Analytical Performance Research Articles

Assessment of homologous recombination deficiency and BRCA status in ovarian cancer: Analytical performance and relevance of a decentralized NGS assay for comprehensive genomic profiling.

3130 Background: Homologous recombination deficiency (HRD) is a complex biomarker with predictive value in ovarian cancer. Understanding both the causes of HRD, such as pathogenic alterations in homologous recombination repair (HRR) genes, and its consequences, like genomic instability (GI), is crucial for exploring various therapeutic strategies, including the potential use of poly (ADP-ribose) polymerase inhibitors (PARPi). This study evaluates the analytical performance and clinical research relevance of the Oncomine™ Comprehensive Assay Plus (OCA Plus), a distributable next-generation sequencing (NGS) research use assay that offers in a single workflow comprehensive genomic profiling, including HRD evaluation. Methods: The OCA Plus panel was used for comprehensive genomic profiling of a series of 299 ovarian cancer research samples from the PAOLA-1 trial, part of the ARCAGY biorepository. Research samples were analyzed to assess agreement with orthogonal method, specifically for BRCA1 and BRCA2 mutational status, GI status and overall HRD status which combined BRCA1/2 mutational status and GI status. GI status was determined using Genomic Instability Metric (GIM), a quantitative method that characterizes unbalanced copy number changes. Progression-free survival (PFS) was retrospectively studied to determine future clinical relevance. Results: The success rate for DNA sequencing was 100%, starting from a minimal sample input of 20ng of genomic DNA isolated from FFPE tissue blocks. The OCA Plus panel provided a detailed genomic profile in a single workflow, achieving high success rates across all biomarkers tested, including single nucleotide variants/indels and HRD (100%). Overall percent agreement (OPA) for HRD status with orthogonal method was 87%. OPA for BRCA1/2 variants was 98%, while OPA for GI status was 80%. PFS analysis demonstrated a significantly better hazard ratio (HR: 0.51, p < .005) for the cases positive for OCA Plus HRD solution compared to the cases negative for the OCA Plus HRD solution (HR: 0.84, p = 0.43). Conclusions: The OCA Plus solution enables robust and reliable comprehensive genomic profiling with high OPA for BRCA1/2 and HRD status compared to commonly used orthogonal method. Albeit additional studies are due, overall, the reported data suggests its future clinical utility in predicting treatment outcomes in ovarian cancer.

Instrumental developments in drift tube ion mobility spectrometry: A review on miniaturization, new manufacturing techniques, and pre-separation.

Drift tube ion mobility spectrometry (DT-IMS) separates ions based on their mobility under weak electric fields and has undergone significant advancements since its introduction as an analytical instrument in 1970. Recent developments in DT-IMS focus on addressing limitations such as low resolving power and challenges in separating complex samples. High-resolution ion shutters, optimized drift tubes, and integrated chip-based pre-separation methods have greatly enhanced analytical performance. Miniaturization has been a key focus, driven by advanced manufacturing techniques and instrumental developments, which have improved portability, reduced costs, and enabled custom designs. Tandem DT-IMS systems further enhance selectivity and structural elucidation, offering expanded capabilities for complex analyses. These innovations have significantly advanced the functionality of DT-IMS, bridging the gap between simple, straightforward designs and high analytical performance. This review highlights the transformative impact of these instrumental developments, miniaturization, and novel manufacturing techniques, positioning DT-IMS as a versatile, high-performance analytical tool for modern workflows.

Alpha test 1 robotic platform for traditional Chinese medicine pretreatment: Automated modular integration and quality control applications.

