High sensitivity phase retrieval method in grating-based x-ray phase contrast imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Zhao; Gao, Kun; Chen, Jian

2015-02-15

Purpose: Grating-based x-ray phase contrast imaging is considered as one of the most promising techniques for future medical imaging. Many different methods have been developed to retrieve phase signal, among which the phase stepping (PS) method is widely used. However, further practical implementations are hindered, due to its complex scanning mode and high radiation dose. In contrast, the reverse projection (RP) method is a novel fast and low dose extraction approach. In this contribution, the authors present a quantitative analysis of the noise properties of the refraction signals retrieved by the two methods and compare their sensitivities. Methods: Using themore » error propagation formula, the authors analyze theoretically the signal-to-noise ratios (SNRs) of the refraction images retrieved by the two methods. Then, the sensitivities of the two extraction methods are compared under an identical exposure dose. Numerical experiments are performed to validate the theoretical results and provide some quantitative insight. Results: The SNRs of the two methods are both dependent on the system parameters, but in different ways. Comparison between their sensitivities reveals that for the refraction signal, the RP method possesses a higher sensitivity, especially in the case of high visibility and/or at the edge of the object. Conclusions: Compared with the PS method, the RP method has a superior sensitivity and provides refraction images with a higher SNR. Therefore, one can obtain highly sensitive refraction images in grating-based phase contrast imaging. This is very important for future preclinical and clinical implementations.« less

Quantitative detection of bovine and porcine gelatin difference using surface plasmon resonance based biosensor

NASA Astrophysics Data System (ADS)

Wardani, Devy P.; Arifin, Muhammad; Suharyadi, Edi; Abraha, Kamsul

2015-05-01

Gelatin is a biopolymer derived from collagen that is widely used in food and pharmaceutical products. Due to some religion restrictions and health issues regarding the gelatin consumption which is extracted from certain species, it is necessary to establish a robust, reliable, sensitive and simple quantitative method to detect gelatin from different parent collagen species. To the best of our knowledge, there has not been a gelatin differentiation method based on optical sensor that could detect gelatin from different species quantitatively. Surface plasmon resonance (SPR) based biosensor is known to be a sensitive, simple and label free optical method for detecting biomaterials that is able to do quantitative detection. Therefore, we have utilized SPR-based biosensor to detect the differentiation between bovine and porcine gelatin in various concentration, from 0% to 10% (w/w). Here, we report the ability of SPR-based biosensor to detect difference between both gelatins, its sensitivity toward the gelatin concentration change, its reliability and limit of detection (LOD) and limit of quantification (LOQ) of the sensor. The sensor's LOD and LOQ towards bovine gelatin concentration are 0.38% and 1.26% (w/w), while towards porcine gelatin concentration are 0.66% and 2.20% (w/w), respectively. The results show that SPR-based biosensor is a promising tool for detecting gelatin from different raw materials quantitatively.

Full skin quantitative optical coherence elastography achieved by combining vibration and surface acoustic wave methods

NASA Astrophysics Data System (ADS)

Li, Chunhui; Guan, Guangying; Huang, Zhihong; Wang, Ruikang K.; Nabi, Ghulam

2015-03-01

By combining with the phase sensitive optical coherence tomography (PhS-OCT), vibration and surface acoustic wave (SAW) methods have been reported to provide elastography of skin tissue respectively. However, neither of these two methods can provide the elastography in full skin depth in current systems. This paper presents a feasibility study on an optical coherence elastography method which combines both vibration and SAW in order to give the quantitative mechanical properties of skin tissue with full depth range, including epidermis, dermis and subcutaneous fat. Experiments are carried out on layered tissue mimicking phantoms and in vivo human forearm and palm skin. A ring actuator generates vibration while a line actuator were used to excited SAWs. A PhS-OCT system is employed to provide the ultrahigh sensitive measurement of the generated waves. The experimental results demonstrate that by the combination of vibration and SAW method the full skin bulk mechanical properties can be quantitatively measured and further the elastography can be obtained with a sensing depth from ~0mm to ~4mm. This method is promising to apply in clinics where the quantitative elasticity of localized skin diseases is needed to aid the diagnosis and treatment.

Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector

PubMed Central

Field, Christopher R.; Lubrano, Adam; Woytowitz, Morgan; Giordano, Braden C.; Rose-Pehrsson, Susan L.

2014-01-01

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples. PMID:25145416

Quantitative detection of trace explosive vapors by programmed temperature desorption gas chromatography-electron capture detector.

PubMed

Field, Christopher R; Lubrano, Adam; Woytowitz, Morgan; Giordano, Braden C; Rose-Pehrsson, Susan L

2014-07-25

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.

The current preference for the immuno-analytical ELISA method for quantitation of steroid hormones (endocrine disruptor compounds) in wastewater in South Africa.

PubMed

Manickum, Thavrin; John, Wilson

2015-07-01

The availability of national test centers to offer a routine service for analysis and quantitation of some selected steroid hormones [natural estrogens (17-β-estradiol, E2; estrone, E1; estriol, E3), synthetic estrogen (17-α-ethinylestradiol, EE2), androgen (testosterone), and progestogen (progesterone)] in wastewater matrix was investigated; corresponding internationally used chemical- and immuno-analytical test methods were reviewed. The enzyme-linked immunosorbent assay (ELISA) (immuno-analytical technique) was also assessed for its suitability as a routine test method to quantitate the levels of these hormones at a sewage/wastewater treatment plant (WTP) (Darvill, Pietermaritzburg, South Africa), over a 2-year period. The method performance and other relevant characteristics of the immuno-analytical ELISA method were compared to the conventional chemical-analytical methodology, like gas/liquid chromatography-mass spectrometry (GC/LC-MS), and GC-LC/tandem mass spectrometry (MSMS), for quantitation of the steroid hormones in wastewater and environmental waters. The national immuno-analytical ELISA technique was found to be sensitive (LOQ 5 ng/L, LOD 0.2-5 ng/L), accurate (mean recovery 96%), precise (RSD 7-10%), and cost-effective for screening and quantitation of these steroid hormones in wastewater and environmental water matrix. A survey of the most current international literature indicates a fairly equal use of the LC-MS/MS, GC-MS/MS (chemical-analytical), and ELISA (immuno-analytical) test methods for screening and quantitation of the target steroid hormones in both water and wastewater matrix. Internationally, the observed sensitivity, based on LOQ (ng/L), for the steroid estrogens E1, E2, EE2, is, in decreasing order: LC-MSMS (0.08-9.54) > GC-MS (1) > ELISA (5) (chemical-analytical > immuno-analytical). At the national level, the routine, unoptimized chemical-analytical LC-MSMS method was found to lack the required sensitivity for meeting environmental requirements for steroid hormone quantitation. Further optimization of the sensitivity of the chemical-analytical LC-tandem mass spectrometry methods, especially for wastewater screening, in South Africa is required. Risk assessment studies showed that it was not practical to propose standards or allowable limits for the steroid estrogens E1, E2, EE2, and E3; the use of predicted-no-effect concentration values of the steroid estrogens appears to be appropriate for use in their risk assessment in relation to aquatic organisms. For raw water sources, drinking water, raw and treated wastewater, the use of bioassays, with trigger values, is a useful screening tool option to decide whether further examination of specific endocrine activity may be warranted, or whether concentrations of such activity are of low priority, with respect to health concerns in the human population. The achievement of improved quantitation limits for immuno-analytical methods, like ELISA, used for compound quantitation, and standardization of the method for measuring E2 equivalents (EEQs) used for biological activity (endocrine: e.g., estrogenic) are some areas for future EDC research.

DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

DOE Office of Scientific and Technical Information (OSTI.GOV)

SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

2004-03-24

Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNAmore » populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.« less

A simple and sensitive method for the determination of fibric acids in the liver by liquid chromatography.

PubMed

Karahashi, Minako; Fukuhara, Hiroto; Hoshina, Miki; Sakamoto, Takeshi; Yamazaki, Tohru; Mitsumoto, Atsushi; Kawashima, Yoichi; Kudo, Naomi

2014-01-01

Fibrates are used in biochemical and pharmacological studies as bioactive tools. Nevertheless, most studies have lacked information concerning the concentrations of fibric acids working inside tissues because a simple and sensitive method is not available for their quantitation. This study aimed to develop a simple and sensitive bioanalytical method for the quantitation of clofibric, bezafibric and fenofibric acids in samples of very small portions of tissues. Fibric acids were extracted into n-hexane-ethyl acetate from tissue homogenates (10 mg of liver, kidney or muscle) or serum (100 µL) and were derivatized with 4-bromomethyl-6,7-dimethoxycoumarin, followed by HPLC with fluorescence detection. These compounds were separated isocratically on a reversed phase with acetonitrile-water. Standard analytical curves were linear over the concentration range of 0.2-20 nmol/10 mg of liver. Precision and accuracy were within acceptable limits. Recovery from liver homogenates ranged from 93.03 to 112.29%. This method enabled the quantitation of fibric acids in 10 mg of liver from rats treated with clofibric acid, bezafibric acid or fenofibrate. From these analytical data, it became clear that there was no large difference in ratio of acyl-CoA oxidase 1 (Acox1) mRNA level to fibric acid content in the liver among the three fibric acids, suggesting that these three fibric acids have similar potency to increase expression of the Acox1 gene, which is a target of peroxisome proliferator-activated receptor α. Thus, the proposed method is a simple, sensitive and reliable tool for the quantitation of fibric acids working in vivo inside livers.

A comprehensive evaluation of various sensitivity analysis methods: A case study with a hydrological model

DOE PAGES

Gan, Yanjun; Duan, Qingyun; Gong, Wei; ...

2014-01-01

Sensitivity analysis (SA) is a commonly used approach for identifying important parameters that dominate model behaviors. We use a newly developed software package, a Problem Solving environment for Uncertainty Analysis and Design Exploration (PSUADE), to evaluate the effectiveness and efficiency of ten widely used SA methods, including seven qualitative and three quantitative ones. All SA methods are tested using a variety of sampling techniques to screen out the most sensitive (i.e., important) parameters from the insensitive ones. The Sacramento Soil Moisture Accounting (SAC-SMA) model, which has thirteen tunable parameters, is used for illustration. The South Branch Potomac River basin nearmore » Springfield, West Virginia in the U.S. is chosen as the study area. The key findings from this study are: (1) For qualitative SA methods, Correlation Analysis (CA), Regression Analysis (RA), and Gaussian Process (GP) screening methods are shown to be not effective in this example. Morris One-At-a-Time (MOAT) screening is the most efficient, needing only 280 samples to identify the most important parameters, but it is the least robust method. Multivariate Adaptive Regression Splines (MARS), Delta Test (DT) and Sum-Of-Trees (SOT) screening methods need about 400–600 samples for the same purpose. Monte Carlo (MC), Orthogonal Array (OA) and Orthogonal Array based Latin Hypercube (OALH) are appropriate sampling techniques for them; (2) For quantitative SA methods, at least 2777 samples are needed for Fourier Amplitude Sensitivity Test (FAST) to identity parameter main effect. McKay method needs about 360 samples to evaluate the main effect, more than 1000 samples to assess the two-way interaction effect. OALH and LPτ (LPTAU) sampling techniques are more appropriate for McKay method. For the Sobol' method, the minimum samples needed are 1050 to compute the first-order and total sensitivity indices correctly. These comparisons show that qualitative SA methods are more efficient but less accurate and robust than quantitative ones.« less

Solid-Phase Radioimmunoassay of Total and Influenza-Specific Immunoglobulin G

PubMed Central

Daugharty, Harry; Warfield, Donna T.; Davis, Marianne L.

1972-01-01

An antigen-antibody system of polystyrene tubes coated with immunoglobulin antibody was used for quantitating immunoglobulins. A similar radioimmunoassay method was adapted for a viral antigen-antibody system. The viral system can be used for quantitating viruses and for measuring virus-specific antibodies by reacting with 125iodine-labeled anti-immunoglobulin G (IgG). Optimal conditions for coating the solid phase, specificity of the immune reaction, and other kinetics and sensitivities of the assay method were investigated. Comparison of direct and indirect methods of assaying for immunoglobulins or viral antibody indicates that the indirect method is more sensitive and can quantitate a minimum of 0.037 μg of IgG per ml. Results of solid-phase radioimmunoassay for influenza antibody correlate well with hemagglutinin antibody titers but not with complement-fixing antibody titers. Radioimmunoassay results for influenza antibody by solid phase are likewise in agreement with results by the carrier precipitate radioimmunoassay method. The simplicity, reproducibility, and versatility of the solid-phase procedure make it diagnostically useful. PMID:5062884

Improved Methods for Capture, Extraction, and Quantitative Assay of Environmental DNA from Asian Bigheaded Carp (Hypophthalmichthys spp.)

PubMed Central

Turner, Cameron R.; Miller, Derryl J.; Coyne, Kathryn J.; Corush, Joel

2014-01-01

Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species. PMID:25474207

Improved methods for capture, extraction, and quantitative assay of environmental DNA from Asian bigheaded carp (Hypophthalmichthys spp.).

PubMed

Turner, Cameron R; Miller, Derryl J; Coyne, Kathryn J; Corush, Joel

2014-01-01

Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.

Critical factors determining the quantification capability of matrix-assisted laser desorption/ionization– time-of-flight mass spectrometry

PubMed Central

Wang, Chia-Chen; Lai, Yin-Hung; Ou, Yu-Meng; Chang, Huan-Tsung; Wang, Yi-Sheng

2016-01-01

Quantitative analysis with mass spectrometry (MS) is important but challenging. Matrix-assisted laser desorption/ionization (MALDI) coupled with time-of-flight (TOF) MS offers superior sensitivity, resolution and speed, but such techniques have numerous disadvantages that hinder quantitative analyses. This review summarizes essential obstacles to analyte quantification with MALDI-TOF MS, including the complex ionization mechanism of MALDI, sensitive characteristics of the applied electric fields and the mass-dependent detection efficiency of ion detectors. General quantitative ionization and desorption interpretations of ion production are described. Important instrument parameters and available methods of MALDI-TOF MS used for quantitative analysis are also reviewed. This article is part of the themed issue ‘Quantitative mass spectrometry’. PMID:27644968

Quantitative assessment of hematopoietic chimerism by quantitative real-time polymerase chain reaction of sequence polymorphism systems after hematopoietic stem cell transplantation.

PubMed

Qin, Xiao-ying; Li, Guo-xuan; Qin, Ya-zhen; Wang, Yu; Wang, Feng-rong; Liu, Dai-hong; Xu, Lan-ping; Chen, Huan; Han, Wei; Wang, Jing-zhi; Zhang, Xiao-hui; Li, Jin-lan; Li, Ling-di; Liu, Kai-yan; Huang, Xiao-jun

2011-08-01

Analysis of changes in recipient and donor hematopoietic cell origin is extremely useful to monitor the effect of hematopoietic stem cell transplantation (HSCT) and sequential adoptive immunotherapy by donor lymphocyte infusions. We developed a sensitive, reliable and rapid real-time PCR method based on sequence polymorphism systems to quantitatively assess the hematopoietic chimerism after HSCT. A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time PCR in 101 HSCT patients with leukemia and other hematological diseases. The chimerism kinetics of bone marrow samples of 8 HSCT patients in remission and relapse situations were followed longitudinally. Recipient genotype discrimination was possible in 97.0% (98 of 101) with a mean number of 2.5 (1-7) informative markers per recipient/donor pair. Using serial dilutions of plasmids containing specific SP markers, the linear correlation (r) of 0.99, the slope between -3.2 and -3.7 and the sensitivity of 0.1% were proved reproducible. By this method, it was possible to very accurately detect autologous signals in the range from 0.1% to 30%. The accuracy of the method in the very important range of autologous signals below 5% was extraordinarily high (standard deviation <1.85%), which might significantly improve detection accuracy of changes in autologous signals early in the post-transplantation course of follow-up. The main advantage of the real-time PCR method over short tandem repeat PCR chimerism assays is the absence of PCR competition and plateau biases, with demonstrated greater sensitivity and linearity. Finally, we prospectively analyzed bone marrow samples of 8 patients who received allografts and presented the chimerism kinetics of remission and relapse situations that illustrated the sensitivity level and the promising clinical application of this method. This SP-based real-time PCR assay provides a rapid, sensitive, and accurate quantitative assessment of mixed chimerism that can be useful in predicting graft rejection and early relapse.

Quantitative methods to direct exploration based on hydrogeologic information

USGS Publications Warehouse

Graettinger, A.J.; Lee, J.; Reeves, H.W.; Dethan, D.

2006-01-01

Quantitatively Directed Exploration (QDE) approaches based on information such as model sensitivity, input data covariance and model output covariance are presented. Seven approaches for directing exploration are developed, applied, and evaluated on a synthetic hydrogeologic site. The QDE approaches evaluate input information uncertainty, subsurface model sensitivity and, most importantly, output covariance to identify the next location to sample. Spatial input parameter values and covariances are calculated with the multivariate conditional probability calculation from a limited number of samples. A variogram structure is used during data extrapolation to describe the spatial continuity, or correlation, of subsurface information. Model sensitivity can be determined by perturbing input data and evaluating output response or, as in this work, sensitivities can be programmed directly into an analysis model. Output covariance is calculated by the First-Order Second Moment (FOSM) method, which combines the covariance of input information with model sensitivity. A groundwater flow example, modeled in MODFLOW-2000, is chosen to demonstrate the seven QDE approaches. MODFLOW-2000 is used to obtain the piezometric head and the model sensitivity simultaneously. The seven QDE approaches are evaluated based on the accuracy of the modeled piezometric head after information from a QDE sample is added. For the synthetic site used in this study, the QDE approach that identifies the location of hydraulic conductivity that contributes the most to the overall piezometric head variance proved to be the best method to quantitatively direct exploration. ?? IWA Publishing 2006.

Quantitative Ultrasound for Measuring Obstructive Severity in Children with Hydronephrosis.

PubMed

Cerrolaza, Juan J; Peters, Craig A; Martin, Aaron D; Myers, Emmarie; Safdar, Nabile; Linguraru, Marius George

2016-04-01

We define sonographic biomarkers for hydronephrotic renal units that can predict the necessity of diuretic nuclear renography. We selected a cohort of 50 consecutive patients with hydronephrosis of varying severity in whom 2-dimensional sonography and diuretic mercaptoacetyltriglycine renography had been performed. A total of 131 morphological parameters were computed using quantitative image analysis algorithms. Machine learning techniques were then applied to identify ultrasound based safety thresholds that agreed with the t½ for washout. A best fit model was then derived for each threshold level of t½ that would be clinically relevant at 20, 30 and 40 minutes. Receiver operating characteristic curve analysis was performed. Sensitivity, specificity and area under the receiver operating characteristic curve were determined. Improvement obtained by the quantitative imaging method compared to the Society for Fetal Urology grading system and the hydronephrosis index was statistically verified. For the 3 thresholds considered and at 100% sensitivity the specificities of the quantitative imaging method were 94%, 70% and 74%, respectively. Corresponding area under the receiver operating characteristic curve values were 0.98, 0.94 and 0.94, respectively. Improvement obtained by the quantitative imaging method over the Society for Fetal Urology grade and hydronephrosis index was statistically significant (p <0.05 in all cases). Quantitative imaging analysis of renal sonograms in children with hydronephrosis can identify thresholds of clinically significant washout times with 100% sensitivity to decrease the number of diuretic renograms in up to 62% of children. Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

The efficacy of semi-quantitative urine protein-to-creatinine (P/C) ratio for the detection of significant proteinuria in urine specimens in health screening settings.

PubMed

Chang, Chih-Chun; Su, Ming-Jang; Ho, Jung-Li; Tsai, Yu-Hui; Tsai, Wei-Ting; Lee, Shu-Jene; Yen, Tzung-Hai; Chu, Fang-Yeh

2016-01-01

Urine protein detection could be underestimated using the conventional dipstick method because of variations in urine aliquots. This study aimed to assess the efficacy of the semi-quantitative urine protein-to-creatinine (P/C) ratio compared with other laboratory methods. Random urine samples were requested from patients undergoing chronic kidney disease screening. Significant proteinuria was determined by the quantitative P/C ratio of at least 150 mg protein/g creatinine. The semi-quantitative P/C ratio, dipstick protein and quantitative protein concentrations were compared and analyzed. In the 2932 urine aliquots, 156 (5.3 %) urine samples were considered as diluted and 60 (39.2 %) were found as significant proteinuria. The semi-quantitative P/C ratio testing had the best sensitivity (70.0 %) and specificity (95.9 %) as well as the lowest underestimation rate (0.37 %) when compared to other laboratory methods in the study. In the semi-quantitative P/C ratio test, 19 (12.2 %) had positive, 52 (33.3 %) had diluted, and 85 (54.5 %) had negative results. Of those with positive results, 7 (36.8 %) were positive detected by traditional dipstick urine protein test, and 9 (47.4 %) were positive detected by quantitative urine protein test. Additionally, of those with diluted results, 25 (48.1 %) had significant proteinuria, and all were assigned as no significant proteinuria by both tests. The semi-quantitative urine P/C ratio is clinically applicable based on its better sensitivity and screening ability for significant proteinuria than other laboratory methods, particularly in diluted urine samples. To establish an effective strategy for CKD prevention, urine protein screening with semi-quantitative P/C ratio could be considered.

Diagnostic accuracy of semi-quantitative and quantitative culture techniques for the diagnosis of catheter-related infections in newborns and molecular typing of isolated microorganisms.

PubMed

Riboli, Danilo Flávio Moraes; Lyra, João César; Silva, Eliane Pessoa; Valadão, Luisa Leite; Bentlin, Maria Regina; Corrente, José Eduardo; Rugolo, Ligia Maria Suppo de Souza; da Cunha, Maria de Lourdes Ribeiro de Souza

2014-05-22

Catheter-related bloodstream infections (CR-BSIs) have become the most common cause of healthcare-associated bloodstream infections in neonatal intensive care units (ICUs). Microbiological evidence implicating catheters as the source of bloodstream infection is necessary to establish the diagnosis of CR-BSIs. Semi-quantitative culture is used to determine the presence of microorganisms on the external catheter surface, whereas quantitative culture also isolates microorganisms present inside the catheter. The main objective of this study was to determine the sensitivity and specificity of these two techniques for the diagnosis of CR-BSIs in newborns from a neonatal ICU. In addition, PFGE was used for similarity analysis of the microorganisms isolated from catheters and blood cultures. Semi-quantitative and quantitative methods were used for the culture of catheter tips obtained from newborns. Strains isolated from catheter tips and blood cultures which exhibited the same antimicrobial susceptibility profile were included in the study as positive cases of CR-BSI. PFGE of the microorganisms isolated from catheters and blood cultures was performed for similarity analysis and detection of clones in the ICU. A total of 584 catheter tips from 399 patients seen between November 2005 and June 2012 were analyzed. Twenty-nine cases of CR-BSI were confirmed. Coagulase-negative staphylococci (CoNS) were the most frequently isolated microorganisms, including S. epidermidis as the most prevalent species (65.5%), followed by S. haemolyticus (10.3%), yeasts (10.3%), K. pneumoniae (6.9%), S. aureus (3.4%), and E. coli (3.4%). The sensitivity of the semi-quantitative and quantitative techniques was 72.7% and 59.3%, respectively, and specificity was 95.7% and 94.4%. The diagnosis of CR-BSIs based on PFGE analysis of similarity between strains isolated from catheter tips and blood cultures showed 82.6% sensitivity and 100% specificity. The semi-quantitative culture method showed higher sensitivity and specificity for the diagnosis of CR-BSIs in newborns when compared to the quantitative technique. In addition, this method is easier to perform and shows better agreement with the gold standard, and should therefore be recommended for routine clinical laboratory use. PFGE may contribute to the control of CR-BSIs by identifying clusters of microorganisms in neonatal ICUs, providing a means of determining potential cross-infection between patients.

Improvement of ion chromatography with ultraviolet photometric detection and comparison with conductivity detection for the determination of serum cations.

PubMed

Shintani, H

1985-05-31

Studies were made of the analytical conditions required for indirect photometric ion chromatography using ultraviolet photometric detection (UV method) for the determination of serum cations following a previously developed serum pre-treatment. The sensitivities of the conductivity detection (CD) and UV methods and the amounts of serum cations determined by both methods were compared. Attempts to improve the sensitivity of the conventional UV method are reported. It was found that the mobile phase previously reported by Small and Miller showed no quantitative response when more than 4 mM copper(II) sulphate pentahydrate was used. As a result, there was no significant difference in the amounts of serum cations shown by the CD and UV methods. However, by adding 0.5-5 mM cobalt(II) sulphate heptahydrate, nickel(II) sulphate hexahydrate, zinc(II) sulphate heptahydrate or cobalt(II) diammonium sulphate hexahydrate to 0.5-1.5 mM copper(II) sulphate pentahydrate, higher sensitivity and a quantitative response were attained.

Development of a quantitative diagnostic method of estrogen receptor expression levels by immunohistochemistry using organic fluorescent material-assembled nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gonda, Kohsuke, E-mail: gonda@med.tohoku.ac.jp; Miyashita, Minoru; Watanabe, Mika

2012-09-28

Highlights: Black-Right-Pointing-Pointer Organic fluorescent material-assembled nanoparticles for IHC were prepared. Black-Right-Pointing-Pointer New nanoparticle fluorescent intensity was 10.2-fold greater than Qdot655. Black-Right-Pointing-Pointer Nanoparticle staining analyzed a wide range of ER expression levels in tissue. Black-Right-Pointing-Pointer Nanoparticle staining enhanced the quantitative sensitivity for ER diagnosis. -- Abstract: The detection of estrogen receptors (ERs) by immunohistochemistry (IHC) using 3,3 Prime -diaminobenzidine (DAB) is slightly weak as a prognostic marker, but it is essential to the application of endocrine therapy, such as antiestrogen tamoxifen-based therapy. IHC using DAB is a poor quantitative method because horseradish peroxidase (HRP) activity depends on reaction time, temperature andmore » substrate concentration. However, IHC using fluorescent material provides an effective method to quantitatively use IHC because the signal intensity is proportional to the intensity of the photon excitation energy. However, the high level of autofluorescence has impeded the development of quantitative IHC using fluorescence. We developed organic fluorescent material (tetramethylrhodamine)-assembled nanoparticles for IHC. Tissue autofluorescence is comparable to the fluorescence intensity of quantum dots, which are the most representative fluorescent nanoparticles. The fluorescent intensity of our novel nanoparticles was 10.2-fold greater than quantum dots, and they did not bind non-specifically to breast cancer tissues due to the polyethylene glycol chain that coated their surfaces. Therefore, the fluorescent intensity of our nanoparticles significantly exceeded autofluorescence, which produced a significantly higher signal-to-noise ratio on IHC-imaged cancer tissues than previous methods. Moreover, immunostaining data from our nanoparticle fluorescent IHC and IHC with DAB were compared in the same region of adjacent tissues sections to quantitatively examine the two methods. The results demonstrated that our nanoparticle staining analyzed a wide range of ER expression levels with higher accuracy and quantitative sensitivity than DAB staining. This enhancement in the diagnostic accuracy and sensitivity for ERs using our immunostaining method will improve the prediction of responses to therapies that target ERs and progesterone receptors that are induced by a downstream ER signal.« less

A new liquid chromatography-mass spectrometry-based method to quantitate exogenous recombinant transferrin in cerebrospinal fluid: a potential approach for pharmacokinetic studies of transferrin-based therapeutics in the central nervous systems.

PubMed

Wang, Shunhai; Bobst, Cedric E; Kaltashov, Igor A

2015-01-01

Transferrin (Tf) is an 80 kDa iron-binding protein that is viewed as a promising drug carrier to target the central nervous system as a result of its ability to penetrate the blood-brain barrier. Among the many challenges during the development of Tf-based therapeutics, the sensitive and accurate quantitation of the administered Tf in cerebrospinal fluid (CSF) remains particularly difficult because of the presence of abundant endogenous Tf. Herein, we describe the development of a new liquid chromatography-mass spectrometry-based method for the sensitive and accurate quantitation of exogenous recombinant human Tf in rat CSF. By taking advantage of a His-tag present in recombinant Tf and applying Ni affinity purification, the exogenous human serum Tf can be greatly enriched from rat CSF, despite the presence of the abundant endogenous protein. Additionally, we applied a newly developed (18)O-labeling technique that can generate internal standards at the protein level, which greatly improved the accuracy and robustness of quantitation. The developed method was investigated for linearity, accuracy, precision, and lower limit of quantitation, all of which met the commonly accepted criteria for bioanalytical method validation.

[Parameter sensitivity of simulating net primary productivity of Larix olgensis forest based on BIOME-BGC model].

PubMed

He, Li-hong; Wang, Hai-yan; Lei, Xiang-dong

2016-02-01

Model based on vegetation ecophysiological process contains many parameters, and reasonable parameter values will greatly improve simulation ability. Sensitivity analysis, as an important method to screen out the sensitive parameters, can comprehensively analyze how model parameters affect the simulation results. In this paper, we conducted parameter sensitivity analysis of BIOME-BGC model with a case study of simulating net primary productivity (NPP) of Larix olgensis forest in Wangqing, Jilin Province. First, with the contrastive analysis between field measurement data and the simulation results, we tested the BIOME-BGC model' s capability of simulating the NPP of L. olgensis forest. Then, Morris and EFAST sensitivity methods were used to screen the sensitive parameters that had strong influence on NPP. On this basis, we also quantitatively estimated the sensitivity of the screened parameters, and calculated the global, the first-order and the second-order sensitivity indices. The results showed that the BIOME-BGC model could well simulate the NPP of L. olgensis forest in the sample plot. The Morris sensitivity method provided a reliable parameter sensitivity analysis result under the condition of a relatively small sample size. The EFAST sensitivity method could quantitatively measure the impact of simulation result of a single parameter as well as the interaction between the parameters in BIOME-BGC model. The influential sensitive parameters for L. olgensis forest NPP were new stem carbon to new leaf carbon allocation and leaf carbon to nitrogen ratio, the effect of their interaction was significantly greater than the other parameter' teraction effect.

Quantitative influence of risk factors on blood glucose level.

PubMed

Chen, Songjing; Luo, Senlin; Pan, Limin; Zhang, Tiemei; Han, Longfei; Zhao, Haixiu

2014-01-01

The aim of this study is to quantitatively analyze the influence of risk factors on the blood glucose level, and to provide theory basis for understanding the characteristics of blood glucose change and confirming the intervention index for type 2 diabetes. The quantitative method is proposed to analyze the influence of risk factors on blood glucose using back propagation (BP) neural network. Ten risk factors are screened first. Then the cohort is divided into nine groups by gender and age. According to the minimum error principle, nine BP models are trained respectively. The quantitative values of the influence of different risk factors on the blood glucose change can be obtained by sensitivity calculation. The experiment results indicate that weight is the leading cause of blood glucose change (0.2449). The second factors are cholesterol, age and triglyceride. The total ratio of these four factors reaches to 77% of the nine screened risk factors. And the sensitivity sequences can provide judgment method for individual intervention. This method can be applied to risk factors quantitative analysis of other diseases and potentially used for clinical practitioners to identify high risk populations for type 2 diabetes as well as other disease.

Orthogonal Comparison of GC-MS and 1H NMR Spectroscopy for Short Chain Fatty Acid Quantitation.

PubMed

Cai, Jingwei; Zhang, Jingtao; Tian, Yuan; Zhang, Limin; Hatzakis, Emmanuel; Krausz, Kristopher W; Smith, Philip B; Gonzalez, Frank J; Patterson, Andrew D

2017-08-01

Short chain fatty acids (SCFAs) are important regulators of host physiology and metabolism and may contribute to obesity and associated metabolic diseases. Interest in SCFAs has increased in part due to the recognized importance of how production of SCFAs by the microbiota may signal to the host. Therefore, reliable, reproducible, and affordable methods for SCFA profiling are required for accurate identification and quantitation. In the current study, four different methods for SCFA (acetic acid, propionic acid, and butyric acid) extraction and quantitation were compared using two independent platforms including gas chromatography coupled with mass spectrometry (GC-MS) and 1 H nuclear magnetic resonance (NMR) spectroscopy. Sensitivity, recovery, repeatability, matrix effect, and validation using mouse fecal samples were determined across all methods. The GC-MS propyl esterification method exhibited superior sensitivity for acetic acid and butyric acid measurement (LOD < 0.01 μg mL -1 , LOQ < 0.1 μg mL -1 ) and recovery accuracy (99.4%-108.3% recovery rate for 100 μg mL -1 SCFA mixed standard spike in and 97.8%-101.8% recovery rate for 250 μg mL -1 SCFAs mixed standard spike in). NMR methods by either quantitation relative to an internal standard or quantitation using a calibration curve yielded better repeatability and minimal matrix effects compared to GC-MS methods. All methods generated good calibration curve linearity (R 2 > 0.99) and comparable measurement of fecal SCFA concentration. Lastly, these methods were used to quantitate fecal SCFAs obtained from conventionally raised (CONV-R) and germ free (GF) mice. Results from global metabolomic analysis of feces generated by 1 H NMR and bomb calorimetry were used to further validate these approaches.

Analysis of Urinary Metabolites of Nerve and Blister Chemical Warfare Agents

DTIC Science & Technology

2014-08-01

of CWAs. The analysis methods use UHPLC-MS/MS in Multiple Reaction Monitoring ( MRM ) mode to enhance the selectivity and sensitivity of the method...Chromatography Mass Spectrometry LOD Limit Of Detection LOQ Limit of Quantitation MRM Multiple Reaction Monitoring MSMS Tandem mass...urine [1]. Those analysis methods use UHPLC- MS/MS in Multiple Reaction Monitoring ( MRM ) mode to enhance the selectivity and sensitivity of the method

Diffusion properties of conventional and calcium-sensitive MRI contrast agents in the rat cerebral cortex.

PubMed

Hagberg, Gisela E; Mamedov, Ilgar; Power, Anthony; Beyerlein, Michael; Merkle, Hellmut; Kiselev, Valerij G; Dhingra, Kirti; Kubìček, Vojtĕch; Angelovski, Goran; Logothetis, Nikos K

2014-01-01

Calcium-sensitive MRI contrast agents can only yield quantitative results if the agent concentration in the tissue is known. The agent concentration could be determined by diffusion modeling, if relevant parameters were available. We have established an MRI-based method capable of determining diffusion properties of conventional and calcium-sensitive agents. Simulations and experiments demonstrate that the method is applicable both for conventional contrast agents with a fixed relaxivity value and for calcium-sensitive contrast agents. The full pharmacokinetic time-course of gadolinium concentration estimates was observed by MRI before, during and after intracerebral administration of the agent, and the effective diffusion coefficient D* was determined by voxel-wise fitting of the solution to the diffusion equation. The method yielded whole brain coverage with a high spatial and temporal sampling. The use of two types of MRI sequences for sampling of the diffusion time courses was investigated: Look-Locker-based quantitative T(1) mapping, and T(1) -weighted MRI. The observation times of the proposed MRI method is long (up to 20 h) and consequently the diffusion distances covered are also long (2-4 mm). Despite this difference, the D* values in vivo were in agreement with previous findings using optical measurement techniques, based on observation times of a few minutes. The effective diffusion coefficient determined for the calcium-sensitive contrast agents may be used to determine local tissue concentrations and to design infusion protocols that maintain the agent concentration at a steady state, thereby enabling quantitative sensing of the local calcium concentration. Copyright © 2014 John Wiley & Sons, Ltd.

Determination of IgE antibodies to the benzylpenicilloyl determinant: a comparison of the sensitivity and specificity of three radio allergo sorbent test methods.

PubMed

Garcia, J J; Blanca, M; Moreno, F; Vega, J M; Mayorga, C; Fernandez, J; Juarez, C; Romano, A; de Ramon, E

1997-01-01

The quantitation of in vitro IgE antibodies to the benzylpenicilloyl determinant (BPO) is a useful tool for evaluating suspected penicillin allergic subjects. Although many different methods have been employed, few studies have compared their diagnostic specificity and sensitivity. In this study, the sensitivity and specificity of three different radio allergo sorbent test (RAST) methods for quantitating specific IgE antibodies to the BPO determinant were compared. Thirty positive control sera (serum samples from penicillin allergic subjects with a positive clinical history and a positive penicillin skin test) and 30 negative control sera (sera from subjects with no history of penicillin allergy and negative skin tests) were tested for BPO-specific IgE antibodies by RAST using three different conjugates coupled to the solid phase: benzylpenicillin conjugated to polylysine (BPO-PLL), benzylpenicillin conjugated to human serum albumin (BPO-HSA), and benzylpenicillin conjugated to an aminospacer (BPO-SP). Receiver operator control curves (ROC analysis) were carried out by determining different cut-off points between positive and negative values. Contingence tables were constructed and sensitivity, specificity, negative predictive values (PV-), and positive predictive values (PV+) were calculated. Pearson correlation coefficients (r) and intraclass correlation coefficients (ICC) were determined and the differences between methods were compared by chi 2 analysis. Analysis of the areas defined by the ROC curves showed statistical differences among the three methods. When cut-off points for optimal sensitivity and specificity were chosen, the BPO-HSA assay was less sensitive and less specific and had a lower PV- and PV+ than the BPO-PLL and BPO-SP assays. Assessment of r and ICC indicated that the correlation was very high, but the concordance between the PLL and SP methods was higher than between the PLL and HSA or SP and HSA methods. We conclude that for quantitating IgE antibodies by RAST to the BPO determinant, BPO-SP or BPO-PLL conjugates offer advantages in sensitivity and specificity compared with BPO-HSA. These results support and extend previous in vitro studies by our group and highlight the importance of the carrier for RAST assays.

The effects of physical activity on impulsive choice: Influence of sensitivity to reinforcement amount and delay

PubMed Central

Strickland, Justin C.; Feinstein, Max A.; Lacy, Ryan T.; Smith, Mark A.

2016-01-01

Impulsive choice is a diagnostic feature and/or complicating factor for several psychological disorders and may be examined in the laboratory using delay-discounting procedures. Recent investigators have proposed using quantitative measures of analysis to examine the behavioral processes contributing to impulsive choice. The purpose of this study was to examine the effects of physical activity (i.e., wheel running) on impulsive choice in a single-response, discrete-trial procedure using two quantitative methods of analysis. To this end, rats were assigned to physical activity or sedentary groups and trained to respond in a delay-discounting procedure. In this procedure, one lever always produced one food pellet immediately, whereas a second lever produced three food pellets after a 0, 10, 20, 40, or 80-second delay. Estimates of sensitivity to reinforcement amount and sensitivity to reinforcement delay were determined using (1) a simple linear analysis and (2) an analysis of logarithmically transformed response ratios. Both analyses revealed that physical activity decreased sensitivity to reinforcement amount and sensitivity to reinforcement delay. These findings indicate that (1) physical activity has significant but functionally opposing effects on the behavioral processes that contribute to impulsive choice and (2) both quantitative methods of analysis are appropriate for use in single-response, discrete-trial procedures. PMID:26964905

Designing novel nano-immunoassays: antibody orientation versus sensitivity

NASA Astrophysics Data System (ADS)

Puertas, S.; Moros, M.; Fernández-Pacheco, R.; Ibarra, M. R.; Grazú, V.; de la Fuente, J. M.

2010-12-01

There is a growing interest in the use of magnetic nanoparticles (MNPs) for their application in quantitative and highly sensitive biosensors. Their use as labels of biological recognition events and their detection by means of some magnetic method constitute a very promising strategy for quantitative high-sensitive lateral-flow assays. In this paper, we report the importance of nanoparticle functionalization for the improvement of sensitivity for a lateral-flow immunoassay. More precisely, we have found that immobilization of IgG anti-hCG through its polysaccharide moieties on MNPs allows more successful recognition of the hCG hormone. Although we have used the detection of hCG as a model in this work, the strategy of binding antibodies to MNPs through its sugar chains reported here is applicable to other antibodies. It has huge potential as it will be very useful for the development of quantitative and high-sensitive lateral-flow assays for its use on human and veterinary, medicine, food and beverage manufacturing, pharmaceutical, medical biologics and personal care product production, environmental remediation, etc.

Protocol for the use of light upon extension real-time PCR for the determination of viral load in HBV infection.

PubMed

Li, Guimin; Li, Wangfeng; Liu, Lixia

2012-01-01

Real-time PCR has engendered wide acceptance for quantitation of hepatitis B virus (HBV) DNA in the blood due to its improved rapidity, sensitivity, reproducibility, and reduced contamination. Here we describe a cost-effective and highly sensitive HBV real-time quantitative assay based on the light upon extension real-time PCR platform and a simple and reliable HBV DNA preparation method using silica-coated magnetic beads.

Diagnostic accuracy of stress perfusion CMR in comparison with quantitative coronary angiography: fully quantitative, semiquantitative, and qualitative assessment.

PubMed

Mordini, Federico E; Haddad, Tariq; Hsu, Li-Yueh; Kellman, Peter; Lowrey, Tracy B; Aletras, Anthony H; Bandettini, W Patricia; Arai, Andrew E

2014-01-01

This study's primary objective was to determine the sensitivity, specificity, and accuracy of fully quantitative stress perfusion cardiac magnetic resonance (CMR) versus a reference standard of quantitative coronary angiography. We hypothesized that fully quantitative analysis of stress perfusion CMR would have high diagnostic accuracy for identifying significant coronary artery stenosis and exceed the accuracy of semiquantitative measures of perfusion and qualitative interpretation. Relatively few studies apply fully quantitative CMR perfusion measures to patients with coronary disease and comparisons to semiquantitative and qualitative methods are limited. Dual bolus dipyridamole stress perfusion CMR exams were performed in 67 patients with clinical indications for assessment of myocardial ischemia. Stress perfusion images alone were analyzed with a fully quantitative perfusion (QP) method and 3 semiquantitative methods including contrast enhancement ratio, upslope index, and upslope integral. Comprehensive exams (cine imaging, stress/rest perfusion, late gadolinium enhancement) were analyzed qualitatively with 2 methods including the Duke algorithm and standard clinical interpretation. A 70% or greater stenosis by quantitative coronary angiography was considered abnormal. The optimum diagnostic threshold for QP determined by receiver-operating characteristic curve occurred when endocardial flow decreased to <50% of mean epicardial flow, which yielded a sensitivity of 87% and specificity of 93%. The area under the curve for QP was 92%, which was superior to semiquantitative methods: contrast enhancement ratio: 78%; upslope index: 82%; and upslope integral: 75% (p = 0.011, p = 0.019, p = 0.004 vs. QP, respectively). Area under the curve for QP was also superior to qualitative methods: Duke algorithm: 70%; and clinical interpretation: 78% (p < 0.001 and p < 0.001 vs. QP, respectively). Fully quantitative stress perfusion CMR has high diagnostic accuracy for detecting obstructive coronary artery disease. QP outperforms semiquantitative measures of perfusion and qualitative methods that incorporate a combination of cine, perfusion, and late gadolinium enhancement imaging. These findings suggest a potential clinical role for quantitative stress perfusion CMR. Copyright © 2014 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

A novel quantitative analysis method of three-dimensional fluorescence spectra for vegetable oils contents in edible blend oil

NASA Astrophysics Data System (ADS)

Xu, Jing; Wang, Yu-Tian; Liu, Xiao-Fei

2015-04-01

Edible blend oil is a mixture of vegetable oils. Eligible blend oil can meet the daily need of two essential fatty acids for human to achieve the balanced nutrition. Each vegetable oil has its different composition, so vegetable oils contents in edible blend oil determine nutritional components in blend oil. A high-precision quantitative analysis method to detect the vegetable oils contents in blend oil is necessary to ensure balanced nutrition for human being. Three-dimensional fluorescence technique is high selectivity, high sensitivity, and high-efficiency. Efficiency extraction and full use of information in tree-dimensional fluorescence spectra will improve the accuracy of the measurement. A novel quantitative analysis is proposed based on Quasi-Monte-Carlo integral to improve the measurement sensitivity and reduce the random error. Partial least squares method is used to solve nonlinear equations to avoid the effect of multicollinearity. The recovery rates of blend oil mixed by peanut oil, soybean oil and sunflower are calculated to verify the accuracy of the method, which are increased, compared the linear method used commonly for component concentration measurement.

Time-Resolved Fluorescent Immunochromatography of Aflatoxin B1 in Soybean Sauce: A Rapid and Sensitive Quantitative Analysis.

PubMed

Wang, Du; Zhang, Zhaowei; Li, Peiwu; Zhang, Qi; Zhang, Wen

2016-07-14

Rapid and quantitative sensing of aflatoxin B1 with high sensitivity and specificity has drawn increased attention of studies investigating soybean sauce. A sensitive and rapid quantitative immunochromatographic sensing method was developed for the detection of aflatoxin B1 based on time-resolved fluorescence. It combines the advantages of time-resolved fluorescent sensing and immunochromatography. The dynamic range of a competitive and portable immunoassay was 0.3-10.0 µg·kg(-1), with a limit of detection (LOD) of 0.1 µg·kg(-1) and recoveries of 87.2%-114.3%, within 10 min. The results showed good correlation (R² > 0.99) between time-resolved fluorescent immunochromatographic strip test and high performance liquid chromatography (HPLC). Soybean sauce samples analyzed using time-resolved fluorescent immunochromatographic strip test revealed that 64.2% of samples contained aflatoxin B1 at levels ranging from 0.31 to 12.5 µg·kg(-1). The strip test is a rapid, sensitive, quantitative, and cost-effective on-site screening technique in food safety analysis.

Sensitive method for the quantitation of droloxifene in plasma and serum by high-performance liquid chromatography employing fluorimetric detection.

PubMed

Tess, D A; Cole, R O; Toler, S M

1995-12-15

A simple and highly sensitive reversed-phase fluorimetric HPLC method for the quantitation of droloxifene from rat, monkey, and human plasma as well as human serum is described. This assay employs solid-phase extraction and has a dynamic range of 25 to 10,000 pg/ml. Sample extraction (efficiencies > 86%) was accomplished using a benzenesulfonic acid (SCX) column with water and methanol rinses. Droloxifene and internal standard were eluted with 1 ml of 3.5% (v/v) ammonium hydroxide (30%) in methanol. Samples were quantitated using post-column UV-photochemical cyclization coupled with fluorimetric detection with excitation and emission wavelengths of 260 nm and 375 nm, respectively. Relative ease of sample extraction and short run times allow for the analysis of approximately 100 samples per day.

Quantitative analysis of ginger components in commercial products using liquid chromatography with electrochemical array detection

PubMed Central

Shao, Xi; Lv, Lishuang; Parks, Tiffany; Wu, Hou; Ho, Chi-Tang; Sang, Shengmin

2010-01-01

For the first time, a sensitive reversed-phase HPLC electrochemical array method has been developed for the quantitative analysis of eight major ginger components ([6]-, [8]-, and [10]-gingerol, [6]-, [8]-, and [10]-shogaol, [6]-paradol, and [1]-dehydrogingerdione) in eleven ginger-containing commercial products. This method was valid with unrivaled sensitivity as low as 7.3 – 20.2 pg of limit of detection and a range of 14.5 to 40.4 pg of limit of quantification. Using this method, we quantified the levels of eight ginger components in eleven different commercial products. Our results found that both levels and ratios among the eight compounds vary greatly in commercial products. PMID:21090746

PALATAL DYSMORPHOGENESIS: QUANTITATIVE RT-PCR

EPA Science Inventory

ABSTRACT

Palatal Dysmorphogenesis : Quantitative RT-PCR

Gary A. Held and Barbara D. Abbott

Reverse transcription PCR (RT-PCR) is a very sensitive method for detecting mRNA in tissue samples. However, as it is usually performed it is does not yield quantitativ...

Low angle light scattering analysis: a novel quantitative method for functional characterization of human and murine platelet receptors.

PubMed

Mindukshev, Igor; Gambaryan, Stepan; Kehrer, Linda; Schuetz, Claudia; Kobsar, Anna; Rukoyatkina, Natalia; Nikolaev, Viacheslav O; Krivchenko, Alexander; Watson, Steve P; Walter, Ulrich; Geiger, Joerg

2012-07-01

Determinations of platelet receptor functions are indispensable diagnostic indicators of cardiovascular and hemostatic diseases including hereditary and acquired receptor defects and receptor responses to drugs. However, presently available techniques for assessing platelet function have some disadvantages, such as low sensitivity and the requirement of large sample sizes and unphysiologically high agonist concentrations. Our goal was to develop and initially characterize a new technique designed to quantitatively analyze platelet receptor activation and platelet function on the basis of measuring changes in low angle light scattering. We developed a novel technique based on low angle light scattering registering changes in light scattering at a range of different angles in platelet suspensions during activation. The method proved to be highly sensitive for simultaneous real time detection of changes in size and shape of platelets during activation. Unlike commonly-used methods, the light scattering method could detect platelet shape change and aggregation in response to nanomolar concentrations of extracellular nucleotides. Furthermore, our results demonstrate that the advantages of the light scattering method make it a choice method for platelet receptor monitoring and for investigation of both murine and human platelets in disease models. Our data demonstrate the suitability and superiority of this new low angle light scattering method for comprehensive analyses of platelet receptors and functions. This highly sensitive, quantitative, and online detection of essential physiological, pathophysiological and pharmacological-response properties of human and mouse platelets is a significant improvement over conventional techniques.

High Throughput Protein Quantitation using MRM Viewer Software and Dynamic MRM on a Triple Quadruple Mass Spectrometer

PubMed Central

Miller, C.; Waddell, K.; Tang, N.

2010-01-01

RP-122 Peptide quantitation using Multiple Reaction Monitoring (MRM) has been established as an important methodology for biomarker verification andvalidation.This requires high throughput combined with high sensitivity to analyze potentially thousands of target peptides in each sample.Dynamic MRM allows the system to only acquire the required MRMs of the peptide during a retention window corresponding to when each peptide is eluting. This reduces the number of concurrent MRM and therefore improves quantitation and sensitivity. MRM Selector allows the user to generate an MRM transition list with retention time information from discovery data obtained on a QTOF MS system.This list can be directly imported into the triple quadrupole acquisition software.However, situations can exist where a) the list of MRMs contain an excess of MRM transitions allowable under the ideal acquisition conditions chosen ( allowing for cycle time and chromatography conditions), or b) too many transitions in a certain retention time region which would result in an unacceptably low dwell time and cycle time.A new tool - MRM viewer has been developed to help users automatically generate multiple dynamic MRM methods from a single MRM list.In this study, a list of 3293 MRM transitions from a human plasma sample was compiled.A single dynamic MRM method with 3293 transitions results in a minimum dwell time of 2.18ms.Using MRM viewer we can generate three dynamic MRM methods with a minimum dwell time of 20ms which can give a better quality MRM quantitation.This tool facilitates both high throughput and high sensitivity for MRM quantitation.

Quantitative analysis of Si1-xGex alloy films by SIMS and XPS depth profiling using a reference material

NASA Astrophysics Data System (ADS)

Oh, Won Jin; Jang, Jong Shik; Lee, Youn Seoung; Kim, Ansoon; Kim, Kyung Joong

2018-02-01

Quantitative analysis methods of multi-element alloy films were compared. The atomic fractions of Si1-xGex alloy films were measured by depth profiling analysis with secondary ion mass spectrometry (SIMS) and X-ray Photoelectron Spectroscopy (XPS). Intensity-to-composition conversion factor (ICF) was used as a mean to convert the intensities to compositions instead of the relative sensitivity factors. The ICFs were determined from a reference Si1-xGex alloy film by the conventional method, average intensity (AI) method and total number counting (TNC) method. In the case of SIMS, although the atomic fractions measured by oxygen ion beams were not quantitative due to severe matrix effect, the results by cesium ion beam were very quantitative. The quantitative analysis results by SIMS using MCs2+ ions are comparable to the results by XPS. In the case of XPS, the measurement uncertainty was highly improved by the AI method and TNC method.

Rapid and sensitive liquid chromatography-tandem mass spectrometric method for the quantitative determination of potentially harmful substance 5,5'-oxydimethylenebis (2-furfural) in traditional Chinese medicine injections.

PubMed

Zang, Qingce; Gao, Yang; Huang, Luojiao; He, Jiuming; Lin, Sheng; Jin, Hongtao; Zhang, Ruiping; Abliz, Zeper

2018-03-01

With the rapid development and wide application of traditional Chinese medicine injection (TCMI), a number of adverse events of some TCMIs have incessantly been reported and have drawn broad attention in recent years. Establishing effective and practical analytical methods for safety evaluation and quality control of TCMI can help to improve the safety of TCMIs in clinical applications. In this study, a sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed and validated for the quantitative determination of potentially harmful substance 5,5'-oxydimethylenebis (2-furfural, OMBF) in TCMI samples. Chromatographic separation was performed on a C18 reversed-phase column (150 mm × 2.1 mm, 5 µm) by gradient elution, using methanol-water containing 0.1% formic acid as mobile phase at the flow rate of 0.3 mL/min. MS/MS detection was performed on a triple quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction-monitoring mode. The method was sensitive with a limit of quantification of 0.3 ng/mL and linear over the range of 0.3-30 ng/mL ( r =0.9998). Intra- and inter-day precision for analyte was <9.52% RSD with recoveries in the range 88.0-109.67% at three concentration levels. The validated method was successfully applied to quantitatively determine the compound OMBF in TCMIs and glucose injections. Our study indicates that this method is simple, sensitive, practicable and reliable, and could be applied for safety evaluation and quality control of TCMIs and glucose injections.

Quantitation of peptides from non-invasive skin tapings using isotope dilution and tandem mass spectrometry.

PubMed

Reisdorph, Nichole; Armstrong, Michael; Powell, Roger; Quinn, Kevin; Legg, Kevin; Leung, Donald; Reisdorph, Rick

2018-05-01

Previous work from our laboratories utilized a novel skin taping method and mass spectrometry-based proteomics to discover clinical biomarkers of skin conditions; these included atopic dermatitis, Staphylococcus aureus colonization, and eczema herpeticum. While suitable for discovery purposes, semi-quantitative proteomics is generally time-consuming and expensive. Furthermore, depending on the method used, discovery-based proteomics can result in high variation and inadequate sensitivity to detect low abundant peptides. Therefore, we strove to develop a rapid, sensitive, and reproducible method to quantitate disease-related proteins from skin tapings. We utilized isotopically-labeled peptides and tandem mass spectrometry to obtain absolute quantitation values on 14 peptides from 7 proteins; these proteins had shown previous importance in skin disease. The method demonstrated good reproducibility, dynamic range, and linearity (R 2  > 0.993) when n = 3 standards were analyzed across 0.05-2.5 pmol. The method was used to determine if differences exist between skin proteins in a small group of atopic versus non-atopic individuals (n = 12). While only minimal differences were found, peptides were detected in all samples and exhibited good correlation between peptides for 5 of the 7 proteins (R 2  = 0.71-0.98). This method can be applied to larger cohorts to further establish the relationships of these proteins to skin disease. Copyright © 2017. Published by Elsevier B.V.

Fast real-time polymerase chain reaction for quantitative detection of Lactobacillus delbrueckii bacteriophages in milk.

PubMed

Martín, Maria Cruz; del Rio, Beatriz; Martínez, Noelia; Magadán, Alfonso H; Alvarez, Miguel A

2008-12-01

One of the main microbiological problems of the dairy industry is the susceptibility of starter bacteria to virus infections. Lactobacillus delbrueckii, a component of thermophilic starter cultures used in the manufacture of several fermented dairy products, including yogurt, is also sensitive to bacteriophage attacks. To avoid the problems associated with these viruses, quick and sensitive detection methods are necessary. In the present study, a fast real-time quantitative polymerase chain reaction assay for the direct detection and quantification of L. delbrueckii phages in milk was developed. A set of primers and a TaqMan MGB probe was designed, based on the lysin gene sequence of different L. delbrueckii phages. The results show the proposed method to be a rapid (total processing time 30 min), specific and highly sensitive technique for detecting L. delbrueckii phages in milk.

Overcoming pain thresholds with multilevel models-an example using quantitative sensory testing (QST) data.

PubMed

Hirschfeld, Gerrit; Blankenburg, Markus R; Süß, Moritz; Zernikow, Boris

2015-01-01

The assessment of somatosensory function is a cornerstone of research and clinical practice in neurology. Recent initiatives have developed novel protocols for quantitative sensory testing (QST). Application of these methods led to intriguing findings, such as the presence lower pain-thresholds in healthy children compared to healthy adolescents. In this article, we (re-) introduce the basic concepts of signal detection theory (SDT) as a method to investigate such differences in somatosensory function in detail. SDT describes participants' responses according to two parameters, sensitivity and response-bias. Sensitivity refers to individuals' ability to discriminate between painful and non-painful stimulations. Response-bias refers to individuals' criterion for giving a "painful" response. We describe how multilevel models can be used to estimate these parameters and to overcome central critiques of these methods. To provide an example we apply these methods to data from the mechanical pain sensitivity test of the QST protocol. The results show that adolescents are more sensitive to mechanical pain and contradict the idea that younger children simply use more lenient criteria to report pain. Overall, we hope that the wider use of multilevel modeling to describe somatosensory functioning may advance neurology research and practice.

Measurement of bone marrow lesions by MR imaging in knee osteoarthritis using quantitative segmentation methods--a reliability and sensitivity to change analysis.

PubMed

Nielsen, Flemming K; Egund, Niels; Peters, David; Jurik, Anne Grethe

2014-12-20

Longitudinal assessment of bone marrow lesions (BMLs) in knee osteoarthritis (KOA) by MRI is usually performed using semi-quantitative grading methods. Quantitative segmentation methods may be more sensitive to detect change over time. The purpose of this study was to evaluate and compare the validity and sensitivity to detect changes of two quantitative MR segmentation methods for measuring BMLs in KOA, one computer assisted (CAS) and one manual (MS) method. Twenty-two patients with KOA confined to the medial femoro-tibial compartment obtained MRI at baseline and follow-up (median 334 days in between). STIR, T1 and fat saturated T1 post-contrast sequences were obtained using a 1.5 T system. The 44 sagittal STIR sequences were assessed independently by two readers for quantification of BML. The signal intensities (SIs) of the normal bone marrow in the lateral femoral condyles and tibial plateaus were used as threshold values. The volume of bone marrow with SIs exceeding the threshold values (BML) was measured in the medial femoral condyle and tibial plateau and related to the total volume of the condyles/plateaus.The 95% limits of agreement at baseline were used to determine the sensitivity to change. The mean threshold values of CAS and MS were almost identical but the absolute and relative BML volumes differed being 1319 mm3/10% and 1828 mm3/15% in the femur and 941 mm3/7% and 2097 mm3/18% in the tibia using CAS and MS, respectively. The BML volumes obtained by CAS and MS were significantly correlated but the tissue changes measured were different. The volume of voxels exceeding the threshold values was measured by CAS whereas MS included intervening voxels with normal SI.The 95% limits of agreement were narrower by CAS than by MS; a significant change of relative BML by CAS was outside the limits of -2.0%-4.7% whereas the limits by MS were -6.9%-8.2%. The BML changed significantly in 13 knees using CAS and in 10 knees by MS. CAS was a reliable method for measuring BML and more sensitive to detect changes over time than MS. The BML volumes measured by the two methods differed but were significantly correlated.

Photogrammetry Applied to Wind Tunnel Testing

NASA Technical Reports Server (NTRS)

Liu, Tian-Shu; Cattafesta, L. N., III; Radeztsky, R. H.; Burner, A. W.

2000-01-01

In image-based measurements, quantitative image data must be mapped to three-dimensional object space. Analytical photogrammetric methods, which may be used to accomplish this task, are discussed from the viewpoint of experimental fluid dynamicists. The Direct Linear Transformation (DLT) for camera calibration, used in pressure sensitive paint, is summarized. An optimization method for camera calibration is developed that can be used to determine the camera calibration parameters, including those describing lens distortion, from a single image. Combined with the DLT method, this method allows a rapid and comprehensive in-situ camera calibration and therefore is particularly useful for quantitative flow visualization and other measurements such as model attitude and deformation in production wind tunnels. The paper also includes a brief description of typical photogrammetric applications to temperature- and pressure-sensitive paint measurements and model deformation measurements in wind tunnels.

Simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues using LC-ESI-MS/MS: An application for pharmacokinetic studies.

PubMed

Chhonker, Yashpal S; Sleightholm, Richard L; Li, Jing; Oupický, David; Murry, Daryl J

2018-01-01

Hydroxychloroquine (HCQ) has been shown to disrupt autophagy and sensitize cancer cells to radiation and chemotherapeutic agents. However, the optimal delivery method, dose, and tumor concentrations required for these effects are not known. This is in part due to a lack of sensitive and reproducible analytical methods for HCQ quantitation in small animals. As such, we developed and validated a selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues. The chromatographic separation and detection of analytes were achieved on a reversed phase Thermo Aquasil C 18 (50×4.6mm, 3μ) column, with gradient elution using 0.2% formic acid and 0.1% formic acid in methanol as mobile phase at a flow rate of 0.5mL/min. Simple protein precipitation was utilized for extraction of analytes from the desired matrix. Analytes were separated and quantitated using MS/MS with an electrospray ionization source in positive multiple reaction monitoring (MRM) mode. The MS/MS response was linear over the concentration range from 1 to 2000ng/mL for all analytes with a correlation coefficient (R 2 ) of 0.998 or better. The within- and between-day precision (relative standard deviation, % RSD) and accuracy were within the acceptable limits per FDA guidelines. The validated method was successfully applied to a preclinical pharmacokinetic mouse study involving low volume blood and tissue samples for hydroxychloroquine and metabolites. Copyright © 2017 Elsevier B.V. All rights reserved.

Qualitative and quantitative analysis of the diuretic component ergone in Polyporus umbellatus by HPLC with fluorescence detection and HPLC-APCI-MS/MS.

PubMed

Zhao, Ying-Yong; Zhao, Ye; Zhang, Yong-Min; Lin, Rui-Chao; Sun, Wen-Ji

2009-06-01

Polyporus umbellatus is a widely used anti-aldosteronic diuretic in Traditional Chinese medicine (TCM). A new, sensitive and selective high-performance liquid chromatography-fluorescence detector (HPLC-FLD) and high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS/MS) method for quantitative and qualitative determination of ergosta-4,6,8(14),22-tetraen-3-one(ergone), which is the main diuretic component, was provided for quality control of P. umbellatus crude drug. The ergone in the ethanolic extract of P. umbellatus was unambiguously characterized by HPLC-APCI, and further confirmed by comparing with a standard compound. The trace ergone was detected by the sensitive and selective HPLC-FLD. Linearity (r2 > 0.9998) and recoveries of low, medium and high concentration (100.5%, 100.2% and 100.4%) were consistent with the experimental criteria. The limit of detection (LOD) of ergone was around 0.2 microg/mL. Our results indicated that the content of ergone in P. umbellatus varied significantly from habitat to habitat with contents ranging from 2.13 +/- 0.02 to 59.17 +/- 0.05 microg/g. Comparison among HPLC-FLD and HPLC-UV or HPLC-APCI-MS/MS demonstrated that the HPLC-FLD and HPLC-APCI-MS/MS methods gave similar quantitative results for the selected herb samples, the HPLC-UV methods gave lower quantitative results than HPLC-FLD and HPLC-APCI-MS/MS methods. The established new HPLC-FLD method has the advantages of being rapid, simple, selective and sensitive, and could be used for the routine analysis of P. umbellatus crude drug.

The effects of physical activity on impulsive choice: Influence of sensitivity to reinforcement amount and delay.

PubMed

Strickland, Justin C; Feinstein, Max A; Lacy, Ryan T; Smith, Mark A

2016-05-01

Impulsive choice is a diagnostic feature and/or complicating factor for several psychological disorders and may be examined in the laboratory using delay-discounting procedures. Recent investigators have proposed using quantitative measures of analysis to examine the behavioral processes contributing to impulsive choice. The purpose of this study was to examine the effects of physical activity (i.e., wheel running) on impulsive choice in a single-response, discrete-trial procedure using two quantitative methods of analysis. To this end, rats were assigned to physical activity or sedentary groups and trained to respond in a delay-discounting procedure. In this procedure, one lever always produced one food pellet immediately, whereas a second lever produced three food pellets after a 0, 10, 20, 40, or 80-s delay. Estimates of sensitivity to reinforcement amount and sensitivity to reinforcement delay were determined using (1) a simple linear analysis and (2) an analysis of logarithmically transformed response ratios. Both analyses revealed that physical activity decreased sensitivity to reinforcement amount and sensitivity to reinforcement delay. These findings indicate that (1) physical activity has significant but functionally opposing effects on the behavioral processes that contribute to impulsive choice and (2) both quantitative methods of analysis are appropriate for use in single-response, discrete-trial procedures. Copyright © 2016 Elsevier B.V. All rights reserved.

Enhanced sensitivity and multiplexing with 2D LC/MRM-MS and labeled standards for deeper and more comprehensive protein quantitation.

PubMed

Percy, Andrew J; Simon, Romain; Chambers, Andrew G; Borchers, Christoph H

2014-06-25

Mass spectrometry (MS)-based protein quantitation is increasingly being employed to verify candidate protein biomarkers. Multiple or selected reaction monitoring-mass spectrometry (MRM-MS or SRM-MS) with isotopically labeled internal standards has proven to be a successful approach in that regard, but has yet to reach its full potential in terms of multiplexing and sensitivity. Here, we report the development of a new MRM method for the quantitation of 253 disease-associated proteins (represented by 625 interference-free peptides) in 13 LC fractions. This 2D RPLC/MRM-MS approach extends the depth and breadth of the assay by 2 orders of magnitude over pre-fractionation-free assays, with 31 proteins below 10 ng/mL and 41 proteins above 10 ng/mL now quantifiable. Standard flow rates are used in both chromatographic dimensions, and up-front depletion or antibody-based enrichment is not required. The LC separations utilize high and low pH conditions, with the former employing an ammonium hydroxide-based eluent, instead of the conventional ammonium formate, resulting in improved LC column lifetime and performance. The high sensitivity (determined concentration range: 15 mg/mL to 452 pg/mL) and robustness afforded by this method makes the full MRM panel, or subsets thereof, useful for the verification of disease-associated plasma protein biomarkers in patient samples. The described research extends the breadth and depth of protein quantitation in undepleted and non-enriched human plasma by employing standard-flow 2D RPLC/MRM-MS in conjunction with a complex mixture of isotopically labeled peptide standards. The proteins quantified are mainly putative biomarkers of non-communicable (i.e., non-infectious) disease (e.g., cardiovascular or cancer), which require pre-clinical verification and validation before clinical implementation. Based on the enhanced sensitivity and multiplexing, this quantitative plasma proteomic method should prove useful in future candidate biomarker verification studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Fluorescence-labeled methylation-sensitive amplified fragment length polymorphism (FL-MS-AFLP) analysis for quantitative determination of DNA methylation and demethylation status.

PubMed

Kageyama, Shinji; Shinmura, Kazuya; Yamamoto, Hiroko; Goto, Masanori; Suzuki, Koichi; Tanioka, Fumihiko; Tsuneyoshi, Toshihiro; Sugimura, Haruhiko

2008-04-01

The PCR-based DNA fingerprinting method called the methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis is used for genome-wide scanning of methylation status. In this study, we developed a method of fluorescence-labeled MS-AFLP (FL-MS-AFLP) analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing method of MS-AFLP analysis. The FL-MS-AFLP analysis enables quantitative evaluation of more than 350 random CpG loci per run. It was shown to allow evaluation of the differences in methylation level of blood DNA of gastric cancer patients and evaluation of hypermethylation and hypomethylation in DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

Characterization and Comparison of Galactomannan Enzyme Immunoassay and Quantitative Real-Time PCR Assay for Detection of Aspergillus fumigatus in Bronchoalveolar Lavage Fluid from Experimental Invasive Pulmonary Aspergillosis

PubMed Central

Francesconi, Andrea; Kasai, Miki; Petraitiene, Ruta; Petraitis, Vidmantas; Kelaher, Amy M.; Schaufele, Robert; Hope, William W.; Shea, Yvonne R.; Bacher, John; Walsh, Thomas J.

2006-01-01

Bronchoalveolar lavage (BAL) is widely used for evaluation of patients with suspected invasive pulmonary aspergillosis (IPA). However, the diagnostic yield of BAL for detection of IPA by culture and direct examination is limited. Earlier diagnosis may be facilitated by assays that can detect Aspergillus galactomannan antigen or DNA in BAL fluid. We therefore characterized and compared the diagnostic yields of a galactomannan enzyme immunoassay (GM EIA), quantitative real-time PCR (qPCR), and quantitative cultures in experiments using BAL fluid from neutropenic rabbits with experimentally induced IPA defined as microbiologically and histologically evident invasion. The qPCR assay targeted the rRNA gene complex of Aspergillus fumigatus. The GM EIA and qPCR assay were characterized by receiver operator curve analysis. With an optimal cutoff of 0.75, the GM EIA had a sensitivity and specificity of 100% in untreated controls. A decline in sensitivity (92%) was observed when antifungal therapy (AFT) was administered. The optimal cutoff for qPCR was a crossover of 36 cycles, with sensitivity and specificity of 80% and 100%, respectively. The sensitivity of qPCR also decreased with AFT to 50%. Quantitative culture of BAL had a sensitivity of 46% and a specificity of 100%. The sensitivity of quantitative culture decreased with AFT to 16%. The GM EIA and qPCR assay had greater sensitivity than culture in detection of A. fumigatus in BAL fluid in experimentally induced IPA (P ± 0.04). Use of the GM EIA and qPCR assay in conjunction with culture-based diagnostic methods applied to BAL fluid could facilitate accurate diagnosis and more-timely initiation of specific therapy. PMID:16825367

Detection of medically important Candida species by absolute quantitation real-time polymerase chain reaction.

PubMed

Than, Leslie Thian Lung; Chong, Pei Pei; Ng, Kee Peng; Seow, Heng Fong

2015-01-01

The number of invasive candidiasis cases has risen especially with an increase in the number of immunosuppressed and immunocom promised patients. The early detection of Candida species which is specific and sensitive is important in determining the correct administration of antifungal drugs to patients. This study aims to develop a method for the detection, identification and quantitation of medically important Candida species through quantitative polymerase chain reaction (qPCR). The isocitrate lyase (ICL) gene which is not found in mammals was chosen as the target gene of real-time PCR. Absolute quantitation of the gene copy number was achieved by constructing the plasmid containing the ICL gene which is used to generate standard curve. Twenty fungal species, two bacterial species and human DNA were tested to check the specificity of the detection method. All eight Candida species were successfully detected, identified and quantitated based on the ICL gene. A seven-log range of the gene copy number and a minimum detection limit of 10(3) copies were achieved. A one-tube absolute quantification real-time PCR that differentiates medically important Candida species via individual unique melting temperature was achieved. Analytical sensitivity and specificity were not compromised.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs.

PubMed

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Zel, Jana; Gruden, Kristina

2008-10-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1-25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

PubMed Central

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Žel, Jana; Gruden, Kristina

2008-01-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification. PMID:18710880

Ultrasound hepatic/renal ratio and hepatic attenuation rate for quantifying liver fat content.

PubMed

Zhang, Bo; Ding, Fang; Chen, Tian; Xia, Liang-Hua; Qian, Juan; Lv, Guo-Yi

2014-12-21

To establish and validate a simple quantitative assessment method for nonalcoholic fatty liver disease (NAFLD) based on a combination of the ultrasound hepatic/renal ratio and hepatic attenuation rate. A total of 170 subjects were enrolled in this study. All subjects were examined by ultrasound and (1)H-magnetic resonance spectroscopy ((1)H-MRS) on the same day. The ultrasound hepatic/renal echo-intensity ratio and ultrasound hepatic echo-intensity attenuation rate were obtained from ordinary ultrasound images using the MATLAB program. Correlation analysis revealed that the ultrasound hepatic/renal ratio and hepatic echo-intensity attenuation rate were significantly correlated with (1)H-MRS liver fat content (ultrasound hepatic/renal ratio: r = 0.952, P = 0.000; hepatic echo-intensity attenuation r = 0.850, P = 0.000). The equation for predicting liver fat content by ultrasound (quantitative ultrasound model) is: liver fat content (%) = 61.519 × ultrasound hepatic/renal ratio + 167.701 × hepatic echo-intensity attenuation rate -26.736. Spearman correlation analysis revealed that the liver fat content ratio of the quantitative ultrasound model was positively correlated with serum alanine aminotransferase, aspartate aminotransferase, and triglyceride, but negatively correlated with high density lipoprotein cholesterol. Receiver operating characteristic curve analysis revealed that the optimal point for diagnosing fatty liver was 9.15% in the quantitative ultrasound model. Furthermore, in the quantitative ultrasound model, fatty liver diagnostic sensitivity and specificity were 94.7% and 100.0%, respectively, showing that the quantitative ultrasound model was better than conventional ultrasound methods or the combined ultrasound hepatic/renal ratio and hepatic echo-intensity attenuation rate. If the (1)H-MRS liver fat content had a value < 15%, the sensitivity and specificity of the ultrasound quantitative model would be 81.4% and 100%, which still shows that using the model is better than the other methods. The quantitative ultrasound model is a simple, low-cost, and sensitive tool that can accurately assess hepatic fat content in clinical practice. It provides an easy and effective parameter for the early diagnosis of mild hepatic steatosis and evaluation of the efficacy of NAFLD treatment.

Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

EPA Science Inventory

Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

Are quantitative sensitivity analysis methods always reliable?

NASA Astrophysics Data System (ADS)

Huang, X.

2016-12-01

Physical parameterizations developed to represent subgrid-scale physical processes include various uncertain parameters, leading to large uncertainties in today's Earth System Models (ESMs). Sensitivity Analysis (SA) is an efficient approach to quantitatively determine how the uncertainty of the evaluation metric can be apportioned to each parameter. Also, SA can identify the most influential parameters, as a result to reduce the high dimensional parametric space. In previous studies, some SA-based approaches, such as Sobol' and Fourier amplitude sensitivity testing (FAST), divide the parameters into sensitive and insensitive groups respectively. The first one is reserved but the other is eliminated for certain scientific study. However, these approaches ignore the disappearance of the interactive effects between the reserved parameters and the eliminated ones, which are also part of the total sensitive indices. Therefore, the wrong sensitive parameters might be identified by these traditional SA approaches and tools. In this study, we propose a dynamic global sensitivity analysis method (DGSAM), which iteratively removes the least important parameter until there are only two parameters left. We use the CLM-CASA, a global terrestrial model, as an example to verify our findings with different sample sizes ranging from 7000 to 280000. The result shows DGSAM has abilities to identify more influential parameters, which is confirmed by parameter calibration experiments using four popular optimization methods. For example, optimization using Top3 parameters filtered by DGSAM could achieve substantial improvement against Sobol' by 10%. Furthermore, the current computational cost for calibration has been reduced to 1/6 of the original one. In future, it is necessary to explore alternative SA methods emphasizing parameter interactions.

A quantitative method for evaluating alternatives. [aid to decision making

NASA Technical Reports Server (NTRS)

Forthofer, M. J.

1981-01-01

When faced with choosing between alternatives, people tend to use a number of criteria (often subjective, rather than objective) to decide which is the best alternative for them given their unique situation. The subjectivity inherent in the decision-making process can be reduced by the definition and use of a quantitative method for evaluating alternatives. This type of method can help decision makers achieve degree of uniformity and completeness in the evaluation process, as well as an increased sensitivity to the factors involved. Additional side-effects are better documentation and visibility of the rationale behind the resulting decisions. General guidelines for defining a quantitative method are presented and a particular method (called 'hierarchical weighted average') is defined and applied to the evaluation of design alternatives for a hypothetical computer system capability.

