Analytical Performance Research Articles

Nanostructured Metal Oxide-Based Electrochemical Biosensors in Medical Diagnosis

Nanostructured metal oxides (NMOs) provide electrical properties such as high surface−to−volume ratio, reaction activity, and good adsorption strength. Furthermore, they serve as a conductive substrate for the immobilization of biomolecules, exhibiting notable biological activity. Capitalizing on these characteristics, they find utility in the development of various electrochemical biosensing devices, elevating the sensitivity and selectivity of such diagnostic platforms. In this review, different types of NMOs, including zinc oxide (ZnO), titanium dioxide (TiO2), iron (II, III) oxide (Fe3O4), nickel oxide (NiO), and copper oxide (CuO); their synthesis methods; and how they can be integrated into biosensors used for medical diagnosis are examined. It also includes a detailed table for the last 10 years covering the morphologies, analysis techniques, analytes, and analytical performances of electrochemical biosensors developed for medical diagnosis.

Comparison of four D-dimer assays in the context of venous thromboembolism in the emergency department.

This observational study conducted across seven emergency care units compares the efficacy of four D-dimer detection methods, namely HemosIL D-dimer HS (HS), HemosIL D-dimer HS-500 (HS-500), VIDAS D-dimer (VIDAS), and HemosIL AcuStar D-dimer (ACUSTAR). The primary focus is on patients with a clinical suspicion of deep venous thrombosis (DVT) or pulmonary embolism (PE). A total of 149 samples were collected from patients with suspected DVT or PE. The confirmation of DVT/PE was based on calf ultrasound or computed tomography-Angiography. Direct comparisons were made between the different detection methods, considering both their analytical performance and clinical utility. Additionally, the impact of an age-adjusted cut-off on the diagnostic accuracy of each method was assessed. The results revealed comparable negative predictive value, sensitivity, and specificity across the methods, with a notable exception of increased specificity for HS compared with HS-500 (50.8% vs. 41.5%, p = 0.03). Further analysis incorporating an age-adjusted cut-off demonstrated a significant improvement in specificity for HS. When using the age-adjusted cut-off, HS exhibited a substantial increase in specificity compared with HS-500 (63.1% vs. 49.2%, p = 0.004) and demonstrated significantly higher specificity compared with VIDAS (63.1% vs. 53.8%, p = 0.04). The study emphasizes the nonuniversal effect of an age-adjusted cut-off and discusses the potential necessity for different cut-off values, particularly in the case of HS-500. These findings contribute to the understanding of D-dimer detection methods in the context of DVT and PE, providing insights into their relative performances and the potential optimization through age-adjusted cut-offs.

An Agile and Accurate Approach for N-Nitrosamines Detection and Quantification in Medicines by DART-MS.

N-nitrosamines (NAs) are prevalent mutagenic impurities in various consumer products. Their discovery in valsartan-containing medicines in 2018 prompted global regulatory agencies to set guidelines on their presence and permissible levels in pharmaceuticals. In order to determine the NAs content in medicines, efficient and sensitive analytical methods have been developed based on mass spectrometry techniques. Direct analysis in real time-mass spectrometry (DART-MS) has emerged as a prominent ambient ionization technique for pharmaceutical analysis due to its high-throughput capability, simplicity, and minimal sample preparation requirements. Thus, in this study DART-MS was evaluated for the screening and quantification of NAs in medicines. DART-MS analyses were conducted in positive ion mode, for both direct tablet analysis and solution analysis. The analytical performance was evaluated regarding linearity, precision, accuracy, limits of detection, and quantification. The DART-MS proved to be suitable for the determination of NAs in medicines, whether through direct tablet analysis or solution analysis. The analytical performance demonstrated linearity in the range from 1.00 to 200.00 ng mL-1, limits of quantification about 1.00 ng mL-1, precision and accuracy lower than 15%, and no significant matrix effect for six drug-related NAs. In conclusion, the DART-MS technique demonstrated to be an alternative method to determine NAs in medicines, aligning with the principles of green chemistry.

