Validation Of Assay Research Articles

Validation of a breast cancer assay for radiotherapy omission: an individual participant data meta-analysis.

There are currently no molecular tests to identify individual breast cancers where radiotherapy (RT) offers no benefit. Profile for the Omission of Local Adjuvant Radiotherapy (POLAR) is a 16-gene molecular signature developed to identify low risk cancers where RT will not further reduce recurrence rates. An individual participant data meta-analysis was performed in 623 cases of node-negative ER+/HER2-negative early breast cancer enrolled in three RT randomized trials for whom primary tumor material was available for analysis. A Cox proportional hazards model on time to locoregional recurrence (LRR) was used to test the interaction between POLAR score and RT. 429 (69%) patients' tumors had a high POLAR score and 194 (31%) had a low score. Patients with high POLAR score had, in the absence of RT, a 10-year cumulative incidence of LRR: 20% (15%-26%) vs 5% (2%-11%) for those with a low score. Patients with a high POLAR score had a large benefit from RT (hazard ratio [HR] for RT vs no RT: 0.37 [0.23-0.60], p < .001). In contrast, there was no evidence of benefit from RT for patients with a low POLAR score (HR: 0.92 [0.42-2.02], p = .832). The test for interaction between RT and POLAR was statistically significant (p = .022). POLAR is not only prognostic for locoregional recurrence but also predictive of benefit from radiotherapy in selected patients. Patients ≥ 50 years with ER+/HER2-negative disease and a low POLAR score could consider omitting adjuvant RT. Further validation in contemporary clinical cohorts is required.

Establishment and validation of a real-time fluorescent PCR freeze-dried type assay for 11 sheep and goats pathogens

Small ruminants are economic mainstay in developing countries because of its direct contribution to food security, but the occurrence of epidemics poses a continuous threat. Early diagnosis can strengthen the prevention of zoonosis. Therefore, a high-sensitivity real-time fluorescent PCR freeze-dried type assay that can be stored and transported at room temperature was developed for 11 sheep and goats pathogens in this study. The results showed that there was no non-specific amplification in systems containing different pathogen positive controls; The areas under the ROC curves were all in the range of 0.99 to 1.00; The LOD counted by Digital PCR were 194, 96, 84, 40, 265, 68, 208, 236, 118, 53 and 723 copies/mL for M. ovis, Pathogenic Leptospira, C. burnetii, BTV, PPRV, Brucella, exJSRV, C. abortus, ORFV, FMDV and CPV, respectively. The coefficients of variation of both intra-group and inter-group replicate tests were less than 5.00%; The accelerated thermostatic lyophilization test was used to predict the validity period; The LOD of each positive pathogen was still detected after 6 days at 56 ℃, suggesting that the validity period can be at least 567 days at room temperature. In this study, a high-sensitivity real-time fluorescent PCR freeze-dried type assay that can be stored and transported at room temperature was developed for 11 sheep and goats pathogens, which were Mycoplasma ovis (M. ovis), Pathogenic Leptospira, Coxiella burnetii (C. burnetii), Bluetongue virus (BTV), Peste des pestis ruminants virus (PPRV), Brucella, exogenous jaagsiekte sheep retrovirus (exJSRV), Chlamydia abortus (C. abortus), Orf virus (ORFV), Foot-and-mouth disease virus (FMDV), and Capripox virus (CPV).

Validation of Serum Tetrahydrocannabinol Assay and Its Clinical Application

Abstract Background The widespread use of marijuana across all age groups in the United States, driven by the legalization of recreational and medical cannabis, has underscored the need for accurate measurement of tetrahydrocannabinol (THC) and its metabolites in blood specimens. This requirement spans both legal and medical contexts, prompting the development of precise diagnostic tools. Methods We developed a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, utilizing a straightforward protein precipitation extraction method to quantify THC and its metabolites: THC, 11-OH-THC, THC-COOH, cannabidiol (CBD), and THC-COOH glucuronide. The assay was rigorously validated for precision, linearity, stability, carryover, and quality assurance to ensure its reliability and accuracy for clinical use. Results Precision studies demonstrated a coefficient of variation ranging from 1.7% to 4.9%. The analytical measurement range was established at 1-100 ng/mL for THC, CBD, and 11-OH-THC, 5-500 ng/mL for THC-COOH, and 10-1000 ng/mL for THC glucuronide, accommodating the broad spectrum of THC/metabolite concentrations observed in our patient cohort. Carryover from all analytes were negligible (&amp;lt;0.01%). Sample stability was confirmed for up to two weeks at -20°C and through three freeze-thaw cycles. Despite a median THC concentration of 5 ng/mL in a limited sample size (N=44), high concentrations of THC and its metabolites correlated with trauma-related incidents and a history of polysubstance abuse in emergency department admissions. Conclusions The LC-MS/MS assay offers superior sensitivity and specificity compared to traditional immunoassays for cannabinoid detection, effectively distinguishing between THC and its metabolites. Its implementation will offer an advanced approach for analyzing the prevalence and clinical implications of THC and metabolite levels in various patient populations, including trauma, pediatric patients, and organ transplant candidates.

