A tandem microextraction method, centrifuge free dispersive liquid-liquid microextraction and thin-film microextraction (DLLME-TFME), was used for analyzing molinate in environmental samples by ion mobility spectrometry (IMS). Considering the IMS as a competitive detection system, coupling these two popular sample preparation methods reduces the effect of solvent interference and improves the sensitivity of the technique.
Trichloromethane and methanol were used as the extraction, and dispersive solvents for the DLLME method and electrospun polyacrylonitrile/copper-benzene-1,4-dicarboxylic acid fibers were used as a sorbent in the TFME method. Some effective experimental variables influencing the extraction efficiency of an analyte such as type and volume of dispersive and extraction solvents, solution pH, ionic strength, sonication time, and extraction time were studied.
The linear dynamic range of 0.5-50 μg L-1 and the limit of detection of 0.1 μg L-1 were obtained under optimized conditions. The relative standard deviations for intra-and inter-day analysis https://biodas.org/ were calculated less than 10%. The present method was used for the determination of molinate in different real samples such as agricultural wastewater, well water, river water, and apple, and the recovery was obtained between 82% and 113%, for the spiked samples.
Acoustofluidic centrifuge for nanoparticle enrichment and separation
Liquid droplets have been studied for decades and have recently experienced renewed attention as a simplified model for numerous fascinating physical phenomena occurring on size scales from the cell nucleus to stellar black holes. Here, we present an acoustofluidic centrifugation technique that leverages an entanglement of acoustic wave actuation and the spin of a fluidic droplet to enable nanoparticle enrichment and separation.
By combining acoustic streaming and droplet spinning, rapid (<1 min) nanoparticle concentration and size-based separation are achieved with a resolution sufficient to identify and isolate exosome subpopulations.
The underlying physical mechanisms have been characterized both numerically and experimentally, and the ability to process biological samples (including DNA segments and exosome subpopulations) has been successfully demonstrated. Together, this acoustofluidic centrifuge overcomes existing limitations in the manipulation of nanoscale (<100 nm) bioparticles and can be valuable for various applications in the fields of biology, chemistry, engineering, material science, and medicine.
Quantitative Evaluation of a Telerobotic System for Vascular Ultrasound Measurement on a Short Arm Human Centrifuge
Artificial Gravity generated by Short Arm Human Centrifuges is a promising multi-system countermeasure for physiological deconditioning during long duration space flights. To allow a continuous assessment of cardiovascular hemodynamics during centrifugation, a telerobotic robotic system holding an ultrasound probe has been installed on a Short Arm Human Centrifuge.
A feasibility study was conducted to define the use capabilities and limitations of such a novel method. The objective of this study is to estimate the reproducibility and precision of remotely controlled vascular ultrasound assessment under centrifugation by assessing peripheral vascular diameter and wall distension. Four repeated centrifugation runs of 5 min, with 2.4 g at feet level, were performed including a 15 min rest between each run for a group of eight healthy male volunteers. Vascular diameter and distention were assessed for the common carotid artery (CCA) and the femoral artery (FA) by ultrasound imaging using a 10 MHz linear array probe (Mylab1, Esaote).
Ultrasound measurements were consecutively performed: a) by an expert user in hand-held mode in standing as well as supine position, b) using the telerobotic arm without centrifugation as baseline and c) using the telerobotic arm during centrifugation. Vascular responses were compared between baseline and under centrifugation. Inter-, intra-registration and group variability have been assessed for hand-held and remotely controlled examination.
The results show that intra-registration variability, σh , was always smaller than inter-registration variability, σm, that is in turned smaller than the inter-subject variability σg (σh < σm < σg). Centrifugation caused no significant changes in CCA diameter but a lower carotid distension compared to manual and robotic ultrasound in supine position (p < 0.05). Femoral diameter was significantly decreased in hypergravity compared to robotic sonography without centrifugation.
A good reproducibility and precision of the remotely controlled vascular ultrasound assessment under centrifugation could be demonstrated. In conclusion, arterial wall dynamics can be precisely assessed for the CCA and femoral artery during centrifugation using a telerobotic ultrasound measurement system. Potential improvements to further enhance reproducibility and safety of the system are discussed.
