bluuebunny/crossref_metadata_2025 huggingface dataset:
B-207 Clinical Evaluation of ExiStation™ FA 96 RV for the detection of human respiratory viruses in human nasopharyngeal swab specimen
Authors: Lee J , Kim J , Lee C , Park H
Published: October 2024

Background

There has been growing demand for fully automated multiplex testing of respiratory viruses in molecular diagnostics following the COVID-19 pandemic. ExiStation™ FA 96 RV developed by BIONEER is a multiplex real-time RT-PCR assay. It is designed for the simultaneous detection and differentiation of SARS-CoV-2, Influenza A, Influenza B, and RSV RNA in human nasopharyngeal swab specimens from individuals showing signs and symptoms of respiratory viral infection. ExiStation™ FA 96 automates the entire process from RNA extraction to real-time RT-PCR for up to 96 samples at once within 100 minutes. Moreover, the system is equipped to decap sample tubes, eliminating the need for handling sample transport media. In this study, the clinical performance of the ExiStation™ FA 96 RV assay was evaluated using nasopharyngeal swab samples.

Methods

Nasopharyngeal swab samples, totaling 859 samples, are collected according to the procedures by the clinical institution in France and preserved in Virus transport media. Once anonymized and randomly allocated, the swab transport media tube, without capping procedure, are directly placed onto the ExiStation™ FA 96. Subsequently, both extraction and real-time RT-PCR are automatically processed. Clinical sensitivity, specificity, and agreement are analyzed by comparing results with confirmatory reagents SARS-CoV-2 RT-qPCR Reagent Kit, PKAmp™ Respiratory SARS-CoV-2 RT-PCR Panel Kit, and Bosphore SARS-CoV-2/Flu/RSV Panel Kit, as well as comparator reagent Allplex™ Respiratory Panel 1 and Allplex™ SARS-CoV-2 Assay.

Results

The comparison between confirmatory reagents and the ExiStation™ FA 96 RV resulted in a clinical sensitivity of at least 96.4% (164/164 for SARS-CoV-2, 64/65 for Influenza A, 74/75 for Influenza B, and 53/55 for RSV) and a clinical specificity of 100% (500/500 for all targets). When compared with the comparator reagent, the overall agreement and Cohen's kappa value were both determined to be above 99.1% and 0.989, respectively (664/664 for SARS-CoV-2, 564/565 for Influenza A, 573/575 for Influenza B, and 550/555 for RSV). Additionally, any discrepant samples identified in comparison with the comparator reagent were verified through sequencing analysis. Upon sequencing analysis on a total of 8 samples and aligning the confirmed results with the analysis content, no alterations observed in the analysis of clinical sensitivity and specificity.

Conclusions

The evaluation of the ExiStation™ FA 96 RV assay showed outstanding clinical performance in detecting four respiratory viruses, SARS-CoV-2, Influenza A, Influenza B, and RSV, with high sensitivity and specificity. This study supports that the ExiStation™ FA 96 RV assay is a reliable tool for simultaneous molecular diagnostics of various respiratory infections in clinical laboratories.
B-208 Room Temperature Stable PCR Reagents Preparation Using Capillary-Mediated Vitrification Process
Authors: Renu S , Peris-Taverner Y , Sharpe J , Amle S , Bronsart L , Mohanty P , Shank-Retzlaff M
Published: October 2024

Background

Polymerase chain reaction (PCR) is a sensitive, and most used molecular technique for the disease diagnosis. Reagents used for PCR is available as a one-step and ready-to-use PCR Master Mix, which contains Taq DNA polymerase, dNTPs, MgC12, PCR reaction buffer. However, to preserve bioactivity, PCR reagents must be stored and distributed as frozen. Desiccation of PCR reagents can avoid cold storage and improper handling of wet reagents and its associated errors. We discovered capillary-mediated vitrification (CMV) processes can stabilize different biomolecules and allow them to be stored at ambient temperature. Here, we hypothesized that the CMV process can preserve and improve PCR reagents thermal stability, which will reduce PCR processing time and improve the testing throughput.

Methods

The CMV process was performed by mixing PCR Master Mix individual or all reagents such as Taq DNA polymerase or Hot Start Taq DNA polymerase, dNTPs, 16S rRNA primers, MgC12, and PCR reaction buffer with an excipient buffer and adding the mixture to a porous scaffold. The loaded scaffolds were placed into our vitrification chamber and dried for 30 min. After drying, the CMV-stabilized sample was tested or packed in Mylar pouches and were stored at ambient and elevated temperatures for 42 days. On the day of testing, the stabilized samples were eluted with nuclease-free water and E. coli genomic DNA was added, and the 50 uL reaction was tested by conventional PCR. The PCR product was visualized under agarose gel electrophoresis.

Results

To assess whether the CMV process maintains PCR reagents activity and not interfering with the assay, the individual PCR reagents and as a Master mix are preserved. The CMV stabilized polymerase enzyme, primer, or dNTPs amplified DNA similar to frozen reagents and produced comparable PCR products. Notably, CMV stabilized PCR Master Mix which contains either Taq DNA polymerase or Hot Start Taq DNA polymerase enzyme, dNTPs, primers, MgC12, and reaction buffer yielded intact amplified PCR product, which is comparable to frozen reagents. Likewise, a high intense PCR product was observed in the stabilized PCR Master Mix reagents when stored at room and elevated temperatures for 42 days.