Sample pretreatment plays an important role in analytical performance, but it is often the rate-limiting step in scientific research. With the development of automation technology, automated sample preparation has become a powerful tool for liberating labor and improving efficiency. In this study, the automatic pretreatment of traditional Chinese medicine test was realized by module combination, and an intelligent robotic platform Alpha Test 1 was developed to decompose the complex preparation method of traditional Chinese medicine into automatic modular components. It includes a multi-channel liquid feeding device, an extraction bottle device, a reflux condensing pipe ring, a reflux extraction and a water bath extraction device. Each module is connected through modular combinations such as liquid injection control, intelligent sorting, and water bath condensation, and the difficulty of manual operation is gradually tackled to achieve the goal of replacing manual labor in the pretreatment process. We selected two representative traditional Chinese medicines, Xianglian Pills and Erzhi Pills, to explore their application in the testing of traditional Chinese medicine. The results of Alpha Test 1 in the identification and content determination met the requirements of Chinese Pharmacopoeia, and the operation was simple, rapid and efficient compared with the manual pretreatment method. This method can meet the requirements of daily laboratory testing and provide technical support for the quality control evaluation and testing of traditional Chinese medicine.

Diagnostic accuracy of self-collected tongue swabs for Mycobacterium tuberculosis complex detection in individuals being assessed for tuberculosis in South Africa using the Xpert MTB/RIF Ultra assay.

Tongue swabs (TS) have shown potential for detecting Mycobacterium tuberculosis complex (MTBC) through downstream molecular testing. Analytical performance varies, depending on the processing protocol and the molecular test used. This study aimed to first investigate the ease of use of TS collection in addition to acceptability by individuals being assessed for tuberculosis and second to determine the performance of self-collected TS on the Xpert MTB/rifampicin (RIF) Ultra (Ultra) assay (Cepheid, Sunnyvale, CA, USA) for MTBC and RIF resistance detection. The ease of use of TS collection and acceptability by study participants was assessed through completion of a survey questionnaire. Analytical performance of self-collected TS on the Ultra assay was determined by comparing results with a liquid culture reference result and with Ultra on sputum. Results were additionally stratified by HIV and smear status. Of the 399 survey respondents, all were happy with the TS collection procedure with minimal discomfort reported. Data analysis was performed on 321 specimens. The sensitivity of the Ultra TS assay for MTBC detection was 78.1% (95% CI, 66.0-87.5%), whereas the specificity was 100% (95% CI, 98.6-100%). Test performance was better in individuals with a higher bacillary load, with some variability. On HIV-positive individuals, TS performance is ∼30% lower than that on sputum but also slightly better than smear microscopy, whereas the performance of TS among HIV-negative individuals is similar to sputum (93% vs. 97%). For RIF resistance profiling, the Ultra TS assay showed 41/41 (100%) concordance with phenotypic drug-susceptibility testing. Although self-collected TS have lower sensitivity compared with sputum, their ease of use and high acceptability make them a valuable additional specimen type for molecular tuberculosis testing. Self-collection can reduce the burden on healthcare workers and increase access to testing, particularly for individuals who are too busy to visit healthcare facilities or fear stigma.

Development and validation of a graphene quantum dot-based sensor for abacavir quantification via fluorescence quenching.

Development and validation of a graphene quantum dot-based sensor for abacavir quantification via fluorescence quenching.

Performance of a 52-gene NGS panel combined with shallow WGS for accurate HRD analysis.