APPROACH: Sensitive Detection of Exosomal Biomarkers by Aptamer-Mediated Proximity Ligation Assay and Time-Resolved Förster Resonance Energy Transfer

Exosomal biomarker detection holds great importance in the field of in vitro diagnostics, offering a non-invasive and highly sensitive approach for early disease detection and personalized treatment. Here, we proposed an “APPROACH” strategy, combining aptamer-mediated proximity ligation assay (PLA) with rolling circle amplification (RCA) and time-resolved Förster resonance energy transfer (TR-FRET) for the sensitive and semi-homogenous detection of exosomal biomarkers. PLA probes consisted of a cholesterol-conjugated oligonucleotide, which anchored to the membrane of an exosome, and a specific aptamer oligonucleotide that recognized a target protein of the exosome; the proximal binding of pairs of PLA probes to the same exosome positioned the oligonucleotides in the vicinity of each other, guiding the hybridization and ligation of two subsequently added backbone and connector oligonucleotides to form a circular DNA molecule. Circular DNA formed from PLA underwent rolling circle amplification (RCA) for signal amplification, and the resulting RCA products were subsequently quantified by TR-FRET. The limits of detection provided by APPROACH for the exosomal biomarkers CD63, PD-L1, and HER2 were 0.46 ng∙μL−1, 0.77 ng∙μL−1, and 1.1 ng∙μL−1, respectively, demonstrating excellent analytical performance with high sensitivity and quantification accuracy. Furthermore, the strategy afforded sensitive detection of exosomal CD63 with a LOD of 1.56 ng∙μL−1 in complex biological matrices, which underscored its anti-interference capability and potential for in vitro detection. The proposed strategy demonstrates wide-ranging applicability in quantifying diverse exosomal biomarkers while exhibiting robust analytical characteristics, including high sensitivity and accuracy.

Determination of Sulfites in Dried Fruits by Paper Spray Ionization Tandem Mass Spectrometry

Sulfite, a widely used food additive, is subject to regulated labeling. The extraction of sulfite as the stable hydroxymethylsulfonate (HMS) form and its quantitative analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been recognized for their good sensitivity, selectivity, and versatility across various food materials. This study aimed to develop a cost-effective and simpler method for sulfite quantitation, while maintaining the superior sensitivity and selectivity of mass spectrometry (MS). To achieve this, we introduced paper spray ionization (PSI), an ambient desorption ionization technique that could achieve the direct measurement of analytes without employing separation. We also employed a novel internal standard (IS) structurally similar to the analyte, replacing the more expensive isotopically labeled IS. Although the PSI-MS/MS method developed in this study exhibited slightly lower analytical performance compared to the conventional LC-MS/MS, it remained effective for sulfite analysis in dried fruits.

Comprehensive Study of Thiazole‐Orange‐based DNA Dyes

The rapid advancement of biotechnology over the past decades has amplified the importance of DNA detection and quantification assays. Many of these assays, such as gel electrophoresis, microscopy, flow cytometry, and the detection of amplification in quantitative polymerase chain reaction (qPCR), rely on the use of DNA‐binding fluorescent dyes. This article presents a comprehensive study of six Thiazole‐Orange‐based fluorescent DNA‐binding dyes: SYBR Safe, SYBR Green, Pico Green, SYTO‐16, SYTO‐9, and the benzothiazole‐based analogue (TOPhBu) of the latter. The selected DNA markers were synthesized at a 10‐milligram scale and characterised spectroscopically to quantify their fluorescence enhancement upon binding to double‐stranded DNA. The ability of the dyes to detect DNA at low concentrations was evaluated using two new metrics, absolute fluorescence enhancement (AFE) and relative fluorescence enhancement (RFE). Quantum chemical calculations shed new light on the mechanism of their fluorogenicity through modelling the excited state behaviour and DNA binding of the dyes. Their analytical performance was further tested in qPCR experiments. The experimental results of this work highlight some important differences in the sensitivity and qPCR efficiency of the studied DNA‐binding dyes which will facilitate the DNA marker selection for analytical purposes and the future development of novel DNA sensors.

