scholarly journals High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tzu-Hsuan Chuang ◽  
Zih-Huei Wu ◽  
Chin-Sheng Kuan ◽  
Meng-Ju Lee ◽  
Chia-Lin Hsieh ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Diploid Cell ◽  
Automatic Identification ◽  
Aneuploid Cell ◽  
Library Preparation ◽  
Preimplantation Genetic Testing ◽  
Thermo Fisher Scientific ◽  
Number Variation ◽  
Chromosomal Duplication ◽  
Fisher Scientific

AbstractThe Ion S5 (Thermo Fisher Scientific) and Miseq (Illumina) NGS systems are both widely used in the clinical laboratories conducting PGT-A. Each system employs discrepant library preparation steps, sequencing principles, and data processing algorithms. The automatic interpretation via Ion Reporter software (Thermo Fisher Scientific) and the manual interpretation via BlueFuse Multi software (Illumina) for chromosomal copy number variation (CNV) represent very different reporting approaches. Thus, it is intriguing to compare their ability of ploidy detection as PGT-A/NGS system. In the present study, four aneuploid cell lines were individually mixed with a diploid cell line at different aneuploid ratios of 0% (0:5), 10% (1:9), 20% (1:4), 40% (2:3), 50% (3:3), 60% (3:2), 80% (4:1) and 100% (5:0) to assess the sensitivity and specificity for whole chromosomal and segmental aneuploidy detection. The clinical biopsies of 107 blastocysts from 46 IVF/PGT-A cycles recruited between December 2019 and February 2020 were used to calculate the concordance. Initially, the pre-amplified products were divided into two aliquots for different library preparation procedures of each system. Applying the same calling criteria, automatic identification was achieved through the Ion Reporter, while well-trained technicians manually identified each sample through the BlueFuse Multi. The results displayed that both systems reliably distinguished chromosomal CNV of the mixtures with at least 10% aneuploidy from karyotypically normal samples ([Ion S5] whole-chromosomal duplication: 2.14 vs. 2.05, p value = 0.009, segmental deletion: 1.88 vs. 2.05, p value = 0.003; [Miseq] whole-chromosomal duplication: 2.12 vs. 2.03, p value = 0.047, segmental deletion: 1.82 vs. 2.03, p value = 0.002). The sensitivity and specificity were comparable between the Ion S5 and Miseq ([sensitivity] 93% vs. 90%, p = 0.78; [specificity] 100% vs. 100%, p value = 1.0). In the 107 clinical biopsies, three displayed chaotic patterns (2.8%), which could not be interpreted for the ploidy. The ploidy concordance was 99.04% (103/104) per embryo and 99.47% (2265/2277) per chromosome pair. Since their ability of detection were proven to be similar, the automatic identification in Ion S5 system presents comparatively faster and more standardized performance.

scholarly journals High Concordance Between Automatic Identification in Ion S5 and Manual Identification in Miseq During Next-Generation Sequencing for Preimplantation Genetic Testing of Aneuploidy

2021 ◽  
Author(s):  
Tzu-Hsuan Chuang ◽  
Zih-Huei Wu ◽  
Chin-Sheng Kuan ◽  
Meng-Ju Lee ◽  
Chia-Lin Hsieh ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Diploid Cell ◽  
P Value ◽  
Automatic Identification ◽  
Aneuploid Cell ◽  
Library Preparation ◽  
Preimplantation Genetic Testing ◽  
Thermo Fisher Scientific ◽  
Chromosomal Duplication ◽  
Fisher Scientific

