Contrived Samples Research Articles

Development of a Fast and Quantitative FLT3-TKD Mutation Prototype Using Lab in a Cartridge™ Technology

Introduction Acute myeloid leukemia (AML) is a heterogenous hematologic malignancy characterized by the rapid proliferation of abnormal myeloid cells in the bone marrow and blood. Fast and reliable diagnostic tests are imperative for the advancement of targeted therapies in the treatment of AML. FLT3 (FMS-like tyrosine kinase3) is one of the most frequent genetic alterations in AML and is present in approximately one-third of newly diagnosed patients. Considering the targeted therapies, such as midostaurin and gilteritinib available for FLT3 mutated AML, accurate and timely diagnosis of FLT3 mutations is vital for guiding treatment decisions and improving patient outcomes. The current diagnostic methods to detect FLT3-TKD mutations require a series of complex steps including blood sample handling, plate-based PCR, enzyme digestion and capillary electrophoresis or sequencing which can be time-consuming and labor-intensive. We have developed and evaluated the performance of a FLT3-TKD prototype using Cepheid's automated cartridge technology that demonstrates significantly less hands-on time and faster turnaround. Objectives We have developed a novel, fast and simplified qPCR-based FLT3-TKD prototype using Cepheid's GeneXpert® automated platform for detecting 14 mutations, including 7 mutations present in codon D835 and another 7 mutations in I836. The FLT3-TKD prototype is an easy-to-use sample in/result out assay with minimal hands-on time and time to results in less than 3 hours, and without the use of restriction enzymes or capillary electrophoresis. This assay is designed for quantitative detection of FLT3-TKD mutated DNA (D835 & I836) using ABL1 as an endogenous control in peripheral blood (PB) of AML patients. This assay utilizes the special chemistry, special design primers, and an optimized wild-type (WT) blocker to specifically detect SNPs and indels. With the combination of WT blocker and unique design of special design primers we were able to selectively amplify the rare mutations in the presence of abundant closely related WT fragments. Results Using this prototype, we were able to detect and quantify all 14 mutations (7 of D835 and 7 of I836) with an estimated LoD of AR 0.02 (Ratio of TKD mutation copies/ABL copies in PB background) in PB samples spiked-in with positive templates. ABL1 serves as a reference gene to normalize the WT DNA present in the background. Plasmid DNA with individual mutations was generated and spiked into the PB to assess the dynamic range of the test. We observed good alignment of dCt-AR (deltaCt-Allelic ratio) regression curve among all 14 mutations within the dynamic range tested (0.02-0.33). With pre-defined thresholds, our assay specificity has been above 95% among 20 screened EDTA donor blood samples. To further assess the sensitivity of this prototype, we also evaluated a CRISPR-CAS engineered cell line for the D835 G>A mutation. Using this cell line, we were successful in detecting the mutation down to AR of 0.008 in a PB background. For quantification analysis we prepared a linear panel of the most prevalent mutation D835(G>T) in the PB background and were able to quantify all mutations within the range of AR 0.008-to 0.83. Conclusions Here we describe the development and evaluation of an automated cartridge-based FLT3-TKD prototype that can detect 14 FLT3-TKD mutations. The cartridge prototype is preloaded with sample processing reagents and qPCR reagents with a turn-around time of less than 3 hours including offboard peripheral blood processing. When tested with contrived samples generated with either plasmid DNA or engineered cell lines spiked into blood, all 14 tested FLT3-TKD mutations were detected within a dynamic range of 0.02 to 0.33 AR with a specificity of > 95% in healthy donor whole blood background. With optimized primer and WT blocker design this prototype showed high sensitivity & specificity. We tested a broader dynamic range for the most prevalent mutation, D835 G>T, and were able to detect from AR of 0.008 to 0.83 AR. Additionally, since the prototype uses the GeneXpert® platform and current universal lysate sample prep procedure, the sample can potentially be used across all Cepheid Hematology Oncology products. This prototype significantly reduces the hands-on time with future potential to provide clinicians with fast and accurate detection of FLT3 mutation status that can aid prognostic evaluation and treatment in AML patients.