TGF-β-mediated crosstalk between TIGIT+ Tregs and CD226+CD8+ T cells in the progression and remission of type 1 diabetes

Type 1 diabetes (T1D) is a chronic autoimmune condition characterized by hyperglycemia resulting from the destruction of insulin-producing β-cells that is traditionally deemed irreversible, but partial remission (PR) with temporary reversal of hyperglycemia is sometimes observed. Here we use single-cell RNA sequencing to delineate the immune cell landscape across patients in different T1D stages. Together with cohort validation and functional assays, we observe dynamic changes in TIGIT+CCR7− Tregs and CD226+CCR7−CD8+ cytotoxic T cells during the peri-remission phase. Machine learning algorithms further identify TIGIT+CCR7− Tregs and CD226+CD8+ T cells as biomarkers for β-cell function decline in a predictive model, while cell communication analysis and in vitro assays suggest that TIGIT+CCR7− Tregs may inhibit CD226+CCR7−CD8+ T cells via TGF-β signaling. Lastly, in both cyclophosphamide-induced and streptozotocin (STZ)-induced mouse diabetes models, CD226 inhibition postpones insulitis onset and reduces hyperglycemia severity. Our results thus identify two interrelated immune cell subsets that may serve as biomarkers for monitoring disease progression and targets for therapeutic intervention of T1D.

Deep Learning-Enhanced Paper-Based Vertical Flow Assay for High-Sensitivity Troponin Detection Using Nanoparticle Amplification.

Successful integration of point-of-care testing (POCT) into clinical settings requires improved assay sensitivity and precision to match laboratory standards. Here, we show how innovations in amplified biosensing, imaging, and data processing, coupled with deep learning, can help improve POCT. To demonstrate the performance of our approach, we present a rapid and cost-effective paper-based high-sensitivity vertical flow assay (hs-VFA) for quantitative measurement of cardiac troponin I (cTnI), a biomarker widely used for measuring acute cardiac damage and assessing cardiovascular risk. The hs-VFA includes a colorimetric paper-based sensor, a portable reader with time-lapse imaging, and computational algorithms for digital assay validation and outlier detection. Operating at the level of a rapid at-home test, the hs-VFA enabled the accurate quantification of cTnI using 50 μL of serum within 15 min per test and achieved a detection limit of 0.2 pg/mL, enabled by gold ion amplification chemistry and time-lapse imaging. It also achieved high precision with a coefficient of variation of <7% and a very large dynamic range, covering cTnI concentrations over 6 orders of magnitude, up to 100 ng/mL, satisfying clinical requirements. In blinded testing, this computational hs-VFA platform accurately quantified cTnI levels in patient samples and showed a strong correlation with the ground truth values obtained by a benchtop clinical analyzer. This nanoparticle amplification-based computational hs-VFA platform can democratize access to high-sensitivity point-of-care diagnostics and provide a cost-effective alternative to laboratory-based biomarker testing.