The FAQUIRE Approach: FAst, QUantitative, hIghly Resolved and sEnsitivity Enhanced 1H, 13C Data.

PubMed

Farjon, Jonathan; Milande, Clément; Martineau, Estelle; Akoka, Serge; Giraudeau, Patrick

2018-02-06

The targeted analysis of metabolites in complex mixtures is a challenging issue. NMR is one of the major tools in this field, but there is a strong need for more sensitive, better-resolved, and faster quantitative methods. In this framework, we introduce the concept of FAst, QUantitative, hIghly Resolved and sEnsitivity enhanced (FAQUIRE) NMR to push forward the limits of metabolite NMR analysis. 2D 1 H, 13 C 2D quantitative maps are promising alternatives for enhancing the spectral resolution but are highly time-consuming because of (i) the intrinsic nature of 2D, (ii) the longer recycling times required for quantitative conditions, and (iii) the higher number of scans needed to reduce the level of detection/quantification to access low concentrated metabolites. To reach this aim, speeding up the recently developed QUantItative Perfected and pUre shifted HSQC (QUIPU HSQC) is an interesting attempt to develop the FAQUIRE concept. Thanks to the combination of spectral aliasing, nonuniform sampling, and variable repetition time, the acquisition time of 2D quantitative maps is reduced by a factor 6 to 9, while conserving a high spectral resolution thanks to a pure shift approach. The analytical potential of the new Quick QUIPU HSQC (Q QUIPU HSQC) is evaluated on a model metabolite sample, and its potential is shown on breast-cell extracts embedding metabolites at millimolar to submillimolar concentrations.

Touch-down reverse transcriptase-PCR detection of IgV(H) rearrangement and Sybr-Green-based real-time RT-PCR quantitation of minimal residual disease in patients with chronic lymphocytic leukemia.

PubMed

Peková, Sona; Marková, Jana; Pajer, Petr; Dvorák, Michal; Cetkovský, Petr; Schwarz, Jirí

2005-01-01

Patients with chronic lymphocytic leukemia (CLL) can relapse even after aggressive therapy and autografts. It is commonly assumed that to prevent relapse the level of minimal residual disease (MRD) should be as low as possible. To evaluate MRD, highly sensitive quantitative assays are needed. The aim of the study was to develop a robust and sensitive method for detection of the clonal immunoglobulin heavy-chain variable (IgV(H)) rearrangement in CLL and to introduce a highly sensitive and specific methodology for MRD monitoring in patients with CLL who undergo intensive treatment. As a prerequisite for MRD detection, touch-down reverse transcriptase (RT)-PCR using degenerate primers were used for the diagnostic identification of (H) gene rearrangement(s). For quantitative MRD detection in 18 patients, we employed a real-time RT-PCR assay (RQ-PCR) making use of patient-specific primers and the cost-saving Sybr-Green reporter dye (SG). For precise calibration of RQ-PCR, patient-specific IgV(H) sequences were cloned. Touch-down RT-PCR with degenerate primers allowed the successful detection of IgV(H) clonal rearrangement(s) in 252 of 257 (98.1%) diagnostic samples. Biallelic rearrangements were found in 27 of 252 (10.7%) cases. Degenerate primers used for the identification of clonal expansion at diagnosis were not sensitive enough for MRD detection. In contrast, our RQ-PCR assay using patient-specific primers and SG reached the sensitivity of 10(-)(6). We demonstrated MRD in each patient tested, including four of four patients in complete remission following autologous hematopoietic stem cell transplantation (HSCT) and three of three following allogeneic 'mini'-HSCT. Increments in MRD might herald relapse; aggressive chemotherapy could induce molecular remission. Our touch-down RT-PCR has higher efficiency to detect clonal IgV(H) rearrangements including the biallelic ones. MRD quantitation of IgV(H) expression using SG-based RQ-PCR represents a highly specific, sensitive, and economic alternative to the current quantitative methods.

Collection of quantitative chemical release field data.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Demirgian, J.; Macha, S.; Loyola Univ.

1999-01-01

Detection and quantitation of chemicals in the environment requires Fourier-transform infrared (FTIR) instruments that are properly calibrated and tested. This calibration and testing requires field testing using matrices that are representative of actual instrument use conditions. Three methods commonly used for developing calibration files and training sets in the field are a closed optical cell or chamber, a large-scale chemical release, and a small-scale chemical release. There is no best method. The advantages and limitations of each method should be considered in evaluating field results. Proper calibration characterizes the sensitivity of an instrument, its ability to detect a component inmore » different matrices, and the quantitative accuracy and precision of the results.« less

A simple and rapid DNA extraction method from whole blood for highly sensitive detection and quantitation of HIV-1 proviral DNA by real-time PCR.

PubMed

McFall, Sally M; Wagner, Robin L; Jangam, Sujit R; Yamada, Douglas H; Hardie, Diana; Kelso, David M

2015-03-01

Early diagnosis and access to treatment for infants with human immunodeficiency virus-1 (HIV-1) is critical to reduce infant mortality. The lack of simple point-of-care tests impedes the timely initiation of antiretroviral therapy. The development of FINA, filtration isolation of nucleic acids, a novel DNA extraction method that can be performed by clinic personnel in less than 2 min has been reported previously. In this report, significant improvements in the DNA extraction and amplification methods are detailed that allow sensitive quantitation of as little as 10 copies of HIV-1 proviral DNA and detection of 3 copies extracted from 100 μl of whole blood. An internal control to detect PCR inhibition was also incorporated. In a preliminary field evaluation of 61 South African infants, the FINA test demonstrated 100% sensitivity and specificity. The proviral copy number of the infant specimens was quantified, and it was established that 100 microliters of whole blood is required for sensitive diagnosis of infants. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

The simultaneous quantitation of ten amino acids in soil extracts by mass fragmentography

NASA Technical Reports Server (NTRS)

Pereira, W. E.; Hoyano, Y.; Reynolds, W. E.; Summons, R. E.; Duffield, A. M.

1972-01-01

A specific and sensitive method for the identification and simultaneous quantitation by mass fragmentography of ten of the amino acids present in soil was developed. The technique uses a computer driven quadrupole mass spectrometer and a commercial preparation of deuterated amino acids is used as internal standards for purposes of quantitation. The results obtained are comparable with those from an amino acid analyzer. In the quadrupole mass spectrometer-computer system up to 25 pre-selected ions may be monitored sequentially. This allows a maximum of 12 different amino acids (one specific ion in each of the undeuterated and deuterated amino acid spectra) to be quantitated. The method is relatively rapid (analysis time of approximately one hour) and is capable of the quantitation of nanogram quantities of amino acids.

Rapid quantitative analysis of 8-iso-prostaglandin-F(2alpha) using liquid chromatography-tandem mass spectrometry and comparison with an enzyme immunoassay method.

PubMed

Dahl, Jeffrey H; van Breemen, Richard B

2010-09-15

A rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the measurement of urinary 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)), a biomarker of lipid peroxidation. Because urine contains numerous F(2) prostaglandin isomers, each with identical mass and similar mass spectrometric fragmentation patterns, chromatographic separation of 8-iso-PGF(2alpha) from its isomers is necessary for its quantitative analysis using MS/MS. We were able to achieve this separation using an isocratic LC method with a run time of less than 9min, which is at least threefold faster than previous methods, while maintaining sensitivity, accuracy, precision, and reliability. The limits of detection and quantitation were 53 and 178pg/ml urine, respectively. We compared our method with a commercially available affinity purification and enzyme immunoassay kit and found both assays to be in agreement. Despite the high sensitivity of the enzyme immunoassay method, it is more expensive and has a narrower dynamic range than LC-MS/MS. Our method was optimized for rapid measurement of 8-iso-PGF(2alpha) in urine, and it is ideally suited for clinical sample analysis. 2010 Elsevier Inc. All rights reserved.

An evaluation of the DRI-ETG EIA method for the determination of ethyl glucuronide concentrations in clinical and post-mortem urine.

PubMed

Turfus, Sophie C; Vo, Tu; Niehaus, Nadia; Gerostamoulos, Dimitri; Beyer, Jochen

2013-06-01

A commercial enzyme immunoassay for the qualitative and semi-quantitative measurement of ethyl glucuronide (EtG) in urine was evaluated. Post-mortem (n=800), and clinical urine (n=200) samples were assayed using a Hitachi 902 analyzer. The determined concentrations were compared with those obtained using a previously published liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of EtG and ethyl sulfate. Using a cut-off of 0.5 µg/ml and LC-MS/MS limit of reporting of 0.1 µg/ml, there was a sensitivity of 60.8% and a specificity of 100% for clinical samples. For post-mortem samples, sensitivity and specificity were 82.4% and 97.1%, respectively. When reducing the cut-off to 0.1 µg/ml, the sensitivity and specificity were 83.3% and 100% for clinical samples whereas for post-mortem samples the sensitivity and specificity were 90.3 % and 88.3 %, respectively. The best trade-offs between sensitivity and specificity for LC-MS/MS limits of reporting of 0.5 and 0.1 µg/ml were achieved when using immunoassay cut-offs of 0.3 and 0.092 µg/ml, respectively. There was good correlation between quantitative results obtained by both methods but analysis of samples by LC-MS/MS gave higher concentrations than by enzyme immunoassay (EIA), with a statistically significant proportional bias (P<0.0001, Deming regression) for both sample types. The immunoassay is reliable for the qualitative and semi-quantitative presumptive detection of ethyl glucuronide in urine. Copyright © 2012 John Wiley & Sons, Ltd.

Polymerase chain displacement reaction.

PubMed

Harris, Claire L; Sanchez-Vargas, Irma J; Olson, Ken E; Alphey, Luke; Fu, Guoliang

2013-02-01

Quantitative PCR assays are now the standard method for viral diagnostics. These assays must be specific, as well as sensitive, to detect the potentially low starting copy number of viral genomic material. We describe a new technique, polymerase chain displacement reaction (PCDR), which uses multiple nested primers in a rapid, capped, one-tube reaction that increases the sensitivity of normal quantitative PCR (qPCR) assays. Sensitivity was increased by approximately 10-fold in a proof-of-principle test on dengue virus sequence. In PCDR, when extension occurs from the outer primer, it displaces the extension strand produced from the inner primer by utilizing a polymerase that has strand displacement activity. This allows a greater than 2-fold increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR. Increased sensitivity in PCDR would be useful in nucleic acid detection for viral diagnostics.

Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level.

PubMed

Zhu, Zhi; Zhang, Wenhua; Leng, Xuefei; Zhang, Mingxia; Guan, Zhichao; Lu, Jiangquan; Yang, Chaoyong James

2012-10-21

Genetic alternations can serve as highly specific biomarkers to distinguish fatal bacteria or cancer cells from their normal counterparts. However, these mutations normally exist in very rare amount in the presence of a large excess of non-mutated analogs. Taking the notorious pathogen E. coli O157:H7 as the target analyte, we have developed an agarose droplet-based microfluidic ePCR method for highly sensitive, specific and quantitative detection of rare pathogens in the high background of normal bacteria. Massively parallel singleplex and multiplex PCR at the single-cell level in agarose droplets have been successfully established. Moreover, we challenged the system with rare pathogen detection and realized the sensitive and quantitative analysis of a single E. coli O157:H7 cell in the high background of 100,000 excess normal K12 cells. For the first time, we demonstrated rare pathogen detection through agarose droplet microfluidic ePCR. Such a multiplex single-cell agarose droplet amplification method enables ultra-high throughput and multi-parameter genetic analysis of large population of cells at the single-cell level to uncover the stochastic variations in biological systems.

Characterization method for relative Raman enhancement for surface-enhanced Raman spectroscopy using gold nanoparticle dimer array

NASA Astrophysics Data System (ADS)

Sugano, Koji; Ikegami, Kohei; Isono, Yoshitada

2017-06-01

In this paper, a characterization method for Raman enhancement for highly sensitive and quantitative surface-enhanced Raman spectroscopy (SERS) is reported. A particle dimer shows a marked electromagnetic enhancement when the particle connection direction is matched to the polarization direction of incident light. In this study, dimers were arrayed by nanotrench-guided self-assembly for a marked total Raman enhancement. By measuring acetonedicarboxylic acid, the fabricated structures were characterized for SERS depending on the polarization angle against the particle connection direction. This indicates that the fabricated structures cause an effective SERS enhancement, which is dominated by the electromagnetic enhancement. Then, we measured 4,4‧-bipyridine, which is a pesticide material, for quantitative analysis. In advance, we evaluated the enhancement of the particle structure by the Raman measurement of acetonedicarboxylic acid. Finally, we compared the Raman intensities of acetonedicarboxylic acid and 4,4‧-bipyridine. Their intensities showed good correlation. The advantage of this method for previously evaluating the enhancement of the substrate was demonstrated. This developed SERS characterization method is expected to be applied to various quantitative trace analyses of molecules with high sensitivity.

Silver Nanoparticle-Based Fluorescence-Quenching Lateral Flow Immunoassay for Sensitive Detection of Ochratoxin A in Grape Juice and Wine.

PubMed

Jiang, Hu; Li, Xiangmin; Xiong, Ying; Pei, Ke; Nie, Lijuan; Xiong, Yonghua

2017-02-28

A silver nanoparticle (AgNP)-based fluorescence-quenching lateral flow immunoassay with competitive format (cLFIA) was developed for sensitive detection of ochratoxin A (OTA) in grape juice and wine samples in the present study. The Ru(phen) 3 2 + -doped silica nanoparticles (RuNPs) were sprayed on the test and control line zones as background fluorescence signals. The AgNPs were designed as the fluorescence quenchers of RuNPs because they can block the exciting light transferring to the RuNP molecules. The proposed method exhibited high sensitivity for OTA detection, with a detection limit of 0.06 µg/L under optimized conditions. The method also exhibited a good linear range for OTA quantitative analysis from 0.08 µg/L to 5.0 µg/L. The reliability of the fluorescence-quenching cLFIA method was evaluated through analysis of the OTA-spiked red grape wine and juice samples. The average recoveries ranged from 88.0% to 110.0% in red grape wine and from 92.0% to 110.0% in grape juice. Meanwhile, less than a 10% coefficient variation indicated an acceptable precision of the cLFIA method. In summary, the new AgNP-based fluorescence-quenching cLFIA is a simple, rapid, sensitive, and accurate method for quantitative detection of OTA in grape juice and wine or other foodstuffs.

Silver Nanoparticle-Based Fluorescence-Quenching Lateral Flow Immunoassay for Sensitive Detection of Ochratoxin A in Grape Juice and Wine

PubMed Central

Jiang, Hu; Li, Xiangmin; Xiong, Ying; Pei, Ke; Nie, Lijuan; Xiong, Yonghua

2017-01-01

A silver nanoparticle (AgNP)-based fluorescence-quenching lateral flow immunoassay with competitive format (cLFIA) was developed for sensitive detection of ochratoxin A (OTA) in grape juice and wine samples in the present study. The Ru(phen)32+-doped silica nanoparticles (RuNPs) were sprayed on the test and control line zones as background fluorescence signals. The AgNPs were designed as the fluorescence quenchers of RuNPs because they can block the exciting light transferring to the RuNP molecules. The proposed method exhibited high sensitivity for OTA detection, with a detection limit of 0.06 µg/L under optimized conditions. The method also exhibited a good linear range for OTA quantitative analysis from 0.08 µg/L to 5.0 µg/L. The reliability of the fluorescence-quenching cLFIA method was evaluated through analysis of the OTA-spiked red grape wine and juice samples. The average recoveries ranged from 88.0% to 110.0% in red grape wine and from 92.0% to 110.0% in grape juice. Meanwhile, less than a 10% coefficient variation indicated an acceptable precision of the cLFIA method. In summary, the new AgNP-based fluorescence-quenching cLFIA is a simple, rapid, sensitive, and accurate method for quantitative detection of OTA in grape juice and wine or other foodstuffs. PMID:28264472

Synthetic internal control sequences to increase negative call veracity in multiplexed, quantitative PCR assays for Phakopsora pachyrhizi

USDA-ARS?s Scientific Manuscript database

Quantitative PCR (Q-PCR) utilizing specific primer sequences and a fluorogenic, 5’-exonuclease linear hydrolysis probe is well established as a detection and identification method for Phakopsora pachyrhizi, the soybean rust pathogen. Because of the extreme sensitivity of Q-PCR, the DNA of a single u...

Sensitive elemental detection using microwave-assisted laser-induced breakdown imaging

NASA Astrophysics Data System (ADS)

Iqbal, Adeel; Sun, Zhiwei; Wall, Matthew; Alwahabi, Zeyad T.

2017-10-01

This study reports a sensitive spectroscopic method for quantitative elemental detection by manipulating the temporal and spatial parameters of laser-induced plasma. The method was tested for indium detection in solid samples, in which laser ablation was used to generate a tiny plasma. The lifetime of the laser-induced plasma can be extended to hundreds of microseconds using microwave injection to remobilize the electrons. In this novel method, temporal integrated signal of indium emission was significantly enhanced. Meanwhile, the projected detectable area of the excited indium atoms was also significantly improved using an interference-, instead of diffraction-, based technique, achieved by directly imaging microwave-enhanced plasma through a novel narrow-bandpass filter, exactly centered at the indium emission line. Quantitative laser-induce breakdown spectroscopy was also recorded simultaneously with the new imaging method. The intensities recorded from both methods exhibit very good mutual linear relationship. The detection intensity was improved to 14-folds because of the combined improvements in the plasma lifetime and the area of detection.

Case-Deletion Diagnostics for Maximum Likelihood Multipoint Quantitative Trait Locus Linkage Analysis

PubMed Central

Mendoza, Maria C.B.; Burns, Trudy L.; Jones, Michael P.

2009-01-01

Objectives Case-deletion diagnostic methods are tools that allow identification of influential observations that may affect parameter estimates and model fitting conclusions. The goal of this paper was to develop two case-deletion diagnostics, the exact case deletion (ECD) and the empirical influence function (EIF), for detecting outliers that can affect results of sib-pair maximum likelihood quantitative trait locus (QTL) linkage analysis. Methods Subroutines to compute the ECD and EIF were incorporated into the maximum likelihood QTL variance estimation components of the linkage analysis program MAPMAKER/SIBS. Performance of the diagnostics was compared in simulation studies that evaluated the proportion of outliers correctly identified (sensitivity), and the proportion of non-outliers correctly identified (specificity). Results Simulations involving nuclear family data sets with one outlier showed EIF sensitivities approximated ECD sensitivities well for outlier-affected parameters. Sensitivities were high, indicating the outlier was identified a high proportion of the time. Simulations also showed the enormous computational time advantage of the EIF. Diagnostics applied to body mass index in nuclear families detected observations influential on the lod score and model parameter estimates. Conclusions The EIF is a practical diagnostic tool that has the advantages of high sensitivity and quick computation. PMID:19172086

Static headspace gas chromatographic method for quantitative determination of residual solvents in pharmaceutical drug substances according to european pharmacopoeia requirements.

PubMed

Otero, Raquel; Carrera, Guillem; Dulsat, Joan Francesc; Fábregas, José Luís; Claramunt, Juan

2004-11-19

A static headspace (HS) gas chromatographic method for quantitative determination of residual solvents in a drug substance has been developed according to European Pharmacopoeia general procedure. A water-dimethylformamide mixture is proposed as sample solvent to obtain good sensitivity and recovery. The standard addition technique with internal standard quantitation was used for ethanol, tetrahydrofuran and toluene determination. Validation was performed within the requirements of ICH validation guidelines Q2A and Q2B. Selectivity was tested for 36 solvents, and system suitability requirements described in the European Pharmacopoeia were checked. Limits of detection and quantitation, precision, linearity, accuracy, intermediate precision and robustness were determined, and excellent results were obtained.

Highly sensitive image-derived indices of water-stressed plants using hyperspectral imaging in SWIR and histogram analysis

PubMed Central

Kim, David M.; Zhang, Hairong; Zhou, Haiying; Du, Tommy; Wu, Qian; Mockler, Todd C.; Berezin, Mikhail Y.

2015-01-01

The optical signature of leaves is an important monitoring and predictive parameter for a variety of biotic and abiotic stresses, including drought. Such signatures derived from spectroscopic measurements provide vegetation indices – a quantitative method for assessing plant health. However, the commonly used metrics suffer from low sensitivity. Relatively small changes in water content in moderately stressed plants demand high-contrast imaging to distinguish affected plants. We present a new approach in deriving sensitive indices using hyperspectral imaging in a short-wave infrared range from 800 nm to 1600 nm. Our method, based on high spectral resolution (1.56 nm) instrumentation and image processing algorithms (quantitative histogram analysis), enables us to distinguish a moderate water stress equivalent of 20% relative water content (RWC). The identified image-derived indices 15XX nm/14XX nm (i.e. 1529 nm/1416 nm) were superior to common vegetation indices, such as WBI, MSI, and NDWI, with significantly better sensitivity, enabling early diagnostics of plant health. PMID:26531782

Technical and financial evaluation of assays for progesterone in canine practice in the UK.

PubMed

Moxon, R; Copley, D; England, G C W

2010-10-02

The concentration of progesterone was measured in 60 plasma samples from bitches at various stages of the oestrous cycle, using commercially available quantitative and semi-quantitative ELISA test kits, as well as by two commercial laboratories undertaking radioimmunoassay (RIA). The RIA, which was assumed to be the 'gold standard' in terms of reliability and accuracy, was the most expensive method when analysing more than one sample per week, and had the longest delay in obtaining results, but had minimal requirements for practice staff time. When compared with the RIA, the quantitative ELISA had a strong positive correlation (r=0.97, P<0.05) and a sensitivity and specificity of 70.6 per cent and 100.0 per cent, respectively, and positive and negative predictive values of 100.0 per cent and 71.0 per cent, respectively, with an overall accuracy of 90.0 per cent. This method was the least expensive when analysing five or more samples per week, but had longer turnaround times than that of the semi-quantitative ELISA and required more staff time. When compared with the RIA, the semi-quantitative ELISA had a sensitivity and specificity of 100.0 per cent and 95.5 per cent, respectively, and positive and negative predictive values of 73.9 per cent and 77.8 per cent, respectively, with an overall accuracy of 89.2 per cent. This method was more expensive than the quantitative ELISA when analysing five or more samples per week, but had the shortest turnaround time and low requirements in terms of staff time.

A minimalist biosensor: Quantitation of cyclic di-GMP using the conformational change of a riboswitch aptamer.

PubMed

Kellenberger, Colleen A; Sales-Lee, Jade; Pan, Yuchen; Gassaway, Madalee M; Herr, Amy E; Hammond, Ming C

2015-01-01

Cyclic di-GMP (c-di-GMP) is a second messenger that is important in regulating bacterial physiology and behavior, including motility and virulence. Many questions remain about the role and regulation of this signaling molecule, but current methods of detection are limited by either modest sensitivity or requirements for extensive sample purification. We have taken advantage of a natural, high affinity receptor of c-di-GMP, the Vc2 riboswitch aptamer, to develop a sensitive and rapid electrophoretic mobility shift assay (EMSA) for c-di-GMP quantitation that required minimal engineering of the RNA.

Sensitive assay, based on hydroxy fatty acids from lipopolysaccharide lipid A, for Gram-negative bacteria in sediments.

PubMed Central

Parker, J H; Smith, G A; Fredrickson, H L; Vestal, J R; White, D C

1982-01-01

Biochemical measures have provided insight into the biomass and community structure of sedimentary microbiota without the requirement of selection by growth or quantitative removal from the sediment grains. This study used the assay of the hydroxy fatty acids released from the lipid A of the lipopolysaccharide in sediments to provide an estimate of the gram-negative bacteria. The method was sensitive to picomolar amounts of hydroxy fatty acids. The recovery of lipopolysaccharide hydroxy fatty acids from organisms added to sediments was quantitative. The lipids were extracted from the sediments with single-phase chloroform-methanol extraction. The lipid-extraction residue was hydrolyzed in 1 N HCl, and the hydroxy fatty acids of the lipopolysaccharide were recovered in chloroform for analysis by gas-liquid chromatography. This method proved to be about fivefold more sensitive than the classical phenol-water or trichloroacetic acid methods when applied to marine sediments. By examination of the patterns of hydroxy fatty acids, it was also possible to help define the community structure of the sedimentary gram-negative bacteria. PMID:6817712

Sensitivity analysis of infectious disease models: methods, advances and their application

PubMed Central

Wu, Jianyong; Dhingra, Radhika; Gambhir, Manoj; Remais, Justin V.

2013-01-01

Sensitivity analysis (SA) can aid in identifying influential model parameters and optimizing model structure, yet infectious disease modelling has yet to adopt advanced SA techniques that are capable of providing considerable insights over traditional methods. We investigate five global SA methods—scatter plots, the Morris and Sobol’ methods, Latin hypercube sampling-partial rank correlation coefficient and the sensitivity heat map method—and detail their relative merits and pitfalls when applied to a microparasite (cholera) and macroparasite (schistosomaisis) transmission model. The methods investigated yielded similar results with respect to identifying influential parameters, but offered specific insights that vary by method. The classical methods differed in their ability to provide information on the quantitative relationship between parameters and model output, particularly over time. The heat map approach provides information about the group sensitivity of all model state variables, and the parameter sensitivity spectrum obtained using this method reveals the sensitivity of all state variables to each parameter over the course of the simulation period, especially valuable for expressing the dynamic sensitivity of a microparasite epidemic model to its parameters. A summary comparison is presented to aid infectious disease modellers in selecting appropriate methods, with the goal of improving model performance and design. PMID:23864497

Apparatus and method for quantitative determination of materials contained in fluids

DOEpatents

Radziemski, Leon J.; Cremers, David A.

1985-01-01

Apparatus and method for near real-time in-situ monitoring of particulates and vapors contained in fluids. Initial filtration of a known volume of the fluid sample is combined with laser-induced dielectric breakdown spectroscopy of the filter employed to obtain qualitative and quantitative information with high sensitivity. Application of the invention to monitoring of beryllium, beryllium oxide, or other beryllium-alloy dusts is demonstrated. Significant shortening of analysis time is achieved from those of the usual chemical techniques of analysis.

Apparatus and method for quantitative determination of materials contained in fluids

DOEpatents

Radziemski, L.J.; Cremers, D.A.

1982-09-07

Apparatus and method for near real-time in-situ monitoring of particulates and vapors contained in fluids are described. Initial filtration of a known volume of the fluid sample is combined with laser-induced dielectric breakdown spectroscopy of the filter employed to obtain qualitative and quantitative information with high sensitivity. Application of the invention to monitoring of beryllium, beryllium oxide, or other beryllium-alloy dusts is shown. Significant shortening of analysis time is achieved from the usual chemical techniques of analysis.

Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions.

PubMed

Belmonte, Frances R; Martin, James L; Frescura, Kristin; Damas, Joana; Pereira, Filipe; Tarnopolsky, Mark A; Kaufman, Brett A

2016-04-28

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error.

Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions

PubMed Central

Belmonte, Frances R.; Martin, James L.; Frescura, Kristin; Damas, Joana; Pereira, Filipe; Tarnopolsky, Mark A.; Kaufman, Brett A.

2016-01-01

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error. PMID:27122135

[The development and validation of the methods for the quantitative determination of sibutramine derivatives in dietary supplements].

PubMed

Stern, K I; Malkova, T L

The objective of the present study was the development and validation of sibutramine demethylated derivatives, desmethyl sibutramine and didesmethyl sibutramine. Gas-liquid chromatography with the flame ionization detector was used for the quantitative determination of the above substances in dietary supplements. The conditions for the chromatographic determination of the analytes in the presence of the reference standard, methyl stearate, were proposed allowing to achieve the efficient separation. The method has the necessary sensitivity, specificity, linearity, accuracy, and precision (on the intra-day and inter-day basis) which suggests its good validation characteristics. The proposed method can be employed in the analytical laboratories for the quantitative determination of sibutramine derivatives in biologically active dietary supplements.

Detection and enumeration of Salmonella enteritidis in homemade ice cream associated with an outbreak: comparison of conventional and real-time PCR methods.

PubMed

Seo, K H; Valentin-Bon, I E; Brackett, R E

2006-03-01

Salmonellosis caused by Salmonella Enteritidis (SE) is a significant cause of foodborne illnesses in the United States. Consumption of undercooked eggs and egg-containing products has been the primary risk factor for the disease. The importance of the bacterial enumeration technique has been enormously stressed because of the quantitative risk analysis of SE in shell eggs. Traditional enumeration methods mainly depend on slow and tedious most-probable-number (MPN) methods. Therefore, specific, sensitive, and rapid methods for SE quantitation are needed to collect sufficient data for risk assessment and food safety policy development. We previously developed a real-time quantitative PCR assay for the direct detection and enumeration of SE and, in this study, applied it to naturally contaminated ice cream samples with and without enrichment. The detection limit of the real-time PCR assay was determined with artificially inoculated ice cream. When applied to the direct detection and quantification of SE in ice cream, the real-time PCR assay was as sensitive as the conventional plate count method in frequency of detection. However, populations of SE derived from real-time quantitative PCR were approximately 1 log higher than provided by MPN and CFU values obtained by conventional culture methods. The detection and enumeration of SE in naturally contaminated ice cream can be completed in 3 h by this real-time PCR method, whereas the cultural enrichment method requires 5 to 7 days. A commercial immunoassay for the specific detection of SE was also included in the study. The real-time PCR assay proved to be a valuable tool that may be useful to the food industry in monitoring its processes to improve product quality and safety.

Simplex and duplex event-specific analytical methods for functional biotech maize.

PubMed

Lee, Seong-Hun; Kim, Su-Jeong; Yi, Bu-Young

2009-08-26

Analytical methods are very important in the control of genetically modified organism (GMO) labeling systems or living modified organism (LMO) management for biotech crops. Event-specific primers and probes were developed for qualitative and quantitative analysis for biotech maize event 3272 and LY 038 on the basis of the 3' flanking regions, respectively. The qualitative primers confirmed the specificity by a single PCR product and sensitivity to 0.05% as a limit of detection (LOD). Simplex and duplex quantitative methods were also developed using TaqMan real-time PCR. One synthetic plasmid was constructed from two taxon-specific DNA sequences of maize and two event-specific 3' flanking DNA sequences of event 3272 and LY 038 as reference molecules. In-house validation of the quantitative methods was performed using six levels of mixing samples, from 0.1 to 10.0%. As a result, the biases from the true value and the relative deviations were all within the range of +/-30%. Limits of quantitation (LOQs) of the quantitative methods were all 0.1% for simplex real-time PCRs of event 3272 and LY 038 and 0.5% for duplex real-time PCR of LY 038. This study reports that event-specific analytical methods were applicable for qualitative and quantitative analysis for biotech maize event 3272 and LY 038.

Application of a SERS-based lateral flow immunoassay strip for the rapid and sensitive detection of staphylococcal enterotoxin B

NASA Astrophysics Data System (ADS)

Hwang, Joonki; Lee, Sangyeop; Choo, Jaebum

2016-06-01

A novel surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay (LFA) biosensor was developed to resolve problems associated with conventional LFA strips (e.g., limits in quantitative analysis and low sensitivity). In our SERS-based biosensor, Raman reporter-labeled hollow gold nanospheres (HGNs) were used as SERS detection probes instead of gold nanoparticles. With the proposed SERS-based LFA strip, the presence of a target antigen can be identified through a colour change in the test zone. Furthermore, highly sensitive quantitative evaluation is possible by measuring SERS signals from the test zone. To verify the feasibility of the SERS-based LFA strip platform, an immunoassay of staphylococcal enterotoxin B (SEB) was performed as a model reaction. The limit of detection (LOD) for SEB, as determined with the SERS-based LFA strip, was estimated to be 0.001 ng mL-1. This value is approximately three orders of magnitude more sensitive than that achieved with the corresponding ELISA-based method. The proposed SERS-based LFA strip sensor shows significant potential for the rapid and sensitive detection of target markers in a simplified manner.A novel surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay (LFA) biosensor was developed to resolve problems associated with conventional LFA strips (e.g., limits in quantitative analysis and low sensitivity). In our SERS-based biosensor, Raman reporter-labeled hollow gold nanospheres (HGNs) were used as SERS detection probes instead of gold nanoparticles. With the proposed SERS-based LFA strip, the presence of a target antigen can be identified through a colour change in the test zone. Furthermore, highly sensitive quantitative evaluation is possible by measuring SERS signals from the test zone. To verify the feasibility of the SERS-based LFA strip platform, an immunoassay of staphylococcal enterotoxin B (SEB) was performed as a model reaction. The limit of detection (LOD) for SEB, as determined with the SERS-based LFA strip, was estimated to be 0.001 ng mL-1. This value is approximately three orders of magnitude more sensitive than that achieved with the corresponding ELISA-based method. The proposed SERS-based LFA strip sensor shows significant potential for the rapid and sensitive detection of target markers in a simplified manner. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07243c

PCR-free quantitative detection of genetically modified organism from raw materials – A novel electrochemiluminescence-based bio-barcode method

PubMed Central

Zhu, Debin; Tang, Yabing; Xing, Da; Chen, Wei R.

2018-01-01

Bio-barcode assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio-barcode assay requires lengthy experimental procedures including the preparation and release of barcode DNA probes from the target-nanoparticle complex, and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio-barcode assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2’2’-bipyridyl) ruthenium (TBR)-labele barcode DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products. PMID:18386909

PCR-free quantitative detection of genetically modified organism from raw materials. An electrochemiluminescence-based bio bar code method.

PubMed

Zhu, Debin; Tang, Yabing; Xing, Da; Chen, Wei R

2008-05-15

A bio bar code assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio bar code assay requires lengthy experimental procedures including the preparation and release of bar code DNA probes from the target-nanoparticle complex and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio bar code assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2,2'-bipyridyl) ruthenium (TBR)-labeled bar code DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products.

Utility of DWI with quantitative ADC values in ovarian tumors: a meta-analysis of diagnostic test performance.

PubMed

Pi, Shan; Cao, Rong; Qiang, Jin Wei; Guo, Yan Hui

2018-01-01

Background Diffusion-weighted imaging (DWI) and quantitative apparent diffusion coefficient (ADC) values are widely used in the differential diagnosis of ovarian tumors. Purpose To assess the diagnostic performance of quantitative ADC values in ovarian tumors. Material and Methods PubMed, Embase, the Cochrane Library, and local databases were searched for studies assessing ovarian tumors using quantitative ADC values. We quantitatively analyzed the diagnostic performances for two clinical problems: benign vs. malignant tumors and borderline vs. malignant tumors. We evaluated diagnostic performances by the pooled sensitivity and specificity values and by summary receiver operating characteristic (SROC) curves. Subgroup analyses were used to analyze study heterogeneity. Results From the 742 studies identified in the search results, 16 studies met our inclusion criteria. A total of ten studies evaluated malignant vs. benign ovarian tumors and six studies assessed malignant vs. borderline ovarian tumors. Regarding the diagnostic accuracy of quantitative ADC values for distinguishing between malignant and benign ovarian tumors, the pooled sensitivity and specificity values were 0.91 and 0.91, respectively. The area under the SROC curve (AUC) was 0.96. For differentiating borderline from malignant tumors, the pooled sensitivity and specificity values were 0.89 and 0.79, and the AUC was 0.91. The methodological quality of the included studies was moderate. Conclusion Quantitative ADC values could serve as useful preoperative markers for predicting the nature of ovarian tumors. Nevertheless, prospective trials focused on standardized imaging parameters are needed to evaluate the clinical value of quantitative ADC values in ovarian tumors.

HPLC method development for evolving applications in the pharmaceutical industry and nanoscale chemistry

NASA Astrophysics Data System (ADS)

Castiglione, Steven Louis

As scientific research trends towards trace levels and smaller architectures, the analytical chemist is often faced with the challenge of quantitating said species in a variety of matricies. The challenge is heightened when the analytes prove to be potentially toxic or possess physical or chemical properties that make traditional analytical methods problematic. In such cases, the successful development of an acceptable quantitative method plays a critical role in the ability to further develop the species under study. This is particularly true for pharmaceutical impurities and nanoparticles (NP). The first portion of the research focuses on the development of a part-per-billion level HPLC method for a substituted phenazine-class pharmaceutical impurity. The development of this method was required due to the need for a rapid methodology to quantitatively determine levels of a potentially toxic phenazine moiety in order to ensure patient safety. As the synthetic pathway for the active ingredient was continuously refined to produce progressively lower amounts of the phenazine impurity, the approach for increasingly sensitive quantitative methods was required. The approaches evolved across four discrete methods, each employing a unique scheme for analyte detection. All developed methods were evaluated with regards to accuracy, precision and linear adherence as well as ancillary benefits and detriments -- e.g., one method in this evolution demonstrated the ability to resolve and detect other species from the phenazine class. The second portion of the research focuses on the development of an HPLC method for the quantitative determination of NP size distributions. The current methodology for the determination of NP sizes employs tunneling electron microscopy (TEM), which requires sample drying without particle size alteration and which, in many cases, may prove infeasible due to cost or availability. The feasibility of an HPLC method for NP size characterizations evolved across three methods, each employing a different approach for size resolution. These methods were evaluated primarily for sensitivity, which proved to be a substantial hurdle to further development, but does not appear to deter future research efforts.

CdSe/ZnS Quantum Dot-Labeled Lateral Flow Strips for Rapid and Quantitative Detection of Gastric Cancer Carbohydrate Antigen 72-4

NASA Astrophysics Data System (ADS)

Yan, Xinyu; Wang, Kan; Lu, Wenting; Qin, Weijian; Cui, Daxiang; He, Jinghua

2016-03-01

Carbohydrate antigen 72-4 (CA72-4) is an important biomarker associated closely with diagnosis and prognosis of early gastric cancer. How to realize quick, sensitive, specific, and quantitative detection of CA72-4 in clinical specimens has become a great requirement. Herein, we reported a CdSe/ZnS quantum dot-labeled lateral flow test strip combined with a charge-coupled device (CCD)-based reader was developed for rapid, sensitive, and quantitative detection of CA72-4. Two mouse monoclonal antibodies (mAbs) against CA72-4 were employed. One of them was coated as a test line, while another mAb was labeled with quantum dots and coated onto conjugate pad. The goat anti-mouse IgG was immobilized as a control line. After sample was added, a sandwich structure was formed with CA72-4 and these two mAbs. The fluorescent signal from quantum dots (QD)-labeled mAb in sandwich structure was related to the amount of detected CA72-4. A CCD-based reader was used to realize quantitative detection of CA72-4. Results showed that developed QD-labeled lateral flow strips to detect CA72-4 biomarker with the sensitivity of 2 IU/mL and 10 min detection time. One hundred sera samples from clinical patients with gastric cancer and healthy people were used to confirm specificity of this strip method; results showed that established strip method own 100 % reproducibility and 100 % specificity compared with Roche electrochemiluminescence assay results. In conclusion, CdSe/ZnS quantum dot-labeled lateral flow strips for detection of CA72-4 could realize rapid, sensitive, and specific detection of clinical samples and could own great potential in clinical translation in near future.

Charged derivatization and on-line solid phase extraction to measure extremely low cortisol and cortisone levels in human saliva with liquid chromatography-tandem mass spectrometry.

PubMed

Magda, Balázs; Dobi, Zoltán; Mészáros, Katalin; Szabó, Éva; Márta, Zoltán; Imre, Tímea; Szabó, Pál T

2017-06-05

The aim of this study was to develop a sensitive, reliable and high-throughput liquid chromatography - electrospray ionization - mass spectrometric (LC-ESI-MS/MS) method for the simultaneous quantitation of cortisol and cortisone in human saliva. Derivatization with 2-hydrazino-1-methylpyridine (HMP) was one of the most challenging aspects of the method development. The reagent was reacting with cortisol and cortisone at 60°C within 1h, giving mono- and bis-hydrazone derivatives. Investigation of derivatization reaction and sample preparation was detailed and discussed. Improvement of method sensitivity was achieved with charged derivatization and use of on-line solid phase extraction (on-line SPE). The lower limit of quantitation (LLOQ) was 5 and 10pg/ml for cortisol and cortisone, respectively. The developed method was subsequently applied to clinical laboratory measurement of cortisol and cortisone in human saliva. Copyright © 2017 Elsevier B.V. All rights reserved.

Portable and sensitive quantitative detection of DNA based on personal glucose meters and isothermal circular strand-displacement polymerization reaction.

PubMed

Xu, Xue-tao; Liang, Kai-yi; Zeng, Jia-ying

2015-02-15

A portable and sensitive quantitative DNA detection method based on personal glucose meters and isothermal circular strand-displacement polymerization reaction was developed. The target DNA triggered target recycling process, which opened capture DNA. The released target then found another capture DNA to trigger another polymerization cycle, which was repeated for many rounds, resulting in the multiplication of the DNA-invertase conjugation on the surface of Streptavidin-MNBs. The DNA-invertase was used to catalyze the hydrolysis of sucrose into glucose for PGM readout. There was a liner relationship between the signal of PGM and the concentration of target DNA in the range of 5.0 to 1000 fM, which is lower than some DNA detection method. In addition, the method exhibited excellent sequence selectivity and there was almost no effect of biological complex to the detection performance, which suggested our method can be successfully applied to DNA detection in real biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Qualitative and quantitative evaluation of a local lymph node assay based on ex vivo interleukin-2 production.

PubMed

Azam, Philippe; Peiffer, Jean-Luc; Ourlin, Jean-Claude; Bonnet, Pierre-Antoine; Tissier, Marie-Hélène; Vian, Laurence; Fabre, Isabelle

2005-01-15

The local lymph node assay (LLNA) is a regular method for the detection of sensitizing chemicals in mice which measures the incorporation of tritiated thymidine in lymph node cells. We have evaluated an alternative to this method based on the interleukin-2 (IL-2) production of lymph node cells. At the mRNA level, no change in the IL-2 gene expression level was detected by real-time PCR analysis. At the protein level, various experimental conditions were checked in order to improve the irritant versus sensitizer discrimination with a restricted set of prototypic compounds. In particular, the use of phytohemagglutinin A (PHA) in an ex vivo cell culture step showed an improvement of both signal and discrimination. In these optimised conditions, a panel of irritants and potency-graded sensitizers was used to assess the performance of the modified method. IFN-gamma production was used as a positive control. For each compound, a dose-response was performed and stimulation indexes (SI) were determined. Effective concentrations (EC) for each sensitizers were then extracted and compared to the literature data of the regular LLNA. The IL-2-based LLNA showed similar performances at both qualitative and quantitative levels compared to regular LLNA.

Quantitative multi-target RNA profiling in Epstein-Barr virus infected tumor cells.

PubMed

Greijer, A E; Ramayanti, O; Verkuijlen, S A W M; Novalić, Z; Juwana, H; Middeldorp, J M

2017-03-01

Epstein-Barr virus (EBV) is etiologically linked to multiple acute, chronic and malignant diseases. Detection of EBV-RNA transcripts in tissues or biofluids besides EBV-DNA can help in diagnosing EBV related syndromes. Sensitive EBV transcription profiling yields new insights on its pathogenic role and may be useful for monitoring virus targeted therapy. Here we describe a multi-gene quantitative RT-PCR profiling method that simultaneously detects a broad spectrum (n=16) of crucial latent and lytic EBV transcripts. These transcripts include (but are not restricted to), EBNA1, EBNA2, LMP1, LMP2, BARTs, EBER1, BARF1 and ZEBRA, Rta, BGLF4 (PK), BXLF1 (TK) and BFRF3 (VCAp18) all of which have been implicated in EBV-driven oncogenesis and viral replication. With this method we determine the amount of RNA copies per infected (tumor) cell in bulk populations of various origin. While we confirm the expected RNA profiles within classic EBV latency programs, this sensitive quantitative approach revealed the presence of rare cells undergoing lytic replication. Inducing lytic replication in EBV tumor cells supports apoptosis and is considered as therapeutic approach to treat EBV-driven malignancies. This sensitive multi-primed quantitative RT-PCR approach can provide broader understanding of transcriptional activity in latent and lytic EBV infection and is suitable for monitoring virus-specific therapy responses in patients with EBV associated cancers. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Comparative evaluation of hepatitis C virus RNA quantitation by branched DNA, NASBA, and monitor assays.

PubMed

Lunel, F; Cresta, P; Vitour, D; Payan, C; Dumont, B; Frangeul, L; Reboul, D; Brault, C; Piette, J C; Huraux, J M

1999-02-01

Several studies have shown a relationship between pretreatment hepatitis C virus (HCV) viral load and the response to interferon (IFN) therapy, creating a need for quantitative HCV-RNA assays. Here, we compared three commercial methods: nucleic acid sequence-based amplification NASBA (Organon), branched DNA 2.0 (bDNA) (Chiron), and Monitor (Roche), with reverse-transcription polymerase chain reaction (RT-PCR) as the reference. We assessed sensitivity and reproducibility on a well-characterized panel of sera (EUROHEP), a Chimp Rodney plasma pool, and samples from IFN-treated and -untreated patients with chronic hepatitis C caused by different HCV genotypes. The reproducibility of the NASBA and bDNA methods was slightly better than that of Monitor, especially for genotypes 2 and 4. NASBA had the highest sensitivity (99% vs. 94% and 88% with Monitor and bDNA, respectively), especially for the follow-up of patients on IFN. NASBA gave the highest HCV-RNA concentrations, which were approximately 10-fold more than with the bDNA assay and 100-fold more than with the Monitor kit. The linearity, tested on the chimp Rodney plasma pool, was better with bDNA for high viral load than with NASBA and Monitor, although for low concentration of HCV RNA, bDNA was negative. Pretreatment viral load was lower in patients who had a sustained virological response to IFN, although the bDNA method was not sensitive enough to quantify all pretreatment samples. This study indicates that gene amplification methods (NASBA or Monitor) have better sensitivity than bDNA assays for quantification of HCV RNA in patients with chronic HCV infection, although the bDNA and NASBA methods are more likely to quantify all genotypes. Prospective studies are needed to demonstrate the usefulness of quantitative assays for the follow-up of patients with chronic hepatitis C.

MBTH: A novel approach to rapid, spectrophotometric quantitation of total algal carbohydrates

DOE PAGES

Van Wychen, Stefanie; Long, William; Black, Stuart K.; ...

2016-11-24

A high-throughput and robust application of the 3-methyl-2-benzothiazolinone hydrazone (MBTH) method was developed for carbohydrate determination in microalgae. The traditional phenol-sulfuric acid method to quantify carbohydrates is strongly affected by algal biochemical components and exhibits a highly variable response to microalgal monosaccharides. We present a novel use of the MBTH method to accurately quantify carbohydrates in hydrolyzate after acid hydrolysis of algal biomass, without a need for neutralization. As a result, the MBTH method demonstrated consistent and sensitive quantitation of algae-specific monosaccharides down to 5 ug mL -1 without interference from other algae acidic hydrolyzate components.

MBTH: A novel approach to rapid, spectrophotometric quantitation of total algal carbohydrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Wychen, Stefanie; Long, William; Black, Stuart K.

A high-throughput and robust application of the 3-methyl-2-benzothiazolinone hydrazone (MBTH) method was developed for carbohydrate determination in microalgae. The traditional phenol-sulfuric acid method to quantify carbohydrates is strongly affected by algal biochemical components and exhibits a highly variable response to microalgal monosaccharides. We present a novel use of the MBTH method to accurately quantify carbohydrates in hydrolyzate after acid hydrolysis of algal biomass, without a need for neutralization. As a result, the MBTH method demonstrated consistent and sensitive quantitation of algae-specific monosaccharides down to 5 ug mL -1 without interference from other algae acidic hydrolyzate components.

A novel Alu-based real-time PCR method for the quantitative detection of plasma circulating cell-free DNA: Sensitivity and specificity for the diagnosis of myocardial infarction

PubMed Central

LOU, XIAOLI; HOU, YANQIANG; LIANG, DONGYU; PENG, LIANG; CHEN, HONGWEI; MA, SHANYUAN; ZHANG, LURONG

2015-01-01

In the present study, we aimed to develop and validate a rapid and sensitive, Alu-based real-time PCR method for the detection of circulating cell-free DNA (cfDNA). This method targeted repetitive elements of the Alu reduplicative elements in the human genome, followed by signal amplification using fluorescence quantification. Standard Alu-puc57 vectors were constructed and 5 pairs of specific primers were designed. Valuation was conducted concerning linearity, variation and recovery. We found 5 linear responses (R1–5=0.998–0.999). The average intra- and inter-assay coefficients of variance were 12.98 and 10.75%, respectively. The recovery was 82.33–114.01%, with a mean recovery index of 101.26%. This Alu-based assay was reliable, accurate and sensitive for the quantitative detection of cfDNA. Plasma from normal controls and patients with myocardial infarction (MI) were analyzed, and the baseline levels of cfDNA were higher in the MI group. The area under the receiver operating characteristic (ROC) curve for Alu1, Alu2, Alu3, Alu4, Alu5 and Alu (Alu1 + Alu2 + Alu3 + Alu4 + Alu5) was 0.887, 0.758, 0.857, 0.940, 0.968 and 0.933, respectively. The optimal cut-off value for Alu1, Alu2, Alu3, Alu4, Alu5 and Alu to predict MI was 3.71, 1.93, 0.22, 3.73, 6.13 and 6.40 log copies/ml. We demonstrate that this new method is a reliable, accurate and sensitive method for the quantitative detection of cfDNA and that it is useful for studying the regulation of cfDNA in certain pathological conditions. Alu4, Alu5 and Alu showed better sensitivity and specificity for the diagnosis of MI compared with cardiac troponin I (cTnI), creatine kinase MB (CK-MB) isoenzyme and lactate dehydrogenase (LDH). Alu5 had the best prognostic ability. PMID:25374065

ADC as a useful diagnostic tool for differentiating benign and malignant vertebral bone marrow lesions and compression fractures: a systematic review and meta-analysis.

PubMed

Suh, Chong Hyun; Yun, Seong Jong; Jin, Wook; Lee, Sun Hwa; Park, So Young; Ryu, Chang-Woo

2018-07-01

To assess the sensitivity and specificity of quantitative assessment of the apparent diffusion coefficient (ADC) for differentiating benign and malignant vertebral bone marrow lesions (BMLs) and compression fractures (CFs) METHODS: An electronic literature search of MEDLINE and EMBASE was conducted. Bivariate modelling and hierarchical summary receiver operating characteristic modelling were performed to evaluate the diagnostic performance of ADC for differentiating vertebral BMLs. Subgroup analysis was performed for differentiating benign and malignant vertebral CFs. Meta-regression analyses according to subject, study and diffusion-weighted imaging (DWI) characteristics were performed. Twelve eligible studies (748 lesions, 661 patients) were included. The ADC exhibited a pooled sensitivity of 0.89 (95% confidence interval [CI] 0.80-0.94) and a pooled specificity of 0.87 (95% CI 0.78-0.93) for differentiating benign and malignant vertebral BMLs. In addition, the pooled sensitivity and specificity for differentiating benign and malignant CFs were 0.92 (95% CI 0.82-0.97) and 0.91 (95% CI 0.87-0.94), respectively. In the meta-regression analysis, the DWI slice thickness was a significant factor affecting heterogeneity (p < 0.01); thinner slice thickness (< 5 mm) showed higher specificity (95%) than thicker slice thickness (81%). Quantitative assessment of ADC is a useful diagnostic tool for differentiating benign and malignant vertebral BMLs and CFs. • Quantitative assessment of ADC is useful in differentiating vertebral BMLs. • Quantitative ADC assessment for BMLs had sensitivity of 89%, specificity of 87%. • Quantitative ADC assessment for CFs had sensitivity of 92%, specificity of 91%. • The specificity is highest (95%) with thinner (< 5 mm) DWI slice thickness.

Comparative evaluation of two Rickettsia typhi-specific quantitative real-time PCRs for research and diagnostic purposes.

PubMed

Papp, Stefanie; Rauch, Jessica; Kuehl, Svenja; Richardt, Ulricke; Keller, Christian; Osterloh, Anke

2017-02-01

Rickettsioses are caused by intracellular bacteria of the family of Rickettsiaceae. Rickettsia (R.) typhi is the causative agent of endemic typhus. The disease occurs worldwide and is one of the most prevalent rickettsioses. Rickettsial diseases, however, are generally underdiagnosed which is mainly due to the lack of sensitive and specific methods. In addition, methods for quantitative detection of the bacteria for research purposes are rare. We established two qPCRs for the detection of R. typhi by amplification of the outer membrane protein B (ompB) and parvulin-type PPIase (prsA) genes. Both qPCRs are specific and exclusively recognize R. typhi but no other rickettsiae including the closest relative, R. prowazekii. The prsA-based qPCR revealed to be much more sensitive than the amplification of ompB and provided highly reproducible results in the detection of R. typhi in organs of infected mice. Furthermore, as a nested PCR the prsA qPCR was applicable for the detection of R. typhi in human blood samples. Collectively, the prsA-based qPCR represents a reliable method for the quantitative detection of R. typhi for research purposes and is a promising candidate for differential diagnosis.

[Clinical significance of monitoring BK polyomavirus in patients after hematopoietic stem cell transplantation].

PubMed

Yin, Chang-Xin; Jiang, Qian-Li; He, Han; Yu, Guo-Pan; Xu, Yue; Meng, Fan-Yi; Yang, Mo

2012-02-01

This study was aimed to establish a method for rapid detecting BK polyomavirus (BKV) and to investigate the feasibility and value used in leukemia patients undergoing hematopoietic stem cell transplantation. Primers were designed according to BKV gene sequence; the quantitative standards for BKV and a real-time fluorescent quantitative PCR for BKV were established. The BKV level in urine samples from 36 patients after hematopoietic stem cell transplantation were detected by established method. The results showed that the standard of reconstructed plasmid and real time fluorescent quantitative PCR method were successfully established, its good specificity, sensitivity and stability were confirmed by experiments. BKV was found in 55.56% of urine samples, and the BKV load in urine was 2.46 × 10(4) - 7.8 × 10(9) copy/ml. It is concluded that the establishment of real-time fluorescent quantitative PCR for BKV detection provides a method for early diagnosis of the patients with hemorrhagic cystitis after hematopoietic stem cell transplantation.

Quantitative diagnostic method for biceps long head tendinitis by using ultrasound.

PubMed

Huang, Shih-Wei; Wang, Wei-Te

2013-01-01

To investigate the feasibility of grayscale quantitative diagnostic method for biceps tendinitis and determine the cut-off points of a quantitative biceps ultrasound (US) method to diagnose biceps tendinitis. Design. Prospective cross-sectional case controlled study. Outpatient rehabilitation service. A total of 336 shoulder pain patients with suspected biceps tendinitis were recruited in this prospective observational study. The grayscale pixel data of the range of interest (ROI) were obtained for both the transverse and longitudinal views of the biceps US. A total of 136 patients were classified with biceps tendinitis, and 200 patients were classified as not having biceps tendinitis based on the diagnostic criteria. Based on the Youden index, the cut-off points were determined as 26.85 for the transverse view and 21.25 for the longitudinal view of the standard deviation (StdDev) of the ROI values, respectively. When the ROI evaluation of the US surpassed the cut-off point, the sensitivity was 68% and the specificity was 90% in the StdDev of the transverse view, and the sensitivity was 81% and the specificity was 73% in the StdDev of the longitudinal view to diagnose biceps tendinitis. For equivocal cases or inexperienced sonographers, our study provides a more objective method for diagnosing biceps tendinitis in shoulder pain patients.

Precise Quantitation of MicroRNA in a Single Cell with Droplet Digital PCR Based on Ligation Reaction.

PubMed

Tian, Hui; Sun, Yuanyuan; Liu, Chenghui; Duan, Xinrui; Tang, Wei; Li, Zhengping

2016-12-06

MicroRNA (miRNA) analysis in a single cell is extremely important because it allows deep understanding of the exact correlation between the miRNAs and cell functions. Herein, we wish to report a highly sensitive and precisely quantitative assay for miRNA detection based on ligation-based droplet digital polymerase chain reaction (ddPCR), which permits the quantitation of miRNA in a single cell. In this ligation-based ddPCR assay, two target-specific oligonucleotide probes can be simply designed to be complementary to the half-sequence of the target miRNA, respectively, which avoids the sophisticated design of reverse transcription and provides high specificity to discriminate a single-base difference among miRNAs with simple operations. After the miRNA-templated ligation, the ddPCR partitions individual ligated products into a water-in-oil droplet and digitally counts the fluorescence-positive and negative droplets after PCR amplification for quantification of the target molecules, which possesses the power of precise quantitation and robustness to variation in PCR efficiency. By integrating the advantages of the precise quantification of ddPCR and the simplicity of the ligation-based PCR, the proposed method can sensitively measure let-7a miRNA with a detection limit of 20 aM (12 copies per microliter), and even a single-base difference can be discriminated in let-7 family members. More importantly, due to its high selectivity and sensitivity, the proposed method can achieve precise quantitation of miRNAs in single-cell lysate. Therefore, the ligation-based ddPCR assay may serve as a useful tool to exactly reveal the miRNAs' actions in a single cell, which is of great importance for the study of miRNAs' biofunction as well as for the related biomedical studies.

A quantitative headspace-solid-phase microextraction-gas chromatography-flame ionization detector method to analyze short chain free fatty acids in rat feces.

PubMed

Fiorini, Dennis; Boarelli, Maria Chiara; Gabbianelli, Rosita; Ballini, Roberto; Pacetti, Deborah

2016-09-01

This study sought to develop and validate a quantitative method to analyze short chain free fatty acids (SCFAs) in rat feces by solid-phase microextraction and gas chromatography (SPME-GC) using the salt mixture ammonium sulfate and sodium dihydrogen phosphate as salting out agent. Conditioning and extraction time, linearity, limits of detection and quantification, repeatability, and recovery were evaluated. The proposed method allows quantification with improved sensitivity as compared with other methods exploiting SPME-GC. The method has been applied to analyze rat fecal samples, quantifying acetic, propionic, isobutyric, butyric, isopentanoic, pentanoic, and hexanoic acids. Copyright © 2016 Elsevier Inc. All rights reserved.

Highly sensitive fluorescence quantitative detection of specific DNA sequences with molecular beacons and nucleic acid dye SYBR Green I.

PubMed

Xiang, Dongshan; Zhai, Kun; Xiang, Wenjun; Wang, Lianzhi

2014-11-01

A highly sensitive fluorescence method of quantitative detection for specific DNA sequence is developed based on molecular beacon (MB) and nucleic acid dye SYBR Green I by synchronous fluorescence analysis. It is demonstrated by an oligonucleotide sequence of wild-type HBV (target DNA) as a model system. In this strategy, the fluorophore of MB is designed to be 6-carboxyfluorescein group (FAM), and the maximum excitation wavelength and maximum emission wavelength are both very close to that of SYBR Green I. In the presence of targets DNA, the MBs hybridize with the targets DNA and form double-strand DNA (dsDNA), the fluorophore FAM is separated from the quencher BHQ-1, thus the fluorophore emit fluorescence. At the same time, SYBR Green I binds to dsDNA, the fluorescence intensity of SYBR Green I is significantly enhanced. When targets DNA are detected by synchronous fluorescence analysis, the fluorescence peaks of FAM and SYBR Green I overlap completely, so the fluorescence signal of system will be significantly enhanced. Thus, highly sensitive fluorescence quantitative detection for DNA can be realized. Under the optimum conditions, the total fluorescence intensity of FAM and SYBR Green I exhibits good linear dependence on concentration of targets DNA in the range from 2×10(-11) to 2.5×10(-9)M. The detection limit of target DNA is estimated to be 9×10(-12)M (3σ). Compared with previously reported methods of detection DNA with MB, the proposed method can significantly enhance the detection sensitivity. Copyright © 2014 Elsevier B.V. All rights reserved.

Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for the detection of the fathead minnow nidovirus.

PubMed

Zhang, Qingli; Standish, Isaac; Winters, Andrew D; Puzach, Corey; Ulferts, Rachel; Ziebuhr, John; Faisal, Mohamed

2014-06-01

Fathead minnow nidovirus (FHMNV) is a serious baitfish-pathogenic virus in North America. Studies to trace the spread of the virus and determine its host range are hampered by the absence of reliable diagnostic assays. In this study, a one-step, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed that targets a region in the FHMNV spike protein gene. The assay was optimized, and the best results were obtained at 8 mM of Mg(2+) with an incubation time of 40 min at 63 °C in the presence of calcein. The analytical sensitivity of the RT-LAMP method was estimated to be as low as 5 viral copies and was 1000-fold more sensitive than the conventional reverse transcription polymerase chain reaction (RT-PCR) method. The diagnostic sensitivity and specificity of the developed RT-LAMP assay versus the RT-PCR assay was 100% and 95.7%, respectively. A quantitative RT-LAMP of FHMNV with a high correlation coefficient (r(2)=0.9926) was also developed and the result of quantitation of viral copies in tissue samples of infected fish showed that the viral loads of the infected fish tissue samples reached up to 4.7×10(10) copies per mg. It is anticipated that the developed RT-LAMP and quantitative RT-LAMP methods will be instrumental for diagnosis and surveillance of FHMNV. Copyright © 2014 Elsevier B.V. All rights reserved.

A model for the characterization of the spatial properties in vestibular neurons

NASA Technical Reports Server (NTRS)

Angelaki, D. E.; Bush, G. A.; Perachio, A. A.

1992-01-01

Quantitative study of the static and dynamic response properties of some otolith-sensitive neurons has been difficult in the past partly because their responses to different linear acceleration vectors exhibited no "null" plane and a dependence of phase on stimulus orientation. The theoretical formulation of the response ellipse provides a quantitative way to estimate the spatio-temporal properties of such neurons. Its semi-major axis gives the direction of the polarization vector (i.e., direction of maximal sensitivity) and it estimates the neuronal response for stimulation along that direction. In addition, the semi-minor axis of the ellipse provides an estimate of the neuron's maximal sensitivity in the "null" plane. In this paper, extracellular recordings from otolith-sensitive vestibular nuclei neurons in decerebrate rats were used to demonstrate the practical application of the method. The experimentally observed gain and phase dependence on the orientation angle of the acceleration vector in a head-horizontal plane was described and satisfactorily fit by the response ellipse model. In addition, the model satisfactorily fits neuronal responses in three-dimensions and unequivocally demonstrates that the response ellipse formulation is the general approach to describe quantitatively the spatial properties of vestibular neurons.

Development of a pressurized liquid extraction-solid-phase extraction followed by liquid chromatography-electrospray ionization tandem mass spectrometry method for the quantitative determination of benzoxazolinones and their degradation products in agricultural soil.

PubMed

Villagrasa, M; Guillamón, M; Navarro, A; Eljarrat, E; Barceló, D

2008-02-01

A new analytical method for the quantitative determination of benzoxazolinones and their degradation products in agricultural soils based on the use of pressurized liquid extraction (PLE) followed by solid-phase extraction (SPE) and then instrumental determination using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) is described. Using this method, the characterization, separation and quantitative detection of a mixture of two benzoxazolinones, benzoxazolin-2-one (BOA) and 6-methoxybenzoxazolin-2-one (MBOA) and their degradation products, 2-aminophenol (APH), N-(2-hydroxyphenyl)malonamic acid (HMPMA), 2-amino-3-H-phenoxazin-3-one (APO), 9-methoxy-2-amino-3-H-phenoxazin-3-one (AMPO), 2-acetylamino-3-H-phenoxazin-3-one (AAPO) and 2-acetylamino-9-methoxy-2-amino-3-H-phenoxazin-3-one (AAMPO) was achieved. The complete LC-ESI-MS-MS precursor-product ion fragmentation pathways for the degradation products of benzoxazolinones are described for the first time. Quantitative analysis was done in the multiple reaction mode using two specific combinations of precursor-product ion transitions for each compound. The optimized method was quality assessed by the measure of parameter as recovery, linearity, sensitivity, repeatability and reproducibility. Recoveries of the analytes ranged from 53 to 123%. The developed method offered improvements to the sensitivity as compared with our previously LC-MS method, with detection limits down to 2.4-21 ng/g of dry weight. This achievement allows us to identify and quantify for the first time degradation products of benzoxazolinones in real agricultural soil samples. Analytes were found in the range of 20.6-149 ng/g dry weight.

A New Kinetic Spectrophotometric Method for the Quantitation of Amorolfine.

PubMed

Soto, César; Poza, Cristian; Contreras, David; Yáñez, Jorge; Nacaratte, Fallon; Toral, M Inés

2017-01-01

Amorolfine (AOF) is a compound with fungicide activity based on the dual inhibition of growth of the fungal cell membrane, the biosynthesis and accumulation of sterols, and the reduction of ergosterol. In this work a sensitive kinetic and spectrophotometric method for the AOF quantitation based on the AOF oxidation by means of KMnO 4 at 30 min (fixed time), pH alkaline, and ionic strength controlled was developed. Measurements of changes in absorbance at 610 nm were used as criterion of the oxidation progress. In order to maximize the sensitivity, different experimental reaction parameters were carefully studied via factorial screening and optimized by multivariate method. The linearity, intraday, and interday assay precision and accuracy were determined. The absorbance-concentration plot corresponding to tap water spiked samples was rectilinear, over the range of 7.56 × 10 -6 -3.22 × 10 -5  mol L -1 , with detection and quantitation limits of 2.49 × 10 -6  mol L -1 and 7.56 × 10 -6  mol L -1 , respectively. The proposed method was successfully validated for the application of the determination of the drug in the spiked tap water samples and the percentage recoveries were 94.0-105.0%. The method is simple and does not require expensive instruments or complicated extraction steps of the reaction product.

Dose limited reliability of quantitative annular dark field scanning transmission electron microscopy for nano-particle atom-counting.

PubMed

De Backer, A; Martinez, G T; MacArthur, K E; Jones, L; Béché, A; Nellist, P D; Van Aert, S

2015-04-01

Quantitative annular dark field scanning transmission electron microscopy (ADF STEM) has become a powerful technique to characterise nano-particles on an atomic scale. Because of their limited size and beam sensitivity, the atomic structure of such particles may become extremely challenging to determine. Therefore keeping the incoming electron dose to a minimum is important. However, this may reduce the reliability of quantitative ADF STEM which will here be demonstrated for nano-particle atom-counting. Based on experimental ADF STEM images of a real industrial catalyst, we discuss the limits for counting the number of atoms in a projected atomic column with single atom sensitivity. We diagnose these limits by combining a thorough statistical method and detailed image simulations. Copyright © 2014 Elsevier B.V. All rights reserved.

A simple and rapid DNA extraction method for Chlamydia trachomatis detection from urogenital swabs.

PubMed

Butzler, Matthew A; Reed, Jennifer L; McFall, Sally M

2017-11-01

A highly sensitive and specific Chlamydia trachomatis (CT) diagnostic test was developed by combining filtration isolation of nucleic acid (FINA) extraction with quantitative polymerase chain reaction including an internal control to identify test inhibition. A pilot study of 40 clinical specimens yielded 100% sensitivity and specificity. Copyright © 2017 Elsevier Inc. All rights reserved.

Assessing direct analysis in real-time-mass spectrometry (DART-MS) for the rapid identification of additives in food packaging.

PubMed

Ackerman, L K; Noonan, G O; Begley, T H

2009-12-01

The ambient ionization technique direct analysis in real time (DART) was characterized and evaluated for the screening of food packaging for the presence of packaging additives using a benchtop mass spectrometer (MS). Approximate optimum conditions were determined for 13 common food-packaging additives, including plasticizers, anti-oxidants, colorants, grease-proofers, and ultraviolet light stabilizers. Method sensitivity and linearity were evaluated using solutions and characterized polymer samples. Additionally, the response of a model additive (di-ethyl-hexyl-phthalate) was examined across a range of sample positions, DART, and MS conditions (temperature, voltage and helium flow). Under optimal conditions, molecular ion (M+H+) was the major ion for most additives. Additive responses were highly sensitive to sample and DART source orientation, as well as to DART flow rates, temperatures, and MS inlet voltages, respectively. DART-MS response was neither consistently linear nor quantitative in this setting, and sensitivity varied by additive. All additives studied were rapidly identified in multiple food-packaging materials by DART-MS/MS, suggesting this technique can be used to screen food packaging rapidly. However, method sensitivity and quantitation requires further study and improvement.

Gold Nanoparticles as a Direct and Rapid Sensor for Sensitive Analytical Detection of Biogenic Amines

NASA Astrophysics Data System (ADS)

El-Nour, K. M. A.; Salam, E. T. A.; Soliman, H. M.; Orabi, A. S.

2017-03-01

A new optical sensor was developed for rapid screening with high sensitivity for the existence of biogenic amines (BAs) in poultry meat samples. Gold nanoparticles (GNPs) with particle size 11-19 nm function as a fast and sensitive biosensor for detection of histamine resulting from bacterial decarboxylation of histidine as a spoilage marker for stored poultry meat. Upon reaction with histamine, the red color of the GNPs converted into deep blue. The appearance of blue color favorably coincides with the concentration of BAs that can induce symptoms of poisoning. This biosensor enables a semi-quantitative detection of analyte in real samples by eye-vision. Quality evaluation is carried out by measuring histamine and histidine using different analytical techniques such as UV-vis, FTIR, and fluorescence spectroscopy as well as TEM. A rapid quantitative readout of samples by UV-vis and fluorescence methods with standard instrumentation were proposed in a short time unlike chromatographic and electrophoretic methods. Sensitivity and limit of detection (LOD) of 6.59 × 10-4 and 0.6 μM, respectively, are determined for histamine as a spoilage marker with a correlation coefficient ( R 2) of 0.993.

A Novel Health Evaluation Strategy for Multifunctional Self-Validating Sensors

PubMed Central

Shen, Zhengguang; Wang, Qi

2013-01-01

The performance evaluation of sensors is very important in actual application. In this paper, a theory based on multi-variable information fusion is studied to evaluate the health level of multifunctional sensors. A novel conception of health reliability degree (HRD) is defined to indicate a quantitative health level, which is different from traditional so-called qualitative fault diagnosis. To evaluate the health condition from both local and global perspectives, the HRD of a single sensitive component at multiple time points and the overall multifunctional sensor at a single time point are defined, respectively. The HRD methodology is emphasized by using multi-variable data fusion technology coupled with a grey comprehensive evaluation method. In this method, to acquire the distinct importance of each sensitive unit and the sensitivity of different time points, the information entropy and analytic hierarchy process method are used, respectively. In order to verify the feasibility of the proposed strategy, a health evaluating experimental system for multifunctional self-validating sensors was designed. The five different health level situations have been discussed. Successful results show that the proposed method is feasible, the HRD could be used to quantitatively indicate the health level and it does have a fast response to the performance changes of multifunctional sensors. PMID:23291576

A new real-time method for investigation of affinity properties and binding kinetics of magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Orlov, Alexey V.; Nikitin, Maxim P.; Bragina, Vera A.; Znoyko, Sergey L.; Zaikina, Marina N.; Ksenevich, Tatiana I.; Gorshkov, Boris G.; Nikitin, Petr I.

2015-04-01

A method for quantitative investigation of affinity constants of receptors immobilized on magnetic nanoparticles (MP) is developed based on spectral correlation interferometry (SCI). The SCI records with a picometer resolution the thickness changes of a layer of molecules or nanoparticles due to a biochemical reaction on a cover slip, averaged over the sensing area. The method is compatible with other types of sensing surfaces employed in biosensing. The measured values of kinetic association constants of magnetic nanoparticles are 4 orders of magnitude higher than those of molecular antibody association with antigen. The developed method also suggests highly sensitive detection of antigens in a wide dynamic range. The limit of detection of 92 pg/ml has been demonstrated for prostate-specific antigen (PSA) with 50-nm MP employed as labels, which produce 3-order amplification of the SCI signals. The calibration curve features high sensitivity (slope) of 3-fold signal raise per 10-fold increase of PSA concentration within 4-order dynamic range, which is an attractive compromise for precise quantitative and highly sensitive immunoassay. The proposed biosensing technique offers inexpensive disposable sensor chips of cover slips and represents an economically sound alternative to traditional immunoassays for disease diagnostics, detection of pathogens in food and environmental monitoring.

Effects of calibration methods on quantitative material decomposition in photon-counting spectral computed tomography using a maximum a posteriori estimator.

PubMed

Curtis, Tyler E; Roeder, Ryan K

2017-10-01

Advances in photon-counting detectors have enabled quantitative material decomposition using multi-energy or spectral computed tomography (CT). Supervised methods for material decomposition utilize an estimated attenuation for each material of interest at each photon energy level, which must be calibrated based upon calculated or measured values for known compositions. Measurements using a calibration phantom can advantageously account for system-specific noise, but the effect of calibration methods on the material basis matrix and subsequent quantitative material decomposition has not been experimentally investigated. Therefore, the objective of this study was to investigate the influence of the range and number of contrast agent concentrations within a modular calibration phantom on the accuracy of quantitative material decomposition in the image domain. Gadolinium was chosen as a model contrast agent in imaging phantoms, which also contained bone tissue and water as negative controls. The maximum gadolinium concentration (30, 60, and 90 mM) and total number of concentrations (2, 4, and 7) were independently varied to systematically investigate effects of the material basis matrix and scaling factor calibration on the quantitative (root mean squared error, RMSE) and spatial (sensitivity and specificity) accuracy of material decomposition. Images of calibration and sample phantoms were acquired using a commercially available photon-counting spectral micro-CT system with five energy bins selected to normalize photon counts and leverage the contrast agent k-edge. Material decomposition of gadolinium, calcium, and water was performed for each calibration method using a maximum a posteriori estimator. Both the quantitative and spatial accuracy of material decomposition were most improved by using an increased maximum gadolinium concentration (range) in the basis matrix calibration; the effects of using a greater number of concentrations were relatively small in magnitude by comparison. The material basis matrix calibration was more sensitive to changes in the calibration methods than the scaling factor calibration. The material basis matrix calibration significantly influenced both the quantitative and spatial accuracy of material decomposition, while the scaling factor calibration influenced quantitative but not spatial accuracy. Importantly, the median RMSE of material decomposition was as low as ~1.5 mM (~0.24 mg/mL gadolinium), which was similar in magnitude to that measured by optical spectroscopy on the same samples. The accuracy of quantitative material decomposition in photon-counting spectral CT was significantly influenced by calibration methods which must therefore be carefully considered for the intended diagnostic imaging application. © 2017 American Association of Physicists in Medicine.

Error analysis applied to several inversion techniques used for the retrieval of middle atmospheric constituents from limb-scanning MM-wave spectroscopic measurements

NASA Technical Reports Server (NTRS)

Puliafito, E.; Bevilacqua, R.; Olivero, J.; Degenhardt, W.

1992-01-01

The formal retrieval error analysis of Rodgers (1990) allows the quantitative determination of such retrieval properties as measurement error sensitivity, resolution, and inversion bias. This technique was applied to five numerical inversion techniques and two nonlinear iterative techniques used for the retrieval of middle atmospheric constituent concentrations from limb-scanning millimeter-wave spectroscopic measurements. It is found that the iterative methods have better vertical resolution, but are slightly more sensitive to measurement error than constrained matrix methods. The iterative methods converge to the exact solution, whereas two of the matrix methods under consideration have an explicit constraint, the sensitivity of the solution to the a priori profile. Tradeoffs of these retrieval characteristics are presented.

Quantitative control of CaCO3 growth on quartz crystal microbalance sensors as a signal amplification method.