Assessment of bridge natural frequency as an indicator of scour using centrifuge modelling
One of the most prevalent causes of bridge failure around the world is “scour”-the gradual erosion of soil around a bridge foundation due to fast-flowing water. A reliable technique for monitoring scour would help bridge engineers take timely countermeasures to safeguard against failure. Although vibration-based techniques for monitoring structural damage have had limited success, primarily due to insufficient sensitivity, these have tended to focus on the detection of local damage. High natural frequency sensitivity has recently been reported for scour damage.
Previous experiments to investigate this have been limited as a result of the cost of full-scale testing and the fact that scaled-down soil-structure models tested outside a centrifuge do not adequately simulate full-scale behaviour. This paper describes the development of what is believed to be the first-ever centrifuge-testing programme to establish the sensitivity of bridge natural frequency to scour. A 1/60 scale model of a two-span integral bridge with 15 m spans was tested at varying levels of scour. For the fundamental mode of vibration, these tests found up to a 40% variation in natural frequency for 30% loss of embedment.
Models of three other types of foundation, which represent a shallow pad foundation, a deep pile bent and a deep monopile, were also tested in the centrifuge at different scour levels. The shallow foundation model showed lower frequency sensitivity to scour than the deep foundation models. Another important finding is that the frequency sensitivity to “global scour” is slightly higher than the sensitivity to “local scour”, for all foundation types. The level of frequency sensitivity (3.1-44% per scour depth equivalent to 30% of embedment of scour) detected in this experiment demonstrates the potential for using natural frequency as an indicator of both local and global scour of bridges, particularly those with deep foundations.
Microliter ultrafast centrifuge platform for size-based particle and cell separation and extraction using novel omnidirectional spiral surface acoustic waves
Asymmetric surface acoustic waves have been shown useful in separating particles and cells in many microfluidics designs, mostly notably sessile microdroplets. However, no one has successfully extracted target particles or cells for later use from such samples. We present a novel omnidirectional spiral surface acoustic wave (OSSAW) design that exploits a new cut of lithium niobate, 152 Y-rotated, to rapidly rotate a microliter sessile drop to ∼10 g, producing efficient multi-size particle separation.
We further extract the separated particles for the first time, demonstrating the ability to target specific particles, for example, platelets from mouse blood for further integrated point-of-care diagnostics. Within ∼5 s of surface acoustic wave actuation, particles with diameter of 5 μm and 1 μm can be separated into two portions with a purity of 83% and 97%, respectively.
Red blood cells and platelets within mouse blood are further demonstrated to be separated with a purity of 93% and 84%, respectively. These advancements potentially provide an effective platform for whole blood separation and point-of-care diagnostics without need for micro or nanoscale fluidic enclosures.
The temperature monitoring in vivo plays a vital role in the investigation of biological processes of organisms and the improvement of disease theranostic methods. The development of lanthanide luminescent nanocomposite-derived temperature probes in vivo allows accurate and reliable interrogation of biological thermodynamic processes due to their superior photostability, high sensitivity, and non-invasive sensing fashion.
This concept presented an overview of the recent development of lanthanide luminescent nanocomposite which are suitable for in vivo temperature monitoring, including the thermometric principles, key features, materials designs as well as their potential biomedical applications for non-invasive temperature detection in the living body. The perspectives of these lanthanide luminescent nanocomposite thermometers https://biodas.org/ for the optimization of temperature monitoring performance and potential future development are also discussed.
Habitat Use and Activity Patterns of Mammals and Birds in Relation to Temperature and Vegetation Cover in the Alpine Ecosystem of Southwestern China with Camera-Trapping Monitoring
The high-altitude ecosystem of the Tibetan Plateau in China is a biodiversity hotspot that provides unique habitats for endemic and relict species along an altitudinal gradient at the eastern edge. Acquiring biodiversity information in this area, where the average altitude is over 4000 m, has been difficult but has been aided by recent developments in non-invasive technology, including infrared-triggered camera trapping.
We used camera trapping to acquire a substantial number of photographic wildlife records in Wolong National Nature Reserve, Sichuan, China, from 2013 to 2016. We collected information of the habitat surrounding the observation sites, resulting in a dataset covering 37 species and 12 environmental factors. We performed a multivariate statistical analysis to discern the dominant environmental factors and cluster the mammals and birds of the ecosystem in order to examine environmental factors contributing to the species’ relative abundance. Species were generalized into three main types, i.e., cold-resistant, phyllophilic, and thermophilic, according to the identifiedkey environmental drivers (i.e., temperature and vegetation) for their abundances.