Copy Number Analysis of Whole Exome Sequencing Data

Abstract Background Genetic alterations implicated in constitutional disorders range in size from single nucleotide substitutions to losses and gains of millions of bases. Whole exome sequencing (WES) captures many clinically relevant variants, but an analytic pipeline optimized for detection of small substitutions, insertions, and deletions may miss pathogenic copy number variants (CNVs). Historically, our laboratory performed separate cytogenetic tests to evaluate for larger genomic imbalances. Addition of copy number analysis to our WES pipeline could improve the assay’s diagnostic yield. This study describes part of our strategy to validate copy number analysis of our constitutional whole exome sequencing data. Methods We identified 18 CNV specimens that underwent both chromosomal microarray analysis (CMA) and WES between 2017 and 2023. Variants included 13 deletions (size range 14Kb-2Mb) and 5 duplications (size range 325Kb-10Mb). The standard method of constitutional copy number analysis in our clinical laboratory is CMA using the Affymetrix Cytoscan HD Array and analysis software. CNVs identified on CMA were reviewed with reference to the Database of Genomic Variants and multiple academic hospital databases. WES was performed on an Illumina sequencing system following capture-based library enrichment with the Integrated DNA Technologies xGen Exome Research, CNV Backbone, Human mtDNA Research, and Human ID Research Panel probes. CNVs identified on WES were called using the Broad Institute’s Genomic Analysis Toolkit (GATK) with a quality score cutoff of 120. Whole exome sequencing data was visualized on the Integrative Genomics Viewer. Results 14 out of 18 CNVs reported on CMA were also detected on WES with a quality score at or above 120 (78%). This includes 10 out of 13 deletions (77%) and 3 out of 5 duplications (60%). For both deletions and duplications, the size ranges of detected variants at or above the cutoff score and undetected variants overlapped. One duplication reported on CMA was detected on WES with a quality score below 120. Visualization of whole exome sequencing data showed variation in probe density that may have affected CNV calling. Conclusions The majority of CNVs reported after CMA (14/18, 78%) were also detected on WES data using a GATK quality score cutoff of 120. The data does not suggest an association between CNV size and detection by WES, although interpretation is limited by the small number of cases. Data visualization may be required to identify some CNVs. Future steps to characterize the limitations of copy number analysis of WES data include assessments of variance in probe density and read depth across the exome. Copy number analysis of WES data from cases with normal CMA results will also be performed for assay validation.

Characterization of the Biological Variability of the Angiome Biomarkers over Time in Healthy Subjects.

Biomarker analyses are an integral part of cancer research. Despite the intense efforts to identify and characterize biomarkers in cancer patients, little is known regarding the natural variation of biomarkers in healthy populations. Here we conducted a clinical study to evaluate the natural variability of biomarkers over time in healthy participants. The angiome multiplex array, a panel of 25 circulating protein biomarkers, was assessed in 28 healthy participants across 8 timepoints over the span of 60 days. We utilized the intraclass correlation coefficient (ICC) to quantify the reliability of the biomarkers. Adjusted ICC values were calculated under the framework of a linear mixed-effects model, taking into consideration age, sex, body mass index (BMI), fasting status, and sampling factors. ICC was calculated to determine the reliability of each biomarker. HGF was the most stable marker (ICC=0.973), while PDGF-BB was the most variable marker (ICC=0.167). In total, ICC analyses revealed that 22 out of 25 measured biomarkers display good (≥0.4) to excellent (>0.75) ICC values. Three markers (PDGF-BB, TGF-1, PDGF-AA) had ICC values <0.4. Greater age was associated with higher IL-6 (p=0.0114). Higher BMI was associated with higher levels of IL-6 (p=0.0003) and VEGF-R3 (p=0.0045). Of the 25 protein biomarkers measured over this short time period, 22 markers were found to have good or excellent ICC values, providing additional validation for this biomarker assay. This data further supports the validation of the angiome biomarker assay and its application as an integrated biomarker in clinical trial testing.

Nonclinical immunogenicity assessment of E3112, a recombinant human hepatocyte growth factor, and its impact on pharmacokinetics in rats and monkeys

E3112 is a recombinant human hepatocyte growth factor currently in development for the treatment of acute liver failure. The assessment of immunogenicity is crucial in the development of biotherapeutics. Consequently, a semi-quantitative assay of anti-drug antibody (ADA) was developed in rat and monkey serum using a ligand binding assay with electrochemiluminescence detection. A standard tiered approach was employed for the immunogenicity assessment, comprising a screening assay and a subsequent confirmatory assay. In the assay validation studies, selectivity, sensitivity, prozone effects, reproducibility, drug tolerance, and stability were evaluated. These assessments were conducted using a surrogate positive control of ADA. The accuracy and precision of the surrogate ADA were within ± 20 % and 20 %, respectively. The stability of ADA was also confirmed under a variety of conditions. The developed assays were successfully employed for the assessment of immunogenicity in rats and monkeys following the administration of a repeated dose of E3112. The administration of E3112 resulted in an increase in ADA levels, with higher levels observed in rats than in monkeys. Systemic exposures of E3112 in rats with higher ADA levels were lower than those with lower ADA, confirming the utility of nonclinical immunogenicity in interpreting pharmacokinetics and its inter-individual variability.

Anti-SARS-CoV-2 IgM Antibody Levels Measured by an In-House ELISA in a Convalescent Latin Population Persist over Time and Exhibit Neutralizing Capacity to Several Variants of Concern.