An Analytical Reliability Performance Assessment of Cell-Free Massive MIMO Systems for 5G and Beyond Cellular Networks

In today‟s realm, the 5G cellular networks offer a new era of fully digital environment by connecting people anywhere anytime. This era requires inspiring demands to the networks in terms of spectral and energy efficiencies, low-latency and ultra-reliable communications. A major development to deal with these unparalleled demands is the introduction of Cell- Free Massive MIMO (CF mMIMO) systems to the cellular network. In recent years, mathematical foundations have been laid for a CF mMIMO communication system to evaluate the spectral and energy efficiencies given the number of access points. Various existing literatures have adopted mathematical models to formulate closed-form expressions for these efficiencies using uncorrelated Rayleigh fading for simple linear processing detectors. In this paper, we try to address a compelling and interesting question which has not received much attention lately. The question is how to evaluate the reliability of a connected user terminal in both a CF mMIMO and conventional mMIMO system? We answer this question by first analyzing the signal models for both systems assuming exact channel estimates and consider an uncorrelated Rayleigh fading channel model. The exact Bit Error Rate, as a reliability assessment, in closed-form are then derived based on strong mathematical foundations, which are simple to numerically evaluate in Matlab R2022, for MPSK and MQAM modulation schemes. The mathematical models presented in this work can be used in 5G and Beyond 5G networks to evaluate the distributions of error rates for a user given the number of Access points or Base Station antennas and modulation scheme. Machine Learning algorithms can be applied to the analytical expressions with a view to predict the error performance of a user terminal given the fading characteristic of the propagation environmen

DEVELOPMENT AND VALIDATION OF A SIMPLE AND COST-EFFECTIVE UV SPECTROPHOTOMETRIC METHOD FOR QUANTIFYING LINEZOLID

Objective: This study focuses on the development and validation of a sensitive, simple, accurate, precise and cost-effective Ultraviolet-Visible (UV) spectrophotometric method for the quantification of Linezolid, a widely used antibiotic in pharmaceutical formulations. Methods: The analysis utilized a solvent system comprising 80% water and 20% methanol (v/v). The absorbance of standard solutions was measured and a calibration curve was constructed. Various analytical performance parameters, including linearity, range, precision, accuracy, Limit of Detection (LOD), Limit of Quantification (LOQ) and ruggedness, were determined following the International Conference on Harmonization (ICH) Q2 (R1) guidelines. Results: The maximum absorption peak (λmax) of Linezolid was determined to be 251 nm in the selected medium. Beer-Lambert’s law was valid in the concentration range of 0.5–9 μg/ml, with a high correlation coefficient (R2) of 0.9955. The proposed method exhibited a recovery ranging from 99.08 to 100.37% with % Relative Standard Deviation (RSD) value consistently below 2%. Conclusion: The study findings confirm the accuracy, precision and reproducibility of the developed method. Additionally, it is characterized by its simplicity, affordability, and time efficiency. Thus, this method can be effectively employed for the quantification of Linezolid in lipid nanoparticles.

A novel NIR fluorescent probe for enhanced β-galactosidase detection and tumor imaging in ovarian cancer models

The advancement of biological imaging techniques critically depends on the development of novel near-infrared (NIR) fluorescent probes. In this study, we introduce a designed NIR fluorescent probe, NRO-βgal, which exhibits a unique off–on response mechanism to β-galactosidase (β-gal). Emitting a fluorescence peak at a wavelength of 670 nm, NRO-βgal showcases a significant Stokes shift of 85 nm, which is indicative of its efficient energy transfer and minimized background interference. The probe achieves a remarkably low in vitro detection limit of 0.2 U/L and demonstrates a rapid response within 10 min, thereby underscoring its exceptional sensitivity, selectivity, and operational swiftness. Such superior analytical performance broadens the horizon for its application in intricate biological imaging studies. To validate the practical utility of NRO-βgal in bio-imaging, we employed ovarian cancer cell and mouse models, where the probe's efficacy in accurately delineating tumor cells was examined. The results affirm NRO-βgal's capability to provide sharp, high-contrast images of tumor regions, thereby significantly enhancing the precision of surgical tumor resection. Furthermore, the probe's potential for real-time monitoring of enzymatic activity in living tissues underscores its utility as a powerful tool for diagnostics in oncology and beyond.

Novel biocompatible N-rich AIE fluorescent probe for live cell imaging and visual onsite detection of uranium.

