scholarly journals A Metric for Quantifying the Resolution of Molecular Assays

2021 ◽  
Author(s):  
Brandon D Wilson ◽  
Michael Eisenstein ◽  
Hyongsok Tom Soh
Keyword(s):  
Molecular Diagnostics ◽  
Limit Of Detection ◽  
Clinical Care ◽  
Glucose Monitoring ◽  
Molecular Assays ◽  
Performance Metric ◽  
Raising Awareness ◽  
Assay Performance ◽  
Molecular Concentration ◽  
Quantitative Performance

Many assay developers focus on limit of detection (LOD) as a primary performance metric, and LOD is indeed useful for assays designed to determine the presence or absence of an analyte. However, LOD is less useful for continuous assays designed to discriminate between concentrations of an analyte-e.g., glucose monitoring in diabetes, where clinical care is guided by particular concentration ranges and thresholds. In such scenarios, it would be valuable to quantify discriminatory resolution-i.e., whether an assay can differentiate 1 pM from 10 pM-but no such standardized metric currently exists. Here, we propose a useful solution, termed "resolution of molecular concentration" (RMC). RMC offers a simple means for characterizing quantitative resolution and quickly comparing the quantitative performance of assays. By raising awareness of the limitations of current metrics for evaluating assay performance, we hope to empower the molecular diagnostics community to evaluate their methods in a more application-appropriate manner.

scholarly journals Considerations for performance metrics of metagenomic next generation sequencing analyses

2020 ◽  
Author(s):  
Jason G. Kralj ◽  
Stephanie L. Servetas ◽  
Samuel P. Forry ◽  
Scott A. Jackson
Keyword(s):  
Performance Metrics ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Ground Truth ◽  
Negative Control ◽  
Fitness For Purpose ◽  
Performance Metric ◽  
The One ◽  
Sensitivity Specificity ◽  
Harmonic Means

AbstractEvaluating the performance of metagenomics analyses has proven a challenge, due in part to limited ground-truth standards, broad application space, and numerous evaluation methods and metrics. Application of traditional clinical performance metrics (i.e. sensitivity, specificity, etc.) using taxonomic classifiers do not fit the “one-bug-one-test” paradigm. Ultimately, users need methods that evaluate fitness-for-purpose and identify their analyses’ strengths and weaknesses. Within a defined cohort, reporting performance metrics by taxon, rather than by sample, will clarify this evaluation. An estimated limit of detection, positive and negative control samples, and true positive and negative true results are necessary criteria for all investigated taxa. Use of summary metrics should be restricted to comparing results of similar cohorts and data, and should employ harmonic means and continuous products for each performance metric rather than arithmetic mean. Such consideration will ensure meaningful comparisons and evaluation of fitness-for-purpose.

scholarly journals A Novel Bead-Based Immunoassay for the Measurement of Heat Shock Proteins 27 and 70

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 863
Author(s):  
Rose Njemini ◽  
Katrijn Verhaeghen ◽  
Tony Mets ◽  
Ilse Weets ◽  
Ivan Bautmans
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Single Molecule ◽  
Limit Of Detection ◽  
Scientific Evidence ◽  
Repeated Testing ◽  
Standard Material ◽  
Coefficients Of Variation ◽  
Assay Performance ◽  
Shock Proteins

Heat shock proteins (HSPs) play an essential role in protecting proteins from denaturation and are implicated in diverse pathophysiological conditions like cardiovascular diseases, cancer, infections, and neurodegenerative diseases. Scientific evidence indicates that if HSP expression falls below a certain level, cells become sensitive to oxidative damage that accelerates protein aggregation diseases. On the other hand, persistently enhanced levels of HSP can lead to inflammatory and oncogenic changes. To date, although techniques for measuring HSPs exist, these assays are limited for use in specific sample types or are time consuming. Therefore, in the present study, we developed a single-molecule assay digital ELISA technology (Single Molecule Array—SIMOA) for the measurement of HSPs, which is time effective and can be adapted to measure multiple analytes simultaneously from a single sample. This technique combines two distinct HSP-specific antibodies that recognize different epitopes on the HSP molecule. A recombinant human HSP protein was used as the standard material. The assay performance characteristics were evaluated by repeated testing of samples spiked with HSP peptide at different levels. The limit of detection was 0.16 and 2 ng/mL for HSP27 and HSP70, respectively. The inter- and intra-assay coefficients of variation were less than 20% in all tested conditions for both HSPs. The HSP levels assayed after serial dilution of samples portrayed dilutional linearity (on average 109%, R2 = 0.998, p < 0.001, for HSP27 and 93%, R2 = 0.994, p < 0.001, for HSP70). A high linear response was also demonstrated with admixtures of plasma exhibiting relatively very low and high levels of HSP70 (R2 = 0.982, p < 0.001). Analyte spike recovery varied between 57% and 95%. Moreover, the relative HSP values obtained using Western blotting correlated significantly with HSP values obtained with the newly developed SIMOA assay (r = 0.815, p < 0.001 and r = 0.895, p < 0.001 for HSP70 and HSP27, respectively), indicating that our method is reliable. In conclusion, the assay demonstrates analytical performance for the accurate assessment of HSPs in various sample types and offers the advantage of a huge range of dilution linearity, indicating that samples with HSP concentration highly above the calibration range can be diluted into range without affecting the precision of the assay.