The coronavirus, SARS-CoV-2, is the causative agent for COVID-19, first registered in Wuhan, China and responsible for more than 6 million deaths worldwide. Currently, RT-PCR is the gold-standard method for diagnosing COVID-19. However, serological tests are needed for screening acute disease diagnosis and screening large populations during the COVID-19 outbreak. Herein, we described the development and validation of an in-house enzyme-linked immunosorbent assay (ELISA) for detecting the levels of anti-spike-1-RBD IgM antibody (CovIgM-ELISA) in well-defined serum/plasma panel for screening and identifying subjects infected with SARS-CoV-2 in a Latin population. In-house CovIgM-ELISA has the format of an indirect ELISA. It was optimized by checkerboard titration using recombinant SARS-CoV-2 spike-S1-RBD protein as an antigen. We found that, compared to the RT-PCR as the standard method, the in-house CovIgM-ELISA displayed sensitivities of 96.15% and 93.22% for samples collected up to 30 or 60 days after infection, respectively, as well as 95.59% specificity with 97.3% accuracy. The agreement kappa value (κ) of our CovIgM-ELISA was substantial when compared to RT-PCR (κ = 0.873) and the anti-SARS-CoV-2 IgM ELISA (InBios Int) (κ = 0.684). The IgM levels detected in the population positively correlated with the neutralizing activity against the wild-type, Alpha and Delta variants of concern, but failed to neutralize Omicron. These data indicate that our in-house CovIgM-ELISA is a compatible performing assay for the detection of SARS-CoV-2 infection.

B-177 Development of a Contrived Fecal Matrix for Clinical Validation of a LC-MS/MS Assay to Quantify Microbiota-derived Metabolites in Stool Specimens

Abstract Background The gut microbiota plays a critical role in health and its disruption can impact clinical outcomes in a variety of circumstances, including graft-versus-host disease following hematopoietic stem cell transplantation, Clostridioides difficile infections, and liver disease. Metagenomic and metabolomic analyses of fecal material is frequently performed in the research setting; however, evaluation of the gut microbiota is not yet incorporated into routine patient care. One barrier to its implementation is the lack of matrix-appropriate materials for validation of clinical assays. While surrogate fecal matrices are being developed for metagenomic sequencing, matrices specific for targeted metabolomics are currently unavailable. In this study, we designed a contrived fecal matrix to validate a new LC-MS/MS method for measurement of microbiota-derived metabolites from stool specimens. Methods Common food products and mucin were individually analyzed by LC-MS/MS for quantities of three microbiota-derived metabolites - butyrate, deoxycholic acid (DCA) and taurocholic acid (TCA). Food items with the lowest endogenous metabolite concentrations were selected to create a contrived fecal matrix with a consistency similar to human stool specimens. Calibrators and quality controls (QC) were then created by spiking butyrate, DCA, and TCA into either the contrived fecal matrix or 50% methanol (MeOH). The internal standards (ISTD) used were d7-butyrate, d4-DCA, and d4-TCA, respectively. Three human stool specimens were selected and spiked at a range of metabolite concentrations to assess matrix effects. All samples, calibrators and controls were homogenized in 80% MeOH extraction solvent containing the ISTDs using beadruptor tubes. The suspension was centrifuged and 25 µL of supernatant was derivatized using 12.5 µL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 12.5 µL 3-nitrophenylhydrazine at 40°C for 30 minutes. For optimal separation of isomers, 2 µL of sample was injected and separated on a CORTECS T3 column (1.6 µm, 2.1 x 100 mm, Waters) at 45°C with a flow rate of 0.350 mL/min and run-time of 20 min using the Exion LC AD (Sciex). Analytes were detected using a Sciex QTRAP 6500 mass spectrometer with an electrospray ionization source. Results A contrived fecal matrix containing a mix of avocado, black beans, gluten-free flour, and cornstarch blended in water and mucin was created. The contrived matrix was confirmed to contain minimal to no endogenous levels of the 3 metabolites of interest: butyrate, DCA, and TCA. Validation of the LC-MS/MS assay using the contrived fecal matrix showed a linear analytical measurement range from 25-2000 µM for butyrate, 1-60 µM for DCA, and 0.5-640 µM for TCA. Intra-assay precision at 7 QC levels for all analytes was &amp;lt;10%. The matrix effects from use of the contrived fecal matrix compared to patient fecal samples were 140-148% for d7-butyrate and 101-112% for d4-DCA across multiple spiked analyte concentrations. Conclusions Our LC-MS/MS method quantifies butyrate, deoxycholic acid, and taurocholic acid, metabolites which serve as indicators of microbiota health in liver disease patients. The contrived fecal matrix provides an additional tool for the development and validation of clinical assays to assess the gut microbiota.