The mammal species with the highest relative abundance were bharal (Pseudois nayaur), Moupin pika (Ochotona thibetana), and Himalayan marmot (Marmota himalayana). The bird species with highest relative abundance were snow partridge (Lerwa lerwa), plain mountain finch (Leucosticte nemoricola), Chinese monal (Lophophorus lhuysii), and alpine accentor (Prunella collaris).
Intraluminal Esophageal TemperatureMonitoring Using the Circa S-Cath™ Temperature Probe to Guide Left Atrial Ablation in Patients with Atrial Fibrillation
Introduction: Radiofrequency catheter ablation is a common treatment for atrial fibrillation (AF), during which thermal esophageal injury may rarely occur and lead to an atrio-esophageal fistula. Therefore, we studied the utility of the Circa S-Cath™ multi-sensor luminal esophageal temperature (LET) probe to prevent esophageal thermal injury.
Methods and results: Thirty-six patients, enrolled prospectively, underwent circumferential or segmental pulmonary vein isolation for treatment of AF. A maximum ablation electrode temperature of 42ºC was programmed for automatic power delivery cutoff. In addition, energy delivery was manually discontinued when the maximum LET on any sensor of the probe rose abruptly (i.e. ˃0.2ºC) or exceeded 39º C. Esophagoscopy was performed immediately after ablation in 18 patients (with the temperature probe still in place) and at approximately 24 hours after ablation in 18 patients.
Esophageal lesions were classified as likely traumatic or thermally related. Of the 36 patients enrolled in the study, 21 had persistent and 15 had paroxysmal AF, average LVEF 57±16% and CHA2DS2VASc score 1.6±1.2 (range 0-4). Average maximum LET was 37.8±1.4ºC, power delivery 31.1±8 watts and ablation electrode temperature 36.4±4.1ºC. Average maximum contact force was 44.5±20.5 grams where measured. Only 1 patient (<3%) had an esophageal lesion that could potentially represent thermal injury and 4 patients (11.1%) had minor traumatic mechanical injury.
Conclusions: LET guided titration of power and duration of energy application, using an insulated multi-sensor esophageal temperature probe, is associated with a low risk of esophageal thermal injury during AF ablation. In only rare cases, LET monitoring resulted in the need to manipulate the esophagus to avoid unacceptable temperature rises, that could not be achieved by adjustment of power and duration of energy application.
Bio-Inspired Microwave Modulator for High-Temperature Electromagnetic Protection, Infrared Stealth and Operating TemperatureMonitoring
High-temperature electromagnetic (EM) protection materials integrated of multiple EM protection mechanisms and functions are regarded as desirable candidates for solving EM interference over a wide temperature range. In this work, a novel microwave modulator is fabricated by introducing carbonyl iron particles (CIP)/resin into channels of carbonized wood (C-wood). Innovatively, the spaced arrangement of two microwave absorbents not only achieves a synergistic enhancement of magnetic and dielectric losses, but also breaks the translational invariance of EM characteristics in the horizontal direction to obtain multiple phase discontinuities in the frequency range of 8.2-18.0 GHz achieving modulation of reflected wave radiation direction.
Accordingly, CIP/C-wood microwave modulator demonstrates the maximum effective bandwidth of 5.2 GHz and the maximum EM protection efficiency over 97% with a thickness of only 1.5 mm in the temperature range 298-673 K. Besides, CIP/C-wood microwave modulator shows stable and low thermal conductivities, as well as monotonic electrical conductivity-temperature characteristics, therefore it can also achieve thermal infrared stealth and working temperature monitoring in wide temperature ranges. This work provides an inspiration for the design of high-temperature EM protection materials with multiple EM protection mechanisms and functions.
Non-Invasive Microwave Hyperthermia and Simultaneous TemperatureMonitoring with a Single Theranostic Applicator
Cancer therapies are constantly evolving. Currently, heating tumor tissue is becoming more accessible as a stand-alone method or in combination with other therapies. Due to its multiple advantages over other heating mechanisms, microwave hyperthermia has recently gained a lot of traction.
In this work, we present a complementary split-ring resonator that is simultaneously excited in two independent frequency bands. With a high-power signal, the applicator is excited and heats the tissue-under-test up to 50°C with an average heating rate of 0.72°C per second. Furthermore, we present a dielectric temperature control system using the same applicator for microwave hyperthermia applications, which currently still requires an additional thermometry system. By exciting the applicator with a low-power signal, we can constantly monitor its resonant frequency.
