scholarly journals Deep Phenotypic Characterisation of CTCs by Combination of Microfluidic Isolation (IsoFlux) and Imaging Flow Cytometry (ImageStream)

Cancers ◽  
2021 ◽  
Vol 13 (24) ◽  
pp. 6386
Author(s):  
Antonio J. Ruiz-Rodríguez ◽  
Maria P. Molina-Vallejo ◽  
Inés Aznar-Peralta ◽  
Cristina González Puga ◽  
Inés Cañas García ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Magnetic Beads ◽  
Limit Of Detection ◽  
Recovery Efficiency ◽  
Analysis Method ◽  
Imaging Flow Cytometry ◽  
Healthy Donors ◽  
Phenotypic Characterisation ◽  
Changes Over Time ◽  
Over Time

The isolation of circulating tumour cells (CTCs) in colorectal cancer (CRC) mostly relies on the expression of epithelial markers such as EpCAM, and phenotypic characterisation is usually performed under fluorescence microscopy with only one or two additional markers. This limits the ability to detect different CTC subpopulations based on multiple markers. The aim of this work was to develop a novel protocol combining two platforms (IsoFluxTM and ImageStream®X) to improve CTC evaluation. Cancer cell lines and peripheral blood from healthy donors were used to evaluate the efficiency of each platform independently and in combination. Peripheral blood was extracted from 16 early CRC patients (before loco-regional surgery) to demonstrate the suitability of the protocol for CTC assessment. Additionally, peripheral blood was extracted from nine patients one month after surgery to validate the utility of our protocol for identifying CTC subpopulation changes over time. Results: Our protocol had a mean recovery efficiency of 69.5% and a limit of detection of at least four cells per millilitre. We developed an analysis method to reduce noise from magnetic beads used for CTC isolation. CTCs were isolated from CRC patients with a median of 37 CTCs (IQ 13.0–85.5) at baseline. CTCs from CRC patients were significantly (p < 0.0001) larger than cytokeratin (CK)-negative cells, and patients were stratified into two groups based on BRAFV600E and PD-L1 expression on CK-positive cells. The changes observed over time included not only the number of CTCs but also their distribution into four different subpopulations defined according to BRAFV600E and PD-L1 positivity. We developed a novel protocol for semi-automatic CTC isolation and phenotypic characterisation by combining two platforms. Assessment of CTCs from early CRC patients using our protocol allowed the identification of two clusters of patients with changing phenotypes over time.

Large Scale Selective Ex Vivo Expansion of CD4+CD25+FOXP3+ Regulatory T Cells from Peripheral Blood.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2344-2344
Author(s):  
Tokiko Nagamura-Inoue ◽  
Kazuo Ogami ◽  
Kazuaki Yokoyama ◽  
Kiyoko Izawa ◽  
Seiichiro Kobayashi ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Large Scale ◽  
Magnetic Beads ◽  
Ex Vivo ◽  
Chronic Gvhd ◽  
Ex Vivo Expansion ◽  
Healthy Donors