Comparison of liquid chromatography-high-resolution tandem mass spectrometry (MS2) and multi-stage mass spectrometry (MS3) for screening toxic natural products

BackgroundLiquid chromatography-high-resolution mass spectrometry (LC-HR-MS) has emerged as a powerful analytical technology for compound screening in clinical toxicology. To evaluate the potential of LC-HR-MS3 in detecting toxic natural products, a spectral library of 85 natural products (79 alkaloids) that contains both MS2 and MS3 mass spectra was constructed and used to identify the natural products. Samples were analyzed using an LC-HR-MS3 method and the generated data were matched to the spectral library to identify the natural products. MethodsTo test the performance of the LC-HR-MS3 method in different sample matrices, the 85 natural product standards were divided into three groups to separate structural isomers and avoid ion suppression effects caused by co-elution of multiple analytes. The grouped analytes were spiked into drug-free serum and drug-free urine to produce contrived clinical samples. ResultsThe compound identification results of the 85 natural products in urine and serum samples were obtained. The match scores using both MS2 and MS3 mass spectra and those using only MS2 mass spectra were compared at 10 different analyte concentrations. The two types of data analysis provided identical identification results for the majority of the analytes (96% in serum, 92% in urine), whereas, for the remaining analytes, the MS2-MS3 tree data analysis had better performance in identifying them at lower concentrations. ConclusionThis study shows that in comparison to LC-HR-MS (MS2), LC-HR-MS3 can increase the performance in identification of a small group of the toxic natural products tested in serum and urine specimens.

A-128 Analytical Performance Evaluation of Assays Used for Iron Studies on the Atellica® CI 1900 Analyzer

Abstract Background The Atellica® CI 1900 Analyzer is an automated, mid-throughput, integrated chemistry and immunoassay analyzer employing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica CH Iron_2 (Iron_2), Total Iron Binding Capacity (TIBC), and Transferrin (Trf) assays* and the Atellica IM Ferritin (Fer) assay on the Atellica CI 1900 Analyzer. Methods The Atellica CI 1900 Iron_2, TIBC, and Trf assays use the same reagents and calibrators as the comparable Atellica chemistry assays. The Atellica CI 1900 Fer assay uses the same reagents and calibrators as the Atellica immunoassay. Precision and method comparison (MC) were used as performance indicators for the Atellica CI 1900 Analyzer. Precision studies were performed according to CLSI EP05-A3 using native and contrived human serum samples. MC studies were performed according to CLSI EP09-A3. Native and contrived human samples were analyzed using the Atellica CH Iron_2, TIBC, and Trf assays on both the Atellica® CH 930 and Atellica CI 1900 Analyzers. For Fer, native and contrived human samples were analyzed using the Atellica IM Fer assay on both the Atellica® IM 1600 and Atellica CI 1900 Analyzers. Results Representative precision and MC results for each assay are listed in the table. For the four assays tested, repeatability and within-lab %CVs were <3.5% and <4.5%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1. Conclusion Evaluation of the Atellica CH Iron_2, TIBC, and Trf and Atellica IM Fer assays using the Atellica CI 1900 Analyzer demonstrated good precision and equivalent performance compared to the same assays on their respective Atellica CH 930 and Atellica IM 1600 Analyzers. *The products mentioned are not commercially available in all countries. Their future availability cannot be guaranteed.

A-218 Analytical Performance Evaluation of Lipid Panel Assays on the Atellica CI 1900 Analyzer

Abstract Background The Atellica® CI 1900 Analyzer is an automated, mid-throughput, integrated chemistry and immunoassay analyzer utilizing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica CH Creatinine (CREA_2)*, Enzymatic Creatinine (ECre_3)*, Urinary/Cerebrospinal Fluid Protein (UCFP)*, and Microalbumin_2 (µALB_2)* assays on the Atellica CI 1900 Analyzer*. Methods The Atellica CI 1900 CH CREA_2, ECre_3, UCFP, and µALB_2 assays use the same reagents and calibrators as the Atellica CH assays. Precision and method comparison (MC) were used as performance indicators for the Atellica CI 1900 Analyzer. Precision studies were performed according to CLSI EP05-A3 using native and contrived human urine samples. One aliquot of each sample pool was tested in duplicate in two runs per day >2 h apart on each analyzer for >20 days. MC studies were performed according to CLSI EP09-A3. Individual native and contrived human urine samples were analyzed using the Atellica CH assays on both the Atellica CH and Atellica CI 1900 Analyzers. Results Representative precision and MC results observed from one reagent lot across indicated sample ranges are listed for each assay in the table below. Over the four assays tested, repeatability and within-lab %CVs were <5.1% and <6.0%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1. Conclusion Evaluation of the Atellica CH CREA_2, ECre_3, UCFP, and µALB_2 assays using the Atellica CI 1900 Analyzer demonstrated good precision and equivalent performance compared to the same assays on the Atellica CH Analyzer. *The products/features mentioned here are not commercially available in all countries. Their future availability cannot be guaranteed.