This resonant frequency depends on the tissue properties, which in turn are temperature-dependent. In the temperature range from 20-50°C, a positive correlation between the temperature and resonant frequency was established.Clinical relevance – Exploiting the dual-band behavior of the complementary split-ring resonator to heat the tissue-under-test while dielectrically monitoring its temperature, creates new possibilities towards a theranostic, non-invasive microwave hyperthermia applicator.
Background: The purpose of this study was to evaluate the efficacy and safety of endovascular brachytherapy (EVBT) combined with transarterial chemoembolization (TACE) for the treatment of hepatocellular carcinoma (HCC) complicated with type III OR IV portal vein tumor thrombosis (PVTT) and to further analyze the prognostic predictors for the patients with HCC and PVTT.
Methods: We retrospectively analyzed the medical records of 54 patients who were diagnosed with HCC complicated with type III or IV PVTT and received EVBT combined with modified TACE treatment from January 2017 to June 2019. Adverse events, treatment response, overall survival (OS), progression-free survival (PFS), and stent patency were analysed to evaluate the efficacy and safety of this treatment. The independent prognostic predictors of OS were also statistically analyzed by the cox regression model.
Results: No adverse events occurred in the enrolled patients receiving EVBT combined with TACE treatment. The objective response and disease control rates were 42.6% and 96.3% respectively within 4 weeks after the treatment. The median OS and PFS were 209 days and 138 days, respectively. Cumulative stent patency rate was 70.4% at the last follow-up. AFP ≥ 400 ng/ml, ECOG PS > 1, Child Pugh grade B, and non-hemihepatic HCC were independent risk predictors to https://biodas.org/ evaluate the OS of HCC patient with type III or IV PVTT.
Conclusions: EVBT combined with TACE was a relatively effective and safe strategy to treat HCC patients with type III or IV PVTT.
Dual immune checkpoint blockade in hepatocellular carcinoma: where do we stand?
Hepatocellular carcinoma (HCC) represents the fourth most common cause of cancer-related death. Surgery, local ablative therapies and liver transplantation are the only potentially curative strategies, but the majority of patients present with advanced disease at diagnosis or develop recurrence after surgery.
In recent years, immunotherapy for HCC has received growing interest, and one of the most promising strategies is the association of two immune checkpoint inhibitors (ICIs), which has already demonstrated its potential in other solid tumors such as melanoma and renal cell carcinoma. Herein, we discuss the role and the biologic rationale of dual immune checkpoint blockade in HCC patients, focusing on the two ICI combinations: nivolumab plus ipilimumab and durvalumab plus tremelimumab.
Exposome and Skin. Part 2. The Influential Role of the Exposome, Beyond UVR, in Actinic Keratosis, Bowen’s Disease and Squamous Cell Carcinoma: A Proposal
Actinic keratosis (AK) is the main risk factor for the development of cutaneous invasive squamous cell carcinoma (SCC). It represents the first sign of severe chronic ultraviolet radiation exposure, which has a clear significant effect. Nevertheless, the skin is exposed to many other exposome factors which should be thoroughly considered. Our aim was to assess the impact of exposome factors other than ultraviolet radiation (UVR) on the etiopathology of AK and Bowen’s disease (BD) and progression of AK to SCC and to design tailored prevention strategies.
We performed an exhaustive literature search in September 2021 through PubMed on the impact of exposome factors other than UVR on AK, BD and SCC. We conducted several parallel searches combining terms of the following topics: AK, BD, SCC and microbiome, hormones, nutrition, alcohol, tobacco, viral infections, chemical contaminants and air pollution. Notably, skin microbiome studies have shown how Staphylococcus aureus infections are associated with AK and AK-to-SCC progression by the production of chronic inflammation. Nutritional studies have demonstrated how a caloric restriction in fat intake, oral nicotinamide and moderate consumption of wine significantly reduce the number of premalignant keratoses and SCC.
Regarding lifestyle factors, both alcohol and smoking are associated with the development of SCC in a dose-dependent manner. Relevant environmental factors are viral infections and chemical contaminants. Human papillomavirus infections induce deregulation of cellular proliferation and are associated with AK, BD and SCC. In addition to outdoor jobs, occupations such as industrial processing and farming also increase the risk of developing keratoses and SCC. The exposome of AK will undoubtedly help the understanding of its etiopathology and possible progression to SCC and will serve as a basis to design tailored prevention strategies.