B-043 Q-nab™ Igg kit: verification and validation of a novel cell-free quantitative neutralizing antibody assay

Abstract Background As the world exits the COVID-19 pandemic era, it is very important to assess population-level and individual seroprotectivity against multiple circulating and emerging SARS-CoV-2 strains, especially in vulnerable populations. Current serological measurements for neutralizing antibodies (NAbs) are either done using complex and time-intensive pseudovirus- or micro-neutralization assays or as a high throughput competitive ELISA assay. These assays, by design, cannot be multiplexed to measure NAb titers against multiple strains and suffer from a limited dynamic range. The Q-NAb IgG Tests, which rely on blocking the non-neutralizing antibodies (NNAbs) using a novel fusion protein, were developed and validated to quantitatively measure NAb titer against the SARS-CoV-2 strains used to develop the first two mRNA vaccines viz. ancestral and BA.4/5 strains. Methods The Q-NAb assay measures the NAb titer in a sample by first sequestering the NNAbs using the fusion protein (made by covalently coupling SARS-CoV-2 BA.4/5 RBD and h-ACE2 protein) thereby enriching the NAbs in the sample. Variant-specific NAbs are then detected using an indirect ELISA where the wells are coated with the variant RBD and yield a signal that is directly proportional to the concentration of NAb. Clinical samples from subjects that have either had a SARS-CoV-2 infection (N=27), taken the initial vaccine series (N=123) and/or the bivalent booster (N=151) and pre-pandemic samples (N=29) were commercially sourced. Q-NAb NAb titers were measured using the ancestral and BA.4/5 kits and were compared against corresponding MN50 values using both parametric and non-parametric models. Single-site precision, low-end sensitivity, dilutional linearity, and interference screening of Q-NAb IgG Kits were evaluated in accordance with appropriate CLSI guidelines. Traceability and total uncertainty of the Q-NAb IgG Kits (Ancestral and BA.4/5) to WHO International Standard 21/340 and 21/338 respectively was established using a calibration hierarchy adapted from ISO 17511:2020. Results Both the ancestral and BA.4/5 Q-NAb IgG Test Kits correlated with MN50 titers with a Spearman correlation coefficient of 0.90 and 0.89 respectively. The total uncertainty in traceability of calibration to WHO International standards 21/340 and 21/338 was found to be less than 10% for both tests. Single site precision across the assay range for BA.4/5 kit was found to be 6-10% with a limit of quantitation of 312 IU/mL. Assays exhibited dilutional linearity across their respective dynamic range and had no significant interference from common endogenous and exogenous substances. Conclusions We have successfully validated the novel Q-NAb IgG neutralization assay platform as individual assays for measuring neutralizing titers against ancestral and BA.4/5 SARS-CoV-2 strains. The assays have expanded dynamic range, excellent analytical performance and correlation to accepted gold-standard microneutralization assays. Both assays are calibrated and traceable to WHO international standards, thereby enabling the compilation and comparison of data from multiple clinical cohorts run across multiple laboratories. The design of the Q-NAb IgG Test kit makes it amenable to multiplexing (for example, on an MSD platform) to assess neutralization titers against multiple variants at the same time. Such a multiplexed platform can be used to assess vaccine efficacy and seroprotection against multiple variants including emerging variants.

B-287 Comparing a Recently FDA-Cleared Free Phenytoin Assay to a Laboratory Developed Test

Abstract 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-273 Clinical Validation of a Novel Lateral Flow Assay for Rapid and Sensitive Urine Fentanyl Screening