PubMed

Wu, Congcong; Sun, Zhaomei; Liu, Li-Shang

2017-07-10

The surface crystallization of CaCO 3 on gold was monitored by a quartz crystal microbalance (QCM). Quantitative control of the grown crystals was realized by adjusting the ratio of two functional groups, -N(CH 3 ) 3 and -COOH, on SAMs. Crystals with uniform size, morphology and polymorphism were obtained. The amount of crystals formed was found to increase with an increase in the -COOH group. The proposed quantitative control of crystallization can be an effective mass amplification strategy for QCM to enhance its assay sensitivity.

Sensitive and selective determination of methylenedioxylated amphetamines by high-performance liquid chromatography with fluorimetric detection.

PubMed

Sadeghipour, F; Veuthey, J L

1997-11-07

A rapid, sensitive and selective liquid chromatographic method with fluorimetric detection was developed for the separation and quantification of four methylenedioxylated amphetamines without interference of other drugs of abuse and common substances found in illicit tablets. The method was validated by examining linearity, precision and accuracy as well as detection and quantification limits. Methylenedioxylated amphetamines were quantified in eight tablets from illicit drug seizures and results were quantitatively compared to HPLC-UV analyses. To demonstrate the better sensitivity of the fluorimetric detection, methylenedioxylated amphetamines were analyzed in serum after a liquid-liquid extraction procedure and results were also compared to HPLC-UV analyses.

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

PubMed

Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

A simple and highly sensitive colorimetric detection method for gaseous formaldehyde.

PubMed

Feng, Liang; Musto, Christopher J; Suslick, Kenneth S

2010-03-31

A colorimetric detection method using amine-functionalized polymer films doped with a pH indicator has been developed for the rapid, sensitive, and quantitative detection of gaseous formaldehyde at concentrations well below the immediately dangerous to life or health (IDLH) limit. In 1 min, visible color changes are easily observed, even down to the permissible exposure limit (PEL) at 750 ppb. The limit of detection is below 50 ppb (7% of the PEL) after 10 min of exposure. This sensor is essentially unaffected by changes in humidity or temperature (4 to 50 degrees C) and is not sensitive to common interferents.

Light scattering application for quantitative estimation of apoptosis

NASA Astrophysics Data System (ADS)

Bilyy, Rostyslav O.; Stoika, Rostyslav S.; Getman, Vasyl B.; Bilyi, Olexander I.

2004-05-01

Estimation of cell proliferation and apoptosis are in focus of instrumental methods used in modern biomedical sciences. Present study concerns monitoring of functional state of cells, specifically the development of their programmed death or apoptosis. The available methods for such purpose are either very expensive, or require time-consuming operations. Their specificity and sensitivity are frequently not sufficient for making conclusions which could be used in diagnostics or treatment monitoring. We propose a novel method for apoptosis measurement based on quantitative determination of cellular functional state taking into account their physical characteristics. This method uses the patented device -- laser microparticle analyser PRM-6 -- for analyzing light scattering by the microparticles, including cells. The method gives an opportunity for quick, quantitative, simple (without complicated preliminary cell processing) and relatively cheap measurement of apoptosis in cellular population. The elaborated method was used for studying apoptosis expression in murine leukemia cells of L1210 line and human lymphoblastic leukemia cells of K562 line. The results obtained by the proposed method permitted measuring cell number in tested sample, detecting and quantitative characterization of functional state of cells, particularly measuring the ratio of the apoptotic cells in suspension.

Investigation of Ion Transmission Effects on Intact Protein Quantification in a Triple Quadrupole Mass Spectrometer

NASA Astrophysics Data System (ADS)

Wang, Evelyn H.; Appulage, Dananjaya Kalu; McAllister, Erin A.; Schug, Kevin A.

2017-09-01

Recently, direct intact protein quantitation using triple quadrupole mass spectrometry (QqQ-MS) and multiple reaction monitoring (MRM) was demonstrated (J. Am. Soc. Mass Spectrom. 27, 886-896 (2016)). Even though QqQ-MS is known to provide extraordinary detection sensitivity for quantitative analysis, we found that intact proteins exhibited a less than 5% ion transmission from the first quadrupole to the third quadrupole mass analyzer in the presence of zero collision energy (ZCE). With the goal to enhance intact protein quantitation sensitivity, ion scattering effects, proton transfer effects, and mass filter resolution widths were examined for their contributions to the lost signal. Protein standards myoglobin and ubiquitin along with small molecules reserpine and vancomycin were analyzed together with various collision induced dissociation (CID) gases (N2, He, and Ar) at different gas pressures. Mass resolution settings played a significant role in reducing ion transmission signal. By narrowing the mass resolution window by 0.35 m/z on each side, roughly 75%-90% of the ion signal was lost. The multiply charged proteins experienced additional proton transfer effects, corresponding to 10-fold signal reduction. A study of increased sensitivity of the method was also conducted with various MRM summation techniques. Although the degree of enhancement was analyte-dependent, an up to 17-fold increase in sensitivity was observed for ubiquitin using a summation of multiple MRM transitions. Biological matrix, human urine, and equine plasma were spiked with proteins to demonstrate the specificity of the method. This study provides additional insight into optimizing the use and sensitivity of QqQ-MS for intact protein quantification. [Figure not available: see fulltext.

Prevalence of distal diabetic polyneuropathy using quantitative sensory methods in a population with diabetes of more than 10 years' disease duration.

PubMed

Miralles-García, José M; de Pablos-Velasco, Pedro; Cabrerizo, Lucio; Pérez, María; López-Gómez, Vanessa

2010-11-01

Results of studies on the prevalence of distal diabetic polyneuropathy (DPN) are contradictory. Conventional methods used for the diagnosis of DPN in clinical practice have limited effectiveness. The present study aimed to assess the prevalence of DPN in a population with long-standing diabetes (more than 10 years disease duration) by measuring vibratory, thermal and tactile sensitivities with quantitative sensory devices, as well as their relationship with associated clinical risk factors. A total of 1011 diabetic patients were evaluated in a multicenter, cross-sectional, observational study. The three sensitivities were assessed by ultrabiothesiometer, aesthesiometer and thermoskin devices, respectively. The prevalence of neuropathic pain was validated by the DN4 questionnaire. Of the 1011 cases included, 400 (39.6%) met the diagnostic criteria of DPN, while no DPN was found in the remaining 611 (60.4%). Of the 400 patients with DPN, 253 (63.2%) showed clinical manifestations, while 147 (36.8%) were diagnosed as subclinical DPN. The prevalence of DPN increased with disease duration. There was a progressive loss of the three sensitivities with increased disease duration, particularly thermal and vibratory sensitivities. This loss was statistically significant for the latter two sensitivities. Among patients with clinical DPN, 84.2% had painful neuropathic symptoms. The prevalence of DPN was positively related to micro- and macroangiopathic complications and with dyslipidemia. This study reveals a high degree of underdiagnosis of DPN, most likely due to the asymptomatic nature of the disease in a considerable proportion of patients. Our observations provide evidence of the usefulness of specific equipment for quantitative and objective assessment of polyneuropathy. Copyright © 2010 SEEN. Published by Elsevier Espana. All rights reserved.

MBTH: A novel approach to rapid, spectrophotometric quantitation of total algal carbohydrates.

PubMed

Van Wychen, Stefanie; Long, William; Black, Stuart K; Laurens, Lieve M L

2017-02-01

A high-throughput and robust application of the 3-methyl-2-benzothiazolinone hydrazone (MBTH) method was developed for carbohydrate determination in microalgae. The traditional phenol-sulfuric acid method to quantify carbohydrates is strongly affected by algal biochemical components and exhibits a highly variable response to microalgal monosaccharides. We present a novel use of the MBTH method to accurately quantify carbohydrates in hydrolyzate after acid hydrolysis of algal biomass, without a need for neutralization. The MBTH method demonstrated consistent and sensitive quantitation of algae-specific monosaccharides down to 5 μg mL -1 without interference from other algae acidic hydrolyzate components. Copyright © 2016 Elsevier Inc. All rights reserved.

A comparison of sorptive extraction techniques coupled to a new quantitative, sensitive, high throughput GC-MS/MS method for methoxypyrazine analysis in wine.

PubMed

Hjelmeland, Anna K; Wylie, Philip L; Ebeler, Susan E

2016-02-01

Methoxypyrazines are volatile compounds found in plants, microbes, and insects that have potent vegetal and earthy aromas. With sensory detection thresholds in the low ng L(-1) range, modest concentrations of these compounds can profoundly impact the aroma quality of foods and beverages, and high levels can lead to consumer rejection. The wine industry routinely analyzes the most prevalent methoxypyrazine, 2-isobutyl-3-methoxypyrazine (IBMP), to aid in harvest decisions, since concentrations decrease during berry ripening. In addition to IBMP, three other methoxypyrazines IPMP (2-isopropyl-3-methoxypyrazine), SBMP (2-sec-butyl-3-methoxypyrazine), and EMP (2-ethyl-3-methoxypyrazine) have been identified in grapes and/or wine and can impact aroma quality. Despite their routine analysis in the wine industry (mostly IBMP), accurate methoxypyrazine quantitation is hindered by two major challenges: sensitivity and resolution. With extremely low sensory detection thresholds (~8-15 ng L(-1) in wine for IBMP), highly sensitive analytical methods to quantify methoxypyrazines at trace levels are necessary. Here we were able to achieve resolution of IBMP as well as IPMP, EMP, and SBMP from co-eluting compounds using one-dimensional chromatography coupled to positive chemical ionization tandem mass spectrometry. Three extraction techniques HS-SPME (headspace-solid phase microextraction), SBSE (stirbar sorptive extraction), and HSSE (headspace sorptive extraction) were validated and compared. A 30 min extraction time was used for HS-SPME and SBSE extraction techniques, while 120 min was necessary to achieve sufficient sensitivity for HSSE extractions. All extraction methods have limits of quantitation (LOQ) at or below 1 ng L(-1) for all four methoxypyrazines analyzed, i.e., LOQ's at or below reported sensory detection limits in wine. The method is high throughput, with resolution of all compounds possible with a relatively rapid 27 min GC oven program. Copyright © 2015 Elsevier B.V. All rights reserved.

Diagnosis of feline leukaemia virus infection by semi-quantitative real-time polymerase chain reaction.

PubMed

Pinches, Mark D G; Helps, Christopher R; Gruffydd-Jones, Tim J; Egan, Kathy; Jarrett, Oswald; Tasker, Séverine

2007-02-01

In this paper the design and use of a semi-quantitative real-time polymerase chain reaction assay (RT-PCR) for feline leukaemia virus (FeLV) provirus is described. Its performance is evaluated against established methods of FeLV diagnosis, including virus isolation and enzyme-linked immunoassay (ELISA) in a population of naturally infected cats. The RT-PCR assay is found to have both a high sensitivity (0.92) and specificity (0.99) when examined by expectation maximisation methods and is also able to detect a large number of cats with low FeLV proviral loads that were negative by other conventional test methods.

Digital PCR analysis of circulating nucleic acids.

PubMed

Hudecova, Irena

2015-10-01

Detection of plasma circulating nucleic acids (CNAs) requires the use of extremely sensitive and precise methods. The commonly used quantitative real-time polymerase chain reaction (PCR) poses certain technical limitations in relation to the precise measurement of CNAs whereas the costs of massively parallel sequencing are still relatively high. Digital PCR (dPCR) now represents an affordable and powerful single molecule counting strategy to detect minute amounts of genetic material with performance surpassing many quantitative methods. Microfluidic (chip) and emulsion (droplet)-based technologies have already been integrated into platforms offering hundreds to millions of nanoliter- or even picoliter-scale reaction partitions. The compelling observations reported in the field of cancer research, prenatal testing, transplantation medicine and virology support translation of this technology into routine use. Extremely sensitive plasma detection of rare mutations originating from tumor or placental cells among a large background of homologous sequences facilitates unraveling of the early stages of cancer or the detection of fetal mutations. Digital measurement of quantitative changes in plasma CNAs associated with cancer or graft rejection provides valuable information on the monitoring of disease burden or the recipient's immune response and subsequent therapy treatment. Furthermore, careful quantitative assessment of the viral load offers great value for effective monitoring of antiviral therapy for immunosuppressed or transplant patients. The present review describes the inherent features of dPCR that make it exceptionally robust in precise and sensitive quantification of CNAs. Moreover, I provide an insight into the types of potential clinical applications that have been developed by researchers to date. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Simultaneous determination of L-tetrahydropalmatine and cocaine in human plasma by simple UPLC-FLD method: application in clinical studies.

PubMed

Yu, Mingming; Hassan, Hazem E; Ibrahim, Ahmed; Bauer, Kenneth S; Kelly, Deanna L; Wang, Jia Bei

2014-08-15

Currently, there are no FDA approved medications for treatment of cocaine addiction underscoring the dire need to develop such a product. There is an accumulating body of evidence that l-tetrahydropalmatine (l-THP), a non-selective dopamine antagonist, can be used for the treatment of cocaine addiction. Indeed, the FDA recently approved its usage in a Phase I study in cocaine abusers and it was indispensable to develop a simple and sensitive method for the simultaneous determination of l-THP and cocaine in human plasma. We developed a UPLC-FLD method for quantitation of these molecules using an ACQUITY BEH C18 column (2.1 mm × 50mm, 1.7 μm) and a mobile phase that consisted of 10mM ammonium phosphate (pH=4.75), methanol, and acetonitrile (v:v:v, 78:16:6). Venlafaxine was used as the internal standard while hexane was used for the liquid-liquid extraction. The flow rate was 0.4 mL/min with fluorescence detection using an excitation wavelength of 230 nm and emission detection wavelength of 315 nm. This method was selective, linear and sensitive with a lower limit of quantification of 2.5 ng/mL for both cocaine and l-THP. The intra-day precision of cocaine and l-THP was <9.50% while the accuracy was <4.29%. The inter-day precision of cocaine and l-THP was <9.14%, and the accuracy was <12.49%. The recovery for cocaine and l-THP ranged from 43.95 to 50.02% and 54.65 to 58.31%, respectively. In comparison to forty reported cocaine quantitation methods this method is simple, sensitive and cost-effective and can be used for simultaneous quantitation of l-THP and cocaine. This method meets the FDA guidelines and can be used in current and future clinical studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Volume dependency for culture of fungi from respiratory secretions and increased sensitivity of Aspergillus quantitative PCR.

PubMed

Fraczek, Marcin G; Kirwan, Marie B; Moore, Caroline B; Morris, Julie; Denning, David W; Richardson, Malcolm D

2014-02-01

Diagnosis of aspergillosis is often difficult. We compared fungal yields from respiratory specimens using the Health Protection Agency standard culture method (BSOP57), a higher volume undiluted culture method Mycology Reference Centre Manchester (MRCM) and Aspergillus quantitative real time polymerase chain reaction (qPCR). Sputum, bronchial aspirate and bronchoalveolar lavage (BAL) samples (total 23) were collected from aspergillosis patients. One fraction of all samples was cultured using the MRCM method, one BSOP57 and one was used for qPCR. The recovery rate for fungi was significantly higher by MRCM (87%) than by BSOP57 (8.7%) from all 23 specimens. Sputum samples were 44% positive by MRCM compared to no fungi isolated (0%) by BSOP57. Bronchial aspirates were 75% positive by MRCM and 0% by BSOP57. BAL samples were positive in 20% by MRCM and 10% by BSOP57. qPCR was always more sensitive than culture (95.6%) from all samples. In general, over 100 mould colonies (81 Aspergillus fumigatus) were grown using the MRCM method compared with only one colony from BSOP57. This study provides a reference point for standardisation of respiratory sample processing in diagnostic laboratories. Culture from higher volume undiluted respiratory specimens has a much higher yield for Aspergillus than BSOP57. qPCR is much more sensitive than culture and the current UK method requires revision. © 2013 Blackwell Verlag GmbH.

Sensitive, Selective Test For Hydrazines

NASA Technical Reports Server (NTRS)

Roundbehler, David; Macdonald, Stephen

1993-01-01

Derivatives of hydrazines formed, then subjected to gas chromatography and detected via chemiluminescence. In method of detecting and quantifying hydrazine vapors, vapors reacted with dinitro compound to enhance sensitivity and selectivity. Hydrazine (HZ), monomethyl hydrazine, (MMH), and unsymmetrical dimethylhydrazine (UDMH) analyzed quantitatively and qualitatively, either alone or in mixtures. Vapors collected and reacted with 2,4-dinitrobenzaldehyde, (DNB), making it possible to concentrate hydrazine in derivative form, thereby increasing sensitivity to low initial concentrations. Increases selectivity because only those constituents of sample reacting with DNB concentrated for analysis.

Harnessing Connectivity in a Large-Scale Small-Molecule Sensitivity Dataset | Office of Cancer Genomics

Cancer.gov

Identifying genetic alterations that prime a cancer cell to respond to a particular therapeutic agent can facilitate the development of precision cancer medicines. Cancer cell-line (CCL) profiling of small-molecule sensitivity has emerged as an unbiased method to assess the relationships between genetic or cellular features of CCLs and small-molecule response. Here, we developed annotated cluster multidimensional enrichment analysis to explore the associations between groups of small molecules and groups of CCLs in a new, quantitative sensitivity dataset.

A Highly Sensitive Method for Quantitative Determination of Abscisic Acid 1

PubMed Central

Michler, Charles H.; Lineberger, R. Daniel; Chism, Grady W.

1986-01-01

An abscisic acid derivative was formed by reaction with pentafluorobenzyl bromide which allowed highly sensitive detection by gas-liquid chromatography with electron capture detection. In comparison to the methyl ester derivative, the pentafluorobenzyl derivative of abscisic acid was four times more sensitive to electron capture detection and was stable at room temperature in the presence of ultraviolet light. Derivatization was rapid and the molecular weight of the new compound was confirmed by gas-liquid chromatography-mass spectrometry. PMID:16665076

A preamplification approach to GMO detection in processed foods.

PubMed

Del Gaudio, S; Cirillo, A; Di Bernardo, G; Galderisi, U; Cipollaro, M

2010-03-01

DNA is widely used as a target for GMO analysis because of its stability and high detectability. Real-time PCR is the method routinely used in most analytical laboratories due to its quantitative performance and great sensitivity. Accurate DNA detection and quantification is dependent on the specificity and sensitivity of the amplification protocol as well as on the quality and quantity of the DNA used in the PCR reaction. In order to enhance the sensitivity of real-time PCR and consequently expand the number of analyzable target genes, we applied a preamplification technique to processed foods where DNA can be present in low amounts and/or in degraded forms thereby affecting the reliability of qualitative and quantitative results. The preamplification procedure utilizes a pool of primers targeting genes of interest and is followed by real-time PCR reactions specific for each gene. An improvement of Ct values was found comparing preamplified vs. non-preamplified DNA. The strategy reported in the present study will be also applicable to other fields requiring quantitative DNA testing by real-time PCR.

Antibodies against toluene diisocyanate protein conjugates. Three methods of measurement.

PubMed

Patterson, R; Harris, K E; Zeiss, C R

1983-12-01

With the use of canine antisera against toluene diisocyanate (TDI)-dog serum albumin (DSA), techniques for measuring antibody against TDI-DSA were evaluated. The use of an ammonium sulfate precipitation assay showed suggestive evidence of antibody binding but high levels of TDI-DSA precipitation in the absence of antibody limit any usefulness of this technique. Double-antibody co-precipitation techniques will measure total antibody or Ig class antibody against 125I-TDI-DSA. These techniques are quantitative. The polystyrene tube radioimmunoassay is a highly sensitive method of detecting and quantitatively estimating IgG antibody. The enzyme linked immunosorbent assay is a rapidly adaptable method for the quantitative estimation of IgG, IgA, and IgM against TDI-homologous proteins. All these techniques were compared and results are demonstrated by using the same serum sample for analysis.

A fast and reliable readout method for quantitative analysis of surface-enhanced Raman scattering nanoprobes on chip surface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Hyejin; Jeong, Sinyoung; Ko, Eunbyeol

2015-05-15

Surface-enhanced Raman scattering techniques have been widely used for bioanalysis due to its high sensitivity and multiplex capacity. However, the point-scanning method using a micro-Raman system, which is the most common method in the literature, has a disadvantage of extremely long measurement time for on-chip immunoassay adopting a large chip area of approximately 1-mm scale and confocal beam point of ca. 1-μm size. Alternative methods such as sampled spot scan with high confocality and large-area scan method with enlarged field of view and low confocality have been utilized in order to minimize the measurement time practically. In this study, wemore » analyzed the two methods in respect of signal-to-noise ratio and sampling-led signal fluctuations to obtain insights into a fast and reliable readout strategy. On this basis, we proposed a methodology for fast and reliable quantitative measurement of the whole chip area. The proposed method adopted a raster scan covering a full area of 100 μm × 100 μm region as a proof-of-concept experiment while accumulating signals in the CCD detector for single spectrum per frame. One single scan with 10 s over 100 μm × 100 μm area yielded much higher sensitivity compared to sampled spot scanning measurements and no signal fluctuations attributed to sampled spot scan. This readout method is able to serve as one of key technologies that will bring quantitative multiplexed detection and analysis into practice.« less

A Method for Comprehensive Glycosite-Mapping and Direct Quantitation of Serum Glycoproteins.

PubMed

Hong, Qiuting; Ruhaak, L Renee; Stroble, Carol; Parker, Evan; Huang, Jincui; Maverakis, Emanual; Lebrilla, Carlito B

2015-12-04

A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and nonspecific enzyme digestions followed by nano liquid chromatography (LC)-chip/quadrupole time-of-flight mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20 min ultra high performance (UP)LC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantitation method that involves protein enrichments. The absolute protein quantitation has a wide linear range (3-4 orders of magnitude) and low limit of quantitation (femtomole level). This rapid and robust quantitation technique, which provides quantitative information for both proteins and glycosylation, will further facilitate disease biomarker discoveries.

Measurement issues associated with quantitative molecular biology analysis of complex food matrices for the detection of food fraud.

PubMed

Burns, Malcolm; Wiseman, Gordon; Knight, Angus; Bramley, Peter; Foster, Lucy; Rollinson, Sophie; Damant, Andrew; Primrose, Sandy

2016-01-07

Following a report on a significant amount of horse DNA being detected in a beef burger product on sale to the public at a UK supermarket in early 2013, the Elliott report was published in 2014 and contained a list of recommendations for helping ensure food integrity. One of the recommendations included improving laboratory testing capacity and capability to ensure a harmonised approach for testing for food authenticity. Molecular biologists have developed exquisitely sensitive methods based on the polymerase chain reaction (PCR) or mass spectrometry for detecting the presence of particular nucleic acid or peptide/protein sequences. These methods have been shown to be specific and sensitive in terms of lower limits of applicability, but they are largely qualitative in nature. Historically, the conversion of these qualitative techniques into reliable quantitative methods has been beset with problems even when used on relatively simple sample matrices. When the methods are applied to complex sample matrices, as found in many foods, the problems are magnified resulting in a high measurement uncertainty associated with the result which may mean that the assay is not fit for purpose. However, recent advances in the technology and the understanding of molecular biology approaches have further given rise to the re-assessment of these methods for their quantitative potential. This review focuses on important issues for consideration when validating a molecular biology assay and the various factors that can impact on the measurement uncertainty of a result associated with molecular biology approaches used in detection of food fraud, with a particular focus on quantitative PCR-based and proteomics assays.

Brain Injury Lesion Imaging Using Preconditioned Quantitative Susceptibility Mapping without Skull Stripping.

PubMed

Soman, S; Liu, Z; Kim, G; Nemec, U; Holdsworth, S J; Main, K; Lee, B; Kolakowsky-Hayner, S; Selim, M; Furst, A J; Massaband, P; Yesavage, J; Adamson, M M; Spincemallie, P; Moseley, M; Wang, Y

2018-04-01

Identifying cerebral microhemorrhage burden can aid in the diagnosis and management of traumatic brain injury, stroke, hypertension, and cerebral amyloid angiopathy. MR imaging susceptibility-based methods are more sensitive than CT for detecting cerebral microhemorrhage, but methods other than quantitative susceptibility mapping provide results that vary with field strength and TE, require additional phase maps to distinguish blood from calcification, and depict cerebral microhemorrhages as bloom artifacts. Quantitative susceptibility mapping provides universal quantification of tissue magnetic property without these constraints but traditionally requires a mask generated by skull-stripping, which can pose challenges at tissue interphases. We evaluated the preconditioned quantitative susceptibility mapping MR imaging method, which does not require skull-stripping, for improved depiction of brain parenchyma and pathology. Fifty-six subjects underwent brain MR imaging with a 3D multiecho gradient recalled echo acquisition. Mask-based quantitative susceptibility mapping images were created using a commonly used mask-based quantitative susceptibility mapping method, and preconditioned quantitative susceptibility images were made using precondition-based total field inversion. All images were reviewed by a neuroradiologist and a radiology resident. Ten subjects (18%), all with traumatic brain injury, demonstrated blood products on 3D gradient recalled echo imaging. All lesions were visible on preconditioned quantitative susceptibility mapping, while 6 were not visible on mask-based quantitative susceptibility mapping. Thirty-one subjects (55%) demonstrated brain parenchyma and/or lesions that were visible on preconditioned quantitative susceptibility mapping but not on mask-based quantitative susceptibility mapping. Six subjects (11%) demonstrated pons artifacts on preconditioned quantitative susceptibility mapping and mask-based quantitative susceptibility mapping; they were worse on preconditioned quantitative susceptibility mapping. Preconditioned quantitative susceptibility mapping MR imaging can bring the benefits of quantitative susceptibility mapping imaging to clinical practice without the limitations of mask-based quantitative susceptibility mapping, especially for evaluating cerebral microhemorrhage-associated pathologies, such as traumatic brain injury. © 2018 by American Journal of Neuroradiology.

Divisional role of quantitative HER2 testing in breast cancer.

PubMed

Yamamoto-Ibusuki, Mutsuko; Yamamoto, Yutaka; Fu, Peifen; Yamamoto, Satoko; Fujiwara, Saori; Honda, Yumi; Iyama, Ken-ichi; Iwase, Hirotaka

2015-03-01

Human epidermal growth factor receptor 2 (HER2) is amplified in human breast cancers in which therapy targeted to HER2 significantly improves patient outcome. We re-visited the use of real-time quantitative polymerase chain reaction (qPCR)-based assays using formalin-fixed paraffin-embedded (FFPE) tissues as alternative methods and investigated their particular clinical relevance. DNA and RNA were isolated from FFPE specimens and HER2 status was assessed by qPCR in 249 consecutive patients with primary breast cancer. Concordance with results forg immunohistochemistry (IHC) and in situ hybridization (ISH), clinical characteristics and survival was assessed. HER2 gene copy number had a stronger correlation with clinicopathological characteristics and excellent concordance with IHC/ISH results (Sensitivity: 96.7 %; concordance: 99.2 %). HER2 gene expression showed inadequate sensitivity, rendering it unsuitable to determine HER2 status (Sensitivity: 46.7 %; concordance: 92.1 %), but lower HER2 gene expression, leading to the classification of many cases as "false negative", contributed to a prediction of better prognosis within the HER2-amplified subpopulation. Quantitative HER2 assessments are suggested to have evolved their accuracy in this decade, which can be a potential alternative for HER2 diagnosis in line with the in situ method, while HER2 gene expression levels could provide additional information regarding prognosis or therapeutic strategy within a HER2-amplified subpopulation.

Development of a SYBR Green I real-time PCR for detection and quantitation of orthopoxvirus by using Ectromelia virus.

PubMed

Cheng, Wenyu; He, Xiaobing; Jia, Huaijie; Chen, Guohua; Wang, Cong; Zhang, Jun; Jing, Zhizhong

2018-04-01

Ectromelia virus (ECTV) is the causative agent of mousepox, which has devastating effects in laboratory-mouse colonies and causes economic loss in biomedical research. More importantly, ECTV has been extensively used as an excellent model for studies of the pathogenesis and immunobiology of human smallpox. A rapid and sensitive SYBR Green I-based real-time PCR assay was developed and used for the detection and quantitation of orthopoxvirus by using ECTV in this study. Primers targeted to the highly conserved region of major core protein P4b gene of orthopoxvirus were designed and the standard plasmid was constructed. This assay was able to detect a minimum of 10 copies of standard DNA and 5 TCID 50 units of ECTV. In addition, no cross-reactions were observed with two DNA viruses, such as herpes simplex virus and swine pseudorabies virus, and one RNA virus, vesicular stomatitis virus. Furthermore, intra- and inter-assay variability data showed that this method had a highly reproducibility and reliability. Moreover, the current assay was faster and had a higher sensitivity for detection of ECTV genomic DNA in cell cultured and clinical test samples. Therefore, the high sensitivity and reproducibility of this SYBR Green real-time PCR approach is a more effective method than the conventional PCR for ECTV diagnosis and quantitation. Copyright © 2017. Published by Elsevier Ltd.

Viral drug sensitivity testing using quantitative PCR: effect of tyrosine kinase inhibitors on polyomavirus BK replication.

PubMed

Randhawa, Parmjeet S; Farasati, Noush A; Huang, Yuchen; Mapara, Markus Y; Shapiro, Ron

2010-12-01

Our objective was to determine whether quantitative polymerase chain reaction (PCR) can be used to measure the effect of tyrosine kinase (TK) inhibition on polyomavirus BK (BKV) replication. The BKV was grown in a cell culture system. The rate of viral replication in the presence or absence of the drug being tested was assessed by amplifying the viral genome using primers directed against the viral capsid 1 protein. Dasatinib, erlotinib, gefitinib, imatinib, sunitinib, and sorafenib all showed antiviral activity at micromolar concentrations. The 50% effective concentration for erlotinib and sorafenib was within blood concentrations readily achieved in human subjects. Quantitative PCR is a convenient method for viral drug sensitivity testing for slow-growing viruses that do not readily produce cytopathic effect. TK inhibitors deserve further consideration as a potential therapeutic option for BKV-associated nephropathy and hemorrhagic cystitis.

Attomole quantitation of protein separations with accelerator mass spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vogel, J S; Grant, P G; Buccholz, B A

2000-12-15

Quantification of specific proteins depends on separation by chromatography or electrophoresis followed by chemical detection schemes such as staining and fluorophore adhesion. Chemical exchange of short-lived isotopes, particularly sulfur, is also prevalent despite the inconveniences of counting radioactivity. Physical methods based on isotopic and elemental analyses offer highly sensitive protein quantitation that has linear response over wide dynamic ranges and is independent of protein conformation. Accelerator mass spectrometry quantifies long-lived isotopes such as 14C to sub-attomole sensitivity. We quantified protein interactions with small molecules such as toxins, vitamins, and natural biochemicals at precisions of 1-5% . Micro-proton-induced-xray-emission quantifies elemental abundancesmore » in separated metalloprotein samples to nanogram amounts and is capable of quantifying phosphorylated loci in gels. Accelerator-based quantitation is a possible tool for quantifying the genome translation into proteome.« less

Race and Ethnicity in Research Methods. Sage Focus Editions, Volume 157.

ERIC Educational Resources Information Center

Stanfield, John H., II, Ed.; Dennis, Rutledge M., Ed.

The contributions in this volume examine the array of methods used in quantitative, qualitative, and comparative and historical research to show how research sensitive to ethnic issues can best be conducted. Rethinking and revising traditional methodologies and applying new ones can portray racial and ethnic issues as they really exist. The…

Target-responsive DNAzyme cross-linked hydrogel for visual quantitative detection of lead.

PubMed

Huang, Yishun; Ma, Yanli; Chen, Yahong; Wu, Xuemeng; Fang, Luting; Zhu, Zhi; Yang, Chaoyong James

2014-11-18

Because of the severe health risks associated with lead pollution, rapid, sensitive, and portable detection of low levels of Pb(2+) in biological and environmental samples is of great importance. In this work, a Pb(2+)-responsive hydrogel was prepared using a DNAzyme and its substrate as cross-linker for rapid, sensitive, portable, and quantitative detection of Pb(2+). Gold nanoparticles (AuNPs) were first encapsulated in the hydrogel as an indicator for colorimetric analysis. In the absence of lead, the DNAzyme is inactive, and the substrate cross-linker maintains the hydrogel in the gel form. In contrast, the presence of lead activates the DNAzyme to cleave the substrate, decreasing the cross-linking density of the hydrogel and resulting in dissolution of the hydrogel and release of AuNPs for visual detection. As low as 10 nM Pb(2+) can be detected by the naked eye. Furthermore, to realize quantitative visual detection, a volumetric bar-chart chip (V-chip) was used for quantitative readout of the hydrogel system by replacing AuNPs with gold-platinum core-shell nanoparticles (Au@PtNPs). The Au@PtNPs released from the hydrogel upon target activation can efficiently catalyze the decomposition of H2O2 to generate a large volume of O2. The gas pressure moves an ink bar in the V-chip for portable visual quantitative detection of lead with a detection limit less than 5 nM. The device was able to detect lead in digested blood with excellent accuracy. The method developed can be used for portable lead quantitation in many applications. Furthermore, the method can be further extended to portable visual quantitative detection of a variety of targets by replacing the lead-responsive DNAzyme with other DNAzymes.

Validation of the Mass-Extraction-Window for Quantitative Methods Using Liquid Chromatography High Resolution Mass Spectrometry.

PubMed

Glauser, Gaétan; Grund, Baptiste; Gassner, Anne-Laure; Menin, Laure; Henry, Hugues; Bromirski, Maciej; Schütz, Frédéric; McMullen, Justin; Rochat, Bertrand

2016-03-15

A paradigm shift is underway in the field of quantitative liquid chromatography-mass spectrometry (LC-MS) analysis thanks to the arrival of recent high-resolution mass spectrometers (HRMS). The capability of HRMS to perform sensitive and reliable quantifications of a large variety of analytes in HR-full scan mode is showing that it is now realistic to perform quantitative and qualitative analysis with the same instrument. Moreover, HR-full scan acquisition offers a global view of sample extracts and allows retrospective investigations as virtually all ionized compounds are detected with a high sensitivity. In time, the versatility of HRMS together with the increasing need for relative quantification of hundreds of endogenous metabolites should promote a shift from triple-quadrupole MS to HRMS. However, a current "pitfall" in quantitative LC-HRMS analysis is the lack of HRMS-specific guidance for validated quantitative analyses. Indeed, false positive and false negative HRMS detections are rare, albeit possible, if inadequate parameters are used. Here, we investigated two key parameters for the validation of LC-HRMS quantitative analyses: the mass accuracy (MA) and the mass-extraction-window (MEW) that is used to construct the extracted-ion-chromatograms. We propose MA-parameters, graphs, and equations to calculate rational MEW width for the validation of quantitative LC-HRMS methods. MA measurements were performed on four different LC-HRMS platforms. Experimentally determined MEW values ranged between 5.6 and 16.5 ppm and depended on the HRMS platform, its working environment, the calibration procedure, and the analyte considered. The proposed procedure provides a fit-for-purpose MEW determination and prevents false detections.

Is quantitative PCR for the pneumolysin (ply) gene useful for detection of pneumococcal lower respiratory tract infection?

PubMed

Abdeldaim, G; Herrmann, B; Korsgaard, J; Olcén, P; Blomberg, J; Strålin, K

2009-06-01

The pneumolysin (ply) gene is widely used as a target in PCR assays for Streptococcus pneumoniae in respiratory secretions. However, false-positive results with conventional ply-based PCR have been reported. The aim here was to study the performance of a quantitative ply-based PCR for the identification of pneumococcal lower respiratory tract infection (LRTI). In a prospective study, fibreoptic bronchoscopy was performed in 156 hospitalized adult patients with LRTI and 31 controls who underwent bronchoscopy because of suspicion of malignancy. Among the LRTI patients and controls, the quantitative ply-based PCR applied to bronchoalveolar lavage (BAL) fluid was positive at >or=10(3) genome copies/mL in 61% and 71% of the subjects, at >or=10(5) genome copies/mL in 40% and 58% of the subjects, and at >or=10(7) genome copies/mL in 15% and 3.2% of the subjects, respectively. Using BAL fluid culture, blood culture, and/or a urinary antigen test, S. pneumoniae was identified in 19 LRTI patients. As compared with these diagnostic methods used in combination, quantitative ply-based PCR showed sensitivities and specificities of 89% and 43% at a cut-off of 10(3) genome copies/mL, of 84% and 66% at a cut-off of 10(5) genome copies/mL, and of 53% and 90% at a cut-off of 10(7) genome copies/mL, respectively. In conclusion, a high cut-off with the quantitative ply-based PCR was required to reach acceptable specificity. However, as a high cut-off resulted in low sensitivity, quantitative ply-based PCR does not appear to be clinically useful. Quantitative PCR methods for S. pneumoniae using alternative gene targets should be evaluated.

Quantitative Mapping of the Spatial Distribution of Nanoparticles in Endo-Lysosomes by Local pH.

PubMed

Wang, Jing; MacEwan, Sarah R; Chilkoti, Ashutosh

2017-02-08

Understanding the intracellular distribution and trafficking of nanoparticle drug carriers is necessary to elucidate their mechanisms of drug delivery and is helpful in the rational design of novel nanoparticle drug delivery systems. The traditional immunofluorescence method to study intracellular distribution of nanoparticles using organelle-specific antibodies is laborious and subject to artifacts. As an alternative, we developed a new method that exploits ratiometric fluorescence imaging of a pH-sensitive Lysosensor dye to visualize and quantify the spatial distribution of nanoparticles in the endosomes and lysosomes of live cells. Using this method, we compared the endolysosomal distribution of cell-penetrating peptide (CPP)-functionalized micelles to unfunctionalized micelles and found that CPP-functionalized micelles exhibited faster endosome-to-lysosome trafficking than unfunctionalized micelles. Ratiometric fluorescence imaging of pH-sensitive Lysosensor dye allows rapid quantitative mapping of nanoparticle distribution in endolysosomes in live cells while minimizing artifacts caused by extensive sample manipulation typical of alternative approaches. This new method can thus serve as an alternative to traditional immunofluorescence approaches to study the intracellular distribution and trafficking of nanoparticles within endosomes and lysosomes.

Near-infrared fluorescence image quality test methods for standardized performance evaluation

NASA Astrophysics Data System (ADS)

Kanniyappan, Udayakumar; Wang, Bohan; Yang, Charles; Ghassemi, Pejhman; Wang, Quanzeng; Chen, Yu; Pfefer, Joshua

2017-03-01

Near-infrared fluorescence (NIRF) imaging has gained much attention as a clinical method for enhancing visualization of cancers, perfusion and biological structures in surgical applications where a fluorescent dye is monitored by an imaging system. In order to address the emerging need for standardization of this innovative technology, it is necessary to develop and validate test methods suitable for objective, quantitative assessment of device performance. Towards this goal, we develop target-based test methods and investigate best practices for key NIRF imaging system performance characteristics including spatial resolution, depth of field and sensitivity. Characterization of fluorescence properties was performed by generating excitation-emission matrix properties of indocyanine green and quantum dots in biological solutions and matrix materials. A turbid, fluorophore-doped target was used, along with a resolution target for assessing image sharpness. Multi-well plates filled with either liquid or solid targets were generated to explore best practices for evaluating detection sensitivity. Overall, our results demonstrate the utility of objective, quantitative, target-based testing approaches as well as the need to consider a wide range of factors in establishing standardized approaches for NIRF imaging system performance.

Rapid Quantitative Analysis of Multiple Explosive Compound Classes on a Single Instrument via Flow-Injection Analysis Tandem Mass Spectrometry.

PubMed

Ostrinskaya, Alla; Kunz, Roderick R; Clark, Michelle; Kingsborough, Richard P; Ong, Ta-Hsuan; Deneault, Sandra

2018-05-24

A flow-injection analysis tandem mass spectrometry (FIA MSMS) method was developed for rapid quantitative analysis of 10 different inorganic and organic explosives. Performance is optimized by tailoring the ionization method (APCI/ESI), de-clustering potentials, and collision energies for each specific analyte. In doing so, a single instrument can be used to detect urea nitrate, potassium chlorate, 2,4,6-trinitrotoluene, 2,4,6-trinitrophenylmethylnitramine, triacetone triperoxide, hexamethylene triperoxide diamine, pentaerythritol tetranitrate, 1,3,5-trinitroperhydro-1,3,5-triazine, nitroglycerin, and octohy-dro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine with sensitivities all in the picogram per milliliter range. In conclusion, FIA APCI/ESI MSMS is a fast (<1 min/sample), sensitive (~pg/mL LOQ), and precise (intraday RSD < 10%) method for trace explosive detection that can play an important role in criminal and attributional forensics, counterterrorism, and environmental protection areas, and has the potential to augment or replace several of the existing explosive detection methods. © 2018 American Academy of Forensic Sciences.

Quantitative molecular analysis in mantle cell lymphoma.

PubMed

Brízová, H; Hilská, I; Mrhalová, M; Kodet, R

2011-07-01

A molecular analysis has three major roles in modern oncopathology--as an aid in the differential diagnosis, in molecular monitoring of diseases, and in estimation of the potential prognosis. In this report we review the application of the molecular analysis in a group of patients with mantle cell lymphoma (MCL). We demonstrate that detection of the cyclin D1 mRNA level is a molecular marker in 98% of patients with MCL. Cyclin D1 quantitative monitoring is specific and sensitive for the differential diagnosis and for the molecular monitoring of the disease in the bone marrow. Moreover, the dynamics of cyclin D1 in bone marrow reflects the disease development and it predicts the clinical course. We employed the molecular analysis for a precise quantitative detection of proliferation markers, Ki-67, topoisomerase IIalpha, and TPX2, that are described as effective prognostic factors. Using the molecular approach it is possible to measure the proliferation rate in a reproducible, standard way which is an essential prerequisite for using the proliferation activity as a routine clinical tool. Comparing with immunophenotyping we may conclude that the quantitative PCR-based analysis is a useful, reliable, rapid, reproducible, sensitive and specific method broadening our diagnostic tools in hematopathology. In comparison to interphase FISH in paraffin sections quantitative PCR is less technically demanding and less time-consuming and furthermore it is more sensitive in detecting small changes in the mRNA level. Moreover, quantitative PCR is the only technology which provides precise and reproducible quantitative information about the expression level. Therefore it may be used to demonstrate the decrease or increase of a tumor-specific marker in bone marrow in comparison with a previously aspirated specimen. Thus, it has a powerful potential to monitor the course of the disease in correlation with clinical data.

A surface enhanced Raman scattering quantitative analytical platform for detection of trace Cu coupled the catalytic reaction and gold nanoparticle aggregation with label-free Victoria blue B molecular probe.

PubMed

Li, Chongning; Ouyang, Huixiang; Tang, Xueping; Wen, Guiqing; Liang, Aihui; Jiang, Zhiliang

2017-01-15

With development of economy and society, there is an urgent need to develop convenient and sensitive methods for detection of Cu 2+ pollution in water. In this article, a simple and sensitive SERS sensor was proposed to quantitative analysis of trace Cu 2+ in water. The SERS sensor platform was prepared a common gold nanoparticle (AuNP)-SiO 2 sol substrate platform by adsorbing HSA, coupling with the catalytic reaction of Cu 2+ -ascorbic acid (H 2 A)-dissolved oxygen, and using label-free Victoria blue B (VBB) as SERS molecular probes. The SERS sensor platform response to the AuNP aggregations by hydroxyl radicals (•OH) oxidizing from the Cu 2+ catalytic reaction, which caused the SERS signal enhancement. Therefore, by monitoring the increase of SERS signal, Cu 2+ in water can be determined accurately. The results show that the SERS sensor platforms owns a linear response with a range from 0.025 to 25μmol/L Cu 2+ , and with a detection limit of 0.008μmol/L. In addition, the SERS method demonstrated good specificity for Cu 2+ , which can determined accurately trace Cu 2+ in water samples, and good recovery and accuracy are obtained for the water samples. With its high selectivity and good accuracy, the sensitive SERS quantitative analysis method is expected to be a promising candidate for determining copper ions in environmental monitoring and food safety. Copyright © 2016 Elsevier B.V. All rights reserved.

Contribution of flow-volume curves to the detection of central airway obstruction*

PubMed Central

Raposo, Liliana Bárbara Perestrelo de Andrade e; Bugalho, António; Gomes, Maria João Marques

2013-01-01

OBJECTIVE: To assess the sensitivity and specificity of flow-volume curves in detecting central airway obstruction (CAO), and to determine whether their quantitative and qualitative criteria are associated with the location, type and degree of obstruction. METHODS: Over a four-month period, we consecutively evaluated patients with bronchoscopy indicated. Over a one-week period, all patients underwent clinical evaluation, flow-volume curve, bronchoscopy, and completed a dyspnea scale. Four reviewers, blinded to quantitative and clinical data, and bronchoscopy results, classified the morphology of the curves. A fifth reviewer determined the morphological criteria, as well as the quantitative criteria. RESULTS: We studied 82 patients, 36 (44%) of whom had CAO. The sensitivity and specificity of the flow-volume curves in detecting CAO were, respectively, 88.9% and 91.3% (quantitative criteria) and 30.6% and 93.5% (qualitative criteria). The most prevalent quantitative criteria in our sample were FEF50%/FIF50% ≥ 1, in 83% of patients, and FEV1/PEF ≥ 8 mL . L–1 . min–1, in 36%, both being associated with the type, location, and degree of obstruction (p < 0.05). There was concordance among the reviewers as to the presence of CAO. There is a relationship between the degree of obstruction and dyspnea. CONCLUSIONS: The quantitative criteria should always be calculated for flow-volume curves in order to detect CAO, because of the low sensitivity of the qualitative criteria. Both FEF50%/FIF50% ≥ 1 and FEV1/PEF ≥ 8 mL . L–1 . min–1 were associated with the location, type and degree of obstruction. PMID:24068266

Quantitation of Melatonin and N-acetylserotonin in Human Plasma by Nanoflow LC-MS/MS and Electrospray LC-MS/MS

PubMed Central

Carter, Melissa D.; Calcutt, M. Wade; Malow, Beth A.; Rose, Kristie L.; Hachey, David L.

2012-01-01

Melatonin (MEL) and its chemical precursor N-acetylserotonin (NAS) are believed to be potential biomarkers for sleep-related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability, and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC-MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1×100 mm, 3.5 μm) or on a polyimide-coated, fused-silica capillary self-packed with 17 cm AquaC18 (3 μm, 125 Å). Quantitation was done using the SRM transitions m/z 233→174 and m/z 219→160 for MEL and NAS, respectively. The analytical response ratio vs. concentration curves were linear for MEL (nanoflow LC: 11.7–1165 pg/mL, LC: 1165–116500 pg/mL) and for NAS (nanoflow LC: 11.0–1095 pg/mL). PMID:22431453

Development and application of UHPLC-MS/MS method for the determination of phenolic compounds in Chamomile flowers and Chamomile tea extracts.

PubMed

Nováková, Lucie; Vildová, Anna; Mateus, Joana Patricia; Gonçalves, Tiago; Solich, Petr

2010-09-15

UHPLC-MS/MS method using BEH C18 analytical column was developed for the separation and quantitation of 12 phenolic compounds of Chamomile (Matricaria recutita L.). The separation was accomplished using gradient elution with mobile phase consisting of methanol and formic acid 0.1%. ESI in both positive and negative ion mode was optimized with the aim to reach high sensitivity and selectivity for quantitation using SRM experiment. ESI in negative ion mode was found to be more convenient for quantitative analysis of all phenolics except of chlorogenic acid and kaempherol, which demonstrated better results of linearity, accuracy and precision in ESI positive ion mode. The results of method validation confirmed, that developed UHPLC-MS/MS method was convenient and reliable for the determination of phenolic compounds in Chamomile extracts with linearity >0.9982, accuracy within 76.7-126.7% and precision within 2.2-12.7% at three spiked concentration levels. Method sensitivity expressed as LOQ was typically 5-20 nmol/l. Extracts of Chamomile flowers and Chamomile tea were subjected to UHPLC-MS/MS analysis. The most abundant phenolic compounds in both Chamomile flowers and Chamomile tea extracts were chlorogenic acid, umbelliferone, apigenin and apigenin-7-glucoside. In Chamomile tea extracts there was greater abundance of flavonoid glycosides such as rutin or quercitrin, while the aglycone apigenin and its glycoside were present in lower amount. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Quantitative assessment model for gastric cancer screening

PubMed Central

Chen, Kun; Yu, Wei-Ping; Song, Liang; Zhu, Yi-Min

2005-01-01

AIM: To set up a mathematic model for gastric cancer screening and to evaluate its function in mass screening for gastric cancer. METHODS: A case control study was carried on in 66 patients and 198 normal people, then the risk and protective factors of gastric cancer were determined, including heavy manual work, foods such as small yellow-fin tuna, dried small shrimps, squills, crabs, mothers suffering from gastric diseases, spouse alive, use of refrigerators and hot food, etc. According to some principles and methods of probability and fuzzy mathematics, a quantitative assessment model was established as follows: first, we selected some factors significant in statistics, and calculated weight coefficient for each one by two different methods; second, population space was divided into gastric cancer fuzzy subset and non gastric cancer fuzzy subset, then a mathematic model for each subset was established, we got a mathematic expression of attribute degree (AD). RESULTS: Based on the data of 63 patients and 693 normal people, AD of each subject was calculated. Considering the sensitivity and specificity, the thresholds of AD values calculated were configured with 0.20 and 0.17, respectively. According to these thresholds, the sensitivity and specificity of the quantitative model were about 69% and 63%. Moreover, statistical test showed that the identification outcomes of these two different calculation methods were identical (P>0.05). CONCLUSION: The validity of this method is satisfactory. It is convenient, feasible, economic and can be used to determine individual and population risks of gastric cancer. PMID:15655813

Sensitive Quantification of Cannabinoids in Milk by Alkaline Saponification-Solid Phase Extraction Combined with Isotope Dilution UPLC-MS/MS.

PubMed

Wei, Binnian; McGuffey, James E; Blount, Benjamin C; Wang, Lanqing

2016-01-01

Maternal exposure to marijuana during the lactation period-either active or passive-has prompted concerns about transmission of cannabinoids to breastfed infants and possible subsequent adverse health consequences. Assessing these health risks requires a sensitive analytical approach that is able to quantitatively measure trace-level cannabinoids in breast milk. Here, we describe a saponification-solid phase extraction approach combined with ultra-high-pressure liquid chromatography-tandem mass spectrometry for simultaneously quantifying Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) in breast milk. We demonstrate for the first time that constraints on sensitivity can be overcome by utilizing alkaline saponification of the milk samples. After extensively optimizing the saponification procedure, the validated method exhibited limits of detections of 13, 4, and 66 pg/mL for THC, CBN, and CBD, respectively. Notably, the sensitivity achieved was significantly improved, for instance, the limits of detection for THC is at least 100-fold more sensitive compared to that previously reported in the literature. This is essential for monitoring cannabinoids in breast milk resulting from passive or nonrecent active maternal exposure. Furthermore, we simultaneously acquired multiple reaction monitoring transitions for 12 C- and 13 C-analyte isotopes. This combined analysis largely facilitated data acquisition by reducing the repetitive analysis rate for samples exceeding the linear limits of 12 C-analytes. In addition to high sensitivity and broad quantitation range, this method delivers excellent accuracy (relative error within ±10%), precision (relative standard deviation <10%), and efficient analysis. In future studies, we expect this method to play a critical role in assessing infant exposure to cannabinoids through breastfeeding.

Nonlinear optical microscopy: use of second harmonic generation and two-photon microscopy for automated quantitative liver fibrosis studies.

PubMed

Sun, Wanxin; Chang, Shi; Tai, Dean C S; Tan, Nancy; Xiao, Guangfa; Tang, Huihuan; Yu, Hanry

2008-01-01

Liver fibrosis is associated with an abnormal increase in an extracellular matrix in chronic liver diseases. Quantitative characterization of fibrillar collagen in intact tissue is essential for both fibrosis studies and clinical applications. Commonly used methods, histological staining followed by either semiquantitative or computerized image analysis, have limited sensitivity, accuracy, and operator-dependent variations. The fibrillar collagen in sinusoids of normal livers could be observed through second-harmonic generation (SHG) microscopy. The two-photon excited fluorescence (TPEF) images, recorded simultaneously with SHG, clearly revealed the hepatocyte morphology. We have systematically optimized the parameters for the quantitative SHG/TPEF imaging of liver tissue and developed fully automated image analysis algorithms to extract the information of collagen changes and cell necrosis. Subtle changes in the distribution and amount of collagen and cell morphology are quantitatively characterized in SHG/TPEF images. By comparing to traditional staining, such as Masson's trichrome and Sirius red, SHG/TPEF is a sensitive quantitative tool for automated collagen characterization in liver tissue. Our system allows for enhanced detection and quantification of sinusoidal collagen fibers in fibrosis research and clinical diagnostics.

Development of an SRM method for absolute quantitation of MYDGF/C19orf10 protein.

PubMed

Dwivedi, Ravi C; Krokhin, Oleg V; El-Gabalawy, Hani S; Wilkins, John A

2016-06-01

To develop a MS-based selected reaction monitoring (SRM) assay for quantitation of myeloid-derived growth factor (MYDGF) formerly chromosome 19 open reading frame (C19orf10). Candidate reporter peptides were identified in digests of recombinant MYDGF. Isotopically labeled forms of these reporter peptides were employed as internal standards for assay development. Two reference peptides were selected SYLYFQTFFK and GAEIEYAMAYSK with respective LOQ of 42 and 380 attomole per injection. Application of the assay to human serum and synovial fluid determined that the assay sensitivity was reduced and quantitation was not achievable. However, the partial depletion of albumin and immunoglobulin from synovial fluids provided estimates of 300-650 femtomoles per injection (0.7-1.6 nanomolar (nM) fluid concentrations) in three of the six samples analyzed. A validated sensitive assay for the quantitation of MYDGF in biological fluids was developed. However, the endogenous levels of MYDGF in such fluids are at or below the current levels of quantitation. The levels of MYDGF are lower than those previously reported using an ELISA. The current results suggest that additional steps may be required to remove high abundance proteins or to enrich MYDGF for SRM-based quantitation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sensitive and quantitative detection of botulinum neurotoxin in neurons derived from mouse embryonic stem cells.

PubMed

Pellett, Sabine; Du, Zhong-wei; Pier, Christina L; Tepp, William H; Zhang, Su-chun; Johnson, Eric A

2011-01-07

Botulinum neurotoxins (BoNTs), the most poisonous protein toxins known, represent a serious bioterrorism threat but are also used as a unique and important bio-pharmaceutical to treat an increasing myriad of neurological disorders. The only currently accepted detection method by the United States Food and Drug Administration for biological activity of BoNTs and for potency determination of pharmaceutical preparations is the mouse bioassay (MBA). Recent advances have indicated that cell-based assays using primary neuronal cells can provide an equally sensitive and robust detection platform as the MBA to reliably and quantitatively detect biologically active BoNTs. This study reports for the first time a BoNT detection assay using mouse embryonic stem cells to produce a neuronal cell culture. The data presented indicate that this assay can reliably detect BoNT/A with a similar sensitivity as the MBA. Published by Elsevier Inc.

Development of a testing method for asbestos fibers in treated materials of asbestos containing wastes by transmission electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamamoto, Takashi, E-mail: tyama@nies.go.jp; Kida, Akiko; Noma, Yukio

Highlights: • A high sensitive and selective testing method for asbestos in treated materials of asbestos containing wastes was developed. • Asbestos can be determined at a limits are a few million fibers per gram and a few μg g{sup −1}. • High temperature melting treatment samples were determined by this method. Asbestos fiber concentration were below the quantitation limit in all samples, and total fiber concentrations were determined as 47–170 × 10{sup 6} g{sup −1}. - Abstract: Appropriate treatment of asbestos-containing wastes is a significant problem. In Japan, the inertization of asbestos-containing wastes based on new treatment processes approvedmore » by the Minister of the Environment is promoted. A highly sensitive method for testing asbestos fibers in inertized materials is required so that these processes can be approved. We developed a method in which fibers from milled treated materials are extracted in water by shaking, and are counted and identified by transmission electron microscopy. Evaluation of this method by using asbestos standards and simulated slag samples confirmed that the quantitation limits are a few million fibers per gram and a few μg/g in a sample of 50 mg per filter. We used this method to assay asbestos fibers in slag samples produced by high-temperature melting of asbestos-containing wastes. Fiber concentrations were below the quantitation limit in all samples, and total fiber concentrations were determined as 47–170 × 10{sup −6} f/g. Because the evaluation of treated materials by TEM is difficult owing to the limited amount of sample observable, this testing method should be used in conjunction with bulk analytical methods for sure evaluation of treated materials.« less

Tellurium: A new sensitive test

USGS Publications Warehouse

Lakin, H.W.; Thompson, C.E.

1963-01-01

A new, extremely sensitive method for the quantitative determination of tellurium is based on the induced precipitation of elemental gold from a 6N HCl solution containing gold chloride, cupric chloride, and hypophosphorous acid; the amount of gold reduced is proportional to the amount of tellurium present. As little as 1 nanogram (1 ?? 70-9 g) of tellurium gives a measurable reaction with 1 mg of gold in 50 ml of solution.

Assessment of cleaning and disinfection in Salmonella-contaminated poultry layer houses using qualitative and semi-quantitative culture techniques.

PubMed

Wales, Andrew; Breslin, Mark; Davies, Robert

2006-09-10

Salmonella infection of laying flocks in the UK is predominantly a problem of the persistent contamination of layer houses and associated wildlife vectors by Salmonella Enteritidis. Methods for its control and elimination include effective cleaning and disinfection of layer houses between flocks, and it is important to be able to measure the success of such decontamination. A method for the environmental detection and semi-quantitative enumeration of salmonellae was used and compared with a standard qualitative method, in 12 Salmonella-contaminated caged layer houses before and after cleaning and disinfection. The quantitative technique proved to have comparable sensitivity to the standard method, and additionally provided insights into the numerical Salmonella challenge that replacement flocks would encounter. Elimination of S. Enteritidis was not achieved in any of the premises examined although substantial reductions in the prevalence and numbers of salmonellae were demonstrated, whilst in others an increase in contamination was observed after cleaning and disinfection. Particular problems with feeders and wildlife vectors were highlighted. The use of a quantitative method assisted the identification of problem areas, such as those with a high initial bacterial load or those experiencing only a modest reduction in bacterial count following decontamination.

Mapping Quantitative Traits in Unselected Families: Algorithms and Examples

PubMed Central

Dupuis, Josée; Shi, Jianxin; Manning, Alisa K.; Benjamin, Emelia J.; Meigs, James B.; Cupples, L. Adrienne; Siegmund, David

2009-01-01

Linkage analysis has been widely used to identify from family data genetic variants influencing quantitative traits. Common approaches have both strengths and limitations. Likelihood ratio tests typically computed in variance component analysis can accommodate large families but are highly sensitive to departure from normality assumptions. Regression-based approaches are more robust but their use has primarily been restricted to nuclear families. In this paper, we develop methods for mapping quantitative traits in moderately large pedigrees. Our methods are based on the score statistic which in contrast to the likelihood ratio statistic, can use nonparametric estimators of variability to achieve robustness of the false positive rate against departures from the hypothesized phenotypic model. Because the score statistic is easier to calculate than the likelihood ratio statistic, our basic mapping methods utilize relatively simple computer code that performs statistical analysis on output from any program that computes estimates of identity-by-descent. This simplicity also permits development and evaluation of methods to deal with multivariate and ordinal phenotypes, and with gene-gene and gene-environment interaction. We demonstrate our methods on simulated data and on fasting insulin, a quantitative trait measured in the Framingham Heart Study. PMID:19278016

The Metabotropic Glutamate Receptor Subtype 5 (mGluR5) Mediates Sensitivity to the Sedative Properties of Ethanol

PubMed Central

Downing, Chris; Marks, Michael J.; Larson, Colin; Johnson, Thomas E.

2010-01-01

Objective Inbred Long-Sleep and Short-Sleep mice (ILS and ISS) were selectively bred for differential sensitivity to the sedative effects of ethanol. Lines of mice derived from these progenitors have been used to identify several Quantitative Trait Loci (QTLs) mediating Loss Of the Righting reflex due to Ethanol (LORE). The present study investigated mGluR5 as a candidate gene underlying Lore7, a QTL mediating differential LORE sensitivity. Methods We used knockout mice, a quantitative complementation test, pharmacological antagonism of mGluR5, real-time quantitative PCR, radioligand binding, DNA sequencing and bioinformatics to examine the role of mGluR5 in ethanol-induced sedation. Results mGluR5 knockout mice had a significantly longer LORE duration than wild-type controls. Administration of the mGluR5 antagonist 2-methyl-6-(phenylethyl)-pyridine (MPEP) had differential effects on LORE in ILS and ISS mice. A quantitative complementation test also supported mGluR5 mediating LORE. Two intronic single-nucleotide polymorphisms in mGluR5 were highly correlated with LORE in recombinant inbred mice derived from a cross between ILS and ISS (LXS RIs). Differences in mGluR5 mRNA level and receptor density were observed between ILS and ISS in distinct brain regions. Finally, data from WebQTL showed that mGluR5 expression was highly correlated with several LORE phenotypes in the LXS RIs. Conclusions Taken together, this data provides convincing evidence that mGluR5 mediates differential sensitivity to the sedative effects of ethanol. Studies from the human literature have also identified MGLUR5 as a potential candidate gene for ethanol sensitivity. PMID:20657349

[Gas chromatography in quantitative analysis of hydrocyanic acid and its salts in cadaveric blood].

PubMed

Iablochkin, V D

2003-01-01

A direct gas chromatography method was designed for the quantitative determination of cyanides (prussic acid) in cadaveric blood. Its sensitivity is 0.05 mg/ml. The routine volatile products, including substances, which emerge due to putrefaction of organic matters, do not affect the accuracy and reproducibility of the method; the exception is H-propanol that was used as the internal standard. The method was used in legal chemical expertise related with acute cyanide poisoning (suicide) as well as with poisoning of products of combustion of nonmetals (foam-rubber). The absolute error does not exceed 10% with a mean quadratic deviation of 0.0029-0.0033 mg.

Determination of alpha-hydroxy acids in cosmetic products by high-performance liquid chromatography with a narrow-bore column.

PubMed

Nicoletti, I; Corradini, C; Cogliandro, E; Cavazza, A

1999-08-01

This paper reports the results of a study carried out to develop a simple, rapid and sensitive method for the separation, identification and quantitative measurement of alpha-hydroxy acids in commercial cosmetics using high-performance liquid chromatography (HPLC). This method is successfully applied to the simultaneous identification and quantitative determination of glycolic, lactic, malic, tartaric and citric acids employing a reversed phase narrow-bore column under isocratic condition and UV detection. The method is validated by determining the precision of replicate analyses and accuracy by analyzing samples with and without adding know amount of the alpha-hydroxy acids. The procedure is suitable for routine analyses of commercial cosmetics.

Automated on-line SPE LC-MS/MS method to quantitate 6beta-hydroxycortisol and cortisol in human urine: use of the 6beta-hydroxycortisol to cortisol ratio as an indicator of CYP3A4 activity.

PubMed

Barrett, Yu Chen; Akinsanya, Billy; Chang, Shu-Ying; Vesterqvist, Ole

2005-07-25

A sensitive method for quantitation of urinary 6beta-hydroxycortisol (6beta-HC) and cortisol using on-line SPE and LC-MS/MS was developed and validated. Human urine samples were injected directly onto an on-line solid phase extraction apparatus, Prospekt-2, followed by HPLC separation and electrospray triple quadrupole LC-MS/MS detection. The inter-day precision for the 6beta-HC:cortisol ratio was 7-9%. The lower limit of quantitation was 1 and 0.2 ng/mL for 6beta-HC and cortisol, respectively. Using the method we observed a diurnal variation on the 6beta-HC:cortisol ratio in healthy volunteers with the maximal ratio observed in the 2-10 pm urine collection period.

Improved profiling of estrogen metabolites by orbitrap LC/MS

PubMed Central

Li, Xingnan; Franke, Adrian A.

2015-01-01

Estrogen metabolites are important biomarkers to evaluate cancer risks and metabolic diseases. Due to their low physiological levels, a sensitive and accurate method is required, especially for the quantitation of unconjugated forms of endogenous steroids and their metabolites in humans. Here, we evaluated various derivatives of estrogens for improved analysis by orbitrap LC/MS in human serum samples. A new chemical derivatization reagent was applied modifying phenolic steroids to form 1-methylimidazole-2-sulfonyl adducts. The method significantly improves the sensitivity 2–100 fold by full scan MS and targeted selected ion monitoring MS over other derivatization methods including, dansyl, picolinoyl, and pyridine-3-sulfonyl products. PMID:25543003

Aptamer-mediated colorimetric method for rapid and sensitive detection of chloramphenicol in food.

PubMed

Yan, Chao; Zhang, Jing; Yao, Li; Xue, Feng; Lu, Jianfeng; Li, Baoguang; Chen, Wei

2018-09-15

We report an aptamer-mediated colorimetric method for sensitive detection of chloramphenicol (CAP). The aptamer of CAP is immobilized by the hybridization with pre-immobilized capture probe in the microtiter plate. The horseradish peroxidase (HRP) is covalently attached to the aptamer by the biotin-streptavidin system for signal production. CAP will preferably bind with aptamer due to the high binding affinity, which attributes to the release of aptamer and HRP and thus, affects the optical signal intensity. Quantitative determination of CAP is successfully achieved in the wide range from 0.001 to 1000 ng/mL with detection limit of 0.0031 ng/mL, which is more sensitive than traditional immunoassays. This method is further validated by measuring the recovery of CAP spiked in two different food matrices (honey and fish). The aptamer-mediated colorimetric method can be a useful protocol for rapid and sensitive screening of CAP, and may be used as an alternative means for traditional immunoassays. Copyright © 2018 Elsevier Ltd. All rights reserved.

Influence of an Education Abroad Program on the Intercultural Sensitivity of STEM Undergraduates: A Mixed Methods Study

ERIC Educational Resources Information Center

Demetry, Chrysanthe; Vaz, Richard F.

2017-01-01

Education abroad programs are becoming more common as a mechanism for developing the global competencies of engineering graduates. An increasing body of research shows that intercultural learning does not occur "de facto" in such programs. This study used quantitative and qualitative methods to explore changes in students' intercultural…

Quantitative relationship between the local lymph node assay and human skin sensitization assays.

PubMed

Schneider, K; Akkan, Z

2004-06-01

The local lymph node assay (LLNA) is a new test method which allows for the quantitative assessment of sensitizing potency in the mouse. Here, we investigate the quantitative correlation between results from the LLNA and two human sensitization tests--specifically, human repeat insult patch tests (HRIPTs) and human maximization tests (HMTs). Data for 57 substances were evaluated, of which 46 showed skin sensitizing properties in human tests, whereas 11 yielded negative results in humans. For better comparability data from mouse and human tests were transformed to applied doses per skin area, which ranged over four orders of magnitude for the substances considered. Regression analysis for the 46 human sensitizing substances revealed a significant positive correlation between the LLNA and human tests. The correlation was better between LLNA and HRIPT data (n=23; r=0.77) than between LLNA and HMT data (n=38; r=0.65). The observed scattering of data points is related to various uncertainties, in part associated with insufficiencies of data from older HMT studies. Predominantly negative results in the LLNA for another 11 substances which showed no skin sensitizing activity in human maximization tests further corroborate the correspondence between LLNA and human tests. Based on this analysis, the LLNA can be considered a reliable basis for relative potency assessments for skin sensitizers. Proposals are made for the regulatory exploitation of the LLNA: four potency groups can be established, and assignment of substances to these groups according to the outcome of the LLNA can be used to characterize skin sensitizing potency in substance-specific assessments. Moreover, based on these potency groups, a more adequate consideration of sensitizing substances in preparations becomes possible. It is proposed to replace the current single concentration limit for skin sensitizers in preparations, which leads to an all or nothing classification of a preparation as sensitizing to skin ("R43") in the European Union, by differentiated concentration limits derived from the limits for the four potency groups.

Quantitative determination of galantamine in human plasma by sensitive liquid chromatography-tandem mass spectrometry using loratadine as an internal standard.

PubMed

Nirogi, Ramakrishna V S; Kandikere, Vishwottam N; Mudigonda, Koteshwara; Maurya, Santosh

2007-02-01

A simple, rapid, sensitive, and selective liquid chromatography-tandem mass spectrometry method is developed and validated for the quantitation of galantamine, an acetylcholinesterase inhibitor in human plasma, using a commercially available compound, loratadine, as the internal standard. Following liquid-liquid extraction, the analytes are separated using an isocratic mobile phase on a reverse-phase C18 column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective (M+H)+ ions, m/z 288 to 213 for galantamine and m/z 383 and 337 for the internal standard. The assay exhibit a linear dynamic range of 0.5-100 ng/mL for galantamine in human plasma. The lower limit of quantitation is 0.5 ng/mL, with a relative standard deviation of less than 8%. Acceptable precision and accuracy are obtained for concentrations over the standard curve range. A run time of 2.5 min for each sample makes it possible to analyze more than 400 human plasma samples per day. The validated method is successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability, or bioequivalence studies.

Magnetoresistive biosensors for quantitative proteomics

NASA Astrophysics Data System (ADS)

Zhou, Xiahan; Huang, Chih-Cheng; Hall, Drew A.

2017-08-01

Quantitative proteomics, as a developing method for study of proteins and identification of diseases, reveals more comprehensive and accurate information of an organism than traditional genomics. A variety of platforms, such as mass spectrometry, optical sensors, electrochemical sensors, magnetic sensors, etc., have been developed for detecting proteins quantitatively. The sandwich immunoassay is widely used as a labeled detection method due to its high specificity and flexibility allowing multiple different types of labels. While optical sensors use enzyme and fluorophore labels to detect proteins with high sensitivity, they often suffer from high background signal and challenges in miniaturization. Magnetic biosensors, including nuclear magnetic resonance sensors, oscillator-based sensors, Hall-effect sensors, and magnetoresistive sensors, use the specific binding events between magnetic nanoparticles (MNPs) and target proteins to measure the analyte concentration. Compared with other biosensing techniques, magnetic sensors take advantage of the intrinsic lack of magnetic signatures in biological samples to achieve high sensitivity and high specificity, and are compatible with semiconductor-based fabrication process to have low-cost and small-size for point-of-care (POC) applications. Although still in the development stage, magnetic biosensing is a promising technique for in-home testing and portable disease monitoring.

Quantitation of sweet steviol glycosides by means of a HILIC-MS/MS-SIDA approach.

PubMed

Well, Caroline; Frank, Oliver; Hofmann, Thomas

2013-11-27

Meeting the rising consumer demand for natural food ingredients, steviol glycosides, the sweet principle of Stevia rebaudiana Bertoni (Bertoni), have recently been approved as food additives in the European Union. As regulatory constraints require sensitive methods to analyze the sweet-tasting steviol glycosides in foods and beverages, a HILIC-MS/MS method was developed enabling the accurate and reliable quantitation of the major steviol glycosides stevioside, rebaudiosides A-F, steviolbioside, rubusoside, and dulcoside A by using the corresponding deuterated 16,17-dihydrosteviol glycosides as suitable internal standards. This quantitation not only enables the analysis of the individual steviol glycosides in foods and beverages but also can support the optimization of breeding and postharvest downstream processing of Stevia plants to produce preferentially sweet and least bitter tasting Stevia extracts.

Quantitative Evaluation of the Use of Actigraphy for Neurological and Psychiatric Disorders

PubMed Central

Song, Yu; Kwak, Shin; Yoshida, Sohei; Yamamoto, Yoshiharu

2014-01-01

Quantitative and objective evaluation of disease severity and/or drug effect is necessary in clinical practice. Wearable accelerometers such as an actigraph enable long-term recording of a patient's movement during activities and they can be used for quantitative assessment of symptoms due to various diseases. We reviewed some applications of actigraphy with analytical methods that are sufficiently sensitive and reliable to determine the severity of diseases and disorders such as motor and nonmotor disorders like Parkinson's disease, sleep disorders, depression, behavioral and psychological symptoms of dementia (BPSD) for vascular dementia (VD), seasonal affective disorder (SAD), and stroke, as well as the effects of drugs used to treat them. We believe it is possible to develop analytical methods to assess more neurological or psychopathic disorders using actigraphy records. PMID:25214709

Development of a method for urine bikunin/urinary trypsin inhibitor (UTI) quantitation and structural characterization: Application to type 1 and type 2 diabetes.

PubMed

Lepedda, Antonio Junior; Nieddu, Gabriele; Rocchiccioli, Silvia; Fresu, Pietro; De Muro, Pierina; Formato, Marilena

2013-12-01

Bikunin is a plasma proteinase inhibitor often associated with inflammatory conditions. It has a half-life of few minutes and it is rapidly excreted into urine as urinary trypsin inhibitor (UTI). UTI levels are usually low in healthy individuals but they can increase up to tenfold in both acute and chronic inflammatory diseases. This article describes a sensitive method for both direct UTI quantitation and structural characterization. UTI purification was performed by anion exchange micro-chromatography followed by SDS-PAGE. A calibration curve for protein quantitation was set up by using a purified UTI fraction. UTI identification and structural characterization was performed by Nano-LC-MS/MS analysis. The method was applied on urine samples from 9 patients with type 1 diabetes, 11 patients with type 2 diabetes, and 28 healthy controls, matched for age and sex with patients, evidencing higher UTI levels in both groups of patients with respect to controls (p < 0.001 and p = 0.001, respectively). Spearman's correlation tests highlighted no association between UTI levels and age in each group tested. Owing to the elevated sensitivity and specificity, the described method allows UTI quantitation from very low quantities of specimen. Furthermore, as UTI concentration is normalized for creatinine level, the analysis could be also performed on randomly collected urine samples. Finally, MS/MS analysis prospects the possibility of characterizing PTM sites potentially able to affect UTI localization, function, and pathophysiological activity. Preliminary results suggest that UTI levels could represent a useful marker of chronic inflammatory condition in type 1 and 2 diabetes. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Clinical neurophysiology and quantitative sensory testing in the investigation of orofacial pain and sensory function.

PubMed

Jääskeläinen, Satu K

2004-01-01

Chronic orofacial pain represents a diagnostic and treatment challenge for the clinician. Some conditions, such as atypical facial pain, still lack proper diagnostic criteria, and their etiology is not known. The recent development of neurophysiological methods and quantitative sensory testing for the examination of the trigeminal somatosensory system offers several tools for diagnostic and etiological investigation of orofacial pain. This review presents some of these techniques and the results of their application in studies on orofacial pain and sensory dysfunction. Clinical neurophysiological investigation has greater diagnostic accuracy and sensitivity than clinical examination in the detection of the neurogenic abnormalities of either peripheral or central origin that may underlie symptoms of orofacial pain and sensory dysfunction. Neurophysiological testing may also reveal trigeminal pathology when magnetic resonance imaging has failed to detect it, so these methods should be considered complementary to each other in the investigation of orofacial pain patients. The blink reflex, corneal reflex, jaw jerk, sensory neurography of the inferior alveolar nerve, and the recording of trigeminal somatosensory-evoked potentials with near-nerve stimulation have all proved to be sensitive and reliable in the detection of dysfunction of the myelinated sensory fibers of the trigeminal nerve or its central connections within the brainstem. With appropriately small thermodes, thermal quantitative sensory testing is useful for the detection of trigeminal small-fiber dysfunction (Adelta and C). In neuropathic conditions, it is most sensitive to lesions causing axonal injury. By combining different techniques for investigation of the trigeminal system, an accurate topographical diagnosis and profile of sensory fiber pathology can be determined. Neurophysiological and quantitative sensory tests have already highlighted some similarities among various orofacial pain conditions and have shown heterogeneity within clinical diagnostic categories. With the aid of neurophysiological recordings and quantitative sensory testing, it is possible to approach a mechanism-based classification of orofacial pain.