scholarly journals Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect Orientia tsutsugamushi DNA

2019 ◽  
Vol 4 (3) ◽  
pp. 113 ◽  
Author(s):  
Chao ◽  
Belinskaya ◽  
Zhang ◽  
Jiang ◽  
Ching
Keyword(s):  
Clinical Utility ◽  
Scrub Typhus ◽  
Limit Of Detection ◽  
Single Copy ◽  
Immunofluorescence Assay ◽  
Clinical Settings ◽  
Orientia Tsutsugamushi ◽  
Multiple Copy ◽  
Molecular Assays ◽  
Copy Gene

Scrub typhus is caused by an obligated intracellular organism, Orientia tsutsugamushi (Orientia). The disease was traditionally thought to be limited in the tsutsugamushi triangle. Recently, scrub typhus has been confirmed in areas outside the triangle. Serological diagnosis of scrub typhus relies on indirect immunofluorescence assay (IFA). Molecular assays such as PCR, qPCR, loop-mediated isothermal amplification, and recombinase polymerase amplification are often targeting a single copy gene. These assays are sensitive and specific, yet they are not broadly used in clinical settings possibly due to low circulating Orientia in blood. In this study, we compared qPCR results using a multiple copy (traD) gene with those using a single copy (47 kDa) gene to assess the improvement of sensitivity and limit of detection. Our results demonstrate that the qPCR using the traD gene provides superior sensitivity in 15 Orientia strains. The limit of detection is below single Orientia genome equivalent and the assay retains specificity with excessive DNA from mouse, chiggers and human. The clinical utility was evaluated using confirmed scrub typhus positive and negative samples. The results show 100% sensitivity and specificity in these samples suggesting that the traD gene qPCR may be useful for clinical diagnosis of Orientia infection.

scholarly journals Predictive Glucose Trends From Continuous Glucose Monitoring: Friend or Foe in Clinical Decision Making?

2019 ◽  
Vol 13 (5) ◽  
pp. 963-966 ◽  
Author(s):  
Kathryn Fantasia ◽  
Katherine Modzelewski ◽  
Devin Steenkamp
Keyword(s):  
Clinical Decision Making ◽  
Clinical Care ◽  
Glucose Monitoring ◽  
Clinical Decision ◽  
The United States ◽  
Rate Of Change ◽  
Inherent Difficulty ◽  
Continuous Glucose Monitor ◽  
Freestyle Libre ◽  
Practical Recommendations

In this commentary, we briefly review the currently recommended approaches to interpretation and management of continuous glucose monitor (CGM) rate of change (ROC) trend arrows and discuss the inherent difficulty in incorporating practical recommendations for their application into routine clinical care. We have limited our review and discussion to the currently available Dexcom G5 and G6 CGM systems and Abbott’s Freestyle Libre flash glucose monitor (FGM) system, as they are the most widely used and currently approved for nonadjunctive use in the United States.

FDA analysis of MRD data in hematologic malignancy applications.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 2541-2541 ◽  
Author(s):  
Nicole Gormley ◽  
Vishal Bhatnagar ◽  
Lori A. Ehrlich ◽  
Bindu Kanapuru ◽  
Hyon-Zu Lee ◽  
...  
Keyword(s):  
Test Performance ◽  
Residual Disease ◽  
Limit Of Detection ◽  
Hematologic Malignancy ◽  
Surrogate Endpoint ◽  
Drug Approval ◽  
Hematologic Malignancies ◽  
Performance Characteristics ◽  
Assay Performance