Conclusions

The study results indicate that the CMV process stabilized and improved the PCR Master Mix reagents activity. The study suggests that the CMV technology has the potential to enable ambient storage and shipping of PCR reagents for molecular diagnostic and other related applications.
B-224 Rapid, Extraction-Free, and Portable Molecular Detection of HIV-1 and HCV Directly From Whole Blood
Authors: Michalak K , Tang D , Nguyen K , Lacap P , Kim J , McLaren P J
Published: October 2024

Background

Early diagnosis of human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) is key to preventing viral transmission and improving linkage to treatment. Isothermal amplification methods, such as reverse transcription looped-mediated amplification (RT-LAMP), have enabled molecular diagnostics to expand beyond centralized laboratories. Despite the advances in molecular point-of-care (POC) testing, many of these tests are costly and still depend on a reliable power-source, specialized equipment, and cold-chain storage, making them impractical for resource-limited settings. To overcome these barriers, there is a need for innovative molecular diagnostics for blood-borne pathogens. Here, we propose a simple, extraction-free, portable workflow for detecting HIV-1 and HCV from whole blood within 40 minutes.

Methods

We developed singleplex RT-LAMP-based tests using modified published primer sets. Contrived whole blood samples containing HIV-1 or HCV virions were diluted in equal parts water and loaded directly into optimized RT-LAMP master mixes. To mitigate cold-chain storage dependence, RT-LAMP reactions were performed using a lyophilized master mix. The reactions were heated for 30 minutes using a hand-held, battery-powered heating device for simultaneous virion lysis and amplification. For simple visual detection, amplification was coupled with a lateral flow-based dipstick test. The analytical sensitivity of each detection test was evaluated using contrived whole blood samples ranging in HIV-1 and HCV 1a viral loads. Additionally, HCV detection was evaluated on genotypes 1b, 2b, and 3a. For result confirmation, a custom CRISPR-Cas12a-based assay was used to verify the presence or absence of pathogen-specific amplicons following amplification.

Results

The diagnostic workflow can be completed within 40 minutes, including less than 10 minutes of hands-on time. At optimal conditions, the HIV-1 and HCV 1a singleplex tests had lower limits of detections of 4.89 log10 cp/mL and 3.77 log10 IU/mL, respectively. Evaluation of the published literature showed that these viral loads are within the ranges observed during acute HIV-1 and HCV infection. HCV detection was also observed for genotypes 1b and 3a, albeit at a lower sensitivity than genotype 1a. No detection was observed for genotype 2b, presumably due to the lower viral loads of the samples. No cross-reactivity between the two tests was observed.

Conclusions

Our proposed workflow allows for rapid detection of blood-borne pathogens without the need for complex equipment or cold-chain storage, showing potential for application in resource-limited settings. Additional validation within a clinical setting using fingerstick blood remains to be tested.
B-243 Validating a highly automated, rapid turnaround urine biomarker assay for diagnosis of acute interstitial nephritis
Authors: Obeid W , Moledina D G , Parikh C R
Published: October 2024

Background

Urinary tumor necrosis factor alpha (TNF-α) and chemokine C-X-C motif ligand 9 (CXCL9) have been identified as potential diagnostic biomarkers for acute interstitial nephritis (AIN). Although validated on standard methods like ELISA and mesoscale discovery (MSD), these platforms have a turnaround time of greater than 4hr. Ella, is a new platform which takes around 90 minutes and is more suited to be used for diagnosis of AIN in hospital setting. However, validation data for urine TNF-α and CXCL-9 in Ella assays are currently unavailable.

Methods

Five samples were selected for dilution-linearity and spike-recovery analysis. At the time of collection, samples were centrifuged at 2,000xg and 4°C for 10 min and the supernatant was aliquoted and stored at -80°C until assayed. We investigated dilution linearity by performing serial dilutions at three levels (Neat, 1:2, 1:4) assayed in duplicate. We performed spike and recovery experiment at three different concentration levels (high, medium, and assay diluent) by adding 5% spike volume of mixed TNF-α and CXCL-9 calibrator or assay diluent to the samples. We defined acceptance when recovery was between 80% - 120%. Intraassay %CV was calculated by averaging %CV of duplicate runs across all experiments. Inter-assay %CV was calculated by averaging %CV three levels of QC over three days. Samples were analyzed on MSD for method comparison and correlation coefficients (Pearson and Spearman) were calculated between the two measurements.

Results

Average recoveries and MSD comparison for TNF-α and CXCL9 are in the table below. Diluting the urine samples 4-fold yielded acceptable recoveries between 80% - 120%. Comparison between MSD and Ella yielded strong correlation for CXCL9 and moderate correlation for TNF-α.

Conclusions

The Ella platform can be used to measure urinary TNF-α and CXCL9 with good precision and acceptable recoveries at 1:4 dilution. Ella measurements compare favorably with those of MSD.
B-244 Analytical Performance and Method Comparison Evaluation of a New High Throughput Fully Automated Plasma GFAP Immunoassay
Authors: Vucetic Z , Szabo M , Salvati M , Schlichtmann B , Patzlaff J , Unruh D , Curtis K , Mediger L , Nichkova-Doseva M
Published: October 2024

Background

Glial Fibrillary Acidic Protein (GFAP) levels are shown to be an indicator of neurologic injury in conditions like Traumatic Brain Injury (TBI) and stroke and as a marker of disease progression in neuromuscular disorders such as multiple sclerosis. Plasma GFAP is also implicated in detecting AD pathology, correlating to the clinical stage of the disease. The performance characteristics of the plasma GFAP immunoassay currently being developed on Beckman Coulter Access 2 and DxI 9000 analyzers are described.