Abstract Background The urgent need for rapid fentanyl detection at the point-of-care originates from its involvement in a high number of overdose deaths, primarily due to illicit use. Given fentanyl's potency compared to traditional opioids, accurate and sensitive detection is critical. Current lateral flow assays (LFA) cannot detect single-digit concentrations and may yield false negatives due to their lower sensitivity. Herein, we evaluate the clinical performance of a novel LFA (InstaStrip Fentanyl Rapid Test (urine)) with 1 ng/mL cutoff for fentanyl and no cross-reactivity for norfentanyl, offering enhanced sensitivity for effective overdose prevention at the ED. Methods A total of 172 urine samples submitted for UDS analysis were used for comparison studies. Fentanyl concentrations were categorized in 5 groups to cover various concentration categories around the cutoff of the LFA (1 ng/mL). These categories are: drug-free (&amp;lt;0.1 ng/mL, n=68), 0.1-0.5 ng/mL (n=14), 0.5-0.9 ng/mL (n=15), 1-1.5 ng/mL (n=9) and &amp;gt; 1.5 ng/mL (n= 66). Concentrations were determined by in-house LC-MS/MS methods validated according to CLIA standards, with fentanyl concentrations quantitated in the ABSciex software. For LFA testing, 150 μL of urine was added to a reagent tube containing dried rabbit anti-fentanyl antibody conjugated gold nanoparticles. After 20 sec of brief shaking, a test strip was inserted into the test tube. After 5 min, qualitative results were read as follow: both the control and test lines were visible in negative results, whereas only the control lines were visible in positive results. Method comparison study of the LFA was conducted by three operators who were blinded to the LC-MS/MS results. Result comparisons were made between the LFA strip and LC-MS/MS results, as well as among different operators for the same samples. Results Of the 75 urine samples with LC-MS/MS fentanyl concentrations ≥ 1 ng/mL, 74 produced consistently positive results using the InstaStrip Fentanyl Rapid Test. Of the 97 urine samples with LC-MS/MS fentanyl concentrations &amp;lt; 1 ng/mL, 96 produced consistently negative results using the InstaStrip Fentanyl Rapid Test. Using the LC-MS/MS as the gold standard method and 1 ng/mL as a cutoff, the clinical sensitivity of InstaStrip Fentanyl Rapid Test was calculated to be 98.67% (95% confidence interval (CI) 92.79-99.97%), and the clinical specificity was 98.97% (95% CI 94.39-99.97%). Consistent results among three operators were obtained for 100% of the “drug free”, “0.1- 0.5 ng/mL” and &amp;gt; 1.5 ng/mL groups, whereas 93.3 % (14 out of 15) and 88.9% (7 out of 8) were obtained for the “0.5-0.9 ng/mL” and “1-1.5 ng/mL” groups, respectively. The two samples with a singlet discrepant result (1 false positive and 1 false negative) in the triplicate experiments contained fentanyl concentrations of 0.9 and 1.0 ng/mL respectively, which are very close to the cutoff value. Conclusions In this study, the InstaStrip Fentanyl Rapid Test (urine) demonstrated high clinical sensitivity (98.67%), high specificity (98.97%) and high reproducibility showing that it may be useful in screening and identifying patients with fentanyl overdose in minutes in an emergency setting. This would facilitate appropriate patient triaging for optimal clinical care.

A-131 An External Laboratory Validation of the Atellica IM Serum Neurofilament Light Chain Assay

Abstract Background Neurofilament light chain (NfL) in serum is a promising biomarker for axonal injury with potential for use in neurodegenerative disease testing, including multiple sclerosis (MS), Alzheimer’s disease, and amyotrophic lateral sclerosis (ALS). This study validated the Siemens Healthineers Atellica® IM Serum Neurofilament Light Chain (sNfL) assay* for research applications. Methods Precision studies were performed using serum and plasma samples. Intra-assay precision comprised three levels of internal assay quality control (QC) and one plasma or serum pool (consisting of serum spiked with CSF). For plasma, one aliquot of each sample was tested in duplicate in two runs per day &amp;gt;2 hours apart for up to &amp;gt;20 days. Serum inter-assay involved three levels of internal assay QC run over 10 days (1 run/day). Sensitivity studies were performed to evaluate the limit of quantitation (LoQ) for serum and plasma using five low concentration samples. Each concentration was tested in 25 replicates for serum, and 50 replicates for plasma. Linearity studies involved five levels, each run three times in duplicate (plasma) or four times (serum). Method comparison studies were performed according to CLSI EP09-A3 using the Atellica® IM NfL laboratory developed test (LDT) offered by Siemens Healthcare Laboratory, LLC. 50 samples within the measuring interval of the LDT for each sample type (plasma and serum) were tested. Results Intra-assay coefficients of variation (%CVs) for internal QC ranged from 1.7-2.5% for concentrations spanning 12.75 to 147.22 pg/mL. Serum and plasma pool intra-assay %CV were 2.5% and 3.4%, respectively. Inter-assay precision of serum %CVs ranged from 2.9-16.1% for 13.4 to 150.8 pg/mL. Plasma inter-assay %CVs demonstrated a slightly improved precision versus serum across runs with %CV ranging from 2.4-12.8% with plasma concentrations ranging from 13.4 to 158.0 pg/mL. Serum and plasma LoQs, defined as the concentration at which CV is &amp;lt;20%, were 2.5 pg/mL (serum/plasma). The assay was linear between 2.5 to 300 pg/mL for serum and plasma. The Atellica IM sNfL assay method comparison to the Atellica IM NfL LDT was analyzed using a Passing-Bablok linear regression model. The assays exhibited acceptable agreement with slopes in the range of 1.12-1.14 across the sample interval tested, 6.4 to 460 pg/mL for serum/plasma. Conclusions This external validation of the Atellica IM sNfL assay demonstrated performance characteristics suitable for research applications. *For Research Use Only. Not for use in diagnostic procedures.

