Established Acceptance Criteria Research Articles

In vitro CAR-T cell killing: validation of the potency assay

For advanced therapy medicinal products, the development and validation of potency assays are required, in accordance with international guidelines, to characterise the product and obtain reliable and consistent data. Our purpose was to validate the killing assay for the evaluation of autologous anti-CD19 chimeric antigen receptor (CAR) T potency. We used CD4 + and CD8 + lymphocytes or anti-CD19 CAR-T cells as effector cells and REH (CD19 +) or MOLM-13 (CD19 −) cell lines as target cells. After co-culturing target and effector cells (1:1 ratio) for 24 h, samples were labelled with 7-AAD, anti-CD3 and anti-CD19 antibodies and the frequency of CD19 + dead cells was evaluated by flow cytometry. In order to verify the CAR-T specificity for the CD19 + target, the co-culture between CAR-T and REH or MOLM-13 at different effector-to-target ratios was scheduled. Moreover, not transduced CD4 + and CD8 + lymphocytes were tested in comparison with CAR-T from the same donor to demonstrate the assay specificity. Linearity and accuracy were evaluated, and established acceptance criteria were compiled for both parameters (r2 ≥ 0.97 for linearity and average relative error ≤ 10% for accuracy). Furthermore, the method was considered robust when performed between 23 and 25 h of co-culture, and the intra-assay, inter-assay and inter-day precision was obtained. Finally, in order to verify the inter-analyst precision, the test was executed by three different operators and the intra-class correlation coefficient was > 0.4 in both cases. In conclusion, we consider this CAR-T potency assay as validated and usable in all steps of product development and quality control.

Integration of protein L-immobilized epoxy magnetic bead capture with LC-MS/MS for therapeutic monoclonal antibody quantification in serum.

In recent years, there has been a growing interest in the thriving monoclonal antibody (mAb) industry due to the wide utilization of mAbs in clinical therapies. Robust and accurate bioanalytical methods are required to enable fast quantification of mAbs in biological matrices, especially in the context of pharmacokinetics (PKs)/pharmacodynamics (PDs) and therapeutic drug monitoring (TDM) studies. In this investigation, we presented a novel immuno-magnetic capture coupled with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method designed for the quantification of immunoglobulin G-kappa-based mAbs in biological fluids. The immunoaffinity absorbent for mAb drug purification was meticulously crafted by immobilizing protein L onto monosize, magnetic poly(glycidyl methacrylate) (m-pGMA) beads, synthesized through dispersion polymerization. The microspheres were acquired with an average size of 1.6 μm, and the optimal binding of mAbs from the aqueous mAb solution was determined to be 45.82 mg g-1. The quantification of mAbs in 10 μL serum samples was achieved through affinity purification using m-pGMA@protein L beads (employing rituximab as an internal standard (IS)), on-bead reduction, and rapid tryptic digestion. Remarkably, the entire process, taking less than 2.5 hours, held significant potential for simplifying pretreatment procedures and minimizing analytical time. Furthermore, the developed method underwent validation in accordance with the European Medicines Agency (EMA) guidelines. The assay demonstrated commendable linearity within the 2-400 μg mL-1 range for both daratumumab and pembrolizumab. Intra- and inter-assay coefficients of variation fell within the range of 0.7% to 13.4%, meeting established acceptance criteria. Other validation parameters also conformed to regulatory standards. Ultimately, the efficacy of the method was substantiated in a pharmacokinetic study following a single-dose intravenous administration to mice, underscoring its applicability and reliability in real-world scenarios.

Results of the IAEA supported national end-to-end audit of the IMRT technique in Poland