Validation of quantitative light-induced fluorescence-digital (QLF-D) for the detection of approximal caries in vitro.

PubMed

Ko, Hae-Youn; Kang, Si-Mook; Kim, Hee Eun; Kwon, Ho-Keun; Kim, Baek-Il

2015-05-01

Detection of approximal caries lesions can be difficult due to their anatomical position. This study aimed to assess the ability of the quantitative light-induced fluorescence-digital (QLF-D) in detecting approximal caries, and to compare the performance with those of the International Caries Detection and Assessment System II (ICDAS II) and digital radiography (DR). Extracted permanent teeth (n=100) were selected and mounted in pairs. The simulation pairs were assessed by one calibrated dentist using each detection method. After all the examinations, the teeth (n=95) were sectioned and examined histologically as gold standard. The modalities were compared in terms of sensitivity, specificity, areas under receiver operating characteristic curves (AUROC) for enamel (D1) and dentine (D3) levels. The intra-examiner reliability was assessed for all modalities. At D1 threshold, the ICDAS II presented the highest sensitivity (0.80) while the DR showed the highest specificity (0.89); however, the methods with the greatest AUC values at D1 threshold were DR and QLF-D (0.80 and 0.80 respectively). At D3 threshold, the methods with the highest sensitivity were ICDAS II and QLF-D (0.64 and 0.64 respectively) while the method with the lowest sensitivity was DR (0.50). However, with regard to the AUC values at D3 threshold, the QLF-D presented the highest value (0.76). All modalities showed to have excellent intra-examiner reliability. The newly developed QLF-D was not only able to detect proximal caries, but also showed to have comparable performance to the visual inspection and radiography in detecting proximal caries. QLF-D has the potential to be a useful detection method for proximal caries. Copyright © 2015 Elsevier Ltd. All rights reserved.

Performance of Two Quantitative PCR Methods for Microbial Source Tracking of Human Sewage and Implications for Microbial Risk Assessment in Recreational Waters

PubMed Central

Staley, Christopher; Gordon, Katrina V.; Schoen, Mary E.

2012-01-01

Before new, rapid quantitative PCR (qPCR) methods for assessment of recreational water quality and microbial source tracking (MST) can be useful in a regulatory context, an understanding of the ability of the method to detect a DNA target (marker) when the contaminant source has been diluted in environmental waters is needed. This study determined the limits of detection and quantification of the human-associated Bacteroides sp. (HF183) and human polyomavirus (HPyV) qPCR methods for sewage diluted in buffer and in five ambient, Florida water types (estuarine, marine, tannic, lake, and river). HF183 was quantifiable in sewage diluted up to 10−6 in 500-ml ambient-water samples, but HPyVs were not quantifiable in dilutions of >10−4. Specificity, which was assessed using fecal composites from dogs, birds, and cattle, was 100% for HPyVs and 81% for HF183. Quantitative microbial risk assessment (QMRA) estimated the possible norovirus levels in sewage and the human health risk at various sewage dilutions. When juxtaposed with the MST marker detection limits, the QMRA analysis revealed that HF183 was detectable when the modeled risk of gastrointestinal (GI) illness was at or below the benchmark of 10 illnesses per 1,000 exposures, but the HPyV method was generally not sensitive enough to detect potential health risks at the 0.01 threshold for frequency of illness. The tradeoff between sensitivity and specificity in the MST methods indicates that HF183 data should be interpreted judiciously, preferably in conjunction with a more host-specific marker, and that better methods of concentrating HPyVs from environmental waters are needed if this method is to be useful in a watershed management or monitoring context. PMID:22885746

Improved phase sensitivity in spectral domain phase microscopy using line-field illumination and self phase-referencing

PubMed Central

Yaqoob, Zahid; Choi, Wonshik; Oh, Seungeun; Lue, Niyom; Park, Yongkeun; Fang-Yen, Christopher; Dasari, Ramachandra R.; Badizadegan, Kamran; Feld, Michael S.

2010-01-01

We report a quantitative phase microscope based on spectral domain optical coherence tomography and line-field illumination. The line illumination allows self phase-referencing method to reject common-mode phase noise. The quantitative phase microscope also features a separate reference arm, permitting the use of high numerical aperture (NA > 1) microscope objectives for high resolution phase measurement at multiple points along the line of illumination. We demonstrate that the path-length sensitivity of the instrument can be as good as 41 pm/Hz, which makes it suitable for nanometer scale study of cell motility. We present the detection of natural motions of cell surface and two-dimensional surface profiling of a HeLa cell. PMID:19550464

I Vivo Quantitative Ultrasound Imaging and Scatter Assessments.

NASA Astrophysics Data System (ADS)

Lu, Zheng Feng

There is evidence that "instrument independent" measurements of ultrasonic scattering properties would provide useful diagnostic information that is not available with conventional ultrasound imaging. This dissertation is a continuing effort to test the above hypothesis and to incorporate quantitative ultrasound methods into clinical examinations for early detection of diffuse liver disease. A well-established reference phantom method was employed to construct quantitative ultrasound images of tissue in vivo. The method was verified by extensive phantom tests. A new method was developed to measure the effective attenuation coefficient of the body wall. The method relates the slope of the difference between the echo signal power spectrum from a uniform region distal to the body wall and the echo signal power spectrum from a reference phantom to the body wall attenuation. The accuracy obtained from phantom tests suggests further studies with animal experiments. Clinically, thirty-five healthy subjects and sixteen patients with diffuse liver disease were studied by these quantitative ultrasound methods. The average attenuation coefficient in normals agreed with previous investigators' results; in vivo backscatter coefficients agreed with the results from normals measured by O'Donnell. Strong discriminating power (p < 0.001) was found for both attenuation and backscatter coefficients between fatty livers and normals; a significant difference (p < 0.01) was observed in the backscatter coefficient but not in the attenuation coefficient between cirrhotic livers and normals. An in vivo animal model of steroid hepatopathy was used to investigate the system sensitivity in detecting early changes in canine liver resulting from corticosteroid administration. The average attenuation coefficient slope increased from 0.7 dB/cm/MHz in controls to 0.82 dB/cm/MHz (at 6 MHz) in treated animals on day 14 into the treatment, and the backscatter coefficient was 26times 10^{ -4}cm^{-1}sr^{-1} in controls compared with 74times 10^{-4}cm^{-1}sr^ {-1} (at 6 MHz) in treated animals. A simplified quantitative approach using video image signals was developed. Results derived both from the r.f. signal analysis and from the video signal analysis are sensitive to the changes in the liver in this animal model.

Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

PubMed Central

Ramírez, Juan Carlos; Cura, Carolina Inés; Moreira, Otacilio da Cruz; Lages-Silva, Eliane; Juiz, Natalia; Velázquez, Elsa; Ramírez, Juan David; Alberti, Anahí; Pavia, Paula; Flores-Chávez, María Delmans; Muñoz-Calderón, Arturo; Pérez-Morales, Deyanira; Santalla, José; Guedes, Paulo Marcos da Matta; Peneau, Julie; Marcet, Paula; Padilla, Carlos; Cruz-Robles, David; Valencia, Edward; Crisante, Gladys Elena; Greif, Gonzalo; Zulantay, Inés; Costales, Jaime Alfredo; Alvarez-Martínez, Miriam; Martínez, Norma Edith; Villarroel, Rodrigo; Villarroel, Sandro; Sánchez, Zunilda; Bisio, Margarita; Parrado, Rudy; Galvão, Lúcia Maria da Cunha; da Câmara, Antonia Cláudia Jácome; Espinoza, Bertha; de Noya, Belkisyole Alarcón; Puerta, Concepción; Riarte, Adelina; Diosque, Patricio; Sosa-Estani, Sergio; Guhl, Felipe; Ribeiro, Isabela; Aznar, Christine; Britto, Constança; Yadón, Zaida Estela; Schijman, Alejandro G.

2015-01-01

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease. PMID:26320872

Simple, Sensitive and Accurate Multiplex Detection of Clinically Important Melanoma DNA Mutations in Circulating Tumour DNA with SERS Nanotags

PubMed Central

Wee, Eugene J.H.; Wang, Yuling; Tsao, Simon Chang-Hao; Trau, Matt

2016-01-01

Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samples where results subsequently validated with ddPCR. With ddPCR-like sensitivity and accuracy yet at the convenience of standard PCR, we believe this multiplex PCR/SERS method could find wide applications in both diagnostics and research. PMID:27446486

Simple, Sensitive and Accurate Multiplex Detection of Clinically Important Melanoma DNA Mutations in Circulating Tumour DNA with SERS Nanotags.

PubMed

Wee, Eugene J H; Wang, Yuling; Tsao, Simon Chang-Hao; Trau, Matt

2016-01-01

Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samples where results subsequently validated with ddPCR. With ddPCR-like sensitivity and accuracy yet at the convenience of standard PCR, we believe this multiplex PCR/SERS method could find wide applications in both diagnostics and research.

Mechanistic applicability domain classification of a local lymph node assay dataset for skin sensitization.

PubMed

Roberts, David W; Patlewicz, Grace; Kern, Petra S; Gerberick, Frank; Kimber, Ian; Dearman, Rebecca J; Ryan, Cindy A; Basketter, David A; Aptula, Aynur O

2007-07-01

The goal of eliminating animal testing in the predictive identification of chemicals with the intrinsic ability to cause skin sensitization is an important target, the attainment of which has recently been brought into even sharper relief by the EU Cosmetics Directive and the requirements of the REACH legislation. Development of alternative methods requires that the chemicals used to evaluate and validate novel approaches comprise not only confirmed skin sensitizers and non-sensitizers but also substances that span the full chemical mechanistic spectrum associated with skin sensitization. To this end, a recently published database of more than 200 chemicals tested in the mouse local lymph node assay (LLNA) has been examined in relation to various chemical reaction mechanistic domains known to be associated with sensitization. It is demonstrated here that the dataset does cover the main reaction mechanistic domains. In addition, it is shown that assignment to a reaction mechanistic domain is a critical first step in a strategic approach to understanding, ultimately on a quantitative basis, how chemical properties influence the potency of skin sensitizing chemicals. This understanding is necessary if reliable non-animal approaches, including (quantitative) structure-activity relationships (Q)SARs, read-across, and experimental chemistry based models, are to be developed.

Validation of HPLC and UV spectrophotometric methods for the determination of meropenem in pharmaceutical dosage form.

PubMed

Mendez, Andreas S L; Steppe, Martin; Schapoval, Elfrides E S

2003-12-04

A high-performance liquid chromatographic method and a UV spectrophotometric method for the quantitative determination of meropenem, a highly active carbapenem antibiotic, in powder for injection were developed in present work. The parameters linearity, precision, accuracy, specificity, robustness, limit of detection and limit of quantitation were studied according to International Conference on Harmonization guidelines. Chromatography was carried out by reversed-phase technique on an RP-18 column with a mobile phase composed of 30 mM monobasic phosphate buffer and acetonitrile (90:10; v/v), adjusted to pH 3.0 with orthophosphoric acid. The UV spectrophotometric method was performed at 298 nm. The samples were prepared in water and the stability of meropenem in aqueous solution at 4 and 25 degrees C was studied. The results were satisfactory with good stability after 24 h at 4 degrees C. Statistical analysis by Student's t-test showed no significant difference between the results obtained by the two methods. The proposed methods are highly sensitive, precise and accurate and can be used for the reliable quantitation of meropenem in pharmaceutical dosage form.

Three Minute Method for Amino Acid Analysis by UHPLC and high resolution quadrupole orbitrap mass spectrometry

PubMed Central

Nemkov, Travis; D'Alessandro, Angelo; Hansen, Kirk C.

2015-01-01

Amino acid analysis is a powerful bioanalytical technique for many biomedical research endeavors, including cancer, emergency medicine, nutrition and neuroscience research. In the present study, we present a three minute analytical method for underivatized amino acid analysis that employs ultra-high performance liquid chromatography and high resolution quadrupole orbitrap mass spectrometry. This method has demonstrated linearity (mM to nM range), reproducibility (intra-day<5%, inter-day<20%), sensitivity (low fmol) and selectivity. Here, we illustrate the rapidity and accuracy of the method through comparison with conventional liquid chromatography-mass spectrometry methods. We further demonstrate the robustness and sensitivity of this method on a diverse range of biological matrices. Using this method we were able to selectively discriminate murine pancreatic cancer cells with and without knocked down expression of Hypoxia Inducible Factor 1α; plasma, lymph and bronchioalveolar lavage fluid samples from control versus hemorrhaged rats; and muscle tissue samples harvested from rats subjected to both low fat and high fat diets. Furthermore, we were able to exploit the sensitivity of the method to detect and quantify the release of glutamate from sparsely isolated murine taste buds. Spiked in light or heavy standards (13C6-arginine, 13C6-lysine, 13C515N2-glutamine) or xenometabolites were used to determine coefficient of variations, confirm linearity of relative quantitation in four different matrices, and overcome matrix effects for absolute quantitation. The presented method enables high-throughput analysis of low abundance samples requiring only one percent of the material extracted from 100,000 cells, 10 μl of biological fluid, or 2 mg of muscle tissue. PMID:26058356

Quantitative Determination of Levonorgestrel in Fish Plasma using UPLC-MS/MS

EPA Science Inventory

In this study, a sensitive high-performance liquid chromatography electrospray tandem mass spectrometric method was developed for the determination of levonorgestrel in fish plasma using levonorgestrel-d6 as an internal standard (IS). In the laboratory, the fish cunner, (Tautogol...

Periodate Oxidation of Vicinal Hydroxyls: Applications to Student Experiments

ERIC Educational Resources Information Center

Pilch, Paul F.; Somerville, Ronald L.

1977-01-01

A quick, sensitive method which is adaptable for quantitating periodate consumption in a variety of undergraduate laboratory contexts is described. A laboratory procedure is also given that may be used for measurements of reaction rates as a function of temperature. (MR)

Enzyme-Encapsulated Liposome-Linked Immunosorbent Assay Enabling Sensitive Personal Glucose Meter Readout for Portable Detection of Disease Biomarkers.

PubMed

Lin, Bingqian; Liu, Dan; Yan, Jinmao; Qiao, Zhi; Zhong, Yunxin; Yan, Jiawei; Zhu, Zhi; Ji, Tianhai; Yang, Chaoyong James

2016-03-23

There is considerable demand for sensitive, selective, and portable detection of disease-associated proteins, particularly in clinical practice and diagnostic applications. Portable devices are highly desired for detection of disease biomarkers in daily life due to the advantages of being simple, rapid, user-friendly, and low-cost. Herein we report an enzyme-encapsulated liposome-linked immunosorbent assay for sensitive detection of proteins using personal glucose meters (PGM) for portable quantitative readout. Liposomes encapsulating a large amount of amyloglucosidase or invertase are surface-coated with recognition elements such as aptamers or antibodies for target recognition. By translating molecular recognition signal into a large amount of glucose with the encapsulated enzyme, disease biomarkers such as thrombin or C-reactive protein (CRP) can be quantitatively detected by a PGM with a high detection limit of 1.8 or 0.30 nM, respectively. With the advantages of portability, ease of use, and low-cost, the method reported here has potential for portable and quantitative detection of various targets for different POC testing scenarios, such as rapid diagnosis in clinic offices, health monitoring at the bedside, and chemical/biochemical safety control in the field.

Detection and quantitation of trace phenolphthalein (in pharmaceutical preparations and in forensic exhibits) by liquid chromatography-tandem mass spectrometry, a sensitive and accurate method.

PubMed

Sharma, Kakali; Sharma, Shiba P; Lahiri, Sujit C

2013-01-01

Phenolphthalein, an acid-base indicator and laxative, is important as a constituent of widely used weight-reducing multicomponent food formulations. Phenolphthalein is an useful reagent in forensic science for the identification of blood stains of suspected victims and for apprehending erring officials accepting bribes in graft or trap cases. The pink-colored alkaline hand washes originating from the phenolphthalein-smeared notes can easily be determined spectrophotometrically. But in many cases, colored solution turns colorless with time, which renders the genuineness of bribe cases doubtful to the judiciary. No method is known till now for the detection and identification of phenolphthalein in colorless forensic exhibits with positive proof. Liquid chromatography-tandem mass spectrometry had been found to be most sensitive, accurate method capable of detection and quantitation of trace phenolphthalein in commercial formulations and colorless forensic exhibits with positive proof. The detection limit of phenolphthalein was found to be 1.66 pg/L or ng/mL, and the calibration curve shows good linearity (r(2) = 0.9974). © 2012 American Academy of Forensic Sciences.

A triplex quantitative real-time PCR assay for differential detection of human adenovirus serotypes 2, 3 and 7.

PubMed

Qiu, Fang-Zhou; Shen, Xin-Xin; Zhao, Meng-Chuan; Zhao, Li; Duan, Su-Xia; Chen, Chen; Qi, Ju-Ju; Li, Gui-Xia; Wang, Le; Feng, Zhi-Shan; Ma, Xue-Jun

2018-05-02

Human adenovirus (HAdV) serotypes 2, 3 and 7 are more prevalent than other serotypes and have been associated with severe pneumonia in pediatric children. Molecular typing of HAdV is not routinely performed in clinical diagnostic laboratories as it is time-consuming and labor-intensive. In the present study, we developed a triplex quantitative real-time PCR assay (tq-PCR) in a single closed tube for differential detection and quantitative analysis of HAdV serotypes 2, 3 and 7. The sensitivity, specificity, reproducibility and clinical performance of tq-PCR were evaluated. The analytical sensitivity of the tq-PCR was 100 copies/reaction for each of HAdV serotypes 2, 3 and 7, and no cross-reaction with other common respiratory viruses or HAdV serotypes 1,4,5,6,31,55 and 57 was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.6% to 3.6%. Of 138 previously-defined HAdV-positive nasopharyngeal aspirates samples tested, the detection agreement between tq-PCR and nested PCR was 96.38% (133/138). The proposed tq-PCR assay is a sensitive, specific and reproducible method and has the potential for clinical use in the rapid and differential detection and quantitation of HAdV serotypes 2, 3 and 7.

Quantitation of TGF-beta1 mRNA in porcine mesangial cells by comparative kinetic RT/PCR: comparison with ribonuclease protection assay and in situ hybridization.

PubMed

Ceol, M; Forino, M; Gambaro, G; Sauer, U; Schleicher, E D; D'Angelo, A; Anglani, F

2001-01-01

Gene expression can be examined with different techniques including ribonuclease protection assay (RPA), in situ hybridisation (ISH), and quantitative reverse transcription-polymerase chain reaction (RT/PCR). These methods differ considerably in their sensitivity and precision in detecting and quantifying low abundance mRNA. Although there is evidence that RT/PCR can be performed in a quantitative manner, the quantitative capacity of this method is generally underestimated. To demonstrate that the comparative kinetic RT/PCR strategy-which uses a housekeeping gene as internal standard-is a quantitative method to detect significant differences in mRNA levels between different samples, the inhibitory effect of heparin on phorbol 12-myristate 13-acetate (PMA)-induced-TGF-beta1 mRNA expression was evaluated by RT/PCR and RPA, the standard method of mRNA quantification, and the results were compared. The reproducibility of RT/PCR amplification was calculated by comparing the quantity of G3PDH and TGF-beta1 PCR products, generated during the exponential phases, estimated from two different RT/PCR (G3PDH, r = 0.968, P = 0.0000; TGF-beta1, r = 0.966, P = 0.0000). The quantitative capacity of comparative kinetic RT/PCR was demonstrated by comparing the results obtained from RPA and RT/PCR using linear regression analysis. Starting from the same RNA extraction, but using only 1% of the RNA for the RT/PCR compared to RPA, significant correlation was observed (r = 0.984, P = 0.0004). Moreover the morphometric analysis of ISH signal was applied for the semi-quantitative evaluation of the expression and localisation of TGF-beta1 mRNA in the entire cell population. Our results demonstrate the close similarity of the RT/PCR and RPA methods in giving quantitative information on mRNA expression and indicate the possibility to adopt the comparative kinetic RT/PCR as reliable quantitative method of mRNA analysis. Copyright 2001 Wiley-Liss, Inc.

Breast cancer Ki67 expression preoperative discrimination by DCE-MRI radiomics features

NASA Astrophysics Data System (ADS)

Ma, Wenjuan; Ji, Yu; Qin, Zhuanping; Guo, Xinpeng; Jian, Xiqi; Liu, Peifang

2018-02-01

To investigate whether quantitative radiomics features extracted from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) are associated with Ki67 expression of breast cancer. In this institutional review board approved retrospective study, we collected 377 cases Chinese women who were diagnosed with invasive breast cancer in 2015. This cohort included 53 low-Ki67 expression (Ki67 proliferation index less than 14%) and 324 cases with high-Ki67 expression (Ki67 proliferation index more than 14%). A binary-classification of low- vs. high- Ki67 expression was performed. A set of 52 quantitative radiomics features, including morphological, gray scale statistic, and texture features, were extracted from the segmented lesion area. Three most common machine learning classification methods, including Naive Bayes, k-Nearest Neighbor and support vector machine with Gaussian kernel, were employed for the classification and the least absolute shrink age and selection operator (LASSO) method was used to select most predictive features set for the classifiers. Classification performance was evaluated by the area under receiver operating characteristic curve (AUC), accuracy, sensitivity and specificity. The model that used Naive Bayes classification method achieved the best performance than the other two methods, yielding 0.773 AUC value, 0.757 accuracy, 0.777 sensitivity and 0.769 specificity. Our study showed that quantitative radiomics imaging features of breast tumor extracted from DCE-MRI are associated with breast cancer Ki67 expression. Future larger studies are needed in order to further evaluate the findings.

Quantitative fluorescence in intracranial tumor: implications for ALA-induced PpIX as an intraoperative biomarker

PubMed Central

Valdés, Pablo A.; Leblond, Frederic; Kim, Anthony; Harris, Brent T.; Wilson, Brian C.; Fan, Xiaoyao; Tosteson, Tor D.; Hartov, Alex; Ji, Songbai; Erkmen, Kadir; Simmons, Nathan E.; Paulsen, Keith D.; Roberts, David W.

2011-01-01

Object Accurate discrimination between tumor and normal tissue is crucial for optimal tumor resection. Qualitative fluorescence of protoporphyrin IX (PpIX), synthesized endogenously following δ-aminolevulinic acid (ALA) administration, has been used for this purpose in high-grade glioma (HGG). The authors show that diagnostically significant but visually imperceptible concentrations of PpIX can be quantitatively measured in vivo and used to discriminate normal from neoplastic brain tissue across a range of tumor histologies. Methods The authors studied 14 patients with diagnoses of low-grade glioma (LGG), HGG, meningioma, and metastasis under an institutional review board–approved protocol for fluorescence-guided resection. The primary aim of the study was to compare the diagnostic capabilities of a highly sensitive, spectrally resolved quantitative fluorescence approach to conventional fluorescence imaging for detection of neoplastic tissue in vivo. Results A significant difference in the quantitative measurements of PpIX concentration occurred in all tumor groups compared with normal brain tissue. Receiver operating characteristic (ROC) curve analysis of PpIX concentration as a diagnostic variable for detection of neoplastic tissue yielded a classification efficiency of 87% (AUC = 0.95, specificity = 92%, sensitivity = 84%) compared with 66% (AUC = 0.73, specificity = 100%, sensitivity = 47%) for conventional fluorescence imaging (p < 0.0001). More than 81% (57 of 70) of the quantitative fluorescence measurements that were below the threshold of the surgeon's visual perception were classified correctly in an analysis of all tumors. Conclusions These findings are clinically profound because they demonstrate that ALA-induced PpIX is a targeting biomarker for a variety of intracranial tumors beyond HGGs. This study is the first to measure quantitative ALA-induced PpIX concentrations in vivo, and the results have broad implications for guidance during resection of intracranial tumors. PMID:21438658

Novel atomic absorption spectrometric and rapid spectrophotometric methods for the quantitation of paracetamol in saliva: application to pharmacokinetic studies.

PubMed

Issa, M M; Nejem, R M; El-Abadla, N S; Al-Kholy, M; Saleh, Akila A

2008-01-01

A novel atomic absorption spectrometric method and two highly sensitive spectrophotometric methods were developed for the determination of paracetamol. These techniques based on the oxidation of paracetamol by iron (III) (method I); oxidation of p-aminophenol after the hydrolysis of paracetamol (method II). Iron (II) then reacts with potassium ferricyanide to form Prussian blue color with a maximum absorbance at 700 nm. The atomic absorption method was accomplished by extracting the excess iron (III) in method II and aspirates the aqueous layer into air-acetylene flame to measure the absorbance of iron (II) at 302.1 nm. The reactions have been spectrometrically evaluated to attain optimum experimental conditions. Linear responses were exhibited over the ranges 1.0-10, 0.2-2.0 and 0.1-1.0 mug/ml for method I, method II and atomic absorption spectrometric method, respectively. A high sensitivity is recorded for the proposed methods I and II and atomic absorption spectrometric method value indicate: 0.05, 0.022 and 0.012 mug/ml, respectively. The limit of quantitation of paracetamol by method II and atomic absorption spectrometric method were 0.20 and 0.10 mug/ml. Method II and the atomic absorption spectrometric method were applied to demonstrate a pharmacokinetic study by means of salivary samples in normal volunteers who received 1.0 g paracetamol. Intra and inter-day precision did not exceed 6.9%.

Novel Atomic Absorption Spectrometric and Rapid Spectrophotometric Methods for the Quantitation of Paracetamol in Saliva: Application to Pharmacokinetic Studies

PubMed Central

Issa, M. M.; Nejem, R. M.; El-Abadla, N. S.; Al-Kholy, M.; Saleh, Akila. A.

2008-01-01

A novel atomic absorption spectrometric method and two highly sensitive spectrophotometric methods were developed for the determination of paracetamol. These techniques based on the oxidation of paracetamol by iron (III) (method I); oxidation of p-aminophenol after the hydrolysis of paracetamol (method II). Iron (II) then reacts with potassium ferricyanide to form Prussian blue color with a maximum absorbance at 700 nm. The atomic absorption method was accomplished by extracting the excess iron (III) in method II and aspirates the aqueous layer into air-acetylene flame to measure the absorbance of iron (II) at 302.1 nm. The reactions have been spectrometrically evaluated to attain optimum experimental conditions. Linear responses were exhibited over the ranges 1.0-10, 0.2-2.0 and 0.1-1.0 μg/ml for method I, method II and atomic absorption spectrometric method, respectively. A high sensitivity is recorded for the proposed methods I and II and atomic absorption spectrometric method value indicate: 0.05, 0.022 and 0.012 μg/ml, respectively. The limit of quantitation of paracetamol by method II and atomic absorption spectrometric method were 0.20 and 0.10 μg/ml. Method II and the atomic absorption spectrometric method were applied to demonstrate a pharmacokinetic study by means of salivary samples in normal volunteers who received 1.0 g paracetamol. Intra and inter-day precision did not exceed 6.9%. PMID:20046743

Evaluation of serological and molecular tests used to identify Toxoplasma gondii infection in pregnant women attended in a public health service in São Paulo state, Brazil.

PubMed

Murata, Fernando Henrique Antunes; Ferreira, Marina Neves; Pereira-Chioccola, Vera Lucia; Spegiorin, Lígia Cosentino Junqueira Franco; Meira-Strejevitch, Cristina da Silva; Gava, Ricardo; Silveira-Carvalho, Aparecida Perpétuo; de Mattos, Luiz Carlos; Brandão de Mattos, Cinara Cássia

2017-09-01

Toxoplasmosis during pregnancy can have severe consequences. The use of sensitive and specific serological and molecular methods is extremely important for the correct diagnosis of the disease. We compared the ELISA and ELFA serological methods, conventional PCR (cPCR), Nested PCR and quantitative PCR (qPCR) in the diagnosis of Toxoplasma gondii infection in pregnant women without clinical suspicion of toxoplasmosis (G1=94) and with clinical suspicion of toxoplasmosis (G2=53). The results were compared using the Kappa index, and the sensitivity, specificity, positive predictive value and negative predictive value were calculated. The results of the serological methods showed concordance between the ELISA and ELFA methods even though ELFA identified more positive cases than ELISA. Molecular methods were discrepant with cPCR using B22/23 primers having greater sensitivity and lower specificity compared to the other molecular methods. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantitative determination of some pharmaceutical piperazine derivatives through complexation with iron(III) chloride.

PubMed

Abou-Attia, F M; Issa, Y M; Abdel-Gawad, F M; Abdel-Hamid, S M

2003-08-01

A simple, accurate and sensitive spectrophotometric method has been developed for the determination of three pharmaceutical piperazine derivatives, namely ketoconazole (KC), trimetazidine hydrochloride (TMH) and piribedil (PD). This method is based on the formation of yellow orange complexes between iron(III) chloride and the investigated drugs. The optimum reaction conditions, spectral characteristics, conditional stability constants and composition of the water soluble complexes have been established. The method permits the determination of KC, TMH and PD over a concentration range 1-15, 1-12 and 1-12 microg ml(-1), respectively. Sandell sensitivity is found to be 0.016, 0.013 and 0.013 microg cm(-2) for KC, TMH and PD, respectively. The method was sensitive, simple, reproducible and accurate within +/-1.5%. The method is applicable to the assay of the three drugs under investigation in different dosage forms and the results are in good agreement with those obtained by the official methods (USP and JP).

Simple and ultra-fast recognition and quantitation of compounded monoclonal antibodies: Application to flow injection analysis combined to UV spectroscopy and matching method.

PubMed

Jaccoulet, E; Schweitzer-Chaput, A; Toussaint, B; Prognon, P; Caudron, E

2018-09-01

Compounding of monoclonal antibody (mAbs) constantly increases in hospital. Quality control (QC) of the compounded mAbs based on quantification and identification is required to prevent potential errors and fast method is needed to manage outpatient chemotherapy administration. A simple and ultra-fast (less than 30 s) method using flow injection analysis associated to least square matching method issued from the analyzer software was performed and evaluated for the routine hospital QC of three compounded mAbs: bevacizumab, infliximab and rituximab. The method was evaluated through qualitative and quantitative parameters. Preliminary analysis of the UV absorption and second derivative spectra of the mAbs allowed us to adapt analytical conditions according to the therapeutic range of the mAbs. In terms of quantitative QC, linearity, accuracy and precision were assessed as specified in ICH guidelines. Very satisfactory recovery was achieved and the RSD (%) of the intermediate precision were less than 1.1%. Qualitative analytical parameters were also evaluated in terms of specificity, sensitivity and global precision through a matrix of confusion. Results showed to be concentration and mAbs dependant and excellent (100%) specificity and sensitivity were reached within specific concentration range. Finally, routine application on "real life" samples (n = 209) from different batch of the three mAbs complied with the specifications of the quality control i.e. excellent identification (100%) and ± 15% of targeting concentration belonging to the calibration range. The successful use of the combination of second derivative spectroscopy and partial least square matching method demonstrated the interest of FIA for the ultra-fast QC of mAbs after compounding using matching method. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of a colloidal gold immunochromatographic strip assay for simple and fast detection of human α-lactalbumin in genetically modified cow milk.

PubMed

Tao, Chenyu; Zhang, Qingde; Feng, Na; Shi, Deshi; Liu, Bang

2016-03-01

The qualitative and quantitative declaration of food ingredients is important to consumers, especially for genetically modified food as it experiences a rapid increase in sales. In this study, we designed an accurate and rapid detection system using colloidal gold immunochromatographic strip assay (GICA) methods to detect genetically modified cow milk. First, we prepared 2 monoclonal antibodies for human α-lactalbumin (α-LA) and measured their antibody titers; the one with the higher titer was used for further experiments. Then, we found the optimal pH value and protein amount of GICA for detection of pure milk samples. The developed strips successfully detected genetically modified cow milk and non-modified cow milk. To determine the sensitivity of GICA, a quantitative ELISA system was used to determine the exact amount of α-LA, and then genetically modified milk was diluted at different rates to test the sensitivity of GICA; the sensitivity was 10 μg/mL. Our results demonstrated that the applied method was effective to detect human α-LA in cow milk. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Quantitation of human milk proteins and their glycoforms using multiple reaction monitoring (MRM).

PubMed

Huang, Jincui; Kailemia, Muchena J; Goonatilleke, Elisha; Parker, Evan A; Hong, Qiuting; Sabia, Rocchina; Smilowitz, Jennifer T; German, J Bruce; Lebrilla, Carlito B

2017-01-01

Human milk plays a substantial role in the child growth, development and determines their nutritional and health status. Despite the importance of the proteins and glycoproteins in human milk, very little quantitative information especially on their site-specific glycosylation is known. As more functions of milk proteins and other components continue to emerge, their fine-detailed quantitative information is becoming a key factor in milk research efforts. The present work utilizes a sensitive label-free MRM method to quantify seven milk proteins (α-lactalbumin, lactoferrin, secretory immunoglobulin A, immunoglobulin G, immunoglobulin M, α1-antitrypsin, and lysozyme) using their unique peptides while at the same time, quantifying their site-specific N-glycosylation relative to the protein abundance. The method is highly reproducible, has low limit of quantitation, and accounts for differences in glycosylation due to variations in protein amounts. The method described here expands our knowledge about human milk proteins and provides vital details that could be used in monitoring the health of the infant and even the mother. Graphical Abstract The glycopeptides EICs generated from QQQ.

Quantitation of human milk proteins and their glycoforms using multiple reaction monitoring (MRM)

PubMed Central

Huang, Jincui; Kailemia, Muchena J.; Goonatilleke, Elisha; Parker, Evan A.; Hong, Qiuting; Sabia, Rocchina; Smilowitz, Jennifer T.; German, J. Bruce

2017-01-01

Human milk plays a substantial role in the child growth, development and determines their nutritional and health status. Despite the importance of the proteins and glycoproteins in human milk, very little quantitative information especially on their site-specific glycosylation is known. As more functions of milk proteins and other components continue to emerge, their fine-detailed quantitative information is becoming a key factor in milk research efforts. The present work utilizes a sensitive label-free MRM method to quantify seven milk proteins (α-lactalbumin, lactoferrin, secretory immunoglobulin A, immunoglobulin G, immunoglobulin M, α1-antitrypsin, and lysozyme) using their unique peptides while at the same time, quantifying their site-specific N-glycosylation relative to the protein abundance. The method is highly reproducible, has low limit of quantitation, and accounts for differences in glycosylation due to variations in protein amounts. The method described here expands our knowledge about human milk proteins and provides vital details that could be used in monitoring the health of the infant and even the mother. PMID:27796459

Determination of alkylphenol and alkylphenolethoxylates in biota by liquid chromatography with detection by tandem mass spectrometry and fluorescence spectroscopy

USGS Publications Warehouse

Schmitz-Afonso, I.; Loyo-Rosales, J.E.; de la Paz Aviles, M.; Rattner, B.A.; Rice, C.P.

2003-01-01

A quantitative method for the simultaneous determination of octylphenol, nonylphenol and the corresponding ethoxylates (1 to 5) in biota is presented. Extraction methods were developed for egg and fish matrices based on accelerated solvent extraction followed by a solid-phase extraction cleanup, using octadecylsilica or aminopropyl cartridges. Identification and quantitation were accomplished by liquid chromatography-electrospray tandem mass spectrometry (LC-MS-MS) and compared to the traditional liquid chromatography with fluorescence spectroscopy detection. LC-MS-MS provides high sensitivity and specificity required for these complex matrices and an accurate quantitation with the use of 13C-labeled internal standards. Quantitation limits by LC-MS-MS ranged from 4 to 12 ng/g in eggs, and from 6 to 22 ng/g in fish samples. These methods were successfully applied to osprey eggs from the Chesapeake Bay and fish from the Great Lakes area. Total levels found in osprey egg samples were up to 18 ng/g wet mass and as high as 8.2 ug/g wet mass in the fish samples.

Evaluation of dried blood spot as an alternative sample collection method for hepatitis C virus RNA quantitation and genotyping using a commercial system.

PubMed

Mahajan, Supriya; Choudhary, Manish Chandra; Kumar, Guresh; Gupta, Ekta

2018-06-01

Dried blood spot (DBS) is a minimally invasive sampling method suitable for sample collection, storage and transportation in resource limited areas. Aim of this study was to compare the diagnostic utility of DBS with plasma sample for HCV RNA quantitation and genotyping using commercial systems. Plasma and DBS card spotted samples were collected from 95 HCV seropositive patients. Both types of samples were subjected to HCV RNA by real-time PCR (Abbott m2000rt, USA). Genotyping was performed using Abbott HCV genotype II kit (Abbott diagnostics, USA) in samples with viral load > 3 log 10  IU/ml. In both plasma and DBS, 14 (14.7%) samples were negative and 81 (85.3%) were positive for HCV RNA. Median viral load in plasma (3.78; range 0-7.43) log 10  IU/ml was comparable to DBS (3.93; range 0-7.24) log 10  IU/ml. DBS demonstrated sensitivity and specificity of 97.5 and 85.7% respectively, with positive predictive value (PPV) of 97.5% and negative predictive value (NPV) of 85.7%. DBS showed good correlation (r 2  = 0.866) and agreement (93.5%) with plasma. Genotyping in 20 patients showed 100% concordance between DBS and plasma samples. DBS showed good sensitivity and specificity as a sampling method for HCV RNA quantitation and genotyping.

The local lymph node assay and skin sensitization testing.

PubMed

Kimber, Ian; Dearman, Rebecca J

2010-01-01

The mouse local lymph node assay (LLNA) is a method for the identification and characterization of skin sensitization hazards. In this context the method can be used both to identify contact allergens, and also determine the relative skin sensitizing potency as a basis for derivation of effective risk assessments.The assay is based on measurement of proliferative responses by draining lymph node cells induced following topical exposure of mice to test chemicals. Such responses are known to be causally and quantitatively associated with the acquisition of skin sensitization and therefore provide a relevant marker for characterization of contact allergic potential.The LLNA has been the subject of exhaustive evaluation and validation exercises and has been assigned Organization for Economic Cooperation and Development (OECD) test guideline 429. Herein we describe the conduct and interpretation of the LLNA.

Quantitation of permethylated N-glycans through multiple-reaction monitoring (MRM) LC-MS/MS.

PubMed

Zhou, Shiyue; Hu, Yunli; DeSantos-Garcia, Janie L; Mechref, Yehia

2015-04-01

The important biological roles of glycans and their implications in disease development and progression have created a demand for the development of sensitive quantitative glycomics methods. Quantitation of glycans existing at low abundance is still analytically challenging. In this study, an N-linked glycans quantitation method using multiple-reaction monitoring (MRM) on a triple quadrupole instrument was developed. Optimum normalized collision energy (CE) for both sialylated and fucosylated N-glycan was determined to be 30%, whereas it was found to be 35% for either fucosylated or sialylated N-glycans. The optimum CE for mannose and complex type N-glycan was determined to be 35%. Additionally, the use of three transitions was shown to facilitate reliable quantitation. A total of 88 N-glycan compositions in human blood serum were quantified using this MRM approach. Reliable detection and quantitation of these glycans was achieved when the equivalence of 0.005 μL of blood serum was analyzed. Accordingly, N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, spanning a dynamic concentration range of three orders of magnitude. MRM was also effectively utilized to quantitatively compare the expression of N-glycans derived from brain-targeting breast carcinoma cells (MDA-MB-231BR) and metastatic breast cancer cells (MDA-MB-231). Thus, the described MRM method of permethylated N-glycan enables a rapid and reliable identification and quantitation of glycans derived from glycoproteins purified or present in complex biological samples.

Quantitation of Permethylated N-Glycans through Multiple-Reaction Monitoring (MRM) LC-MS/MS

NASA Astrophysics Data System (ADS)

Zhou, Shiyue; Hu, Yunli; DeSantos-Garcia, Janie L.; Mechref, Yehia

2015-04-01

The important biological roles of glycans and their implications in disease development and progression have created a demand for the development of sensitive quantitative glycomics methods. Quantitation of glycans existing at low abundance is still analytically challenging. In this study, an N-linked glycans quantitation method using multiple-reaction monitoring (MRM) on a triple quadrupole instrument was developed. Optimum normalized collision energy (CE) for both sialylated and fucosylated N-glycan was determined to be 30%, whereas it was found to be 35% for either fucosylated or sialylated N-glycans. The optimum CE for mannose and complex type N-glycan was determined to be 35%. Additionally, the use of three transitions was shown to facilitate reliable quantitation. A total of 88 N-glycan compositions in human blood serum were quantified using this MRM approach. Reliable detection and quantitation of these glycans was achieved when the equivalence of 0.005 μL of blood serum was analyzed. Accordingly, N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, spanning a dynamic concentration range of three orders of magnitude. MRM was also effectively utilized to quantitatively compare the expression of N-glycans derived from brain-targeting breast carcinoma cells (MDA-MB-231BR) and metastatic breast cancer cells (MDA-MB-231). Thus, the described MRM method of permethylated N-glycan enables a rapid and reliable identification and quantitation of glycans derived from glycoproteins purified or present in complex biological samples.

Variable selection based near infrared spectroscopy quantitative and qualitative analysis on wheat wet gluten

NASA Astrophysics Data System (ADS)

Lü, Chengxu; Jiang, Xunpeng; Zhou, Xingfan; Zhang, Yinqiao; Zhang, Naiqian; Wei, Chongfeng; Mao, Wenhua

2017-10-01

Wet gluten is a useful quality indicator for wheat, and short wave near infrared spectroscopy (NIRS) is a high performance technique with the advantage of economic rapid and nondestructive test. To study the feasibility of short wave NIRS analyzing wet gluten directly from wheat seed, 54 representative wheat seed samples were collected and scanned by spectrometer. 8 spectral pretreatment method and genetic algorithm (GA) variable selection method were used to optimize analysis. Both quantitative and qualitative model of wet gluten were built by partial least squares regression and discriminate analysis. For quantitative analysis, normalization is the optimized pretreatment method, 17 wet gluten sensitive variables are selected by GA, and GA model performs a better result than that of all variable model, with R2V=0.88, and RMSEV=1.47. For qualitative analysis, automatic weighted least squares baseline is the optimized pretreatment method, all variable models perform better results than those of GA models. The correct classification rates of 3 class of <24%, 24-30%, >30% wet gluten content are 95.45, 84.52, and 90.00%, respectively. The short wave NIRS technique shows potential for both quantitative and qualitative analysis of wet gluten for wheat seed.

ASSESSMENT OF DIFFERENTIAL GENE EXPRESSION IN SHEEPSHEAD MINNOWS EXPOSED TO ENVIRONMENTAL ESTROGENS

EPA Science Inventory

Gene arrays and quantitative real-time PCR (Q-PRC) are sensitive methods for assessing exposure of fish and other wildlife to environmental contaminants by measuring changes in gene expression. Several genes normally induced by estradiol in female fish, those for vitellogenins (...

Quantum dots assisted laser desorption/ionization mass spectrometric detection of carbohydrates: qualitative and quantitative analysis.

PubMed

Bibi, Aisha; Ju, Huangxian

2016-04-01

A quantum dots (QDs) assisted laser desorption/ionization mass spectrometric (QDA-LDI-MS) strategy was proposed for qualitative and quantitative analysis of a series of carbohydrates. The adsorption of carbohydrates on the modified surface of different QDs as the matrices depended mainly on the formation of hydrogen bonding, which led to higher MS intensity than those with conventional organic matrix. The effects of QDs concentration and sample preparation method were explored for improving the selective ionization process and the detection sensitivity. The proposed approach offered a new dimension to the application of QDs as matrices for MALDI-MS research of carbohydrates. It could be used for quantitative measurement of glucose concentration in human serum with good performance. The QDs served as a matrix showed the advantages of low background, higher sensitivity, convenient sample preparation and excellent stability under vacuum. The QDs assisted LDI-MS approach has promising application to the analysis of carbohydrates in complex biological samples. Copyright © 2016 John Wiley & Sons, Ltd.

Acoustics based assessment of respiratory diseases using GMM classification.

PubMed

Mayorga, P; Druzgalski, C; Morelos, R L; Gonzalez, O H; Vidales, J

2010-01-01

The focus of this paper is to present a method utilizing lung sounds for a quantitative assessment of patient health as it relates to respiratory disorders. In order to accomplish this, applicable traditional techniques within the speech processing domain were utilized to evaluate lung sounds obtained with a digital stethoscope. Traditional methods utilized in the evaluation of asthma involve auscultation and spirometry, but utilization of more sensitive electronic stethoscopes, which are currently available, and application of quantitative signal analysis methods offer opportunities of improved diagnosis. In particular we propose an acoustic evaluation methodology based on the Gaussian Mixed Models (GMM) which should assist in broader analysis, identification, and diagnosis of asthma based on the frequency domain analysis of wheezing and crackles.

Highly sensitive quantitative PCR for the detection and differentiation of Pseudogymnoascus destructans and other Pseudogymnoascus species.

PubMed

Shuey, Megan M; Drees, Kevin P; Lindner, Daniel L; Keim, Paul; Foster, Jeffrey T

2014-03-01

White-nose syndrome is a fungal disease that has decimated bat populations across eastern North America. Identification of the etiologic agent, Pseudogymnoascus destructans (formerly Geomyces destructans), in environmental samples is essential to proposed management plans. A major challenge is the presence of closely related species, which are ubiquitous in many soils and cave sediments and often present in high abundance. We present a dual-probe real-time quantitative PCR assay capable of detecting and differentiating P. destructans from closely related fungi in environmental samples from North America. The assay, based on a single nucleotide polymorphism (SNP) specific to P. destructans, is capable of rapid low-level detection from various sampling media, including sediment, fecal samples, wing biopsy specimens, and skin swabs. This method is a highly sensitive, high-throughput method for identifying P. destructans, other Pseudogymnoascus spp., and Geomyces spp. in the environment, providing a fundamental component of research and risk assessment for addressing this disease, as well as other ecological and mycological work on related fungi.

Electrostatic Effects in Filamentous Protein Aggregation

PubMed Central

Buell, Alexander K.; Hung, Peter; Salvatella, Xavier; Welland, Mark E.; Dobson, Christopher M.; Knowles, Tuomas P.J.

2013-01-01

Electrostatic forces play a key role in mediating interactions between proteins. However, gaining quantitative insights into the complex effects of electrostatics on protein behavior has proved challenging, due to the wide palette of scenarios through which both cations and anions can interact with polypeptide molecules in a specific manner or can result in screening in solution. In this article, we have used a variety of biophysical methods to probe the steady-state kinetics of fibrillar protein self-assembly in a highly quantitative manner to detect how it is modulated by changes in solution ionic strength. Due to the exponential modulation of the reaction rate by electrostatic forces, this reaction represents an exquisitely sensitive probe of these effects in protein-protein interactions. Our approach, which involves a combination of experimental kinetic measurements and theoretical analysis, reveals a hierarchy of electrostatic effects that control protein aggregation. Furthermore, our results provide a highly sensitive method for the estimation of the magnitude of binding of a variety of ions to protein molecules. PMID:23473495

Chemotyping the distribution of vitamin D metabolites in human serum

NASA Astrophysics Data System (ADS)

Müller, Miriam J.; Stokes, Caroline S.; Lammert, Frank; Volmer, Dietrich A.

2016-02-01

Most studies examining the relationships between vitamin D and disease or health focus on the main 25-hydroxyvitamin D3 (25(OH)D3) metabolite, thus potentially overlooking contributions and dynamic effects of other vitamin D metabolites, the crucial roles of several of which have been previously demonstrated. The ideal assay would determine all relevant high and low-abundant vitamin D species simultaneously. We describe a sensitive quantitative assay for determining the chemotypes of vitamin D metabolites from serum after derivatisation and ultra-high performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (UHPLC-ESI-MS/MS). We performed a validation according to the ‘FDA Guidance for Industry Bioanalytical Method Validation’. The proof-of-concept of the method was then demonstrated by following the metabolite concentrations in patients with chronic liver diseases (CLD) during the course of a vitamin D supplementation study. The new quantitative profiling assay provided highly sensitive, precise and accurate chemotypes of the vitamin D metabolic process rather than the usually determined 25(OH)D3 concentrations.

Qualitative PCR method for Roundup Ready soybean: interlaboratory study.

PubMed

Kodama, Takashi; Kasahara, Masaki; Minegishi, Yasutaka; Futo, Satoshi; Sawada, Chihiro; Watai, Masatoshi; Akiyama, Hiroshi; Teshima, Reiko; Kurosawa, Yasunori; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

2011-01-01

Quantitative and qualitative methods based on PCR have been developed for genetically modified organisms (GMO). Interlaboratory studies were previously conducted for GMO quantitative methods; in this study, an interlaboratory study was conducted for a qualitative method for a GM soybean, Roundup Ready soy (RR soy), with primer pairs designed for the quantitative method of RR soy studied previously. Fourteen laboratories in Japan participated. Each participant extracted DNA from 1.0 g each of the soy samples containing 0, 0.05, and 0.10% of RR soy, and performed PCR with primer pairs for an internal control gene (Le1) and RR soy followed by agarose gel electrophoresis. The PCR product amplified in this PCR system for Le1 was detected from all samples. The sensitivity, specificity, and false-negative and false-positive rates of the method were obtained from the results of RR soy detection. False-negative rates at the level of 0.05 and 0.10% of the RR soy samples were 6.0 and 2.3%, respectively, revealing that the LOD of the method was somewhat below 0.10%. The current study demonstrated that the qualitative method would be practical for monitoring the labeling system of GM soy in kernel lots.

Quantitative nanoimmunosensor based on dark-field illumination with enhanced sensitivity and on-off switching using scattering signals.

PubMed

Lee, Seungah; Nan, He; Yu, Hyunung; Kang, Seong Ho

2016-05-15

A nanoimmunosensor based on wavelength-dependent dark-field illumination with enhanced sensitivity was used to detect a disease-related protein molecule at zeptomolar (zM) concentrations. The assay platform of 100-nm gold nanospots could be selectively acquired using the wavelength-dependence of enhanced scattering signals from antibody-conjugated plasmonic silver nanoparticles (NPs) with on-off switching using optical filters. Detection of human thyroid-stimulating hormone (hTSH) at a sensitivity of 100 zM, which corresponds to 1-2 molecules per gold spot, was possible within a linear range of 100 zM-100 fM (R=0.9968). A significantly enhanced sensitivity (~4-fold) was achieved with enhanced dark-field illumination compared to using a total internal reflection fluorescence immunosensor. Immunoreactions were confirmed via optical axial-slicing based on the spectral characteristics of two plasmonic NPs. This method of using wavelength-dependent dark-field illumination had an enhanced sensitivity and a wide, linear dynamic range of 100 zM-100 fM, and was an effective tool for quantitatively detecting a single molecule on a nanobiochip for molecular diagnostics. Copyright © 2016 Elsevier B.V. All rights reserved.

Method of detecting and counting bacteria

NASA Technical Reports Server (NTRS)

Picciolo, G. L.; Chappelle, E. W. (Inventor)

1976-01-01

An improved method is provided for determining bacterial levels, especially in samples of aqueous physiological fluids. The method depends on the quantitative determination of bacterial adenosine triphosphate (ATP) in the presence of nonbacterial ATP. The bacterial ATP is released by cell rupture and is measured by an enzymatic bioluminescent assay. A concentration technique is included to make the method more sensitive. It is particularly useful where the fluid to be measured contains an unknown or low bacteria count.

Precise quantitation of 136 urinary proteins by LC/MRM-MS using stable isotope labeled peptides as internal standards for biomarker discovery and/or verification studies.

PubMed

Percy, Andrew J; Yang, Juncong; Hardie, Darryl B; Chambers, Andrew G; Tamura-Wells, Jessica; Borchers, Christoph H

2015-06-15

Spurred on by the growing demand for panels of validated disease biomarkers, increasing efforts have focused on advancing qualitative and quantitative tools for more highly multiplexed and sensitive analyses of a multitude of analytes in various human biofluids. In quantitative proteomics, evolving strategies involve the use of the targeted multiple reaction monitoring (MRM) mode of mass spectrometry (MS) with stable isotope-labeled standards (SIS) used for internal normalization. Using that preferred approach with non-invasive urine samples, we have systematically advanced and rigorously assessed the methodology toward the precise quantitation of the largest, multiplexed panel of candidate protein biomarkers in human urine to date. The concentrations of the 136 proteins span >5 orders of magnitude (from 8.6 μg/mL to 25 pg/mL), with average CVs of 8.6% over process triplicate. Detailed here is our quantitative method, the analysis strategy, a feasibility application to prostate cancer samples, and a discussion of the utility of this method in translational studies. Copyright © 2015 Elsevier Inc. All rights reserved.

Micelle Enhanced Fluorimetric and Thin Layer Chromatography Densitometric Methods for the Determination of (±) Citalopram and its S – Enantiomer Escitalopram

PubMed Central

Taha, Elham A.; Salama, Nahla N.; Wang, Shudong

2009-01-01

Two sensitive and validated methods were developed for determination of a racemic mixture citalopram and its enantiomer S-(+) escitalopram. The first method was based on direct measurement of the intrinsic fluorescence of escitalopram using sodium dodecyl sulfate as micelle enhancer. This was further applied to determine escitalopram in spiked human plasma, as well as in the presence of common and co-administerated drugs. The second method was TLC densitometric based on various chiral selectors was investigated. The optimum TLC conditions were found to be sensitive and selective for identification and quantitative determination of enantiomeric purity of escitalopram in drug substance and drug products. The method can be useful to investigate adulteration of pure isomer with the cheap racemic form. PMID:19652757

Micelle enhanced fluorimetric and thin layer chromatography densitometric methods for the determination of (+/-) citalopram and its S-enantiomer escitalopram.

PubMed

Taha, Elham A; Salama, Nahla N; Wang, Shudong

2009-04-07

Two sensitive and validated methods were developed for determination of a racemic mixture citalopram and its enantiomer S-(+) escitalopram. The first method was based on direct measurement of the intrinsic fluorescence of escitalopram using sodium dodecyl sulfate as micelle enhancer. This was further applied to determine escitalopram in spiked human plasma, as well as in the presence of common and co-administrated drugs. The second method was TLC densitometric based on various chiral selectors was investigated. The optimum TLC conditions were found to be sensitive and selective for identification and quantitative determination of enantiomeric purity of escitalopram in drug substance and drug products. The method can be useful to investigate adulteration of pure isomer with the cheap racemic form.

Detection of soy proteins in processed foods: literature overview and new experimental work.

PubMed

Koppelman, Stef J; Lakemond, Catriona M M; Vlooswijk, Riek; Hefle, Susan L

2004-01-01

Several tests for the detection of soy proteins in foods have been described in the literature, and some are commercially available. This article gives an overview of these methods and discusses the advantages and disadvantages of each individual method. Based on the conclusions of this inventory, an experimental approach was designed to improve the sensitivity of measuring soy protein in processed foods. The aimed sensitivity is 10 ppm (10 microg soy protein in 1 g solid sample), which is over 100-fold lower than presently available tests. The aimed sensitivity is this low because levels of food allergens at 10 ppm and above may provoke reactions in food allergic persons. Native soybean meal, soy protein isolate, soy protein concentrate, and textured soy flakes were used as test materials. Several extraction procedures were compared and a new method using high pH was selected. Polyclonal antibodies were raised in rabbits and goats, and immunopurified antibodies were used in sandwich and inhibition enzyme-linked immunosorbent assay (ELISA). Extraction at pH 12 resulted in good yields for all tested samples, both quantitatively (Bradford) and qualitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunopurified rabbit antibodies against this extract used in a competition ELISA format resulted in a sensitive test with a detection limit of 0.02 microg/mL, corresponding to 0.4 microg/g (0.4 ppm) in food samples. Cross-reactivity with some main food ingredients was measured and appeared to be negative in all cases. The presently developed test is applicable for soy ingredients and soy-containing foods that are processed in different ways. The limit of quantitation is 1 ppm, which is an enormous improvement over earlier described methods.

Comparative Evaluation of Preliminary Screening Methods for Colorectal Cancer in a Mass Program.

PubMed

Ye, Ding; Huang, Qiuchi; Li, Qilong; Jiang, Xiyi; Mamat, Mayila; Tang, Mengling; Wang, Jianbing; Chen, Kun

2017-09-01

The fecal immunochemical test (FIT) has been widely used in preliminary screening for colorectal cancer (CRC). The high-risk factor questionnaire (HRFQ) and quantitative risk-assessment method (QRAM) are recommended for estimating the risk of CRC qualitatively and quantitatively in China. We aimed to prospectively compare the diagnostic values of CRC preliminary screening methods to identify which method is preferable as a screening strategy. Individuals aged 40-74 years old were enrolled in a mass CRC screening program from January 1, 2007 to December 31, 2014, in Jiashan County, Zhejiang Province, China. FIT of two stool specimens at 1-week intervals was performed by laboratory personnel and face-to-face interviews were conducted by trained investigators. Screening data in the program were linked to a CRC surveillance and registry system, and CRC cases reported in the system were regarded as true patients. A total of 96,043 subjects were included. The sensitivity and specificity of FIT for detecting CRC cases were 75.49% (95% CI 69.84-80.39) and 90.36% (95% CI 90.17-90.54), respectively. QRAM was more sensitive (p < 0.001) and less specific (p < 0.001) than HRFQ. The sensitivity and specificity of FIT along with HRFQ were 86.56% (95% CI 81.81-90.22) and 81.37% (95% CI 81.12-81.62), and those of FIT along with QRAM were 88.93% (95% CI 84.47-92.23) and 73.95% (95% CI 73.67-74.23). Our findings suggest that CRC preliminary screening with FIT and QRAM in parallel has high sensitivity and satisfactory specificity, and is a useful strategy in mass screening programs.

Multidimensional NMR approaches towards highly resolved, sensitive and high-throughput quantitative metabolomics.

PubMed

Marchand, Jérémy; Martineau, Estelle; Guitton, Yann; Dervilly-Pinel, Gaud; Giraudeau, Patrick

2017-02-01

Multi-dimensional NMR is an appealing approach for dealing with the challenging complexity of biological samples in metabolomics. This article describes how spectroscopists have recently challenged their imagination in order to make 2D NMR a powerful tool for quantitative metabolomics, based on innovative pulse sequences combined with meticulous analytical chemistry approaches. Clever time-saving strategies have also been explored to make 2D NMR a high-throughput tool for metabolomics, relying on alternative data acquisition schemes such as ultrafast NMR. Currently, much work is aimed at drastically boosting the NMR sensitivity thanks to hyperpolarisation techniques, which have been used in combination with fast acquisition methods and could greatly expand the application potential of NMR metabolomics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Apricot DNA as an indicator for persipan: detection and quantitation in marzipan using ligation-dependent probe amplification.

PubMed

Luber, Florian; Demmel, Anja; Hosken, Anne; Busch, Ulrich; Engel, Karl-Heinz

2012-06-13

The confectionery ingredient marzipan is exclusively prepared from almond kernels and sugar. The potential use of apricot kernels, so-called persipan, is an important issue for the quality assessment of marzipan. Therefore, a ligation-dependent probe amplification (LPA) assay was developed that enables a specific and sensitive detection of apricot DNA, as an indicator for the presence of persipan. The limit of detection was determined to be 0.1% persipan in marzipan. The suitability of the method was confirmed by the analysis of 20 commercially available food samples. The integration of a Prunus -specific probe in the LPA assay as a reference allowed for the relative quantitation of persipan in marzipan. The limit of quantitation was determined to be 0.5% persipan in marzipan. The analysis of two self-prepared mixtures of marzipan and persipan demonstrated the applicability of the quantitation method at concentration levels of practical relevance for quality control.

Quantitative analysis of the effect of climate change and human activities on runoff in the Liujiang River Basin

NASA Astrophysics Data System (ADS)

LI, X.

2017-12-01

Abstract: As human basic and strategic natural resources, Water resources have received an unprecedented challenge under the impacts of global climate change. Analyzing the variation characteristics of runoff and the effect of climate change and human activities on runoff could provide the basis for the reasonable utilization and management of water resources. Taking the Liujiang River Basin as the research object, the discharge data of hydrological station and meteorological data at 24 meteorological stations in the Guangxi Province as the basis, the variation characteristics of runoff and precipitation in the Liujiang River Basin was analyzed, and the quantitatively effect of climate change and human activities on runoff was proposed. The results showed that runoff and precipitation in the Liujiang River Basin had an increasing trend from 1964 to 2006. Using the method of accumulative anomaly and the orderly cluster method, the runoff series was divided into base period and change period. BP - ANN model and sensitivity coefficient method were used for quantifying the influences of climate change and human activities on runoff. We found that the most important factor which caused an increase trend of discharges in the Liujiang River Basin was precipitation. Human activities were also important factors which influenced the intra-annual distribution of runoff. Precipitation had a more sensitive influence to runoff variation than potential evaporation in the Liujiang River Basin. Key words: Liujiang River Basin, climate change, human activities, BP-ANN, sensitivity coefficient method

Development and validation of sensitive LC/MS/MS method for quantitative bioanalysis of levonorgestrel in rat plasma and application to pharmacokinetics study.

PubMed

Ananthula, Suryatheja; Janagam, Dileep R; Jamalapuram, Seshulatha; Johnson, James R; Mandrell, Timothy D; Lowe, Tao L

2015-10-15

Rapid, sensitive, selective and accurate LC/MS/MS method was developed for quantitative determination of levonorgestrel (LNG) in rat plasma and further validated for specificity, linearity, accuracy, precision, sensitivity, matrix effect, recovery efficiency and stability. Liquid-liquid extraction procedure using hexane:ethyl acetate mixture at 80:20 v:v ratio was employed to efficiently extract LNG from rat plasma. Reversed phase Luna column C18(2) (50×2.0mm i.d., 3μM) installed on a AB SCIEX Triple Quad™ 4500 LC/MS/MS system was used to perform chromatographic separation. LNG was identified within 2min with high specificity. Linear calibration curve was drawn within 0.5-50ng·mL(-1) concentration range. The developed method was validated for intra-day and inter-day accuracy and precision whose values fell in the acceptable limits. Matrix effect was found to be minimal. Recovery efficiency at three quality control (QC) concentrations 0.5 (low), 5 (medium) and 50 (high) ng·mL(-1) was found to be >90%. Stability of LNG at various stages of experiment including storage, extraction and analysis was evaluated using QC samples, and the results showed that LNG was stable at all the conditions. This validated method was successfully used to study the pharmacokinetics of LNG in rats after SubQ injection, providing its applicability in relevant preclinical studies. Copyright © 2015 Elsevier B.V. All rights reserved.

LC-MS/MS method development for quantitative analysis of acetaminophen uptake by the aquatic fungus Mucor hiemalis.

PubMed

Esterhuizen-Londt, Maranda; Schwartz, Katrin; Balsano, Evelyn; Kühn, Sandra; Pflugmacher, Stephan

2016-06-01

Acetaminophen is a pharmaceutical, frequently found in surface water as a contaminant. Bioremediation, in particular, mycoremediation of acetaminophen is a method to remove this compound from waters. Owing to the lack of quantitative analytical method for acetaminophen in aquatic organisms, the present study aimed to develop a method for the determination of acetaminophen using LC-MS/MS in the aquatic fungus Mucor hiemalis. The method was then applied to evaluate the uptake of acetaminophen by M. hiemalis, cultured in pellet morphology. The method was robust, sensitive and reproducible with a lower limit of quantification of 5 pg acetaminophen on column. It was found that M. hiemalis internalize the pharmaceutical, and bioaccumulate it with time. Therefore, M. hiemalis was deemed a suitable candidate for further studies to elucidate its pharmaceutical tolerance and the longevity in mycoremediation applications. Copyright © 2016 Elsevier Inc. All rights reserved.

Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation.

PubMed

Böttcher, S; Ritgen, M; Pott, C; Brüggemann, M; Raff, T; Stilgenbauer, S; Döhner, H; Dreger, P; Kneba, M

2004-10-01

The clinically most suitable method for minimal residual disease (MRD) detection in chronic lymphocytic leukemia is still controversial. We prospectively compared MRD assessment in 158 blood samples of 74 patients with CLL after stem cell transplantation (SCT) using four-color flow cytometry (MRD flow) in parallel with consensus IgH-PCR and ASO IgH real-time PCR (ASO IgH RQ-PCR). In 25 out of 106 samples (23.6%) with a polyclonal consensus IgH-PCR pattern, MRD flow still detected CLL cells, proving higher sensitivity of flow cytometry over PCR-genescanning with consensus IgH-primers. Of 92 samples, 14 (15.2%) analyzed in parallel by MRD flow and by ASO IgH RQ-PCR were negative by our flow cytometric assay but positive by PCR, thus demonstrating superior sensitivity of RQ-PCR with ASO primers. Quantitative MRD levels measured by both methods correlated well (r=0.93). MRD detection by flow and ASO IgH RQ-PCR were equally suitable to monitor MRD kinetics after allogeneic SCT, but the PCR method detected impending relapses after autologous SCT earlier. An analysis of factors that influence sensitivity and specificity of flow cytometry for MRD detection allowed to devise further improvements of this technique.

Micro-droplet Digital Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction Technologies Provide Highly Sensitive and Accurate Detection of Zika Virus.

PubMed

Hui, Yuan; Wu, Zhiming; Qin, Zhiran; Zhu, Li; Liang, Junhe; Li, Xujuan; Fu, Hanmin; Feng, Shiyu; Yu, Jianhai; He, Xiaoen; Lu, Weizhi; Xiao, Weiwei; Wu, Qinghua; Zhang, Bao; Zhao, Wei

2018-06-01

The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus (ZIKV) and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction (ddPCR) and real-time quantitative PCR (RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold (Ct) value was linear from 10 1 to 10 8  copy/μL, with a standard curve R 2 of 0.999 and amplification efficiency of 92.203%; however, a concentration as low as 1 copy/μL could not be detected. In comparison with RT-qPCR, the ddPCR method resulted in a linear range of 10 1 -10 4  copy/μL and was able to detect concentrations as low as 1 copy/μL. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples (above 10 1  copy/μL), while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.

Utility of mass spectrometry in the diagnosis of prion diseases

USDA-ARS?s Scientific Manuscript database

We developed a sensitive mass spectrometry-based method of quantitating the prions present in a variety of mammalian species. Calibration curves relating the area ratios of the selected analyte peptides and their oxidized analogs to their homologous stable isotope labeled internal standards were pre...

METHODOLOGICAL APPROACH FOR MEASURING PRIORITY DBPS IN REVERSE OSMOSIS CONCENTRATED DRINKING WATER

EPA Science Inventory

Many disinfection by-products (DBPs) are formed when drinking water is chlorinated, but only a few are routinely measured or regulated. Various studies have revealed a plethora of DBPs for which sensitive and quantitative analytical methods have always been a major limiting facto...

SENSITIVITY ANALYSIS OF THE APPLICATION OF CHEMICAL EXPOSURE CRITERIA FOR COMPARING SITES AND WATERSHEDS

EPA Science Inventory

A methodology was developed for deriving quantitative exposure criteria useful for comparing a site or watershed to a reference condition. The prototype method used indicators of exposures to oil contamination and combustion by-products, naphthalene and benzo(a)pyrene metabolites...

A sensitive LC-MS/MS method for the simultaneous determination of amoxicillin and ambroxol in human plasma with segmental monitoring.

PubMed

Dong, Xin; Ding, Li; Cao, Xiaomei; Jiang, Liyuan; Zhong, Shuisheng

2013-04-01

Amoxicillin (AMO) degrades in plasma at room temperature and readily undergoes hydrolysis by the plasma amidase. In this paper, a novel, rapid and sensitive LC-MS/MS method operated in segmental and multiple reaction monitoring has been developed for the simultaneous determination of amoxicillin and ambroxol in human plasma. The degradation of amoxicillin in plasma was well prevented by immediate addition of 20 μL glacial acetic acid to 200 μL aliquot of freshly collected plasma samples before storage at -80°C. The sensitivity of the method was improved with segmental monitoring of the analytes, and lower limits of quantitation of 0.5 ng/mL for ambroxol and 5 ng/mL for amoxicillin were obtained. The sensitivity of our method was five times better than those of the existing methods. Furthermore, the mass response saturation problem with amoxicillin was avoided by diluting the deproteinized plasma samples with water before injection into the LC-MS/MS system. The method was successfully employed in a pharmacokinetic study of the compound amoxicillin and ambroxol hydrochloride tablets. Copyright © 2012 John Wiley & Sons, Ltd.

[Sensitivity to change].

PubMed

Igl, W; Zwingmann, C; Faller, H

2005-04-01

In rehabilitation research patient questionnaires are widely used for evaluative purposes, i. e. to measure improvements or deteriorations over time. This is only possible if the questionnaires applied appropriately reflect "true" change over time, i. e. they have to be sensitive to change. The aim of this paper is to point out the importance of the "sensitivity to change" concept for evaluative assessment tools and evaluative studies, respectively, considering quality of life research as an example. Various qualitative aspects, e. g. scaling of response options of assessment tools, are covered as well as quantitative methods, i. e. study designs and indices. Furthermore, recommendations for interpretation are given.

Evaluation of Legionella pneumophila contamination in Italian hotel water systems by quantitative real-time PCR and culture methods.

PubMed

Bonetta, Sa; Bonetta, Si; Ferretti, E; Balocco, F; Carraro, E

2010-05-01

This study was designed to define the extent of water contamination by Legionella pneumophila of certain Italian hotels and to compare quantitative real-time PCR with the conventional culture method. Nineteen Italian hotels of different sizes were investigated. In each hotel three hot water samples (boiler, room showers, recycling) and one cold water sample (inlet) were collected. Physico-chemical parameters were also analysed. Legionella pneumophila was detected in 42% and 74% of the hotels investigated by the culture method and by real-time PCR, respectively. In 21% of samples analysed by the culture method, a concentration of >10(4) CFU l(-1) was found, and Leg. pneumophila serogroup 1 was isolated from 10.5% of the hotels. The presence of Leg. pneumophila was significantly influenced by water sample temperature, while no association with water hardness or residual-free chlorine was found. This study showed a high percentage of buildings colonized by Leg. pneumophila. Moreover, real-time PCR proved to be sensitive enough to detect lower levels of contamination than the culture method. This study indicates that the Italian hotels represent a possible source of risk for Legionnaires' disease and confirms the sensitivity of the molecular method. To our knowledge, this is the first report to demonstrate Legionella contamination in Italian hotels using real-time PCR and culture methods.

A sensitive and rapid assay for 4-aminophenol in paracetamol drug and tablet formulation, by flow injection analysis with spectrophotometric detection.

PubMed

Bloomfield, M S

2002-12-06

4-Aminophenol (4AP) is the primary degradation product of paracetamol which is limited at a low level (50 ppm or 0.005% w/w) in the drug substance by the European, United States, British and German Pharmacopoeias, employing a manual colourimetric limit test. The 4AP limit is widened to 1000 ppm or 0.1% w/w for the tablet product monographs, which quote the use of a less sensitive automated HPLC method. The lower drug substance specification limit is applied to our products, (50 ppm, equivalent to 25 mug 4AP in a tablet containing 500-mg paracetamol) and the pharmacopoeial HPLC assay was not suitable at this low level due to matrix interference. For routine analysis a rapid, automated assay was required. This paper presents a highly sensitive, precise and automated method employing the technique of Flow Injection (FI) analysis to quantitatively assay low levels of this degradant. A solution of the drug substance, or an extract of the tablets, containing 4AP and paracetamol is injected into a solvent carrier stream and merged on-line with alkaline sodium nitroprusside reagent, to form a specific blue derivative which is detected spectrophotometrically at 710 nm. Standard HPLC equipment is used throughout. The procedure is fully quantitative and has been optimised for sensitivity and robustness using a multivariate experimental design (multi-level 'Central Composite' response surface) model. The method has been fully validated and is linear down to 0.01 mug ml(-1). The approach should be applicable to a range of paracetamol products.

Oxidation of methionine 216 in sheep and elk prion protein is highly dependent upon the amino acid at position 218 but is not important for prion propagation

USDA-ARS?s Scientific Manuscript database

We developed a sensitive mass spectrometry-based method of quantitating the prions present in elk and sheep. Calibration curves relating the area ratios of the selected analyte peptides and their homologous stable isotope labeled internal standards were prepared. This method was compared to the ELIS...

[Comparison of two quantitative methods of endobronchial ultrasound real-time elastography for evaluating intrathoracic lymph nodes].

PubMed

Mao, X W; Yang, J Y; Zheng, X X; Wang, L; Zhu, L; Li, Y; Xiong, H K; Sun, J Y

2017-06-12

Objective: To compare the clinical value of two quantitative methods in analyzing endobronchial ultrasound real-time elastography (EBUS-RTE) images for evaluating intrathoracic lymph nodes. Methods: From January 2014 to April 2014, EBUS-RTE examination was performed in patients who received EBUS-TBNA examination in Shanghai Chest Hospital. Each intrathoracic lymph node had a selected EBUS-RTE image. Stiff area ratio and mean hue value of region of interest (ROI) in each image were calculated respectively. The final diagnosis of lymph node was based on the pathologic/microbiologic results of EBUS-TBNA, pathologic/microbiologic results of other examinations and clinical following-up. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy were evaluated for distinguishing malignant and benign lesions. Results: Fifty-six patients and 68 lymph nodes were enrolled in this study, of which 35 lymph nodes were malignant and 33 lymph nodes were benign. The stiff area ratio and mean hue value of benign and malignant lesions were 0.32±0.29, 0.62±0.20 and 109.99±28.13, 141.62±17.52, respectively, and statistical differences were found in both of those two methods ( t =-5.14, P <0.01; t =-5.53, P <0.01). The area under curves was 0.813, 0.814 in stiff area ratio and mean hue value, respectively. The optimal diagnostic cut-off value of stiff area ratio was 0.48, and the sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 82.86%, 81.82%, 82.86%, 81.82% and 82.35%, respectively. The optimal diagnostic cut-off value of mean hue value was 126.28, and the sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 85.71%, 75.76%, 78.95%, 83.33% and 80.88%, respectively. Conclusion: Both the stiff area ratio and mean hue value methods can be used for analyzing EBUS-RTE images quantitatively, having the value of differentiating benign and malignant intrathoracic lymph nodes, and the stiff area ratio is better than the mean hue value between the two methods.

Stepwise sensitivity analysis from qualitative to quantitative: Application to the terrestrial hydrological modeling of a Conjunctive Surface-Subsurface Process (CSSP) land surface model

NASA Astrophysics Data System (ADS)

Gan, Yanjun; Liang, Xin-Zhong; Duan, Qingyun; Choi, Hyun Il; Dai, Yongjiu; Wu, Huan

2015-06-01

An uncertainty quantification framework was employed to examine the sensitivities of 24 model parameters from a newly developed Conjunctive Surface-Subsurface Process (CSSP) land surface model (LSM). The sensitivity analysis (SA) was performed over 18 representative watersheds in the contiguous United States to examine the influence of model parameters in the simulation of terrestrial hydrological processes. Two normalized metrics, relative bias (RB) and Nash-Sutcliffe efficiency (NSE), were adopted to assess the fit between simulated and observed streamflow discharge (SD) and evapotranspiration (ET) for a 14 year period. SA was conducted using a multiobjective two-stage approach, in which the first stage was a qualitative SA using the Latin Hypercube-based One-At-a-Time (LH-OAT) screening, and the second stage was a quantitative SA using the Multivariate Adaptive Regression Splines (MARS)-based Sobol' sensitivity indices. This approach combines the merits of qualitative and quantitative global SA methods, and is effective and efficient for understanding and simplifying large, complex system models. Ten of the 24 parameters were identified as important across different watersheds. The contribution of each parameter to the total response variance was then quantified by Sobol' sensitivity indices. Generally, parameter interactions contribute the most to the response variance of the CSSP, and only 5 out of 24 parameters dominate model behavior. Four photosynthetic and respiratory parameters are shown to be influential to ET, whereas reference depth for saturated hydraulic conductivity is the most influential parameter for SD in most watersheds. Parameter sensitivity patterns mainly depend on hydroclimatic regime, as well as vegetation type and soil texture. This article was corrected on 26 JUN 2015. See the end of the full text for details.