e17570 Background: Homologous recombination deficiency (HRD) is a pivotal biomarker for predicting response to PARP inhibitors in multiple cancers. This study evaluates the analytical performance of a 53-gene somatic NGS panel combined with low-pass whole genome sequencing (shallow WGS, sWGS) for HRD assessment. Methods: In the present study, 36 FFPE tissues from ovarian cancer patients were analyzed for 52 genes implicated in DNA repair pathways, along with sWGS for the evaluation of tumor genomic instability (GI). Libraries were generated with the KAPA HyperPlus Kit (Roche) and sequenced in duplicate using the Avity sequencing instrument (Element Bioscience) and the DNBSEQ-G400 NGS platform, subsequent to conversion for compatibility with MGI chemistry. HRD positivity was defined as either the presence of pathogenic BRCA1/2 mutations or high GI. Data analysis was performed using the SeqOne platform. The performance of the sWGS assay in detecting GI was initially assessed using the OncoScan CNV assay for 15 samples, as well as 4 reference materials with known HRD status. The efficacy of sWGS was also evaluated by comparing its agreement with the GI scores obtained from the validated HRD test (Myriad MyChoice) in 21 samples. Results: A 93% agreement was observed between the sWGS assay and OncoScan, demonstrating strong concordance. Additionally, the assay accurately evaluated the HRD status in all 4 reference samples with known values. The results obtained were highly similar between the two sequencing platforms. In addition, shallow NGS achieved >95% overall percentage agreement (OPA) with the validated HRD test for genomic instability score (Table 1). 5 of the 6 samples with discordant GI findings between Myriad and OncoScan aligned with the Myriad results when analyzed with sWGS. This implies that sWGS has the potential to resolve the discrepancies that were observed with OncoScan, thereby indicating its reliability as a robust alternative for HRD analysis. Conclusions: The assay demonstrated robust performance in detecting HRD-associated genomic signatures, supporting its applicability in clinical use. The reliability of the NGS assay for clinical use is guaranteed by its high concordance (95%) with the validated test, which entails the incorporation of shallow WGS for HRD analysis. Concordance between Myriad MyChoice and shallow WGS. Validated HRD Test HRD + HRD - PPV NPV OPA r Shallow WGS HRD HRD + 13 0 100% 88% 95.24% 0.9014 HRD - 1 7 p< .00001

An automated liquid-liquid extraction platform for high-throughput sample preparation of urinary phthalate metabolites in human biomonitoring.

An automated liquid-liquid extraction platform for high-throughput sample preparation of urinary phthalate metabolites in human biomonitoring.

Computational studies for the development of extracellular vesicle-based biosensors.

Computational studies for the development of extracellular vesicle-based biosensors.

Laser-induced graphene electrode modified by platinum nanoparticle/zein/gelatin/glucose oxidase for non-invasive glucose sensor in multiple biofluids.

Laser-induced graphene electrode modified by platinum nanoparticle/zein/gelatin/glucose oxidase for non-invasive glucose sensor in multiple biofluids.

An electrochemiluminescence biosensor based on metal porphyrin luminophore and covalent organic framework for the sensitive detection of ctDNA in non-small cell lung cancer.

An electrochemiluminescence biosensor based on metal porphyrin luminophore and covalent organic framework for the sensitive detection of ctDNA in non-small cell lung cancer.

Harmonisation of the diagnostic performances of serological ELISA tests for C. burnetii in ruminants in the absence of a gold standard: Optimal cut-offs and performances reassessment.

Harmonisation of the diagnostic performances of serological ELISA tests for C. burnetii in ruminants in the absence of a gold standard: Optimal cut-offs and performances reassessment.

Quantification of trimethylamine-N-oxide (TMAO) and its main related trimethylammonium-containing compounds in human plasma by LC-MS/MS.

Quantification of trimethylamine-N-oxide (TMAO) and its main related trimethylammonium-containing compounds in human plasma by LC-MS/MS.

Evaluating the performance of the Xpert HPV assay in detecting HPV positive cases in Morocco.

Evaluating the performance of the Xpert HPV assay in detecting HPV positive cases in Morocco.

Assessment of accuracy, clinical validity, and analytical linearity in point-of-care glucose monitoring devices for diabetes mellitus: A systematic review and meta-analysis.

Assessment of accuracy, clinical validity, and analytical linearity in point-of-care glucose monitoring devices for diabetes mellitus: A systematic review and meta-analysis.

Analytical Performance of Plazomicin E-strips (Ezy MIC™) Compared with Broth Micro-dilution

Analytical Performance of Plazomicin E-strips (Ezy MIC™) Compared with Broth Micro-dilution

Impact of analytical bias on machine learning models for sepsis prediction using laboratory data