B-151 Development and Validation of Plasma Catecholamines by Mass Spectrometry

Abstract Background Our established method for measuring plasma catecholamines utilized a Shimadzu HPLC coupled to an ESA Coulochem III electrochemical detector. Chromatographic run times were long, resulting in slow turnaround times. Instrument maintenance was labor intensive, and the assay was prone to interferences. To overcome these limitations, we changed the method to liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements. Methods After the addition of a labeled internal standard for each analyte, samples are loaded onto 50 mg of activated alumina at pH 8.6, utilizing a Tris-HCl, sodium carbonate and EDTA buffer. After incubation the alumina is then washed with 50% acetonitrile followed by water. Elution off the alumina is performed with 2% acetic acid. Derivatization is then performed on the eluate utilizing acetaldehyde and a cyanoborohydride buffer. Following derivatization, the samples are analyzed for epinephrine, norepinephrine, and dopamine by LC-MS/MS using a Sciex 7500 coupled to a ThermoFisher Scientific TLX-4 equipped with Vanquish pumps. Chromatographic separation was obtained using a Phenomenex Kinetex 2.6 um BiPhenyl 50x3 mm analytical column. Results Runtimes were significantly shorter than with the previous method, and no interferences were observed. Assay validation consisted of: intra/inter assay precision, accuracy, recovery, linearity, carryover, limit of detection (LOD), and interferences. Three levels were assessed for intra assay precision and each of the analytes monitored yielded %CVs of less than 6%. Four levels were assessed for inter assay precision and each of the analytes monitored yielded %CVs of less than 10%. When using linear regression analysis to compare results with the previous clinical assay, the slope, y-intercept and R2 were the following; dopamine: 1.06, 2.74, 0.978; epinephrine: 0.905, 2.20, 0.989; norepinephrine: 0.919, 9.75, 0.953. Spike and recovery from five samples yielded a mean average recovery of 93.4% for dopamine, 100% for epinephrine and 98.1% for norepinephrine. For linearity 4 samples were diluted and the mean % difference from expected was -1.66% for dopamine, -0.25% for epinephrine and 1.78% for norepinephrine. Blanks were injected following the high standard and carryover was negligible. LOD was determined by running a low sample pool along with a blank over 20 assays. The LOD was determined to be less than the LLOQ. Sixty-two common drugs/vitamins along with varying levels of hemolysis, lipemia, and bilirubin were also tested to ensure no interferences were seen. Conclusions The LC-MS/MS method exceeded the performance of the historical Shimdazu HPLC system with ESA Coluochem III electrochemical detector method. We anticipate that the better analytical performance will improve the testing quality of our patients.

Developmental validation of a multiplex qPCR assay for simultaneous quantification of nuclear and mitochondrial DNA

Quantification of human DNA is key in forensic genetics. A more accurate estimate of the amount of DNA is essential for planning and optimising genotyping assays, as is evaluating the presence of PCR inhibitory substances and DNA degradation status. Multiplex qPCR assays are helpful in forensics because they can quantify different targets simultaneously, thus saving valuable samples, time, and labour. The aim of this study was to highlight the challenges in the developmental validation of a multiplex real-time PCR assay and the drawbacks encountered in translating a previously described and validated assay (SD quants) to a different technology by modifying the dye probes and reagent mix to be used in a different instrument. We developed a TaqMan probe-based multiplex qPCR using reagents and fluorescent probes adapted for the Rotor-Gene 6000 instrument (QIAGEN, Hilden, Germany). The initial assay combined two mitochondrial DNA (mtDNA) and two nuclear DNA (nDNA) targets, with amplification products of different sizes (mtDNA = 69 and 143bp; nDNA = 71 and 181bp), to estimate the DNA degradation status and an internal positive control (IPC) to detect potential inhibitors. During the initial testing of the assay, we observed an interaction between the 69bp mtDNA target and the 71bp nDNA target probe, and experiments were conducted to resolve this issue without success. We removed the small nDNA target (71bp) and changed from a 5-plex to a 4-plex qPCR assay (qMIND). The final tetraplex assay was tested on 105 forensic samples and/or small amounts of degraded DNA, such as bones, teeth, fingernails, formalin-fixed paraffin-embedded tissues (FFPE), and hair shaft samples. The quantification results were compared with data acquired from the same samples using another commercially available quantification system commonly used in forensic laboratories. In addition, the short tandem repeat (STR) profiles were investigated to determine their correlation with the quantitative values obtained. Overall, the qPCR assay was robust and reliable for DNA quantification in samples commonly used in forensic practice.