Methods

The prototype GFAP assay is a one-step sandwich assay utilizing an anti-GFAP mouse monoclonal (MAb) antibody/alkaline phosphatase conjugate and paramagnetic particles coated with a complementary anti-GFAP mouse MAb. Sample and reactants are incubated and washed, and then a chemiluminescent substrate is added. The light generated is directly proportional to the GFAP concentration in the sample. The assay time to the first result is ∼30 minutes. Analytical performance evaluation and method comparison across different platforms was performed. 43 EDTA plasma samples, consisting of 19 Alzheimer’s Disease (AD) patient samples and 24 age-matched healthy normal samples, were evaluated.

Results

The prototype GFAP assay has a targeted analytical measuring range of 1.0 pg/ml to approximately 700.0 pg/ml (up to 10,000 pg/ml). All normal samples were quantified with 90% measuring below 20.0 pg/ml and a low dose coefficient of variation <4.0%. A comparison of the prototype GFAP assay on DxI 9000 and Access 2 analyzers shows a Passing-Bablok slope of 0.94 (R=0.992) and an intercept of 0.37 pg/ml. Concordance analysis shows excellent agreement between the Access 2 and DxI 9000 assays. The dose ratio of the median of AD over the median of normal samples for Access 2 and DxI 9000 is 2.31 and 2.41, respectively, with AD patient median dose values of 14.3 pg/ml and 14.6 pg/mL, respectively, and normal sample median dose values of 6.2 pg/ml and 6.0 pg/mL, respectively.

Conclusions

The prototype GFAP assay provides fast, highly sensitive, and precise results in an automated immunoassay on the Beckman Coulter Access 2 and DxI 9000 platforms. The prototype plasma GFAP assay holds promise for diagnosing and monitoring various neurodegenerative diseases, including AD.
B-246 Pab to mab conversion via mass spectrometry: direct antibody discovery from serum
Authors: Crha J , Han X , Le Bihan T , Nunez De Villavicencio Diaz T , Ma B
Published: October 2024

Background

Navigating the intricacies of discovering unique and functional antibody binders for in vitro diagnostic development comes with several technological challenges, such as limited diversity of antibody repertoires, functional screening challenges, and the absence of suitable in vitro models. A proteomics-driven strategy to antibody discovery is a promising solution to overcome these obstacles by facilitating polyclonal to monoclonal antibody conversion.

De novo polyclonal antibody sequencing utilizes mass spectrometry and machine-learning driven bioinformatics to obtain complete antibody sequences from immunoserum or purified protein samples. This approach investigates the entire circulating antibody repertoire at the protein level, thereby broadening the spectrum of potential candidates. It further facilitates functional selection by enabling antibody purification and enrichment techniques. This capability facilitates the identification of antibodies possessing unique functions, crucial for subsequent downstream development.

We demonstrate the application and de novo polyclonal antibody sequencing using mass spectrometry and machine learning bioinformatics to enable the next generation of antibody discovery.

Methods

In this study, we employed a novel mass spectrometry-based approach, REpAb, to de novo sequence polyclonal antibodies directly from the immunized serum of animals or human patients. REpAb integrates mass spectrometry with machine learning-based bioinformatics to de novo sequence and assemble monoclonal antibodies from a polyclonal mixture. Protein lysates underwent analysis using an Orbitrap EclipseTM Series instrument (ThermoFisher Scientific, CA, US) coupled with the LC Evosep One (Evosep, Denmark).

Results

De novo polyclonal sequencing with REpAb yielded unique antibody binders not found in splenocytes or peripheral blood mononuclear cells (PBMCs). De novo antibody sequencing-discovered antibody sequences from naturally exposed humans showed unrestricted germline usage, compared to restricted germline used in antigen-immunized animals. Additionally, a proteomics-based approach for antibody discovery enables functional antibody selection through standard protein A/G purification, followed by negative and positive selection enrichment strategies to isolate antibody clones specific to the region of interest on the antigen.

Conclusions

Sequencing antibodies at the protein level directly reflects the circulating antibody repertoires, which expands the antibody diversity from the BCR repertoire. Proteomic-based antibody discovery empowers the functional selection of antibodies via meticulously designed enrichment strategies. This approach allows for the identification of a diverse pool of candidates possessing specific functions, such as anti-idiotypic features.
B-253 Using biomarkers in recurrent renal stone formers to detect silent stones
Authors: Gohel M , Ng C
Published: October 2024

Background

Glycosaminoglycans (GAGs) protect cells from binding of calcium oxalate (CaOx) crystals to prevent CaOx stone formation. Hyaluronan (HA) is a non-sulfated GAG with crystallization-promoting activity during renal stone formation. The excretion of urinary GAGs and HA were measured in stone-formers (SF), post-treated SF, and normal controls to reveal their relationship with renal stone disease.

Methods

Three groups were included - active SF, post-treated SF (SF after extracorporeal shock wave lithotripsy) and normal controls with 40 subjects in each group. Early morning urine was collected for each subject. The hexuronate content of the GAGs were measured by carbazole reaction and values for GAGs were standardized against creatinine. Individual GAGs extracts were then digested sequentially with Streptomyces hyaluronidase and chondroitinase ABC to yield the HA disaccharides for analysis by high performance liquid chromatography.

Results

Hexuronate content of GAGs were in the order of post-treated SF < SF << normal controls. SF had an enhanced urinary excretion of HA with no increase of urinary HA in post-treated SF group. However, both the SF and post-treated SF groups had increased proportion of HA (of total GAGs) compared to normal controls. Active-SF and post-treated SF groups had lower total GAGs content but increased proportion of HA than that of the normal controls indicating that urinary GAGs and HA are probably protective/risk factors, respectively, for renal stone disease.