Abstract CD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in allograft- and self-tolerance and thus have the potential for therapeutic application in immunological and allergic disorders. However, the frequencies of Treg in peripheral blood are very low. Here we attempted the ex vivo expansion of Treg to enable the adaptive immunoregulatory therapy in humans. CD4+ T cells from peripheral blood of healthy donors or patients with chronic graft-versus-host disease (GVHD) were isolated by anti-CD4 monoclonal antibody (MAb)-conjugated magnetic beads, and cultured using a plastic plate coated with anti-CD28 and anti-CD3-MAbs in the medium containing recombinant human (rh) IL-2 and rhTGF-b. After one week of culture, expanding cells were once detached from the plate and subjected to the fresh medium including rhIL-2 and rhTGF-b but not MAbs. After 2-weeks of culture, phenotypic and functional analyses were performed. Mixed lymphocyte reaction was done using CFSE-labeled responder T cells and autologous or allogeneic dendritic cells (DC) with or without expanded Treg-rich populations. Xenogeneic -GVHD in NOD-Scid mice was induced by the injection of human T cells expressing luciferace transgene, followed by in vivo bioluminescence imaging (BLI) analysis using a CCD camera. Our expansion procedure with TGF-b yielded 45-83% purity of Foxp3+CD25+CD4+Treg co-expressing CTLA-4, CD54 and GITR, while 8-42% purity without TGF-b(p&lt;0.001). These cell populations also displayed CD45RO+CD45RA−CD26high+ memory phenotype. An average expansion rate of Treg was 62,200 fold (25,500–97,900) in healthy donors during the culture periods (n=5). Thus, we obtained an average of 4.7x108 Treg from the initial number of 5x105 CD4+ T cells in peripheral blood. Additionally, from peripheral CD4+ T cells in patients with chronic GVHD, Treg could be expanded equivalently to healthy donors. The resulting Treg-rich populations inhibited the proliferative response of CFSE-labeled T cells to autologous and allogeneic DC (Figure 1). The ex vivo expanded Treg-rich populations had the inhibitory effect on xeno-reactive T cells expressing luciferase transgene in a xenogeneic GVHD model (Figure 2). Our procedure has allowed efficient ex vivo expansion of Treg-rich populations from a small volume of peripheral blood, and will be applicable to clinical use. Figure 1. MLR inhibited by expanded Treg Figure 1. MLR inhibited by expanded Treg Figure 2. Xenogeneic GVHD diminished by expanded Treg. Figure 2. Xenogeneic GVHD diminished by expanded Treg.

scholarly journals Development and optimization of a new method for direct extraction of SARS-CoV-2 RNA from municipal wastewater using magnetic beads

2020 ◽  
Author(s):  
Ana L. Parra Guardado ◽  
Crystal L. Sweeney ◽  
Emalie Hayes ◽  
Benjamin F. Trueman ◽  
Yannan Huang ◽  
...  
Keyword(s):  
Municipal Wastewater ◽  
Magnetic Beads ◽  
Limit Of Detection ◽  
Direct Method ◽  
Rna Extraction ◽  
Magnetic Bead ◽  
Sample Volume ◽  
Recovery Efficiency ◽  
Extraction Protocol ◽  
Wastewater Samples

AbstractThe use of magnetic beads in the extraction of nucleic acids from wastewater is presented as an approach to simplify extraction techniques for the detection of SARS-CoV-2 viral fragments in wastewater. In particular, this paper describes the development and optimization of a direct method for extracting SARS-CoV-2 RNA from municipal wastewater using magnetic beads. The recovery efficiency of the method using Accuplex SARS-CoV-2 Positive Reference Material (ASCV-2) was examined. Method factors assessed were sample volume, concentration of magnetic bead mix, elution temperature, and water matrix (deionized (DI) water and wastewater). The combination of optimized method parameters that resulted in the highest RNA recovery in both DI water (26.0 ± 0.8%) and wastewater (11.8 ± 1.4%) was a sample volume of 1.0 mL, a magnetic beads concentration of 100 µL mL-1 sample, and an elution temperature of 60 °C. The performance of this optimized method was further assessed in recovery experiments using wastewater samples spiked at 1.8×106 and 1.8×104 gene copies L-1 (GU L-1) with Gamma Inactivated SARS-COV-2 (GI-SCV-2) and 1.0×106 and 1.0×104 infectious units L-1 of Human Coronavirus 229E (HCV 229E) as viral surrogates. Recoveries of 86.1 and 4.6% were achieved for wastewater samples spiked with GI-SCV-2 at low and high concentrations, respectively. In assessing the effects of wastewater pre-filtration and addition of DL-Dithiothreitol (DTT, used to inactivate RNases that may degrade RNA) on recovery efficiency of ASCV-2, the magnetic bead-based extraction protocol performed optimally with unfiltered wastewater without DTT (recovery = 17.4 ± 0.4%). The method limit of detection (MLOD) for ASCV-2 recovered from pre-filtered wastewater was determined to be 4.6×104 GU L-1 (95% degree of confidence). Using this optimized magnetic bead-based extraction protocol, the presence of SARS-CoV-2 RNA was verified in wastewater collected from sewershed locations in Atlantic Canada. This emerging RNA extraction method is direct, rapid, and does not require the use of specialized equipment, thus offering advantageous application for laboratories with limited resources. As such, this method is an indispensable tool in the monitoring of wastewater for SARS-CoV-2 to potentially understand COVID-19 infection occurrence within communities and inform public health leaders.Graphical Abstract