A Genome-Wide Investigation of Effects of Aberrant DNA Methylation on the Usage of Alternative Promoters in Hepatocellular Carcinoma
Background: The alternative usage of promoters provides a way to regulate gene expression, has a significant influence on the transcriptome, and contributes to the cellular transformation of cancer. However, the function of alternative promoters (APs) in hepatocellular carcinoma (HCC) has not been systematically studied yet. In addition, the potential mechanism of regulation to the usage of APs remains unclear. DNA methylation, one of the most aberrant epigenetic modifications in cancers, is known to regulate transcriptional activity. Whether DNA methylation regulates the usage of APs needs to be explored. Here, we aim to investigate the effects of DNA methylation on usage of APs in HCC.
Methods: Promoter activities were calculated based on RNA-seq data. Functional enrichment analysis was implemented to conduct GO terms. Correlation tests were used to detect the correlation between promoter activity and methylation status. The LASSO regression model was used to generate a diagnostic model. Kaplan-Meier analysis was used to compare the overall survival between high and low methylation groups. RNA-seq and whole-genome bisulfite sequencing (WGBS) in HCC samples were performed to validate the correlation of promoter activity and methylation.
Results: We identified 855 APs in total, which could be well used to distinguish cancer from normal samples. The correlation of promoter activity and DNA methylation in APs was observed, and the APs with negative correlation were defined as methylation-regulated APs (mrAPs). Six mrAPs were identified to generate a diagnostic model with good performance (AUC = 0.97). Notably, the majority of mrAPs had CpG sites that could be used to predict clinical outcomes by methylation status. Finally, we verified 85.6% of promoter activity variation and 92.3% of methylation changes in our paired RNA-seq and WGBS samples, respectively. The negative correlation between promoter activity and methylation status was further confirmed in our HCC samples.
Conclusion: The aberrant methylation status plays a critical role in the precision usage of APs in HCC, which sheds light on the mechanism of cancer development and provides a new insight into cancer screening and treatment.
A case of locally advanced adenosquamous carcinoma of the cecum with long-term survival
A 63-year-old woman was admitted to our hospital with a right lower abdominal mass and general fatigue. Preoperative examination suggested a large ovarian tumor or cecal carcinoma. However, her intraoperative diagnosis was colon cancer; we therefore performed an ileocecal resection with oophorectomy. The tumor was pathologically diagnosed as adenosquamous carcinoma T4bN1M-stage IIIa.
We administrated CapeOX adjuvant chemotherapy for 6 months. Adenosquamous carcinoma is extremely rare, at around 0.1% of all colorectal cancers, and usually has a poor prognosis. The patient is still alive without recurrence after 84 post-operative months, even with later developments of metachronous early colorectal cancer and breast cancer. We herein report a rare case of cecal ASC with good prognosis.
Background: Several studies have compared the use of dried blot spot (DBS) as an alternative to plasma specimens, mainly using Whatman 903 cards as filter paper. The aim of this study was to evaluate the use of Whatman FTA card (FTA card) specimens for HIV-1 viral load testing compared to plasma specimens using two real-time PCR assays manufactured by Roche and Abbott.
Methodology: A cross-sectional study was conducted between April 2017 and September 2017 on HIV-1 patients admitted to Yalgado Ouédraogo Teaching Hospital. Paired FTA cards and plasma specimens were collected and analysed using the Abbott Real-Time HIV-1 assay (Abbott) and COBAS AmpliPrep/COBAS TaqMan v2.0 (Roche).
Results: In total, 107 patients were included. No statistical differences (P>0.05) were observed between the mean viral loads obtained from the FTA cards and those of the plasma specimens using the Roche and Abbott assays. In total, 29 samples with Roche and 15 samples with Abbott assay showed discrepant results. At viral loads of ≤1000 copies ml-1, the sensitivity and specificity of the FTA cards were 78.6 and 100% with Roche, and 92.3 and 95.9% with Abbott, respectively. Both the Roche and Abbott assays showed good correlation and agreement between the FTA cards and plasma values.
Conclusion: Our study demonstrates the feasibility of using FTA card filter paper for HIV-1 viral load testing. However, further studies will be required https://biodas.org/ for the validation of the use of FTA card filter paper in HIV-1 treatment monitoring.
Stability of Human Immunodeficiency Virus Serological Markers in Samples Collected as HemaSpot and Whatman 903 Dried Blood Spots.