Conclusions

The higher recurrence rate of renal stone disease may be due to the presence of sub-species of GAGs, HA, that becomes an accidental participant for renal stone formation. Concomitantly, other species of charged sulfated GAGs, which are protective to the cells and prevent crystal binding, are found to be in lesser content in active SF and post-treated SF. Clinicians can use these two markers, which are easily detectable (by ELISA) for prevention and monitoring of silent renal stone formation.
B-257 Dont go breaking my heart: use and misuse of beta-blockers in clinical and forensic casework (2019-2023)
Authors: Midthun K M
Published: October 2024

Background

Beta-adrenergic antagonists, or beta-blockers, are commonly prescribed for the treatment and prevention of a variety of cardiovascular issues and other health conditions. These include, but are not limited to heart failure, heart rhythm disturbances, high blood pressure, hypertension, anxiety, asthma, glaucoma, and migraines. Beta-blockers work by blocking access to beta-receptors, which then enhance or inhibit cellular activity and result in dilating blood vessels and relaxing the heart muscle. When misused, accidentally or intentionally, serious adverse effects including dizziness, seizure, heart failure, QTc prolongation, coma, and death can occur. In routine toxicology testing, beta-blockers are often reported qualitatively, which likely underestimates the reporting of related adverse events. We performed a retrospective review to evaluate quantitative levels of beta-blockers in blood and identify trends of potential misuse.

Methods

We queried our laboratory’s data management system from 2019-2023 for reported findings of beta-blockers in blood. Analytes were quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results were analyzed using Microsoft Office software.

Results

Blood results and a comparison of known therapeutic and adverse concentrations are shown in Table 1. Age and sex were reported in 50% and 74% of cases, respectively. Average age ranged from 43 years old (propranolol) to 59 years old (atenolol and metoprolol), without major differences between sexes. Overall use of beta-blockers was split evenly between men and women. Importantly, each analyte provided examples which far exceeded known therapeutic levels, with several cases submitted as potential overdoses.

Conclusions

Since beta-blockers are generally reported qualitatively during routine toxicology testing, concentrations are unknown. Quantitative testing has shown that beta-blocker concentrations often exceed known therapeutic ranges, which can lead to adverse effects and fatal outcomes. Physicians and death investigators should be aware of these adverse effects and the need for specialized testing in the event of suspected beta-blocker toxicity.
B-258 Rapid clozapine blood levels on the Beckman Coulter DxC 500 AU
Authors: Fritz J , Holler A , Wisnoski J , Hilaire M , Salamone S
Published: October 2024

Background

Clozapine is the most effective drug for treatment-resistant schizophrenia. Multiple guidelines strongly recommend clozapine therapeutic drug monitoring (TDM). Measuring clozapine blood levels provides essential information when there are questions of medication adherence, efficacy, toxicity, medication interactions, or concern over other factors that may influence clozapine levels. Turnaround time is essential for effective clinical decisions. However, with liquid-chromatography/mass spectrometry, clozapine levels can take days to report results. To address the medical need for a faster turnaround time, a rapid clozapine immunoassay was developed for automated clinical chemistry analyzers. The immunoassay measuring range is based on established APA Practice Guidelines and ASCP/AGNP Consensus Statement. We developed an analyzer application for the DxC 500 AU (time to first result < 9 minutes) to expand the availability of rapid clozapine blood level testing.

Methods

The performance of the FDA-cleared clozapine assay was evaluated on the DxC 500 AU according to CLSI guidelines. Precision testing was performed with three clozapine spiked pools over five days with five replicates per sample. Precision sample recovery was assessed to their theoretical spike concentration. Linearity was verified with five spiked serum samples covering the measuring range. Method comparison was performed with 40 remnant patient samples vs. the Beckman Coulter AU480.

Results

Repeatability CV was ≤5.5% and within-laboratory precision CV was ≤6.1%. Individual deviation was ±20% and mean deviation was ±2% for all samples. Deviation from linearity was <3%. Deming regression statistics for the method comparison were a slope of 0.987, an intercept of 5.7, and an R of 0.9948. The methods are statistically equivalent: the 99% confidence limits contained 1 and 0 for the slope and intercept respectively.

Conclusions

The precision, recovery, linearity, and method comparison results for clozapine on the DxC 500 AU demonstrate that the analyzer can be used to generate robust results for clozapine.
B-259 Cannabinoid Prevalence in Umbilical Cord Tissue Toxicity Testing
Authors: Reinhart A , Kacinko S L , Midthun K M
Published: October 2024

Background

Cannabis is rapidly becoming legalized in the United States. While its effects on the developing fetus are not fully understood, current recommendations advise against cannabis use and exposure during pregnancy due to its association with negative outcomes, such as preterm birth, fetal growth restriction, low birth weight, and developmental deficits. If drug exposure is suspected, umbilical cord tissue (UCT) testing is growing in frequency as a tissue of choice when testing infants. Neonatal testing protocols vary between hospitals, including both targeted or universal collection and targeted or universal testing of neonatal specimens. Based on the current environment of legalization, we were curious to see what the changes in cannabis use were over the previous five years, from 2019-2023.

Methods

We queried our laboratory information system for reported findings from screening and confirmatory umbilical cord testing, both of which were analyzed via liquid chromatography tandem-mass spectrometry (LC-MS/MS). Results were analyzed using R coding software.