B-332 Evaluation of the Analytical Performance of Seven Therapeutic Drug Monitoring Assays on the Atellica® CI 1900 Analyzer

Abstract Background The Atellica® CI 1900 Analyzer is an automated, mid-throughput integrated chemistry and immunoassay analyzer employing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica CH Vancomycin (Vanc), Carbamazepine (Carb), Gentamicin (Gent), Phenytoin (Phny), Tobramycin (Tob), Valproic Acid (VPA), and Theophylline (Theo) assays* on the Atellica CI 1900 Analyzer. Methods The Atellica CI 1900 Vanc, Carb, Gent, Phny, Tob, VPA, and Theo assays use the same reagents and calibrators as the Atellica chemistry assays. Precision and method comparison (MC) were used as performance indicators for the Atellica CI 1900 Analyzer. Precision studies were performed according to CLSI EP05-A3 using native and contrived human serum samples. One aliquot of each sample pool was tested in duplicate in two runs per day ≥2 h apart on each analyzer for ≥20 days. MC studies were performed according to CLSI EP09-A3. Individual native and contrived human serum samples were analyzed using the Atellica chemistry assays on both the Atellica CH and Atellica CI 1900 Analyzers. Precision and MC were evaluated with three reagent lots on one system. Results Representative precision and MC results observed from one reagent lot across indicated sample ranges are listed in the table below for each assay. Over the seven assays tested, repeatability and within-lab % CVs were <6.5% and <11.0%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1. Conclusion Evaluation of the Atellica CH Vanc, Carb, Gent, Phny, Tob, VPA, and Theo assays using the Atellica CI 1900 Analyzer demonstrated good precision and equivalent performance compared to the same assays on the Atellica CH 930 Analyzer.*The products/features mentioned here are not commercially available in all countries. Their future availability cannot be guaranteed.

A-125 Analytical Performance of Chemistry Assays Comprising the Basic Metabolic Panel on the Atellica CI 1900 Analyzer

Abstract Background The Atellica® CI 1900 Analyzer is an automated, mid-throughput, integrated chemistry and immunoassay analyzer utilizing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica CH Urea Nitrogen (UN_c), Creatinine (CREA_2), Enzymatic Creatinine (ECre_3), A-LYTE™ Multisensor (IMT Na K Cl), Carbon Dioxide (CO2_c), Glucose Oxidase (GluO), Glucose Hexokinase_3 (GluH_3), and Calcium_2 (CA_2) assays* on the Atellica CI 1900 Analyzer*. Methods The Atellica CI 1900 CH UN_c, CREA_2, ECre_3, IMT Na K Cl, CO2_c, GluO, GluH_3, and CA_2 assays use the same reagents and calibrators as the Atellica CH assays. Precision and method comparison (MC) were used as performance indicators for the Atellica CI 1900 Analyzer. Precision studies were performed according to CLSI EP05-A3 using native and contrived human serum samples. One aliquot of each sample pool was tested in duplicate in two runs per day >2 h apart on each analyzer for >20 days. MC studies were performed according to CLSI EP09-A3. Individual native and contrived human serum samples were analyzed using the Atellica CH assays on both the Atellica CH and Atellica CI 1900 Analyzers. Results Representative precision and MC results are listed for each assay in the table. Over the 10 assays tested, repeatability and within-lab %CVs were <5.0% and <6.0%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1. Conclusion Evaluation of the Atellica CH UN_c, CREA_2, ECre_3, IMT Na K Cl, CO2_c, GluO, GluH_3, and CA_2 assays using the Atellica CI 1900 Analyzer demonstrated good precision and equivalent performance compared to the same assays on the Atellica CH Analyzer. *The products mentioned are not commercially available in all countries. Their future availability cannot be guaranteed.

A-105 Analytical Performance Evaluation of Four General Chemistry Assays on the Atellica CI 1900 Analyzer Used to Assess Kidney Function

Abstract Background The Atellica® CI 1900 Analyzer is an automated, mid-throughput, integrated chemistry and immunoassay analyzer utilizing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica CH Creatinine (CREA_2)*, Enzymatic Creatinine (ECre_3)*, Urinary/Cerebrospinal Fluid Protein (UCFP)*, and Microalbumin_2 (µALB_2)* assays on the Atellica CI 1900 Analyzer*. Methods The Atellica CI 1900 CH CREA_2, ECre_3, UCFP, and µALB_2 assays use the same reagents and calibrators as the Atellica CH assays. Precision and method comparison (MC) were used as performance indicators for the Atellica CI 1900 Analyzer. Precision studies were performed according to CLSI EP05-A3 using native and contrived human urine samples. One aliquot of each sample pool was tested in duplicate in two runs per day >2 h apart on each analyzer for >20 days. MC studies were performed according to CLSI EP09-A3. Individual native and contrived human urine samples were analyzed using the Atellica CH assays on both the Atellica CH and Atellica CI 1900 Analyzers. Results Representative precision and MC results observed from one reagent lot across indicated sample ranges are listed for each assay in the table below. Over the four assays tested, repeatability and within-lab %CVs were <5.1% and <6.0%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1. Conclusion Evaluation of the Atellica CH CREA_2, ECre_3, UCFP, and µALB_2 assays using the Atellica CI 1900 Analyzer demonstrated good precision and equivalent performance compared to the same assays on the Atellica CH Analyzer. *The products/features mentioned here are not commercially available in all countries. Their future availability cannot be guaranteed.