A sensitive and biocompatible N-rich probe for rapid visualuranium detectionwas constructed by grafting two trianiline groups to 2,6-bis(aminomethyl)pyridine. Possessing excellent aggregation-induced emission (AIE) property and the advantages to form multidentate chelate with U selectively, the probe has been applied successfully to visualize uranium in complex environmental water samples and living cells, demonstrating outstanding anti-interference ability against large equivalent of different ions over a wide effective pH range. A large linear range (1.0 × 10-7-9.0 × 10-7mol/L) and low detection limit (72.6nmol/L, 17.28ppb) were achieved for the visual determination of uranium. The recognition mechanism, photophysical properties, analytical performance and cytotoxicity were systematically investigated, demonstrating highpotential for fast risk assessment of uranium pollution in field and in vivo.

Flow cytometry for meningeal infiltration in B acute lymphoblastic leukemia in a low middle income country.

Meningeal infiltration in children with B acute lymphoblastic leukemia is one of the most serious complications. Timely diagnosis not only significantly enhances treatment efficacy but also leads to improve patient outcome and reduce risk of relapse. This is particularly crucial in low to middle income countries facing health constraints, where optimizing resources is essential. Conventional cytology (CC) study of cerebrospinal fluid (CSF) is considered in different countries to be the Gold-standard despite its low sensitivity (< 50%). The study of CSF by multiparametric flow cytometry (MFC) appears to be an alternative. The aim of our study was to assess MFC analytical performance compared with CC. Our cross sectional study was conducted over a six-month period in the biological hematology department. CSF samples underwent analysis for the presence of blasts using both CC and MFC. Cytological slides of the CSF were prepared by cytocentrifugation in a Shandon Cytospin 4™. Flow cytometric analysis was performed on the BD FACSLyric™ flow cytometer. All statistical analyses were performed using SPSS version 21.0 (SPSS Inc.). Agreement between the two methods was made using the Kappa index and χ2 test. This study was approved by the local ethics committee. Sixty CSF samples from 39 children with B acute lymphoblastic leukemia were analyzed. Meningeal infiltration was detected respectively in 20% of cases by MFC and 5% of cases by CC, with a significant difference p = 0.006. Comparing the two methods, the Kappa coefficient was 0.35, indicating weak agreement between the two methods. Moreover, MFC positivity was higher even for hypocellular samples. Of the 51 hypocellular samples, eight were positive by MFC while they were negative by CC. MFC shows better sensitivity while retaining good specificity for the detection of meningeal involvement. MFC could therefore be a complementary method to CC for detecting blast cells in the central nervous system.

An antifouling electrochemical biosensor using self-signal for Salmonella typhimurium direct detection in food sample

Eating food contaminated by foodborne pathogens can lead to illness. The development of electrochemical sensors for pathogen detection has received widespread attention. However, the analytical performance of electrochemical sensors is inevitably affected by the non-specific adsorption of molecules in the sample. Moreover, the external signal probes might be affected by the complex components in the sample accompanied with signal suppression. This work presents an electrochemical aptasensor for Salmonella typhimurium detection based on the self-signal of poly-xanthurenic acid and the antifouling ability of chondroitin sulfate. The detection time was 60 min. The linear range was from 101 to 107 CFU/mL, and the detection limit was 3 CFU/mL. The biosensors presented good repeatability and storage stability. And the biosensors has been successfully applied in milk and orange juice. This strategy is expected to be applied in the design of other antifouling biosensors, to achieve rapid detection of pathogens and ensure food safety.

Eco-friendly fabrication of silver nanoparticle-decorated electrodes for aqueous bisphenol A sensing

This work fabricated an electrochemical sensor for detecting bisphenol A (BPA) by employing silver nanoparticles (AgNPs) synthesized through an eco-friendly approach. The AgNPs were produced using parsley leaf extract, which acted as a reducing and stabilizing agent in the synthesis process. The AgNPs synthesized using the green approach exhibited an average size of 15.2 ± 2.1 nm. The AgNP-modified screen-printed electrode (SPE) exhibited excellent electrocatalytic activity towards BPA oxidation, with an enhanced peak current of 28.6 μA compared to 5.2 μA for the bare SPE. The optimized sensor exhibited impressive analytical performance, boasting a broad linear range spanning 0.05–5 μM for BPA detection. Remarkably, it achieved a low limit of detection of 0.02 μM, coupled with high sensitivity. Furthermore, the sensor demonstrated commendable reproducibility, with a RSD of 3.6%, and exceptional repeatability, evidenced by an RSD of 2.4%. Upon evaluating the sensor's performance in analyzing real water specimens, the recoveries oscillated between 95.8% and 103.5%, accompanied by RSDs not exceeding 4.2%, thereby demonstrating its successful application for BPA detection. The eco-friendly synthesis approach and superior analytical performance make the AgNP-modified SPE a promising tool for on-site environmental monitoring and drinking water analysis.