Results

Overall, annually, 2019-2023 positivity rates for testing in general ranged from 41% - 44% positivity and maintained at 44% from 2020 onwards (n=90384 total workorders 2019-2023, n=40551 total positive workorders). Cannabinoids were starkly the most common drug found, accounting for 59-63% of all positive workorders annually from 2019-2023. There were no statistically significant differences in the cannabinoid positivity rates between 2019-2023. Of note, cannabinoid-only (delta-9-carboxy THC) workorders accounted for 28% of all positive workorders (n=25962) during that time frame. This was followed by methamphetamine-only (n=2485 workorders) and buprenorphine-only (n=1765 workorders). The combination of cannabinoids and methamphetamine, the most common drug combination found in UCT testing, had a range of positive results from 2019 - 2023 of 2.9 - 3.6% (n=1327 workorders). Cannabinoids and cocaine, the second most prevalent drug combination, ranged in positivity from 1.3 - 1.6% from 2019-2023 (n=616 workorders).

Conclusions

Cannabinoid exposure is prevalent in pregnant individuals undergoing UCT testing. Physicians should take care to keep informed on the impacts of cannabis exposure on the developing fetus and educating clients on the effects of cannabis exposure during pregnancy.
B-262 Validation of a point-of-care lateral flow immunoassay for urine drug testing prior to application in an outpatient rapid access addiction medicine clinic
Authors: Bohn M , Delaney S , Jung B , Lamb W , Leung F
Published: October 2024

Background

Point-of-care (POC) urine drug testing is a useful adjunct to self-reporting in rapid access addiction medicine settings to immediately guide patient management. However, available POC immunoassays have limitations including cross-reactivity with unrelated compounds or low sensitivity that may cause false results. Here, we assessed the performance of a multi-drug test panel by comparing against gold-standard liquid chromatography tandem mass spectrometry (LC-MS/MS) testing.

Methods

102 residual urine specimens were assayed using a competitive lateral flow immunoassay (LFA) for 10 drugs: 2-ethylene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP, methadone metabolite), buprenorphine, morphine, hydromorphone, oxycodone, fentanyl, cocaine, methamphetamine, amphetamine, and benzodiazepines (BTNX Rapid ResponseTM Multi-Drug Panel). Results were compared to those obtained by LC-MS/MS (n=67, 66%) or kinetic interaction of microparticles in solution automated immunoassay (KIMS) (Roche Diagnostics, n=35, 33%). Broad spectrum LC-MS/MS results were reviewed in entirety for discordant cases, particularly in false positives to identify the presence of any known cross-reacting compounds.

Results

Of 10 drugs evaluated, four demonstrated ≥95% agreement with LC-MS/MS or KIMS immunoassay (EDDP, buprenorphine, oxycodone, methamphetamine). Fentanyl demonstrated the highest false negative rate of 44% (LFA cut-off: 10 ng/mL) followed by amphetamines (22%, cut-off: 1000 ng/mL). Morphine and hydromorphone demonstrated false positive rates of 14% and 18%, respectively, with most cases likely due to cross-reacting opiate or opioid metabolites. Benzodiazepines (target: Oxazepam) demonstrated false positive and negative rates of 13% and 24%, respectively.

Conclusions

To our knowledge, this is the first study to evaluate the performance of the BTNX multi-drug test panel against definitive testing in urine samples. While good concordance was observed for most drugs, high rates of discordant results for fentanyl and others emphasize the need for confirmatory testing by methods with higher sensitivity and specificity. Careful consideration prior to implementation would be essential, including physician education, interpretative comments, and training resources.
B-263 Analytical Performance Evaluation of Eleven Drug of Abuse Assays on the Atellica CI Analyzer
Authors: Lewisch S , Gupta P , Varhue W , Arrode-Bruses G , Snyder J
Published: October 2024

Background

The Atellica® CI Analyzer is an automated, high-throughput integrated chemistry and immunoassay analyzer utilizing both Atellica® CH and Atellica® IM Assays. This study evaluated the analytical performance of the Atellica CH Amphetamines (Amp), Barbiturates (Brb), Benzodiazepine (Bnz), Cannabinoids THC (Thc), Cocaine Metabolite (Coc), Ecstasy (Xtc), Methadone (Mdn), Opiates (Op), Oxycodone (OXY), Phencyclidine (Pcp), and Propoxyphene (Ppx) Assays on the Atellica CI Analyzer.

Methods

Precision and method comparison (MC) studies were used as performance indicators. Precision studies were performed according to CLSI EP05-A3 using quality control (QC) samples consisting of contrived human urine samples. One aliquot of each QC was tested in duplicate in two runs per day ≥2 hours apart on each analyzer for ≥ 20 days. Precision for each assay was evaluated with one reagent lot on one system. Method comparison studies were performed with three reagent lots according to CLSI EP12-A2. Individual native and contrived human urine samples were analyzed using the Atellica CH Amp, Barb, Bnz, Thc, Coc, Xtc, Mdn, Op, OXY, Pcp, and Ppx Assays on both the Atellica CH and Atellica CI Analyzers. The results were assessed based on analyte values used for distinguishing positive (value ≥cutoff) and negative (value <cutoff) specimens.

Results

As shown in table below, repeatability and within-lab %CVs based on manufacturer arbitrary units (mAU) were <0.8% and <3.2%, respectively or qualitative interpretation for each replicate remained unchanged for all 80 precision testing measurements (OXY, PcP). Qualitative accuracy assessed by concordance analysis demonstrated ≥95% agreement between the Atellica CI Analyzer and Atellica CH Analyzer.