Quantitative imaging assay for NF-κB nuclear translocation in primary human macrophages

PubMed Central

Noursadeghi, Mahdad; Tsang, Jhen; Haustein, Thomas; Miller, Robert F.; Chain, Benjamin M.; Katz, David R.

2008-01-01

Quantitative measurement of NF-κB nuclear translocation is an important research tool in cellular immunology. Established methodologies have a number of limitations, such as poor sensitivity, high cost or dependence on cell lines. Novel imaging methods to measure nuclear translocation of transcriptionally active components of NF-κB are being used but are also partly limited by the need for specialist imaging equipment or image analysis software. Herein we present a method for quantitative detection of NF-κB rel A nuclear translocation, using immunofluorescence microscopy and the public domain image analysis software ImageJ that can be easily adopted for cellular immunology research without the need for specialist image analysis expertise and at low cost. The method presented here is validated by demonstrating the time course and dose response of NF-κB nuclear translocation in primary human macrophages stimulated with LPS, and by comparison with a commercial NF-κB activation reporter cell line. PMID:18036607

Recent trends in high spin sensitivity magnetic resonance

NASA Astrophysics Data System (ADS)

Blank, Aharon; Twig, Ygal; Ishay, Yakir

2017-07-01

Magnetic resonance is a very powerful methodology that has been employed successfully in many applications for about 70 years now, resulting in a wealth of scientific, technological, and diagnostic data. Despite its many advantages, one major drawback of magnetic resonance is its relatively poor sensitivity and, as a consequence, its bad spatial resolution when examining heterogeneous samples. Contemporary science and technology often make use of very small amounts of material and examine heterogeneity on a very small length scale, both of which are well beyond the current capabilities of conventional magnetic resonance. It is therefore very important to significantly improve both the sensitivity and the spatial resolution of magnetic resonance techniques. The quest for higher sensitivity led in recent years to the development of many alternative detection techniques that seem to rival and challenge the conventional ;old-fashioned; induction-detection approach. The aim of this manuscript is to briefly review recent advances in the field, and to provide a quantitative as well as qualitative comparison between various detection methods with an eye to future potential advances and developments. We first offer a common definition of sensitivity in magnetic resonance to enable proper quantitative comparisons between various detection methods. Following that, up-to-date information about the sensitivity capabilities of the leading recently-developed detection approaches in magnetic resonance is provided, accompanied by a critical comparison between them and induction detection. Our conclusion from this comparison is that induction detection is still indispensable, and as such, it is very important to look for ways to significantly improve it. To do so, we provide expressions for the sensitivity of induction-detection, derived from both classical and quantum mechanics, that identify its main limiting factors. Examples from current literature, as well as a description of new ideas, show how these limiting factors can be mitigated to significantly improve the sensitivity of induction detection. Finally, we outline some directions for the possible applications of high-sensitivity induction detection in the field of electron spin resonance.

Detection of anti-Yta antibodies using a sensitive and specific enzyme-linked immunosorbent assay.

PubMed

Geen, J; Hullin, D A; Hogg, S I

1999-01-01

A specific, sensitive and semi-quantitative enzyme-linked immunosorbent assay (ELISA) is described to detect anti-Yta antibodies in human serum. Recombinant acetylcholinesterase (AChE E.C.3.1.1.7) was employed as the coating antigen in the microtitre plate and horseradish peroxidase (HRP)-conjugated specific antibody (IgG) was used as the secondary antibody. The method developed showed excellent sensitivity, detecting a titre > 1 in 600,000 (3.5 ng/mL mouse IgG protein) for mouse monoclonal (mMAb) anti-AChE antibody. No cross-reaction was seen with other common blood group antibodies, confirming the specificity of the method. The recombinant antigen's AChE phenotype was confirmed as Yta, as no reaction was detected with anti-Ytb-positive sera. The ELISA method correlated closely with the established serological grading system used routinely in blood transfusion laboratories.

Quantitative risk assessment for skin sensitization: Success or failure?

PubMed

Kimber, Ian; Gerberick, G Frank; Basketter, David A

2017-02-01

Skin sensitization is unique in the world of toxicology. There is a combination of reliable, validated predictive test methods for identification of skin sensitizing chemicals, a clearly documented and transparent approach to risk assessment, and effective feedback from dermatology clinics around the world delivering evidence of the success or failure of the hazard identification/risk assessment/management process. Recent epidemics of contact allergy, particularly to preservatives, have raised questions of whether the safety/risk assessment process is working in an optimal manner (or indeed is working at all!). This review has as its focus skin sensitization quantitative risk assessment (QRA). The core toxicological principles of QRA are reviewed, and evidence of use and misuse examined. What becomes clear is that skin sensitization QRA will only function adequately if two essential criteria are met. The first is that QRA is applied rigourously, and the second is that potential exposure to the sensitizing substance is assessed adequately. This conclusion will come as no surprise to any toxicologist who appreciates the basic premise that "risk = hazard x exposure". Accordingly, use of skin sensitization QRA is encouraged, not least because the essential feedback from dermatology clinics can be used as a tool to refine QRA in situations where this risk assessment tool has not been properly used. Copyright © 2016 Elsevier Inc. All rights reserved.

Real-time quantitative PCR of Staphylococcus aureus and application in restaurant meals.

PubMed

Berrada, H; Soriano, J M; Mañes, J; Picó, Y

2006-01-01

Staphylococcus aureus is considered the second most common pathogen to cause outbreaks of food poisoning, exceeded only by Campylobacter. Consumption of foods containing this microorganism is often identified as the cause of illness. In this study, a rapid, reliable, and sensitive real-time quantitative PCR was developed and compared with conventional culture methods. Real-time quantitative PCR was carried out by purifying DNA extracts of S. aureus with a Staphylococcus sample preparation kit and quantifying it in the LightCycler system with hybridization probes. The assay was linear from a range of 10 to 10(6) S. aureus cells (r2 > 0.997). The PCR reaction presented an efficiency of >85%. Accuracy of the PCR-based assay, expressed as percent bias, was around 13%, and the precision, expressed as a percentage of the coefficient of variation, was 7 to 10%. Intraday and interday variability were studied at 10(2) CFU/g and was 12 and 14%, respectively. The proposed method was applied to the analysis of 77 samples of restaurant meals in Valencia (Spain). In 11.6% of samples S. aureus was detected by real-time quantitative PCR, as well as by the conventional microbiological method. An excellent correspondence between real-time quantitative PCR and microbiological numbers (CFU/g) was observed with deviations of < 28%.

Comparison of three commercially available fit-test methods.

PubMed

Janssen, Larry L; Luinenburg, D Michael; Mullins, Haskell E; Nelson, Thomas J

2002-01-01

American National Standards Institute (ANSI) standard Z88.10, Respirator Fit Testing Methods, includes criteria to evaluate new fit-tests. The standard allows generated aerosol, particle counting, or controlled negative pressure quantitative fit-tests to be used as the reference method to determine acceptability of a new test. This study examined (1) comparability of three Occupational Safety and Health Administration-accepted fit-test methods, all of which were validated using generated aerosol as the reference method; and (2) the effect of the reference method on the apparent performance of a fit-test method under evaluation. Sequential fit-tests were performed using the controlled negative pressure and particle counting quantitative fit-tests and the bitter aerosol qualitative fit-test. Of 75 fit-tests conducted with each method, the controlled negative pressure method identified 24 failures; bitter aerosol identified 22 failures; and the particle counting method identified 15 failures. The sensitivity of each method, that is, agreement with the reference method in identifying unacceptable fits, was calculated using each of the other two methods as the reference. None of the test methods met the ANSI sensitivity criterion of 0.95 or greater when compared with either of the other two methods. These results demonstrate that (1) the apparent performance of any fit-test depends on the reference method used, and (2) the fit-tests evaluated use different criteria to identify inadequately fitting respirators. Although "acceptable fit" cannot be defined in absolute terms at this time, the ability of existing fit-test methods to reject poor fits can be inferred from workplace protection factor studies.

Characterization of copy numbers of 16S rDNA and 16S rRNA of Candidatus Liberibacter asiaticus and the implication in detection in planta using quantitative PCR.

PubMed

Kim, Jeong-Soon; Wang, Nian

2009-03-06

Citrus Huanglongbing (HLB) is one of the most devastating diseases on citrus and is associated with Candidatus Liberibacter spp.. The pathogens are phloem limited and have not been cultured in vitro. The current management strategy of HLB is to remove infected citrus trees and reduce psyllid populations with insecticides to prevent the spreading. This strategy requires sensitive and reliable diagnostic methods for early detection. We investigated the copy numbers of the 16S rDNA and 16S rRNA of the HLB pathogen and the implication of improving the diagnosis of HLB for early detection using Quantitative PCR. We compared the detection of HLB with different Quantitative PCR based methods with primers/probe targeting either 16S rDNA, beta-operon DNA, 16S rRNA, or beta-operon RNA. The 16S rDNA copy number of Ca. Liberibacter asiaticus was estimated to be three times of that of the beta-operon region, thus allowing detection of lower titer of Ca. L. asiaticus. Quantitative reverse transcriptional PCR (QRT-PCR) indicated that the 16S rRNA averaged 7.83 times more than that of 16S rDNA for the same samples. Dilution analysis also indicates that QRT-PCR targeting 16S rRNA is 10 time more sensitive than QPCR targeting 16S rDNA. Thus QRT-PCR was able to increase the sensitivity of detection by targeting 16S rRNA. Our result indicates that Candidatus Liberibacter asiaticus contains three copies of 16S rDNA. The copy number of 16S rRNA of Ca. L. asiaticus in planta averaged about 7.8 times of 16S rDNA for the same set of samples tested in this study. Detection sensitivity of HLB could be improved through the following approaches: using 16S rDNA based primers/probe in the QPCR assays; and using QRT-PCR assays targeting 16S rRNA.

Quantitative estimation of α-PVP metabolites in urine by GC-APCI-QTOFMS with nitrogen chemiluminescence detection based on parent drug calibration.

PubMed

Mesihää, Samuel; Rasanen, Ilpo; Ojanperä, Ilkka

2018-05-01

Gas chromatography (GC) hyphenated with nitrogen chemiluminescence detection (NCD) and quadrupole time-of-flight mass spectrometry (QTOFMS) was applied for the first time to the quantitative analysis of new psychoactive substances (NPS) in urine, based on the N-equimolar response of NCD. A method was developed and validated to estimate the concentrations of three metabolites of the common stimulant NPS α-pyrrolidinovalerophenone (α-PVP) in spiked urine samples, simulating an analysis having no authentic reference standards for the metabolites and using the parent drug instead for quantitative calibration. The metabolites studied were OH-α-PVP (M1), 2″-oxo-α-PVP (M3), and N,N-bis-dealkyl-PVP (2-amino-1-phenylpentan-1-one; M5). Sample preparation involved liquid-liquid extraction with a mixture of ethyl acetate and butyl chloride at a basic pH and subsequent silylation of the sec-hydroxyl and prim-amino groups of M1 and M5, respectively. Simultaneous compound identification was based on the accurate masses of the protonated molecules for each compound by QTOFMS following atmospheric pressure chemical ionization. The accuracy of quantification of the parent-calibrated NCD method was compared with that of the corresponding parent-calibrated QTOFMS method, as well as with a reference QTOFMS method calibrated with the authentic reference standards. The NCD method produced an equally good accuracy to the reference method for α-PVP, M3 and M5, while a higher negative bias (25%) was obtained for M1, best explainable by recovery and stability issues. The performance of the parent-calibrated QTOFMS method was inferior to the reference method with an especially high negative bias (60%) for M1. The NCD method enabled better quantitative precision than the QTOFMS methods To evaluate the novel approach in casework, twenty post- mortem urine samples previously found positive for α-PVP were analyzed by the parent calibrated NCD method and the reference QTOFMS method. The highest difference in the quantitative results between the two methods was only 33%, and the NCD method's precision as the coefficient of variation was better than 13%. The limit of quantification for the NCD method was approximately 0.25μg/mL in urine, which generally allowed the analysis of α-PVP and the main metabolite M1. However, the sensitivity was not sufficient for the low concentrations of M3 and M5. Consequently, while having potential for instant analysis of NPS and metabolites in moderate concentrations without reference standards, the NCD method should be further developed for improved sensitivity to be more generally applicable. Copyright © 2018 Elsevier B.V. All rights reserved.

Quantitation of melatonin and n-acetylserotonin in human plasma by nanoflow LC-MS/MS and electrospray LC-MS/MS.

PubMed

Carter, Melissa D; Calcutt, M Wade; Malow, Beth A; Rose, Kristie L; Hachey, David L

2012-03-01

Melatonin (MEL) and its chemical precursor N-acetylserotonin (NAS) are believed to be potential biomarkers for sleep-related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC-MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies, and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays, plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1 × 100  mm, 3.5 µm) or on a polyimide-coated, fused-silica capillary self-packed with 17 cm AquaC18 (3 µm, 125 Å). Quantitation was done using the SRM transitions m/z 233 → 174 and m/z 219 → 160 for MEL and NAS, respectively. The analytical response ratio versus concentration curves were linear for MEL (nanoflow LC: 11.7-1165  pg/mL, LC: 1165-116,500  pg/mL) and for NAS (nanoflow LC: 11.0-1095  pg/mL). Copyright © 2012 John Wiley & Sons, Ltd.

Current methods and advances in bone densitometry

NASA Technical Reports Server (NTRS)

Guglielmi, G.; Gluer, C. C.; Majumdar, S.; Blunt, B. A.; Genant, H. K.

1995-01-01

Bone mass is the primary, although not the only, determinant of fracture. Over the past few years a number of noninvasive techniques have been developed to more sensitively quantitate bone mass. These include single and dual photon absorptiometry (SPA and DPA), single and dual X-ray absorptiometry (SXA and DXA) and quantitative computed tomography (QCT). While differing in anatomic sites measured and in their estimates of precision, accuracy, and fracture discrimination, all of these methods provide clinically useful measurements of skeletal status. It is the intent of this review to discuss the pros and cons of these techniques and to present the new applications of ultrasound (US) and magnetic resonance (MRI) in the detection and management of osteoporosis.

High PRF ultrafast sliding compound doppler imaging: fully qualitative and quantitative analysis of blood flow

NASA Astrophysics Data System (ADS)

Kang, Jinbum; Jang, Won Seuk; Yoo, Yangmo

2018-02-01

Ultrafast compound Doppler imaging based on plane-wave excitation (UCDI) can be used to evaluate cardiovascular diseases using high frame rates. In particular, it provides a fully quantifiable flow analysis over a large region of interest with high spatio-temporal resolution. However, the pulse-repetition frequency (PRF) in the UCDI method is limited for high-velocity flow imaging since it has a tradeoff between the number of plane-wave angles (N) and acquisition time. In this paper, we present high PRF ultrafast sliding compound Doppler imaging method (HUSDI) to improve quantitative flow analysis. With the HUSDI method, full scanline images (i.e. each tilted plane wave data) in a Doppler frame buffer are consecutively summed using a sliding window to create high-quality ensemble data so that there is no reduction in frame rate and flow sensitivity. In addition, by updating a new compounding set with a certain time difference (i.e. sliding window step size or L), the HUSDI method allows various Doppler PRFs with the same acquisition data to enable a fully qualitative, retrospective flow assessment. To evaluate the performance of the proposed HUSDI method, simulation, in vitro and in vivo studies were conducted under diverse flow circumstances. In the simulation and in vitro studies, the HUSDI method showed improved hemodynamic representations without reducing either temporal resolution or sensitivity compared to the UCDI method. For the quantitative analysis, the root mean squared velocity error (RMSVE) was measured using 9 angles (-12° to 12°) with L of 1-9, and the results were found to be comparable to those of the UCDI method (L  =  N  =  9), i.e.  ⩽0.24 cm s-1, for all L values. For the in vivo study, the flow data acquired from a full cardiac cycle of the femoral vessels of a healthy volunteer were analyzed using a PW spectrogram, and arterial and venous flows were successfully assessed with high Doppler PRF (e.g. 5 kHz at L  =  4). These results indicate that the proposed HUSDI method can improve flow visualization and quantification with a higher frame rate, PRF and flow sensitivity in cardiovascular imaging.

High PRF ultrafast sliding compound doppler imaging: fully qualitative and quantitative analysis of blood flow.

PubMed

Kang, Jinbum; Jang, Won Seuk; Yoo, Yangmo

2018-02-09

Ultrafast compound Doppler imaging based on plane-wave excitation (UCDI) can be used to evaluate cardiovascular diseases using high frame rates. In particular, it provides a fully quantifiable flow analysis over a large region of interest with high spatio-temporal resolution. However, the pulse-repetition frequency (PRF) in the UCDI method is limited for high-velocity flow imaging since it has a tradeoff between the number of plane-wave angles (N) and acquisition time. In this paper, we present high PRF ultrafast sliding compound Doppler imaging method (HUSDI) to improve quantitative flow analysis. With the HUSDI method, full scanline images (i.e. each tilted plane wave data) in a Doppler frame buffer are consecutively summed using a sliding window to create high-quality ensemble data so that there is no reduction in frame rate and flow sensitivity. In addition, by updating a new compounding set with a certain time difference (i.e. sliding window step size or L), the HUSDI method allows various Doppler PRFs with the same acquisition data to enable a fully qualitative, retrospective flow assessment. To evaluate the performance of the proposed HUSDI method, simulation, in vitro and in vivo studies were conducted under diverse flow circumstances. In the simulation and in vitro studies, the HUSDI method showed improved hemodynamic representations without reducing either temporal resolution or sensitivity compared to the UCDI method. For the quantitative analysis, the root mean squared velocity error (RMSVE) was measured using 9 angles (-12° to 12°) with L of 1-9, and the results were found to be comparable to those of the UCDI method (L  =  N  =  9), i.e.  ⩽0.24 cm s -1 , for all L values. For the in vivo study, the flow data acquired from a full cardiac cycle of the femoral vessels of a healthy volunteer were analyzed using a PW spectrogram, and arterial and venous flows were successfully assessed with high Doppler PRF (e.g. 5 kHz at L  =  4). These results indicate that the proposed HUSDI method can improve flow visualization and quantification with a higher frame rate, PRF and flow sensitivity in cardiovascular imaging.

Sensitive, rapid, quantitative and in vitro method for the detection of biologically active staphylococcal enterotoxin type E

USDA-ARS?s Scientific Manuscript database

Staphylococcus aureus is a major bacterial pathogen which causes clinical infections and food poisoning. This bacterium produces a group of enterotoxins (SEs). These enterotoxins have two separate but related biological activities. They cause gastroenteritis and function as superantigens that activa...

Measuring changes in transmission of neglected tropical diseases, malaria, and enteric pathogens from quantitative antibody levels

PubMed Central

van der Laan, Mark J.; Hubbard, Alan E.; Steel, Cathy; Kubofcik, Joseph; Hamlin, Katy L.; Moss, Delynn M.; Nutman, Thomas B.; Priest, Jeffrey W.; Lammie, Patrick J.

2017-01-01

Background Serological antibody levels are a sensitive marker of pathogen exposure, and advances in multiplex assays have created enormous potential for large-scale, integrated infectious disease surveillance. Most methods to analyze antibody measurements reduce quantitative antibody levels to seropositive and seronegative groups, but this can be difficult for many pathogens and may provide lower resolution information than quantitative levels. Analysis methods have predominantly maintained a single disease focus, yet integrated surveillance platforms would benefit from methodologies that work across diverse pathogens included in multiplex assays. Methods/Principal findings We developed an approach to measure changes in transmission from quantitative antibody levels that can be applied to diverse pathogens of global importance. We compared age-dependent immunoglobulin G curves in repeated cross-sectional surveys between populations with differences in transmission for multiple pathogens, including: lymphatic filariasis (Wuchereria bancrofti) measured before and after mass drug administration on Mauke, Cook Islands, malaria (Plasmodium falciparum) before and after a combined insecticide and mass drug administration intervention in the Garki project, Nigeria, and enteric protozoans (Cryptosporidium parvum, Giardia intestinalis, Entamoeba histolytica), bacteria (enterotoxigenic Escherichia coli, Salmonella spp.), and viruses (norovirus groups I and II) in children living in Haiti and the USA. Age-dependent antibody curves fit with ensemble machine learning followed a characteristic shape across pathogens that aligned with predictions from basic mechanisms of humoral immunity. Differences in pathogen transmission led to shifts in fitted antibody curves that were remarkably consistent across pathogens, assays, and populations. Mean antibody levels correlated strongly with traditional measures of transmission intensity, such as the entomological inoculation rate for P. falciparum (Spearman’s rho = 0.75). In both high- and low transmission settings, mean antibody curves revealed changes in population mean antibody levels that were masked by seroprevalence measures because changes took place above or below the seropositivity cutoff. Conclusions/Significance Age-dependent antibody curves and summary means provided a robust and sensitive measure of changes in transmission, with greatest sensitivity among young children. The method generalizes to pathogens that can be measured in high-throughput, multiplex serological assays, and scales to surveillance activities that require high spatiotemporal resolution. Our results suggest quantitative antibody levels will be particularly useful to measure differences in exposure for pathogens that elicit a transient antibody response or for monitoring populations with very high- or very low transmission, when seroprevalence is less informative. The approach represents a new opportunity to conduct integrated serological surveillance for neglected tropical diseases, malaria, and other infectious diseases with well-defined antigen targets. PMID:28542223

Quantitation of Permethylated N-Glycans through Multiple-Reaction Monitoring (MRM) LC-MS/MS

PubMed Central

Zhou, Shiyue; Hu, Yunli; DeSantos-Garcia, Janie L.; Mechref, Yehia

2015-01-01

The important biological roles of glycans and their implications in disease development and progression have created a demand for the development of sensitive quantitative glycomics methods. Quantitation of glycans existing at low abundance is still analytically challenging. In this study, an N-linked glycans quantitation method using multiple reaction monitoring (MRM) on a triple quadrupole instrument was developed. Optimum normalized collision energy (CE) for both sialylated and fucosylated N-glycan structures was determined to be 30% while it was found to be 35% for either fucosylated or sialylated structures The optimum CE for mannose and complex type N-glycan structures was determined to be 35%. Additionally, the use of three transitions was shown to facilitate reliable quantitation. A total of 88 N-glycan structures in human blood serum were quantified using this MRM approach. Reliable detection and quantitation of these structures was achieved when the equivalence of 0.005 μL of blood serum was analyzed. Accordingly, N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, spanning a dynamic concentration range of three orders of magnitudes. MRM was also effectively utilized to quantitatively compare the expression of N-glycans derived from brain-targeting breast carcinoma cells (MDA-MB-231BR) and metastatic breast cancer cells (MDA-MB-231). Thus, the described MRM method of permethylated N-glycan structures enables a rapid and reliable identification and quantitation of glycans derived from glycoproteins purified or present in complex biological samples. PMID:25698222

Comparative Evaluation of Four Real-Time PCR Methods for the Quantitative Detection of Epstein-Barr Virus from Whole Blood Specimens.

PubMed

Buelow, Daelynn; Sun, Yilun; Tang, Li; Gu, Zhengming; Pounds, Stanley; Hayden, Randall

2016-07-01

Monitoring of Epstein-Barr virus (EBV) load in immunocompromised patients has become integral to their care. An increasing number of reagents are available for quantitative detection of EBV; however, there are little published comparative data. Four real-time PCR systems (one using laboratory-developed reagents and three using analyte-specific reagents) were compared with one another for detection of EBV from whole blood. Whole blood specimens seeded with EBV were used to determine quantitative linearity, analytical measurement range, lower limit of detection, and CV for each assay. Retrospective testing of 198 clinical samples was performed in parallel with all methods; results were compared to determine relative quantitative and qualitative performance. All assays showed similar performance. No significant difference was found in limit of detection (3.12-3.49 log10 copies/mL; P = 0.37). A strong qualitative correlation was seen with all assays that used clinical samples (positive detection rates of 89.5%-95.8%). Quantitative correlation of clinical samples across assays was also seen in pairwise regression analysis, with R(2) ranging from 0.83 to 0.95. Normalizing clinical sample results to IU/mL did not alter the quantitative correlation between assays. Quantitative EBV detection by real-time PCR can be performed over a wide linear dynamic range, using three different commercially available reagents and laboratory-developed methods. EBV was detected with comparable sensitivity and quantitative correlation for all assays. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

The local lymph node assay in 2014.

PubMed

Basketter, David A; Gerberick, G Frank; Kimber, Ian

2014-01-01

Toxicology endeavors to predict the potential of materials to cause adverse health (and environmental) effects and to assess the risk(s) associated with exposure. For skin sensitizers, the local lymph node assay was the first method to be fully and independently validated, as well as the first to offer an objective end point with a quantitative measure of sensitizing potency (in addition to hazard identification). Fifteen years later, it serves as the primary standard for the development of in vitro/in chemico/in silico alternatives.

Dependency of plasmon resonance sensitivity of colloidal gold nanoparticles on the identity of surrounding ionic media

NASA Astrophysics Data System (ADS)

Mehrdel, B.; Aziz, A. Abdul

2018-03-01

The plasmon resonance sensitivity of gold nanoparticles (AuNPs) in sodium chloride (NaCl) liquid in near-infrared to the visible spectral region was investigated. The correlation between NaCl concentration and refractive index was analyzed using concentration dependency and Lorenz-Lorenz methods. The first derivative method was applied to the measured absorption spectra to quantitatively evaluate the plasmon resonance sensitivity. To understand the influence of the identity of the surrounding medium on the plasmon resonance sensitivity, experiments were repeated by replacing NaCl with sodium hydroxide (NaOH), followed by phosphate buffered saline (PBS). Experimental results showed that NaCl is the most effective ionic surrounding medium, which gives prominent plasmon resonance response. AuNPs size can have a significant influence on the plasmon resonance sensitivity. For tiny AuNPs (∼10 nm AuNPs), the plasmon resonance is insensitive to the identity of the surrounding medium due to their low cross-section value.

Characterization and measurement of natural gas trace constituents. Volume 1. Arsenic. Final report, June 1989-October 1993

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chao, S.S.; Attari, A.

1995-01-01

The discovery of arsenic compounds, as alkylarsines, in natural gas prompted this research program to develop reliable measurement techniques needed to assess the efficiency of removal processes for these environmentally sensitive substances. These techniques include sampling, speciation, quantitation and on-line instrumental methods for monitoring the total arsenic concentration. The current program has yielded many products, including calibration standards, arsenic-specific sorbents, sensitive analytical methods and instrumentation. Four laboratory analytical methods have been developed and successfully employed for arsenic determination in natural gas. These methods use GC-AED and GC-MS instruments to speciate alkylarsines, and peroxydisulfate extraction with FIAS, special carbon sorbent withmore » XRF and an IGT developed sorbent with GFAA for total arsenic measurement.« less

Evaluation of quantitative PCR measurement of bacterial colonization of epithelial cells.

PubMed

Schmidt, Marcin T; Olejnik-Schmidt, Agnieszka K; Myszka, Kamila; Borkowska, Monika; Grajek, Włodzimierz

2010-01-01

Microbial colonization is an important step in establishing pathogenic or probiotic relations to host cells and in biofilm formation on industrial or medical devices. The aim of this work was to verify the applicability of quantitative PCR (Real-Time PCR) to measure bacterial colonization of epithelial cells. Salmonella enterica and Caco-2 intestinal epithelial cell line was used as a model. To verify sensitivity of the assay a competition of the pathogen cells to probiotic microorganism was tested. The qPCR method was compared to plate count and radiolabel approach, which are well established techniques in this area of research. The three methods returned similar results. The best quantification accuracy had radiolabel method, followed by qPCR. The plate count results showed coefficient of variation two-times higher than this of qPCR. The quantitative PCR proved to be a reliable method for enumeration of microbes in colonization assay. It has several advantages that make it very useful in case of analyzing mixed populations, where several different species or even strains can be monitored at the same time.

A selective and sensitive method for quantitation of lysergic acid diethylamide (LSD) in whole blood by gas chromatography-ion trap tandem mass spectrometry.

PubMed

Libong, Danielle; Bouchonnet, Stéphane; Ricordel, Ivan

2003-01-01

A gas chromatography-ion trap tandem mass spectrometry (GC-ion trap MS-MS) method for detection and quantitation of LSD in whole blood is presented. The sample preparation process, including a solid-phase extraction step with Bond Elut cartridges, was performed with 2 mL of whole blood. Eight microliters of the purified extract was injected with a cold on-column injection method. Positive chemical ionization was performed using acetonitrile as reagent gas; LSD was detected in the MS-MS mode. The chromatograms obtained from blood extracts showed the great selectivity of the method. GC-MS quantitation was performed using lysergic acid methylpropylamide as the internal standard. The response of the MS was linear for concentrations ranging from 0.02 ng/mL (detection threshold) to 10.0 ng/mL. Several parameters such as the choice of the capillary column, the choice of the internal standard and that of the ionization mode (positive CI vs. EI) were rationalized. Decomposition pathways under both ionization modes were studied. Within-day and between-day stability were evaluated.

Report on the analysis of common beverages spiked with gamma-hydroxybutyric acid (GHB) and gamma-butyrolactone (GBL) using NMR and the PURGE solvent-suppression technique.

PubMed

Lesar, Casey T; Decatur, John; Lukasiewicz, Elaan; Champeil, Elise

2011-10-10

In forensic evidence, the identification and quantitation of gamma-hydroxybutyric acid (GHB) in "spiked" beverages is challenging. In this report, we present the analysis of common alcoholic beverages found in clubs and bars spiked with gamma-hydroxybutyric acid (GHB) and gamma-butyrolactone (GBL). Our analysis of the spiked beverages consisted of using (1)H NMR with a water suppression method called Presaturation Utilizing Relaxation Gradients and Echoes (PURGE). The following beverages were analyzed: water, 10% ethanol in water, vodka-cranberry juice, rum and coke, gin and tonic, whisky and diet coke, white wine, red wine, and beer. The PURGE method allowed for the direct identification and quantitation of both compounds in all beverages except red and white wine where small interferences prevented accurate quantitation. The NMR method presented in this paper utilizes PURGE water suppression. Thanks to the use of a capillary internal standard, the method is fast, non-destructive, sensitive and requires no sample preparation which could disrupt the equilibrium between GHB and GBL. Published by Elsevier Ireland Ltd.

A model to estimate insulin sensitivity in dairy cows.

PubMed

Holtenius, Paul; Holtenius, Kjell

2007-10-11

Impairment of the insulin regulation of energy metabolism is considered to be an etiologic key component for metabolic disturbances. Methods for studies of insulin sensitivity thus are highly topical. There are clear indications that reduced insulin sensitivity contributes to the metabolic disturbances that occurs especially among obese lactating cows. Direct measurements of insulin sensitivity are laborious and not suitable for epidemiological studies. We have therefore adopted an indirect method originally developed for humans to estimate insulin sensitivity in dairy cows. The method, "Revised Quantitative Insulin Sensitivity Check Index" (RQUICKI) is based on plasma concentrations of glucose, insulin and free fatty acids (FFA) and it generates good and linear correlations with different estimates of insulin sensitivity in human populations. We hypothesized that the RQUICKI method could be used as an index of insulin function in lactating dairy cows. We calculated RQUICKI in 237 apparently healthy dairy cows from 20 commercial herds. All cows included were in their first 15 weeks of lactation. RQUICKI was not affected by the homeorhetic adaptations in energy metabolism that occurred during the first 15 weeks of lactation. In a cohort of 24 experimental cows fed in order to obtain different body condition at parturition RQUICKI was lower in early lactation in cows with a high body condition score suggesting disturbed insulin function in obese cows. The results indicate that RQUICKI might be used to identify lactating cows with disturbed insulin function.

A review on recent developments in mass spectrometry instrumentation and quantitative tools advancing bacterial proteomics.

PubMed

Van Oudenhove, Laurence; Devreese, Bart

2013-06-01

Proteomics has evolved substantially since its early days, some 20 years ago. In this mini-review, we aim to provide an overview of general methodologies and more recent developments in mass spectrometric approaches used for relative and absolute quantitation of proteins. Enhancement of sensitivity of the mass spectrometers as well as improved sample preparation and protein fractionation methods are resulting in a more comprehensive analysis of proteomes. We also document some upcoming trends for quantitative proteomics such as the use of label-free quantification methods. Hopefully, microbiologists will continue to explore proteomics as a tool in their research to understand the adaptation of microorganisms to their ever changing environment. We encourage them to incorporate some of the described new developments in mass spectrometry to facilitate their analyses and improve the general knowledge of the fascinating world of microorganisms.

Quantitative determination of Auramine O by terahertz spectroscopy with 2DCOS-PLSR model

NASA Astrophysics Data System (ADS)

Zhang, Huo; Li, Zhi; Chen, Tao; Qin, Binyi

2017-09-01

Residues of harmful dyes such as Auramine O (AO) in herb and food products threaten the health of people. So, fast and sensitive detection techniques of the residues are needed. As a powerful tool for substance detection, terahertz (THz) spectroscopy was used for the quantitative determination of AO by combining with an improved partial least-squares regression (PLSR) model in this paper. Absorbance of herbal samples with different concentrations was obtained by THz-TDS in the band between 0.2THz and 1.6THz. We applied two-dimensional correlation spectroscopy (2DCOS) to improve the PLSR model. This method highlighted the spectral differences of different concentrations, provided a clear criterion of the input interval selection, and improved the accuracy of detection result. The experimental result indicated that the combination of the THz spectroscopy and 2DCOS-PLSR is an excellent quantitative analysis method.

Quantitative studies of sulphate conjugation by isolated rat liver cells using [35S]sulphate.

PubMed

Dawson, J; Knowles, R G; Pogson, C I

1991-06-21

We have developed a simple, rapid and sensitive method for the study of sulphate conjugation in isolated liver cells based on the incorporation of 35S from [35S]sulphate. Excess [35S]sulphate is removed by a barium precipitation procedure, leaving [35S]sulphate conjugates in solution. We have used this method to examine the kinetics of sulphation of N-acetyl-p-aminophenol (acetaminophen), 4-nitrophenol and 1-naphthol in isolated rat liver cells. The efficiency of recovery of the sulphate conjugates was greater than 86%. The method is applicable to the quantitative study of sulphate conjugation of any substrate which forms a sulphate conjugate that is soluble in the presence of barium, without the need for standards or radiolabelled sulphate acceptors.

A facile one-step fluorescence method for the quantitation of low-content single base deamination impurity in synthetic oligonucleotides.

PubMed

Su, Xiaoye; Liang, Ruiting; Stolee, Jessica A

2018-06-05

Oligonucleotides are being researched and developed as potential drug candidates for the treatment of a broad spectrum of diseases. The characterization of antisense oligonucleotide (ASO) impurities caused by base mutations (e.g. deamination) which are closely related to the target ASO is a significant analytical challenge. Herein, we describe a novel one-step method, utilizing a strategy that combines fluorescence-ON detection with competitive hybridization, to achieve single base mutation quantitation in extensively modified synthetic ASOs. Given that this method is highly specific and sensitive (LoQ = 4 nM), we envision that it will find utility for screening other impurities as well as sequencing modified oligonucleotides. Copyright © 2018 Elsevier B.V. All rights reserved.

Quantitative analysis of benzodiazepines in vitreous humor by high-performance liquid chromatography

PubMed Central

Bazmi, Elham; Behnoush, Behnam; Akhgari, Maryam; Bahmanabadi, Leila

2016-01-01

Objective: Benzodiazepines are frequently screened drugs in emergency toxicology, drugs of abuse testing, and in forensic cases. As the variations of benzodiazepines concentrations in biological samples during bleeding, postmortem changes, and redistribution could be biasing forensic medicine examinations, hence selecting a suitable sample and a validated accurate method is essential for the quantitative analysis of these main drug categories. The aim of this study was to develop a valid method for the determination of four benzodiazepines (flurazepam, lorazepam, alprazolam, and diazepam) in vitreous humor using liquid–liquid extraction and high-performance liquid chromatography. Methods: Sample preparation was carried out using liquid–liquid extraction with n-hexane: ethyl acetate and subsequent detection by high-performance liquid chromatography method coupled to diode array detector. This method was applied to quantify benzodiazepines in 21 authentic vitreous humor samples. Linear curve for each drug was obtained within the range of 30–3000 ng/mL with coefficient of correlation higher than 0.99. Results: The limit of detection and quantitation were 30 and 100 ng/mL respectively for four drugs. The method showed an appropriate intra- and inter-day precision (coefficient of variation < 10%). Benzodiazepines recoveries were estimated to be over 80%. The method showed high selectivity; no additional peak due to interfering substances in samples was observed. Conclusion: The present method was selective, sensitive, accurate, and precise for the quantitative analysis of benzodiazepines in vitreous humor samples in forensic toxicology laboratory. PMID:27635251

Quantitating Human Optic Disc Topography

NASA Astrophysics Data System (ADS)

Graebel, William P.; Cohan, Bruce E.; Pearch, Andrew C.

1980-07-01

A method is presented for quantitatively expressing the topography of the human optic disc, applicable in a clinical setting to the diagnosis and management of glaucoma. Pho-tographs of the disc illuminated by a pattern of fine, high contrast parallel lines are digitized. From the measured deviation of the lines as they traverse the disc surface, disc topography is calculated, using the principles of optical sectioning. The quantitators applied to express this topography have the the following advantages : sensitivity to disc shape; objectivity; going beyond the limits of cup-disc ratio estimates and volume calculations; perfect generality in a mathematical sense; an inherent scheme for determining a non-subjective reference frame to compare different discs or the same disc over time.

Multiplexed, quantitative, and targeted metabolite profiling by LC-MS/MRM.

PubMed

Wei, Ru; Li, Guodong; Seymour, Albert B

2014-01-01

Targeted metabolomics, which focuses on a subset of known metabolites representative of biologically relevant metabolic pathways, is a valuable tool to discover biomarkers and link disease phenotypes to underlying mechanisms or therapeutic modes of action. A key advantage of targeted metabolomics, compared to discovery metabolomics, is its immediate readiness for extracting biological information derived from known metabolites and quantitative measurements. However, simultaneously analyzing hundreds of endogenous metabolites presents a challenge due to their diverse chemical structures and properties. Here we report a method which combines different chromatographic separation conditions, optimal ionization polarities, and the most sensitive triple-quadrupole MS-based data acquisition mode, multiple reaction monitoring (MRM), to quantitatively profile 205 endogenous metabolites in 10 min.

Complementary techniques: validation of gene expression data by quantitative real time PCR.

PubMed

Provenzano, Maurizio; Mocellin, Simone

2007-01-01

Microarray technology can be considered the most powerful tool for screening gene expression profiles of biological samples. After data mining, results need to be validated with highly reliable biotechniques allowing for precise quantitation of transcriptional abundance of identified genes. Quantitative real time PCR (qrt-PCR) technology has recently reached a level of sensitivity, accuracy and practical ease that support its use as a routine bioinstrumentation for gene level measurement. Currently, qrt-PCR is considered by most experts the most appropriate method to confirm or confute microarray-generated data. The knowledge of the biochemical principles underlying qrt-PCR as well as some related technical issues must be beard in mind when using this biotechnology.

THE BORON-CURCUMIN COMPLEX IN TRACE BORON DETERMINATIONS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heyes, M.R.; Metcalfe, J.

1963-01-01

A simple and robust method for the formation of the complex of boron with curcumin is described. The sensitivity of the method is 6.6 x 10/sup -5/ g/cm/sup 2/. Formation of the complex is believed to be quantitative under the conditions used and some evidence is given for a 1: 3 boron; curcumin ratio. Methods are outlined for the determination of boron in a number of metals, compounds, and organic materials. (auth)

Pseudotargeted MS Method for the Sensitive Analysis of Protein Phosphorylation in Protein Complexes.

PubMed

Lyu, Jiawen; Wang, Yan; Mao, Jiawei; Yao, Yating; Wang, Shujuan; Zheng, Yong; Ye, Mingliang

2018-05-15

In this study, we presented an enrichment-free approach for the sensitive analysis of protein phosphorylation in minute amounts of samples, such as purified protein complexes. This method takes advantage of the high sensitivity of parallel reaction monitoring (PRM). Specifically, low confident phosphopeptides identified from the data-dependent acquisition (DDA) data set were used to build a pseudotargeted list for PRM analysis to allow the identification of additional phosphopeptides with high confidence. The development of this targeted approach is very easy as the same sample and the same LC-system were used for the discovery and the targeted analysis phases. No sample fractionation or enrichment was required for the discovery phase which allowed this method to analyze minute amount of sample. We applied this pseudotargeted MS method to quantitatively examine phosphopeptides in affinity purified endogenous Shc1 protein complexes at four temporal stages of EGF signaling and identified 82 phospho-sites. To our knowledge, this is the highest number of phospho-sites identified from the protein complexes. This pseudotargeted MS method is highly sensitive in the identification of low abundance phosphopeptides and could be a powerful tool to study phosphorylation-regulated assembly of protein complex.

Rapid and Highly Sensitive Detection of Lead Ions in Drinking Water Based on a Strip Immunosensor

PubMed Central

Kuang, Hua; Xing, Changrui; Hao, Changlong; Liu, Liqiang; Wang, Libing; Xu, Chuanlai

2013-01-01

In this study, we have first developed a rapid and sensitive strip immunosensor based on two heterogeneously-sized gold nanoparticles (Au NPs) probes for the detection of trace lead ions in drinking water. The sensitivity was 4-fold higher than that of the conventional LFA under the optimized conditions. The visual limit of detection (LOD) of the amplified method for qualitative detection lead ions was 2 ng/mL and the LOD for semi-quantitative detection could go down to 0.19 ng/mL using a scanning reader. The method suffered from no interference from other metal ions and could be used to detect trace lead ions in drinking water without sample enrichment. The recovery of the test samples ranged from 96% to 103%. As the detection method could be accomplished within 15 min, this method could be used as a potential tool for preliminary monitoring of lead contamination in drinking water. PMID:23539028

GC-MS quantitation of fragrance compounds suspected to cause skin reactions. 1.

PubMed

Chaintreau, Alain; Joulain, Daniel; Marin, Christophe; Schmidt, Claus-Oliver; Vey, Matthias

2003-10-22

Recent changes in European legislation require monitoring of 24 volatile compounds in perfumes as they might elicit skin sensitization. This paper reports a GC-MS quantitation procedure for their determination in fragrance concentrates. GC and MS conditions were optimized for a routine use: analysis within 30 min, solvent and internal standard selection, and stock solution stability. Calibration curves were linear in the range of 2-100 mg/L with coefficients of determination in excess of 0.99. The method was tested using real perfumes spiked with known amounts of reference compounds.

[Development of ELISA-kit of quantitative analysis for Zearalenone].

PubMed

Wang, Yu-ping; Ji, Rong; Jiang, Tao; Kang, Wei-jun

2006-03-01

To develope a rapid, sensitive, quantitative ELISA-kit for Zearalenone and determine zearalenone in cereals. On the base of monoclonal antibodies against ZEN, apply indirect ELISA to study the performance parameter of the kit. The limited concentration of detection of the ELISA-kit was 1ng/ml, linear range was 1-200 ng/ml, the linear equation was Y = 0.99 - 0.40 x (R2 = 0.99). The inhibition concentration of 50% against ZEN was 16.3 ng/ml. The average recovery rate of spiked corn and wheat was 96.5% and 95.5%, respectively, the coefficient of variant was 13.2% and 10.9%, respectively. The kit can be stored at 4 degrees C over 6 months. The cross reaction rate with the other mycotoxins was less than 1%, and coefficient of variant within-laboratory and between-laboratory was less than 15% and less than 20%, respectively. Detecting the VICAM sample with ELISA method and HPLC method, the results were within the range of the sample, and there was no statistic difference between the two methods. This ELISA-kit was quick, sensitive, stable and specific and can be used to determine ZEN in cereals.

Quantitative methylation-sensitive arbitrarily primed PCR method to determine differential genomic DNA methylation in Down Syndrome.

PubMed

Chango, Abalo; Abdennebi-Najar, Latifa; Tessier, Frederic; Ferré, Séverine; Do, Sergio; Guéant, Jean-Louis; Nicolas, Jean Pierre; Willequet, Francis

2006-10-20

Relative levels of DNA hypermethylation were quantified in DS individuals using a new method based on a combination of methylation-sensitive arbitrarily primed polymerase chain reaction (MS-AP-PCR) and quantification of DNA fragments with the Agilent 2100 bioanalyzer. Four of the DS individuals had low plasma total homocysteine (tHcy) level (4.3 +/- 0.3 micromol/l) and 4 other had high-tHcy level (14.1 +/- 0.9 micromol/l). Eight healthy control individuals were matched to the DS cases for age, sex, and tHcy levels. We have identified and quantified six hypermethylated fragments. Their sizes ranged from 230-bp to 700-bp. In cases and controls, low-tHcy did not affect methylation level of identified fragments, mean methylation values were 68.0 +/- 39.7% and 52.1 +/- 40.3%, respectively. DNA methylation in DS individuals did not change significantly (59.7+/-34.5%) in response to high-tHcy level in contrast to controls (23.4 +/- 17.7%, P = 0.02). Further, the quantitative MS-AP-PCR using this microfludic system is a useful method for determining differential genomic DNA methylation.

Direct and sensitive determination of glyphosate and aminomethylphosphonic acid in environmental water samples by high performance liquid chromatography coupled to electrospray tandem mass spectrometry.

PubMed

Guo, Hongyue; Riter, Leah S; Wujcik, Chad E; Armstrong, Daniel W

2016-04-22

A novel method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was developed for the sensitive determination of glyphosate and its major degradation product, AMPA in environmental water samples. The method involves the use of MS compatible mobile phases (0.1% formic acid in water and acetonitrile) for HPLC and direct analysis of water samples without sample derivatization. The method has been validated in different types of water matrices (drinking, surface and groundwater) by accuracy and precision studies with samples spiked at 0.1, 7.5 and 90 ppb. All mean accuracy values ranged from 85% to 112% for glyphosate and AMPA using both primary and secondary quantitative ion transitions (RSD ≤ 10%). Moreover, both primary and secondary ion transitions for glyphosate and AMPA can achieve the quantitation limits at 0.1 ppb. The linear dynamic range of the calibration curves were from 0.1 to 100 ppb for each analyte at each ion transitions with correlation coefficient higher than 0.997. Copyright © 2016 Elsevier B.V. All rights reserved.

Retrieval of complex χ(2) parts for quantitative analysis of sum-frequency generation intensity spectra

PubMed Central

Hofmann, Matthias J.; Koelsch, Patrick

2015-01-01

Vibrational sum-frequency generation (SFG) spectroscopy has become an established technique for in situ surface analysis. While spectral recording procedures and hardware have been optimized, unique data analysis routines have yet to be established. The SFG intensity is related to probing geometries and properties of the system under investigation such as the absolute square of the second-order susceptibility χ(2)2. A conventional SFG intensity measurement does not grant access to the complex parts of χ(2) unless further assumptions have been made. It is therefore difficult, sometimes impossible, to establish a unique fitting solution for SFG intensity spectra. Recently, interferometric phase-sensitive SFG or heterodyne detection methods have been introduced to measure real and imaginary parts of χ(2) experimentally. Here, we demonstrate that iterative phase-matching between complex spectra retrieved from maximum entropy method analysis and fitting of intensity SFG spectra (iMEMfit) leads to a unique solution for the complex parts of χ(2) and enables quantitative analysis of SFG intensity spectra. A comparison between complex parts retrieved by iMEMfit applied to intensity spectra and phase sensitive experimental data shows excellent agreement between the two methods. PMID:26450297

Molecular diagnosis of toxoplasmosis in immunocompromised patients.

PubMed

Robert-Gangneux, Florence; Belaz, Sorya

2016-08-01

Toxoplasmosis in immunocompromised patients is associated with a high mortality rate. Molecular techniques are important tools to diagnose acute disease in immunocompromised patients, but there are various methods with variable efficiency. Some of them have been validated for the diagnosis of congenital toxoplasmosis, but the impact of their use has not been evaluated in immunocompromised patients. Toxoplasmosis is of increasing importance in non-HIV immunocompromised patients. In addition, the picture of disease shows greater severity in South America, both in immunocompetent study participants and in congenitally infected infants. These epidemiological differences could influence the sensitivity of diagnostic methods. This review analyzes recent data on molecular diagnosis and compares them with older ones, in light of progress gained in molecular techniques and of recent epidemiological findings. Most recent studies were conducted in South America and used PCR targeting the B1 gene. PCR on blood could allow diagnosing a significant proportion of patients with ocular toxoplasmosis in Brazil. Quantitative PCR methods with specific probes should be used to improve sensitivity and warrant specificity. Performance of quantitative PCR targeting the repeated 529 bp sequence for the diagnosis of toxoplasmosis in immunocompromised patients needs evaluation in field studies in South America and in western countries.

Leaf epidermis images for robust identification of plants

PubMed Central

da Silva, Núbia Rosa; Oliveira, Marcos William da Silva; Filho, Humberto Antunes de Almeida; Pinheiro, Luiz Felipe Souza; Rossatto, Davi Rodrigo; Kolb, Rosana Marta; Bruno, Odemir Martinez

2016-01-01

This paper proposes a methodology for plant analysis and identification based on extracting texture features from microscopic images of leaf epidermis. All the experiments were carried out using 32 plant species with 309 epidermal samples captured by an optical microscope coupled to a digital camera. The results of the computational methods using texture features were compared to the conventional approach, where quantitative measurements of stomatal traits (density, length and width) were manually obtained. Epidermis image classification using texture has achieved a success rate of over 96%, while success rate was around 60% for quantitative measurements taken manually. Furthermore, we verified the robustness of our method accounting for natural phenotypic plasticity of stomata, analysing samples from the same species grown in different environments. Texture methods were robust even when considering phenotypic plasticity of stomatal traits with a decrease of 20% in the success rate, as quantitative measurements proved to be fully sensitive with a decrease of 77%. Results from the comparison between the computational approach and the conventional quantitative measurements lead us to discover how computational systems are advantageous and promising in terms of solving problems related to Botany, such as species identification. PMID:27217018

Qualitative and Quantitative Analysis of the Major Constituents in Chinese Medical Preparation Lianhua-Qingwen Capsule by UPLC-DAD-QTOF-MS

PubMed Central

Jia, Weina; Wang, Chunhua; Wang, Yuefei; Pan, Guixiang; Jiang, Miaomiao; Li, Zheng; Zhu, Yan

2015-01-01

Lianhua-Qingwen capsule (LQC) is a commonly used Chinese medical preparation to treat viral influenza and especially played a very important role in the fight against severe acute respiratory syndrome (SARS) in 2002-2003 in China. In this paper, a rapid ultraperformance liquid chromatography coupled with diode-array detector and quadrupole time-of-flight mass spectrometry (UPLC-DAD-QTOF-MS) method was established for qualitative and quantitative analysis of the major constituents of LQC. A total of 61 compounds including flavonoids, phenylpropanoids, anthraquinones, triterpenoids, iridoids, and other types of compounds were unambiguously or tentatively identified by comparing the retention times and accurate mass measurement with reference compounds or literature data. Among them, twelve representative compounds were further quantified as chemical markers in quantitative analysis, including salidroside, chlorogenic acid, forsythoside E, cryptochlorogenic acid, amygdalin, sweroside, hyperin, rutin, forsythoside A, phillyrin, rhein, and glycyrrhizic acid. The UPLC-DAD method was evaluated with linearity, limit of detection (LOD), limit of quantification (LOQ), precision, stability, repeatability, and recovery tests. The results showed that the developed quantitative method was linear, sensitive, and precise for the quality control of LQC. PMID:25654135

Hypersensitive Detection and Quantitation of BoNT/A by IgY Antibody against Substrate Linear-Peptide

PubMed Central

Li, Tao; Liu, Hao; Cai, Kun; Tian, Maoren; Wang, Qin; Shi, Jing; Gao, Xiang; Wang, Hui

2013-01-01

Botulinum neurotoxin A (BoNT/A), the most acutely poisonous substance to humans known, cleave its SNAP-25 substrate with high specificity. Based on the endopeptidase activity, different methods have been developed to detect BoNT/A, but most lack ideal reproducibility or sensitivity, or suffer from long-term or unwanted interferences. In this study, we developed a simple method to detect and quantitate trace amounts of botulinum neurotoxin A using the IgY antibody against a linear-peptide substrate. The effects of reaction buffer, time, and temperature were analyzed and optimized. When the optimized assay was used to detect BoNT/A, the limit of detection of the assay was 0.01 mouse LD50 (0.04 pg), and the limit of quantitation was 0.12 mouse LD50/ml (0.48 pg). The findings also showed favorable specificity of detecting BoNT/A. When used to detect BoNT/A in milk or human serum, the proposed assay exhibited good quantitative accuracy (88% < recovery < 111%; inter- and intra-assay CVs < 18%). This method of detection took less than 3 h to complete, indicating that it can be a valuable method of detecting BoNT/A in food or clinical diagnosis. PMID:23555605

Hypersensitive detection and quantitation of BoNT/A by IgY antibody against substrate linear-peptide.

PubMed

Li, Tao; Liu, Hao; Cai, Kun; Tian, Maoren; Wang, Qin; Shi, Jing; Gao, Xiang; Wang, Hui

2013-01-01

Botulinum neurotoxin A (BoNT/A), the most acutely poisonous substance to humans known, cleave its SNAP-25 substrate with high specificity. Based on the endopeptidase activity, different methods have been developed to detect BoNT/A, but most lack ideal reproducibility or sensitivity, or suffer from long-term or unwanted interferences. In this study, we developed a simple method to detect and quantitate trace amounts of botulinum neurotoxin A using the IgY antibody against a linear-peptide substrate. The effects of reaction buffer, time, and temperature were analyzed and optimized. When the optimized assay was used to detect BoNT/A, the limit of detection of the assay was 0.01 mouse LD50 (0.04 pg), and the limit of quantitation was 0.12 mouse LD50/ml (0.48 pg). The findings also showed favorable specificity of detecting BoNT/A. When used to detect BoNT/A in milk or human serum, the proposed assay exhibited good quantitative accuracy (88% < recovery < 111%; inter- and intra-assay CVs < 18%). This method of detection took less than 3 h to complete, indicating that it can be a valuable method of detecting BoNT/A in food or clinical diagnosis.

Benchmarking quantitative label-free LC-MS data processing workflows using a complex spiked proteomic standard dataset.

PubMed

Ramus, Claire; Hovasse, Agnès; Marcellin, Marlène; Hesse, Anne-Marie; Mouton-Barbosa, Emmanuelle; Bouyssié, David; Vaca, Sebastian; Carapito, Christine; Chaoui, Karima; Bruley, Christophe; Garin, Jérôme; Cianférani, Sarah; Ferro, Myriam; Van Dorssaeler, Alain; Burlet-Schiltz, Odile; Schaeffer, Christine; Couté, Yohann; Gonzalez de Peredo, Anne

2016-01-30

Proteomic workflows based on nanoLC-MS/MS data-dependent-acquisition analysis have progressed tremendously in recent years. High-resolution and fast sequencing instruments have enabled the use of label-free quantitative methods, based either on spectral counting or on MS signal analysis, which appear as an attractive way to analyze differential protein expression in complex biological samples. However, the computational processing of the data for label-free quantification still remains a challenge. Here, we used a proteomic standard composed of an equimolar mixture of 48 human proteins (Sigma UPS1) spiked at different concentrations into a background of yeast cell lysate to benchmark several label-free quantitative workflows, involving different software packages developed in recent years. This experimental design allowed to finely assess their performances in terms of sensitivity and false discovery rate, by measuring the number of true and false-positive (respectively UPS1 or yeast background proteins found as differential). The spiked standard dataset has been deposited to the ProteomeXchange repository with the identifier PXD001819 and can be used to benchmark other label-free workflows, adjust software parameter settings, improve algorithms for extraction of the quantitative metrics from raw MS data, or evaluate downstream statistical methods. Bioinformatic pipelines for label-free quantitative analysis must be objectively evaluated in their ability to detect variant proteins with good sensitivity and low false discovery rate in large-scale proteomic studies. This can be done through the use of complex spiked samples, for which the "ground truth" of variant proteins is known, allowing a statistical evaluation of the performances of the data processing workflow. We provide here such a controlled standard dataset and used it to evaluate the performances of several label-free bioinformatics tools (including MaxQuant, Skyline, MFPaQ, IRMa-hEIDI and Scaffold) in different workflows, for detection of variant proteins with different absolute expression levels and fold change values. The dataset presented here can be useful for tuning software tool parameters, and also testing new algorithms for label-free quantitative analysis, or for evaluation of downstream statistical methods. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product.

PubMed

Fazelahi, Mansoureh; Kia, Vahid; Kaghazian, Hooman; Paryan, Mahdi

2017-11-26

Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli's and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (10 1 to 10 7 copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.

Optimization and Validation of a Sensitive Method for HPLC-PDA Simultaneous Determination of Torasemide and Spironolactone in Human Plasma using Central Composite Design.

PubMed

Subramanian, Venkatesan; Nagappan, Kannappan; Sandeep Mannemala, Sai

2015-01-01

A sensitive, accurate, precise and rapid HPLC-PDA method was developed and validated for the simultaneous determination of torasemide and spironolactone in human plasma using Design of experiments. Central composite design was used to optimize the method using content of acetonitrile, concentration of buffer and pH of mobile phase as independent variables, while the retention factor of spironolactone, resolution between torasemide and phenobarbitone; and retention time of phenobarbitone were chosen as dependent variables. The chromatographic separation was achieved on Phenomenex C(18) column and the mobile phase comprising 20 mM potassium dihydrogen ortho phosphate buffer (pH-3.2) and acetonitrile in 82.5:17.5 v/v pumped at a flow rate of 1.0 mL min(-1). The method was validated according to USFDA guidelines in terms of selectivity, linearity, accuracy, precision, recovery and stability. The limit of quantitation values were 80 and 50 ng mL(-1) for torasemide and spironolactone respectively. Furthermore, the sensitivity and simplicity of the method suggests the validity of method for routine clinical studies.

Culturally Sensitive Parent Education: A Critical Review of Quantitative Research.

ERIC Educational Resources Information Center

Gorman, Jean Cheng; Balter, Lawrence

1997-01-01

Critically reviews the quantitative literature on culturally sensitive parent education programs, discussing issues of research methodology and program efficacy in producing change among ethnic minority parents and their children. Culturally sensitive programs for African American and Hispanic families are described in detail. Methodological flaws…

Optical demodulation system for digitally encoded suspension array in fluoroimmunoassay

NASA Astrophysics Data System (ADS)

He, Qinghua; Li, Dongmei; He, Yonghong; Guan, Tian; Zhang, Yilong; Shen, Zhiyuan; Chen, Xuejing; Liu, Siyu; Lu, Bangrong; Ji, Yanhong

2017-09-01

A laser-induced breakdown spectroscopy and fluorescence spectroscopy-coupled optical system is reported to demodulate digitally encoded suspension array in fluoroimmunoassay. It takes advantage of the plasma emissions of assembled elemental materials to digitally decode the suspension array, providing a more stable and accurate recognition to target biomolecules. By separating the decoding procedure of suspension array and adsorption quantity calculation of biomolecules into two independent channels, the cross talk between decoding and label signals in traditional methods had been successfully avoided, which promoted the accuracy of both processes and realized more sensitive quantitative detection of target biomolecules. We carried a multiplexed detection of several types of anti-IgG to verify the quantitative analysis performance of the system. A limit of detection of 1.48×10-10 M was achieved, demonstrating the detection sensitivity of the optical demodulation system.

Development of an UPLC-MS/MS micromethod for quantitation of cinitapride in plasma and its application in a pharmacokinetic interaction trial.

PubMed

Marcelín-Jiménez, Gabriel; Contreras, Leticia; Esquivel, Javier; Ávila, Óscar; Batista, Dany; Ángeles, Alionka P; García-González, Alberto

2017-03-01

Cinitapride (CIN) is a benzamide-derived molecule used for the treatment of gastroesophageal reflux and dyspepsia. Its pharmacokinetics are controversial due to the use of supratherapeutic doses and the lack of sensitive methodology. Therefore, a sensitive and accurate micromethod was developed for its quantitation in human plasma. CIN was extracted from 300 µl of heparinized plasma by liquid-liquid extraction using cisapride as internal standard, and analyzed with an ultra performance liquid chromatograph employing positive multiple-reaction monitoring-MS. The method proved to be rapid, accurate and stable within a range between 50 and 2000 pg/ml and was successfully validated and applied in a pharmacokinetic interaction trial, where it was demonstrated that oral co-administration of simethicone does not modify the bioavailability of CIN.

Improved micromethod for mezlocillin quantitation in serum and urine by high-pressure liquid chromatography.

PubMed Central

Fiore, D; Auger, F A; Drusano, G L; Dandu, V R; Lesko, L J

1984-01-01

A rapid, sensitive, and specific method of analysis for mezlocillin in serum and urine by high-pressure liquid chromatography is described. A solid-phase extraction column was used to remove interfering substances from samples before chromatography. Quantitation included the use of an internal standard, nafcillin. Mezlocillin was chromatographed with a phosphate buffer-acetonitrile (73:27) mobile phase and a C-18 reverse-phase column and detected at a wavelength of 220 nm. The assay had a sensitivity of 1.6 micrograms/ml and a linearity of up to 600 micrograms/ml and 16 mg/ml in serum and urine, respectively, with only 0.1 ml of sample. The interday and intraday coefficients of variation for replicate analyses of spiked serum and urine specimens were less than 6.5%. PMID:6517560

Measurement of plasma hydrogen sulfide in vivo and in vitro

PubMed Central

Shen, Xinggui; Pattillo, Christopher B.; Pardue, Sibile; Bir, Shyamal C.; Wang, Rui; Kevil, Christopher G.

2015-01-01

The gasotransmitter hydrogen sulfide is known to regulate multiple cellular functions during normal and pathophysiological states. However, a paucity of concise information exists regarding quantitative amounts of hydrogen sulfide involved in physiological and pathological responses. This is primarily due to disagreement among various methods employed to measure free hydrogen sulfide. In this article, we describe a very sensitive method of measuring the presence of H2S in plasma down to nanomolar levels, using monobromobimane (MBB). The current standard assay using methylene blue provides erroneous results that do not actually measure H2S. The method presented herein involves derivatization of sulfide with excess MBB in 100 mM Tris–HCl buffer (pH 9.5, 0.1 mM DTPA) for 30 min in 1% oxygen at room temperature. The fluorescent product sulfide-dibimane (SDB) is analyzed by RP-HPLC using an eclipse XDB-C18 (4.6×250 mm) column with gradient elution by 0.1% (v/v) trifluoroacetic acid in acetonitrile. The limit of detection for sulfide-dibimane is 2 nM and the SDB product is very stable over time, allowing batch storage and analysis. In summary, our MBB method is suitable for sensitive quantitative measurement of free hydrogen sulfide in multiple biological samples such as plasma, tissue and cell culture lysates, or media. PMID:21276849

Simultaneous quantitation of oxidized and reduced glutathione via LC-MS/MS: An insight into the redox state of hematopoietic stem cells.

PubMed

Carroll, Dustin; Howard, Diana; Zhu, Haining; Paumi, Christian M; Vore, Mary; Bondada, Subbarao; Liang, Ying; Wang, Chi; St Clair, Daret K

2016-08-01

Cellular redox balance plays a significant role in the regulation of hematopoietic stem-progenitor cell (HSC/MPP) self-renewal and differentiation. Unregulated changes in cellular redox homeostasis are associated with the onset of most hematological disorders. However, accurate measurement of the redox state in stem cells is difficult because of the scarcity of HSC/MPPs. Glutathione (GSH) constitutes the most abundant pool of cellular antioxidants. Thus, GSH metabolism may play a critical role in hematological disease onset and progression. A major limitation to studying GSH metabolism in HSC/MPPs has been the inability to measure quantitatively GSH concentrations in small numbers of HSC/MPPs. Current methods used to measure GSH levels not only rely on large numbers of cells, but also rely on the chemical/structural modification or enzymatic recycling of GSH and therefore are likely to measure only total glutathione content accurately. Here, we describe the validation of a sensitive method used for the direct and simultaneous quantitation of both oxidized and reduced GSH via liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) in HSC/MPPs isolated from bone marrow. The lower limit of quantitation (LLOQ) was determined to be 5.0ng/mL for GSH and 1.0ng/mL for GSSG with lower limits of detection at 0.5ng/mL for both glutathione species. Standard addition analysis utilizing mouse bone marrow shows that this method is both sensitive and accurate with reproducible analyte recovery. This method combines a simple extraction with a platform for the high-throughput analysis, allows for efficient determination of GSH/GSSG concentrations within the HSC/MPP populations in mouse, chemotherapeutic treatment conditions within cell culture, and human normal/leukemia patient samples. The data implicate the importance of the modulation of GSH/GSSG redox couple in stem cells related diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Diagnostic performance of semi-quantitative and quantitative stress CMR perfusion analysis: a meta-analysis.

PubMed

van Dijk, R; van Assen, M; Vliegenthart, R; de Bock, G H; van der Harst, P; Oudkerk, M

2017-11-27

Stress cardiovascular magnetic resonance (CMR) perfusion imaging is a promising modality for the evaluation of coronary artery disease (CAD) due to high spatial resolution and absence of radiation. Semi-quantitative and quantitative analysis of CMR perfusion are based on signal-intensity curves produced during the first-pass of gadolinium contrast. Multiple semi-quantitative and quantitative parameters have been introduced. Diagnostic performance of these parameters varies extensively among studies and standardized protocols are lacking. This study aims to determine the diagnostic accuracy of semi- quantitative and quantitative CMR perfusion parameters, compared to multiple reference standards. Pubmed, WebOfScience, and Embase were systematically searched using predefined criteria (3272 articles). A check for duplicates was performed (1967 articles). Eligibility and relevance of the articles was determined by two reviewers using pre-defined criteria. The primary data extraction was performed independently by two researchers with the use of a predefined template. Differences in extracted data were resolved by discussion between the two researchers. The quality of the included studies was assessed using the 'Quality Assessment of Diagnostic Accuracy Studies Tool' (QUADAS-2). True positives, false positives, true negatives, and false negatives were subtracted/calculated from the articles. The principal summary measures used to assess diagnostic accuracy were sensitivity, specificity, andarea under the receiver operating curve (AUC). Data was pooled according to analysis territory, reference standard and perfusion parameter. Twenty-two articles were eligible based on the predefined study eligibility criteria. The pooled diagnostic accuracy for segment-, territory- and patient-based analyses showed good diagnostic performance with sensitivity of 0.88, 0.82, and 0.83, specificity of 0.72, 0.83, and 0.76 and AUC of 0.90, 0.84, and 0.87, respectively. In per territory analysis our results show similar diagnostic accuracy comparing anatomical (AUC 0.86(0.83-0.89)) and functional reference standards (AUC 0.88(0.84-0.90)). Only the per territory analysis sensitivity did not show significant heterogeneity. None of the groups showed signs of publication bias. The clinical value of semi-quantitative and quantitative CMR perfusion analysis remains uncertain due to extensive inter-study heterogeneity and large differences in CMR perfusion acquisition protocols, reference standards, and methods of assessment of myocardial perfusion parameters. For wide spread implementation, standardization of CMR perfusion techniques is essential. CRD42016040176 .

Hematocrit Measurement with R2* and Quantitative Susceptibility Mapping in Postmortem Brain.

PubMed

Walsh, A J; Sun, H; Emery, D J; Wilman, A H

2018-05-24

Noninvasive venous oxygenation quantification with MR imaging will improve the neurophysiologic investigation and the understanding of the pathophysiology in neurologic diseases. Available MR imaging methods are limited by sensitivity to flow and often require assumptions of the hematocrit level. In situ postmortem imaging enables evaluation of methods in a fully deoxygenated environment without flow artifacts, allowing direct calculation of hematocrit. This study compares 2 venous oxygenation quantification methods in in situ postmortem subjects. Transverse relaxation (R2*) mapping and quantitative susceptibility mapping were performed on a whole-body 4.7T MR imaging system. Intravenous measurements in major draining intracranial veins were compared between the 2 methods in 3 postmortem subjects. The quantitative susceptibility mapping technique was also applied in 10 healthy control subjects and compared with reference venous oxygenation values. In 2 early postmortem subjects, R2* mapping and quantitative susceptibility mapping measurements within intracranial veins had a significant and strong correlation ( R 2 = 0.805, P = .004 and R 2 = 0.836, P = .02). Higher R2* and susceptibility values were consistently demonstrated within gravitationally dependent venous segments during the early postmortem period. Hematocrit ranged from 0.102 to 0.580 in postmortem subjects, with R2* and susceptibility as large as 291 seconds -1 and 1.75 ppm, respectively. Measurements of R2* and quantitative susceptibility mapping within large intracranial draining veins have a high correlation in early postmortem subjects. This study supports the use of quantitative susceptibility mapping for evaluation of in vivo venous oxygenation and postmortem hematocrit concentrations. © 2018 by American Journal of Neuroradiology.

Detection of Echinoderm Microtubule Associated Protein Like 4-Anaplastic Lymphoma Kinase Fusion Genes in Non-small Cell Lung Cancer Clinical Samples by a Real-time Quantitative Reverse Transcription Polymerase Chain Reaction Method.

PubMed

Zhao, Jing; Zhao, Jin-Yin; Chen, Zhi-Xia; Zhong, Wei; Li, Long-Yun; Liu, Li-Cheng; Hu, Xiao-Xu; Chen, Wei-Jun; Wang, Meng-Zhao

2016-12-20

Objective To establish a real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) for the rapid, sensitive, and specific detection of echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion genes in non-small cell lung cancer. Methods The specific primers for the four variants of EML4-ALK fusion genes (V1, V2, V3a, and V3b) and Taqman fluorescence probes for the detection of the target sequences were carefully designed by the Primer Premier 5.0 software. Then, using pseudovirus containing EML4-ALK fusion genes variants (V1, V2, V3a, and V3b) as the study objects, we further analyzed the lower limit, sensitivity, and specificity of this method. Finally, 50 clinical samples, including 3 ALK-fluorescence in situ hybridization (FISH) positive specimens, were collected and used to detect EML4-ALK fusion genes using this method. Results The lower limit of this method for the detection of EML4-ALK fusion genes was 10 copies/μl if no interference of background RNA existed. Regarding the method's sensitivity, the detection resolution was as high as 1% and 0.5% in the background of 500 and 5000 copies/μl wild-type ALK gene, respectively. Regarding the method's specificity, no non-specific amplification was found when it was used to detect EML4-ALK fusion genes in leukocyte and plasma RNA samples from healthy volunteers. Among the 50 clinical samples, 47 ALK-FISH negative samples were also negative. Among 3 ALK-FISH positive samples, 2 cases were detected positive using this method, but another was not detected because of the failure of RNA extraction. Conclusion The proposed qRT-PCR assay for the detection of EML4-ALK fusion genes is rapid, simple, sensitive, and specific, which is deserved to be validated and widely used in clinical settings.

Phase calibration target for quantitative phase imaging with ptychography.

PubMed

Godden, T M; Muñiz-Piniella, A; Claverley, J D; Yacoot, A; Humphry, M J

2016-04-04

Quantitative phase imaging (QPI) utilizes refractive index and thickness variations that lead to optical phase shifts. This gives contrast to images of transparent objects. In quantitative biology, phase images are used to accurately segment cells and calculate properties such as dry mass, volume and proliferation rate. The fidelity of the measured phase shifts is of critical importance in this field. However to date, there has been no standardized method for characterizing the performance of phase imaging systems. Consequently, there is an increasing need for protocols to test the performance of phase imaging systems using well-defined phase calibration and resolution targets. In this work, we present a candidate for a standardized phase resolution target, and measurement protocol for the determination of the transfer of spatial frequencies, and sensitivity of a phase imaging system. The target has been carefully designed to contain well-defined depth variations over a broadband range of spatial frequencies. In order to demonstrate the utility of the target, we measure quantitative phase images on a ptychographic microscope, and compare the measured optical phase shifts with Atomic Force Microscopy (AFM) topography maps and surface profile measurements from coherence scanning interferometry. The results show that ptychography has fully quantitative nanometer sensitivity in optical path differences over a broadband range of spatial frequencies for feature sizes ranging from micrometers to hundreds of micrometers.

Three-phase bone scintigraphy for diagnosis of Charcot neuropathic osteoarthropathy in the diabetic foot - does quantitative data improve diagnostic value?

PubMed

Fosbøl, M; Reving, S; Petersen, E H; Rossing, P; Lajer, M; Zerahn, B

2017-01-01

To investigate whether inclusion of quantitative data on blood flow distribution compared with visual qualitative evaluation improve the reliability and diagnostic performance of 99 m Tc-hydroxymethylene diphosphate three-phase bone scintigraphy (TPBS) in patients suspected for charcot neuropathic osteoarthropathy (CNO) of the foot. A retrospective cohort study of TPBS performed on 148 patients with suspected acute CNO referred from a single specialized diabetes care centre. The quantitative blood flow distribution was calculated based on the method described by Deutsch et al. All scintigraphies were re-evaluated by independent, blinded observers twice with and without quantitative data on blood flow distribution at ankle and focus level, respectively. The diagnostic validity of TPBS was determined by subsequent review of clinical data and radiological examinations. A total of 90 patients (61%) had confirmed diagnosis of CNO. The sensitivity, specificity and accuracy of three-phase bone scintigraphy without/with quantitative data were 89%/88%, 58%/62% and 77%/78%, respectively. The intra-observer agreement improved significantly by adding quantitative data in the evaluation (Kappa value 0·79/0·94). The interobserver agreement was not significantly improved. Adding quantitative data on blood flow distribution in the interpretation of TBPS improves intra-observer variation, whereas no difference in interobserver variation was observed. The sensitivity of TPBS in the diagnosis of CNO is high, but holds limited specificity. Diagnostic performance does not improve using quantitative data in the evaluation. This may be due to the reference intervals applied in the study or the absence of a proper gold standard diagnostic procedure for comparison. © 2015 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd.