A-124 Evaluation of the Analytical Performance of 10 General Chemistry Assays on the Atellica® CI 1900 Analyzer

Abstract Background The Atellica® CI 1900 Analyzer is an automated, mid-throughput, integrated chemistry and immunoassay analyzer employing both Atellica chemistry and Atellica immuno assays. This study was designed to evaluate the analytical performance of the Atellica CH Alkaline Phosphatase concentrated (ALP_2c), Direct Bilirubin 2 (DBil_2), Total Bilirubin_2 (TBil_2), Albumin (Alb), Albumin BCP (AlbP), Total Protein II (TP), Aspartate Aminotransferase (AST), Aspartate Aminotransferase P5P (ASTPLc), Alanine Aminotransferase (ALT), and Alanine Aminotransferase P5P (ALTPLc) assays* on the Atellica CI 1900 Analyzer. Methods The Atellica CI 1900 ALP_2c, DBil_2, TBil_2, Alb, AlbP, TP, AST, ASTPLc, ALT, and ALTPLc assays use the same reagents and calibrators as the Atellica Chemistry assays. Precision and method comparison (MC) were used as performance indicators for the Atellica CI 1900 Analyzer. Precision studies were performed according to CLSI EP05-A3 using native and contrived human serum samples. One aliquot of each sample pool was tested in duplicate in two runs per day >2 h apart on each analyzer for >20 days. MC studies were performed according to CLSI EP09-A3. Individual native and contrived human serum samples were analyzed using the Atellica CH assays on both the Atellica® CH 930 and Atellica CI 1900 Analyzers. Results Representative precision and MC results are listed for each assay in the table below. For the nine assays tested, repeatability and within-lab %CVs were <6.0% and <9.0%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1. Conclusion Evaluation of the Atellica CH ALP_2c, DBil_2, TBil_2, Alb, AlbP, TP, AST, ASTPLc, ALT, and ALTPLc assays using the Atellica CI 1900 Analyzer demonstrated acceptable precision and equivalent performance compared to the same assays on the Atellica CH 930 Analyzer. *The products mentioned are not commercially available in all countries. Their future availability cannot be guaranteed.

B-379 Analytical Performance of Two Tumor Marker Immunoassays on the Atellica CI 1900 Analyzer

Abstract Background The Atellica® CI 1900 Analyzer is an automated, mid-throughput, integrated chemistry and immunoassay analyzer utilizing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica IM Carcinoembryonic Antigen (CEA) and Alpha Fetoprotein (AFP) assays* on the Atellica CI 1900 Analyzer. Methods The Atellica CI 1900 CEA and AFP assays use the same reagents and calibrators as the comparable Atellica IM assays. Precision and method comparison (MC) were used as performance indicators for the Atellica CI 1900 Analyzer. Precision studies were performed according to CLSI EP05-A3 using quality control, native, and contrived human serum samples. One aliquot of each sample pool was tested in duplicate in two runs per day ≥2 h apart on each analyzer for ≥20 days. MC studies were performed according to CLSI EP09-A3. Individual native and contrived human serum samples were analyzed using the Atellica IM assays on both the Atellica IM and Atellica CI 1900 Analyzers. Results Representative precision and MC results observed for each assay across indicated sample ranges are listed in the table. Slopes determined by the Deming linear regression model were approximately equal to 1. Conclusion Evaluation of the CEA and AFP assays using the Atellica CI 1900 Analyzer demonstrated good precision and equivalent performance compared to the same assays on the Atellica IM Analyzer. *The products/features mentioned here are not commercially available in all countries. Their future availability cannot be guaranteed.