Conclusions

Evaluation of the Atellica CH Amp, Barb, Bnz, Thc, Coc, Xtc, Mdn, Op, OXY, Pcp, and Ppx Assays using the Atellica CI Analyzer demonstrated acceptable precision and equivalent performance compared to the same assays on the Atellica CH Analyzer.
B-277 Detectable Levels of Toxic Metals in Children's Playgrounds
Authors: Oyelakin-Ogunbileje J O , Oyelakin S T , Sadagopa R V
Published: October 2024

Background

Metal-contaminated soil poses significant health risks to kids because they are more sensitive to heavy metal exposure in the environment. Studies have shown that continuous exposure to metals can increase the risk of metal accumulation in blood and various body organs. This study determines the levels of some toxic metals in various playgrounds in our community.

Methods

We determined the levels of lead (Pb), chromium (Cr), cadmium (Cd), and copper (Cu) in six playgrounds (parks) (T-RP, T-GP, T-TX, L-HH, L-WH, L-SPLX) using graphite furnace atomic absorption spectrophotometry (GF-AAS) technique. Soil samples were collected at least one foot below the soil surface, and metal was extracted using ultra-pure nitric before analysis using GF-AAS. Data were analyzed using analysis of variance (ANOVA).

Results

The results showed significant detectable levels of the metals in all playgrounds evaluated in this study. L-WH Park in League City has the highest detectable levels of Pb (12.43 ± 46 mg/Kg, P<0.05) compared to other children playgrounds examined in this study (T-RP (3.93 ± 3.97 mg/Kg), T-GP (7.09 ± 2.79 mg/Kg), T-TX (6.05 ± 1.92 mg/Kg), L-HH (4.68 ± 0.76 mg/Kg), L-SPLX (2.64 ± 1.63 mg/Kg). The concentration of detectable cadmium in T-RP park (0.16 ± 0.11 mg/Kg) was significantly higher than all the other five children's playgrounds studied L-WH (0.02 ± 0.01 ), T-GP (0.05 ± 0.04 mg/Kg), T-TX (0.02 ± 0.01 mg/Kg), L-HH (0.04 ± 0.01 mg/Kg), L-SPLX (0.09 ± 0.04 mg/Kg) (P<0.05).

Conclusions

The playgrounds contain detectable toxic metals that might pose health risks to children in the community. Though the lead levels are below the CDC-recommended lead soil concentration in urban areas, they still pose health risks to children in the environment when continuously exposed to the playground. The concentrations of Cu and Cr were not statistically different among the playgrounds. The detection of Pb and Cd highlights the potential health risks to children. Overall, this study shows that metal levels in playgrounds should be public information available to parents.
B-280 Nmr-based biomaker measurement determines high-dose methotrexate elimination rates in lymphoma and leukemia treatment
Authors: Robertson A , Einhell S , Schwäble Santamaría A , Grassi M , Pukrop T , Schiffer E
Published: October 2024

Background

The cytotoxic methotrexate (MTX) is used in high doses in lymphoma and acute lymphocytic leukemia (ALL) treatment. A therapeutic plasma dose range is crucial to minimize MTX accumulation and prolonged exposure, which can lead to myelosuppression and other adverse effects. Determining MTX elimination prior to drug administration is difficult, given its non-linear clearance. Here we present a model to predict MTX elimination rates based on metabolite measurement in patients receiving high-dose methotrexate, using nuclear-magnetic resonance (NMR) spectroscopy.

Methods

A series of 26 residual sera from patients receiving MTX infusions as per treatment protocols for CNS lymphoma (MATRIX protocol, 3.5 g/m2 over four hours) and ALL (GMALL protocol, 1.5 g/m2 over 24 hours) were collected from patients at the University Hospital Regensburg at four timepoints (t0, t24, t42, and t48). Serum was NMR-measured in five replicates. NMR spectra for each sample were characterized and relevant parameters extracted. First order elimination constants were modeled for two MTX dosage regimes (<4 g, ≥4 g) using characterized NMR features from samples measured at t0.

Results

Metabolomic and lipidomic biomarkers measured at baseline (t0) were able to accurately predict MTX elimination rate constant k, determined by fitting MTX levels at a t24, t42, and t48, using first order c(t)∼c0*e(-kt) kinetic function (>4 g: R2 = 0.475, p-value < 0.05; ≤4 g: R2 = 0.918, p-value < 0.01) (Fig. A). Results were cross-validated with five-time repeated three-fold cross-validation. Performance values are given as means of the folds.

Conclusions

NMR-measured biomarkers can predict dose-dependent MTX elimination rates in patients receiving high-dose MTX, presenting a novel method to predict MTX clearance, allowing for personalized dose-monitoring and minimization of adverse events. Validation with a larger cohort is required.
B-287 Comparing a Recently FDA-Cleared Free Phenytoin Assay to a Laboratory Developed Test
Authors: Zakrajsek J A , Zakrajsek J T , McShane A J
Published: October 2024

Background

Phenytoin is an anti-convulsant drug that is commonly prescribed to epileptic patients to control seizures. The drug circulates in two forms: protein-bound (90%), and the free, biologically active form (10%). In certain circumstances, such as protein-binding disruption, hypoalbuminemia, and drug-drug interactions, the total phenytoin serum concentration may not accurately reflect active phenytoin levels. Therefore, a free phenytoin level (i.e. non-protein bound) may be indicated. Until recent FDA-clearance, we utilized an in-house, laboratory-development test (LDT) to deliver these needed results. The purpose of this study was to validate the FDA-cleared assay and compare its performance characteristics to a free phenytoin LDT.