Comparison of two devices for measuring exercise transcutaneous oxygen pressures in patients with claudication

VASA ◽  
2015 ◽  
Vol 44 (5) ◽  
pp. 355-362 ◽  
Author(s):  
Marie Urban ◽  
Alban Fouasson-Chailloux ◽  
Isabelle Signolet ◽  
Christophe Colas Ribas ◽  
Mathieu Feuilloy ◽  
...  
Keyword(s):  
Oxygen Pressure ◽  
Optical Sensors ◽  
Chemical Sensors ◽  
Transcutaneous Oxygen ◽  
Starting Values ◽  
Two Systems ◽  
Transcutaneous Oxygen Pressure ◽  
New Sensors ◽  
Changes Over Time ◽  
Over Time

Abstract. Summary: Background: We aimed at estimating the agreement between the Medicap® (photo-optical) and Radiometer® (electro-chemical) sensors during exercise transcutaneous oxygen pressure (tcpO2) tests. Our hypothesis was that although absolute starting values (tcpO2rest: mean over 2 minutes) might be different, tcpO2-changes over time and the minimal value of the decrease from rest of oxygen pressure (DROPmin) results at exercise shall be concordant between the two systems. Patients and methods: Forty seven patients with arterial claudication (65 + / - 7 years) performed a treadmill test with 5 probes each of the electro-chemical and photo-optical devices simultaneously, one of each system on the chest, on each buttock and on each calf. Results: Seventeen Medicap® probes disconnected during the tests. tcpO2rest and DROPmin values were higher with Medicap® than with Radiometer®, by 13.7 + / - 17.1 mm Hg and 3.4 + / - 11.7 mm Hg, respectively. Despite the differences in absolute starting values, changes over time were similar between the two systems. The concordance between the two systems was approximately 70 % for classification of test results from DROPmin. Conclusions: Photo-optical sensors are promising alternatives to electro-chemical sensors for exercise oximetry, provided that miniaturisation and weight reduction of the new sensors are possible.

Neuropsychological Symptoms in Posttraumatic Stress Disorder and Changes Over Time

2007 ◽  
Author(s):  
Miranda Olff ◽  
Mirjam Nijdam ◽  
Kristin Samuelson ◽  
Julia Golier ◽  
Mariel Meewisse ◽  
...  
Keyword(s):  
Posttraumatic Stress Disorder ◽  
Posttraumatic Stress ◽  
Stress Disorder ◽  
Neuropsychological Symptoms ◽  
Changes Over Time ◽  
Over Time

Changes Over Time in Adaptive Behavior Functioning of Twice-Exceptional Children

2010 ◽  
Author(s):  
Rebecca D. Stinson ◽  
Zachary Sussman ◽  
Megan Foley Nicpon ◽  
Allison L. Allmon ◽  
Courtney Cornick ◽  
...  
Keyword(s):  
Adaptive Behavior ◽  
Exceptional Children ◽  
Twice Exceptional ◽  
Changes Over Time ◽  
Over Time

Kategorisierung der Schädelform des Cavalier King Charles Spaniels

2019 ◽  
Vol 47 (02) ◽  
pp. 133-133
Keyword(s):  
Pilot Study ◽  
Cavalier King Charles Spaniel ◽  
Conformation Changes ◽  
Veterinary Record ◽  
Changes Over Time ◽  
Over Time

Knowler SP, Gillstedt L, Mitchell TJ et al. Pilot study of head conformation changes over time in the Cavalier King Charles spaniel breed. Veterinary Record 2019. doi:10.1136/vr.105135.

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