The dosimetry audit services were established in Poland in 1991, since then new audits have been introduced. The recently developed IAEA audit methodology for IMRT H&N treatments was tested nationally. Anthropomorphic SHANE phantom (CIRS) was used to perform measurements in 8 hospitals which voluntarily participated in the study. Each participant had to complete successfully pre-visit activities to take part in an onsite visit. During the visit, auditors together with the local staff, did a CT scan using local protocol, recalculated the plan and verified all the relevant parameters and performed measurements with an ionization chamber and films in SHANE. The adoption of IAEA methodology to the national circumstances was done with no major issues. Participants plans were verified and the results of ionization chamber were all within the 5 % tolerance limit for PTV (max 4,5%) and 7 % for OAR (max 5,3%). Film global gamma results (3 %, 3 mm, 90 % acceptance limit) were within 91,5–99,7% range. The IAEA established acceptance criteria which were achievable for most tests except for CTtoRED conversion curve. The locally performed study allowed establishing new limits. The audit gave interesting results and showed that the procedure is very thorough and capable to identify issues related with suboptimal treatment preparation and delivery. The new limits for CTtoRED conversion curve were adopted for national study. Such an audit gives an opportunity to verify the quality of locally implemented procedures and should be available for Polish hospitals on a daily basis.

Determination of benzodiazepines and Z-hypnotic drugs in whole blood samples by GC–MS/MS: Method development, validation and application

Benzodiazepines (BZDs) and Z-drugs are widely used as anxiolytics, sedative hypnotics, anticonvulsants and muscle relaxants. “Designer benzodiazepines” (DBZDs) are a new psychoactive substance class consisting of benzodiazepine derivatives that are not allowed for medical use and are known for being used recreationally. From a toxicologist standpoint, the huge number of such substances implicate a necessity for developing fast and reliable methods to identify and quantify these substances in biological matrices, especially in blood.In this study, a method based on gas chromatography–tandem mass spectrometry (GC–MS/MS) was developed and validated for the determination of 28 benzodiazepine derivatives and 3 Z- drugs. Liquid–liquid extraction requiring the use of a 0.5 mL sample of whole blood and 1 mL of ethyl acetate was applied. No derivatization was necessary to obtain sensitivity and selectivity.All validation data met the established acceptance criteria in accordance with international forensic toxicology guidelines. The method was linear within the tested range of 1–100 ng/mL, 1–200 ng/mL, 1–250 ng/mL, 5–200 ng/mL and 10–250 ng/mL depending on the analyte. The LOD was in the range of 0.09 to 0.66 ng/mL, while the LOQ was between 1 and 10 ng/mL. The calculated mean accuracy ranged between 85.1% and 114.4% for intraday replicates and between 85.2% and 111.2% for interday replicates. Intraday precision (as CVs) ranged from 0.2% to 12.5%, while interday precision ranged from 2.0% to 14.5%. Recovery was between 83.8% and 111.2%.This study was the first attempt to apply GC–MS/MS for the multianalyte determination of benzodiazepines and Z-drugs without analyte derivatization. The developed method proved to be sensitive, specific, simple and fast. The analysis in MRM mode gives high sensitivity and selectivity. Thus, the presented method can be successfully utilized for clinical and forensic toxicology analysis, for interpretation ofresults and for the preparation of forensic toxicological opinions for courts of law.

Bridging the Gap: Assessing Material Performance of Laboratory Specimens and Concrete Structures.

Chloride ingress and carbonation pose a significant risk of steel rebar corrosion in concrete structures. Various models exist to simulate the initiation phase of rebar corrosion, addressing both carbonation and chloride ingress mechanisms separately. These models also consider the environmental loads and material resistances, typically determined through laboratory testing based on specific standards. However, recent findings show significant differences between material resistances obtained from standardized laboratory specimens and those extracted from real structures, with the latter exhibiting inferior performance on average. To address this issue, a comparative study was conducted between laboratory specimens and on-site test walls or slabs, all cast using the same concrete batch. This study encompassed five construction sites featuring different concrete compositions. While laboratory specimens adhered to European curing standards, the walls were subjected to formwork curing for a predetermined period (typically 7 days) to simulate practical conditions. In some instances, a portion of the test walls/slabs received only one day of surface curing to emulate inadequate curing conditions. Subsequent testing of compressive strength and resistance to chloride ingress revealed that field specimens exhibited lower material resistance compared to their laboratory counterparts. This trend was also observed in the modulus of elasticity and carbonation rate. Notably, shorter curing periods further compromised performance, particularly resistance to chloride ingress and carbonation. These findings highlight the importance of establishing acceptance criteria not only for concrete delivered to construction sites but also for ensuring the quality of the actual structure.