Abstract Objectives Machine learning (ML) models, using laboratory data, support early sepsis prediction. However, analytical bias in laboratory measurements can compromise their performance and validity in real-world settings. We aimed to evaluate how analytically acceptable bias may affect the validity and generalizability of ML models trained on laboratory data. Methods A support vector machine model (SVM) for sepsis prediction was developed using complete blood count and erythrocyte sedimentation rate data from outpatients (CS, n=104) and patients from acute inflammatory status wards (SS, n=107). Twenty-six combinations were derived by white blood cells (WBC), platelets (PLT), and erythrocyte sedimentation rate (ESR) biases from analytical performance specifications (APS). The diagnostic performances of the 26 conditions tested were compared to the original dataset. Results SVM performance of the original dataset was AUC 90.6 % [95 %CI: 80.6–98.7 %]. Minimum, desirable and optimum acceptable biases for WBC were 7.7 , 5.1 and 2.6 %, respectively, for PLT were 6.7 , 4.5 and 2.2 %, respectively and for ESR were 31.6 , 21.1 and 10.5 %, respectively. Across all conditions, AUC varied from 89.8 % [95 %CI: 79.0–97.7 %] (for PLT bias −6.7 %), to 89.5 % [95 %CI: 79.1–98.0 %] (for ESR Bias +31.6 %) to 90.4 % [95 %CI: 79.3–98.4 %] (for WBC Bias −5.1 %). Using a combination of biases, the lowest AUC was 87.8 % [95 %CI: 75.9–96.6 %]. No statistically significant differences were observed for AUC (p>0.05). Conclusions Bias can influence model performance depending on the parameters and their combinations. Developing new validation strategies to assess the impact of analytical bias on laboratory data in ML models could improve their reliability.

Detection of HPV-16 by a Simple and Cost-Effective DNA Probe: Polyadenine-Polythymine-Decorated Gold Nanoparticles (PolyA-PolyT@AuNPs).

HPV-16 identification is crucial for point-of-care molecular diagnosis of cervical cancer. However, the diagnostic methods currently in use are unable to combine both high analytical performance and cost-efficient medical diagnosis. To circumvent this, we have developed a novel nanobiosensor for diagnosis of the HPV-16 L1 gene sequence through colorimetric methods. Using a unique conjugation technique of polyvalent DNA to gold nanoparticles (AuNPs), quickly synthesizing AuNPs synthesis in situ at the particular probe-polyadenine-polythymine (A15-T10) strands was developed. Transmission electron microscopy (TEM), dynamic light scattering (DLS), gel electrophoresis, UV-Vis spectroscopy, and visual detection demonstrate that the Poly(A)15-(T)10 strand can create separate anisotropic AuNPs so that a thick layer of DNA is functionalized on each AuNP, therefore generating polyvalent (p)DNA-AuNPs. Moreover, the hybridization test, UV-Vis spectroscopy, TEM, visual detection, and DLS results further confirmed the innovative sensing applicability, showing more excellent attractiveness of pDNA-AuNPs conjugation in diagnostics in biomedicine and particular sequence detection, such as double-stranded DNA (dsDNA) by employing probe-polyA-polyT extra-strands that are in harmony with the intended sequence. This sensor demonstrated good performance levels with a sensitivity of up to 1.9nM for pUCm-T. Additionally, the sensor showed intense discrimination against actual clinically collected HPV-16 samples, consistent stability, and good repeatability.

Simultaneous Determination of Glibenclamide, Metoprolol Tartrate, and Phenol Red Using HPLC-PDA: Application in Rat Intestinal Permeability Studies and Its Greenness Assessment Using NEMI, Analytical Eco-Scale, GAPI, MoGAPI, AGREE, AGREEprep, RAPI, BAGI, and CACI.

ABSTRACTThis study presents the development and validation of an environmentally friendly HPLC‐PDA method for the simultaneous determination of glibenclamide (GLB), metoprolol tartrate (MET), and phenol red (PR). The method was optimized to ensure high trueness, precision, and robustness, adhering to ICH guidelines. The pKa of GLB was determined using reverse‐phase HPLC under varying pH conditions, providing valuable insights into its physicochemical properties and enhancing its pharmaceutical formulation potential. Intestinal permeability studies of GLB, conducted via the in situ Single Pass Intestinal Perfusion (SPIP) technique, validated the method's applicability in biological systems. In addition to analytical performance, the environmental sustainability of the method was assessed using green metrics, including the Analytical Eco‐Scale, GAPI, MoGAPI, AGREE, AGREEprep RAPI, BAGI, and CACI. The method achieved high scores, reflecting its alignment with the principles of green analytical chemistry and its minimal ecological impact. This dual focus on analytical rigor and environmental responsibility emphasizes the method's potential for broader applications in pharmaceutical and biomedical research. The findings of this study contribute to the ongoing efforts in developing sustainable analytical methodologies. Future research could explore its applicability to other pharmaceutical compounds and expand its use in complex matrices, further promoting environmentally conscious practices in analytical chemistry.