Abstract The Ion S5 (Thermo Fisher Scientific) and Miseq (Illumina) NGS systems are both widely used in the clinical laboratories conducting PGT-A. Each system employs discrepant library preparation steps, sequencing principles, and data processing algorithms. The automatic interpretation via Ion Reporter software (Thermo Fisher Scientific) and the manual interpretation via BlueFuse Multi software (Illumina) for chromosomal copy number variation (CNV) represent very different reporting approaches. Thus, it is intriguing to compare their ability of ploidy detection as PGT-A/NGS system. In the present study, four aneuploid cell lines were individually mixed with a diploid cell line at different aneuploid ratios of 0% (0:5), 10% (1:9), 20% (1:4), 40% (2:3), 50% (3:3), 60% (3:2), 80% (4:1) and 100% (5:0) to assess the sensitivity and specificity for whole chromosomal and segmental aneuploidy detection. The clinical biopsies of 107 blastocysts from 46 IVF/PGT-A cycles recruited between December 2019 and February 2020 were used to calculate the concordance. Initially, the pre-amplified products were divided into two aliquots for different library preparation procedures of each system. Applying with the same calling criteria, automatic identification was achieved through the Ion Reporter, while well-trained technicians manually identified each sample through the BlueFuse Multi. The results displayed that both systems reliably distinguished chromosomal CNV of the mixtures with at least 10% aneuploidy from karyotypically normal samples ([Ion S5] whole-chromosomal duplication: 2.14 vs. 2.05, p-value=0.009, segmental deletion: 1.88 vs. 2.05, p-value=0.003; [Miseq] whole-chromosomal duplication: 2.12 vs. 2.03, p-value=0.047, segmental deletion: 1.82 vs. 2.03, p-value=0.002). The sensitivity and specificity were comparable between the Ion S5 and Miseq ([sensitivity] 93% vs. 90%, p=0.78; [specificity] 100% vs. 100%, p-value=1.0). In the 107 clinical biopsies, three displayed chaotic patterns (2.8%), which could not be interpreted for the ploidy. The ploidy concordance was 99.04% (103/104) per embryo and 99.47% (2265/2277) per chromosome pair. Since their ability of detection were proven to be similar, the automatic identification in Ion S5 system presents comparatively faster and more standardized performance.

scholarly journals Evaluation of a Rapid Automated Next Generation Sequencing Assay for Precision Medicine in Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4444-4444
Author(s):  
Georgia M. Andrew ◽  
Laura W. Dillon ◽  
Maria Saeed ◽  
Ting-Chia Chang ◽  
Srikrishna K. Narava ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Targeted Therapies ◽  
Myeloid Leukemia ◽  
Turnaround Time ◽  
Ion Torrent ◽  
Library Preparation ◽  
Thermo Fisher Scientific ◽  
Generation Sequencing ◽  
Fisher Scientific ◽  
Acute Myeloid