Development and validation of a peptide-mapping procedure for a novel C1 esterase inhibitor

Peptide mapping is a key method for studying the primary structure of proteins. With its sensitivity to the slightest changes in the covalent structure of a protein, this method is applicable both to medicinal product identification at the control stage and to production process stability monitoring.The aim of the study was to develop and validate a peptide-mapping procedure for the identification of a novel highly glycosylated recombinant C1 esterase inhibitor.Materials and methods. The authors studied recombinant human C1 esterase inhibitor products and trypsin. The study involved peptide mapping using reverse-phase high-performance liquid chromatography and high-resolution mass spectrometry. The following statistics were calculated to evaluate the results: mean, standard deviation, and coefficient of variation. The validation parameters included specificity, precision, and robustness.Results. The authors tested several variants of sample preparation for tryptic digests, including additional N-glycanase treatment and complete deglycosylation, and established the optimal conditions for sample preparation and chromatographic separation of C1 esterase inhibitor peptides to obtain consistent chromatographic profiles (peptide maps). The authors identified characteristic peaks and the corresponding relative retention time and area ranges. The absolute retention time of the second (characteristic) peak was approximately 16.5–16.9 minutes. The relative retention times were 2.14–2.21 for peak 9, 2.55–2.64 for peak 12, 2.97–3.14 for peak 14, 3.11–3.29 for peak 15, and 6.20–6.63 for peak 28.Conclusions. The authors developed a peptide-mapping procedure for C1 esterase inhibitors and optimised the conditions to achieve an over 18-hour reduction in sample preparation time. This procedure met the established acceptance criteria for specificity, precision, and robustness.

A unified design for validation of methods for quality control of blood components

Quality assurance of transfusion media is a fundamental principle of the functioning of all blood service institutions, allowing guaranteeing the safety of the recipient. An integral part of the procuring process of blood components is their laboratory testing, which results reliability and reproducibility can be proved by carrying out of validation of analytical methods. The aim of the study was to define the rules for planning and carrying out validation tests of methods for quality control of blood components, including verification of their compliance with established acceptance criteria. Material and methods. The analysis of the literature about the safety of blood components and the quality of laboratory tests was carried out with an assessment of the applied significance of the information proved. Results and discussion. As a result of the work done, a unified model for confirming of the accuracy of determining of the safety indicators of transfusion media was created, which contributes to improving of the quality of donor biomaterial and the effectiveness of therapy. Conclusions. The presented validation design is relevant for medical organizations working in the sphere of blood donation and its components.

Mastery Level of Higher – Order Thinking Skills (HOTS) High School Students in Mathematical Learning: Systematic Literature Survey

This study is a systematic literature review of 18 study articles focusing on the level of mastery of high school students' HOTS in Mathematics subjects. The study was conducted to identify students' level of mastery in Mathematics subjects, identify the challenges and constraints faced by students in the process mastery of HOTS in Mathematics subjects and identifying factors that influence the level of mastery of HOTS among students. This study was conducted based on Preferred Reporting Items for Systematic Reviews and Meta-Analyses PRISMA guidelines. Two main bases were used in this study, namely Scopus and Google Scholar and found 18 study articles selected for analysis based on established acceptance criteria by the researcher. Based on the analysis of the findings, the researchers devised four main themes highlighted in each selected study article. There are 3 study articles containing themes of HOTS’ mastery levels and influencing factors, 9 study articles that use diverse learning approaches and its influence on the dominance of HOTS. Next the theme of the relationship of the level of mastery of HOTS with the level of student achievement contains 2 study articles and 4 of the 18 selected articles contain the theme of perception and students' readiness towards HOTS. Through the findings of this study, we can conclude that studies focusing on the full level of mastery of HOTS are still under-implemented. Hence, through the review of this systematic literature, it is hoped that further studies can be conducted based on the study of this literature.