Progranulin measurement with a new automated method: a step forward in the diagnostic approach to neurodegenerative disorders.

Mutations in the GRN gene encoded glycoprotein progranulin (PGRN), cause 5-10 % of all cases of frontotemporal lobar degeneration (FTLD). The aim of our study was to verify the analytical and clinical performance of an automated chemiluminescent immunoassay method for PGRN measurement recently developed (Chorus Evo, Diesse Diagnostica, Italy). Five plasma pools and residual plasma samples (K2EDTA) from 25 control subjects (11 males, 62-79 years; 14 females, 54-76 years) and 151 patients (70 males, 53-81 years; 81 females, 44-82 years) with different neurodegenerative disorders (NDs), were assayed. In 61 out of 151 patients, genetic GRN screening was carried. Within-run imprecision (CV%) ranged from 3.8 % (11.5 pg/L) to 10.8 % (2.5 pg/L), and between-run, from 5.6 % (68.7 pg/L) to 10.7 % (2.8 pg/L). At genetic screening, 3 out of 61patients were classified as GRN+ carriers, 18 as "other mutations" and 40 as "no-mutations" carriers. The PGRN median level in GRN+ carriers (15.9 pg/L) was significantly lower than that in control subjects (32.8 pg/L; p=0.006), in GRN- (27.50 pg/L; p=0.007), in other mutation carriers (24.80 pg/L; p=0.05) and in NDs patients (22.40 pg/L; p=0.05) ROC analysis, demonstrates the accuracy of progranulin levels in discriminating between "GRN+" and "GRN-" carriers (AUC 0.985) as well as "GRN+" and "other mutations" carriers (AUC 0.870). The new automated progranulin method, for robust analytical performance, is suitable for use in the clinical setting, supporting clinicians in making a differential diagnosis in patients with neurodegenerative disorder.

Evaluation of AUTION EYE AI-4510 flow cell morphology analyzer for counting particles in urine.

We evaluated the performance of a novel flow cell morphology analyzer AUTION EYE AI-4510 for counting particles in urine. Analytical performance was assessed according to the EFLM European Urinalysis Guideline 2023. Trueness was compared by analyzing 1.012 fresh urine samples with the AUTION EYE AI-4510 (ARKRAY, Inc., Kyoto, Japan) against phase-contrast visual microscopy. Poisson statistics were utilized in assessment of imprecision of particle counts both with quality control material and patient specimens. Relative imprecision against theoretical Poisson imprecision, R(CV), was estimated to be 1.1 for red blood cells (RBC), 1.0 for white blood cells (WBC), 0.9 for squamous epithelial cells (SEC) and 1.1 for bacteria. The agreement with visual microscopy (Cohen's weighted kappa) was 0.93 for RBC, 0.95 for WBC, 0.90 for SEC, 0.79 for non-squamous epithelial cells (NSEC), 0.67 for combined casts, 0.90 for crystals and 0.88 for bacteria. No clinically significant bias was observed. Limits of quantitation at CV=30 % reached 4×106/L for RBC and 5×106/L for WBC. Differentiation of urinary crystals was improved as compared to previous data on digital cuvette imaging. The ARKRAY AUTION EYE AI-4510 provided a desirable imprecision, met the criteria for linearity, LoQ and carry-over, and showed an optimum comparison to visual microscopy for RBC, WBC, SEC and crystals as defined in the EFLM European Urinalysis Guideline 2023. The identification of kidney damage is recommended to be improved by using user-defined review rules. Performance of bacteria counting needs to be confirmed against urine bacterial cultures.