Abstract Background: In the last decade there have been significant advances in diagnosing and classifying adult acute myeloid leukemia (AML) based on genomic profiling, enabling risk-stratification and targeted therapies. In 2017 the US FDA approved the first gene mutation targeted therapies for AML with multiple additional targeted therapies since approved or in development. Given the typical acuity of AML at initial presentation however and the current turnaround time for next-generation sequencing (NGS) assays, most patients will start definitive initial therapy before all potentially targetable mutations are known. There is, therefore, a significant need for a fast molecular genotyping test to determine eligibility for personalized therapy in AML. The NCI Myeloid Assay (NMA) is a comprehensive targeted NGS assay on the Ion Torrent Genexus System, a fully automated platform that provides a rapid turnaround time from specimen receipt to clinical reporting. NMA utilizes Thermo Fisher Scientific's Oncomine Myeloid Assay GX and appears ideally suited for use in upcoming AML targeted therapy trials but has yet to be extensively tested in a cohort of AML patient diagnostic samples and compared to a standard targeted "myeloid panel" NGS assay platform (s-NGS). Methods: DNA samples (n=173) extracted from pretreatment bone marrow and/or peripheral blood of adult patients (n=112) diagnosed with de novo AML or high-risk myelodysplastic syndrome (MDS), were blindly tested in parallel using the NMA and s-NGS assays. For the NMA assay, 27.75ng of DNA was put into the Genexus System. All runs, controls, and samples were first analyzed for sequencing quality using established quality control (QC) metrics to assess pass/fail status. For all samples that passed QC metrics, variant results generated by the Ion Torrent Genexus pipeline were manually reviewed prior to being called true positive variants. For the s-NGS, using the ArcherDx Myeloid VariantPlex assay, a DNA input of 50ng was used for library preparation on a dual pre- and post-PCR separated automated liquid-handling workflow. Resulting libraries were sequencing on the Novaseq 6000 (Illumina) and the data analyzed using the Archer Analysis software and filtered as previously described (PMID: 34258102). Results from the two assays were compared for mutations with a variant allele fraction (VAF) >5% occurring in genes of interest in small molecule targeted clinical trials including: FLT3, IDH1, IDH2, JAK2, KIT, NPM1, NRAS, KRAS, and TP53. For FLT3-ITD comparison, the presence or absence of a call by the assay was used. Results: Utilizing a 5% VAF reporting threshold, a total of 171 and 174 variants were detected by NMA and s-NGS assays, respectively. A high rate of concordance was observed between the assays, with NMA detecting 96% of s-NGS variants and s-NGS detecting 95% of NMA variants. The VAF of detected single nucleotide variants was highly correlated (r=0.9848, P<0.0001, Figure 1A). NPM1 mutation VAF values trended lower by s-NGS compared to NMA. We investigated the discordant calls (n=15 total in 11 patients). One patient was correctly identified as having an NRAS p.Gly12 mutation by both approaches, but the resulting mutation was incorrectly annotated by the s-NGS pipeline. Samples from two patients (including one with both blood and marrow tested) were correctly identified as being FLT3 tyrosine kinase domain mutated by both sequencing approaches, although only the major of two missense variants identified by s-NGS was reported by the NMA pipeline. None of these patients, however, would be misclassified. The remaining 11 discordant calls were false negatives (including 6 variants detected by s-NGS but not by NMA). All of these "edge case" variants were detectable by lowering the VAF reporting threshold below 5% (Figure 1B). Conclusions: NMA is an automated sample-to-results workflow that can identify myeloid disorder-associated genomic variants in less than 48 hours from library preparation to clinical reporting. We show that NMA is highly concordant with a standard DNA NGS assay for detecting mutations within recurrently mutated AML genes. Accurate rapid genotyping is required for assignment to initial treatment with targeted therapy, and this technology may be a valuable tool for upcoming clinical trials for patients with myeloid malignancies. Figure 1 Figure 1. Disclosures Zhang: Thermo Fisher Scientific: Current Employment. Sedova: Thermo Fisher Scientific: Current Employment. Huang: Thermo Fisher Scientific: Current Employment. Mittal: Thermo Fisher Scientific: Current Employment. Hatch: Thermo Fisher Scientific: Current Employment. Ni: Thermo Fisher Scientific: Current Employment. Kaznadzey: Thermo Fisher Scientific: Current Employment. Sadis: Thermo Fisher Scientific: Current Employment. Smith: Thermo Fisher Scientific: Current Employment. Williams: Illumina: Other: CRADA. Hourigan: Sellas: Research Funding.

scholarly journals POS0009 THE RELATIONSHIP BETWEEN DIFFERENT IGG AND IGA ANTI-MODIFIED PROTEIN AUTOANTIBODIES IN RHEUMATOID ARTHRITIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 206.1-207
Author(s):  
C. Grönwall ◽  
L. Liljefors ◽  
H. Bang ◽  
A. Hensvold ◽  
M. Hansson ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Disease Activity ◽  
Comprehensive Evaluation ◽  
Patient Characteristics ◽  
Post Translational Modifications ◽  
Cell Clones ◽  
Thermo Fisher Scientific ◽  
History Of ◽  
The Relationship ◽  
Fisher Scientific