Touch–based potentiometric sensors for simultaneous detection of urea and ammonium from fingertip sweat

Urea is a biomarker for diagnosis and management of multiple disorders. The measurement of urea could help in the diagnosis of patients with kidney dysfunction. Despite their attractive analytical performances, the existing urea measurement methods are still limited to sophisticated and time–consuming or require invasive blood sampling. Herein, a reliable noninvasive, simple, and disposable touch–based potentiometric sensor is fabricated for rapid monitoring of fingertip sweat urea. Novel touch–based urea detection relies on immobilized urease recognition and a solid–contact ammonium–ion–selective electrode, along with a screen–printed carbon working electrode modified carboxylated carbon nanotubes for enhancing sensor’s sensitivity. The fingertip sweat under sedentary rest conditions can be instantaneously collected on customized porous polyvinyl alcohol hydrogel transporting sweat to working electrode surface where the hydrolysis of urea is catalyzed, generating bicarbonate and ammonium. The resulting ammonium can be detected with ion–selective transducer, enabling indirect monitoring of sweat urea. Potential interference from co–existing ammonium in human sweat is addressed by developing a dual disposable potentiometric sensor, consisting of urea and ammonium sensors on a single strip platform allowing for simultaneous touch–based detection of sweat urea and ammonium. The resulting dual disposable sensing system offers rapid urea and ammonium monitoring within a group of healthy human subjects, along with a good correlation with R2 = 0.974 between the sweat urea and capillary blood urea levels. Additional confirmation among a broad sample of individuals along with the validation of sensor data through centralized laboratory tests will establish the practicality of simultaneously monitoring urea and ammonium levels.

Automated H2O2 monitoring during photo-Fenton processes using an Arduino self-assembled automatic system

A cheap and easy to use Arduino self-assembled automatic system was employed to continuously monitor the hydrogen peroxide consumption during the photo-Fenton degradation of caffeine, selected as model target compound. The automatic system made it possible to measure the H2O2 concentration in the reaction cell via a colorimetric reaction and to take samples for HPLC analysis minimising the operator manual intervention and exposure to UV radiation. The obtained results were compared in terms of LOD and LOQ with H2O2 measurements manually performed using UV-Vis spectrophotometry, evidencing better analytical performance when using the automatic system; LOD and LOQ were respectively 0.032mM and 0.106mM for the automatic system against 0.064mM and 0.213mM for UV-Vis spectrophotometry. Furthermore, the photo-Fenton treatment was optimised by means of a Design of Experiments (DoE) investigating the effect of added H2O2 concentration, iron concentration and caffeine initial concentration on system performances. The use of the automatic device for such monitoring provided several advantages: automation (with consequent reduction of the workload), measurement increased precision, reduced reagents consumption and waste production in agreement with the principles of Green Analytical Chemistry.

Tailoring supramolecular solvents with phosphoryl groups for highly efficient extraction of chlorophenols in natural waters

BackgroundChlorophenols are routinely determined in aquatic systems to check compliance with the restrictive international legislations set for protection of human and aquatic life. Their control requires affordable analytical methods, particularly in labs at low- and medium-income countries. Liquid chromatography-UV detection is a convenient technique for this purpose, but the availability of suitable sample processing remains pending. Organic solvents are inefficient for extracting the whole range of chlorophenols whereas solid-phase extractions are expensive and labour-intensive. So, an efficient, fast and cheap extraction of chlorophenols, amenable to any lab, would help to cope with their worldwide analytical control in natural waters. ResultsA supramolecular solvent (SUPRAS) was tailored for providing mixed interaction mechanisms aimed at the efficient extraction of chlorophenols prior to LC-UV. The SUPRAS was synthesized from the self-assembly of hexylphosphonic acid under acidic conditions and consisted of sponge-like nanostructures made up of amphiphile and water. The phosphoryl (PO) group was selected as the major driver of the extraction because of its ability to act as halogen and hydrogen bond acceptor for chlorophenols. Additional interactions were hydrogen bonds from O–H amphiphilic groups and the surrounding water, and dispersion and CH-π interactions in the hydrocarbon chains. The number of binding sites in the SUPRAS could be modulated by addition of salt. The SUPRAS formed in situ in the sample, the extraction took 5 min, the concentration factor was around 220, quantification limits (0.1–0.3 μg L−1) were below the EU standards, and the method worked for natural waters. SignificanceA fast, low-cost, and organic solvent-free sample processing only requiring conventional lab equipment (stirrers and centrifuges) provided SUPRAS extracts that could be directly analyzed by LC-UV. SUPRAS synthesis occurred spontaneously in the water sample under addition of hexylphosphonic acid and the whole process required low skills. The method meets the analytical and operational performances for the analytical control of chlorophenols in natural waters and it is within the reach of any lab.