Control of Organic Impurities in Semisynthetic Antibiotics

In 2012, the European Medicines Agency (EMA) adopted a guideline, which divided all antibiotics into groups according to the manufacturing process and established acceptance criteria for organic impurities for each of the groups.The aim of the study was to justify the requirements and methodological approaches to setting the limits for organic impurities in semisynthetic antibiotics.Materials and methods: the authors analysed the requirements established by the leading world pharmacopoeias and the State Pharmacopoeia of the Russian Federation regarding the control of organic impurities in semisynthetic antibiotics, using the example of four semisynthetic antibiotics: doxycyline hyclate, clarithromycin, meropenem, and ceftriaxone. The study used the methods of comparative analysis and content analysis. Results: the study demonstrated that the organic impurity profiles of the analysed active substances and the corresponding finished medicinal products often differ significantly across the leading pharmacopoeias, either qualitatively or quantitatively. The Russian, European, and United States pharmacopoeias provide for the use of impurity reference standards in the test procedures for the determination of impurities in active substances of the semisynthetic antibiotics in question, whereas the Japanese Pharmacopoeia allows the use of non-compendial reference substances in the assessment of the chromatographic system separation power.Conclusions: the ability of a pharmacopoeial text to cover a variety of medicinal products coming to the Russian market from different countries has become a vital issue. This includes covering the impurity determination procedures, reference standards, and limits used, because general-purpose methods and limits do not always allow for correct assessment of impurity profiles in substances produced by different manufacturing processes. The current USP practice is to include various impurity control procedures in monographs on medicinal products, and the limits may also vary. This approach may be applied in the State Pharmacopoeia of the Russian Federation as well.

A Review on Method Development, Validation, Optimization and Applications of HPLC

Abstract: High-Performance Liquid Chromatography (HPLC) is a technique used to separate components from the mixture. The technology consists of two phases, the stationary phase, and the mobile phase. Component separation is based on differences in partition coefficients in two phases. The present review article focuses on the principle, types, instrumentation, process validation, application, advantages, disadvantages of HPLC. HPLC methods development and validation play important roles in the discovery, development, manufacture of pharmaceutical drugs, and various other studies related to humans and animals. An analytical procedure is developed to test a defining characteristic of the drug substance or drug product against established acceptance criteria for that characteristic. Validation of HPLC method as per ICH Guidelines covers all the performance characteristics of validation, like Accuracy, precision, specificity, linearity, range, the limit of detection, the limit of quantification, robustness, and system suitability testing. Keywords: HPLC, Types, Method validation, Method development, Instrumentation, and Applications.

Validated GC–MS method for determination of bisphenol a and its five analogues in dietary and nutritional supplements

Bisphenol A (BPA) and its analogues showing structural and functional similarity to BPA are commonly applied in various industrial applications and thus are becoming ubiquitous in the environment. At the same time there is increasing scientific evidence that exposure to these chemicals may lead to adverse health effects in human and wildlife. In recent years dietary and nutritional supplements dedicated for athletes have become more popular and are widely used even by people who are not professionals. This study presents the development, optimization and validation of an analytical procedure for determination of six bisphenol analogues in dietary supplements using gas chromatography – mass spectrometry technique. All validation parameters met the established acceptance criteria in accordance with international guidelines. The method was linear within the tested range of 50–1000 ng/mL, the limit of quantitation was set as the lowest calibration point 50 ng/mL, detection limit for each bisphenol was calculated as standard estimation error on the basis of the linearity testing and was in the range of 8.73–15.44 ng/mL. Method trueness, accuracy (within and between runs) and precision were also verified and were in the range of 78.8 – 100.4%, 94.3 – 103.1%,0.5 – 9.6% respectively.The developed procedure was successfully applied for real samples analysis, namely for fifteen sport supplements of different composition and designated for various purposes, i.e. for increasing effectiveness, promoting muscle recovery and endurance, reducing tiredness and fatigue or increasing immune ability. For the majority of samples, bisphenols were either not detected or detected at the LOD level except bisphenol A which was quantitated in few samples at LOQ level or higher (in the range 0.852–2.892 ng/mg)The field of bisphenol analogues analytics has increased in recent years due to law regulations becoming more strict, thus the development of new analytical tools for quality control of dietary products is needed and fully justified.

METHOD COMPARISON FOR MEASUREMENT OF SYMMETRIC DIMETHYLARGININE IN TIGERS (PANTHERA TIGRIS).