Background:Seropositive rheumatoid arthritis (RA) is characterized by the presence of rheumatoid factor (RF) and anti-citrullinated protein autoantibodies (ACPA) with different fine-specificities. Yet, other serum anti-modified protein autoantibodies (AMPA), e.g. anti-carbamylated (Carb), anti-acetylated (KAc), and anti-malondialdehyde acetaldehyde (MAA) modified protein antibodies, have been described. By using RA patient single-cell derived monoclonal antibodies we have previously shown that individual ACPA clones recognize small distinct citrulline-containing epitopes giving them extensive multireactivity when these epitopes are found in many peptides and proteins. Moreover, certain CCP2+ multireactive ACPA clones bind also to cabamylated and acetylated autoantigens [1].Objectives:To provide a comprehensive evaluation of serum IgG and IgA autoreactivity to different post-translational modifications in RA.Methods:We analyzed 30 different IgG and IgA AMPA reactivities to modified antigens by ELISA and autoantigen arrays, in N=1985 newly diagnosed RA patients and population controls. The study utilized both previously established (i.e IgG and IgA CCP2; IgG ACPA fine-specificities; IgG anti-Carb fibrinogen and Carb FCS; IgG and IgA Cit/Carb/KAc/Orn(Ac)-vimentin), and novel assays (e.g. IgG anti-MAA and IgG anti-acetylated histones). Association with patient characteristics such as smoking and disease activity were explored. The newly developed assays were also evaluated in SLE disease controls and CCP2+ RA-risk individuals without arthritis.Results:Carb and KAc reactivities by different assays were primarily seen in patients also positive for citrulline-reactivity. Modified vimentin (mod-Vim) peptides were used for direct comparison of different AMPA reactivities, revealing that IgA AMPA recognizing mod-Vim was mainly detected in subsets of patients with high IgG anti-Cit-Vim levels and a history of smoking. IgG acetylation reactivity was mainly detected in a subset of patients with Cit and Carb reactivity. Anti-acetylated histone 2B reactivity was RA-specific and associated with high anti-CCP2 IgG levels, multiple ACPA fine-specificities, and smoking. This reactivity was also found to be present in CCP2+ RA-risk individuals without arthritis. Our data further demonstrate that IgG autoreactivity to MAA was increased in RA compared to controls with highest levels in CCP2+ RA, but was not RA-specific, and showed low correlation with other AMPA. Anti-MAA was instead associated with disease activity and was not significantly increased in CCP2+ individuals at risk of RA. Notably, RA patients could be subdivided into four different subsets based on their AMPA IgG and IgA reactivity profiles.Conclusion:We conclude that autoantibodies exhibiting different patterns of ACPA fine-specificities as well as Carb and KAc reactivity are present in RA and may be derived from multireactive B-cell clones. Anti-Carb and anti-KAc could be considered reactivities within the “Cit-umbrella” similar to ACPA fine-specificities, while MAA is distinctly different.References:[1]Sahlström P, Hansson M, Steen J, Amara K, Titcombe PJ, Forsström B, Stålesen R, Israelsson L, Piccoli L, Lundberg K, Klareskog L, Mueller DL, Catrina AI, Skriner K, Malmström V, Grönwall C. Different Hierarchies of Anti-Modified Protein Autoantibody Reactivities in Rheumatoid Arthritis. Arthritis Rheumatol. 2020 Oct;72(10):1643-1657. PMID: 32501655Caroline Grönwall: None declared, Lisa Liljefors: None declared, Holger Bang Employee of: Employee at ORGENTEC Diagnostika GmbH, Aase Hensvold: None declared, Monika Hansson: None declared, Linda Mathsson-Alm Employee of: Employee at Thermo Fisher Scientific, Lena Israelsson: None declared, Anna Svärd: None declared, Cyril CLAVEL: None declared, Elisabet Svenungsson: None declared, Iva Gunnarsson: None declared, Guy Serre: None declared, Saedis Saevarsdottir: None declared, Alf Kastbom: None declared, Lars Alfredsson: None declared, Vivianne Malmström: None declared, Johan Rönnelid: None declared, Anca Catrina: None declared, Karin Lundberg: None declared, Lars Klareskog: None declared

Évaluation du CDx90® (Microgenics Thermo Fisher Scientific) pour le dosage urinaire des drogues