Evaluation of a combined triple method to detect causative HPV in oral and oropharyngeal squamous cell carcinomas: p16 Immunohistochemistry, Consensus PCR HPV-DNA, and In Situ Hybridization

PubMed Central

2012-01-01

Background Recent emerging evidences identify Human Papillomavirus (HPV) related Head and Neck squamous cell carcinomas (HN-SCCs) as a separate subgroup among Head and Neck Cancers with different epidemiology, histopathological characteristics, therapeutic response to chemo-radiation treatment and clinical outcome. However, there is not a worldwide consensus on the methods to be used in clinical practice. The endpoint of this study was to demonstrate the reliability of a triple method which combines evaluation of: 1. p16 protein expression by immunohistochemistry (p16-IHC); 2. HPV-DNA genotyping by consensus HPV-DNA PCR methods (Consensus PCR); and 3 viral integration into the host by in situ hybridization method (ISH). This triple method has been applied to HN-SCC originated from oral cavity (OSCC) and oropharynx (OPSCC), the two anatomical sites in which high risk (HR) HPVs have been clearly implicated as etiologic factors. Methylation-Specific PCR (MSP) was performed to study inactivation of p16-CDKN2a locus by epigenetic events. Reliability of multiple methods was measured by Kappa statistics. Results All the HN-SCCs confirmed HPV positive by PCR and/or ISH were also p16 positive by IHC, with the latter showing a very high level of sensitivity as single test (100% in both OSCC and OPSCC) but lower specificity level (74% in OSCC and 93% in OPSCC). Concordance analysis between ISH and Consensus PCR showed a faint agreement in OPSCC (κ = 0.38) and a moderate agreement in OSCC (κ = 0.44). Furthermore, the addition of double positive score (ISHpositive and Consensus PCR positive) increased significantly the specificity of HR-HPV detection on formalin-fixed paraffin embedded (FFPE) samples (100% in OSCC and 78.5% in OPSCC), but reduced the sensitivity (33% in OSCC and 60% in OPSCC). The significant reduction of sensitivity by the double method was compensated by a very high sensitivity of p16-IHC detection in the triple approach. Conclusions Although HR-HPVs detection is of utmost importance in clinical settings for the Head and Neck Cancer patients, there is no consensus on which to consider the 'golden standard' among the numerous detection methods available either as single test or combinations. Until recently, quantitative E6 RNA PCR has been considered the 'golden standard' since it was demonstrated to have very high accuracy level and very high statistical significance associated with prognostic parameters. In contrast, quantitative E6 DNA PCR has proven to have very high level of accuracy but lesser prognostic association with clinical outcome than the HPV E6 oncoprotein RNA PCR. However, although it is theoretically possible to perform quantitative PCR detection methods also on FFPE samples, they reach the maximum of accuracy on fresh frozen tissue. Furthermore, worldwide diagnostic laboratories have not all the same ability to analyze simultaneously both FFPE and fresh tissues with these quantitative molecular detection methods. Therefore, in the current clinical practice a p16-IHC test is considered as sufficient for HPV diagnostic in accordance with the recently published Head and Neck Cancer international guidelines. Although p16-IHC may serve as a good prognostic indicator, our study clearly demonstrated that it is not satisfactory when used exclusively as the only HPV detecting method. Adding ISH, although known as less sensitive than PCR-based detection methods, has the advantage to preserve the morphological context of HPV-DNA signals in FFPE samples and, thus increase the overall specificity of p16/Consensus PCR combination tests. PMID:22376902

Quantification of the methylation status of the PWS/AS imprinted region: comparison of two approaches based on bisulfite sequencing and methylation-sensitive MLPA.

PubMed

Dikow, Nicola; Nygren, Anders Oh; Schouten, Jan P; Hartmann, Carolin; Krämer, Nikola; Janssen, Bart; Zschocke, Johannes

2007-06-01

Standard methods used for genomic methylation analysis allow the detection of complete absence of either methylated or non-methylated alleles but are usually unable to detect changes in the proportion of methylated and unmethylated alleles. We compare two methods for quantitative methylation analysis, using the chromosome 15q11-q13 imprinted region as model. Absence of the non-methylated paternal allele in this region leads to Prader-Willi syndrome (PWS) whilst absence of the methylated maternal allele results in Angelman syndrome (AS). A proportion of AS is caused by mosaic imprinting defects which may be missed with standard methods and require quantitative analysis for their detection. Sequence-based quantitative methylation analysis (SeQMA) involves quantitative comparison of peaks generated through sequencing reactions after bisulfite treatment. It is simple, cost-effective and can be easily established for a large number of genes. However, our results support previous suggestions that methods based on bisulfite treatment may be problematic for exact quantification of methylation status. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) avoids bisulfite treatment. It detects changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in one simple reaction. Once established in a laboratory setting, the method is more accurate, reliable and less time consuming.

Sensitivity and specificity enhanced enzyme-linked immunosorbent assay by rational hapten modification and heterogeneous antibody/coating antigen combinations for the detection of melamine in milk, milk powder and feed samples.

PubMed

Cao, Biyun; Yang, Hong; Song, Juan; Chang, Huafang; Li, Shuqun; Deng, Anping

2013-11-15

The adulteration of food products with melamine has led to an urgent requirement for sensitive, specific, rapid and reliable quantitative/screening methods. To enhance the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) for the detection of melamine in milk, milk powder and feed samples, rational hapten modification and heterogeneous antibody/coating antigen combinations were adopted. Three melamine derivatives with different length of carboxylic spacer at the end were synthesized and linked to carrier proteins for the production of immunogens and coating antigens. Monoclonal antibody against melamine was produced by hybridoma technology. Under optimal experimental conditions, the standard curves of the ELISAs for melamine were constructed in range of 0.1-100 ng mL(-1). The sensitivity was 10-300 times enhanced compared to those in the published literatures. The cross-reactivity values of the ELISAs also demonstrated the assays exhibited high specificity. Five samples were spiked with melamine at different concentrations and detected by the ELISA. The recovery rates of 72.8-123.0% and intra-assay coefficients of variation of 0.8-18.9% (n=3) were obtained. The ELISA for milk sample was confirmed by high-performance liquid chromatography with a high correlation coefficient of 0.9902 (n=6). The proposed ELISA was proven to be a feasible quantitative/screening method for melamine analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

Analytical Glycobiology at High Sensitivity: Current Approaches and Directions

PubMed Central

Novotny, Milos V.; Alley, William R.; Mann, Benjamin F.

2013-01-01

This review summarizes the analytical advances made during the last several years in the structural and quantitative determinations of glycoproteins in complex biological mixtures. The main analytical techniques used in the fields of glycomics and glycoproteomics involve different modes of mass spectrometry and their combinations with capillary separation methods such as microcolumn liquid chromatography and capillary electrophoresis. The needs for high-sensitivity measurements have been emphasized in the oligosaccharide profiling used in the field of biomarker discovery through MALDI mass spectrometry. High-sensitivity profiling of both glycans and glycopeptides from biological fluids and tissue extracts has been aided significantly through lectin preconcentration and the uses of affinity chromatography. PMID:22945852

Trimethylation enhancement using diazomethane (TrEnDi): rapid on-column quaternization of peptide amino groups via reaction with diazomethane significantly enhances sensitivity in mass spectrometry analyses via a fixed, permanent positive charge.

PubMed

Wasslen, Karl V; Tan, Le Hoa; Manthorpe, Jeffrey M; Smith, Jeffrey C

2014-04-01

Defining cellular processes relies heavily on elucidating the temporal dynamics of proteins. To this end, mass spectrometry (MS) is an extremely valuable tool; different MS-based quantitative proteomics strategies have emerged to map protein dynamics over the course of stimuli. Herein, we disclose our novel MS-based quantitative proteomics strategy with unique analytical characteristics. By passing ethereal diazomethane over peptides on strong cation exchange resin within a microfluidic device, peptides react to contain fixed, permanent positive charges. Modified peptides display improved ionization characteristics and dissociate via tandem mass spectrometry (MS(2)) to form strong a2 fragment ion peaks. Process optimization and determination of reactive functional groups enabled a priori prediction of MS(2) fragmentation patterns for modified peptides. The strategy was tested on digested bovine serum albumin (BSA) and successfully quantified a peptide that was not observable prior to modification. Our method ionizes peptides regardless of proton affinity, thus decreasing ion suppression and permitting predictable multiple reaction monitoring (MRM)-based quantitation with improved sensitivity.

Quantitative fucK gene polymerase chain reaction on sputum and nasopharyngeal secretions to detect Haemophilus influenzae pneumonia.

PubMed

Abdeldaim, Guma M K; Strålin, Kristoffer; Olcén, Per; Blomberg, Jonas; Mölling, Paula; Herrmann, Björn

2013-06-01

A quantitative polymerase chain reaction (PCR) for the fucK gene was developed for specific detection of Haemophilus influenzae. The method was tested on sputum and nasopharyngeal aspirate (NPA) from 78 patients with community-acquired pneumonia (CAP). With a reference standard of sputum culture and/or serology against the patient's own nasopharyngeal isolate, H. influenzae etiology was detected in 20 patients. Compared with the reference standard, fucK PCR (using the detection limit 10(5) DNA copies/mL) on sputum and NPA showed a sensitivity of 95.0% (19/20) in both cases, and specificities of 87.9% (51/58) and 89.5% (52/58), respectively. In a receiver operating characteristic curve analysis, sputum fucK PCR was found to be significantly superior to sputum P6 PCR for detection of H. influenzae CAP. NPA fucK PCR was positive in 3 of 54 adult controls without respiratory symptoms. In conclusion, quantitative fucK real-time PCR provides a sensitive and specific identification of H. influenzae in respiratory secretions. Copyright © 2013 Elsevier Inc. All rights reserved.

TH-AB-209-09: Quantitative Imaging of Electrical Conductivity by VHF-Induced Thermoacoustics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patch, S; Hull, D; See, W

Purpose: To demonstrate that very high frequency (VHF) induced thermoacoustics has the potential to provide quantitative images of electrical conductivity in Siemens/meter, much as shear wave elastography provides tissue stiffness in kPa. Quantitatively imaging a large organ requires exciting thermoacoustic pulses throughout the volume and broadband detection of those pulses because tomographic image reconstruction preserves frequency content. Applying the half-wavelength limit to a 200-micron inclusion inside a 7.5 cm diameter organ requires measurement sensitivity to frequencies ranging from 4 MHz down to 10 kHz, respectively. VHF irradiation provides superior depth penetration over near infrared used in photoacoustics. Additionally, VHF signalmore » production is proportional to electrical conductivity, and prostate cancer is known to suppress electrical conductivity of prostatic fluid. Methods: A dual-transducer system utilizing a P4-1 array connected to a Verasonics V1 system augmented by a lower frequency focused single element transducer was developed. Simultaneous acquisition of VHF-induced thermoacoustic pulses by both transducers enabled comparison of transducer performance. Data from the clinical array generated a stack of 96-images with separation of 0.3 mm, whereas the single element transducer imaged only in a single plane. In-plane resolution and quantitative accuracy were measured at isocenter. Results: The array provided volumetric imaging capability with superior resolution whereas the single element transducer provided superior quantitative accuracy. Combining axial images from both transducers preserved resolution of the P4-1 array and improved image contrast. Neither transducer was sensitive to frequencies below 50 kHz, resulting in a DC offset and low-frequency shading over fields of view exceeding 15 mm. Fresh human prostates were imaged ex vivo and volumetric reconstructions reveal structures rarely seen in diagnostic images. Conclusion: Quantitative whole-organ thermoacoustic tomography will be feasible by sparsely interspersing transducer elements sensitive to the low end of the ultrasonic range.« less

The environment of x ray selected BL Lacs: Host galaxies and galaxy clustering

NASA Technical Reports Server (NTRS)

Wurtz, Ron; Stocke, John T.; Ellingson, Erica; Yee, Howard K. C.

1993-01-01

Using the Canada-France-Hawaii Telescope, we have imaged a complete, flux-limited sample of Einstein Medium Sensitivity Survey BL Lacertae objects in order to study the properties of BL Lac host galaxies and to use quantitative methods to determine the richness of their galaxy cluster environments.

[Determination of arbutin in apple juice concentrate by ultra performance liquid chromatography with electrospray ionization tandem mass spectrometry].

PubMed

Kong, Xianghong; He, Qiang; Yue, Aishan; Wu, Shuangmin; Li, Jianhua

2010-06-01

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS) method was developed for the determination of arbutin in apple juice concentrate. Samples were diluted with water, then cleaned-up with a PS-DVB column. Quantitation was carried out using an external standard method. UPLC was performed on an Eclipse Plus C, column (100 mm x 2.1 mm, 1.8 microm) using a gradient solvent system (methanol-water). MS/MS was performed with multiple reaction monitoring (MRM) mode. The detection limit of arbutin was 0.02 mg/L. The method showed good linear relationship at the range of 0.04-2.0 mg/L. The recoveries ranged from 75.2% to 102.7% with relative standard deviations (RSDs) less than 8.9%. The method is simple, fast and sensitive. It's suitable for quantitative and qualitative analysis of arbutin in apple juice concentrate.

Development of an Analytical Method for the Determination of Amoxicillin in Commercial Drugs and Wastewater Samples, and Assessing its Stability in Simulated Gastric Digestion.

PubMed

Unutkan, Tugçe; Bakirdere, Sezgin; Keyf, Seyfullah

2018-01-01

A highly sensitive analytical HPLC-UV method was developed for the determination of amoxicillin in drugs and wastewater samples at a single wavelength (230 nm). In order to substantially predict the in vivo behavior of amoxicillin, drug samples were subjected to simulated gastric conditions. The calibration plot of the method was linear from 0.050 to 500 mg L-1 with a correlation coefficient of 0.9999. The limit of detection and limit of quantitation were found to be 16 and 54 μg L-1, respectively. The percentage recovery of amoxicillin in wastewater was found to be 97.0 ± 1.6%. The method was successfully applied for the qualitative and quantitative determination of amoxicillin in drug samples including tablets and suspensions. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Highly Sensitive Quantitative PCR for the Detection and Differentiation of Pseudogymnoascus destructans and Other Pseudogymnoascus Species

PubMed Central

Shuey, Megan M.; Drees, Kevin P.; Lindner, Daniel L.; Keim, Paul

2014-01-01

White-nose syndrome is a fungal disease that has decimated bat populations across eastern North America. Identification of the etiologic agent, Pseudogymnoascus destructans (formerly Geomyces destructans), in environmental samples is essential to proposed management plans. A major challenge is the presence of closely related species, which are ubiquitous in many soils and cave sediments and often present in high abundance. We present a dual-probe real-time quantitative PCR assay capable of detecting and differentiating P. destructans from closely related fungi in environmental samples from North America. The assay, based on a single nucleotide polymorphism (SNP) specific to P. destructans, is capable of rapid low-level detection from various sampling media, including sediment, fecal samples, wing biopsy specimens, and skin swabs. This method is a highly sensitive, high-throughput method for identifying P. destructans, other Pseudogymnoascus spp., and Geomyces spp. in the environment, providing a fundamental component of research and risk assessment for addressing this disease, as well as other ecological and mycological work on related fungi. PMID:24375140

Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction.

PubMed

Abdeldaim, Guma M K; Strålin, Kristoffer; Kirsebom, Leif A; Olcén, Per; Blomberg, Jonas; Herrmann, Björn

2009-08-01

A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 10(4) DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

Ultrasensitive Hybridization-Based ELISA Method for the Determination of Phosphorodiamidate Morpholino Oligonucleotides in Biological samples.

PubMed

Burki, Umar; Straub, Volker

2017-01-01

Determining the concentration of oligonucleotide in biological samples such as tissue lysate and serum is essential for determining the biodistribution and pharmacokinetic profile, respectively. ELISA-based assays have shown far greater sensitivities compared to other methods such as HPLC and LC/MS. Here, we describe a novel ultrasensitive hybridization-based ELISA method for quantitating morpholino oligonucleotides in mouse tissue lysate and serum samples. The assay has a linear detection range of 5-250 pM (R2 > 0.99).

Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application

PubMed Central

Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

2011-01-01

Background Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. Methods The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. Conclusions The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. Significance The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food. PMID:21283808

The agony of choice in dermatophyte diagnostics-performance of different molecular tests and culture in the detection of Trichophyton rubrum and Trichophyton interdigitale.

PubMed

Kupsch, C; Ohst, T; Pankewitz, F; Nenoff, P; Uhrlaß, S; Winter, I; Gräser, Y

2016-08-01

Dermatophytosis caused by dermatophytes of the genera Trichophyton and Microsporum belong to the most frequent mycoses worldwide. Molecular detection methods proved to be highly sensitive and enable rapid and accurate detection of dermatophyte species from clinical specimens. For the first time, we compare the performance of different molecular methods with each other and with conventional diagnostics in the detection of dermatophytoses caused by Trichophyton rubrum and Trichophyton interdigitale in clinical specimens (nail, skin and hair). The compared molecular methods comprise two already published PCR-ELISAs, a published quantitative RT-PCR as well as a newly developed PCR-ELISA targeting the internal transcribed spacer region. We investigated the sensitivity of the assays by analysing 375 clinical samples. In 148 specimens (39.5%) a positive result was gained in at least one of the four molecular tests or by culture, but the number of detected agents differed significantly between some of the assays. The most sensitive assay, a PCR-ELISA targeting a microsatellite region, detected 81 T. rubrum infections followed by an internal transcribed spacer PCR-ELISA (60), quantitative RT-PCR (52) and a topoisomerase II PCR-ELISA (51), whereas cultivation resulted in T. rubrum identification in 37 samples. The pros and cons of all four tests in routine diagnostics are discussed. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

An autoanalyzer test for the quantitation of platelet-associated IgG

NASA Technical Reports Server (NTRS)

Levitan, Nathan; Teno, Richard A.; Szymanski, Irma O.

1986-01-01

A new quantitative antiglobulin consumption (QAC) test for the measurement of platelet-associated IgG is described. In this test washed platelets are incubated with anti-IgG at a final dilution of 1:2 million. The unneutralized fraction of anti-IgG remaining in solution is then measured with an Autoanalyzer and soluble IgG is used for calibration. The dose-response curves depicting the percent neutralization of anti-IgG by platelets and by soluble IgG were compared in detail and found to be nearly identical, indicating that platelet-associated IgG can be accurately quantitated by this method. The mean IgG values were 2287 molecules/platelet for normal adults and 38,112 molecules/platelet for ITP patients. The Autoanalyzer QAC test is a sensitive and reproducible assay for the quantitation of platelet-associated IgG.

Recombinant plasmid-based quantitative Real-Time PCR analysis of Salmonella enterica serotypes and its application to milk samples.

PubMed

Gokduman, Kurtulus; Avsaroglu, M Dilek; Cakiris, Aris; Ustek, Duran; Gurakan, G Candan

2016-03-01

The aim of the current study was to develop, a new, rapid, sensitive and quantitative Salmonella detection method using a Real-Time PCR technique based on an inexpensive, easy to produce, convenient and standardized recombinant plasmid positive control. To achieve this, two recombinant plasmids were constructed as reference molecules by cloning the two most commonly used Salmonella-specific target gene regions, invA and ttrRSBC. The more rapid detection enabled by the developed method (21 h) compared to the traditional culture method (90 h) allows the quantitative evaluation of Salmonella (quantification limits of 10(1)CFU/ml and 10(0)CFU/ml for the invA target and the ttrRSBC target, respectively), as illustrated using milk samples. Three advantages illustrated by the current study demonstrate the potential of the newly developed method to be used in routine analyses in the medical, veterinary, food and water/environmental sectors: I--The method provides fast analyses including the simultaneous detection and determination of correct pathogen counts; II--The method is applicable to challenging samples, such as milk; III--The method's positive controls (recombinant plasmids) are reproducible in large quantities without the need to construct new calibration curves. Copyright © 2016 Elsevier B.V. All rights reserved.

Nature of a Red Cell Sensitizing Substance from Streptococci1

PubMed Central

Jackson, Robert W.; Moskowitz, Merwin

1966-01-01

Jackson, Robert W. (Purdue University, Lafayette, Ind.), and Merwin Moskowitz. Nature of a red cell sensitizing substance from streptococci. J. Bacteriol. 91:2205–2209. 1966.—A method for purifying a streptococcal antigen which sensitizes red cells to agglutination by antiserum is described. The antigen, when purified by this method, is almost exclusively composed of glycerophosphate and d-alanine. The ratio of alanine to glycerophosphate varies from 1:5 to 1:3. The glycerophosphate is polymerized and is thus a teichoic acid. The polyglycerophosphate appears to be the antigenic determinant for agglutination. d-Alanine is readily removed by mild base and appears to be necessary for the attachment of the teichoic acid to red cells. Quantitative removal of alanine does not affect the ability of the polymer to absorb antibody from serum. PMID:5329284

Determination of 3D optic axis orientation in cartilage by polarization-sensitive optical coherence tomography

NASA Astrophysics Data System (ADS)

Ugryumova, Nadya; Bonesi, Marco; Matcher, Stephen J.

2008-02-01

Polarization-sensitive optical coherence tomography has been used to solve fast-axis fibre orientation in three dimension space. Previously we have demonstrated that the apparent variations in polar angle orientation of collagen fibers along sagittal ridge of equine third metacarpophalangeal joint exist. A quantitative method based on multiple angles of illumination has been proposed to determine the polar angle of the collagen fibers. This method however ignored the full 3D structure by assuming that the collagen fibers long-axis lay within the plane of incidence. A new quantitative method based on the theory of light propagation in uniaxial materials is described which avoids this assumption. To test this method we have performed control experiments on a sample of equine tendon (this tissue has well defined c-axis lying along the long-axis of the tendon). Several samples of tendon were cut to achieve a planar surface inclined at -20° to the long axis. Additional 30° rotation provided non-zero azimuthal angle. The surface was then imaged using incident beam angles -40°, -20°, 0, +20°, +40° in two orthogonal planes. Values for both the polar and azimuthal angles were then derived using a numerical optimisation procedure. Results agreed qualitatively with the nominal values but suggested that the accuracy was limited by our method of determining the apparent birefringence.

Determination of 3D optic axis orientation in cartilage by polarization-sensitive optical coherence tomography

NASA Astrophysics Data System (ADS)

Ugryumova, Nadya; Matcher, Stephen J.

2007-02-01

Polarization-sensitive optical coherence tomography has been used to solve fast-axis fibre orientation in three dimension space. Previously we have demonstrated that the apparent variations in polar angle orientation of collagen fibers along sagittal ridge of equine third metacarpophalangeal joint exist. A quantitative method based on multiple angles of illumination has been proposed to determine the polar angle of the collagen fibers. This method however ignored the full 3-D structure by assuming that the collagen fibers long-axis lay within the plane of incidence. A new quantitative method based on the theory of light propagation in uniaxial materials is described which avoids this assumption. To test this method we have performed control experiments on a sample of equine tendon (this tissue has well defined c-axis lying along the long-axis of the tendon). Several samples of tendon were cut to achieve a planar surface inclined at -20° to the long axis. Additional 30° rotation provided non-zero azimuthal angle. The surface was then imaged using incident beam angles -40°, -20°, 0, +20°, +40° in two orthogonal planes. Values for both the polar and azimuthal angles were then derived using a numerical optimisation procedure. Results agreed qualitatively with the nominal values but suggested that the accuracy was limited by our method of determining the apparent birefringence.

Quantitation of low molecular weight sugars by chemical derivatization-liquid chromatography/multiple reaction monitoring/mass spectrometry.

PubMed

Han, Jun; Lin, Karen; Sequria, Carita; Yang, Juncong; Borchers, Christoph H

2016-07-01

A new method for the separation and quantitation of 13 mono- and disaccharides has been developed by chemical derivatization/ultra-HPLC/negative-ion ESI-multiple-reaction monitoring MS. 3-Nitrophenylhydrazine (at 50°C for 60 min) was shown to be able to quantitatively derivatize low-molecular weight (LMW) reducing sugars. The nonreducing sugar, sucrose, was not derivatized. A pentafluorophenyl-bonded phase column was used for the chromatographic separation of the derivatized sugars. This method exhibits femtomole-level sensitivity, high precision (CVs of ≤ 4.6%) and high accuracy for the quantitation of LMW sugars in wine. Excellent linearity (R(2) ≥ 0.9993) and linear ranges of ∼500-fold for disaccharides and ∼1000-4000-fold for monosaccharides were achieved. With internal calibration ((13) C-labeled internal standards), recoveries were between 93.6% ± 1.6% (xylose) and 104.8% ± 5.2% (glucose). With external calibration, recoveries ranged from 82.5% ± 0.8% (ribulose) to 105.2% ± 2.1% (xylulose). Quantitation of sugars in two red wines and two white wines was performed using this method; quantitation of the central carbon metabolism-related carboxylic acids and tartaric acid was carried out using a previously established derivatization procedure with 3-nitrophenylhydrazine as well. The results showed that these two classes of compounds-both of which have important organoleptic properties-had different compositions in red and white wines. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative imaging biomarkers: the application of advanced image processing and analysis to clinical and preclinical decision making.

PubMed

Prescott, Jeffrey William

2013-02-01

The importance of medical imaging for clinical decision making has been steadily increasing over the last four decades. Recently, there has also been an emphasis on medical imaging for preclinical decision making, i.e., for use in pharamaceutical and medical device development. There is also a drive towards quantification of imaging findings by using quantitative imaging biomarkers, which can improve sensitivity, specificity, accuracy and reproducibility of imaged characteristics used for diagnostic and therapeutic decisions. An important component of the discovery, characterization, validation and application of quantitative imaging biomarkers is the extraction of information and meaning from images through image processing and subsequent analysis. However, many advanced image processing and analysis methods are not applied directly to questions of clinical interest, i.e., for diagnostic and therapeutic decision making, which is a consideration that should be closely linked to the development of such algorithms. This article is meant to address these concerns. First, quantitative imaging biomarkers are introduced by providing definitions and concepts. Then, potential applications of advanced image processing and analysis to areas of quantitative imaging biomarker research are described; specifically, research into osteoarthritis (OA), Alzheimer's disease (AD) and cancer is presented. Then, challenges in quantitative imaging biomarker research are discussed. Finally, a conceptual framework for integrating clinical and preclinical considerations into the development of quantitative imaging biomarkers and their computer-assisted methods of extraction is presented.

A simplified implementation of edge detection in MATLAB is faster and more sensitive than fast fourier transform for actin fiber alignment quantification.

PubMed

Kemeny, Steven Frank; Clyne, Alisa Morss

2011-04-01

Fiber alignment plays a critical role in the structure and function of cells and tissues. While fiber alignment quantification is important to experimental analysis and several different methods for quantifying fiber alignment exist, many studies focus on qualitative rather than quantitative analysis perhaps due to the complexity of current fiber alignment methods. Speed and sensitivity were compared in edge detection and fast Fourier transform (FFT) for measuring actin fiber alignment in cells exposed to shear stress. While edge detection using matrix multiplication was consistently more sensitive than FFT, image processing time was significantly longer. However, when MATLAB functions were used to implement edge detection, MATLAB's efficient element-by-element calculations and fast filtering techniques reduced computation cost 100 times compared to the matrix multiplication edge detection method. The new computation time was comparable to the FFT method, and MATLAB edge detection produced well-distributed fiber angle distributions that statistically distinguished aligned and unaligned fibers in half as many sample images. When the FFT sensitivity was improved by dividing images into smaller subsections, processing time grew larger than the time required for MATLAB edge detection. Implementation of edge detection in MATLAB is simpler, faster, and more sensitive than FFT for fiber alignment quantification.

The LLNA: A Brief Review of Recent Advances and Limitations

PubMed Central

Anderson, Stacey E.; Siegel, Paul D.; Meade, B. J.

2011-01-01

Allergic contact dermatitis is the second most commonly reported occupational illness, accounting for 10% to 15% of all occupational diseases. This highlights the importance of developing rapid and sensitive methods for hazard identification of chemical sensitizers. The murine local lymph node assay (LLNA) was developed and validated for the identification of low molecular weight sensitizing chemicals. It provides several benefits over other tests for sensitization because it provides a quantitative endpoint, dose-responsive data, and allows for prediction of potency. However, there are also several concerns with this assay including: levels of false positive responses, variability due to vehicle, and predictivity. This report serves as a concise review which briefly summarizes the progress, advances and limitations of the assay over the last decade. PMID:21747867

Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.

PubMed

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-04-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis.

Printing 2-Dimentional Droplet Array for Single-Cell Reverse Transcription Quantitative PCR Assay with a Microfluidic Robot

PubMed Central

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-01-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis. PMID:25828383

Study on the Multi-marker Components Quantitative HPLC Fingerprint of the Compound Chinese Medicine Wuwei Changyanning Granule

PubMed Central

Yang, Xian; Yang, Shui-Ping; Zhang, Xue; Yu, Xiao-Dong; He, Qi-Yi; Wang, Bo-Chu

2014-01-01

The aim of this paper is to develop a rapid and highly sensitive quantitative HPLC fingerprint method with multiple indicators by using the Compound Chinese Medicine Wuwei Changyanning granule and 5 herbs in the prescription. The quantitative fingerprint chromatogram with multiple indicators was investigated. і)6 compositions included rutin, gallic acid, chlorogenic acid, atractylenolide Ⅰ, pachymic acid and apigenin, which originated from 5 herbs respectively, were selected as quantitative compositions, and their contents were determined using HPLC from 11 batches granules and the corresponding 5 medicinal materials. ⅱ) The precision, stability and repeatability of fingerprinting were investigated. In addition, common peaks number, the percentage of non-common peaks and similarity were also studied. Among them, 21 common peaks in the granule could find the source of peaks from the 5 herbs, among of 10 peaks from Niuerfeng, 9 peaks from Laliao, 3 peaks from Baishu, 3 peaks from Fuling and 5 peaks from Guanghuoxiang. The results showed that the identification method of fingerprinting was reliable. PMID:25587307

Graphene oxide-coated stir bar sorptive extraction of trace aflatoxins from soy milk followed by high performance liquid chromatography-laser-induced fluorescence detection.

PubMed

Ma, Haiyan; Ran, Congcong; Li, Mengjiao; Gao, Jinglin; Wang, Xinyu; Zhang, Lina; Bian, Jing; Li, Junmei; Jiang, Ye

2018-04-01

Mycotoxins are potential food pollutants produced by fungi. Among them, aflatoxins (AFs) are the most toxic. Therefore, AFs were selected as models, and a sensitive, simple and green graphene oxide (GO)-based stir bar sorptive extraction (SBSE) method was developed for extraction and determination of AFs with high performance liquid chromatography-laser-induced fluorescence detector (HPLC-LIF). This method improved the sensitivity of AFs detection and solved the deposition difficulty of the direct use of GO as adsorbent. Several parameters including a spiked amount of NaCl, stirring rate, extraction time and desorption time were investigated. Under optimal conditions, the quantitative method had low limits of detection of 2.4-8.0 pg/mL, which were better than some reported AFs analytical methods. The developed method has been applied to soy milk samples with good recoveries ranging from 80.5 to 102.3%. The prepared GO-based SBSE can be used as a sensitive screening technique for detecting AFs in soy milk.

Differential diagnosis of solitary pulmonary nodules based on 99mTc-EDDA/HYNIC-TOC scintigraphy: the effect of tumour size on the optimal method of image assessment.

PubMed

Płachcińska, Anna; Mikołajczak, Renata; Kozak, Józef; Rzeszutek, Katarzyna; Kuśmierek, Jacek

2006-09-01

The aim of the study was to determine an optimal method for the evaluation of scintigrams obtained with (99m)Tc-EDDA/HYNIC-TOC for the purpose of differential diagnosis of solitary pulmonary nodules (SPNs) and to assess the diagnostic value of the method. Eighty-five patients (48 males and 37 females, mean age 57 years, range 34-78 years) were enrolled in the study. Patients underwent (99m)Tc-EDDA/HYNIC-TOC scintigraphy for the purpose of differential diagnosis of SPNs (size between 1 and 4 cm). Images of all patients were evaluated visually in a prospective manner. Positive scintigraphic results were found in 37 out of 40 (93%) patients with malignant SPNs including 34 out of 35 (97%) patients with primary lung carcinoma. Two remaining false negative cases turned out to be metastatic lesions of malignant melanoma and leiomyosarcoma. Among 45 benign tumours, negative results were obtained in 31 cases (69%) and positive results in 14. The accuracy of the method was 80%. Analysis of the results of the visual assessment of scintigrams revealed a significantly higher frequency of false positive results among larger nodules (diameter at least 1.4 cm). Uptake of the tracer in those nodules was therefore assessed semi-quantitatively (using the tumour-to-background ratio), in expectation of an improvement in the low specificity of the visual method. The semi-quantitative assessment reduced the total number of false positive results in a subgroup of larger nodules from 13 to six, while preserving the high sensitivity of the method. The combination of visual analysis (for lesions smaller than 1.4 cm in diameter) and semi-quantitative assessment (for larger lesions) provided a high sensitivity of the method and significantly improved its specificity (84%) and accuracy (88%) in comparison with visual analysis (p<0.05).

[The role of endotracheal aspirate culture in the diagnosis of ventilator-associated pneumonia: a meta analysis].

PubMed

Wang, Fei; He, Bei

2013-01-01

To investigate the role of endotracheal aspirate (EA) culture in the diagnosis and antibiotic management in ventilator-associated pneumonia (VAP). We searched CNKI, Wanfang, PUBMED and EMBASE databases published from January 1990 to December 2011, to find relevant literatures on VAP microbiological diagnostic techniques including EA and bronchoalveolar lavage (BALF). The following key words were used: ventilator associated pneumonia, diagnosis and adult. Meta-analysis was performed and the sensitivity and specificity of EA on VAP diagnosis were calculated. Our literature search identified 1665 potential articles, 8 of which fulfilled our selection criteria including 561 patients with paired cultures. Using BALF quantitative culture as reference standard, the sensitivity and specificity of EA were 72% and 71%. When considering quantitative culture of EA only, the sensitivity and specificity improved to 90% and 65%, while the positive and the negative predictive values were 68% and 89% respectively. However, the sensitivity and specificity of semi-quantitative culture of EA were only 50% and 80%, with a positive predictive value of 77% and a negative predictive value of 58% respectively. EA culture had relatively poor sensitivity and specificity, although quantitative culture of EA only could improve the sensitivity. Initiating therapy on the basis of EA quantitative culture may still result in excessive antibiotic usage. Our data suggested that EA could provide some information for clinical decision but could not replace the role of BALF quantitative culture in VAP diagnosis.

Measurement of sulfur-containing compounds involved in the metabolism and transport of cysteamine and cystamine. Regional differences in cerebral metabolism☆

PubMed Central

Pinto, John T.; Khomenko, Tetyana; Szabo, Sandor; McLaren, Gordon D.; Denton, Travis T.; Krasnikov, Boris F.; Jeitner, Thomas M.; Cooper, Arthur J.L.

2009-01-01

An HPLC method with coulometric detection is presented for the quantitation of cysteamine, cystamine, thialysine, glutathione, glutathione disulfide and an oxidized metabolite of thialysine [S-(2-aminoethyl)-l-cysteine ketimine decarboxylated dimer (AECK-DD)]. The advantage of coulometric detection is that derivatization is unnecessary if the analyte is redox sensitive. The method was used to quantitate several sulfur-containing compounds in plasma and brain following gavage feeding of cysteamine to rats. Cysteamine, cystamine, thialysine and AECK-DD were detected in the brains of these animals. Interestingly, cysteamine treatment resulted in greatly elevated levels of cerebral methionine, despite the fact that cysteamine is not a precursor of methionine. PMID:19523884

Optimized and validated flow-injection spectrophotometric analysis of topiramate, piracetam and levetiracetam in pharmaceutical formulations.

PubMed

Hadad, Ghada M; Abdel-Salam, Randa A; Emara, Samy

2011-12-01

Application of a sensitive and rapid flow injection analysis (FIA) method for determination of topiramate, piracetam, and levetiracetam in pharmaceutical formulations has been investigated. The method is based on the reaction with ortho-phtalaldehyde and 2-mercaptoethanol in a basic buffer and measurement of absorbance at 295 nm under flow conditions. Variables affecting the determination such as sample injection volume, pH, ionic strength, reagent concentrations, flow rate of reagent and other FIA parameters were optimized to produce the most sensitive and reproducible results using a quarter-fraction factorial design, for five factors at two levels. Also, the method has been optimized and fully validated in terms of linearity and range, limit of detection and quantitation, precision, selectivity and accuracy. The method was successfully applied to the analysis of pharmaceutical preparations.

A novel approach for the quantitation of carbohydrates in mash, wort, and beer with RP-HPLC using 1-naphthylamine for precolumn derivatization.

PubMed

Rakete, Stefan; Glomb, Marcus A

2013-04-24

A novel universal method for the determination of reducing mono-, di-, and oligosaccharides in complex matrices on RP-HPLC using 1-naphthylamine for precolumn derivatization with sodium cyanoborhydride was established to study changes in the carbohydrate profile during beer brewing. Fluorescence and mass spectrometric detection enabled very sensitive analyses of beer-relevant carbohydrates. Mass spectrometry additionally allowed the identification of the molecular weight and thereby the degree of polymerization of unknown carbohydrates. Thus, carbohydrates with up to 16 glucose units were detected. Comparison demonstrated that the novel method was superior to fluorophore-assisted carbohydrate electrophoresis (FACE). The results proved the HPLC method clearly to be more powerful in regard to sensitivity and resolution. Analogous to FACE, this method was designated fluorophore-assisted carbohydrate HPLC (FAC-HPLC).

Combined visual and semi-quantitative assessment of 123I-FP-CIT SPECT for the diagnosis of dopaminergic neurodegenerative diseases.

PubMed

Ueda, Jun; Yoshimura, Hajime; Shimizu, Keiji; Hino, Megumu; Kohara, Nobuo

2017-07-01

Visual and semi-quantitative assessments of 123 I-FP-CIT single-photon emission computed tomography (SPECT) are useful for the diagnosis of dopaminergic neurodegenerative diseases (dNDD), including Parkinson's disease, dementia with Lewy bodies, progressive supranuclear palsy, multiple system atrophy, and corticobasal degeneration. However, the diagnostic value of combined visual and semi-quantitative assessment in dNDD remains unclear. Among 239 consecutive patients with a newly diagnosed possible parkinsonian syndrome who underwent 123 I-FP-CIT SPECT in our medical center, 114 patients with a disease duration less than 7 years were diagnosed as dNDD with the established criteria or as non-dNDD according to clinical judgment. We retrospectively examined their clinical characteristics and visual and semi-quantitative assessments of 123 I-FP-CIT SPECT. The striatal binding ratio (SBR) was used as a semi-quantitative measure of 123 I-FP-CIT SPECT. We calculated the sensitivity and specificity of visual assessment alone, semi-quantitative assessment alone, and combined visual and semi-quantitative assessment for the diagnosis of dNDD. SBR was correlated with visual assessment. Some dNDD patients with a normal visual assessment had an abnormal SBR, and vice versa. There was no statistically significant difference between sensitivity of the diagnosis with visual assessment alone and semi-quantitative assessment alone (91.2 vs. 86.8%, respectively, p = 0.29). Combined visual and semi-quantitative assessment demonstrated superior sensitivity (96.7%) to visual assessment (p = 0.03) or semi-quantitative assessment (p = 0.003) alone with equal specificity. Visual and semi-quantitative assessments of 123 I-FP-CIT SPECT are helpful for the diagnosis of dNDD, and combined visual and semi-quantitative assessment shows superior sensitivity with equal specificity.

The 'sniffer-patch' technique for detection of neurotransmitter release.

PubMed

Allen, T G

1997-05-01

A wide variety of techniques have been employed for the detection and measurement of neurotransmitter release from biological preparations. Whilst many of these methods offer impressive levels of sensitivity, few are able to combine sensitivity with the necessary temporal and spatial resolution required to study quantal release from single cells. One detection method that is seeing a revival of interest and has the potential to fill this niche is the so-called 'sniffer-patch' technique. In this article, specific examples of the practical aspects of using this technique are discussed along with the procedures involved in calibrating these biosensors to extend their applications to provide quantitative, in addition to simple qualitative, measurements of quantal transmitter release.

Determination of capsaicinoids in topical cream by liquid-liquid extraction and liquid chromatography.

PubMed

Kaale, Eliangiringa; Van Schepdael, Ann; Roets, Eugène; Hoogmartens, Jos

2002-11-07

A reversed-phase liquid chromatography (LC) method has been developed, optimised and validated for the separation and quantitation of capsaicin (CP) and dihydrocapsaicin (DHCP) in a topical cream formulation. Sample preparation involves liquid-liquid extraction prior to LC analysis. The method uses a Hypersil C(18) BDS, 5 micrometer, 250x4.6 mm I.D. column maintained at 35 degrees C. The mobile phase comprises methanol, water, acetonitrile (ACN) and acetic acid (47:42:10:1, v/v/v/v) at a flow rate of 1.0 ml/min. Robustness was evaluated by performing a central composite face-centred design (CCF) experiment. The method shows good selectivity, linearity, sensitivity and repeatability. The conditions allow the separation and quantitation of CP and DHCP without interference from the other substances contained in the cream.

A Ratiometric Threshold for Determining Presence of Cancer During Fluorescence-guided Surgery

PubMed Central

Warram, Jason M; de Boer, Esther; Moore, Lindsay S.; Schmalbach, Cecelia E; Withrow, Kirk P; Carroll, William R; Richman, Joshua S; Morlandt, Anthony B; Brandwein-Gensler, Margaret; Rosenthal, Eben L

2015-01-01

Background&Objective Fluorescence-guided imaging to assist in identification of malignant margins has the potential to dramatically improve oncologic surgery. However a standardized method for quantitative assessment of disease-specific fluorescence has not been investigated. Introduced here is a ratiometric threshold derived from mean fluorescent tissue intensity that can be used to semi-quantitatively delineate tumor from normal tissue. Methods Open-field and a closed-field imaging devices were used to quantify fluorescence in punch biopsy tissues sampled from primary tumors collected during a phase 1 trial evaluating the safety of cetuximab-IRDye800 in patients (n=11) undergoing surgical intervention for head and neck cancer. Fluorescence ratios were calculated using mean fluorescence intensity (MFI) from punch biopsy normalized by MFI of patient-matched tissues. Ratios were compared to pathological assessment and a ratiometric threshold was established to predict presence of cancer. Results During open-field imaging using an intraoperative device, the threshold for muscle normalized tumor fluorescence was found to be 2.7, which produced a sensitivity of 90.5% and specificity of 78.6% for delineating disease tissue. The skin-normalized threshold generated greater sensitivity (92.9%) and specificity (81.0%). Conclusion Successful implementation of a semi-quantitative threshold can provide a scientific methodology for delineating disease from normal tissue during fluorescence-guided resection of cancer. PMID:26074273

Characterization of Cerebral White Matter Properties Using Quantitative Magnetic Resonance Imaging Stains

PubMed Central

Hurley, Samuel A.; Samsonov, Alexey A.; Adluru, Nagesh; Hosseinbor, Ameer Pasha; Mossahebi, Pouria; Tromp, Do P.M.; Zakszewski, Elizabeth; Field, Aaron S.

2011-01-01

Abstract The image contrast in magnetic resonance imaging (MRI) is highly sensitive to several mechanisms that are modulated by the properties of the tissue environment. The degree and type of contrast weighting may be viewed as image filters that accentuate specific tissue properties. Maps of quantitative measures of these mechanisms, akin to microstructural/environmental-specific tissue stains, may be generated to characterize the MRI and physiological properties of biological tissues. In this article, three quantitative MRI (qMRI) methods for characterizing white matter (WM) microstructural properties are reviewed. All of these measures measure complementary aspects of how water interacts with the tissue environment. Diffusion MRI, including diffusion tensor imaging, characterizes the diffusion of water in the tissues and is sensitive to the microstructural density, spacing, and orientational organization of tissue membranes, including myelin. Magnetization transfer imaging characterizes the amount and degree of magnetization exchange between free water and macromolecules like proteins found in the myelin bilayers. Relaxometry measures the MRI relaxation constants T1 and T2, which in WM have a component associated with the water trapped in the myelin bilayers. The conduction of signals between distant brain regions occurs primarily through myelinated WM tracts; thus, these methods are potential indicators of pathology and structural connectivity in the brain. This article provides an overview of the qMRI stain mechanisms, acquisition and analysis strategies, and applications for these qMRI stains. PMID:22432902

A novel quantified bitterness evaluation model for traditional Chinese herbs based on an animal ethology principle.

PubMed

Han, Xue; Jiang, Hong; Han, Li; Xiong, Xi; He, Yanan; Fu, Chaomei; Xu, Runchun; Zhang, Dingkun; Lin, Junzhi; Yang, Ming

2018-03-01

Traditional Chinese herbs (TCH) are currently gaining attention in disease prevention and health care plans. However, their general bitter taste hinders their use. Despite the development of a variety of taste evaluation methods, it is still a major challenge to establish a quantitative detection technique that is objective, authentic and sensitive. Based on the two-bottle preference test (TBP), we proposed a novel quantitative strategy using a standardized animal test and a unified quantitative benchmark. To reduce the difference of results, the methodology of TBP was optimized. The relationship between the concentration of quinine and animal preference index (PI) was obtained. Then the PI of TCH was measured through TBP, and bitterness results were converted into a unified numerical system using the relationship of concentration and PI. To verify the authenticity and sensitivity of quantified results, human sensory testing and electronic tongue testing were applied. The quantified results showed a good discrimination ability. For example, the bitterness of Coptidis Rhizoma was equal to 0.0579 mg/mL quinine, and Nelumbinis Folium was equal to 0.0001 mg/mL. The validation results proved that the new assessment method for TCH was objective and reliable. In conclusion, this study provides an option for the quantification of bitterness and the evaluation of taste masking effects.

Quantitative detection of type A staphylococcal enterotoxin by Laurell electroimmunodiffusion.

PubMed

Gasper, E; Heimsch, R C; Anderson, A W

1973-03-01

The detection of staphylococcal enterotoxin A by the quantitative technique of electroimmunodiffusion is described. High dilutions of type-specific rabbit antiserum were used in 1% agarose gels, 1 mm thick, and prepared in 0.05-mug barbital buffer, pH 8.6. Volumes of 10 muliters containing 1.5 to 10 ng of toxin were electrophoresed out of 4-mm diameter wells at 5 mA/cm width of gel. The precipitin cones formed were made visible by first immersing the agarose gels in 0.2 M NaCl and then overlaying the surface with the purified globulin fraction of sheep serum against rabbit globulin, followed by soaking of the gels in 1% aqueous cadmium acetate and staining with 0.1% thiazine red in 1% glacial acetic acid. Fully extended cones, 4 to 23 mm in length depending on toxin concentration and antiserum dilution, were developed in 2 to 5 h of electrophoresis, and visualization was achieved within 2 to 3 h. Because the method is qualitative, quantitative, simple, rapid, and sensitive, it offers a practical tool for the detection of small amounts of bacterial toxins in contaminated foods. The method should also qualify as a sensitive detection device in biochemical procedures which attempt to trace, detect, and identify biological substances in nanogram quantities, provided these substances are antigenic and capable of forming a precipitate with their specific antibodies.

A new method to calibrate the absolute sensitivity of a soft X-ray streak camera

NASA Astrophysics Data System (ADS)

Yu, Jian; Liu, Shenye; Li, Jin; Yang, Zhiwen; Chen, Ming; Guo, Luting; Yao, Li; Xiao, Shali

2016-12-01

In this paper, we introduce a new method to calibrate the absolute sensitivity of a soft X-ray streak camera (SXRSC). The calibrations are done in the static mode by using a small laser-produced X-ray source. A calibrated X-ray CCD is used as a secondary standard detector to monitor the X-ray source intensity. In addition, two sets of holographic flat-field grating spectrometers are chosen as the spectral discrimination systems of the SXRSC and the X-ray CCD. The absolute sensitivity of the SXRSC is obtained by comparing the signal counts of the SXRSC to the output counts of the X-ray CCD. Results show that the calibrated spectrum covers the range from 200 eV to 1040 eV. The change of the absolute sensitivity in the vicinity of the K-edge of the carbon can also be clearly seen. The experimental values agree with the calculated values to within 29% error. Compared with previous calibration methods, the proposed method has several advantages: a wide spectral range, high accuracy, and simple data processing. Our calibration results can be used to make quantitative X-ray flux measurements in laser fusion research.

Validated reversed phase LC method for quantitative analysis of polymethoxyflavones in citrus peel extracts.

PubMed

Wang, Zhenyu; Li, Shiming; Ferguson, Stephen; Goodnow, Robert; Ho, Chi-Tang

2008-01-01

Polymethoxyflavones (PMFs), which exist exclusively in the citrus genus, have biological activities including anti-inflammatory, anticarcinogenic, and antiatherogenic properties. A validated RPLC method was developed for quantitative analysis of six major PMFs, namely nobiletin, tangeretin, sinensetin, 5,6,7,4'-tetramethoxyflavone, 3,5,6,7,3',4'-hexamethoxyflavone, and 3,5,6,7,8,3',4'-heptamethoxyflavone. The polar embedded LC stationary phase was able to fully resolve the six analogues. The developed method was fully validated in terms of linearity, accuracy, precision, sensitivity, and system suitability. The LOD of the method was calculated as 0.15 microg/mL and the recovery rate was between 97.0 and 105.1%. This analytical method was successfully applied to quantify the individual PMFs in four commercially available citrus peel extracts (CPEs). Each extract shows significant difference in the PMF composition and concentration. This method may provide a simple, rapid, and reliable tool to help reveal the correlation between the bioactivity of the PMF extracts and the individual PMF content.

Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders

PubMed Central

Marquet, Pierre; Depeursinge, Christian; Magistretti, Pierre J.

2014-01-01

Abstract. Quantitative phase microscopy (QPM) has recently emerged as a new powerful quantitative imaging technique well suited to noninvasively explore a transparent specimen with a nanometric axial sensitivity. In this review, we expose the recent developments of quantitative phase-digital holographic microscopy (QP-DHM). Quantitative phase-digital holographic microscopy (QP-DHM) represents an important and efficient quantitative phase method to explore cell structure and dynamics. In a second part, the most relevant QPM applications in the field of cell biology are summarized. A particular emphasis is placed on the original biological information, which can be derived from the quantitative phase signal. In a third part, recent applications obtained, with QP-DHM in the field of cellular neuroscience, namely the possibility to optically resolve neuronal network activity and spine dynamics, are presented. Furthermore, potential applications of QPM related to psychiatry through the identification of new and original cell biomarkers that, when combined with a range of other biomarkers, could significantly contribute to the determination of high risk developmental trajectories for psychiatric disorders, are discussed. PMID:26157976

Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders.

PubMed

Marquet, Pierre; Depeursinge, Christian; Magistretti, Pierre J

2014-10-01

Quantitative phase microscopy (QPM) has recently emerged as a new powerful quantitative imaging technique well suited to noninvasively explore a transparent specimen with a nanometric axial sensitivity. In this review, we expose the recent developments of quantitative phase-digital holographic microscopy (QP-DHM). Quantitative phase-digital holographic microscopy (QP-DHM) represents an important and efficient quantitative phase method to explore cell structure and dynamics. In a second part, the most relevant QPM applications in the field of cell biology are summarized. A particular emphasis is placed on the original biological information, which can be derived from the quantitative phase signal. In a third part, recent applications obtained, with QP-DHM in the field of cellular neuroscience, namely the possibility to optically resolve neuronal network activity and spine dynamics, are presented. Furthermore, potential applications of QPM related to psychiatry through the identification of new and original cell biomarkers that, when combined with a range of other biomarkers, could significantly contribute to the determination of high risk developmental trajectories for psychiatric disorders, are discussed.

Determination of ametryn herbicide by bioassay and gas chromatography-mass spectrometry in analysis of residues in drinking water.

PubMed

Queiroz, R H; Lanchote, V L; Bonato, P S; Tozato, E; de Carvalho, D; Gomes, M A; Cerdeira, A L

1999-06-01

A simple, rapid and quantitative bioassay method was compared to a gas chromatography/mass spectrometry (GC/MS) procedure for the analysis of ametryn in surface and groundwater. This method was based on the activity of ametryn in inhibiting the growth of the primary root and shoot of germinating letuce, Lactuca sativa L. seed. The procedure was sensitive to 0.01 microgram/l and was applicable from this concentration up to 0.6 microgram/l. Initial surface sterilization of the seed, selection of pregerminated seed of certain root lengths and special equipment are not necessary. So, we concluded that the sensitivity of the bioassay method is compatible with the chromatographic method (GC-MS). However, the study of the correlation between methods suggests that the bioassay should be used only as a screening technique for the evaluation of ametryn residues in water.

Simultaneous detection and quantitation of organic impurities in methamphetamine by ultra-high-performance liquid chromatography-tandem mass spectrometry, a complementary technique for methamphetamine profiling.

PubMed

Li, Li; Brown, Jaclyn L; Toske, Steven G

2018-04-06

The analysis of organic impurities plays an important role in the impurity profiling of methamphetamine, which in turn provides valuable information about methamphetamine manufacturing, in particular its synthetic route, chemicals, and precursors used. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) is ideally suited for this purpose due to its excellent sensitivity, selectivity, and wide linear range in multiple reaction monitoring (MRM) mode. In this study, a dilute-and-shoot UHPLC-MS/MS method was developed for the simultaneous identification and quantitation of 23 organic manufacturing impurities in illicit methamphetamine. The developed method was validated in terms of stability, limit of detection (LOD), lower limit of quantification (LLOQ), accuracy, and precision. More than 100 illicitly prepared methamphetamine samples were analyzed. Due to its ability to detect ephedrine/pseudoephedrine and its high sensitivity for critical target markers (eg, chloro-pseudoephedrine, N-cyclohexylamphetamine, and compounds B and P), more impurities and precursor/pre-precursors were identified and quantified versus the current procedure by gas chromatography-mass spectrometry (GC-MS). Consequently, more samples could be classified by their synthetic routes. However, the UHPLC-MS/MS method has difficulty in detecting neutral and untargeted emerging manufacturing impurities and can therefore only serve as a complement to the current method. Despite this deficiency, the quantitative information acquired by the presented UHPLC-MS/MS methodology increased the sample discrimination power, thereby enhancing the capacity of methamphetamine profiling program (MPP) to conduct sample-sample comparisons. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Quantifying spontaneous metastasis in a syngeneic mouse melanoma model using real time PCR.

PubMed

Deng, Wentao; McLaughlin, Sarah L; Klinke, David J

2017-08-07

Modeling metastasis in vivo with animals is a priority for both revealing mechanisms of tumor dissemination and developing therapeutic methods. While conventional intravenous injection of tumor cells provides an efficient and consistent system for studying tumor cell extravasation and colonization, studying spontaneous metastasis derived from orthotopic tumor sites has the advantage of modeling more aspects of the metastatic cascade, but is challenging as it is difficult to detect small numbers of metastatic cells. In this work, we developed an approach for quantifying spontaneous metastasis in the syngeneic mouse B16 system using real time PCR. We first transduced B16 cells with lentivirus expressing firefly luciferase Luc2 gene for bioluminescence imaging. Next, we developed a real time quantitative PCR (qPCR) method for the detection of luciferase-expressing, metastatic tumor cells in mouse lungs and other organs. To illustrate the approach, we quantified lung metastasis in both spontaneous and experimental scenarios using B16F0 and B16F10 cells in C57BL/6Ncrl and NOD-Scid Gamma (NSG) mice. We tracked B16 melanoma metastasis with both bioluminescence imaging and qPCR, which were found to be self-consistent. Using this assay, we can quantitatively detect one Luc2 positive tumor cell out of 10 4 tissue cells, which corresponds to a metastatic burden of 1.8 × 10 4 metastatic cells per whole mouse lung. More importantly, the qPCR method was at least a factor of 10 more sensitive in detecting metastatic cell dissemination and should be combined with bioluminescence imaging as a high-resolution, end-point method for final metastatic cell quantitation. Given the rapid growth of primary tumors in many mouse models, assays with improved sensitivity can provide better insight into biological mechanisms that underpin tumor metastasis.

An improved method to measure nitrate/nitrite with an NO-selective electrochemical sensor

PubMed Central

Boo, Yong Chool; Tressel, Sarah L.; Jo, Hanjoong

2007-01-01

Nitric oxide produced from nitric oxide synthase(s) is an important cell signaling molecule in physiology and pathophysiology. In the present study, we describe a very sensitive and convenient analytical method to measure NOx (nitrite plus nitrate) in culture media by employing an ultra-sensitive nitric oxide-selective electrochemical sensor which became commercially available recently. An aliquot of conditioned culture media was first treated with nitrate reductase/NADPH/glucose-6-phosphate dehydrogenase/glucose-6-phosphate to convert nitrate to nitrite quantitatively. The nitrite (that is present originally plus the reduced nitrate) was then reduced to equimolar NO in an acidic iodide bath while NO was being detected by the sensor. This analytical method appears to be very useful to assess basal and stimulated NO release from cultured cells. PMID:17056288

Molecular sensitivity threshold of wet mount and an immunochromatographic assay evaluated by quantitative real-time PCR for diagnosis of Trichomonas vaginalis infection in a low-risk population of childbearing women.

PubMed

Leli, Christian; Castronari, Roberto; Levorato, Lucia; Luciano, Eugenio; Pistoni, Eleonora; Perito, Stefano; Bozza, Silvia; Mencacci, Antonella

2016-06-01

Vaginal trichomoniasis is a sexually transmitted infection caused by Trichomonas vaginalis, a flagellated protozoan. Diagnosis of T. vaginalis infection is mainly performed by wet mount microscopy, with a sensitivity ranging from 38% to 82%, compared to culture, still considered the gold standard. Commercial immunochromatographic tests for monoclonal-antibody-based detection have been introduced as alternative methods for diagnosis of T. vaginalis infection and have been reported in some studies to be more sensitive than wet mount. Real-time PCR methods have been recently developed, with optimal sensitivity and specificity. The aim of this study was to evaluate whether there is a molecular sensitivity threshold for both wet mount and imunochromatographic assays. To this aim, a total of 1487 low-risk childbearing women (median age 32 years, interquartile range 27-37) were included in the study, and underwent vaginal swab for T. vaginalis detection by means of a quantitative real-time PCR assay, wet mount and an immunochromatographic test. Upon comparing the results, prevalence values observed were 1.3% for real-time PCR, 0.5% for microscopic examination, and 0.8% for the immunochromatographic test. Compared to real-time PCR, wet mount sensitivity was 40% (95% confidence interval 19.1% to 63.9%) and specificity was 100% (95% CI 99.7% to 100%). The sensitivity and specificity of the immunochromatographic assay were 57.9% (95% CI 33.5% to 79.8%) and 99.9% (95% CI 99.6% to 100%), respectively. Evaluation of the wet mount results and those of immunochromatographic assay detection in relation to the number of T. vaginalis DNA copies detected in vaginal samples showed that the lower identification threshold for both wet mount (chi-square 6.1; P = 0.016) and the immunochromatographic assay (chi-square 10.7; P = 0.002) was ≥100 copies of T. vaginalis DNA/5 mcl of eluted DNA.

Hepatitis C Virus RNA Real-Time Quantitative RT-PCR Method Based on a New Primer Design Strategy.

PubMed

Chen, Lida; Li, Wenli; Zhang, Kuo; Zhang, Rui; Lu, Tian; Hao, Mingju; Jia, Tingting; Sun, Yu; Lin, Guigao; Wang, Lunan; Li, Jinming

2016-01-01

Viral nucleic acids are unstable when improperly collected, handled, and stored, resulting in decreased sensitivity of currently available commercial quantitative nucleic acid testing kits. Using known unstable hepatitis C virus RNA, we developed a quantitative RT-PCR method based on a new primer design strategy to reduce the impact of nucleic acid instability on nucleic acid testing. The performance of the method was evaluated for linearity, limit of detection, precision, specificity, and agreement with commercial hepatitis C virus assays. Its clinical application was compared to that of two commercial kits--Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) and Kehua. The quantitative RT-PCR method delivered a good performance, with a linearity of R(2) = 0.99, a total limit of detection (genotypes 1 to 6) of 42.6 IU/mL (95% CI, 32.84 to 67.76 IU/mL), a CV of 1.06% to 3.34%, a specificity of 100%, and a high concordance with the CAP/CTM assay (R(2) = 0.97), with a means ± SD value of -0.06 ± 1.96 log IU/mL (range, -0.38 to 0.25 log IU/mL). The method was superior to commercial assays in detecting unstable hepatitis C virus RNA (P < 0.05). This quantitative RT-PCR method can effectively eliminate the influence of RNA instability on nucleic acid testing. The principle of primer design strategy may be applied to the detection of other RNA or DNA viruses. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Quantitative analysis of [Dmt(1)]DALDA in ovine plasma by capillary liquid chromatography-nanospray ion-trap mass spectrometry.

PubMed

Wan, Haibao; Umstot, Edward S; Szeto, Hazel H; Schiller, Peter W; Desiderio, Dominic M

2004-04-15

The synthetic opioid peptide analog Dmt-D-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA; [Dmt= 2',6'-dimethyltyrosine) is a highly potent and selective mu opioid-receptor agonist. A very sensitive and robust capillary liquid chromatography/nanospray ion-trap (IT) mass spectrometry method has been developed to quantify [Dmt(1)]DALDA in ovine plasma, using deuterated [Dmt(1)]DALDA as the internal standard. The standard MS/MS spectra of d(0)- and d(5)-[Dmt(1)]DALDA were obtained, and the collision energy was experimentally optimized to 25%. The product ion [ M + 2H-NH(3)](2+) (m/z 312.2) was used to identify and to quantify the synthetic opioid peptide analog in ovine plasma samples. The MS/MS detection sensitivity for [Dmt(1)]DALDA was 625 amol. A calibration curve was constructed, and quantitative analysis was performed on a series of ovine plasma samples.

Micro-machined calorimetric biosensors

DOEpatents

Doktycz, Mitchel J.; Britton, Jr., Charles L.; Smith, Stephen F.; Oden, Patrick I.; Bryan, William L.; Moore, James A.; Thundat, Thomas G.; Warmack, Robert J.

2002-01-01

A method and apparatus are provided for detecting and monitoring micro-volumetric enthalpic changes caused by molecular reactions. Micro-machining techniques are used to create very small thermally isolated masses incorporating temperature-sensitive circuitry. The thermally isolated masses are provided with a molecular layer or coating, and the temperature-sensitive circuitry provides an indication when the molecules of the coating are involved in an enthalpic reaction. The thermally isolated masses may be provided singly or in arrays and, in the latter case, the molecular coatings may differ to provide qualitative and/or quantitative assays of a substance.

Quantitative evaluation of skeletal muscle defects in second harmonic generation images.

PubMed

Liu, Wenhua; Raben, Nina; Ralston, Evelyn

2013-02-01

Skeletal muscle pathologies cause irregularities in the normally periodic organization of the myofibrils. Objective grading of muscle morphology is necessary to assess muscle health, compare biopsies, and evaluate treatments and the evolution of disease. To facilitate such quantitation, we have developed a fast, sensitive, automatic imaging analysis software. It detects major and minor morphological changes by combining texture features and Fourier transform (FT) techniques. We apply this tool to second harmonic generation (SHG) images of muscle fibers which visualize the repeating myosin bands. Texture features are then calculated by using a Haralick gray-level cooccurrence matrix in MATLAB. Two scores are retrieved from the texture correlation plot by using FT and curve-fitting methods. The sensitivity of the technique was tested on SHG images of human adult and infant muscle biopsies and of mouse muscle samples. The scores are strongly correlated to muscle fiber condition. We named the software MARS (muscle assessment and rating scores). It is executed automatically and is highly sensitive even to subtle defects. We propose MARS as a powerful and unbiased tool to assess muscle health.

Quantitative evaluation of skeletal muscle defects in second harmonic generation images

NASA Astrophysics Data System (ADS)

Liu, Wenhua; Raben, Nina; Ralston, Evelyn

2013-02-01

Skeletal muscle pathologies cause irregularities in the normally periodic organization of the myofibrils. Objective grading of muscle morphology is necessary to assess muscle health, compare biopsies, and evaluate treatments and the evolution of disease. To facilitate such quantitation, we have developed a fast, sensitive, automatic imaging analysis software. It detects major and minor morphological changes by combining texture features and Fourier transform (FT) techniques. We apply this tool to second harmonic generation (SHG) images of muscle fibers which visualize the repeating myosin bands. Texture features are then calculated by using a Haralick gray-level cooccurrence matrix in MATLAB. Two scores are retrieved from the texture correlation plot by using FT and curve-fitting methods. The sensitivity of the technique was tested on SHG images of human adult and infant muscle biopsies and of mouse muscle samples. The scores are strongly correlated to muscle fiber condition. We named the software MARS (muscle assessment and rating scores). It is executed automatically and is highly sensitive even to subtle defects. We propose MARS as a powerful and unbiased tool to assess muscle health.

Measurement of lidocaine and 2,6-dimethylaniline in minipig plasma, skin, and dermal tapes using UHPLC with electrospray MS/MS.

PubMed

Li, Qian; Magers, Tobias; King, Brad; Engel, Brian J; Bakhtiar, Ray; Green, Charisse; Shoup, Ronald

2018-06-15

Sensitive LC-MS/MS methods were developed to measure lidocaine and its metabolite 2,6-dimethylaniline (2,6-DMA) with application to transdermal studies. The methods for lidocaine in minipig plasma, tissue biopsies, and dermal tapes utilized mixed mode/SCX solid phase extraction, with lower quantitation limits of 25 pg/mL in plasma, 15 ng/g tissue, and 5 ng/tape. 2,6-DMA was measured in plasma and skin tissue homogenates by ultrafiltration and (for tissue) by further derivatization with 4-methoxybenzoyl chloride to form the corresponding benzamide derivative, which extended the lower limit of quantitation to 200 pg/mL. The methods allowed local measurement of lidocaine in stratum corneum, punch biopsies, and plasma and of 2,6-DMA in plasma and biopsies obtained from minipigs dosed with experimental transdermal formulations. Quantitation limits were approximately 7-fold lower than previously reported for lidocaine and 3-fold lower for 2,6-DMA. Copyright © 2018 Elsevier B.V. All rights reserved.

Determination of Grayanotoxins from Rhododendron brachycarpum in Dietary Supplements and Homemade Wine by Liquid Chromatography-Quadrupole Time-of-Flight-Mass Spectrometry and Liquid Chromatography-Tandem Mass Spectrometry.

PubMed

Hwang, Taeik; Noh, Eunyoung; Jeong, Ji Hye; Park, Sung-Kwan; Shin, Dongwoo; Kang, Hoil

2018-02-28

A sensitive and specific high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS) method combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the determination of grayanotoxins I and III in dietary supplements and homemade wine. Grayanotoxins I and III were successfully extracted using solid-phase extraction cartridges, characterized by LC-QTOF-MS, and quantitated by LC-MS/MS. The LC-MS/MS calibration curves were linear over concentrations of 10-100 ng/mL (grayanotoxin I) and 20-400 ng/mL (grayanotoxin III). Grayanotoxins I and III were found in 51 foodstuffs, with quantitative determinations revealing total toxin concentrations of 18.4-101 000 ng/mL (grayanotoxin I) and 15.3-56 000 ng/mL (grayanotoxin III). The potential of the validated method was demonstrated by successful quantitative analysis of grayanotoxins I and III in dietary supplements and homemade wine; the method appears suitable for the routine detection of grayanotoxins I and III from Rhododendron brachycarpum.

Quantitative determination of major active components in Ginkgo biloba dietary supplements by liquid chromatography/mass spectrometry.

PubMed

Ding, Shujing; Dudley, Ed; Plummer, Sue; Tang, Jiandong; Newton, Russell P; Brenton, A Gareth

2006-01-01

A reversed-phase high-performance liquid chromatography/electrospray ionisation mass spectrometry (RP-HPLC/ESI-MS) method was developed and validated for the simultaneous determination of ten major active components in Ginkgo biloba extract (bilobalide, ginkgolides A, B, C, quercetin, kaempferol, isorhamnetin, rutin hydrate, quercetin-3-beta-D-glucoside and quercitrin hydrate) which have not been previously reported to be quantified in a single analysis. The ten components exhibit baseline separation in 50 min by C18 chromatography using a water/1:1 (v/v) methanol/acetonitrile gradient. Quantitation was performed using negative ESI-MS in selected ion monitoring (SIM) mode. Good reproducibility and recovery were obtained by this method. The sensitivity of both UV and different mass spectrometry modes (full scan, selected ion monitoring (SIM), and selected reaction monitoring (SRM)) were compared and both quantitation with and without internal standard were evaluated. The analysis of Ginkgo biloba commercial products showed remarkable variations in the rutin and quercetin content as well as the terpene lactone contents although all the products satisfy the conventional quality control method. Copyright 2006 John Wiley & Sons, Ltd.

Diagnostic accuracy of quantitative real-time PCR assay versus clinical and Gram stain identification of bacterial vaginosis.

PubMed

Menard, J-P; Mazouni, C; Fenollar, F; Raoult, D; Boubli, L; Bretelle, F

2010-12-01

The purpose of this investigation was to determine the diagnostic accuracy of quantitative real-time polymerase chain reaction (PCR) assay in diagnosing bacterial vaginosis versus the standard methods, the Amsel criteria and the Nugent score. The Amsel criteria, the Nugent score, and results from the molecular tool were obtained independently from vaginal samples of 163 pregnant women who reported abnormal vaginal symptoms before 20 weeks gestation. To determine the performance of the molecular tool, we calculated the kappa value, sensitivity, specificity, and positive and negative predictive values. Either or both of the Amsel criteria (≥3 criteria) and the Nugent score (score ≥7) indicated that 25 women (15%) had bacterial vaginosis, and the remaining 138 women did not. DNA levels of Gardnerella vaginalis or Atopobium vaginae exceeded 10(9) copies/mL or 10(8) copies/mL, respectively, in 34 (21%) of the 163 samples. Complete agreement between both reference methods and high concentrations of G. vaginalis and A. vaginae was found in 94.5% of women (154/163 samples, kappa value = 0.81, 95% confidence interval 0.70-0.81). The nine samples with discordant results were categorized as intermediate flora by the Nugent score. The molecular tool predicted bacterial vaginosis with a sensitivity of 100%, a specificity of 93%, a positive predictive value of 73%, and a negative predictive value of 100%. The quantitative real-time PCR assay shows excellent agreement with the results of both reference methods for the diagnosis of bacterial vaginosis.

Ultrasensitive Genotypic Detection of Antiviral Resistance in Hepatitis B Virus Clinical Isolates▿ †

PubMed Central

Fang, Jie; Wichroski, Michael J.; Levine, Steven M.; Baldick, Carl J.; Mazzucco, Charles E.; Walsh, Ann W.; Kienzle, Bernadette K.; Rose, Ronald E.; Pokornowski, Kevin A.; Colonno, Richard J.; Tenney, Daniel J.

2009-01-01

Amino acid substitutions that confer reduced susceptibility to antivirals arise spontaneously through error-prone viral polymerases and are selected as a result of antiviral therapy. Resistance substitutions first emerge in a fraction of the circulating virus population, below the limit of detection by nucleotide sequencing of either the population or limited sets of cloned isolates. These variants can expand under drug pressure to dominate the circulating virus population. To enhance detection of these viruses in clinical samples, we established a highly sensitive quantitative, real-time allele-specific PCR assay for hepatitis B virus (HBV) DNA. Sensitivity was accomplished using a high-fidelity DNA polymerase and oligonucleotide primers containing locked nucleic acid bases. Quantitative measurement of resistant and wild-type variants was accomplished using sequence-matched standards. Detection methodology that was not reliant on hybridization probes, and assay modifications, minimized the effect of patient-specific sequence polymorphisms. The method was validated using samples from patients chronically infected with HBV through parallel sequencing of large numbers of cloned isolates. Viruses with resistance to lamivudine and other l-nucleoside analogs and entecavir, involving 17 different nucleotide substitutions, were reliably detected at levels at or below 0.1% of the total population. The method worked across HBV genotypes. Longitudinal analysis of patient samples showed earlier emergence of resistance on therapy than was seen with sequencing methodologies, including some cases of resistance that existed prior to treatment. In summary, we established and validated an ultrasensitive method for measuring resistant HBV variants in clinical specimens, which enabled earlier, quantitative measurement of resistance to therapy. PMID:19433559

Purity assessment of recombinant human granulocyte colony-stimulating factor in finished drug product by capillary zone electrophoresis.

PubMed

Benković, Goran; Skrlin, Ana; Madić, Tomislav; Debeljak, Zeljko; Medić-Šarić, Marica

2014-09-01

Current methods for determination of impurities with different charge-to-volume ratio are limited especially in terms of sensitivity and precision. The main goal of this research was to establish a quantitative method for determination of impurities with charges differing from that of recombinant human granulocyte colony-stimulating factor (rhG-CSF, filgrastim) with superior precision and sensitivity compared to existing methods. A CZE method has been developed, optimized, and validated for a purity assessment of filgrastim in liquid pharmaceutical formulations. Optimal separation of filgrastim from the related impurities with different charges was achieved on a 50 μm id fused-silica capillary of a total length of 80.5 cm. A BGE that contains 100 mM phosphoric acid adjusted to pH 7.0 with triethanolamine was used. The applied voltage was 20 kV while the temperature was maintained at 25°C. UV detection was set to 200 nm. Method was validated in terms of selectivity/specificity, linearity, precision, LOD, LOQ, stability, and robustness. Linearity was observed in the concentration range of 6-600 μg/mL and the LOQ was determined to be 0.3% relative to the concentration of filgrastim of 0.6 mg/mL. Other validation parameters were also found to be acceptable; thus the method was successfully applied for a quantitative purity assessment of filgrastim in a finished drug product. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrodialytic in-line preconcentration for ionic solute analysis.

PubMed

Ohira, Shin-Ichi; Yamasaki, Takayuki; Koda, Takumi; Kodama, Yuko; Toda, Kei

2018-04-01

Preconcentration is an effective way to improve analytical sensitivity. Many types of methods are used for enrichment of ionic solute analytes. However, current methods are batchwise and include procedures such as trapping and elution. In this manuscript, we propose in-line electrodialytic enrichment of ionic solutes. The method can enrich ionic solutes within seconds by quantitative transfer of analytes from the sample solution to the acceptor solution under an electric field. Because of quantitative ion transfer, the enrichment factor (the ratio of the concentration in the sample and to that in the obtained acceptor solution) only depends on the flow rate ratio of the sample solution to the acceptor solution. The ratios of the concentrations and flow rates are equal for ratios up to 70, 20, and 70 for the tested ionic solutes of inorganic cations, inorganic anions, and heavy metal ions, respectively. The sensitivity of ionic solute determinations is also improved based on the enrichment factor. The method can also simultaneously achieve matrix isolation and enrichment. The method was successively applied to determine the concentrations of trace amounts of chloroacetic acids in tap water. The regulated concentration levels cannot be determined by conventional high-performance liquid chromatography with ultraviolet detection (HPLC-UV) without enrichment. However, enrichment with the present method is effective for determination of tap water quality by improving the limits of detection of HPLC-UV. The standard addition test with real tap water samples shows good recoveries (94.9-109.6%). Copyright © 2017 Elsevier B.V. All rights reserved.

The future of targeted peptidomics.

PubMed

Findeisen, Peter

2013-12-01

Targeted MS is becoming increasingly important for sensitive and specific quantitative detection of proteins and respective PTMs. In this article, Ceglarek et al. [Proteomics Clin. Appl. 2013, 7, 794-801] present an LC-MS-based method for simultaneous quantitation of seven apolipoproteins in serum specimens. The assay fulfills many necessities of routine diagnostic applications, namely, low cost, high throughput, and good reproducibility. We anticipate that validation of new biomarkers will speed up with this technology and the palette of laboratory-based diagnostic tools will hopefully be augmented significantly in the near future. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Highly sensitive transient absorption imaging of graphene and graphene oxide in living cells and circulating blood.