Renal disease is well documented in nondomestic felids and is monitored and diagnosed by serum concentration of blood urea nitrogen, creatinine, and phosphorous. Symmetric dimethylarginine (SDMA) has proven to be an earlier and more sensitive biomarker for the assessment of glomerular filtration rate. Although SDMA is commonly measured in nondomestic felids, information concerning the validity of the assay is lacking. The purpose of the study was to perform a method comparison between high-throughput immunoassay and the reference method, liquid chromatography-tandem mass spectrometry (LC-MS/MS), to quantify SDMA concentrations in tiger blood samples. Concentrations of SDMA were measured for 81 individual tiger samples. The SDMA immunoassay demonstrated excellent correlation to the LC-MS/MS reference method. A Passing and Bablok linear regression analysis had a slope of 1.03 (95% CI, 0.99-1.11), an intercept of 1.64 (95% CI, 0.46-2.34), and a Pearson R= 0.99. The mean bias was 1.53 µg/dl (95% CI, 0.63-2.42 µg/dl), and the limit of agreement was ±7.96 µg/dl. The degree of bias is within established acceptance criteria of 1-3 µg/dl for the immunoassay. Although this study provides good evidence of the utility of the immunoassay to measure SDMA in tiger serum and plasma, further assay validation is recommended.

Retention and impairment of neurocognitive functions in mild cognitive impairment and Alzheimer's disease with a comprehensive neuropsychological test.

AimMATRICS Consensus Cognitive Battery was developed by the National Institute of Mental Health to establish acceptance criteria for measuring cognitive changes in schizophrenia and can be used to assess cognitive functions in other psychiatric disorders. We used a Japanese version of MATRICS Consensus Cognitive Battery to explore the changes in multiple cognitive functions in patients with mild cognitive impairment and mild Alzheimer's disease.MethodsWe administered the Japanese version of MATRICS Consensus Cognitive Battery to 11 patients with mild cognitive impairment (MCI), 11 patients with Alzheimer's disease, and 27 healthy controls. All Japanese versions of MATRICS Consensus Cognitive Battery domain scores were converted to t‐scores using sample means and standard deviations and were compared for significant performance differences among healthy control, MCI, and mild Alzheimer's disease groups.ResultsCompared with healthy controls, patients with MCI and mild Alzheimer's disease demonstrated the same degree of impairment to processing speed, verbal learning, and visual learning. Reasoning and problem‐solving showed significant impairments only in mild Alzheimer's disease. Verbal and visual abilities in working memory showed different performances in the MCI and mild Alzheimer's disease groups, with the Alzheimer's disease group demonstrating significantly more deficits in these domains. No significant difference was found among the groups in attention/vigilance and social cognition.ConclusionsThe Japanese version of MATRICS Consensus Cognitive Battery can be used to elucidate the characteristics of cognitive dysfunction of normal aging, MCI, and mild dementia in clinical practice.

Development and Validation of an Ion-Pair HPLC-UV Method for the Quantitation of Quinoline and Indoloquinoline Alkaloids in Herbal and Pharmaceutical Antimalarial Formulations