2010 ◽  
Vol 25 (2) ◽  
pp. 118-122
Author(s):  
K. Barrial ◽  
T. Le Bricon ◽  
F. Courtier ◽  
M.-H. Tourvieille ◽  
S. Hilaire ◽  
...  
Keyword(s):  
Thermo Fisher Scientific ◽  
Fisher Scientific

scholarly journals Immune Repertoire Analysis of Multiple Myeloma Research Samples Using NGS Characterization of Multiple B Cell Receptors in a Single Reaction

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1881-1881
Author(s):  
Geoffrey Lowman ◽  
Landon Pastushok ◽  
Karen Mochoruk ◽  
Wayne Hill ◽  
Michelle Toro ◽  
...  
Keyword(s):  
B Cell ◽  
Light Chain ◽  
Somatic Hypermutation ◽  
Cell Receptor ◽  
Clonal Lineage ◽  
Repertoire Analysis ◽  
Targeted Ngs ◽  
Thermo Fisher Scientific ◽  
Fisher Scientific ◽  
Single Reaction

Abstract Introduction B cell repertoire analysis by next-generation sequencing (NGS) is at the forefront of leukemia and lymphoma research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to other methods. Importantly, in research of post-germinal B cell disorders, such as multiple myeloma (MM), NGS methods allow for the study of clonal lineage based on somatic hypermuation patterns. Current targeted NGS assays require multiple libraries to survey each B cell receptor chain (IGH, IgK, IgL), and this fact is highlighted when initial clonality detection fails due to mutations under primer binding sites. This issue can be especially true in MM which has a high rate of SHM. To address these issues, we have developed an assay for B cell analysis, based on Ion AmpliSeq™ technology, which enables efficient detection of IGH, IgK, and IgL chain rearrangements in a single reaction. Methods The B cell pan-clonality panel (Oncomine™ BCR Pan-Clonality Assay) targets the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IgK, IgL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. To maximize sensitivity, we included primers to amplify IgK loci rearrangements involving Kappa deletion element and the constant region intron. To evaluate assay performance, we conducted reproducibility studies and clonality assessment using gDNA from a total of 45 MM research samples. All MM cases examined in this work were confirmed clonal previously by light chain restriction via flow cytometry or IHC/ISH in tissue sections - 16 of the 45 MM samples were identified as lambda light chain restricted. For comparison, a small cohort of 12 B-ALL samples were also included in the study. Sequencing and repertoire analyses were performed using the Ion GeneStudio S5 System and Ion Reporter 5.16 analysis software. Results Clonality assessment of MM clinical research samples show an 93% overall positive detection rate by an assay which combines the IGH, IgK, and IgL chains in a single reaction using published guidelines for clonality assignment. Thirty-four of 45 samples show positive detection of an IGH rearrangement, while 41 of 45 showed positive detection of at least one light chain receptor. In total, 42 of 45 samples were deemed clonal by the single tube assay based on detection for one or more receptor. Clonality results for this sample set are well correlated with orthogonal data from flow, IHC/ISH, or alternate NGS assays. A clonal lambda light chain was identified in 14 of 16 samples determined to be lambda restricted by flow cytometry. In two of the lambda restricted samples only a clonal lambda rearrangement was identified, showing the benefit of including primers targeting both the kappa and lambda light chains in a pan-clonality NGS assay. Both the MM and B-ALL cohorts were evaluated for biased IGHV gene usage. IGHV3-11 was observed in 5 of 45 MM and 5 of 12 B-ALL samples. IGHV4-34, typically linked to autoreactive antibodies and underrepresented in germinal center and memory B-cells, was nonetheless found in 5 of 45 MM samples surveyed. Estimates of somatic hypermutation rates were calculated using the BCR pan-clonality assay. Most MM samples, as expected, contained some somatic hypermutation with 6 of 45 samples showing greater than 10% mutation rates. Automated lineage analysis, based on somatic hypermuation signatures within each sample, identified 8 of 45 MM samples which contained 5 or more clones in the primary clonal lineage, with one case containing a lineage with 23 clones. Two MM samples showed no somatic hypermutation as measured using the FR3 primers contained in the BCR pan-clonality assay. These samples were also evaluated using an FR1-J targeted NGS assay, which confirmed relatively low mutation rates for these MM samples at 0.44% and 1.3%, respectively. Conclusions These results demonstrate the utility of a novel assay for combined repertoire analysis of B cell receptor heavy and light chains in a single library preparation reaction. We expect this assay to simplify laboratory workflows and including analysis tools such as automated somatic hypermutation rate calculation and clonal lineage identification may open new paths for research in lymphoid cell disorders. For research use only. Disclosures Lowman: Thermo Fisher Scientific: Current Employment. Toro: Thermo Fisher Scientific: Current Employment. Pickle: Thermo Fisher Scientific: Current Employment. Ostresh: Thermo Fisher Scientific: Current Employment. Sarda: Thermo Fisher Scientific: Current Employment. Yang: Thermo Fisher Scientific: Current Employment.