Dried blood spots (DBS) are frequently used in clinical testing for biosurveillance, infectious disease and confirmatory testing, and clinical trials, particularly for populations in remote areas. The HemaSpot-HF blood collection device (HS) provides an alternative format to the Whatman 903 cards (903) to simplify sample collection and processing. In this study, the performance of the HS was compared to that of the 903 using previously characterized clinical specimens and HIV seroconversion panels known to exhibit markers of early human immunodeficiency virus (HIV) infection.
HS and 903 samples were prepared and tested by Bio-Rad GS HIV Combo Ag/Ab enzyme immunoassay (EIA), GS HIV-1/-2 Plus O EIA, GS HIV-1 Western blot, and HIV-1 Geenius assays. Both HS and 903 performed well for up to 6 months at room temperature, but a marked loss of Western blot and low titer antibody signals from early infection samples was observed in samples stored for 180 days at elevated (37 to 45°C) temperatures and high humidity (95%).
HemaSpot samples placed in sealed bags with additional desiccant were protected from degradation and showed improved signal recovery relative to that of the 903. HS was easier to use than the 903 and showed higher sensitivity and reproducibility for early infection samples and improved stability.
Whatman Protein Saver Cards for Storage and Detection of Parasitic Enteropathogens.
Current methods to identify the etiology of diarrhea require laboratory facilities for storage of pathogens, which is often challenging in low-resource settings. This study evaluated the efficacy of a low-cost method for preserving stool specimens for the detection of parasitic enteropathogens using Whatman 903 protein saver cards (Sigma-Aldrich, St. Louis, MO). Stool samples known to be positive by multiplex real-time polymerase chain reaction for Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica parasites were preserved on 232 Whatman cards.
DNA was then extracted from cards using Chelex and Qiagen extraction protocols, and tested for these parasites using multiplex real-time PCR. We included stool samples known to have a higher parasite load (cycle threshold [ct]-value < 30) and those with a lower parasite load (ct values 30-35). Sensitivities and specificities were determined using DNA extracted directly from whole stool samples using Qiagen kits (QIAGEN, Hilden, Germany). For whole stool samples with ct values < 30, preserved directly on Whatman 903 protein saver cards for Giardia analysis, the sensitivity was 100% for both Qiagen and Chelex DNA extraction.
For E. histolytica, this was 100% for sensitivity for Qiagen and 80% for Chelex DNA extractions, and for Cryptosporidium, this was 80% for Qiagen and 50% for Chelex DNA extraction. The specificity was 100% for all parasites for all extraction procedures. Given the high sensitivity for stool samples with higher parasite loads, we recommend the use of the Whatman 903 protein saver card for preserving fecal specimens for the analysis of Giardia and E. histolytica using Qiagen DNA extractions in low-resource settings.
Comparison of stool collection and storage on Whatman FTA Elute cards versus frozen stool for enteropathogen detection using the TaqMan Array Card PCR assay.
The use of Polymerase Chain Reaction (PCR) assays for pathogen detection in travelers’ diarrhea (TD) field studies is limited by the on-site processing and storage requirements for fecal specimens. The objectives of this investigation were to i) characterize the pathogen distribution in deployed military personnel with TD using the TaqMan® Array Card PCR (TAC) on frozen stool and diarrheal smears on Whatman FTA Elute cards (FTA cards), and to ii) compare TAC detection of enteropathogen targets using smeared FTA cards and frozen stool, using TAC on frozen stool as the ‘reference standard’.
Stool samples, obtained from active duty personnel with acute TD enrolled in a field trial, were smeared onto FTA cards and stored at room temperature. A corresponding aliquot of stool was frozen in a cryovial. FTA cards and frozen stool samples were tested at a central lab, using a customized TAC for detection of TD pathogens. 187 paired frozen stool samples and smeared FTA cards were stored for a median of 712 days (IQR 396-750) before testing. Overall detection rates were 78.6% for frozen stool and 73.2% for FTA cards. Diarrheagenic Escherichia coli were the most common bacteria identified. Using the TAC results on frozen stool as the reference, the overall sensitivity and specificity of TAC on FTA cards was 72.9% and 98.0% respectively.
TAC on FTA cards demonstrated a decrease in sensitivity with increasing frozen stool quantification cycle (Cq) (90.0% in FTA cards with a corresponding frozen stool Cq < 30, and 72.9% in samples with a corresponding frozen stool Cq < 35). Our findings support the use and further development of FTA cards in combination with a quantitative PCR assay for enteropathogen detection in TD field studies.