PubMed

Li, Junjie; Zhang, Weixia; Chung, Ting-Fung; Slipchenko, Mikhail N; Chen, Yong P; Cheng, Ji-Xin; Yang, Chen

2015-07-23

We report a transient absorption (TA) imaging method for fast visualization and quantitative layer analysis of graphene and GO. Forward and backward imaging of graphene on various substrates under ambient condition was imaged with a speed of 2 μs per pixel. The TA intensity linearly increased with the layer number of graphene. Real-time TA imaging of GO in vitro with capability of quantitative analysis of intracellular concentration and ex vivo in circulating blood were demonstrated. These results suggest that TA microscopy is a valid tool for the study of graphene based materials.

Measures of fish behavior as indicators of sublethal toxicosis during standard toxicity tests

USGS Publications Warehouse

Little, E.E.; DeLonay, A.J.

1996-01-01

Behavioral functions essential for growth and survival can be dramatically altered by sublethal exposure to toxicants. Measures of these behavioral responses are effective in detecting adverse effects of sublethal contaminant exposure. Behavioral responses of fishes can be qualitatively and quantitatively evaluated during routine toxicity tests. At selected intervals of exposure, qualitative evaluations are accomplished through direct observations, whereas video recordings are used for quantitative evaluations. Standardized procedures for behavioral evaluation are readily applicable to different fish species and provide rapid, sensitive, and ecologically relevant assessments of sublethal exposure. The methods are readily applied to standardized test protocols.

Taqman real-time quantitative PCR for identification of western flower thrip (Frankliniella occidentalis) for plant quarantine

PubMed Central

Huang, K. S.; Lee, S. E.; Yeh, Y.; Shen, G. S.; Mei, E.; Chang, C. M.

2010-01-01

Western flower thrip (Frankliniella occidentalis) is a major global pest of agricultural products. It directly damages crops through feeding, oviposition activity or transmission of several plant viruses. We describe a Taqman real-time quantitative PCR detection system, which can rapidly identify F. occidentalis from thrips larvae to complement the traditional morphological identification. The data showed that our detection system targeted on the ribosomal RNA gene regions of F. occidentalis has high sensitivity and specificity. The rapid method can be used for on-site testing of samples at ports-of-entry in the future. PMID:20129946

Taqman real-time quantitative PCR for identification of western flower thrip (Frankliniella occidentalis) for plant quarantine.

PubMed

Huang, K S; Lee, S E; Yeh, Y; Shen, G S; Mei, E; Chang, C M

2010-08-23

Western flower thrip (Frankliniella occidentalis) is a major global pest of agricultural products. It directly damages crops through feeding, oviposition activity or transmission of several plant viruses. We describe a Taqman real-time quantitative PCR detection system, which can rapidly identify F. occidentalis from thrips larvae to complement the traditional morphological identification. The data showed that our detection system targeted on the ribosomal RNA gene regions of F. occidentalis has high sensitivity and specificity. The rapid method can be used for on-site testing of samples at ports-of-entry in the future.

BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks.

PubMed

Yan, Winston X; Mirzazadeh, Reza; Garnerone, Silvano; Scott, David; Schneider, Martin W; Kallas, Tomasz; Custodio, Joaquin; Wernersson, Erik; Li, Yinqing; Gao, Linyi; Federova, Yana; Zetsche, Bernd; Zhang, Feng; Bienko, Magda; Crosetto, Nicola

2017-05-12

Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.

Non-radioactive detection of trinucleotide repeat size variability.

PubMed

Tomé, Stéphanie; Nicole, Annie; Gomes-Pereira, Mario; Gourdon, Genevieve

2014-03-06

Many human diseases are associated with the abnormal expansion of unstable trinucleotide repeat sequences. The mechanisms of trinucleotide repeat size mutation have not been fully dissected, and their understanding must be grounded on the detailed analysis of repeat size distributions in human tissues and animal models. Small-pool PCR (SP-PCR) is a robust, highly sensitive and efficient PCR-based approach to assess the levels of repeat size variation, providing both quantitative and qualitative data. The method relies on the amplification of a very low number of DNA molecules, through sucessive dilution of a stock genomic DNA solution. Radioactive Southern blot hybridization is sensitive enough to detect SP-PCR products derived from single template molecules, separated by agarose gel electrophoresis and transferred onto DNA membranes. We describe a variation of the detection method that uses digoxigenin-labelled locked nucleic acid probes. This protocol keeps the sensitivity of the original method, while eliminating the health risks associated with the manipulation of radiolabelled probes, and the burden associated with their regulation, manipulation and waste disposal.

Simultaneous Quantitation of Advanced Glycation End Products in Soy Sauce and Beer by Liquid Chromatography-Tandem Mass Spectrometry without Ion-Pair Reagents and Derivatization.

PubMed

Nomi, Yuri; Annaka, Hironori; Sato, Shinji; Ueta, Etsuko; Ohkura, Tsuyoshi; Yamamoto, Kazuhiro; Homma, Seiichi; Suzuki, Emiko; Otsuka, Yuzuru

2016-11-09

The aim of this study was to develop a simple and sensitive method to analyze several advanced glycation end products (AGEs) simultaneously using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to apply this method to the quantitation of AGEs in brown-colored foods. The developed method enabled to separate and quantitate simultaneously seven AGEs, and was applied to the determination of free AGEs contained in various kinds of soy sauce and beer. The major AGEs in soy sauce and beer were N ε -carboxymethyllysine (CML), N ε -carboxyethyllysine (CEL), and N δ -(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine (MG-H1). Using the developed LC-MS/MS method, recovery test on soy sauce and beer samples showed the recovery values of 85.3-103.9% for CML, 95.9-107.4% for CEL, and 69.5-123.2% for MG-H1. In particular, it is the first report that free CML, CEL, and MG-H1 were present in beer. Furthermore, long-term storage and heating process of soy sauce increased CML and MG-H1.

Improvements to direct quantitative analysis of multiple microRNAs facilitating faster analysis.

PubMed

Ghasemi, Farhad; Wegman, David W; Kanoatov, Mirzo; Yang, Burton B; Liu, Stanley K; Yousef, George M; Krylov, Sergey N

2013-11-05

Studies suggest that patterns of deregulation in sets of microRNA (miRNA) can be used as cancer diagnostic and prognostic biomarkers. Establishing a "miRNA fingerprint"-based diagnostic technique requires a suitable miRNA quantitation method. The appropriate method must be direct, sensitive, capable of simultaneous analysis of multiple miRNAs, rapid, and robust. Direct quantitative analysis of multiple microRNAs (DQAMmiR) is a recently introduced capillary electrophoresis-based hybridization assay that satisfies most of these criteria. Previous implementations of the method suffered, however, from slow analysis time and required lengthy and stringent purification of hybridization probes. Here, we introduce a set of critical improvements to DQAMmiR that address these technical limitations. First, we have devised an efficient purification procedure that achieves the required purity of the hybridization probe in a fast and simple fashion. Second, we have optimized the concentrations of the DNA probe to decrease the hybridization time to 10 min. Lastly, we have demonstrated that the increased probe concentrations and decreased incubation time removed the need for masking DNA, further simplifying the method and increasing its robustness. The presented improvements bring DQAMmiR closer to use in a clinical setting.

Quantitative analysis of periodontal pathogens by ELISA and real-time polymerase chain reaction.

PubMed

Hamlet, Stephen M

2010-01-01

The development of analytical methods enabling the accurate identification and enumeration of bacterial species colonizing the oral cavity has led to the identification of a small number of bacterial pathogens that are major factors in the etiology of periodontal disease. Further, these methods also underpin more recent epidemiological analyses of the impact of periodontal disease on general health. Given the complex milieu of over 700 species of microorganisms known to exist within the complex biofilms found in the oral cavity, the identification and enumeration of oral periodontopathogens has not been an easy task. In recent years however, some of the intrinsic limitations of the more traditional microbiological analyses previously used have been overcome with the advent of immunological and molecular analytical methods. Of the plethora of methodologies reported in the literature, the enzyme-linked immunosorbent assay (ELISA), which combines the specificity of antibody with the sensitivity of simple enzyme assays and the polymerase chain reaction (PCR), has been widely utilized in both laboratory and clinical applications. Although conventional PCR does not allow quantitation of the target organism, real-time PCR (rtPCR) has the ability to detect amplicons as they accumulate in "real time" allowing subsequent quantitation. These methods enable the accurate quantitation of as few as 10(2) (using rtPCR) to 10(4) (using ELISA) periodontopathogens in dental plaque samples.

Evaluation of the clinical sensitivity for the quantification of human immunodeficiency virus type 1 RNA in plasma: Comparison of the new COBAS TaqMan HIV-1 with three current HIV-RNA assays--LCx HIV RNA quantitative, VERSANT HIV-1 RNA 3.0 (bDNA) and COBAS AMPLICOR HIV-1 Monitor v1.5.

PubMed

Katsoulidou, Antigoni; Petrodaskalaki, Maria; Sypsa, Vana; Papachristou, Eleni; Anastassopoulou, Cleo G; Gargalianos, Panagiotis; Karafoulidou, Anastasia; Lazanas, Marios; Kordossis, Theodoros; Andoniadou, Anastasia; Hatzakis, Angelos

2006-02-01

The COBAS TaqMan HIV-1 test (Roche Diagnostics) was compared with the LCx HIV RNA quantitative assay (Abbott Laboratories), the Versant HIV-1 RNA 3.0 (bDNA) assay (Bayer) and the COBAS Amplicor HIV-1 Monitor v1.5 test (Roche Diagnostics), using plasma samples of various viral load levels from HIV-1-infected individuals. In the comparison of TaqMan with LCx, TaqMan identified as positive 77.5% of the 240 samples versus 72.1% identified by LCx assay, while their overall agreement was 94.6% and the quantitative results of samples that were positive by both methods were strongly correlated (r=0.91). Similarly, in the comparison of TaqMan with bDNA 3.0, both methods identified 76.3% of the 177 samples as positive, while their overall agreement was 95.5% and the quantitative results of samples that were positive by both methods were strongly correlated (r=0.95). Finally, in the comparison of TaqMan with Monitor v1.5, TaqMan identified 79.5% of the 156 samples as positive versus 80.1% identified by Monitor v1.5, while their overall agreement was 95.5% and the quantitative results of samples that were positive by both methods were strongly correlated (r=0.96). In conclusion, the new COBAS TaqMan HIV-1 test showed excellent agreement with other widely used commercially available tests for the quantitation of HIV-1 viral load.

Comparisons of Robustness and Sensitivity between Cancer and Normal Cells by Microarray Data

PubMed Central

Chu, Liang-Hui; Chen, Bor-Sen

2008-01-01

Robustness is defined as the ability to uphold performance in face of perturbations and uncertainties, and sensitivity is a measure of the system deviations generated by perturbations to the system. While cancer appears as a robust but fragile system, few computational and quantitative evidences demonstrate robustness tradeoffs in cancer. Microarrays have been widely applied to decipher gene expression signatures in human cancer research, and quantification of global gene expression profiles facilitates precise prediction and modeling of cancer in systems biology. We provide several efficient computational methods based on system and control theory to compare robustness and sensitivity between cancer and normal cells by microarray data. Measurement of robustness and sensitivity by linear stochastic model is introduced in this study, which shows oscillations in feedback loops of p53 and demonstrates robustness tradeoffs that cancer is a robust system with some extreme fragilities. In addition, we measure sensitivity of gene expression to perturbations in other gene expression and kinetic parameters, discuss nonlinear effects in feedback loops of p53 and extend our method to robustness-based cancer drug design. PMID:19259409

Development and validation of an UPLC-MS/MS method for the quantification of tamoxifen and its main metabolites in human scalp hair.

PubMed

Drooger, Jan C; Jager, Agnes; Lam, Mei-Ho; den Boer, Mathilda D; Sleijfer, Stefan; Mathijssen, Ron H J; de Bruijn, Peter

2015-10-10

The aim of this study was to validate an earlier developed high-performance highly sensitive ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method for quantification of tamoxifen and its three main metabolites (N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen) in scalp hair. This non-invasive method might, by segmental analysis of hair, be useful in the determination of the concentration of drugs and its metabolites over time, which can be used to study a wide variety of clinical relevant questions. Hair samples (150-300 hair strands, cut as close to the scalp as possible from the posterior vertex region of the head) were collected from female patients taking tamoxifen 20mg daily (n=19). The analytes were extracted using a liquid-liquid extraction procedure with carbonate buffer at pH 8.8 and a mixture of n-hexane/isopropranol method, followed by UPLC-MS/MS chromatography, based on an earlier validated method. The calibration curves were linear in the range of 1.00-200 pmol for tamoxifen and N-desmethyl-tamoxifen, with lower limit of quantitation of 1.00 pmol and 0.100-20.0 pmol with lower limit of quantitation of 0.100 pmol for endoxifen and 4-hydroxy-tamoxifen. Assay performance was fair with a within-run and between-run variability less than 9.24 at the three quality control samples and less than 15.7 for the lower limit of quantitation. Importantly, a steep linear decline was observed from distal to proximal hair segments. Probably, this is due to UV exposure as we showed degradation of tamoxifen and its metabolites after exposure to UV-light. Furthermore, higher concentrations of tamoxifen were found in black hair samples compared to blond and brown hair samples. We conclude that measurement of the concentration of tamoxifen and its main metabolites in hair is possible, with the selective, sensitive, accurate and precise UPLC-MS/MS method. However, for tamoxifen, it seems not possible to determine exposure over time with segmental analysis of hair, probably largely due to the effect of UV irradiation. Further research should therefore focus on quantification of other anticancer drugs, in segmented scalp hair, that are less sensitive to UV irradiation. Copyright © 2015 Elsevier B.V. All rights reserved.

Measuring changes in transmission of neglected tropical diseases, malaria, and enteric pathogens from quantitative antibody levels.

PubMed

Arnold, Benjamin F; van der Laan, Mark J; Hubbard, Alan E; Steel, Cathy; Kubofcik, Joseph; Hamlin, Katy L; Moss, Delynn M; Nutman, Thomas B; Priest, Jeffrey W; Lammie, Patrick J

2017-05-01

Serological antibody levels are a sensitive marker of pathogen exposure, and advances in multiplex assays have created enormous potential for large-scale, integrated infectious disease surveillance. Most methods to analyze antibody measurements reduce quantitative antibody levels to seropositive and seronegative groups, but this can be difficult for many pathogens and may provide lower resolution information than quantitative levels. Analysis methods have predominantly maintained a single disease focus, yet integrated surveillance platforms would benefit from methodologies that work across diverse pathogens included in multiplex assays. We developed an approach to measure changes in transmission from quantitative antibody levels that can be applied to diverse pathogens of global importance. We compared age-dependent immunoglobulin G curves in repeated cross-sectional surveys between populations with differences in transmission for multiple pathogens, including: lymphatic filariasis (Wuchereria bancrofti) measured before and after mass drug administration on Mauke, Cook Islands, malaria (Plasmodium falciparum) before and after a combined insecticide and mass drug administration intervention in the Garki project, Nigeria, and enteric protozoans (Cryptosporidium parvum, Giardia intestinalis, Entamoeba histolytica), bacteria (enterotoxigenic Escherichia coli, Salmonella spp.), and viruses (norovirus groups I and II) in children living in Haiti and the USA. Age-dependent antibody curves fit with ensemble machine learning followed a characteristic shape across pathogens that aligned with predictions from basic mechanisms of humoral immunity. Differences in pathogen transmission led to shifts in fitted antibody curves that were remarkably consistent across pathogens, assays, and populations. Mean antibody levels correlated strongly with traditional measures of transmission intensity, such as the entomological inoculation rate for P. falciparum (Spearman's rho = 0.75). In both high- and low transmission settings, mean antibody curves revealed changes in population mean antibody levels that were masked by seroprevalence measures because changes took place above or below the seropositivity cutoff. Age-dependent antibody curves and summary means provided a robust and sensitive measure of changes in transmission, with greatest sensitivity among young children. The method generalizes to pathogens that can be measured in high-throughput, multiplex serological assays, and scales to surveillance activities that require high spatiotemporal resolution. Our results suggest quantitative antibody levels will be particularly useful to measure differences in exposure for pathogens that elicit a transient antibody response or for monitoring populations with very high- or very low transmission, when seroprevalence is less informative. The approach represents a new opportunity to conduct integrated serological surveillance for neglected tropical diseases, malaria, and other infectious diseases with well-defined antigen targets.

Spectroflurimetric estimation of the new antiviral agent ledipasvir in presence of sofosbuvir

NASA Astrophysics Data System (ADS)

Salama, Fathy M.; Attia, Khalid A.; Abouserie, Ahmed A.; El-Olemy, Ahmed; Abolmagd, Ebrahim

2018-02-01

A spectroflurimetric method has been developed and validated for the selective quantitative determination of ledipasvir in presence of sofosbuvir. In this method the native fluorescence of ledipasvir in ethanol at 405 nm was measured after excitation at 340 nm. The proposed method was validated according to ICH guidelines and show high sensitivity, accuracy and precision. Furthermore this method was successfully applied to the analysis of ledipasvir in pharmaceutical dosage form without interference from sofosbuvir and other additives and the results were statistically compared to a reported method and found no significant difference.

Safe, rapid, and sensitive method of quantitating and distinguishing among Shiga toxins in complex media and human serum

USDA-ARS?s Scientific Manuscript database

Shiga toxins are primarily responsible for the virulence associated with Shiga toxin producing Escherichia coli (STEC) infections. The expression of the Shiga toxins is controlled by a phage that infects the host. More than one phage can infect a host and the host can inactivate infecting phages. T...

Short communication: Improved method for centrifugal recovery of bacteria from raw milk applied to sensitive real-time quantitative PCR detection of Salmonella spp

USDA-ARS?s Scientific Manuscript database

Centrifugation of milk is widely used as a separation/concentration step in assays for pathogenic microorganisms. Separation of the cream and liquid supernate from the pellet containing sedimented solids, somatic cells and microorganisms eliminates many interfering substances, and resuspension of th...

Quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals. A novel labeling method in study of membranes and surface systems.

PubMed

Papper, V; Medvedeva, N; Fishov, I; Likhtenshtein, G I

2000-01-01

We proposed a new method for the study of molecular dynamics and fluidity of the living and model biomembranes and surface systems. The method is based on the measurements of the sensitized photoisomerization kinetics of a photochrome probe. The cascade triplet cis-trans photoisomerization of the excited stilbene derivative sensitized with the excited triplet Erythrosin B has been studied in a model liposome membrane. The photoisomerization reaction is depressed with nitroxide radicals quenching the excited triplet state of the sensitizer. The enhanced fluorescence polarization of the stilbene probe incorporated into liposome membranes indicates that the stilbene molecules are squeezed in a relatively viscous media of the phospholipids. Calibration of the "triple" cascade system is based on a previously proposed method that allows the measurement of the product of the quenching rate constant and the sensitizer's triplet lifetime, as well as the quantitative detection of the nitroxide radicals in the vicinity of the membrane surface. The experiment was conducted using the constant-illumination fluorescence technique. Sensitivity of the method using a standard commercial spectrofluorimeter is about 10(-12) mol of fluorescence molecules per sample and can be improved using an advanced fluorescence technique. The minimal local concentration of nitroxide radicals or any other quenchers being detected is about 10(-5) M. This method enables the investigation of any chemical and biological surface processes of microscopic scale when the minimal volume is about 10(-3) microL or less.

Meta-analysis of diagnostic accuracy studies in mental health

PubMed Central

Takwoingi, Yemisi; Riley, Richard D; Deeks, Jonathan J

2015-01-01

Objectives To explain methods for data synthesis of evidence from diagnostic test accuracy (DTA) studies, and to illustrate different types of analyses that may be performed in a DTA systematic review. Methods We described properties of meta-analytic methods for quantitative synthesis of evidence. We used a DTA review comparing the accuracy of three screening questionnaires for bipolar disorder to illustrate application of the methods for each type of analysis. Results The discriminatory ability of a test is commonly expressed in terms of sensitivity (proportion of those with the condition who test positive) and specificity (proportion of those without the condition who test negative). There is a trade-off between sensitivity and specificity, as an increasing threshold for defining test positivity will decrease sensitivity and increase specificity. Methods recommended for meta-analysis of DTA studies --such as the bivariate or hierarchical summary receiver operating characteristic (HSROC) model --jointly summarise sensitivity and specificity while taking into account this threshold effect, as well as allowing for between study differences in test performance beyond what would be expected by chance. The bivariate model focuses on estimation of a summary sensitivity and specificity at a common threshold while the HSROC model focuses on the estimation of a summary curve from studies that have used different thresholds. Conclusions Meta-analyses of diagnostic accuracy studies can provide answers to important clinical questions. We hope this article will provide clinicians with sufficient understanding of the terminology and methods to aid interpretation of systematic reviews and facilitate better patient care. PMID:26446042

Sensitive spectrophotometric determination of aceclofenac following azo dye formation with 4-carboxyl-2,6-dinitrobenzene diazonium ion.

PubMed

Aderibigbe, Segun A; Adegoke, Olajire A; Idowu, Olakunle S; Olaleye, Sefiu O

2012-01-01

The study is a description of a sensitive spectrophotometric determination of aceclofenac following azo dye formation with 4-carboxyl-2,6-dinitrobenzenediazonium ion (CDNBD). Spot test and thin layer chromatography revealed the formation of a new compound distinct from CDNBD and aceclofenac. Optimization studies established a reaction time of 5 min at 30 degrees C after vortex mixing the drug/CDNBD for 10 s. An absorption maximum of 430 nm was selected as analytical wavelength. A linear response was observed over 1.2-4.8 μg/mL of aceclofenac with a correlation coefficient of 0.9983 and the drug combined with CDNBD at stoichiometric ratio of 2 : 1. The method has a limit of detection of 0.403 μg/mL, limit of quantitation of 1.22 μg/mL and is reproducible over a three day assessment. The method gave Sandell's sensitivity of 3.279 ng/cm2. Intra- and inter-day accuracies (in terms of errors) were less than 6% while precisions were of the order of 0.03-1.89% (RSD). The developed spectrophotometric method is of equivalent accuracy (p > 0.05) with British Pharmacopoeia, 2010 potentiometric method. It has the advantages of speed, simplicity, sensitivity and more affordable instrumentation and could found application as a rapid and sensitive analytical method of aceclofenac. It is the first described method by azo dye derivatization for the analysis of aceclofenac in bulk samples and dosage forms.

Quantitation of zolpidem in biological fluids by electro-driven microextraction combined with HPLC-UV analysis.

PubMed

Yaripour, Saeid; Mohammadi, Ali; Esfanjani, Isa; Walker, Roderick B; Nojavan, Saeed

2018-01-01

In this study, for the first time, an electro-driven microextraction method named electromembrane extraction combined with a simple high performance liquid chromatography and ultraviolet detection was developed and validated for the quantitation of zolpidem in biological samples. Parameters influencing electromembrane extraction were evaluated and optimized. The membrane consisted of 2-ethylhexanol immobilized in the pores of a hollow fiber. As a driving force, a 150 V electric field was applied to facilitate the analyte migration from the sample matrix to an acceptor solution through a supported liquid membrane. The pHs of donor and acceptor solutions were optimized to 6.0 and 2.0, respectively. The enrichment factor was obtained >75 within 15 minutes. The effect of carbon nanotubes (as solid nano-sorbents) on the membrane performance and EME efficiency was evaluated. The method was linear over the range of 10-1000 ng/mL for zolpidem (R 2 >0.9991) with repeatability ( %RSD) between 0.3 % and 7.3 % ( n = 3). The limits of detection and quantitation were 3 and 10 ng/mL, respectively. The sensitivity of HPLC-UV for the determination of zolpidem was enhanced by electromembrane extraction. Finally, the method was employed for the quantitation of zolpidem in biological samples with relative recoveries in the range of 60-79 %.

Quantitation of zolpidem in biological fluids by electro-driven microextraction combined with HPLC-UV analysis

PubMed Central

Yaripour, Saeid; Mohammadi, Ali; Esfanjani, Isa; Walker, Roderick B.; Nojavan, Saeed

2018-01-01

In this study, for the first time, an electro-driven microextraction method named electromembrane extraction combined with a simple high performance liquid chromatography and ultraviolet detection was developed and validated for the quantitation of zolpidem in biological samples. Parameters influencing electromembrane extraction were evaluated and optimized. The membrane consisted of 2-ethylhexanol immobilized in the pores of a hollow fiber. As a driving force, a 150 V electric field was applied to facilitate the analyte migration from the sample matrix to an acceptor solution through a supported liquid membrane. The pHs of donor and acceptor solutions were optimized to 6.0 and 2.0, respectively. The enrichment factor was obtained >75 within 15 minutes. The effect of carbon nanotubes (as solid nano-sorbents) on the membrane performance and EME efficiency was evaluated. The method was linear over the range of 10-1000 ng/mL for zolpidem (R2 >0.9991) with repeatability ( %RSD) between 0.3 % and 7.3 % (n = 3). The limits of detection and quantitation were 3 and 10 ng/mL, respectively. The sensitivity of HPLC-UV for the determination of zolpidem was enhanced by electromembrane extraction. Finally, the method was employed for the quantitation of zolpidem in biological samples with relative recoveries in the range of 60-79 %. PMID:29805344

Calibration methods influence quantitative material decomposition in photon-counting spectral CT

NASA Astrophysics Data System (ADS)

Curtis, Tyler E.; Roeder, Ryan K.

2017-03-01

Photon-counting detectors and nanoparticle contrast agents can potentially enable molecular imaging and material decomposition in computed tomography (CT). Material decomposition has been investigated using both simulated and acquired data sets. However, the effect of calibration methods on material decomposition has not been systematically investigated. Therefore, the objective of this study was to investigate the influence of the range and number of contrast agent concentrations within a modular calibration phantom on quantitative material decomposition. A commerciallyavailable photon-counting spectral micro-CT (MARS Bioimaging) was used to acquire images with five energy bins selected to normalize photon counts and leverage the contrast agent k-edge. Material basis matrix values were determined using multiple linear regression models and material decomposition was performed using a maximum a posteriori estimator. The accuracy of quantitative material decomposition was evaluated by the root mean squared error (RMSE), specificity, sensitivity, and area under the curve (AUC). An increased maximum concentration (range) in the calibration significantly improved RMSE, specificity and AUC. The effects of an increased number of concentrations in the calibration were not statistically significant for the conditions in this study. The overall results demonstrated that the accuracy of quantitative material decomposition in spectral CT is significantly influenced by calibration methods, which must therefore be carefully considered for the intended diagnostic imaging application.

Assessment of partial coalescence in whippable oil-in-water food emulsions.

PubMed

Petrut, Raul Flaviu; Danthine, Sabine; Blecker, Christophe

2016-03-01

Partial coalescence influences to a great extent the properties of final food products such as ice cream and whipped toppings. In return, the partial coalescence occurrence and development are conditioned, in such systems, by the emulsion's intrinsic properties (e.g. solid fat content, fat crystal shape and size), formulation (e.g. protein content, surfactants presence) and extrinsic factors (e.g. cooling rate, shearing). A set of methods is available for partial coalescence investigation and quantification. These methods are critically reviewed in this paper, balancing the weaknesses of the methods in terms of structure alteration (for turbidity, dye dilution, etc.) and assumptions made for mathematical models (for particle size determination) with their advantages (good repeatability, high sensitivity, etc.). With the methods proposed in literature, the partial coalescence investigations can be conducted quantitatively and/or qualitatively. Good correlation were observed between some of the quantitative methods such as dye dilution, calorimetry, fat particle size; while a poor correlation was found in the case of solvent extraction method with other quantitative methods. The most suitable way for partial coalescence quantification was implied to be the fat particle size method, which would give results with a high degree of confidence if used in combination with a microscopic technique for the confirmation of partial coalescence as the main destabilization mechanism. Copyright © 2015 Elsevier B.V. All rights reserved.

High-throughput detection and screening of plants modified by gene editing using quantitative real-time polymerase chain reaction.

PubMed

Peng, Cheng; Wang, Hua; Xu, Xiaoli; Wang, Xiaofu; Chen, Xiaoyun; Wei, Wei; Lai, Yongmin; Liu, Guoquan; Godwin, Ian Douglas; Li, Jieqin; Zhang, Ling; Xu, Junfeng

2018-05-15

Gene editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been developed to detect gene-edited organisms, these techniques are time and labour intensive. Meanwhile, few studies have investigated high-throughput detection and screening strategies for plants modified by gene editing. In this study, we developed a simple, sensitive and high-throughput quantitative real-time (qPCR)-based method. The qPCR-based method exploits two differently labelled probes that are placed within one amplicon at the gene editing target site to simultaneously detect the wild-type and a gene-edited mutant. We showed that the qPCR-based method can accurately distinguish CRISPR/Cas9-induced mutants from the wild-type in several different plant species, such as Oryza sativa, Arabidopsis thaliana, Sorghum bicolor, and Zea mays. Moreover, the method can subsequently determine the mutation type by direct sequencing of the qPCR products of mutations due to gene editing. The qPCR-based method is also sufficiently sensitive to distinguish between heterozygous and homozygous mutations in T 0 transgenic plants. In a 384-well plate format, the method enabled the simultaneous analysis of up to 128 samples in three replicates without handling the post-polymerase chain reaction (PCR) products. Thus, we propose that our method is an ideal choice for screening plants modified by gene editing from many candidates in T 0 transgenic plants, which will be widely used in the area of plant gene editing. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

The parallel reaction monitoring method contributes to a highly sensitive polyubiquitin chain quantification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsuchiya, Hikaru; Tanaka, Keiji, E-mail: tanaka-kj@igakuken.or.jp; Saeki, Yasushi, E-mail: saeki-ys@igakuken.or.jp

2013-06-28

Highlights: •The parallel reaction monitoring method was applied to ubiquitin quantification. •The ubiquitin PRM method is highly sensitive even in biological samples. •Using the method, we revealed that Ufd4 assembles the K29-linked ubiquitin chain. -- Abstract: Ubiquitylation is an essential posttranslational protein modification that is implicated in a diverse array of cellular functions. Although cells contain eight structurally distinct types of polyubiquitin chains, detailed function of several chain types including K29-linked chains has remained largely unclear. Current mass spectrometry (MS)-based quantification methods are highly inefficient for low abundant atypical chains, such as K29- and M1-linked chains, in complex mixtures thatmore » typically contain highly abundant proteins. In this study, we applied parallel reaction monitoring (PRM), a quantitative, high-resolution MS method, to quantify ubiquitin chains. The ubiquitin PRM method allows us to quantify 100 attomole amounts of all possible ubiquitin chains in cell extracts. Furthermore, we quantified ubiquitylation levels of ubiquitin-proline-β-galactosidase (Ub-P-βgal), a historically known model substrate of the ubiquitin fusion degradation (UFD) pathway. In wild-type cells, Ub-P-βgal is modified with ubiquitin chains consisting of 21% K29- and 78% K48-linked chains. In contrast, K29-linked chains are not detected in UFD4 knockout cells, suggesting that Ufd4 assembles the K29-linked ubiquitin chain(s) on Ub-P-βgal in vivo. Thus, the ubiquitin PRM is a novel, useful, quantitative method for analyzing the highly complicated ubiquitin system.« less

Leukotriene B4 catabolism: quantitation of leukotriene B4 and its omega-oxidation products by reversed-phase high-performance liquid chromatography.

PubMed

Shak, S

1987-01-01

LTB4 and its omega-oxidation products may be rapidly, sensitively, and specifically quantitated by the methods of solid-phase extraction and reversed-phase high-performance liquid chromatography (HPLC), which are described in this chapter. Although other techniques, such as radioimmunoassay or gas chromatography-mass spectrometry, may be utilized for quantitative analysis of the lipoxygenase products of arachidonic acid, only the technique of reversed-phase HPLC can quantitate as many as 10 metabolites in a single analysis, without prior derivatization. In this chapter, we also reviewed the chromatographic theory which we utilized in order to optimize reversed-phase HPLC analysis of LTB4 and its omega-oxidation products. With this information and a gradient HPLC system, it is possible for any investigator to develop a powerful assay for the potent inflammatory mediator, LTB4, or for any other lipoxygenase product of arachidonic acid.

Gold nanoparticle-based enzyme-linked antibody-aptamer sandwich assay for detection of Salmonella Typhimurium.

PubMed

Wu, Wenhe; Li, Jun; Pan, Dun; Li, Jiang; Song, Shiping; Rong, Mingge; Li, Zixi; Gao, Jimin; Lu, Jianxin

2014-10-08

Enzyme-linked immunosorbent assay (ELISA) provides a convenient means for the detection of Salmonella enterica serovar Typhimurium (STM), which is important for rapid diagnosis of foodborne pathogens. However, conventional ELISA is limited by antibody-antigen immunoreactions and suffers from poor sensitivity and tedious sample pretreatment. Therefore, development of novel ELISA remains challenging. Herein, we designed a comprehensive strategy for rapid, sensitive, and quantitative detection of STM with high specificity by gold nanoparticle-based enzyme-linked antibody-aptamer sandwich (nano-ELAAS) method. STM was captured and preconcentrated from samples with aptamer-modified magnetic particles, followed by binding with detector antibodies. Then nanoprobes carrying a large amount of reporter antibodies and horseradish peroxidase molecules were used for colorimetric signal amplification. Under the optimized reaction conditions, the nano-ELAAS assay had a quantitative detection range from 1 × 10(3) to 1 × 10(8) CFU mL(-1), a limit of detection of 1 × 10(3) CFU mL(-1), and a selectivity of >10-fold for STM in samples containing other bacteria at higher concentration with an assay time less than 3 h. In addition, the developed nanoprobes were improved in terms of detection range and/or sensitivity when compared with two commercial enzyme-labeled antibody signal reporters. Finally, the nano-ELAAS method was demonstrated to work well in milk samples, a common source of STM contamination.

Temporal Lobe Epilepsy: Quantitative MR Volumetry in Detection of Hippocampal Atrophy

PubMed Central

Farid, Nikdokht; Girard, Holly M.; Kemmotsu, Nobuko; Smith, Michael E.; Magda, Sebastian W.; Lim, Wei Y.; Lee, Roland R.

2012-01-01

Purpose: To determine the ability of fully automated volumetric magnetic resonance (MR) imaging to depict hippocampal atrophy (HA) and to help correctly lateralize the seizure focus in patients with temporal lobe epilepsy (TLE). Materials and Methods: This study was conducted with institutional review board approval and in compliance with HIPAA regulations. Volumetric MR imaging data were analyzed for 34 patients with TLE and 116 control subjects. Structural volumes were calculated by using U.S. Food and Drug Administration–cleared software for automated quantitative MR imaging analysis (NeuroQuant). Results of quantitative MR imaging were compared with visual detection of atrophy, and, when available, with histologic specimens. Receiver operating characteristic analyses were performed to determine the optimal sensitivity and specificity of quantitative MR imaging for detecting HA and asymmetry. A linear classifier with cross validation was used to estimate the ability of quantitative MR imaging to help lateralize the seizure focus. Results: Quantitative MR imaging–derived hippocampal asymmetries discriminated patients with TLE from control subjects with high sensitivity (86.7%–89.5%) and specificity (92.2%–94.1%). When a linear classifier was used to discriminate left versus right TLE, hippocampal asymmetry achieved 94% classification accuracy. Volumetric asymmetries of other subcortical structures did not improve classification. Compared with invasive video electroencephalographic recordings, lateralization accuracy was 88% with quantitative MR imaging and 85% with visual inspection of volumetric MR imaging studies but only 76% with visual inspection of clinical MR imaging studies. Conclusion: Quantitative MR imaging can depict the presence and laterality of HA in TLE with accuracy rates that may exceed those achieved with visual inspection of clinical MR imaging studies. Thus, quantitative MR imaging may enhance standard visual analysis, providing a useful and viable means for translating volumetric analysis into clinical practice. © RSNA, 2012 Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12112638/-/DC1 PMID:22723496

[Modernized study on eye's signs of blood-stasis syndrome].

PubMed

Wu, Rui; Xie, Jian-xiang; Zhao, Feng-da

2011-03-01

To make out a computerized formula to diagnose eye's signs of blood-stasis syndrome (BSS), and to improve the previous diagnostic methods by naked eyes. The formula was created by detecting and analyzing the changes of eye's signs in 544 patients (261 of non-BSS and 283 of BSS) quantitatively, adopting computer's color scale principle. And the sensitivity, specificity and accuracy of the formula were verified in 382 patients (97 non-BSS and 285 of BSS). The computerized integral was compared with the naked eye integral, and the normal reference value was calculated with percentile. Various observatory indices of eye's sign were positively correlated with BSS. The specificity of the computerized method was 83.5%, and the diagnostic sensitivity was 89.8%, the accuracy 88.2%, and the correct index 0.733. Comparisons between the computerized integral method and the naked eye integral method showed significant difference in patients of non-BSS or of BSS in various degrees (including mild, moderate and severe) (P < 0.01). The reference value of the naked eye method was below 15. The computerized formula of eye's signs has higher specificity and sensitivity in the diagnosis of BSS, while the naked eye integral method is proved to be useful.

Wide-Field Imaging of Single-Nanoparticle Extinction with Sub-nm2 Sensitivity

NASA Astrophysics Data System (ADS)

Payne, Lukas M.; Langbein, Wolfgang; Borri, Paola

2018-03-01

We report on a highly sensitive wide-field imaging technique for quantitative measurement of the optical extinction cross section σext of single nanoparticles. The technique is simple and high speed, and it enables the simultaneous acquisition of hundreds of nanoparticles for statistical analysis. Using rapid referencing, fast acquisition, and a deconvolution analysis, a shot-noise-limited sensitivity down to 0.4 nm2 is achieved. Measurements on a set of individual gold nanoparticles of 5 nm diameter using this method yield σext=(10.0 ±3.1 ) nm2, which is consistent with theoretical expectations and well above the background fluctuations of 0.9 nm2 .

Imaging alpha particle detector

DOEpatents

Anderson, David F.

1985-01-01

A method and apparatus for detecting and imaging alpha particles sources is described. A conducting coated high voltage electrode (1) and a tungsten wire grid (2) constitute a diode configuration discharge generator for electrons dislodged from atoms or molecules located in between these electrodes when struck by alpha particles from a source (3) to be quantitatively or qualitatively analyzed. A thin polyester film window (4) allows the alpha particles to pass into the gas enclosure and the combination of the glass electrode, grid and window is light transparent such that the details of the source which is imaged with high resolution and sensitivity by the sparks produced can be observed visually as well. The source can be viewed directly, electronically counted or integrated over time using photographic methods. A significant increase in sensitivity over other alpha particle detectors is observed, and the device has very low sensitivity to gamma or beta emissions which might otherwise appear as noise on the alpha particle signal.

Imaging alpha particle detector

DOEpatents

Anderson, D.F.

1980-10-29

A method and apparatus for detecting and imaging alpha particles sources is described. A dielectric coated high voltage electrode and a tungsten wire grid constitute a diode configuration discharge generator for electrons dislodged from atoms or molecules located in between these electrodes when struck by alpha particles from a source to be quantitatively or qualitatively analyzed. A thin polyester film window allows the alpha particles to pass into the gas enclosure and the combination of the glass electrode, grid and window is light transparent such that the details of the source which is imaged with high resolution and sensitivity by the sparks produced can be observed visually as well. The source can be viewed directly, electronically counted or integrated over time using photographic methods. A significant increase in sensitivity over other alpha particle detectors is observed, and the device has very low sensitivity to gamma or beta emissions which might otherwise appear as noise on the alpha particle signal.

Measurements of carbon-14 with cavity ring-down spectroscopy

DOE PAGES

McCartt, A. D.; Ognibene, T.; Bench, G.; ...

2015-06-13

Accelerator Mass Spectrometry (AMS) is the most sensitive method for quantitation of 14C in biological samples. This technology has been used in a variety of low dose, human health related studies over the last 20 years when very high sensitivity was needed. AMS helped pioneer these scientific methods, but its expensive facilities and requirements for highly trained technical staff have limited their proliferation. Quantification of 14C by cavity ring-down spectroscopy (CRDS) offers an approach that eliminates many of the shortcomings of an accelerator-based system and would supplement the use of AMS in biomedical research. Our initial prototype, using a non-idealmore » wavelength laser and under suboptimal experimental conditions, has a 3.5-modern, 1-σ precision for detection of milligram-sized, carbon-14-elevated samples. Furthermore, these results demonstrate proof of principle and provided a starting point for the development of a spectrometer capable of biologically relevant sensitivities.« less

A Quantitative Comparison of Calibration Methods for RGB-D Sensors Using Different Technologies.

PubMed

Villena-Martínez, Víctor; Fuster-Guilló, Andrés; Azorín-López, Jorge; Saval-Calvo, Marcelo; Mora-Pascual, Jeronimo; Garcia-Rodriguez, Jose; Garcia-Garcia, Alberto

2017-01-27

RGB-D (Red Green Blue and Depth) sensors are devices that can provide color and depth information from a scene at the same time. Recently, they have been widely used in many solutions due to their commercial growth from the entertainment market to many diverse areas (e.g., robotics, CAD, etc.). In the research community, these devices have had good uptake due to their acceptable levelofaccuracyformanyapplicationsandtheirlowcost,butinsomecases,theyworkatthelimitof their sensitivity, near to the minimum feature size that can be perceived. For this reason, calibration processes are critical in order to increase their accuracy and enable them to meet the requirements of such kinds of applications. To the best of our knowledge, there is not a comparative study of calibration algorithms evaluating its results in multiple RGB-D sensors. Specifically, in this paper, a comparison of the three most used calibration methods have been applied to three different RGB-D sensors based on structured light and time-of-flight. The comparison of methods has been carried out by a set of experiments to evaluate the accuracy of depth measurements. Additionally, an object reconstruction application has been used as example of an application for which the sensor works at the limit of its sensitivity. The obtained results of reconstruction have been evaluated through visual inspection and quantitative measurements.

A rapid method for determining salinomycin and monensin sensitivity in Eimeria tenella.

PubMed

Jenkins, M C; O'Brien, C N; Fuller, L; Mathis, G F; Fetterer, R

2014-12-15

Standard methods of determining the ionophore sensitivity of Eimeria rely on infecting chickens with an isolate or a mixture of Eimeria spp. oocysts in the presence of different anti-coccidial drugs. The purpose of this study was to develop a rapid in vitro method for assessing salinomycin and monensin sensitivity in Eimeria tenella. Cultures of MDBK cells were grown to 85% confluency, and then inoculated with excysted E. tenella laboratory strain (APU-1) sporozoites in the presence of different concentrations of salinomycin or monensin. At various timepoints, the monolayers were fixed for counting intraceullar sporozoites, or were subjected to DNA extraction, followed by molecular analysis using quantitative (qPCR) or semi-quantitative PCR (sqPCR). Preliminary experiments showed that 24h was the optimum time for harvesting the E. tenella-infected cell cultures. The average number of E. tenella sporozoites relative to untreated controls displayed a linear decrease between 0.3 and 33.0 μg/ml salinomycin and between 0.3 and 3.3 μg/ml monensin. A similar pattern was observed in the relative amount of E. tenella DNA as measured by sqPCR. A linear decrease in the relative amount of E. tenella DNA was observed over the entire range of salinomycin and monensin concentrations as measured by qPCR possibly reflecting the greater sensitivity of this assay. Comparison of sporozoite counting, sqPCR, and qPCR signals using a criterion of 50% inhibition in sporozoite numbers or level of PCR amplification product showed good agreement between the three assays. E. tenella field isolates (FS-1 and FS-2) displaying resistance to salinomycin and monensin were evaluated in the in vitro assay using qPCR and sqPCR. Compared to E. tenella APU-1, the E. tenella FS-1 and FS-2 isolates showed higher levels of E. tenella DNA at 24h by both qPCR and sqPCR. This in vitro assay represents a significant advance in developing rapid, cost-effective methods for assessing ionophore sensitivity in E. tenella. Published by Elsevier B.V.

The Lumipulse G HBsAg-Quant assay for screening and quantification of the hepatitis B surface antigen.

PubMed

Yang, Ruifeng; Song, Guangjun; Guan, Wenli; Wang, Qian; Liu, Yan; Wei, Lai

2016-02-01

Qualitative HBsAg assay is used to screen HBV infection for decades. The utility of quantitative assay is also rejuvenated recently. We aimed to evaluate and compare the performance of a novel ultra-sensitive and quantitative assay, the Lumipulse assay, with the Architect and Elecsys assays. As screening methods, specificity was compared using 2043 consecutive clinical routine samples. As quantitative assays, precision and accuracy were assessed. Sera from 112 treatment-naïve chronic hepatitis B patients, four patients undergoing antiviral therapy and one patient with acute infection were tested to compare the correlations. Samples with concurrent HBsAg/anti-HBs were also quantified. The Lumipulse assay precisely quantified ultra-low level of HBsAg (0.004 IU/mL). It identified additional 0.98% (20/2043) clinical samples with trance amount of HBsAg. Three assays displayed excellent linear correlations irrespective of genotypes and S-gene mutations (R(2)>0.95, P<0.0001), while minor quantitative biases existed. The Lumipulse assay did not yield higher HBsAg concentrations in samples with concomitant anti-HBs. Compared with other assays, the Lumipulse assay is sensitive and specific for detecting HBsAg. The interpretation of the extremely low-level results, however, is challenging. Quantitative HBsAg results by different assays are highly correlated, but they should be interpreted interchangeably only after conversion to eliminate the biases. Copyright © 2015 Elsevier B.V. All rights reserved.

A Flow Cytometry-Based Assay for Quantifying Non-Plaque Forming Strains of Yellow Fever Virus

PubMed Central

Hammarlund, Erika; Amanna, Ian J.; Dubois, Melissa E.; Barron, Alex; Engelmann, Flora; Messaoudi, Ilhem; Slifka, Mark K.

2012-01-01

Primary clinical isolates of yellow fever virus can be difficult to quantitate by standard in vitro methods because they may not form discernable plaques or induce a measurable cytopathic effect (CPE) on cell monolayers. In our hands, the Dakar strain of yellow fever virus (YFV-Dakar) could not be measured by plaque assay (PA), focus-forming assay (FFA), or by measurement of CPE. For these reasons, we developed a YFV-specific monoclonal antibody (3A8.B6) and used it to optimize a highly sensitive flow cytometry-based tissue culture limiting dilution assay (TC-LDA) to measure levels of infectious virus. The TC-LDA was performed by incubating serial dilutions of virus in replicate wells of C6/36 cells and stained intracellularly for virus with MAb 3A8.B6. Using this approach, we could reproducibly quantitate YFV-Dakar in tissue culture supernatants as well as from the serum of viremic rhesus macaques experimentally infected with YFV-Dakar. Moreover, the TC-LDA approach was >10-fold more sensitive than standard plaque assay for quantitating typical plaque-forming strains of YFV including YFV-17D and YFV-FNV (French neurotropic vaccine). Together, these results indicate that the TC-LDA technique is effective for quantitating both plaque-forming and non-plaque-forming strains of yellow fever virus, and this methodology may be readily adapted for the study and quantitation of other non-plaque-forming viruses. PMID:23028428

A flow cytometry-based assay for quantifying non-plaque forming strains of yellow fever virus.

PubMed

Hammarlund, Erika; Amanna, Ian J; Dubois, Melissa E; Barron, Alex; Engelmann, Flora; Messaoudi, Ilhem; Slifka, Mark K

2012-01-01

Primary clinical isolates of yellow fever virus can be difficult to quantitate by standard in vitro methods because they may not form discernable plaques or induce a measurable cytopathic effect (CPE) on cell monolayers. In our hands, the Dakar strain of yellow fever virus (YFV-Dakar) could not be measured by plaque assay (PA), focus-forming assay (FFA), or by measurement of CPE. For these reasons, we developed a YFV-specific monoclonal antibody (3A8.B6) and used it to optimize a highly sensitive flow cytometry-based tissue culture limiting dilution assay (TC-LDA) to measure levels of infectious virus. The TC-LDA was performed by incubating serial dilutions of virus in replicate wells of C6/36 cells and stained intracellularly for virus with MAb 3A8.B6. Using this approach, we could reproducibly quantitate YFV-Dakar in tissue culture supernatants as well as from the serum of viremic rhesus macaques experimentally infected with YFV-Dakar. Moreover, the TC-LDA approach was >10-fold more sensitive than standard plaque assay for quantitating typical plaque-forming strains of YFV including YFV-17D and YFV-FNV (French neurotropic vaccine). Together, these results indicate that the TC-LDA technique is effective for quantitating both plaque-forming and non-plaque-forming strains of yellow fever virus, and this methodology may be readily adapted for the study and quantitation of other non-plaque-forming viruses.

Shot noise-limited Cramér-Rao bound and algorithmic sensitivity for wavelength shifting interferometry

NASA Astrophysics Data System (ADS)

Chen, Shichao; Zhu, Yizheng

2017-02-01

Sensitivity is a critical index to measure the temporal fluctuation of the retrieved optical pathlength in quantitative phase imaging system. However, an accurate and comprehensive analysis for sensitivity evaluation is still lacking in current literature. In particular, previous theoretical studies for fundamental sensitivity based on Gaussian noise models are not applicable to modern cameras and detectors, which are dominated by shot noise. In this paper, we derive two shot noiselimited theoretical sensitivities, Cramér-Rao bound and algorithmic sensitivity for wavelength shifting interferometry, which is a major category of on-axis interferometry techniques in quantitative phase imaging. Based on the derivations, we show that the shot noise-limited model permits accurate estimation of theoretical sensitivities directly from measured data. These results can provide important insights into fundamental constraints in system performance and can be used to guide system design and optimization. The same concepts can be generalized to other quantitative phase imaging techniques as well.

Simplified dichromated gelatin hologram recording process

NASA Technical Reports Server (NTRS)

Georgekutty, Tharayil G.; Liu, Hua-Kuang

1987-01-01

A simplified method for making dichromated gelatin (DCG) holographic optical elements (HOE) has been discovered. The method is much less tedious and it requires a period of processing time comparable with that for processing a silver halide hologram. HOE characteristics including diffraction efficiency (DE), linearity, and spectral sensitivity have been quantitatively investigated. The quality of the holographic grating is very high. Ninety percent or higher diffraction efficiency has been achieved in simple plane gratings made by this process.

Quantitative mass spectrometric analysis of glycoproteins combined with enrichment methods.

PubMed

Ahn, Yeong Hee; Kim, Jin Young; Yoo, Jong Shin

2015-01-01

Mass spectrometry (MS) has been a core technology for high sensitive and high-throughput analysis of the enriched glycoproteome in aspects of quantitative assays as well as qualitative profiling of glycoproteins. Because it has been widely recognized that aberrant glycosylation in a glycoprotein may involve in progression of a certain disease, the development of efficient analysis tool for the aberrant glycoproteins is very important for deep understanding about pathological function of the glycoprotein and new biomarker development. This review first describes the protein glycosylation-targeting enrichment technologies mainly employing solid-phase extraction methods such as hydrizide-capturing, lectin-specific capturing, and affinity separation techniques based on porous graphitized carbon, hydrophilic interaction chromatography, or immobilized boronic acid. Second, MS-based quantitative analysis strategies coupled with the protein glycosylation-targeting enrichment technologies, by using a label-free MS, stable isotope-labeling, or targeted multiple reaction monitoring (MRM) MS, are summarized with recent published studies. © 2014 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc.

Development of high-throughput and high sensitivity capillary gel electrophoresis platform method for Western, Eastern, and Venezuelan equine encephalitis (WEVEE) virus like particles (VLPs) purity determination and characterization.

PubMed

Gollapudi, Deepika; Wycuff, Diane L; Schwartz, Richard M; Cooper, Jonathan W; Cheng, K C

2017-10-01

In this paper, we describe development of a high-throughput, highly sensitive method based on Lab Chip CGE-SDS platform for purity determination and characterization of virus-like particle (VLP) vaccines. A capillary gel electrophoresis approach requiring about 41 s per sample for analysis and demonstrating sensitivity to protein initial concentrations as low as 20 μg/mL, this method has been used previously to evaluate monoclonal antibodies, but this application for lot release assay of VLPs using this platform is unique. The method was qualified and shown to be accurate for the quantitation of VLP purity. Assay repeatability was confirmed to be less than 2% relative standard deviation of the mean (% RSD) with interday precision less than 2% RSD. The assay can evaluate purified VLPs in a concentration range of 20-249 μg/mL for VEE and 20-250 μg/mL for EEE and WEE VLPs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A GC-MS method for the detection and quantitation of ten major drugs of abuse in human hair samples.

PubMed

Orfanidis, A; Mastrogianni, O; Koukou, A; Psarros, G; Gika, H; Theodoridis, G; Raikos, N

2017-03-15

A sensitive analytical method has been developed in order to identify and quantify major drugs of abuse (DOA), namely morphine, codeine, 6-monoacetylmorphine, cocaine, ecgonine methyl ester, benzoylecgonine, amphetamine, methamphetamine, methylenedioxymethamphetamine and methylenedioxyamphetamine in human hair. Samples of hair were extracted with methanol under ultrasonication at 50°C after a three step rinsing process to remove external contamination and dirt hair. Derivatization with BSTFA was selected in order to increase detection sensitivity of GC/MS analysis. Optimization of derivatization parameters was based on experiments for the selection of derivatization time, temperature and volume of derivatising agent. Validation of the method included evaluation of linearity which ranged from 2 to 350ng/mg of hair mean concentration for all DOA, evaluation of sensitivity, accuracy, precision and repeatability. Limits of detection ranged from 0.05 to 0.46ng/mg of hair. The developed method was applied for the analysis of hair samples obtained from three human subjects and were found positive in cocaine, and opiates. Published by Elsevier B.V.

Analysis of glycosaminoglycan-derived disaccharides by capillary electrophoresis using laser-induced fluorescence detection

PubMed Central

Chang, Yuqing; Yang, Bo; Zhao, Xue; Linhardt, Robert J.

2012-01-01

A quantitative and highly sensitive method for the analysis of glycosaminoglycan (GAG)-derived disaccharides is presented that relies on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. This method enables complete separation of seventeen GAG-derived disaccharides in a single run. Unsaturated disaccharides were derivatized with 2-aminoacridone (AMAC) to improve sensitivity. The limit of detection was at the attomole level and about 100-fold more sensitive than traditional CE-ultraviolet detection. A CE separation timetable was developed to achieve complete resolution and shorten analysis time. The RSD of migration time and peak areas at both low and high concentrations of unsaturated disaccharides are all less than 2.7% and 3.2%, respectively, demonstrating that this is a reproducible method. This analysis was successfully applied to cultured Chinese hamster ovary cell samples for determination of GAG disaccharides. The current method simplifies GAG extraction steps, and reduces inaccuracy in calculating ratios of heparin/heparan sulfate to chondroitin sulfate/dermatan sulfate, resulting from the separate analyses of a single sample. PMID:22609076

Quantitation of zoledronic acid in murine bone by liquid chromatography coupled with tandem mass spectrometry.

PubMed

Raccor, Brianne S; Sun, Jianxun; Lawrence, Ross F; Li, Lei; Zhang, Hai; Somerman, Martha J; Totah, Rheem A

2013-09-15

An in vitro method for extraction and quantification of zoledronic acid (ZA) from murine bone was developed. Whole mouse bones were incubated in ZA solutions with predetermined concentrations and bound ZA was subsequently extracted from bone with phosphoric acid and derivatized using trimethylsilyl diazomethane (TMS-DAM). ZA tetra-methyl phosphonate was quantified by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). This resulted in a sensitive, accurate, and precise method that was linear over three orders of magnitude (0.0250-50.0μg/mL ZA). For quality control (QC) samples, intra-and inter-day coefficients of variance were calculated and were less than 10%. This method was then applied to an in vivo model to quantitate ZA from the femur and mandible of three mice treated with ZA for two weeks. The mean ZA extracted from the mandible was four fold higher than that extracted from the femur (3.06±0.52 vs. 0.76±0.09ng/mg, respectively) indicating that ZA did not distribute equally in the skeleton and had a preference to the mandible. In conclusion, a highly sensitive method to measure ZA from mouse skeleton was developed, which can be easily adapted to multiple mammalian models including humans receiving ZA treatment. Copyright © 2013 Elsevier B.V. All rights reserved.

Novel quantitative real-time LCR for the sensitive detection of SNP frequencies in pooled DNA: method development, evaluation and application.

PubMed

Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

2011-01-19

Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food.

Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation.

PubMed

Yegnasubramanian, Srinivasan; Lin, Xiaohui; Haffner, Michael C; DeMarzo, Angelo M; Nelson, William G

2006-02-09

Hypermethylation of CpG island (CGI) sequences is a nearly universal somatic genome alteration in cancer. Rapid and sensitive detection of DNA hypermethylation would aid in cancer diagnosis and risk stratification. We present a novel technique, called COMPARE-MS, that can rapidly and quantitatively detect CGI hypermethylation with high sensitivity and specificity in hundreds of samples simultaneously. To quantitate CGI hypermethylation, COMPARE-MS uses real-time PCR of DNA that was first digested by methylation-sensitive restriction enzymes and then precipitated by methyl-binding domain polypeptides immobilized on a magnetic solid matrix. We show that COMPARE-MS could detect five genome equivalents of methylated CGIs in a 1000- to 10,000-fold excess of unmethylated DNA. COMPARE-MS was used to rapidly quantitate hypermethylation at multiple CGIs in >155 prostate tissues, including benign and malignant prostate specimens, and prostate cell lines. This analysis showed that GSTP1, MDR1 and PTGS2 CGI hypermethylation as determined by COMPARE-MS could differentiate between malignant and benign prostate with sensitivities >95% and specificities approaching 100%. This novel technology could significantly improve our ability to detect CGI hypermethylation.

Safety and Utility of Quantitative Sensory Testing among Adults with Sickle Cell Disease: Indicators of Neuropathic Pain?

PubMed Central

Ezenwa, Miriam O.; Molokie, Robert E.; Wang, Zaijie Jim; Yao, Yingwei; Suarez, Marie L.; Pullum, Cherese; Schlaeger, Judith M.; Fillingim, Roger B.; Wilkie, Diana J.

2014-01-01

Objectives Pain is the hallmark symptom of sickle cell disease (SCD), yet the types of pain that these patients experience, and the underlying mechanisms, have not been well characterized. The study purpose was to determine the safety and utility of a mechanical and thermal quantitative sensory testing (QST) protocol and the feasibility of utilizing neuropathic pain questionnaires among adults with SCD. Methods A convenience sample (N=25, 18 women, mean age 38.5 ± 12.5 [20–58 years]) completed self-report pain and quality-of-life tools. Subjects also underwent testing with the TSA-II NeuroSensory Analyzer and calibrated von Frey microfilaments. Results We found that the QST protocol was safe and did not stimulate a SCD pain crisis. There was evidence of central sensitization (n=15), peripheral sensitization (n=1), a mix of central and peripheral sensitization (n=8), or no sensitization (n=1). The neuropathic pain self-report tools were feasible with evidence of construct validity; 40% of the subjects reported S-LANSS scores that were indicative of neuropathic pain and had evidence of central, peripheral or mixed sensitization. Discussion The QST protocol can be safely conducted in adults with SCD and provides evidence of central or peripheral sensitization, which is consistent with a neuropathic component to SCD pain. These findings are novel, warrant a larger confirmatory study, and indicate the need for normative QST data from African American adults and older adults. PMID:25581383

Cleaning verification: A five parameter study of a Total Organic Carbon method development and validation for the cleaning assessment of residual detergents in manufacturing equipment.

PubMed

Li, Xue; Ahmad, Imad A Haidar; Tam, James; Wang, Yan; Dao, Gina; Blasko, Andrei

2018-02-05

A Total Organic Carbon (TOC) based analytical method to quantitate trace residues of clean-in-place (CIP) detergents CIP100 ® and CIP200 ® on the surfaces of pharmaceutical manufacturing equipment was developed and validated. Five factors affecting the development and validation of the method were identified: diluent composition, diluent volume, extraction method, location for TOC sample preparation, and oxidant flow rate. Key experimental parameters were optimized to minimize contamination and to improve the sensitivity, recovery, and reliability of the method. The optimized concentration of the phosphoric acid in the swabbing solution was 0.05M, and the optimal volume of the sample solution was 30mL. The swab extraction method was 1min sonication. The use of a clean room, as compared to an isolated lab environment, was not required for method validation. The method was demonstrated to be linear with a correlation coefficient (R) of 0.9999. The average recoveries from stainless steel surfaces at multiple spike levels were >90%. The repeatability and intermediate precision results were ≤5% across the 2.2-6.6ppm range (50-150% of the target maximum carry over, MACO, limit). The method was also shown to be sensitive with a detection limit (DL) of 38ppb and a quantitation limit (QL) of 114ppb. The method validation demonstrated that the developed method is suitable for its intended use. The methodology developed in this study is generally applicable to the cleaning verification of any organic detergents used for the cleaning of pharmaceutical manufacturing equipment made of electropolished stainless steel material. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative evaluation improves specificity of myocardial perfusion SPECT in the assessment of functionally significant intermediate coronary artery stenoses: a comparative study with fractional flow reserve measurements.

PubMed

Sahiner, Ilgin; Akdemir, Umit O; Kocaman, Sinan A; Sahinarslan, Asife; Timurkaynak, Timur; Unlu, Mustafa

2013-02-01

Myocardial perfusion SPECT (MPS) is a noninvasive method commonly used for assessment of the hemodynamic significance of intermediate coronary stenoses. Fractional flow reserve (FFR) measurement is a well-validated invasive method used for the evaluation of intermediate stenoses. We aimed to determine the association between MPS and FFR findings in intermediate degree stenoses and evaluate the added value of quantification in MPS. Fifty-eight patients who underwent intracoronary pressure measurement in the catheterization laboratory to assess the physiological significance of intermediate (40-70%) left anterior descending (LAD) artery lesions, and who also underwent stress myocardial perfusion SPECT either for the assessment of an intermediate stenosis or for suspected coronary artery disease were analyzed retrospectively in the study. Quantitative analysis was performed using the 4DMSPECT program, with visual assessment performed by two experienced nuclear medicine physicians blinded to the angiographic findings. Summed stress scores (SSS) and summed difference scores (SDS) in the LAD artery territory according to the 20 segment model were calculated. A summed stress score of ≥ 3 and an SDS of ≥ 2 were assumed as pathologic, indicating significance of the lesion; a cutoff value of 0.75 was used to define abnormal FFR. Both visual and quantitative assessment results were compared with FFR using Chi-square (χ²) test. The mean time interval between two studies was 13 ± 11 days. FFR was normal in 45 and abnormal in 13 patients. Considering the FFR results as the gold standard method for assessing the significance of the lesion, the sensitivity and specificity of quantitative analysis determining the abnormal flow reserve were 85 and 84%, respectively, while visual analysis had a sensitivity of 77% and a specificity of 51%. There was a good agreement between the observers (κ = 0.856). Summed stress and difference scores demonstrated moderate inverse correlations with FFR values (r = -0.542, p < 0.001 and r = -0.506, p < 0.001, respectively). Quantitative analysis of the myocardial perfusion SPECT increases the specificity in evaluating the significance of intermediate degree coronary lesions.

Computer-based objective quantitative assessment of pulmonary parenchyma via x-ray CT

NASA Astrophysics Data System (ADS)

Uppaluri, Renuka; McLennan, Geoffrey; Sonka, Milan; Hoffman, Eric A.

1998-07-01

This paper is a review of our recent studies using a texture- based tissue characterization method called the Adaptive Multiple Feature Method. This computerized method is automated and performs tissue classification based upon the training acquired on a set of representative examples. The AMFM has been applied to several different discrimination tasks including normal subjects, subjects with interstitial lung disease, smokers, asbestos-exposed subjects, and subjects with cystic fibrosis. The AMFM has also been applied to data acquired using different scanners and scanning protocols. The AMFM has shown to be successful and better than other existing techniques in discriminating the tissues under consideration. We demonstrate that the AMFM is considerably more sensitive and specific in characterizing the lung, especially in the presence of mixed pathology, as compared to more commonly used methods. Evidence is presented suggesting that the AMFM is highly sensitive to some of the earliest disease processes.

Mass spectrometric-based stable isotopic 2-aminobenzoic acid glycan mapping for rapid glycan screening of biotherapeutics.

PubMed

Prien, Justin M; Prater, Bradley D; Qin, Qiang; Cockrill, Steven L

2010-02-15

Fast, sensitive, robust methods for "high-level" glycan screening are necessary during various stages of a biotherapeutic product's lifecycle, including clone selection, process changes, and quality control for lot release testing. Traditional glycan screening involves chromatographic or electrophoretic separation-based methods, and, although reproducible, these methods can be time-consuming. Even ultrahigh-performance chromatographic and microfluidic integrated LC/MS systems, which work on the tens of minute time scale, become lengthy when hundreds of samples are to be analyzed. Comparatively, a direct infusion mass spectrometry (MS)-based glycan screening method acquires data on a millisecond time scale, exhibits exquisite sensitivity and reproducibility, and is amenable to automated peak annotation. In addition, characterization of glycan species via sequential mass spectrometry can be performed simultaneously. Here, we demonstrate a quantitative high-throughput MS-based mapping approach using stable isotope 2-aminobenzoic acid (2-AA) for rapid "high-level" glycan screening.

Quantitative analysis of PEG-functionalized colloidal gold nanoparticles using charged aerosol detection.

PubMed

Smith, Mackensie C; Crist, Rachael M; Clogston, Jeffrey D; McNeil, Scott E

2015-05-01

Surface characteristics of a nanoparticle, such as functionalization with polyethylene glycol (PEG), are critical to understand and achieve optimal biocompatibility. Routine physicochemical characterization such as UV-vis spectroscopy (for gold nanoparticles), dynamic light scattering, and zeta potential are commonly used to assess the presence of PEG. However, these techniques are merely qualitative and are not sensitive enough to distinguish differences in PEG quantity, density, or presentation. As an alternative, two methods are described here which allow for quantitative measurement of PEG on PEGylated gold nanoparticles. The first, a displacement method, utilizes dithiothreitol to displace PEG from the gold surface. The dithiothreitol-coated gold nanoparticles are separated from the mixture via centrifugation, and the excess dithiothreitol and dissociated PEG are separated through reversed-phase high-performance liquid chromatography (RP-HPLC). The second, a dissolution method, utilizes potassium cyanide to dissolve the gold nanoparticles and liberate PEG. Excess CN(-), Au(CN)2 (-), and free PEG are separated using RP-HPLC. In both techniques, the free PEG can be quantified against a standard curve using charged aerosol detection. The displacement and dissolution methods are validated here using 2-, 5-, 10-, and 20-kDa PEGylated 30-nm colloidal gold nanoparticles. Further value in these techniques is demonstrated not only by quantitating the total PEG fraction but also by being able to be adapted to quantitate the free unbound PEG and the bound PEG fractions. This is an important distinction, as differences in the bound and unbound PEG fractions can affect biocompatibility, which would not be detected in techniques that only quantitate the total PEG fraction.

Quantitative and multiplexed detection for blood typing based on quantum dot-magnetic bead assay.

PubMed

Xu, Ting; Zhang, Qiang; Fan, Ya-Han; Li, Ru-Qing; Lu, Hua; Zhao, Shu-Ming; Jiang, Tian-Lun

2017-01-01

Accurate and reliable blood grouping is essential for safe blood transfusion. However, conventional methods are qualitative and use only single-antigen detection. We overcame these limitations by developing a simple, quantitative, and multiplexed detection method for blood grouping using quantum dots (QDs) and magnetic beads. In the QD fluorescence assay (QFA), blood group A and B antigens were quantified using QD labeling and magnetic beads, and the blood groups were identified according to the R value (the value was calculated with the fluorescence intensity from dual QD labeling) of A and B antigens. The optimized performance of QFA was established by blood typing 791 clinical samples. Quantitative and multiplexed detection for blood group antigens can be completed within 35 min with more than 10 5 red blood cells. When conditions are optimized, the assay performance is satisfactory for weak samples. The coefficients of variation between and within days were less than 10% and the reproducibility was good. The ABO blood groups of 791 clinical samples were identified by QFA, and the accuracy obtained was 100% compared with the tube test. Receiver-operating characteristic curves revealed that the QFA has high sensitivity and specificity toward clinical samples, and the cutoff points of the R value of A and B antigens were 1.483 and 1.576, respectively. In this study, we reported a novel quantitative and multiplexed method for the identification of ABO blood groups and presented an effective alternative for quantitative blood typing. This method can be used as an effective tool to improve blood typing and further guarantee clinical transfusion safety.

Identification and measurement of beta-lactam antibiotic residues in milk: integration of screening kits with liquid chromatography.

PubMed

Harik-Khan, R; Moats, W A

1995-01-01

A procedure for identifying and quantitating violative beta-lactams in milk is described. This procedure integrates beta-lactam residue detection kits with the multiresidue automated liquid chromatographic (LC) cleanup method developed in our laboratory. Spiked milk was deproteinized, extracted, and subjected to reversed-phase LC using a gradient program that concentrated the beta-lactams. Amoxicillin, ampicillin, cephapirin, ceftiofur, cloxacillin, and penicillin G were, thus, separated into 5 fractions that were subsequently tested for activity by using 4 kits. beta-lactams in the positive fractions were quantitated by analytical LC methods developed in our laboratory. The LC cleanup method separated beta-lactam antibiotics from each other and from interferences in the matrix and also concentrated the antibiotics, thus increasing the sensitivity of the kits to the beta-lactam antibiotics. The procedure facilitated the task of identifying and measuring the beta-lactam antibiotics that may be present in milk samples.

Development and validation of a reversed-phase fluorescence HPLC method for determination of bucillamine in human plasma using pre-column derivatization with monobromobimane.

PubMed

Lee, Kang Choon; Chun, Young Goo; Kim, Insoo; Shin, Beom Soo; Park, Eun-Seok; Yoo, Sun Dong; Youn, Yu Seok

2009-07-15

A simple, specific and sensitive derivatization with monobromobimane (mBrB) and the corresponding HPLC-fluorescence quantitation method for the analysis of bucillamine in human plasma was developed and validated. The analytical procedure involves a simple protein precipitation, pre-column fluorescence derivatization, and separation by reversed-phase high performance liquid chromatography (RP-HPLC). The calibration curve showed good linearity over a wide concentration range (50 ng/mL to 10 microg/mL) in human plasma (r(2)=0.9998). The lower limit of quantitation (LLOQ) was 50 ng/mL. The average precision and accuracy at LLOQ were within 6.3% and 107.6%, respectively. This method was successfully applied to a pharmacokinetic study after oral administration of a dose (300 mg) of bucillamine to 20 healthy Korean volunteers.

Quantitative proteome analysis using isobaric peptide termini labeling (IPTL).

PubMed

Arntzen, Magnus O; Koehler, Christian J; Treumann, Achim; Thiede, Bernd

2011-01-01

The quantitative comparison of proteome level changes across biological samples has become an essential feature in proteomics that remains challenging. We have recently introduced isobaric peptide termini labeling (IPTL), a novel strategy for isobaric quantification based on the derivatization of peptide termini with complementary isotopically labeled reagents. Unlike non-isobaric quantification methods, sample complexity at the MS level is not increased, providing improved sensitivity and protein coverage. The distinguishing feature of IPTL when comparing it to more established isobaric labeling methods (iTRAQ and TMT) is the presence of quantification signatures in all sequence-determining ions in MS/MS spectra, not only in the low mass reporter ion region. This makes IPTL a quantification method that is accessible to mass spectrometers with limited capabilities in the low mass range. Also, the presence of several quantification points in each MS/MS spectrum increases the robustness of the quantification procedure.

Detection of lysergic acid diethylamide (LSD) in urine by gas chromatography-ion trap tandem mass spectrometry.

PubMed

Sklerov, J H; Kalasinsky, K S; Ehorn, C A

1999-10-01

A confirmatory method for the detection and quantitation of lysergic acid diethylamide (LSD) is presented. The method employs gas chromatography-tandem mass spectrometry (GC-MS-MS) using an internal ionization ion trap detector for sensitive MS-MS-in-time measurements of LSD extracted from urine. Following a single-step solid-phase extraction of 5 mL of urine, underivatized LSD can be measured with limits of quantitation and detection of 80 and 20 pg/mL, respectively. Temperature-programmed on-column injections of urine extracts were linear over the concentration range 20-2000 pg/mL (r2 = 0.999). Intraday and interday coefficients of variation were < 6% and < 13%, respectively. This procedure has been applied to quality-control specimens and LSD-positive samples in this laboratory. Comparisons with alternate GC-MS methods and extraction procedures are discussed.

Theory and preliminary experimental verification of quantitative edge illumination x-ray phase contrast tomography.

PubMed

Hagen, C K; Diemoz, P C; Endrizzi, M; Rigon, L; Dreossi, D; Arfelli, F; Lopez, F C M; Longo, R; Olivo, A

2014-04-07

X-ray phase contrast imaging (XPCi) methods are sensitive to phase in addition to attenuation effects and, therefore, can achieve improved image contrast for weakly attenuating materials, such as often encountered in biomedical applications. Several XPCi methods exist, most of which have already been implemented in computed tomographic (CT) modality, thus allowing volumetric imaging. The Edge Illumination (EI) XPCi method had, until now, not been implemented as a CT modality. This article provides indications that quantitative 3D maps of an object's phase and attenuation can be reconstructed from EI XPCi measurements. Moreover, a theory for the reconstruction of combined phase and attenuation maps is presented. Both reconstruction strategies find applications in tissue characterisation and the identification of faint, weakly attenuating details. Experimental results for wires of known materials and for a biological object validate the theory and confirm the superiority of the phase over conventional, attenuation-based image contrast.

Metabolite fingerprinting of Camptotheca acuminata and the HPLC-ESI-MS/MS analysis of camptothecin and related alkaloids.

PubMed

Montoro, Paola; Maldini, Mariateresa; Piacente, Sonia; Macchia, Mario; Pizza, Cosimo

2010-01-20

The major phytochemical constituents, namely, alkaloids, flavonoids and ellagic acid derivatives, of leaves of Camptotheca acuminata were identified using high performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ESI-MS) in extracts of plants cultivated in Italy and collected at different growth stages. Alkaloids related to camptothecin were identified and quantified by HPLC coupled with ESI-tandem mass spectrometry (MS/MS) employing, respectively, an ion trap and a triple quadrupole mass analyser. The fragmentation patterns of alkaloids related to camptothecin were analysed and a specific Multiple Reaction Monitoring HPLC-MS/MS method was developed for the quantitative determination of these constituents. The described method provides high sensitivity and specificity for the characterisation and quantitative determination of the alkaloids in C. acuminata.

An MRM-based workflow for absolute quantitation of lysine-acetylated metabolic enzymes in mouse liver.

PubMed

Xu, Leilei; Wang, Fang; Xu, Ying; Wang, Yi; Zhang, Cuiping; Qin, Xue; Yu, Hongxiu; Yang, Pengyuan

2015-12-07

As a key post-translational modification mechanism, protein acetylation plays critical roles in regulating and/or coordinating cell metabolism. Acetylation is a prevalent modification process in enzymes. Protein acetylation modification occurs in sub-stoichiometric amounts; therefore extracting biologically meaningful information from these acetylation sites requires an adaptable, sensitive, specific, and robust method for their quantification. In this work, we combine immunoassays and multiple reaction monitoring-mass spectrometry (MRM-MS) technology to develop an absolute quantification for acetylation modification. With this hybrid method, we quantified the acetylation level of metabolic enzymes, which could demonstrate the regulatory mechanisms of the studied enzymes. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of protein acetylation in physiology and pathophysiology.