Cost-effectiveness of genetic testing of endocrine tumor patients using a comprehensive hereditary cancer gene panel.

Heterogenous clinical manifestations, overlapping phenotypes and complex genetic backgrounds are common in patients with endocrine tumors. There are no comprehensive recommendations for genetic testing and counselling of these patients compared to other hereditary cancer syndromes. The application of multigene panel testing is common in clinical genetic laboratories, but their performance for patients with endocrine tumors has not been assessed. As a national reference center, we prospectively tested the diagnostic utility and cost-efficiency of a multigene panel covering 113 genes representing genetic susceptibility for solid tumors. 1279 patients (including 96 cases with endocrine tumors) were evaluated between October 2021 and December 2022 who were suspected to have hereditary tumor syndromes. The analytical performance of the hereditary cancer panel was suitable for diagnostic testing. Clinical diagnosis was confirmed in 24% (23/96); incidental findings in genes not associated with the patient's phenotype were identified in 5% (5/96). A further 7% of pathogenic/likely pathogenic variants were detected in genes with potential genetic susceptibility roles but currently no clear clinical consequence. Cost-benefit analysis showed that the application of a more comprehensive gene panel in a diagnostic laboratory yielded a shorter turnaround time and provided additional genetic results with the same cost and workload. Using comprehensive multigene panel results in faster turnaround time and cost-efficiently identifies genetic alterations in hereditary endocrine tumor syndromes. Incidentally identified variants in patients with poor prognoses may serve as a potential therapeutic target in tumors where therapeutic possibilities are limited.

Exploring highly electro-active zinc peroxide nanorod for selective detection of hydrazine

Hydrazine (N2H4), widely used as a raw material for producing synthetic catalysts, agricultural chemicals, and pharmaceutical products, has had an irreversible impact on air, water, and soil in the environment because of its high toxicity. To prevent making it lethal contamination should be monitored and detected. Here in this work, for the first time, Zinc peroxide (ZnO2) nanorods (NRs) were synthesized using a simple hydrothermal method to modify the gold electrode (AuE) for the fabrication of the ZnO2@AuE sensor. The fabricated sensor was successfully applied for the selective and efficient detection of hydrazine based on the chronoamperometry technique. According to theoretical calculations, the metal peroxide nanorods result in high oxygen vacancies, surface defects, and Zn ratios. This triggers substantial electron transfer, effectively enhancing the conductivity, electrocatalytic, and stability properties of the AuE with economic benefits. The effect of surface modification on the bare electrode was analyzed using the EIS technique. The ZnO2/Au electrode's electrochemical behavior towards hydrazine was studied using cyclic voltammetry (CV) at a scan rate of 70 mVs−1. Further, chronoamperometry was applied to investigate the effect of time, pH, nanorod concentration, and scan rate on the analytical performance parameters of the electrode ZnO2@AuE. The developed sensor (ZnO2/AuE) demonstrated an excellent detection limit (LOD) of 4.09 nM and good sensitivity of 2.4 × 10−2 µAµM−1cm−2 towards the oxidation of hydrazine having a fast response time (within 5 s). The developed sensors promise reproducible results along with good stability, which makes them effective for the electrochemical sensing of hydrazine. Such outstanding sensing performance is attributed to the tailored direct electrochemical redox surface of ZnO2, which led to selective sensing of hydrazine in tap, distilled, and sewage water. Keeping these features into consideration, our developed sensor can be adopted for real applications, especially for environmental surveillance.