Methods

Validation of the FDA-cleared assay was performed on an automated chemistry analyzer (Roche Diagnostics, cobas c502), utilizing a kinetic interaction of microparticles in a solution (KIMS) method. Both the FDA and LDT assay used the same free phenytoin reagent, however the calibrators and pipetting parameters for each assay were different. FDA assay used a six-point calibration at concentrations of: 0, 2.5, 5, 10, 20, 40 µg/mL (Preciset TDM I Calibrators), whereas the LDT assay used a six-point calibration at concentrations of: 0.0, 0.5, 1.0, 2.0, 3.0, 4.0 µg/mL (cobas FP Free Phenytoin Calibrators). The following performance characteristics were determined for the FDA-cleared assay and compared to the current LDT: analytical measurement range, precision, accuracy, and maximum dilution. Free forms of the drug were obtained as a filtrate through centrifugation using a molecular weight cut-off filter (30 kDa, Millipore), spun at 3600 rpm for twenty minutes.

Results

Conclusions

The FDA-cleared free phenytoin assay is suitable for use in the clinical laboratory producing similar results to our LDT. Switching to the FDA-cleared assay reduces the regulatory burden and eliminates the need for transferring reagent into open channel cassettes and manual dilutions.
B-295 Sensitive and Rapid Homogeneous Immunoassay for the Detection of Zolpidem and its Major Metabolite in Urine
Authors: Hennessey V , Chung K , Singh R , Houts T , O’Malley R
Published: October 2024

Background

Zolpidem, a schedule IV controlled substance under the Controlled Substances Act, is commonly known as Ambien and Ambien CR, a prescription only treatment for insomnia. Zolpidem is one of the most common prescribed sleep aids in the U.S. due to its rapid sedative effects. The recommended duration of treatment is between two days and four weeks, as long-term use of zolpidem increases the risk of habit formation. Addiction to zolpidem can cause severe side effects, including memory loss, delusion, and in extreme cases, fatal overdose. From 2005-2010, Zolpidem-related emergency room visits doubled, underscoring the need for accurate testing as zolpidem testing plays an important role in cases of misuse, diversion, and forensics. The sole commercially available homogeneous immunoassay for the detection of zolpidem in urine has a relatively high cutoff at 20 ng/mL and poor cross-reactivity (0.02%) to major metabolite zolpidem 4-carboxylic acid. ARK Diagnostics has developed the ARK™ Zolpidem Assay to detect zolpidem at a cutoff concentration of 10 ng/mL with high cross-reactivity to its metabolite, Zolpidem phenyl 4-carboxylic acid and no cross-reactivity to zaleplon and zopiclone at concentrations below 100,000 ng/mL.

Methods

The ARK™ Zolpidem Assay is a liquid stable homogeneous enzyme immunoassay, consisting of two reagents, with a cutoff concentration of 10 ng/mL and semi-quantitative range up to 40 ng/mL. Preliminary performance characterization for this assay was evaluated on the Beckman Coulter AU680 Automated Clinical Chemistry Analyzer. Precision, analytical recovery, specificity, Histogram Overlap Analysis of ±50% controls and the cutoff, and method comparison with LC-MS/MS were evaluated.

Results

In semi-quantitative mode, total precision ranged from 2.6 to 3.1% CV. Spiked recovery of zolpidem ranged from 88.6% (2.5 ng/mL) to 96.8% (10 ng/mL). The major metabolite, zolpidem phenyl-4-carboxylic acid, showed an approximate equivalence to the 10 ng/mL zolpidem cutoff at 30 ng/mL (33.3% cross-reactivity). Histogram overlap analysis showed no overlap between cutoff and control levels. Method correlation with LC-MS/MS using authentic urine samples showed an excellent agreement with a specificity of 100% and sensitivity of 100% (15 positives and 100 negatives).

Conclusions

The ARK™ Zolpidem Assay measures zolpidem and its major metabolite zolpidem phenyl-4-carboxylic acid in human urine with acceptable performance. The assay is sensitive, rapid, and applicable to a wide range of clinical chemistry analyzers.
B-299 Comparison of a Recently FDA-Cleared Urine Benzodiazepine Screening Assay with β-Glucuronidase Hydrolysis to Liquid Chromatography-Mass Spectrometry
Authors: Zakrajsek J T , Zakrajsek J A , Spiesman A M , McShane A J
Published: October 2024

Background

Benzodiazepines are a class of prescribed psychoactive drugs that act as a depressant on the central nervous system. Over the last decade, the fast-acting sedative effects of these drugs have led to misuse, therefore making detection important for monitoring proper drug compliance. Immunoassays on urine samples are commonly used as a screening method and the results are confirmed using high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). The metabolites of benzodiazepine in urine are predominantly excreted as glucuronides which may lead to false negative results when utilizing immunoassay-based methods. The purpose of this study was to compare a new generation of benzodiazepine reagent, that includes a β-glucuronidase enzyme to increase sensitivity, to LC-MS/MS. Secondly, the positivity rate was compared before and after the increased sensitivity screening assay was implemented.

Methods

Validation of benzodiazepine II assay was performed on an automated chemistry analyzer (Roche Diagnostics, cobas c502), utilizing a kinetic interaction of microparticles in a solution (KIMS) method. A total of thirty urine samples that screened positive on the benzodiazepine II assay and thirty samples that screened negative, were compared with LC-MS/MS. The LC-MS/MS panel included α- hydroxytriazolam, 7- aminoclonazepam, nordiazepam, temazepam, oxazepam, lorazepam, and α-hydroxyalprazolam. The lower limit of quantitation was 40 ng/mL for all compounds except α-hydroxyalprazolam which was 60 ng/mL. The cutoff concentration for the benzodiazepine II assay was 200 ng/mL. Three months of data was also collected before and after converting to the new generation of benzodiazepine assay.