The agar diffusion scratch assay - A novel method to assess the bioactive and cytotoxic potential of new materials and compounds

PubMed Central

Pusnik, Mascha; Imeri, Minire; Deppierraz, Grégoire; Bruinink, Arie; Zinn, Manfred

2016-01-01

A profound in vitro evaluation not only of the cytotoxic but also of bioactive potential of a given compound or material is crucial for predicting potential effects in the in vivo situation. However, most of the current methods have weaknesses in either the quantitative or qualitative assessment of cytotoxicity and/or bioactivity of the test compound. Here we describe a novel assay combining the ISO 10993-5 agar diffusion test and the scratch also termed wound healing assay. In contrast to these original tests this assay is able to detect and distinguish between cytotoxic, cell migration modifying and cytotoxic plus cell migration modifying compounds, and this at higher sensitivity and in a quantitative way. PMID:26861591

Assessing the activity of sarcoidosis: quantitative /sup 67/Ga-citrate imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fajman, W.A.; Greenwald, L.V.; Staton, G.

1984-04-01

Three different methods of quantitating /sup 67/Ga-citrate lung images - a visual index, a computer-assisted index, and the total-lung-to-background ratio - were compared in 71 studies of patients with biopsy-proven sarcoidosis. Fifty consecutive cases were analyzed independently by two different observers using all three methods. In these studies, each index was correlated with the cell differential in the bronchoalveolar lavage fluid. The total-lung-to-background ratio proved to be the simplest to perform; correlated best with the original visual index and the percentage of lymphocytes obtained in bronchoalveolar lavage fluid. Sensitivity for detecting active disease was 84% compared with 64% and 58%more » for the visual and computer-assisted indices, respectively, with no sacrifice in specificity.« less

Separation and simultaneous quantitation of PGF2α and its epimer 8-iso-PGF2α using modifier-assisted differential mobility spectrometry tandem mass spectrometry.

PubMed

Liang, Chunsu; Sun, Hui; Meng, Xiangjun; Yin, Lei; Fawcett, J Paul; Yu, Huaidong; Liu, Ting; Gu, Jingkai

2018-03-01

Because many therapeutic agents are contaminated by epimeric impurities or form epimers as a result of metabolism, analytical tools capable of determining epimers are increasingly in demand. This article is a proof-of-principle report of a novel DMS-MS/MS method to separate and simultaneously quantify epimers, taking PGF2 α and its 8-epimer, 8- iso -PGF2 α , as an example. Good accuracy and precision were achieved in the range of 10-500 ng/mL with a run time of only 1.5 min. Isopropanol as organic modifier facilitated a good combination of sensitivity and separation. The method is the first example of the quantitation of epimers without chromatographic separation.

Quantitative validation of a nonlinear histology-MRI coregistration method using Generalized Q-sampling Imaging in complex human cortical white matter

PubMed Central

Gangolli, Mihika; Holleran, Laurena; Kim, Joong Hee; Stein, Thor D.; Alvarez, Victor; McKee, Ann C.; Brody, David L.

2017-01-01

Advanced diffusion MRI methods have recently been proposed for detection of pathologies such as traumatic axonal injury and chronic traumatic encephalopathy which commonly affect complex cortical brain regions. However, radiological-pathological correlations in human brain tissue that detail the relationship between the multi-component diffusion signal and underlying pathology are lacking. We present a nonlinear voxel based two dimensional coregistration method that is useful for matching diffusion signals to quantitative metrics of high resolution histological images. When validated in ex vivo human cortical tissue at a 250 × 250 × 500 micron spatial resolution, the method proved robust in correlations between generalized q-sampling imaging and histologically based white matter fiber orientations, with r = 0.94 for the primary fiber direction and r = 0.88 for secondary fiber direction in each voxel. Importantly, however, the correlation was substantially worse with reduced spatial resolution or with fiber orientations derived using a diffusion tensor model. Furthermore, we have detailed a quantitative histological metric of white matter fiber integrity termed power coherence capable of distinguishing between architecturally complex but intact white matter from disrupted white matter regions. These methods may allow for more sensitive and specific radiological-pathological correlations of neurodegenerative diseases affecting complex gray and white matter. PMID:28365421

A quantitative method for residues of macrolide antibiotics in porcine kidney by liquid chromatography/tandem mass spectrometry.

PubMed

Dickson, Leslie C; O'Byrne, Collin; Chan, Wayne

2012-01-01

An LC/MS/MS-based multiresidue quantitative method was developed for the macrolides erythromycin A, neospiramycin I, oleandomycin, spiramycin I, tilmicosin, and tylosin A in porcine kidney tissues. The Canadian Food Inspection Agency (CFIA) had as part of its analytical scope an LC/UV method for quantification of residues of two macrolide antibiotics, tilmicosin and tylosin A, in the kidney, liver, and muscle of cattle, swine, and poultry. The method could not reliably detect concentrations below 10 microg/kg. To increase the scope of the CFIA's analytical capabilities, a sensitive multiresidue quantitative method for macrolide residues in food animal tissues was required. Porcine kidney samples were extracted with acetonitrile and alkaline buffer and cleaned-up using silica-based C18 SPE cartridges. Sample extracts were analyzed using LC/MS/MS with positive electrospray ionization. Fitness for purpose was verified in a single-laboratory validation study using a second analyst. The working analytical range was 5 to 50 microg/kg. LOD and LOQ were 0.5 to 0.6 microg/kg and 1.5 to 3.0 microg/kg, respectively. Limits of identification were 0.5 to 2.0 microg/kg. Relative intermediate precisions were 8 to 17%. Average absolute recoveries were 68 to 76%.

Mini-FLOTAC and Kato-Katz: helminth eggs watching on the shore of Lake Victoria.

PubMed

Barda, Beatrice; Zepherine, Henry; Rinaldi, Laura; Cringoli, Giuseppe; Burioni, Roberto; Clementi, Massimo; Albonico, Marco

2013-07-31

One of the challenges for monitoring helminth control programmes based on preventive chemotherapy is the lack of a copro-parasitological gold-standard method that combines good sensitivity with quantitative performance, low cost, and easy-to-learn technique.The aim of our study was to evaluate and compare, the WHO recommended quantitative diagnostic technique (Kato-Katz) and the Mini-FLOTAC. Mini-FLOTAC is an innovative method based on floatation of helminths eggs with two different solutions (FS2 and FS7) using a close system (Fill-FLOTAC) with 5% fixative. Kato-Katz was performed following WHO recommendation. The study was carried out in a rural part of Tanzania, close to Lake Victoria, where the laboratory facilities are fairly scarce, and the basic technique used in the local laboratory (direct smear) was taken as reference standard. 201 children were screened for intestinal helminths and 91% of them were found to be positive. The agreement among the three techniques was calculated with k Cohen coefficient and was fairly good (k = 0.4), although the Mini-FLOTAC results were more sensitive for hookworm (98%) with FS2, and for S.mansoni (90%) with FS7 followed by Kato-Katz (91% and 60% respectively) and direct smear (30% and 10% respectively). A good agreement was found between Mini-FLOTAC and Kato-Katz (k = 0.81) with FS7 (k = 0.76) for hookworm diagnosis and a fairly good one for S.mansoni diagnosis (k = 0.5). For both infections we had a poor agreement between the two quantitative techniques and the direct smear (k<0.3). Kato-Katz diagnosed a higher number of eggs (calculated by arithmetic mean) both for hookworm (455 vs 424 EPG) and for S.mansoni (71 vs 58 EPG) compared with the Mini-FLOTAC, but the differences were not significant (p = 0.4). Mini-FLOTAC is a promising technique, comparable and as sensitive as the Kato-Katz, which is the recommended method in intestinal helminthology for monitoring helminth control programmes. A comparative advantage of the Mini-FLOTAC is that it comprises of a closed system with preserved samples that both protects the operators and allows subsequent examination of the samples. Further studies are needed to validate the mini-FLOTAC with other quantitative techniques (McMaster) and in different settings where other soil-transmitted helminths are also endemic.

Using an innovative mixed method methodology to investigate the appropriateness of a quantitative instrument in an African context: Antiretroviral treatment and quality of life.

PubMed

Greeff, Minrie; Chepuka, Lignet M; Chilemba, Winnie; Chimwaza, Angela F; Kululanga, Lucy I; Kgositau, Mabedi; Manyedi, Eva; Shaibu, Sheila; Wright, Susan C D

2014-01-01

The relationship between quality of life (QoL) and antiretroviral treatment (ART) has mainly been studied using quantitative scales often not appropriate for use in other contexts and without taking peoples' lived experiences into consideration. Sub-Saharan Africa has the highest incidence of HIV and AIDS yet there is paucity in research done on QoL. This research report is intended to give an account of the use of a mixed method convergent parallel design as a novice approach to evaluate an instrument's context specificity, appropriateness and usefulness in another context for which it was designed. Data were collected through a qualitative exploration of the experiences of QoL of people living with HIV or AIDS (PLHA) in Africa since being on ART, as well as the quantitative measurements obtained from the HIV/AIDS-targeted quality of life (HAT-QoL) instrument. This study was conducted in three African countries. Permission and ethical approval to conduct the study were obtained. Purposive voluntary sampling was used to recruit PLHA through mediators working in community-based HIV/AIDS organisations and health clinics. Interviews were analysed through open coding and the quantitative data through descriptive statistics and the Cronbach's alpha coefficient. A much wider range and richness of experiences were expressed than measured by the HAT-QoL instrument. Although an effective instrument for use in the USA, it was found not to be sensitive, appropriate and useful in an African context in its present form. The recommendations focus on adapting the instrument using the data from the in-depth interviews or to develop a context-sensitive instrument that could measure QoL of PLHA in Africa.

Influence of Ultrasonic Nonlinear Propagation on Hydrophone Calibration Using Two-Transducer Reciprocity Method

NASA Astrophysics Data System (ADS)

Yoshioka, Masahiro; Sato, Sojun; Kikuchi, Tsuneo; Matsuda, Yoichi

2006-05-01

In this study, the influence of ultrasonic nonlinear propagation on hydrophone calibration by the two-transducer reciprocity method is investigated quantitatively using the Khokhlov-Zabolotskaya-Kuznetsov (KZK) equation. It is proposed that the correction for the diffraction and attenuation of ultrasonic waves used in two-transducer reciprocity calibration can be derived using the KZK equation to remove the influence of nonlinear propagation. The validity of the correction is confirmed by comparing the sensitivities calibrated by the two-transducer reciprocity method and laser interferometry.

Radiobacteriolysis: a New Technique Using Chromium-51 for Assaying Anti- Vibrio cholerae Antibodies

PubMed Central

Blachman, Uzy; Clark, W. R.; Pickett, M. J.

1973-01-01

A new method for detecting and quantitating antibodies against Vibrio cholerae is described. The reaction involves the release of radiochromium from prelabeled vibrios in the presence of specific antibody and complement. The entire assay can be completed within 5 hr. The method is highly reproducible, immunologically specific, temperature- and complement-dependent, and significantly more sensitive than other methods currently used for titration of anti-Vibrio cholerae antibodies. The technique is also potentially applicable to titration of antibodies against other gram-negative bacteria. PMID:4570279

The quantitative insulin sensitivity check index is not able to detect early metabolic alterations in young patients with polycystic ovarian syndrome.

PubMed

Angioni, Stefano; Sanna, Stefania; Magnini, Roberta; Melis, Gian Benedetto; Fulghesu, Anna Maria

2011-07-01

To verify whether QUICKY is a suitable method for the identification of metabolic deterioration in normal weight patients affected by polycystic ovarian syndrome (PCOS). Prospective clinical study. Seventy-nine PCOS normal weight adolescent subjects, 50 eumenorrheic, normal weight, non-hirsute controls matched for age and BMI. Quantitative insulin sensitivity check index (QUICKY) and integrated secretory area under the curve of insulin values (I-AUC) during oral glucose tolerance test were calculated. Seventy-nine PCOS and 50 controls were studied. Normal insulin sensitivity was defined as upper control 95th percentile by QUICKY values <0.31, I-AUC at 180 min < 16,645. When applying the calculated I-AUC cut-off, 41 PCOS were classified as normoinsulinemic and 38 as hyperinsulinemic, whereas using the calculated QUICKY cut-off, only 19 PCOS could be classified as insulin resistant (IR). Fifteen out of the 60 non-IR PCOS presented hyperinsulinemia; fasting glucose and insulin levels and QUICKY were not sufficient to identify these subjects. Thus, QUICKY displayed a low sensitivity (44%) and specificity (91%) in the diagnosis of the metabolic disorder disclosed by I-AUC. CONCLUSIONS.: In young normal weight patients with PCOS the prevalence of early alterations of insulin metabolism are not detectable by QUICKY studies.

Development and validation of a simple and sensitive method for quantification of levodopa and carbidopa in rat and monkey plasma using derivatization and UPLC-MS/MS.

PubMed

Junnotula, Venkatraman; Licea-Perez, Hermes

2013-05-01

A simple, selective, and sensitive quantitative method has been developed for the simultaneous determination of levodopa and carbidopa in rat and monkey plasma by protein precipitation using acetonitrile containing the derivatizing reagent, flourescamine. Derivatized products of levodopa and carbidopa were separated on a BEH C18 column (2.1 mm × 50 mm; 1.7 μm particle size) using ultra high performance liquid chromatography (UHPLC) without any further purification. Tandem mass spectrometry (MS/MS) was used for detection. The method was validated over the concentration range of 5-5000 ng/mL and 3-3000 ng/mL for levodopa and carbidopa, respectively in rat and monkey plasma. Due to the poor stability of the investigated analytes in biological matrices, a mixture of sodium metabisulfite and hydrazine dihydrochloride was used as a stabilizer. This method was successfully utilized to support pharmacokinetic studies in both species. The results from assay validations and incurred samples re-analysis show that the method is selective, sensitive and robust. To our knowledge, this is the first UHPLC-MS/MS based method that utilizes derivatization with fluorescamine and provides adequate sensitivity for both levodopa and carbidopa with 50 μL of sample and a run time of 3.5 min. Copyright © 2013 Elsevier B.V. All rights reserved.

Highly sensitive catalytic spectrophotometric determination of ruthenium

NASA Astrophysics Data System (ADS)

Naik, Radhey M.; Srivastava, Abhishek; Prasad, Surendra

2008-01-01

A new and highly sensitive catalytic kinetic method (CKM) for the determination of ruthenium(III) has been established based on its catalytic effect on the oxidation of L-phenylalanine ( L-Pheala) by KMnO 4 in highly alkaline medium. The reaction has been followed spectrophotometrically by measuring the decrease in the absorbance at 526 nm. The proposed CKM is based on the fixed time procedure under optimum reaction conditions. It relies on the linear relationship where the change in the absorbance (Δ At) versus added Ru(III) amounts in the range of 0.101-2.526 ng ml -1 is plotted. Under the optimum conditions, the sensitivity of the proposed method, i.e. the limit of detection corresponding to 5 min is 0.08 ng ml -1, and decreases with increased time of analysis. The method is featured with good accuracy and reproducibility for ruthenium(III) determination. The ruthenium(III) has also been determined in presence of several interfering and non-interfering cations, anions and polyaminocarboxylates. No foreign ions interfered in the determination ruthenium(III) up to 20-fold higher concentration of foreign ions. In addition to standard solutions analysis, this method was successfully applied for the quantitative determination of ruthenium(III) in drinking water samples. The method is highly sensitive, selective and very stable. A review of recently published catalytic spectrophotometric methods for the determination of ruthenium(III) has also been presented for comparison.

Quantitative Determination of Bioactive Constituents in Noni Juice by High-performance Liquid Chromatography with Electrospray Ionization Triple Quadrupole Mass Spectrometry

PubMed Central

Yan, Yongqiu; Lu, Yu; Jiang, Shiping; Jiang, Yu; Tong, Yingpeng; Zuo, Limin; Yang, Jun; Gong, Feng; Zhang, Ling; Wang, Ping

2018-01-01

Background: Noni juice has been extensively used as folk medicine for the treatment of arthritis, infections, analgesic, colds, cancers, and diabetes by Polynesians for many years. Due to the lack of standard scientific evaluation methods, various kinds of commercial Noni juice with different quality and price were available on the market. Objective: To establish a sensitive, reliable, and accurate high-performance liquid chromatography with electrospray ionization triple quadrupole mass spectrometry (HPLC-ESI-MS/MS) method for separation, identification, and simultaneous quantitative analysis of bioactive constituents in Noni juice. Materials and Methods: The analytes and eight batches of commercially available samples from different origins were separated and analyzed by the HPLC-ESI-MS/MS method on an Agilent ZORBAX SB-C18 (150 mm × 4.6 mm i.d., 5 μm) column using a gradient elution of acetonitrile-methanol-0.05% glacial acetic acid in water (v/v) at a constant flow rate of 0.5 mL/min. Results: Seven components were identification and all of the assay parameters were within the required limits. Components were within the correlation coefficient values (R2 ≥ 0.9993) at the concentration ranges tested. The precision of the assay method was <0.91% and the repeatability between 1.36% and 3.31%. The accuracy varied from 96.40% to 103.02% and the relative standard deviations of stability were <3.91%. Samples from the same origin showed similar content while different origins showed significant different result. Conclusions: The developed methods would provide a reliable basis and be useful in the establishment of a rational quality control standard of Noni juice. SUMMARY Separation, identification, and simultaneous quantitative analysis method of seven bioactive constituents in Noni juice is originally developed by high-performance liquid chromatography with electrospray ionization triple quadrupole mass spectrometryThe presented method was successfully applied to the quality control of eight batches of commercially available samples of Noni juiceThis method is simple, sensitive, reliable, accurate, and efficient method with strong specificity, good precision, and high recovery rate and provides a reliable basis for quality control of Noni juice. Abbreviations used: HPLC-ESI-MS/MS: High-performance liquid chromatography with electrospray ionization triple quadrupole mass spectrometry, LOD: Limit of detection, LOQ: Limit of quantitation, S/N: Signal-to-noise ratio, RSD: Relative standard deviations, DP: Declustering potential, CE: Collision energy, MRM: Multiple reaction monitoring, RT: Retention time. PMID:29576704

A diameter-sensitive flow entropy method for reliability consideration in water distribution system design

NASA Astrophysics Data System (ADS)

Liu, Haixing; Savić, Dragan; Kapelan, Zoran; Zhao, Ming; Yuan, Yixing; Zhao, Hongbin

2014-07-01

Flow entropy is a measure of uniformity of pipe flows in water distribution systems. By maximizing flow entropy one can identify reliable layouts or connectivity in networks. In order to overcome the disadvantage of the common definition of flow entropy that does not consider the impact of pipe diameter on reliability, an extended definition of flow entropy, termed as diameter-sensitive flow entropy, is proposed. This new methodology is then assessed by using other reliability methods, including Monte Carlo Simulation, a pipe failure probability model, and a surrogate measure (resilience index) integrated with water demand and pipe failure uncertainty. The reliability assessment is based on a sample of WDS designs derived from an optimization process for each of the two benchmark networks. Correlation analysis is used to evaluate quantitatively the relationship between entropy and reliability. To ensure reliability, a comparative analysis between the flow entropy and the new method is conducted. The results demonstrate that the diameter-sensitive flow entropy shows consistently much stronger correlation with the three reliability measures than simple flow entropy. Therefore, the new flow entropy method can be taken as a better surrogate measure for reliability and could be potentially integrated into the optimal design problem of WDSs. Sensitivity analysis results show that the velocity parameters used in the new flow entropy has no significant impact on the relationship between diameter-sensitive flow entropy and reliability.

Dual function microscope for quantitative DIC and birefringence imaging

NASA Astrophysics Data System (ADS)

Li, Chengshuai; Zhu, Yizheng

2016-03-01

A spectral multiplexing interferometry (SXI) method is presented for integrated birefringence and phase gradient measurement on label-free biological specimens. With SXI, the retardation and orientation of sample birefringence are simultaneously encoded onto two separate spectral carrier waves, generated by a crystal retarder oriented at a specific angle. Thus sufficient information for birefringence determination can be obtained from a single interference spectrum, eliminating the need for multiple acquisitions with mechanical rotation or electrical modulation. In addition, with the insertion of a Nomarski prism, the setup can then acquire quantitative differential interference contrast images. Red blood cells infected by malaria parasites are imaged for birefringence retardation as well as phase gradient. The results demonstrate that the SXI approach can achieve both quantitative phase imaging and birefringence imaging with a single, high-sensitivity system.

Highly sensitive electrochemical detection of human telomerase activity based on bio-barcode method.

PubMed

Li, Ying; Liu, Bangwei; Li, Xia; Wei, Qingli

2010-07-15

In the present study, an electrochemical method for highly sensitive detection of human telomerase activity was developed based on bio-barcode amplification assay. Telomerase was extracted from HeLa cells, then the extract was mixed with telomerase substrate (TS) primer to perform extension reaction. The extension product was hybridized with the capture DNA immobilized on the Au electrode and then reacted with the signal DNA on Au nanoparticles to form a sandwich hybridization mode. Electrochemical signals were generated by chronocoulometric interrogation of [Ru(NH(3))(6)](3+) that quantitatively binds to the DNA on Au nanoparticles via electrostatic interaction. This method can detect the telomerase activity from as little as 10 cultured cancer cells without the polymerase chain reaction (PCR) amplification of telomerase extension product. Copyright (c) 2010 Elsevier B.V. All rights reserved.

[A quantitative real time polymerase chain reaction for detection of HBV covalently closed circular DNA in livers of the HBV infected patients].

PubMed

Wang, Mei-Rong; Qiu, Ning; Lu, Shi-Chun; Xiu, Dian-Rong; Yu, Jian-Guo; Li, Tong; Liu, Xue-En; Zhuang, Hui

2011-05-01

To establish and optimize a sensitive and specific quantitative real-time polymerase chain reaction (PCR) method for detection of hepatitis B virus covalently closed circular DNA (HBV cccDNA) in liver tissue. Specific primers and probes were designed to detect HBV DNA (tDNA) and cccDNA. A series of plasmids (3.44 × 10(0) - 3.44 × 10(9) copies/µl) containing a full double-stranded copies of HBV genome (genotype C) were used to establish the standard curve of real-time PCR. Liver samples of 33 patients with HBV related hepatocellular carcinoma (HCC), 13 Chronic hepatitis B patients (CHB) and 10 non-HBV patients were collected to verify the sensitivity and specificity of the assay. A fraction of extracted DNA was digested with a Plasmid-Safe ATP-dependent Dnase (PSAD) for HBV cccDNA detection and the remaining was used for tDNA and β-globin detection. The amount (copies/cell) of HBV cccDNA and tDNA were measured by a real-time PCR, using β-globin housekeeping gene as a quantitation standard. The standard curves of real-time PCR with a linear range of 3.44 × 10(0) to 3.44 × 10(9) copies/µl were established for detecting HBV cccDNA and tDNA, and both of the lowest detection limits of HBV cccDNA and tDNA were 3.44 × 10(0) copies/µl. The lowest quantitation levels of HBV cccDNA in liver tissues tested in 33 HBV related HCC patients and 13 CHB patients were 0.003 copies/cell and 0.031 copies/cell, respectively. HBV cccDNA and tDNA in liver tissue of 10 non-HBV patient appeared to be negative. The true positive rate was increasing through the digestion of HBV DNA by PSAD, and the analytic specificity of cccDNA detection improved by 7.24 × 10(2) times. Liver tissues of 2 patients were retested 5 times in the PCR for detecting cccDNA and the coefficient of variations on cycle threshold (Ct) were between 0.224% - 0.609%. A highly sensitive and specific quantitative real time PCR method for the detection of HBV cccDNA in liver tissue was established and could be used for clinical and epidemiological studies.

Highly Sensitive and Automated Surface Enhanced Raman Scattering-based Immunoassay for H5N1 Detection with Digital Microfluidics.

PubMed

Wang, Yang; Ruan, Qingyu; Lei, Zhi-Chao; Lin, Shui-Chao; Zhu, Zhi; Zhou, Leiji; Yang, Chaoyong

2018-04-17

Digital microfluidics (DMF) is a powerful platform for a broad range of applications, especially immunoassays having multiple steps, due to the advantages of low reagent consumption and high automatization. Surface enhanced Raman scattering (SERS) has been proven as an attractive method for highly sensitive and multiplex detection, because of its remarkable signal amplification and excellent spatial resolution. Here we propose a SERS-based immunoassay with DMF for rapid, automated, and sensitive detection of disease biomarkers. SERS tags labeled with Raman reporter 4-mercaptobenzoic acid (4-MBA) were synthesized with a core@shell nanostructure and showed strong signals, good uniformity, and high stability. A sandwich immunoassay was designed, in which magnetic beads coated with antibodies were used as solid support to capture antigens from samples to form a beads-antibody-antigen immunocomplex. By labeling the immunocomplex with a detection antibody-functionalized SERS tag, antigen can be sensitively detected through the strong SERS signal. The automation capability of DMF can greatly simplify the assay procedure while reducing the risk of exposure to hazardous samples. Quantitative detection of avian influenza virus H5N1 in buffer and human serum was implemented to demonstrate the utility of the DMF-SERS method. The DMF-SERS method shows excellent sensitivity (LOD of 74 pg/mL) and selectivity for H5N1 detection with less assay time (<1 h) and lower reagent consumption (∼30 μL) compared to the standard ELISA method. Therefore, this DMF-SERS method holds great potentials for automated and sensitive detection of a variety of infectious diseases.

Use of local noise power spectrum and wavelet analysis in quantitative image quality assurance for EPIDs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Soyoung

Purpose: To investigate the use of local noise power spectrum (NPS) to characterize image noise and wavelet analysis to isolate defective pixels and inter-subpanel flat-fielding artifacts for quantitative quality assurance (QA) of electronic portal imaging devices (EPIDs). Methods: A total of 93 image sets including custom-made bar-pattern images and open exposure images were collected from four iViewGT a-Si EPID systems over three years. Global quantitative metrics such as modulation transform function (MTF), NPS, and detective quantum efficiency (DQE) were computed for each image set. Local NPS was also calculated for individual subpanels by sampling region of interests within each subpanelmore » of the EPID. The 1D NPS, obtained by radially averaging the 2D NPS, was fitted to a power-law function. The r-square value of the linear regression analysis was used as a singular metric to characterize the noise properties of individual subpanels of the EPID. The sensitivity of the local NPS was first compared with the global quantitative metrics using historical image sets. It was then compared with two commonly used commercial QA systems with images collected after applying two different EPID calibration methods (single-level gain and multilevel gain). To detect isolated defective pixels and inter-subpanel flat-fielding artifacts, Haar wavelet transform was applied on the images. Results: Global quantitative metrics including MTF, NPS, and DQE showed little change over the period of data collection. On the contrary, a strong correlation between the local NPS (r-square values) and the variation of the EPID noise condition was observed. The local NPS analysis indicated image quality improvement with the r-square values increased from 0.80 ± 0.03 (before calibration) to 0.85 ± 0.03 (after single-level gain calibration) and to 0.96 ± 0.03 (after multilevel gain calibration), while the commercial QA systems failed to distinguish the image quality improvement between the two calibration methods. With wavelet analysis, defective pixels and inter-subpanel flat-fielding artifacts were clearly identified as spikes after thresholding the inversely transformed images. Conclusions: The proposed local NPS (r-square values) showed superior sensitivity to the noise level variations of individual subpanels compared with global quantitative metrics such as MTF, NPS, and DQE. Wavelet analysis was effective in detecting isolated defective pixels and inter-subpanel flat-fielding artifacts. The proposed methods are promising for the early detection of imaging artifacts of EPIDs.« less

Retinal status analysis method based on feature extraction and quantitative grading in OCT images.

PubMed

Fu, Dongmei; Tong, Hejun; Zheng, Shuang; Luo, Ling; Gao, Fulin; Minar, Jiri

2016-07-22

Optical coherence tomography (OCT) is widely used in ophthalmology for viewing the morphology of the retina, which is important for disease detection and assessing therapeutic effect. The diagnosis of retinal diseases is based primarily on the subjective analysis of OCT images by trained ophthalmologists. This paper describes an OCT images automatic analysis method for computer-aided disease diagnosis and it is a critical part of the eye fundus diagnosis. This study analyzed 300 OCT images acquired by Optovue Avanti RTVue XR (Optovue Corp., Fremont, CA). Firstly, the normal retinal reference model based on retinal boundaries was presented. Subsequently, two kinds of quantitative methods based on geometric features and morphological features were proposed. This paper put forward a retinal abnormal grading decision-making method which was used in actual analysis and evaluation of multiple OCT images. This paper showed detailed analysis process by four retinal OCT images with different abnormal degrees. The final grading results verified that the analysis method can distinguish abnormal severity and lesion regions. This paper presented the simulation of the 150 test images, where the results of analysis of retinal status showed that the sensitivity was 0.94 and specificity was 0.92.The proposed method can speed up diagnostic process and objectively evaluate the retinal status. This paper aims on studies of retinal status automatic analysis method based on feature extraction and quantitative grading in OCT images. The proposed method can obtain the parameters and the features that are associated with retinal morphology. Quantitative analysis and evaluation of these features are combined with reference model which can realize the target image abnormal judgment and provide a reference for disease diagnosis.

Characterization of Infrastructure Materials using Nonlinear Ultrasonics

NASA Astrophysics Data System (ADS)

Liu, Minghe

In order to improve the safety, reliability, cost, and performance of civil and mechanical structures/components, it is necessary to develop techniques that are capable of characterizing and quantifying the amount of distributed damage in engineering materials before any detectable discontinuities (cracks, delaminations, voids, etc.) appear. In this dissertation, novel nonlinear ultrasonic NDE methods are developed and applied to characterize cumulative damage such as fatigue damage in metallic materials and degradation of cement-based materials due to chemical reactions. First, nonlinear Rayleigh surface waves are used to measure the near-surface residual stresses in shot-peened aluminum alloy (AA 7075) samples. Results show that the nonlinear Rayleigh wave is very sensitive to near-surface residual stresses, and has the potential to quantitatively detect them. Second, a novel two-wave mixing method is theoretically developed and numerically verified. This method is then successfully applied to detect the fatigue damage in aluminum alloy (AA 6061) samples subjected to monotonic compression. In addition to its high sensitivity to fatigue damage, this collinear wave mixing method allows the measurement over a specific region of interest in the specimen, and this capability makes it possible to obtain spatial distribution of fatigue damage through the thickness direction of the sample by simply timing the transducers. Third, the nonlinear wave mixing method is used to characterize the degradation of cement-based materials caused by alkali-silica reaction (ASR). It is found that the nonlinear ultrasonic method is sensitive to detect ASR damage at very early stage, and has the potential to identify the different damage stages. Finally, a micromechanics-based chemo-mechanical model is developed which relates the acoustic nonlinearity parameter to ASR damage. This model provides a way to quantitatively predict the changes in the acoustic nonlinearity parameter due to ASR damage, which can be used to guide experimental measurements for nondestructive evaluation of ASR damage.

Development of a Quantitative Sandwich Enzyme-Linked Immunosorbent Assay for Detecting the MPT64 Antigen of Mycobacterium tuberculosis

PubMed Central

Ji, Mijung; Cho, Byungki; Cho, Young Shik; Park, Song-Yong; Cho, Sang-Nae

2014-01-01

Purpose Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. Materials and Methods The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. Results The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×104 CFU/mL and 2.0×106 CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). Conclusion The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis. PMID:24719143

Ultrasound-Based Quantification of Vitreous Floaters Correlates with Contrast Sensitivity and Quality of Life

PubMed Central

Mamou, Jonathan; Wa, Christianne A.; Yee, Kenneth M. P.; Silverman, Ronald H.; Ketterling, Jeffrey A.; Sadun, Alfredo A.; Sebag, J.

2015-01-01

Purpose. Clinical evaluation of floaters lacks quantitative assessment of vitreous structure. This study used quantitative ultrasound (QUS) to measure vitreous opacities. Since floaters reduce contrast sensitivity (CS) and quality of life (Visual Function Questionnaire [VFQ]), it is hypothesized that QUS will correlate with CS and VFQ in patients with floaters. Methods. Twenty-two eyes (22 subjects; age = 57 ± 19 years) with floaters were evaluated with Freiburg acuity contrast testing (FrACT; %Weber) and VFQ. Ultrasonography used a customized probe (15-MHz center frequency, 20-mm focal length, 7-mm aperture) with longitudinal and transverse scans taken in primary gaze and a horizontal longitudinal scan through premacular vitreous in temporal gaze. Each scan set had 100 frames of log-compressed envelope data. Within each frame, two regions of interest (ROIs) were analyzed (whole-central and posterior vitreous) to yield three parameters (energy, E; mean amplitude, M; and percentage of vitreous filled by echodensities, P50) averaged over the entire 100-frame dataset. Statistical analyses evaluated E, M, and P50 correlations with CS and VFQ. Results. Contrast sensitivity ranged from 1.19%W (normal) to 5.59%W. All QUS parameters in two scan positions within the whole-central ROI correlated with CS (R > 0.67, P < 0.001). P50 in the nasal longitudinal position had R = 0.867 (P < 0.001). Correlations with VFQ ranged from R = 0.52 (P < 0.013) to R = 0.65 (P < 0.001). Conclusions. Quantitative ultrasound provides quantitative measures of vitreous echodensity that correlate with CS and VFQ, providing objective assessment of vitreous structure underlying the functional disturbances induced by floaters, useful to quantify vitreous disease severity and the response to therapy. PMID:25613948

Enhanced analytical sensitivity of a quantitative PCR for CMV using a modified nucleic-acid extraction procedure.

PubMed

Ferreira-Gonzalez, A; Yanovich, S; Langley, M R; Weymouth, L A; Wilkinson, D S; Garrett, C T

2000-01-01

Accurate and rapid diagnosis of CMV disease in immunocompromised individuals remains a challenge. Quantitative polymerase chain reaction (QPCR) methods for detection of CMV in peripheral blood mononuclear cells (PBMC) have improved the positive and negative predictive value of PCR for diagnosis of CMV disease. However, detection of CMV in plasma has demonstrated a lower negative predictive value for plasma as compared with PBMC. To enhance the sensitivity of the QPCR assay for plasma specimens, plasma samples were centrifuged before nucleic-acid extraction and the extracted DNA resolubilized in reduced volume. Optimization of the nucleic-acid extraction focused on decreasing or eliminating the presence of inhibitors in the pelleted plasma. Quantitation was achieved by co-amplifying an internal quantitative standard (IS) with the same primer sequences as CMV. PCR products were detected by hybridization in a 96-well microtiter plate coated with a CMV or IS specific probe. The precision of the QPCR assay for samples prepared from untreated and from pelleted plasma was then assessed. The coefficient of variation for both types of samples was almost identical and the magnitude of the coefficient of variations was reduced by a factor of ten if the data were log transformed. Linearity of the QPCR assay extended over a 3.3-log range for both types of samples but the range of linearity for pelleted plasma was 20 to 40,000 viral copies/ml (vc/ml) in contrast to 300 to 400,000 vc/ml for plasma. Thus, centrifugation of plasma before nucleic-acid extraction and resuspension of extracted CMV DNA in reduced volume enhanced the analytical sensitivity approximately tenfold over the dynamic range of the assay. Copyright 2000 Wiley-Liss, Inc.

Community sensitization and decision-making for trial participation: a mixed-methods study from The Gambia.

PubMed

Dierickx, Susan; O'Neill, Sarah; Gryseels, Charlotte; Immaculate Anyango, Edna; Bannister-Tyrrell, Melanie; Okebe, Joseph; Mwesigwa, Julia; Jaiteh, Fatou; Gerrets, René; Ravinetto, Raffaella; D'Alessandro, Umberto; Peeters Grietens, Koen

2017-08-16

Ensuring individual free and informed decision-making for research participation is challenging. It is thought that preliminarily informing communities through 'community sensitization' procedures may improve individual decision-making. This study set out to assess the relevance of community sensitization for individual decision-making in research participation in rural Gambia. This anthropological mixed-methods study triangulated qualitative methods and quantitative survey methods in the context of an observational study and a clinical trial on malaria carried out by the Medical Research Council Unit Gambia. Although 38.7% of the respondents were present during sensitization sessions, 91.1% of the respondents were inclined to participate in the trial when surveyed after the sensitization and prior to the informed consent process. This difference can be explained by the informal transmission of information within the community after the community sensitization, expectations such as the benefits of participation based on previous research experiences, and the positive reputation of the research institute. Commonly mentioned barriers to participation were blood sampling and the potential disapproval of the household head. Community sensitization is effective in providing first-hand, reliable information to communities as the information is cascaded to those who could not attend the sessions. However, further research is needed to assess how the informal spread of information further shapes people's expectations, how the process engages with existing social relations and hierarchies (e.g. local political power structures; permissions of heads of households) and how this influences or changes individual consent. © 2017 The Authors Developing World Bioethics Published by John Wiley & Sons Ltd.

GIS coupled Multiple Criteria based Decision Support for Classification of Urban Coastal Areas in India

NASA Astrophysics Data System (ADS)

Dhiman, R.; Kalbar, P.; Inamdar, A. B.

2017-12-01

Coastal area classification in India is a challenge for federal and state government agencies due to fragile institutional framework, unclear directions in implementation of costal regulations and violations happening at private and government level. This work is an attempt to improvise the objectivity of existing classification methods to synergies the ecological systems and socioeconomic development in coastal cities. We developed a Geographic information system coupled Multi-criteria Decision Making (GIS-MCDM) approach to classify urban coastal areas where utility functions are used to transform the costal features into quantitative membership values after assessing the sensitivity of urban coastal ecosystem. Furthermore, these membership values for costal features are applied in different weighting schemes to derive Coastal Area Index (CAI) which classifies the coastal areas in four distinct categories viz. 1) No Development Zone, 2) Highly Sensitive Zone, 3) Moderately Sensitive Zone and 4) Low Sensitive Zone based on the sensitivity of urban coastal ecosystem. Mumbai, a coastal megacity in India is used as case study for demonstration of proposed method. Finally, uncertainty analysis using Monte Carlo approach to validate the sensitivity of CAI under specific multiple scenarios is carried out. Results of CAI method shows the clear demarcation of coastal areas in GIS environment based on the ecological sensitivity. CAI provides better decision support for federal and state level agencies to classify urban coastal areas according to the regional requirement of coastal resources considering resilience and sustainable development. CAI method will strengthen the existing institutional framework for decision making in classification of urban coastal areas where most effective coastal management options can be proposed.

Increased sensitivity of OSHA method analysis of diacetyl and 2,3-pentanedione in air

PubMed Central

LeBouf, Ryan; Simmons, Michael

2018-01-01

Gas chromatography/mass spectrometry (GC/MS) operated in selected ion monitoring mode was used to enhance the sensitivity of OSHA Methods 1013/1016 for measuring diacetyl and 2,3-pentanedione in air samples. The original methods use flame ionization detection which cannot achieve the required sensitivity to quantify samples at or below the NIOSH recommended exposure limits (REL: 5 ppb for diacetyl and 9.3 ppb for 2,3-pentanedione) when sampling for both diacetyl and 2,3-pentanedione. OSHA Method 1012 was developed to measure diacetyl at lower levels but requires an electron capture detector, and a sample preparation time of 36 hours. Using GC/MS allows detection of these two alpha-diketones at lower levels than OSHA Method 1012 for diacetyl and OSHA Method 1016 for 2,3-pentanedione. Acetoin and 2,3-hexanedione may also be measured using this technique. Method quantification limits were 1.1 ppb for diacetyl (22% of the REL), 1.1 ppb for 2,3-pentanedione (12% of the REL), 1.1 ppb for 2,3-hexanedione, and 2.1 ppb for acetoin. Average extraction efficiencies above the limit of quantitation were 100% for diacetyl, 92% for 2,3-pentanedione, 89% for 2,3-hexanedione, and 87% for acetoin. Mass spectrometry with OSHA Methods 1013/1016 could be used by analytical laboratories to provide more sensitive and accurate measures of exposure to diacetyl and 2,3-pentanedione. PMID:27792470

Increased sensitivity of OSHA method analysis of diacetyl and 2,3-pentanedione in air.

PubMed

LeBouf, Ryan; Simmons, Michael

2017-05-01

Gas chromatography/mass spectrometry (GC/MS) operated in selected ion monitoring mode was used to enhance the sensitivity of OSHA Methods 1013/1016 for measuring diacetyl and 2,3-pentanedione in air samples. The original methods use flame ionization detection which cannot achieve the required sensitivity to quantify samples at or below the NIOSH recommended exposure limits (REL: 5 ppb for diacetyl and 9.3 ppb for 2,3-pentanedione) when sampling for both diacetyl and 2,3-pentanedione. OSHA Method 1012 was developed to measure diacetyl at lower levels but requires an electron capture detector, and a sample preparation time of 36 hours. Using GC/MS allows detection of these two alpha-diketones at lower levels than OSHA Method 1012 for diacetyl and OSHA Method 1016 for 2,3-pentanedione. Acetoin and 2,3-hexanedione may also be measured using this technique. Method quantification limits were 1.1 ppb for diacetyl (22% of the REL), 1.1 ppb for 2,3-pentanedione (12% of the REL), 1.1 ppb for 2,3-hexanedione, and 2.1 ppb for acetoin. Average extraction efficiencies above the limit of quantitation were 100% for diacetyl, 92% for 2,3-pentanedione, 89% for 2,3-hexanedione, and 87% for acetoin. Mass spectrometry with OSHA Methods 1013/1016 could be used by analytical laboratories to provide more sensitive and accurate measures of exposure to diacetyl and 2,3-pentanedione.

Quantitation Error in 1H MRS Caused by B1 Inhomogeneity and Chemical Shift Displacement.

PubMed

Watanabe, Hidehiro; Takaya, Nobuhiro

2017-11-08

The quantitation accuracy in proton magnetic resonance spectroscopy ( 1 H MRS) improves at higher B 0 field. However, a larger chemical shift displacement (CSD) and stronger B 1 inhomogeneity exist. In this work, we evaluate the quantitation accuracy for the spectra of metabolite mixtures in phantom experiments at 4.7T. We demonstrate a position-dependent error in quantitation and propose a correction method by measuring water signals. All experiments were conducted on a whole-body 4.7T magnetic resonance (MR) system with a quadrature volume coil for transmission and reception. We arranged three bottles filled with metabolite solutions of N-acetyl aspartate (NAA) and creatine (Cr) in a vertical row inside a cylindrical phantom filled with water. Peak areas of three singlets of NAA and Cr were measured on three 1 H spectra at three volume of interests (VOIs) inside three bottles. We also measured a series of water spectra with a shifted carrier frequency and measured a reception sensitivity map. The ratios of NAA and Cr at 3.92 ppm to Cr at 3.01 ppm differed amongst the three VOIs in peak area, which leads to a position-dependent error. The nature of slope depicting the relationship between peak areas and the shifted values of frequency was like that between the reception sensitivities and displacement at every VOI. CSD and inhomogeneity of reception sensitivity cause amplitude modulation along the direction of chemical shift on the spectra, resulting in a quantitation error. This error may be more significant at higher B 0 field where CSD and B 1 inhomogeneity are more severe. This error may also occur in reception using a surface coil having inhomogeneous B 1 . Since this type of error is around a few percent, the data should be analyzed with greater attention while discussing small differences in the studies of 1 H MRS.

A new kind of metal detector based on chaotic oscillator

NASA Astrophysics Data System (ADS)

Hu, Wenjing

2017-12-01

The sensitivity of a metal detector greatly depends on the identification ability to weak signals from the probe. In order to improve the sensitivity of metal detectors, this paper applies the Duffing chaotic oscillator to metal detectors based on its characteristic which is very sensitive to weak periodic signals. To make a suitable Duffing system for detectors, this paper computes two Lyapunov characteristics exponents of the Duffing oscillator, which help to obtain the threshold of the Duffing system in the critical state accurately and give quantitative criteria for chaos. Meanwhile, a corresponding simulation model of the chaotic oscillator is made by the Simulink tool box of Matlab. Simulation results shows that Duffing oscillator is very sensitive to sinusoidal signals in high frequency cases. And experimental results show that the measurable diameter of metal particles is about 1.5mm. It indicates that this new method can feasibly and effectively improve the metal detector sensitivity.

Sensitivity analysis, calibration, and testing of a distributed hydrological model using error‐based weighting and one objective function

USGS Publications Warehouse

Foglia, L.; Hill, Mary C.; Mehl, Steffen W.; Burlando, P.

2009-01-01

We evaluate the utility of three interrelated means of using data to calibrate the fully distributed rainfall‐runoff model TOPKAPI as applied to the Maggia Valley drainage area in Switzerland. The use of error‐based weighting of observation and prior information data, local sensitivity analysis, and single‐objective function nonlinear regression provides quantitative evaluation of sensitivity of the 35 model parameters to the data, identification of data types most important to the calibration, and identification of correlations among parameters that contribute to nonuniqueness. Sensitivity analysis required only 71 model runs, and regression required about 50 model runs. The approach presented appears to be ideal for evaluation of models with long run times or as a preliminary step to more computationally demanding methods. The statistics used include composite scaled sensitivities, parameter correlation coefficients, leverage, Cook's D, and DFBETAS. Tests suggest predictive ability of the calibrated model typical of hydrologic models.

Robust detection of rare species using environmental DNA: The importance of primer specificity

Treesearch

Taylor M. Wilcox; Kevin S. McKelvey; Michael K. Young; Stephen F. Jane; Winsor H. Lowe; Andrew R. Whiteley; Michael K. Schwartz

2013-01-01

Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probebased chemistries may represent a particularly powerful tool because of the method's sensitivity, specificity, and potential to quantify target DNA. However, there has been little work understanding the performance of these assays in the presence...

A novel strategy for rapid detection of NT-proBNP

NASA Astrophysics Data System (ADS)

Cui, Qiyao; Sun, Honghao; Zhu, Hui

2017-09-01

In order to establish a simple, rapid, sensitive, and specific quantitative assay to detect the biomarkers of heart failure, in this study, biotin-streptavidin technology was employed with fluorescence immunochromatographic assay to detect the concentration of the biomarkers in serum, and this method was applied to detect NT-proBNP, which is valuable for diagnostic evaluation of heart failure.

An efficient spectrofluorimetric method adopts doxazosin, terazosin and alfuzosin coupling with orthophthalaldehyde: Application in human plasma

NASA Astrophysics Data System (ADS)

Omar, Mahmoud A.; Mohamed, Abdel-Maaboud I.; Derayea, Sayed M.; Hammad, Mohamed A.; Mohamed, Abobakr A.

2018-04-01

A new, selective and sensitive spectrofluorimetric method was designed for the quantitation of doxazosin (DOX), terazosin (TER) and alfuzosin (ALF) in their dosage forms and human plasma. The method adopts efficient derivatization of the studied drugs with ortho-phthalaldehyde (OPA), in the presence of 2-mercaptoethanol in borate buffer (pH 9.7) to generate a highly fluorescent isoindole derivatives, which can strongly enhance the fluorescence intensities of the studied drugs, allowing their sensitive determination at 430 nm after excitation at 337 nm. The fluorescence-concentration plots were rectilinear over the ranges (10.0-400.0) ng/mL. Detection and quantification limits were found to be (0.52-3.88) and (1.59-11.76) ng/mL, respectively. The proposed method was validated according to ICH guidelines, and successfully applied for the determination of pharmaceutical preparations of the studied drugs. Moreover, the high sensitivity of the proposed method permits its successful application to the analysis of the studied drugs in spiked human plasma with % recovery (96.12 ± 1.34-100.66 ± 0.57, n = 3). A proposal for the reaction mechanism was presented.

Recent advances in rice genome and chromosome structure research by fluorescence in situ hybridization (FISH).

PubMed

Ohmido, Nobuko; Fukui, Kiichi; Kinoshita, Toshiro

2010-01-01

Fluorescence in situ hybridization (FISH) is an effective method for the physical mapping of genes and repetitive DNA sequences on chromosomes. Physical mapping of unique nucleotide sequences on specific rice chromosome regions was performed using a combination of chromosome identification and highly sensitive FISH. Increases in the detection sensitivity of smaller DNA sequences and improvements in spatial resolution have ushered in a new phase in FISH technology. Thus, it is now possible to perform in situ hybridization on somatic chromosomes, pachytene chromosomes, and even on extended DNA fibers (EDFs). Pachytene-FISH allows the integration of genetic linkage maps and quantitative chromosome maps. Visualization methods using FISH can reveal the spatial organization of the centromere, heterochromatin/euchromatin, and the terminal structures of rice chromosomes. Furthermore, EDF-FISH and the DNA combing technique can resolve a spatial distance of 1 kb between adjacent DNA sequences, and the detection of even a 300-bp target is now feasible. The copy numbers of various repetitive sequences and the sizes of various DNA molecules were quantitatively measured using the molecular combing technique. This review describes the significance of these advances in molecular cytology in rice and discusses future applications in plant studies using visualization techniques.

Real-time quantitative PCR detection of circulating tumor cells using tag DNA mediated signal amplification strategy.

PubMed

Mei, Ting; Lu, Xuewen; Sun, Ning; Li, Xiaomei; Chen, Jitao; Liang, Min; Zhou, Xinke; Fang, Zhiyuan

2018-06-05

The level of circulating tumor cell (CTCs) is a reliable marker for tumor burden and malignant progression. Quantification of CTCs remains technically challenging due to the rarity of these cells in peripheral blood. In the present study, we established a real-time quantitative PCR (Q-PCR) based method for sensitive detection of CTCs without DNA extraction. Blood sample was first turned to erythrocyte lyses and then incubated with two antibodies, tag-DNA modified CK-19 antibody and magnetic beads conjugated EpCAM antibody. Tumor cells were further enriched by magnetic separation. Tag-DNA that immobilized on tumor cells through CK-19 antibodies were also retrieved, which was further quantified by Q-PCR. This assay was able to detect single tumor cell in a 5 mL blood sample. The detection rate of clinical tumor blood sample was 92.3%. Furthermore, CTC count in patient was correlated with tumor stage and tumor status. The signal amplification was based on tag DNA rather than tumor gene, which was independent of nucleic acid extraction. With high sensitivity and convenience, this method can be a good alternative for the determination of cancer progress. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid and sensitive quantitation of the antiproliferative agent mitoguazone in small volumes of plasma by high-performance liquid chromatography with ultraviolet detection.

PubMed

Cheng, Ching-Ling; Lin, E Gin; Chou, Chen-Hsi

2003-08-15

Mitoguazone is an antiproliferative agent used in chemotherapy. This study describes a simple and sensitive high-performance liquid chromatographic method for the determination of mitoguazone in 100 microl of plasma. Samples were deproteinized with 100 microl of a solution of internal standard (amiloride, 10 microg/ml) in acetonitrile. An aliquot of the supernatant was injected onto the column. HPLC separation was achieved on a silica column with the mobile phase of methanol-50 mM potassium phosphate buffer (pH 3)-triethylamine (80:20:0.3, v/v), at a flow-rate of 1 ml/min. The eluent was detected at 320 nm. The retention time was about 5.5 min for amiloride and 12 min for mitoguazone. No endogenous substances were found to interfere. Calibration curves were linear from 0.25 to 50 microg/ml. The absolute recoveries of mitoguazone and amiloride were both greater than 84%. The limit of quantitation was 0.25 microg/ml. The intra- and inter-day precision (expressed as RSD) was 5.8%, or less, and the accuracy was 94.7% of the nominal concentration. The method is suitable in pharmacokinetic investigation and monitoring mitoguazone concentration.

Single molecule quantitation and sequencing of rare translocations using microfluidic nested digital PCR.

PubMed

Shuga, Joe; Zeng, Yong; Novak, Richard; Lan, Qing; Tang, Xiaojiang; Rothman, Nathaniel; Vermeulen, Roel; Li, Laiyu; Hubbard, Alan; Zhang, Luoping; Mathies, Richard A; Smith, Martyn T

2013-09-01

Cancers are heterogeneous and genetically unstable. New methods are needed that provide the sensitivity and specificity to query single cells at the genetic loci that drive cancer progression, thereby enabling researchers to study the progression of individual tumors. Here, we report the development and application of a bead-based hemi-nested microfluidic droplet digital PCR (dPCR) technology to achieve 'quantitative' measurement and single-molecule sequencing of somatically acquired carcinogenic translocations at extremely low levels (<10(-6)) in healthy subjects. We use this technique in our healthy study population to determine the overall concentration of the t(14;18) translocation, which is strongly associated with follicular lymphoma. The nested dPCR approach improves the detection limit to 1×10(-7) or lower while maintaining the analysis efficiency and specificity. Further, the bead-based dPCR enabled us to isolate and quantify the relative amounts of the various clonal forms of t(14;18) translocation in these subjects, and the single-molecule sensitivity and resolution of dPCR led to the discovery of new clonal forms of t(14;18) that were otherwise masked by the conventional quantitative PCR measurements. In this manner, we created a quantitative map for this carcinogenic mutation in this healthy population and identified the positions on chromosomes 14 and 18 where the vast majority of these t(14;18) events occur.

Evaluation of a rapid, quantitative real-time PCR method for enumeration of pathogenic Candida cells in water

USGS Publications Warehouse

Brinkman, Nichole E.; Haugland, Richard A.; Wymer, Larry J.; Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Vesper, Stephen J.

2003-01-01

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5′ nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

A quantitative PCR assay for the detection and quantification of Babesia bovis and B. bigemina.

PubMed

Buling, A; Criado-Fornelio, A; Asenzo, G; Benitez, D; Barba-Carretero, J C; Florin-Christensen, M

2007-06-20

The haemoparasites Babesia bovis and Babesia bigemina affect cattle over vast areas of the tropics and temperate parts of the world. Microscopic examination of blood smears allows the detection of clinical cases of babesiosis, but this procedure lacks sensitivity when parasitaemia levels are low. In addition, differentiating between similar haemoparasites can be very difficult. Molecular diagnostic procedures can, however, overcome these problems. This paper reports a quantitative PCR (qPCR) assay involving the use of SYBR Green. Based on the amplification of a small fragment of the cytochrome b gene, this method shows both high sensitivity and specificity, and allows quantification of parasite DNA. In tests, reproducible quantitative results were obtained over the range of 0.1 ng to 0.1 fg of parasite DNA. Melting curve analysis differentiated between B. bovis and B. bigemina. To assess the performance of the new qPCR procedure it was used to screen for babesiosis in 40 cows and 80 horses. B. bigemina was detected in five cows (three of these were also found to be positive by standard PCR techniques targeting the 18S rRNA gene). In addition, B. bovis was detected in one horse and B. bigemina in two horses using the proposed method, while none was found positive by ribosomal standard PCR. The sequences of the B. bigemina cytochrome b and 18S rRNA genes were completely conserved in isolates from Spain and Argentina, while those of B. bovis showed moderate polymorphism.

Multiparametric MRI Assessment of Human Articular Cartilage Degeneration: Correlation with Quantitative Histology and Mechanical Properties

PubMed Central

Rautiainen, Jari; Nissi, Mikko J.; Salo, Elli-Noora; Tiitu, Virpi; Finnilä, Mikko A.J.; Aho, Olli-Matti; Saarakkala, Simo; Lehenkari, Petri; Ellermann, Jutta; Nieminen, Miika T.

2014-01-01

Purpose To evaluate the sensitivity of quantitative MRI techniques (T1, T1,Gd, T2, continous wave (CW) T1ρ dispersion, adiabatic T1ρ, adiabatic T2ρ, RAFF and inversion-prepared magnetization transfer (MT)) for assessment of human articular cartilage with varying degrees of natural degeneration. Methods Osteochondral samples (n = 14) were obtained from the tibial plateaus of patients undergoing total knee replacement. MRI of the specimens was performed at 9.4 T and the relaxation time maps were evaluated in the cartilage zones. For reference, quantitative histology, OARSI grading and biomechanical measurements were performed and correlated with MRI findings. Results All MRI parameters, except T1,Gd, showed statistically significant differences in tangential and full-thickness ROIs between early and advanced osteoarthritis (OA) groups, as classified by OARSI grading. CW-T1ρ showed significant dispersion in all ROIs and featured classical laminar structure of cartilage with spin-lock powers below 1000 Hz. Adiabatic T1ρ, T2ρ, CW-T1ρ, MT and RAFF correlated strongly with OARSI grade and biomechanical parameters. Conclusion MRI parameters were able to differentiate between early and advanced OA. Furthermore, rotating frame methods, namely adiabatic T1ρ, adiabatic T2ρ, CW-T1ρ and RAFF, as well as MT experiment correlated strongly with biomechanical parameters and OARSI grade, suggesting high sensitivity of the parameters for cartilage degeneration. PMID:25104181

Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer

NASA Astrophysics Data System (ADS)

Wang, Evelyn H.; Combe, Peter C.; Schug, Kevin A.

2016-05-01

Methods that can efficiently and effectively quantify proteins are needed to support increasing demand in many bioanalytical fields. Triple quadrupole mass spectrometry (QQQ-MS) is sensitive and specific, and it is routinely used to quantify small molecules. However, low resolution fragmentation-dependent MS detection can pose inherent difficulties for intact proteins. In this research, we investigated variables that affect protein and fragment ion signals to enable protein quantitation using QQQ-MS. Collision induced dissociation gas pressure and collision energy were found to be the most crucial variables for optimization. Multiple reaction monitoring (MRM) transitions for seven standard proteins, including lysozyme, ubiquitin, cytochrome c from both equine and bovine, lactalbumin, myoglobin, and prostate-specific antigen (PSA) were determined. Assuming the eventual goal of applying such methodology is to analyze protein in biological fluids, a liquid chromatography method was developed. Calibration curves of six standard proteins (excluding PSA) were obtained to show the feasibility of intact protein quantification using QQQ-MS. Linearity (2-3 orders), limits of detection (0.5-50 μg/mL), accuracy (<5% error), and precision (1%-12% CV) were determined for each model protein. Sensitivities for different proteins varied considerably. Biological fluids, including human urine, equine plasma, and bovine plasma were used to demonstrate the specificity of the approach. The purpose of this model study was to identify, study, and demonstrate the advantages and challenges for QQQ-MS-based intact protein quantitation, a largely underutilized approach to date.

Detection of nucleophosmin 1 mutations by quantitative real-time polymerase chain reaction versus capillary electrophoresis: a comparative study.

PubMed

Barakat, Fareed H; Luthra, Rajyalakshmi; Yin, C Cameron; Barkoh, Bedia A; Hai, Seema; Jamil, Waqar; Bhakta, Yaminiben I; Chen, Su; Medeiros, L Jeffrey; Zuo, Zhuang

2011-08-01

Nucleophosmin 1 (NPM1) is the most commonly mutated gene in acute myeloid leukemia. Detection of NPM1 mutations is useful for stratifying patients for therapy, predicting prognosis, and assessing for minimal residual disease. Several methods have been developed to rapidly detect NPM1 mutations in genomic DNA and/or messenger RNA specimens. To directly compare a quantitative real-time polymerase chain reaction (qPCR) assay with a widely used capillary electrophoresis assay for detecting NPM1 mutations. We adopted and modified a qPCR assay designed to detect the 6 most common NPM1 mutations and performed the assay in parallel with capillary electrophoresis assay in 207 bone marrow aspirate or peripheral blood samples from patients with a range of hematolymphoid neoplasms. The qPCR assay demonstrated a higher analytical sensitivity than the capillary electrophoresis 1/1000 versus 1/40, respectively. The capillary electrophoresis assay generated 10 equivocal results that needed to be repeated, whereas the qPCR assay generated only 1 equivocal result. After test conditions were optimized, the qPCR and capillary electrophoresis methods produced 100% concordant results, 85 positive and 122 negative. Given the higher analytical sensitivity and specificity of the qPCR assay, that assay is less likely to generate equivocal results than the capillary electrophoresis assay. Moreover, the qPCR assay is quantitative, faster, cheaper, less prone to contamination, and well suited for monitoring minimal residual disease.

Comparison of quantitative and qualitative tests for glucose-6-phosphate dehydrogenase deficiency in the neonatal period.

PubMed

Keihanian, F; Basirjafari, S; Darbandi, B; Saeidinia, A; Jafroodi, M; Sharafi, R; Shakiba, M

2017-06-01

Considering the high prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency among newborns, different screening methods have been established in various countries. In this study, we aimed to assess the prevalence of G6PD deficiency among newborns in Rasht, Iran, and compare G6PD activity in cord blood samples, using quantitative and qualitative tests. This cross-sectional, prospective study was performed at five largest hospitals in Rasht, Guilan Province, Iran. The screening tests were performed for all the newborns, referred to these hospitals. Specimens were characterized in terms of G6PD activity under ultraviolet light, using the kinetic method and the qualitative fluorescent spot test (FST). We also determined the sensitivity, specificity, negative predictive value, and positive predictive value of the qualitative assay. Blood samples were collected from 1474 newborns. Overall, 757 (51.4%) subjects were male. As the findings revealed, 1376 (93.4%) newborns showed normal G6PD activity, while 98 (6.6%) had G6PD deficiency. There was a significant difference in the mean G6PD level between males and females (P = 0.0001). Also, a significant relationship was detected between FST results and the mean values obtained in the quantitative test (P < 0.0001). According to the present study, FST showed acceptable sensitivity and specificity for G6PD activity, although it appeared inefficient for diagnostic purposes in some cases. © 2017 John Wiley & Sons Ltd.

Perspectives on Non-Animal Alternatives for Assessing Sensitization Potential in Allergic Contact Dermatitis

PubMed Central

Sharma, Nripen S.; Jindal, Rohit; Mitra, Bhaskar; Lee, Serom; Li, Lulu; Maguire, Tim J.; Schloss, Rene; Yarmush, Martin L.

2014-01-01

Skin sensitization remains a major environmental and occupational health hazard. Animal models have been used as the gold standard method of choice for estimating chemical sensitization potential. However, a growing international drive and consensus for minimizing animal usage have prompted the development of in vitro methods to assess chemical sensitivity. In this paper, we examine existing approaches including in silico models, cell and tissue based assays for distinguishing between sensitizers and irritants. The in silico approaches that have been discussed include Quantitative Structure Activity Relationships (QSAR) and QSAR based expert models that correlate chemical molecular structure with biological activity and mechanism based read-across models that incorporate compound electrophilicity. The cell and tissue based assays rely on an assortment of mono and co-culture cell systems in conjunction with 3D skin models. Given the complexity of allergen induced immune responses, and the limited ability of existing systems to capture the entire gamut of cellular and molecular events associated with these responses, we also introduce a microfabricated platform that can capture all the key steps involved in allergic contact sensitivity. Finally, we describe the development of an integrated testing strategy comprised of two or three tier systems for evaluating sensitization potential of chemicals. PMID:24741377

A New Method for Assessing How Sensitivity and Specificity of Linkage Studies Affects Estimation

PubMed Central

Moore, Cecilia L.; Amin, Janaki; Gidding, Heather F.; Law, Matthew G.

2014-01-01

Background While the importance of record linkage is widely recognised, few studies have attempted to quantify how linkage errors may have impacted on their own findings and outcomes. Even where authors of linkage studies have attempted to estimate sensitivity and specificity based on subjects with known status, the effects of false negatives and positives on event rates and estimates of effect are not often described. Methods We present quantification of the effect of sensitivity and specificity of the linkage process on event rates and incidence, as well as the resultant effect on relative risks. Formulae to estimate the true number of events and estimated relative risk adjusted for given linkage sensitivity and specificity are then derived and applied to data from a prisoner mortality study. The implications of false positive and false negative matches are also discussed. Discussion Comparisons of the effect of sensitivity and specificity on incidence and relative risks indicate that it is more important for linkages to be highly specific than sensitive, particularly if true incidence rates are low. We would recommend that, where possible, some quantitative estimates of the sensitivity and specificity of the linkage process be performed, allowing the effect of these quantities on observed results to be assessed. PMID:25068293

To list or not to list? The value and detriment of freelisting in ethnobotanical studies.

PubMed

Zambrana, Narel Y Paniagua; Bussmann, Rainer W; Hart, Robbie E; Huanca, Araceli L Moya; Soria, Gere Ortiz; Vaca, Milton Ortiz; Álvarez, David Ortiz; Morán, Jorge Soria; Morán, María Soria; Chávez, Saúl; Moreno, Bertha Chávez; Moreno, Gualberto Chávez; Roca, Oscar; Siripi, Erlin

2018-04-01

Although freelisting and semi-structured interviews are widespread methods in ethnobotany, few studies quantitatively examine how these methods may bias results. Using a comprehensive ethnobotanical inventory of palm species, uses and names in the Chácobo tribe of Bolivia, we show that interviews elicit more items than freelists, but the effect is sensitive to sample size, item type and data categorization. This implies that even subtle methodological choices may greatly affect reported results.

Quantitative Determination of Bioactive Constituents in Noni Juice by High-performance Liquid Chromatography with Electrospray Ionization Triple Quadrupole Mass Spectrometry.

PubMed

Yan, Yongqiu; Lu, Yu; Jiang, Shiping; Jiang, Yu; Tong, Yingpeng; Zuo, Limin; Yang, Jun; Gong, Feng; Zhang, Ling; Wang, Ping

2018-01-01

Noni juice has been extensively used as folk medicine for the treatment of arthritis, infections, analgesic, colds, cancers, and diabetes by Polynesians for many years. Due to the lack of standard scientific evaluation methods, various kinds of commercial Noni juice with different quality and price were available on the market. To establish a sensitive, reliable, and accurate high-performance liquid chromatography with electrospray ionization triple quadrupole mass spectrometry (HPLC-ESI-MS/MS) method for separation, identification, and simultaneous quantitative analysis of bioactive constituents in Noni juice. The analytes and eight batches of commercially available samples from different origins were separated and analyzed by the HPLC-ESI-MS/MS method on an Agilent ZORBAX SB-C 18 (150 mm × 4.6 mm i.d., 5 μm) column using a gradient elution of acetonitrile-methanol-0.05% glacial acetic acid in water (v/v) at a constant flow rate of 0.5 mL/min. Seven components were identification and all of the assay parameters were within the required limits. Components were within the correlation coefficient values ( R 2 ≥ 0.9993) at the concentration ranges tested. The precision of the assay method was <0.91% and the repeatability between 1.36% and 3.31%. The accuracy varied from 96.40% to 103.02% and the relative standard deviations of stability were <3.91%. Samples from the same origin showed similar content while different origins showed significant different result. The developed methods would provide a reliable basis and be useful in the establishment of a rational quality control standard of Noni juice. Separation, identification, and simultaneous quantitative analysis method of seven bioactive constituents in Noni juice is originally developed by high-performance liquid chromatography with electrospray ionization triple quadrupole mass spectrometryThe presented method was successfully applied to the quality control of eight batches of commercially available samples of Noni juiceThis method is simple, sensitive, reliable, accurate, and efficient method with strong specificity, good precision, and high recovery rate and provides a reliable basis for quality control of Noni juice. Abbreviations used: HPLC-ESI-MS/MS: High-performance liquid chromatography with electrospray ionization triple quadrupole mass spectrometry, LOD: Limit of detection, LOQ: Limit of quantitation, S/N: Signal-to-noise ratio, RSD: Relative standard deviations, DP: Declustering potential, CE: Collision energy, MRM: Multiple reaction monitoring, RT: Retention time.

Finding the bottom and using it

PubMed Central

Sandoval, Ruben M.; Wang, Exing; Molitoris, Bruce A.

2014-01-01

Maximizing 2-photon parameters used in acquiring images for quantitative intravital microscopy, especially when high sensitivity is required, remains an open area of investigation. Here we present data on correctly setting the black level of the photomultiplier tube amplifier by adjusting the offset to allow for accurate quantitation of low intensity processes. When the black level is set too high some low intensity pixel values become zero and a nonlinear degradation in sensitivity occurs rendering otherwise quantifiable low intensity values virtually undetectable. Initial studies using a series of increasing offsets for a sequence of concentrations of fluorescent albumin in vitro revealed a loss of sensitivity for higher offsets at lower albumin concentrations. A similar decrease in sensitivity, and therefore the ability to correctly determine the glomerular permeability coefficient of albumin, occurred in vivo at higher offset. Finding the offset that yields accurate and linear data are essential for quantitative analysis when high sensitivity is required. PMID:25313346

Ratiometric Raman Spectroscopy for Quantification of Protein Oxidative Damage

PubMed Central

Jiang, Dongping; Yanney, Michael; Zou, Sige; Sygula, Andrzej

2009-01-01

A novel ratiometric Raman spectroscopic (RMRS) method has been developed for quantitative determination of protein carbonyl levels. Oxidized bovine serum albumin (BSA) and oxidized lysozyme were used as model proteins to demonstrate this method. The technique involves conjugation of protein carbonyls with dinitrophenyl hydrazine (DNPH), followed by drop coating deposition Raman spectral acquisition (DCDR). The RMRS method is easy to implement as it requires only one conjugation reaction, a single spectral acquisition, and does not require sample calibration. Characteristic peaks from both protein and DNPH moieties are obtained in a single spectral acquisition, allowing the protein carbonyl level to be calculated from the peak intensity ratio. Detection sensitivity for the RMRS method is ~0.33 pmol carbonyl/measurement. Fluorescence and/or immunoassay based techniques only detect a signal from the labeling molecule and thus yield no structural or quantitative information for the modified protein while the RMRS technique provides for protein identification and protein carbonyl quantification in a single experiment. PMID:19457432

Polarization sensitive optical coherence tomography – a review [Invited

PubMed Central

de Boer, Johannes F.; Hitzenberger, Christoph K.; Yasuno, Yoshiaki

2017-01-01

Optical coherence tomography (OCT) is now a well-established modality for high-resolution cross-sectional and three-dimensional imaging of transparent and translucent samples and tissues. Conventional, intensity based OCT, however, does not provide a tissue-specific contrast, causing an ambiguity with image interpretation in several cases. Polarization sensitive (PS) OCT draws advantage from the fact that several materials and tissues can change the light’s polarization state, adding an additional contrast channel and providing quantitative information. In this paper, we review basic and advanced methods of PS-OCT and demonstrate its use in selected biomedical applications. PMID:28663869

Quantitative polarized light microscopy using spectral multiplexing interferometry.

PubMed

Li, Chengshuai; Zhu, Yizheng

2015-06-01

We propose an interferometric spectral multiplexing method for measuring birefringent specimens with simple configuration and high sensitivity. The retardation and orientation of sample birefringence are simultaneously encoded onto two spectral carrier waves, generated interferometrically by a birefringent crystal through polarization mixing. A single interference spectrum hence contains sufficient information for birefringence determination, eliminating the need for mechanical rotation or electrical modulation. The technique is analyzed theoretically and validated experimentally on cellulose film. System simplicity permits the possibility of mitigating system birefringence background. Further analysis demonstrates the technique's exquisite sensitivity as high as ∼20  pm for retardation measurement.

X-ray fluorescence holography studies for a Cu3Au crystal

NASA Astrophysics Data System (ADS)

Dąbrowski, K. M.; Dul, D. T.; Jaworska-Gołąb, T.; Rysz, J.; Korecki, P.

2015-12-01

In this work we show that performing a numerical correction for beam attenuation and indirect excitation allows one to fully restore element sensitivity in the three-dimensional reconstruction of the atomic structure. This is exemplified by a comparison of atomic images reconstructed from holograms measured for ordered and disordered phases of a Cu3Au crystal that clearly show sensitivity to changes in occupancy of the atomic sites. Moreover, the numerical correction, which is based on quantitative methods of X-ray fluorescence spectroscopy, was extended to take into account the influence of a disturbed overlayer in the sample.

A non-contact, thermal noise based method for the calibration of lateral deflection sensitivity in atomic force microscopy.

PubMed

Mullin, Nic; Hobbs, Jamie K

2014-11-01

Calibration of lateral forces and displacements has been a long standing problem in lateral force microscopies. Recently, it was shown by Wagner et al. that the thermal noise spectrum of the first torsional mode may be used to calibrate the deflection sensitivity of the detector. This method is quick, non-destructive and may be performed in situ in air or liquid. Here we make a full quantitative comparison of the lateral inverse optical lever sensitivity obtained by the lateral thermal noise method and the shape independent method developed by Anderson et al. We find that the thermal method provides accurate results for a wide variety of rectangular cantilevers, provided that the geometry of the cantilever is suitable for torsional stiffness calibration by the torsional Sader method, in-plane bending of the cantilever may be eliminated or accounted for and that any scaling of the lateral deflection signal between the measurement of the lateral thermal noise and the measurement of the lateral deflection is eliminated or corrected for. We also demonstrate that the thermal method may be used to characterize the linearity of the detector signal as a function of position, and find a deviation of less than 8% for the instrument used.

Analytical methods for quantitation of prenylated flavonoids from hops.

PubMed

Nikolić, Dejan; van Breemen, Richard B

2013-01-01

The female flowers of hops ( Humulus lupulus L.) are used as a flavoring agent in the brewing industry. There is growing interest in possible health benefits of hops, particularly as estrogenic and chemopreventive agents. Among the possible active constituents, most of the attention has focused on prenylated flavonoids, which can chemically be classified as prenylated chalcones and prenylated flavanones. Among chalcones, xanthohumol (XN) and desmethylxanthohumol (DMX) have been the most studied, while among flavanones, 8-prenylnaringenin (8-PN) and 6-prenylnaringenin (6-PN) have received the most attention. Because of the interest in medicinal properties of prenylated flavonoids, there is demand for accurate, reproducible and sensitive analytical methods to quantify these compounds in various matrices. Such methods are needed, for example, for quality control and standardization of hop extracts, measurement of the content of prenylated flavonoids in beer, and to determine pharmacokinetic properties of prenylated flavonoids in animals and humans. This review summarizes currently available analytical methods for quantitative analysis of the major prenylated flavonoids, with an emphasis on the LC-MS and LC-MS-MS methods and their recent applications to biomedical research on hops. This review covers all methods in which prenylated flavonoids have been measured, either as the primary analytes or as a part of a larger group of analytes. The review also discusses methodological issues relating to the quantitative analysis of these compounds regardless of the chosen analytical approach.

Quantitative estimation of gymnemagenin in Gymnema sylvestre extract and its marketed formulations using the HPLC-ESI-MS/MS method.

PubMed

Kamble, Bhagyashree; Gupta, Ankur; Patil, Dada; Janrao, Shirish; Khatal, Laxman; Duraiswamy, B

2013-02-01

Gymnema sylvestre, with gymnemic acids as active pharmacological constituents, is a popular ayurvedic herb and has been used to treat diabetes, as a remedy for cough and as a diuretic. However, very few analytical methods are available for quality control of this herb and its marketed formulations. To develop and validate a new, rapid, sensitive and selective HPLC-ESI (electrospray ionisation)-MS/MS method for quantitative estimation of gymnemagenin in G. sylvestre and its marketed formulations. HPLC-ESI-MS/MS method using a multiple reactions monitoring mode was used for quantitation of gymnemagenin. Separation was carried out on a Luna C-18 column using gradient elution of water and methanol (with 0.1% formic acid and 0.3% ammonia). The developed method was validated as per International Conference on Harmonisation Guideline ICH-Q2B and found to be accurate, precise and linear over a relatively wide range of concentrations (5.280-305.920 ng/mL). Gymnemagenin contents were found from 0.056 ± 0.002 to 4.77 ± 0.59% w/w in G. sylvestre and its marketed formulations. The method established is simple, rapid, with high sample throughput, and can be used as a tool for quality control of G. sylvestre and its formulations. Copyright © 2012 John Wiley & Sons, Ltd.