Quinine- and cryptolepine-based antimalarials serve as valuable alternatives to artemisinin-based combination therapies (ACTs) in Ghana. Their use, however, is associated with adulteration and substandard quality challenges. An HPLC method targeting quinoline and indoloquinoline antimalarial alkaloids was developed, validated, and applied to evaluate herbal and pharmaceutical antimalarial formulations (HPAFs) and starting materials (APIs). The separation/quantitation of the alkaloids (including quinine, quinidine, cinchonine, cinchonidine, dihydroquinine, dihydroquinidine, and cryptolepine) was achieved on a Zorbax SB-CN column (250 mm × 4.6 mm, 5 μm), with an isocratic elution system of methanol: trifluoroacetic acid (0.1%, v/v) (15 : 85, v/v) at 1.5 mL/min and 223 nm. Method validation was according to ICH Q2(R1) guidelines. It was then used to assess the quality of APIs (n = 3) and HPAFs (n = 44) including quinine-based pharmaceutical antimalarial formulations (QBPAFs) (n = 23) and herbal antimalarial products (HAMPs). The method was found to be specific, selective, accurate, precise, and robust toward the alkaloids with linearity achieved within specified concentration ranges (r2 > 0.995 for all analytes). Analyte stability ranged between 6 and 12 hours. All the APIs contained quinine <99.0%–101.0%, with dihydroquinine and cinchonidine at levels compliant with the established acceptance criteria. The QBPAFs had quinine content ranging between 50.2% and 151.2%, with 43.5% (n = 10/23) of them complying with the acceptance criteria. The related alkaloids observed in the QBPAFs included quinidine (56.5%, n = 13/23), dihydroquinine (100%, n = 23/23), dihydroquinidine (21.7%, n = 5/23), cinchonine (17.4%, n = 4/23), and cinchonidine (95.7%, n = 22/23). For the HAMPs, 81.0% (n = 17/21) were adulterated with quinine (0.59 ± 0.04 mg/10 mL–86.03 ± 0.02 mg/10 mL). Cryptolepine was identified in 19% (n = 4/21) of the HAMPs with concentration ranging between 43.99 ± 0.43 μg/mL and 747.86 ± 0.34 μg/mL. In conclusion, the application of the ion-pair HPLC method targeting quinoline and indoloquinoline antimalarials has demonstrated the presence of quality and poor-quality HPAFs on the Ghanaian market.

Estrogenic Activity of Tetrazole Derivatives Bearing Bisphenol Structures: Computational Studies, Synthesis, and In Vitro Assessment.

The classification of bisphenol A (BPA) as an industrial endocrine disruptor has led to a ban of this ubiquitous critical starting material from food and medical applications. Thus, scientists worldwide are researching to develop non-ER binding starting compounds to fulfill unmet market needs. In line with this trending research topic, the current paper highlights the development of tetrazole derivatives bearing a bisphenol structure (TbB) as a novel weak binder or potential inactive to the estrogen receptor (ER) and androgen receptor (AR). The structure and ligand-based approach supported by binding affinity analysis, electrostatic complementarity, ADMET prediction, and in silico studies identified TbBs as privileged substitutes for BPA. Five TbB ligands were successfully synthesized and subjected to biological testing comprising radioligand competitive binding and functional cellular assays. The measured IC50 value for BPA was found to be 0.24 μM, whereas all the inhibitions were less than 15% for the two TbB ligands, 223-2 and 223-3. As these TbB ligands did not meet the established acceptance criteria of 50% inhibition, they are considered as extremely weak binders to ERα. Steric clashes, the desolvation effect, and the increased total polar surface area (TPSA) of TbB ligands in the hydrophobic binding site are hypothesized to be possible reasons for low binding. Modeling studies complemented by bioassays highlight TbB compounds as privileged prospective BPA replacements. However, more research on TbB ligand toxicity is needed to understand and substantiate that the adverse effects on the hormonal system, for example, via metabolic activation, are not elicited.

Archaeomagnetic evidence of a likely earlier occupation of “El Caracol” lava flow (Zacapu Malpaís, Western Mesoamerica)

A comprehensive archaeomagnetic investigation was carried out on seven ceramic fragments recovered at the "El Caracol" lava flow in the Zacapu Malpaís –Michoacan state, Mexico –. A full set of magnetic-mineralogy experiments (encompassing thermomagnetic curves, hysteresis loops, backfield curves, and isothermal remanent magnetization acquisition measurements) were carried out. The experimental methodology also considers magneto-chemical alteration detection, cooling rate, and remanence anisotropy effects to ensure reliable archaeointensity determinations. Five of the seven ceramic fragments – a pipe (potsherd I), vessel's supports (potsherd II and V), a vessel's wall-fragment (potsherd III), and a pipe's nozzle (potsherd VII) – yielded archaeointensity results within the established acceptance criteria to guarantee quality data. Thermal demagnetization of potsherds I, II, V, and VII shows between two and three magnetization components: one associated with potsherd's fabrication, the second with sample's reheating, and the third one, when present, with a viscous component. For each of the first two components, an intensity value was calculated. Potsherds I, II, III, and VII yielded archaeointensity values between 30 and 40 µT, while potsherd V had a much lower value <20 µT. Although secular variation models do not predict such low intensities, similar values have been reported for the center and south of Mexico. Archaeomagnetic dating was carried out with two regional Paleosecular Variation Curves. The "El Caracol" lava flow is an area with human occupation reported from AD 1200. The results obtained in this work, on the contrary, envision the possibility of an early occupation of the site.