2541 Background: There is considerable interest in the use of minimal residual disease (MRD) in clinical trials of hematologic malignancies, especially as a potential surrogate endpoint to expedite drug approval. Although surrogacy has not yet been established in most hematologic malignancies (except CML), MRD data has been submitted in applications to the agency to allow for further characterization of the product’s activity. Here we describe the new drug applications (NDA) or biologics licensing applications (BLA) that included MRD data and provide an analysis of the MRD data and the Agency’s decisions. Methods: The authors reviewed internal databases for all original and supplemental NDA and BLA applications submitted to the Division of Hematology Products (DHP) between 2014 and 2016 that pertained to malignant hematologic conditions. The applications were reviewed for inclusion of MRD data. The clinical reviews and prescribing information (PI) of the identified applications were reviewed for assessment of MRD data and FDA’s findings. Results: There were 34 NDAs or BLAs submitted between 2014-2016. Of these, 13 (38%) included MRD data, in the following diseases: CML, CLL, ALL, and MM. MRD data was added to the PI in 6 cases (46%), not included in 4 (31%), and was not proposed for inclusion in 3 (23%). Among the 6 cases in which MRD data was included in the PI, 5 used PCR testing and 1 used flow cytometry. Among the 4 in which MRD data was not included in the PI, the identified issues included: missing data among those in CR, incomplete test performance characteristics data (e.g.-limit of detection), disparate sample sources (blood, bone marrow), high amount of test failure rates (i.e.-inability to identify a clonal rearrangement), lack of test validation in the proposed disease setting, and inappropriate planned statistical analyses. Conclusions: Nearly 40% of applications submitted to DHP between 2014 and 2016 included MRD data. While the data submitted was deemed adequate for inclusion in the PI in 46% of cases, 31% of applications contained MRD data that the Agency deemed un-interpretable. Data collection and assay performance characteristics should be of significant rigor and completeness to allow for comprehensive review.

Development of Lateral Flow Immunochromatographic Strips for Micropollutants Screening Using Colorants of Aptamer Functionalized Nanogold Particles Part I Methodology and Optimization

2018 ◽  
Vol 101 (5) ◽  
pp. 1402-1407 ◽  
Author(s):  
Shuai Zhao ◽  
Shan Zhang ◽  
Sai Wang ◽  
Jiahui Liu ◽  
Yiyang Dong
Keyword(s):  
Reading Time ◽  
Limit Of Detection ◽  
Blocking Agent ◽  
Rapid Identification ◽  
Lateral Flow ◽  
Immunochromatographic Strip ◽  
Assay Performance ◽  
Nanogold Particles ◽  
Visual Limit ◽  
Optimized Conditions

Abstract A methodology of lateral flow immunochromatographic strip based on aptamer was developed for on-site detection of the small molecule micropollutants. In the present study, we try for the first time to investigate the feasibility of developing a strip assay for the analysis of micropollutants as methodological prototypes by combining the high selectivity and affinity of aptamers with the unique optical properties of nanogolds. This quantitative method was based on the competition for the aptamer between targets and DNA probes. Crucial parameters that might influence the sensitivity, such as the size of nanogolds, amount of aptamer, type and pH of streptavidin, type of nitrocellulose (NC) membrane, blocking procedure, and reading time, were systematically investigated to obtain the optimum assay performance. With the optimized conditions [nanogolds 25 nm, 50 μM aptamer, pH 8 of GSA (a type of streptavidin named “SA Gold,” which is a sulfhydrylization streptavidin), Millipore HFC 135 NC membrane, 1% bovine serum albumin as the blocking agent and added in the running buffer and sample pad soakage agents, and 20 min reading time] the aptamer-based lateral flow assay will show a low visual limit of detection and scanning reader LOD. The strip for on-site screening using colorants of aptamer functionalized nanogold particles did not require any complicated equipment and was a potential portable tool for rapid identification of micropollutants.

scholarly journals Verification and Validation of SARS-CoV-2 Assay Performance on the Abbott m2000 and Alinity m Systems

2021 ◽  
Author(s):  
Julie W. Hirschhorn ◽  
April Kegl ◽  
Tanisha Dickerson ◽  
W. Bailey Glen ◽  
Gang Xu ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Clinical Performance ◽  
Cycle Number ◽  
Percent Agreement ◽  
Assay Performance ◽  
Diagnostic Panel ◽  
Highly Correlated ◽  
Clinical Verification ◽  
Bridging Study ◽  
Bridging Studies