ADLM Guidance Document on Laboratory Diagnosis of Respiratory Viruses.

Respiratory viral infections are among the most frequent infections experienced worldwide. The COVID-19 pandemic has highlighted the need for testing and currently several tests are available for the detection of a wide range of viruses. These tests vary widely in terms of the number of viral pathogens included, viral markers targeted, regulatory status, and turnaround time to results, as well as their analytical and clinical performance. Given these many variables, selection and interpretation of testing requires thoughtful consideration. The current guidance document is the authors' expert opinion based on the preponderance of available evidence to address key questions related to best practices for laboratory diagnosis of respiratory viral infections including who to test, when to test, and what tests to use. An algorithm is proposed to help laboratories decide on the most appropriate tests to use for the diagnosis of respiratory viral infections.

Abstract PO3-07-03: Development &amp; Validation of an AI-supported Workflow for Clinical Scoring of HER2, ER, PR &amp; Ki67 Immunohistochemistry in Breast Cancer Tissue

Abstract Background: Immunohistochemical evaluation of HER2 status, the hormone receptors ER and PR, and the proliferation marker Ki67 forms part of the routine clinical diagnostic pathway for invasive breast carcinomas, and is the cornerstone of treatment stratification, informing both prognosis and patient management. Pathologist scoring of immunohistochemistry (IHC) at the microscope is time-consuming and prone to significant inter- and intra-observer variability. We developed HALO Breast AI, a decision-support system designed to improve efficiency and diagnostic accuracy through automating whole slide image (WSI) scoring. Here, we present preliminary results of a validation study of HALO Breast AI. Methods: HALO Breast AI was developed with routine diagnostic cases sourced from three institutes. The algorithm was trained using 107,755 pathologist-reviewed annotations to identify and threshold DAB-positive tumor cells within automatically segmented tumor regions. Internal validation was conducted on 80 unseen WSI, using 60,012 pathologist-reviewed annotations to assess analytical performance. Comparison of the algorithm scores to the mode of 3 expert pathologists (where at least 2 out of 3 agreed) was used to assess consensus agreement. Clinical performance and generalizability were assessed by comparing the algorithm scores to clinical data from two independent external institutes across 200 unseen WSI (n=50 per marker) from institute one and 300 unseen WSI (n=100 per marker [ ER, PR &amp; HER2, only]) from institute 2. Results: The median image F1-score for tumor classification was 0.91, while the median image F1-score for cell level validation was 0.96. Internal validation showed agreement between HALO Breast AI and the mode of 3 expert pathologists of 100% for ER, 90% for PR, 95% for Ki67 and 90% for HER2. High concordance was measured between the algorithm and the pathologists’ scores, with Light’s kappa of 0.91 for ER, 0.85 for PR, 0.79 for HER2 and 0.82 for Ki67. Performance on WSI obtained from external institute one, showed agreement between the clinical score obtained from HALO Breast AI and the clinical data of 96% for ER, 94% for PR, 84% for HER2 and 84% for Ki67. External institute two showed agreement of 90% for ER, 90% for PR and 83% for HER2. Of the ER &amp; PR cases that were in disagreement at the 1% clinical cut-off, the algorithm percent-positive scores were within a 1-3% range in 5 out of the 5 from institute 1 and 14 out of the 20 from institute 2. So, although on a categorical scale the AI assigned category disagreed with the clinical category, the results were close on a continuous scale. Conclusions: HALO Breast AI accurately detects tumor regions and tumor cells within breast cancer tissue and demonstrates high clinical agreement when scoring routine diagnostic IHC. Additionally, HALO Breast AI shows good generalisability, with a consistently strong performance across inherent variability that exists between external, independent data sets. Computer-aided diagnostic tools such as HALO Breast AI have the potential to support pathologists in the diagnostic setting by improving workflow efficiency and standardising results. Citation Format: Meredith Lodge, Alastair Ironside, Ashley Graham, Antonio Polonia, Stefan Reinhard, Wiebke Solass, Inti Zlobec, Peter Caie. Development &amp; Validation of an AI-supported Workflow for Clinical Scoring of HER2, ER, PR &amp; Ki67 Immunohistochemistry in Breast Cancer Tissue [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-07-03.