Development and Validation of an Enzyme-Linked Immunosorbent Assay (ELISA)-Based Method for Quantitative Evaluation of Urinary Extracellular Vesicles as Biomarkers for CKD

Development and Validation of an Enzyme-Linked Immunosorbent Assay (ELISA)-Based Method for Quantitative Evaluation of Urinary Extracellular Vesicles as Biomarkers for CKD

Magnetic 3D bioprinting of skeletal muscle spheroid for a spheroid-based screening assay

Over the past decade, there has been a rapid development in the use of magnetic three dimensional (3D) based cell culture systems. Concerning the skeletal muscle, 3D culture systems can provide biological insights for translational clinical research in the fields of muscle physiology and metabolism. These systems can enhance the cell culture environment by improving spatially-oriented cellular assemblies and morphological features closely mimicking the in vivo tissues/organs, since they promote strong interactions between cells and the extracellular matrix (ECM). However, the time-consuming and complex nature of 3D traditional culture techniques pose a challenge to the widespread adoption of 3D systems. Herein, a bench protocol is presented for creating an innovative, promptly assembled and user-friendly culture platform for the magnetic 3D bioprinting of skeletal muscle spheroids. Our protocol findings revealed consistent morphological outcomes and the functional development of skeletal muscle tissue, as evidenced by the expression of muscle-specific contractile proteins and myotubes and the responsiveness to stimulation with cholinergic neurotransmitters. This proof-of-concept protocol confirmed the future potential for further validation and application of spheroid-based assays in human skeletal muscle research.

MicroRNA-324 mediates bone homeostasis and the regulation of osteoblast and osteoclast differentiation and activity

MicroRNAs (miRNAs) modulate the expression of other RNA molecules. One miRNA can target many transcripts, allowing each miRNA to play key roles in many biological pathways. Defects in bone homeostasis result in common age-related diseases including osteoporosis. Serum levels of miR-324-3p positively correlate with several features of bone maintenance. In contrast here, using in vivo micro-computed tomography and histology, global miR-324-null mice demonstrated increased bone mineral density and both trabecular and cortical thickness, with effect magnitudes increasing with age. The bone marrow of miR-324-null mice had reduced lipid content while TRAP staining revealed a decrease in osteoclasts, with histomorphometry demonstrating an increased rate of bone formation. Ex vivo assays showed that the high bone mass phenotype of miR-324-null mice resulted from both increased osteoblast activity and decreased osteoclastogenesis. RNA-seq analysis of osteoblasts, osteoclasts and bone marrow macrophages and target validation assays identified that the osteoclast fusion regulator Pin1 and the master osteogenic regulator Runx2 were targets of miR-324-5p in osteoclast lineage cells and osteoblasts, respectively. Indeed, in vitro Runx2 overexpression recapitulated the increased osteogenesis and decreased adipogenesis phenotype observed in vivo by the loss of miR-324. Overall, these data demonstrate the importance of miR-324 in bone homeostasis by regulating aspects of both bone formation and remodelling. Elucidation of pathways regulated by miR-324 offer promise for the treatment of bone diseases such as osteoporosis.

Evaluation of C-C Motif Chemokine Receptor 5 (CCR5) as a Sample Adequacy Control in HPV Molecular Diagnostics.

Background: Reliable Human Papillomavirus (HPV) testing and genotyping are essential for quality assurance in HPV-based primary screening, disease management and for monitoring the impact of HPV vaccination. The clinical validation of HPV molecular diagnostic assays has significantly contributed to these objectives; however, little emphasis has been placed on assuring sample quality. This study aimed to evaluate the accuracy of sample cellularity assessment using the C-C Motif Chemokine Receptor 5 (CCR5) gene target as a marker of sample adequacy in molecular diagnostics. Methods: Jurkat cell line samples were counted using both a Thoma cell-counting chamber and Fluorescence-Activated Cell Sorting (FACS). Jurkat cell line samples at three different concentrations were subsequently evaluated using the OncoPredict HPV Quality Control (QC) real-time PCR assay, employing CCR5 for molecular cellularity quantification. Results: The cellularity values obtained were comparable across the three different methods for all dilutions of the cell line tested. Conclusions: The results obtained from this study show that CCR5 represents a promising molecular marker for the accurate quantification of sample cellularity, confirming its use as a reliable sample adequacy control, thus reducing the risk of "false-negative" results.