We verified the analytical performance of the Abbott RealTime SARS-CoV-2 assay on the m2000 system and compared its clinical performance to the CDC 2019-nCoV Real-Time PCR Diagnostic Panel and the ThermoFisher TaqPath RT-PCR COVID-19 kit. We also performed a bridging study comparing the RealTime SARS-CoV-2 assay with the new Abbott Alinity m SARS-CoV-2 assay. A number of standards, reference materials, and commercially available controls were used for the analytical verification to confirm the limit of detection, linearity, and reproducibility. We used nasopharyngeal (NP) swab specimens collected in saline for the clinical verification and bridging studies. Overall, we found 91.2% positive percent agreement (PPA) (95% CI 76.2 to 98.14%) and a 100% negative percent agreement (NPA) (95% CI 97.97 to 100%) between the results of the RealTime SARS-CoV-2 and CDC tests with 217 NP specimens (P=0.13). We found a PPA of 100% (95% CI 90.26 to 100%) and a NPA of 95.15% (95% CI 83.47 to 99.4%) between the results of the RealTime and TaqPath tests with 77 NP specimens (P=0.24). Finally, we tested 203 NP swab specimens for SARS-CoV-2 on the m2000 on the Alinity m systems. The PPA and NPA were 92.2% (95% CI, 85.3 to 96.59%) and 92% (95% PI, 84.8 to 96.5%), respectively (P=0.4). Although cycle number (Cn) values obtained for the concordant positive samples were highly correlated (R2, 0.95), the Cn values were on average 14.14 higher on the Alinity m system due to the unread cycles with the RealTime SARS-CoV-2 assay.

scholarly journals Chitozyme: First Peroxidase-like Activity of Chitosan for Multiplexed Visual Detection of H2O2, Glucose and Lactate on Paper-based Device

2017 ◽  
Author(s):  
Syed Rahin Ahmed ◽  
Xuan Weng ◽  
Suresh Neethirajan
Keyword(s):  
Visual Detection ◽  
Limit Of Detection ◽  
Natural Polymer ◽  
Multiplex Detection ◽  
Practical Applications ◽  
Naked Eye ◽  
Pharmaceutical Technology ◽  
Assay Performance ◽  
Cost Efficient ◽  
Amplification Strategy

AbstractVisual read-out diagnostics tools are promising candidates for field applicable medical devices. Current colorimetric biosensors require introduction of natural enzymes or nanozymes, which has some serious drawbacks for practical applications. Chitosan, a natural polymer, provides safe and efficient compound in medical and pharmaceutical technology. Herein, we report on a simple, cost-efficient, field-portable, environmental friendly and ultra-sensitive multiplex detection platform based on peroxidase-like activity of chitosan in the presence of 3,3’,5,5’-Tetramethylbenzidine (TMBZ) and H2O2. This straight forward signal amplification strategy was successfully applied to detect H2O2, glucose and lactate with the limit of detection (LOD) of 2.64 pM, 0.104 μM and 2.8 nM respectively, represents the lowest LOD of H2O2, glucose and lactate with visual read-out. The chitosan-based assay performance was also retained in complex biological media for glucose and lactate detection. Furthermore, the proposed assay was successfully demonstrated as a paper-based colorimetric biosensor. Most importantly, the simplicity, biocompatibility and sensitivity of the proposed assay will open new doors for instrument free naked eye visual detection of H2O2, glucose and lactate detection.

scholarly journals Continuous Glucose Monitoring in the Management of Neonates with Persistent Hypoglycaemia and Congenital Hyperinsulinism

2021 ◽  
Author(s):  
Myat Win ◽  
Rowan Beckett ◽  
Lynn Thomson ◽  
Ajay Thankamony ◽  
Kathy Beardsall
Keyword(s):  
Real Time ◽  
Preterm Infants ◽  
Continuous Glucose Monitoring ◽  
Clinical Care ◽  
Neurodevelopmental Outcome ◽  
Glucose Monitoring ◽  
Relative Difference ◽  
Congenital Hyperinsulinism ◽  
Glucose Levels ◽  
Sensor Glucose

Abstract Background Persistent hypoglycaemia is common in the newborn and is associated with poor neurodevelopmental outcome. Adequate monitoring is critical in prevention, but is dependent on frequent, often hourly blood sampling. Continuous glucose monitoring (CGM) is increasingly being used in children with type 1 diabetes mellitus, but use in neonatology remains limited. We aimed to introduce real-time CGM to provide insights into patterns of dysglycaemia and to support the management of persistent neonatal hypoglycaemia. Methods This is a single centre retrospective study of real-time CGM use over a 4-year period in babies with persistent hypoglycaemia. Results CGMs were inserted in 14 babies: 8 term and 6 preterm infants, 9 with evidence of congenital hyperinsulinism (CHI). A total of 224 days of data were collected demonstrating marked fluctuations in glucose levels in babies with CHI, with a higher sensor glucose SD (1.52±0.79 mmol/l vs 0.77±0.22mmol/l) in infants with CHI compared to preterm infants. A total of 1254 paired glucose values (CGM and blood) were compared and gave a mean absolute relative difference (MARD) of 11%. Conclusion CGM highlighted the challenges of preventing hypoglycaemia in these babies when using intermittent blood glucose levels alone, and the potential application of CGM as an adjunct to clinical care.

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