Application of planimetric analysis in geological research – experiments and observations

Planimetric analysis is a well-known and commonly used method for determining the content of a given component in the area of the analyzed surface. In geology, it is used to determine both the mineral and maceral composition, and its results are helpful in solving a wide range of research problems. The aim of this study was to investigate the relationships between the obtained results of planimetric analysis and the density of the measurement grids, which can be adapted to specific situations in geological samples in order to optimize the ratio of the accuracy of the result to the time of analysis. The research was divided into two stages. Models were used in the first stage, while in the second, coal samples were investigated. In the first stage, a virtual grid of measurement points with dimensions of 100 by 100 points was created, which gave a total of 10 000 points. After creating the measurement grid model, 102 scenarios were established, differing in the content of the analyzed component A (0.1%, 0.5% and from 1 to 100%). In each of the adopted scenarios, 100 combinations were carried out so that the arrangement of points on the measurement grid corresponding to component A was random. Then, the number of measuring points was reduced several times from the original 10 000 to 100 and each time content of component A was calculated. The obtained average component A content in most cases turned out to be only slightly different from the original, which cannot be stated in the case of both minimum and maximum values – here a wider range of results is observed. In the second stage, 3 coal samples of varying maceral composition complexity were investigated and a measurement grid of 100 by 100 points was used. The original grid density was gradually reduced (down to 144 points) and maceral composition was calculated each time. Results obtained from models as well as from samples show a clear trend of decreasing accuracy with decreasing density of the measuring grid. The obtained results were also analyzed in terms of the established acceptance criteria for which the content of the component was assumed to be different from its content based on a grid of 10 000 points by up to 5%, 10% or 30% of the original value. The analysis of the relationship between the accuracy of the obtained results and the time of conducting the analysis seems to indicate that the choice of the measurement grid based on 500 points is optimal.

Abstract 2239: Comprehensive validation of RNA sequencing for clinical NGS fusion genes and RNA expression reporting

Abstract Tumor genome sequencing has emerged as a powerful tool for identifying biomarkers for targeted cancer therapies. While DNA sequencing is a well-established method and considered a gold standard, RNA sequencing (RNA-seq) can identify anomalies in gene transcription, regulation of gene expression, and gene fusions, which have critical diagnostic and therapeutic impacts. Tempus Labs is CLIA-certified, CAP-accredited and offers several clinically validated NGS assays for solid tumor and hematological malignancy testing, including the xT targeted 648-gene oncology panel and the xE whole-exome ~20,000 gene panel. The Tempus whole-transcriptome assay by hybrid-capture NGS has been enhanced to improve coverage, focusing on fusion genes and RNA expression calls. We clinically and analytically validated fusion calls for gene rearrangement detection and clinical reporting, and analytically validated RNA expression counts for research purposes only. For RNA fusion (translocation) calling, we sequenced 96 tumor samples, including formalin-fixed, paraffin-embedded (FFPE) tissue, whole blood, and bone marrow samples from 12 cancer types; 82 of these samples contained reportable fusions. Of these, 86 samples were positive for RNA fusions, including 62 positive samples with targeted fusion breakpoints (i.e., utilizing custom probes designed to the breakpoints and spiked-in at hybridization) and 24 positive samples with untargeted fusion breakpoints (i.e., utilizing no custom probes). Targeted fusion accuracy was evaluated against the first version of the RNA sequencing assay. The first version of this assay was evaluated with cell line controls. In addition, validation included a specificity test and a rolling validation of fusions via DNA sequencing (positive controls). The concordance for targeted fusions was 100%, with 99.9% sensitivity and 99.9% specificity. For untargeted fusions, the overall concordance was 97%, with 97% sensitivity. For RNA expression calling, we conducted a clinical linearity study using 88 samples and testing 18 genes to measure concordance between qPCR ΔCT values and normalized gene expression levels. Of these, 15/18 genes met the established acceptance criteria of R &amp;gt; 0.75. In conclusion, the whole-transcriptome by hybrid-capture NGS assay offers clinically and analytically validated unbiased detection of common and novel gene rearrangements, as well as analytically validated gene expression data for comprehensive research analyses. Citation Format: Jun Hu, Jerod Parsons, Brittany Mineo, Josh SK Bell, Jenna Malinauskas, Joshua Drews, Jack Michuda, Calvin McCarter, Rasika Dhond, Jack C. Tyndall, Nithya Sreenivasan, Sheeri Hanjra, Shannon Gallagher, Ankit Jambusaria, Naihui Zhou, Catherine Igartua, Nike Beaubier, Richard A. Blidner, Robert Tell. Comprehensive validation of RNA sequencing for clinical NGS fusion genes and RNA expression reporting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2239.