Results

Conclusions

The addition of β-glucuronidase to the benzodiazepine II assay allowed for high sensitivity when detecting urine benzodiazepine metabolites. With the increased sensitivity, almost a doubling of positivity rate was observed. The overall high diagnostic accuracy of the test makes it a reliable screening method for urine benzodiazepine detection.
B-300 Fentanyl and Xylazine Positivity Patterns at a Northeast U.S. Children’s Hospital
Authors: Poventud-Fuentes I , Unsihuay Vila D M , Osterhoudt K , Patel K
Published: October 2024

Background

In the U.S., fentanyl mixed with xylazine has been designated as an emerging health threat. Information on whether xylazine is present with fentanyl poisoning could help better navigate the management of children exposed to fentanyl mixed with xylazine. The detection of xylazine can be a marker used to differentiate medical vs non-medical fentanyl exposure. We aimed to explore the trends in positivity rates of fentanyl and/or xylazine at an urban pediatric tertiary care hospital.

Methods

We conducted a retrospective analysis from October 2021 - January 2024 of fentanyl positive urine drug screen (UDS) results by immunoassay performed in-house, and the reflex confirmatory testing by liquid chromatography-tandem mass spectrometry (LC-MS/MS), performed by an external laboratory. When available, xylazine detection results by gas chromatography mass spectrometry (GC-MS) were also included in the analysis. A chart review of fentanyl positive UDS cases was conducted to determine if fentanyl was medically administered prior to the UDS. We evaluated the positivity rate of fentanyl, the co-detection of xylazine, and the positivity patterns based on age, sex, and collection department.

Results

Fentanyl positive tests encompassed 3.4% (227/6632) of all UDS performed at our institution and 11.0% (227/2058) of all positive UDS tests, with medical fentanyl administration prior to UDS documented in 76.2% (173/227) of cases. 217/227 samples had confirmatory testing with 96.8% (210/217) confirmed by LC-MS/MS. The median age of fentanyl positives was 7 years (41.9% ≤ 3 years old) and 57.3% were male. Fentanyl-only positive UDS were seen in 33.0% (75/227) of all fentanyl positives and 45.6% (108/227) were also positive for benzodiazepines. 68.7% (156/227) were collected in the intensive care units, and 19.8% (45/227) were from the emergency department. 46.7% (106/227) of fentanyl positive cases had concurrent GC-MS results available. Xylazine was detected in 13.2% (14/106) of these cases, with 13 cases reported as unintentional exposure on patients' chart, and 12 cases having a fentanyl-only positive UDS. The median age of xylazine positives was 2 years (71.4% ≤ 3 years old, age ranged from 19 days to 14 years) and 78.6% were male. 7/14 fentanyl mixed with xylazine cases were collected in the emergency department and 7/14 were collected in intensive care. 2/14 of the xylazine positive cases had medical fentanyl administration prior to UDS.

Conclusions

Detection and management of fentanyl poisoning, especially when mixed with xylazine, remains a challenge in children. Presence of xylazine is a distinct marker of exposure to non-medical fentanyl. Continued surveillance of these cases could shed light on patterns that could aid further the management of affected children and contribute to harm reduction efforts.
B-309 Evaluation of the Analytical Performance of Breast and Ovarian Cancer Antigen Assays on the Atellica CI Analyzer
Authors: Quintanilla M , Rybin A , Frenna J , Arrode-Bruses G , Leipold H
Published: October 2024

Background

The Atellica® CI Analyzer is an automated, high-throughput integrated chemistry and immunoassay analyzer utilizing both Atellica® CH and Atellica® IM Assays. This study evaluated the analytical performance of the Atellica IM breast cancer antigen CA 15-3™ (CA 15-3) and the ovarian cancer antigen CA 125II™ (CA 125II) Assays on the Atellica CI Analyzer.

Methods

Precision studies were performed according to CLSI EP05-A3 using native and contrived human serum samples as well as contrived horse serum samples for CA 15-3. One aliquot of each sample pool was tested in duplicate in two runs per day ≥2 hours apart on each analyzer for ≥20 days. Precision studies were evaluated with one reagent lot on two systems. Method comparison studies were performed using three reagent lots according to CLSI EP09c. Individual native and contrived human serum samples were analyzed using the Atellica IM CA 15-3 and CA 125II Assays on the Atellica IM Analyzer and the Atellica CI Analyzer. LoB, LoD, and LoQ were determined as described in CLSI EP17-A2.

Results

Representative precision and method comparison results for each assay are listed in the table. For the two assays tested, repeatability and within-lab %CVs were <2.4% and <6.9%, respectively. Slopes determined by the weighted Deming linear regression model were approximately 1. Detection capability for the Atellica IM CA 15-3 Assay on the Atellica CI Analyzer was estimated to be 1.0 U/mL, 2.0 U/mL and 3.0 U/mL for LoB, LoD, and LoQ, respectively. When using the Atellica IM CA 125II Assay, detection capability was estimated to be 2.0 U/mL, 3.0 U/mL and 3.0 U/mL for LoB, LoD, and LoQ, respectively.

Conclusions

Evaluation of the Atellica IM CA 15-3 and CA 125II Assays using the Atellica CI Analyzer demonstrated good precision and equivalent performance compared to the same assays on the Atellica IM Analyzer.