Vol 9 (1) ◽  
pp. 13-19
Author(s):  
David Lu ◽  
Rachel Krupa ◽  
Melissa Harvey ◽  
Ryon P. Graf ◽  
Nicole Schreiber ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Control Cell ◽  
Circulating Tumor Cell ◽  
Cancer Center ◽  
Cell Detection ◽  
Castration Resistant Prostate Cancer ◽  
Nccn Guidelines ◽  
Castration Resistant ◽  
Validation Testing

Introduction: Here we describe the development of a protein immunofluorescent assay for the detection of nuclear-localized androgen receptor variant 7 (AR-V7) protein within circulating tumor cells (CTCs) identified in patient blood samples. Used in the clinic, the test result serves as a validated biomarker of futility for patients with progressing metastatic castration-resistant prostate cancer (mCRPC) who are treated with androgen receptor targeted therapies (AATT) in whom nuclear-localized AR-V7 CTCs are identified and have received level 2A evidence in the 2019 National Cancer Center Network (NCCN) guidelines (v1.0). Methods: Assay development was completed on the Epic Sciences rare cell detection platform using control cell lines of known AR-V7 status and clinical testing of mCRPC patient samples obtained at the decision point in management. Results and conclusions: Using these samples, all assay parameters, scoring criteria, and clinical cutoffs for positivity were prospectively selected and locked. After assay lock, blinded clinical validation testing was initiated on multiple, independent, clinical cohorts as reported by Scher et al (JAMA Oncol. 2016;2:1441-1449; JAMA Oncol. 2018;4:1179-1186) and Armstrong et al (J Clin Oncol. 2019;37:1120-1129).

scholarly journals The Maternal Leucocytes in Thrombophilia and Hypothyroidism and their Influence on Fetal Cells

2018 ◽  
Vol 0 (0) ◽  
Author(s):  
Tanja R. Novakovic ◽  
Zana C. Dolicanin ◽  
Goran M. Babic ◽  
Natasa Z. Djordjevic
Keyword(s):  
Pregnant Women ◽  
Placental Abruption ◽  
Fetal Growth Retardation ◽  
Inherited Thrombophilia ◽  
Percentage Distribution ◽  
Fetal Cells ◽  
Hypertensive Disorders ◽  
Increased Risk ◽  
Β Hcg ◽  
Cytogenetic Methods

AbstractThe literature data show that thrombophilia and maternal dysfunction of thyroid gland during pregnancy are associated with an increased risk of miscarriage, placental abruption, hypertensive disorders and fetal growth retardation. It was shown that thyroid hormones and hypercoagulable states influence onto a leucocyte activity. The aim of this study has been to investigate maternal leucocytes changes and their correlation with frequency of fetal cells micronuclei in pregnant women with thrombophilia and hypothyroidism. Th e samples of blood and amniotic fl uid were collected from healthy pregnant women and pregnant women with inherited thrombophilia and hypothyroidism (16 - 18 weeks of gestation). Hematological characteristics were determined by using standard hematological methods. The frequency of micronuclei was determined in fetal cells after amniocentesis by using standard cytogenetic methods. The results of this study showed significant higher levels of β-hCG, number of monocytes and eosinophils in blood of pregnant women with thrombophilia. A large number of eosinophils was documented in blood of pregnant women with hypothyroidism. Increased percentage distribution of eosinophils and basophils is shown in both investigated groups of pregnant women. The increased fetal cells micronuclei frequency and their correlation with percentage distribution of eosinophils and basophils were indicated in pregnant women with hypothyroidism. The obtained results suggest that an increased percentage of eosinophils and basophils in pregnant women with hypothyroidism contribute to a formation of micronuclei in fetal cells.

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