Optimization of hemolysis, icterus and lipemia interference thresholds for 35 clinical chemistry assays

ObjectivesInterference of chemistry assays by hemolysis, icterus and lipemia (HIL) was investigated on the Abbott Alinity c system. We sought to empirically establish optimized HIL index thresholds for the purposes of reporting HIL interference in a hospital laboratory and advising clinicians on the interpretation of laboratory results in the presence of hemolysis, icterus or lipemia. MethodsHIL index values measured by spectrophotometry were compared with concentrations of hemoglobin, bilirubin and Intralipid. HIL interference of 35 Abbott Alinity chemistry assays was subsequently investigated by pairwise comparison of test results in pooled serum or plasma with those in test preparations spiked with hemolysate, bilirubin or Intralipid. Data generated from the interference experiments were critically assessed according to assay-specific acceptance criteria adapted from multiple sources, and optimized thresholds for HIL indices were established. ResultsCorrelations between HIL index values and their corresponding concentrations of hemoglobin, bilirubin and Intralipid were, in general, very good within the ranges of interferent concentrations tested. Hemolysis significantly affected 12 of 35 assays, whereas bilirubin and Intralipid interfered with four and three assays, respectively. Both the direction and magnitude of Intralipid interference with the direct bilirubin assay were dependent on the concentrations of the analyte. ConclusionsHIL interference of the Abbott Alinity clinical chemistry assays investigated in this study was not uncommon. At present, there are no universally accepted criteria for defining significant assay interference for clinical practice. In establishing acceptance criteria for defining assay interference, each assay should be assessed according to both analytical criteria and clinical relevance.

The performance of food safety management system in relation to the microbiological safety of salmon nigiri sushi: A multiple case study in a Japanese chain restaurant

The study's objective was to evaluate the performance of the food safety management system (FSMS) in relation to the microbiological food safety (FS) output of Salmon Nigiri Sushi in the selected outlets of a Japanese chain restaurant. Two outlets that are FSMS certified with ISO 22000 and two outlets that are FSMS not certified with ISO 22000 were selected. Microbiological Assessment Scheme was used in the study of evaluating the performance of the FSMS, in which samples were collected from the selected critical sampling locations and analysed for Bacillus cereus, Vibrio parahaemolyticus, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Coliform, Total Plate Count (TPC), and Yeast and Mould Count. The microbial counts were compared against the relevant established acceptance criteria to determine FS level and consequently calculate the FS output. The overall FS output of the two outlets that are non-ISO 22000-certified FSMS was 2–3 (moderate to a good level) and 2 (moderate level) in contrast to ISO 22000-certified FSMS outlets that was 2–3 (moderate to a good level) and 1 to 2 (poor to a moderate level). None of the outlets was able to achieve their FS objectives. Bacillus cereus, Vibrio parahaemolyticus, and Listeria monocytogenes were not detected, and therefore, the FS level was determined at level 3 (good result) for all outlets. The FS level for TPC and E. coli was determined at level 1 (low result) due to high E. coli counts (even in filtered tap water), which lead to ineffectiveness in cleaning and sanitation of food contact surfaces, including hand washing. It appeared the performance of FSMS in relation to the microbiological FS outputs of Salmon Nigiri Sushi of outlets that are non-ISO 22000-certified FSMS were better than outlets that are ISO 22000-certified FSMS.