scholarly journals Does the Pre-Ovulatory Pig Oviduct Rule Sperm Capacitation In Vivo Mediating Transcriptomics of Catsper Channels?

2020 ◽  
Vol 21 (5) ◽  
pp. 1840 ◽  
Author(s):  
Cristina A. Martinez ◽  
Manuel Alvarez-Rodriguez ◽  
Dominic Wright ◽  
Heriberto Rodriguez-Martinez
Keyword(s):  
Ex Vivo ◽  
Female Genital Tract ◽  
Thermo Fisher Scientific ◽  
Positive Regulators ◽  
Porcine Gene ◽  
Single Fertilization ◽  
Boar Spermatozoa ◽  
Go Terms ◽  
Fisher Scientific

Spermatozoa need to conduct a series of biochemical changes termed capacitation in order to fertilize. In vivo, capacitation is sequentially achieved during sperm transport and interaction with the female genital tract, by mechanisms yet undisclosed in detail. However, when boar spermatozoa are stored in the tubal reservoir pre-ovulation, most appear to be in a non-capacitated state. This study aimed at deciphering the transcriptomics of capacitation-related genes in the pig pre-ovulatory oviduct, following the entry of semen or of sperm-free seminal plasma (SP). Ex-vivo samples of the utero-tubal junction (UTJ) and isthmus were examined with a microarray chip (GeneChip® Porcine Gene 1.0 ST Array, Thermo Fisher Scientific) followed by bioinformatics for enriched analysis of functional categories (GO terms) and restrictive statistics. The results confirmed that entry of semen or of relative amounts of sperm-free SP modifies gene expression of these segments, pre-ovulation. It further shows that enriched genes are differentially associated with pathways relating to sperm motility, acrosome reaction, single fertilization, and the regulation of signal transduction GO terms. In particular, the pre-ovulation oviduct stimulates the Catsper channels for sperm Ca2+ influx, with AKAPs, CATSPERs, and CABYR genes being positive regulators while PKIs and CRISP1 genes appear to be inhibitors of the process. We postulate that the stimulation of PKIs and CRISP1 genes in the pre-ovulation sperm reservoir/adjacent isthmus, mediated by SP, act to prevent premature massive capacitation prior to ovulation.

203 SINGLE-STEP GENE EDITING OF 3 XENOANTIGENS IN PORCINE FIBROBLASTS USING PROGRAMMABLE NUCLEASES

2017 ◽  
Vol 29 (1) ◽  
pp. 210 ◽  
Author(s):  
A. Perota ◽  
I. Lagutina ◽  
C. Quadalti ◽  
R. Duchi ◽  
P. Turini ◽  
...  
Keyword(s):  
Gene Editing ◽  
Magnetic Beads ◽  
Single Step ◽  
High Rate ◽  
Pcr Analysis ◽  
Thermo Fisher Scientific ◽  
Protective Genes ◽  
Pcr Products ◽  
Programmable Nucleases ◽  
Fisher Scientific