Detection of anti-hepatitis C virus and hepatitis C virus RNA in dried blood spot specimens using Whatman No. 1 filter paper.
Dried blood spot (DBS) specimen simplifies blood collection, processing, storage and shipment and may reduce the cost of testing for hepatitis C virus (HCV) infection. We wanted to see if DBS using a cheap filter paper is reliable alternative to serum for detection of anti-HCV and HCV RNA.
At a tertiary care hospital in Northeast India, we collected 91 paired DBS and serum specimens from patients at risk of HCV infection from July 2014 to June 2015. DBS was collected on Whatman No. 1 filter paper. After processing, the specimens were subjectedto anti-HCV detection by a third-generation Enzyme-Linked Immunosorbent Assay (ELISA). The reactive DBS and serum specimens were further subjected to HCV RNA detection by polymerase chain reaction. The results were analysed in paired screen-positive study design.
Anti-HCV was detected in 9 (9.9%) DBS specimens and 10 (10.9%) serum specimens. There was statistically significant (P < 0.0001) correlation between the optical density values of DBS and serum specimens (Pearson r = 0.9181, 95% confidence interval: 0.8781-0.9453). HCV RNA was detected in 5/9 (55.6%) reactive DBS and 9/10 (90.0%) reactive serum specimens. There was no correlation between HCV RNA levels in the DBS and the serum specimens. The relative sensitivity rate and the relative false-positive rate of DBS anti-HCV ELISA were 0.89 and 1.00, respectively.
DBS using Whatman No. 1 filter paper is quite reliable as serum for detection of anti-HCV. It can be useful in effective surveillance. However, it is not suitable for confirmation of chronic HCV infection.
Quality management of nucleic acid beginning materials is crucial to make sure the success of downstream experiments. Especially, Next Generation Sequencing (NGS) developed to a robust device in virtually all genetic analysis and diagnostic areas. Due to the institution of low enter library protocols for NGS workflows sequencing of cell-free DNA (cfDNA) grew to become doable.
Since the downstream purposes are sometimes time-consuming and costly, tight QC steps are required to make sure that samples are match for functions. These QC steps may be carried out with automated electrophoresis programs. Different cell-free DNA samples had been evaluated for Sample high quality with an Agilent 4200 TapeStation system and the Agilent Cell-free DNA ScreenTape assay. Depending on preanalytical pattern therapy or extraction strategies the standard of cfDNA can fluctuate.
The outcomes embrace a rating to qualify cfDNA samples in response to their contamination stage with excessive molecular weight materials. This permits defining a threshold for goal pattern qualification previous to librarypreparation. Moreover, correct quantification of cfDNA samples is crucial to find out appropriate enter quantities for cfDNA librarypreparation previous to sequencing.
Quality management of cfDNA is crucial to make sure the success of downstream experiments. Automated electrophoresis programs standardize pattern high quality management and allow goal pattern integrity evaluation in addition to the institution of high quality thresholds.
Automated Workflow for Somatic and Germline Next Generation Sequencing Analysis in Routine Clinical Cancer Diagnostics
Thanks to customized drugs developments and collaborations between trade, scientific analysis teams and regulatory companies, subsequent technology sequencing (NGS) is popping into a standard observe quicker than one might have initially anticipated.
When contemplating scientific purposes of NGS in oncology, a fast workflow for DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue samples, in addition to producing prime quality librarypreparation, may be actual challenges. Here we take into account these targets and the way making use of efficient automation expertise to NGS workflows could assist enhance yield, timing and quality-control. We firstly evaluated DNA restoration from archived FFPE blocks from three totally different guide extraction strategies and two automated extraction workstations.
The workflow was then applied to somatic (lung/colon panel) and germline (BRCA1/2) librarypreparation for NGS evaluation exploiting two automated workstations. All industrial kits gave good ends in phrases of DNA yield and high quality.
On the opposite hand, the automated workstation workflow has been confirmed to be a sound computerized extraction system to acquire prime quality DNA appropriate for NGS evaluation (lung/colon Ampli-seq panel).
Moreover, it may be effectively built-in with an open liquid dealing with platform to supply high-quality libraries from germline DNA with extra reproducibility and excessive protection for focused sequences in much less time (BRCA1/2). The introduction of automation in routine workflow results in an enchancment of NGS standardization and elevated scale up of pattern preparations, lowering labor and timing, with optimization of reagents and administration.