Programmable nucleases (ZFN, Tal Effector Nucleases, and CRISPR) opened a new era for mammal genome editing, in particular for the pigs used for xenotransplantation. Multiple gene editing events are required both for knockout (KO) of xenoantigens and for targeted integration of human protective genes (Perota et al. 2016 J. Genet. Genomics 43, 233–23). The objective of the present work was to edit selected pig lines to KO the enzymes coding for the most relevant xenoantigens (i.e. GGTA1, CMAH, and B4GalNT2), combining Talens and CRISPR/Cas9 technologies to magnetic beads selection (Li et al. 2013 Xenotransplantation 22, 20–31). Primary porcine adult fibroblasts were transfected using Nucleofector (V-024 program). In a single reaction 2 × 106 fibroblasts were co-transfected using 2 different sets of TALENS (4 μg/set) specific for CMAH (Conchon et al., 2013) and GGTA1 (Perota et al., 2015) genes together with B4GalNT2-specific CRISPR/Cas9 expression vector (2 μg; pX330-B4GalNT2; Estrada et al., 2015). Eight days post-transfection (DPT), Gal–/– cells were selected initially using biotin-conjugated IB4 lectin (Sigma, St. Louis, MO, USA) and magnetic beads (Dynabeads M-280, Thermo Fisher Scientific, Waltham, MA, USA). The selected cells were then plated on 150-mm Petri dishes (200 cells/dish) and cultured for 10 days. Selected colonies were expanded for PCR analysis and cryopreserved for somatic cell nuclear transfer (SCNT). All colonies were analysed by PCR for CMAH gene and their resulting products were digested with HindIII (HindIII-RFLP). Colonies that lost wild-type HindIII as a consequence of Talens effected deletion were PCR characterised for GGTA1, selecting those that had detectable Indels after gel electrophoresis and finally analysed by PCR for B4GalNT2. All PCR products were validated by sequencing for all the 3 genes of interest (TopoTA, Thermo Fisher Scientific). Selected colonies were used as nuclear donors for SCNT (Lagutina et al., 2006). Eight DPT we obtained 3.45 ×106 cells. About 6.0 × 103 Gal-negative cells (0.17%) were collected from the supernatant after magnetic beads separation. Eighteen DPT, 120 colonies were picked up and their HindIII-RFLP analyses on CMAH gene revealed that 22 colonies (18.3%) were KO for both CMAH alleles. Of these 22 colonies following electrophoretic analyses of GGTA1-PCR products, 13 colonies had detectable Indels. These 13 colonies were finally PCR analysed and sequenced for B4GalNT2 and sequenced. Final sequencing results confirmed that 2 colonies (1.6%) resulted in KO for the 3 genes. Three different zona-free SCNT experiments were done and 579 reconstructed embryos were obtained. On Day 7, 322 morulae or blastocysts (56%) were transferred in 3 synchronised sows and 2 (66%) became pregnant. In conclusion, after gene editing with programmable nucleases, combining beads-mediated selection with well-designed molecular analyses, we developed a multistep assay that can be used efficiently to detect desired gene edited events in cell colonies suitable for the SCNT. Embryos generated after SCNT were able to establish pregnancies at a high rate. This work is supported by European FP7 grants Translink (n° 603049) and Xenoislet (n° 601827).

scholarly journals Monitoraggio terapeutico del levetiracetam (Keppra®): confronto tra un nuovo metodo immunologico e il metodo di riferimento in cromatografia liquida ad alta prestazione

2015 ◽  
Vol 4 (1) ◽  
Author(s):  
V. Bianchi ◽  
F. Martino ◽  
S. Piccinini ◽  
A. Pinca ◽  
G. Sida ◽  
...  
Keyword(s):  
Sono Stati ◽  
Thermo Fisher Scientific ◽  
Fisher Scientific

Obiettivi: Il monitoraggio terapeutico levetiracetam, nuovo farmaco anti-convulsivante può essere indicato in pazienti le cui condizioni potrebbero alterare le caratteristiche farmacocinetiche, nella personalizzazione della singola posologia e nella valutazione della compliance del paziente. In questo studio, per il dosaggio del levetiracetam viene confrontato il metodo validato Bio-Rad HPLC attualmente in uso con il metodo ARKTM immunologico con strumenti statistici avanzati. Metodi: Le concentrazioni di levetiracetam di 63 campioni sono stati determinati utilizzando: 1) "Levetiracetam mediante HPLC" kit da Bio-Rad (Hercules, CA) sul sistema HPLC Agilent 1100 e 2) immunologico "ARKTM Levetiracetam" di ARK Diagnostics Inc. (Fremont, CA) sulla piattaforma CDx90 da Thermo Fisher Scientific Inc. Risultati: L’imprecisione e il bias intra-laboratorio del nuovo metodo, valutati su un periodo di 20 giorni, sono stati rispettivamente 7,4% e 0,5% a 7,5 μg/mL, 4,5% e 1,9% a 30 μg/mL, 3,1% e 2,0% a 75 μg /mL. Analisi di regressione Passign-Bablok (X: Bio- Rad, Y: Ark) ha mostrato una intercetta non significativo di 0,16 (95% CI -0.55- 0.72) e una pendenza marginalmente significativa di 0,95 (95% CI ,90-0,99) che suggerisce un minimo errore sistematico proporzionale. Anche l’analisi Bland- Altman ha mostrato un minimo bias sistematico di 1,0 mg/mL (95% CI 0,32-1,69) con il 95% delle differenze HPLC-Ark vanno da -4.3 (95% CI -5,52 - (-) 3.16) e 6,3 (95% CI 5,16-7,52). I dati hanno mostrato che i valori ottenuti dai due metodi sono da considerarsi identici sia entro l’imprecisione intrinseca così come per le specifiche di qualità analitiche (errore massimo ammissibile il 15%). Conclusioni: Il nuovo metodo ARKTM sulla piattaforma CDx è accettabile e può essere utilizzato per misurare la concentrazione di routine levetiracetam. In particolare, poiché il metodo immunologico abbassa drasticamente il TAT è più facile ed immediato l’aggiustamento della posologia nei pazienti critici.

scholarly journals Differential Expression of Inflammation-Related Genes in Down Syndrome Patients with or without Periodontal Disease

2019 ◽  
Vol 2019 ◽  
pp. 1-6
Author(s):  
M. Baus-Domínguez ◽  
R. Gómez-Díaz ◽  
D. Torres-Lagares ◽  
J. R. Corcuera-Flores ◽  
J. C. Ruiz-Villandiego ◽  
...  
Keyword(s):  
Gene Expression ◽  
Down Syndrome ◽  
Periodontal Disease ◽  
Differential Expression ◽  
Clinical Feature ◽  
Related Gene ◽  
Thermo Fisher Scientific ◽  
Oral Inflammation ◽  
Fisher Scientific

Aim. Aware that Down Syndrome patients present among their clinical characteristics impaired immunity, the aim of this study is to identify the statistically significant differences in inflammation-related gene expression by comparing Down Syndrome patients with Periodontal Disease (DS+PD+) with Down Syndrome patients without Periodontal Disease (DS+PD-), and their relationship with periodontitis as a chronic oral inflammatory clinical feature. Materials and Methods. Case study and controls on eleven Down Syndrome patients (DS+PD+ vs. DS+PD-). RNA was extracted from peripheral blood using a Qiagen PAXgene Blood miRNA Kit when performing an oral examination. A search for candidate genes (92 selected) was undertaken on the total genes obtained using a Scientific GeneChip® Scanner 3000 (Thermo Fisher Scientific) and Clariom S solutions for human, mouse, and rat chips, with more than 20,000 genes annotated for measuring expression levels. Results. Of the 92 inflammation-related genes taken initially, four genes showed a differential expression across both groups with a p value of <0.05 from the data obtained using RNA processing of the patient sample. Said genes were TNFSF13B (p=0.0448), ITGB2 (p=0.0033), ANXA3 (p=0.0479), and ANXA5 (p=0.016). Conclusions. There are differences in inflammation-related gene expression in Down Syndrome patients when comparing patients who present a state of chronic oral inflammation with patients with negative rates of periodontal disease.

Sign in / Sign up

Export Citation Format

Share Document