A-129 Analytical Performance Evaluation of Vitamin D, Intact Parathyroid Hormone, and Inorganic Phosphorus on the Atellica CI 1900 Analyzer

Abstract Background The Atellica® CI 1900 Analyzer is an automated, mid-throughput, integrated chemistry and immunoassay analyzer utilizing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica IM Vitamin D (VitD) and Intact Parathyroid Hormone (PTH) and Atellica CH Inorganic Phosphorus (IP) assays* on the Atellica CI 1900 Analyzer*. Methods The Atellica CI 1900 VitD, PTH, and IP assays use the same reagents and calibrators as the Atellica IM VitD and PTH and CH IP assays. Precision and method comparison (MC) were used as performance indicators for the Atellica CI 1900 Analyzer. Precision studies were performed according to CLSI EP05-A3 using native and contrived human serum (VitD and IP) or plasma (PTH) samples. One aliquot of each sample pool was tested in duplicate in two runs per day >2 h apart on each analyzer for >20 days. MC studies were performed according to CLSI EP09-A3. Native and contrived human samples were analyzed using the Atellica IM VitD and PTH assays on both the Atellica IM and Atellica CI 1900 Analyzers. For IP, native and contrived human samples were analyzed using the Atellica CH IP assay on both the Atellica CH and Atellica CI 1900 Analyzers. Results Representative precision and MC results for each assay across indicated sample ranges are listed in the table. For the three assays tested, repeatability and within-lab %CVs were <4.5% and <10%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1. Conclusion Evaluation of the Atellica IM VitD and PTH and Atellica CH IP assays using the Atellica CI 1900 Analyzer demonstrated good precision and equivalent performance compared to the same assays on their respective Atellica IM and Atellica CH Analyzers. *The products/features mentioned are not commercially available in all countries. Their future availability cannot be guaranteed.

B-042 Evaluation of the Analytical Performance of Three Prostate-specific Antigen Immunoassays on the Atellica CI 1900 Analyzer

Abstract Background The Atellica® CI 1900 Analyzer is an automated, mid-throughput, integrated chemistry and immunoassay analyzer employing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica IM Prostate-Specific Antigen (PSA)*, Complexed Prostate-Specific Antigen (cPSA)*, and Free Prostate-Specific Antigen (fPSA)* assays on the Atellica CI 1900 Analyzer. Methods The Atellica CI 1900 PSA, cPSA, and fPSA assays use the same reagents and calibrators as the comparable Atellica IM assays. Limit of quantitation (LoQ), precision, and method comparison (MC) were used as performance indicators for the Atellica CI 1900 Analyzer. LoQ studies were performed in accordance with CLSI EP17-A2 using native and contrived human serum samples. Precision studies were performed according to CLSI EP05-A3 using contrived human serum samples. One aliquot of each sample pool was tested in duplicate in two runs per day >2 hours apart on each analyzer for >20 days. MC studies were performed according to CLSI EP09-A3. Individual native human serum samples were analyzed using the Atellica IM assays on both the Atellica IM and Atellica CI 1900 Analyzers. Results LoQ is defined as the lowest amount of analyte in a sample at which the within-laboratory %CV is ≤20%. Representative precision and MC results for each assay across indicated sample ranges are listed in the table. Over the three assays tested, repeatability and within-lab %CVs were ≤8.0% and ≤15.5%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1. Conclusion Evaluation of the Atellica IM PSA, cPSA, and fPSA assays using the Atellica CI 1900 Analyzer demonstrated good precision and equivalent performance compared to the same assays on the Atellica IM Analyzer. *The products/features mentioned here are not commercially available in all countries. Their future availability cannot be guaranteed. fPSA assay is not available for sale in US.

B-231 Development and Evaluation of a Lyophilized Mpox Virus qPCR Assay

Abstract Background Increased cases of monkeypox virus (MPXV) have been reported to WHO from 110 countries. As of January 16, 2023, a total of 84 733 laboratory confirmed and 1354 probable cases, including 80 deaths, have been reported to WHO. The US is assessed as high-risk region. Real-time PCR, with its sensitivity and specificity, is the preferred lab test technique. Since gene mutation has been observed in MPXV, developing assays with more than one target was recommended by the FDA. Methods A lyophilized qPCR multiplex assay was developed for MPXV detection. This assay contains primers and (1) MGB probe targeting highly conserved MPXV sequence (GeneBank AF380138 F3L), (2) BHQ probe targeting non-variola orthopoxvirus (OPX) sequence, and (3) a probe targeting a selected human house-keeping gene as an internal control. Furthermore, external positive and negative controls were included. A MPXV positive clinical sample from human skin lesions was used for the assay validation. The viral copy number of the sample was determined as 1×107 copies/mL using the standard curve method. Two hundred µL of samples were processed in the KingFisherTM Flex system. The PCR reaction consisted of 5 µL of DNA sample and 20 µL reaction mix and was run on 7500 Fast Dx Real-Time PCR System (Life Technologies). The limit of detection (LoD) was determined as the concentration at which at least 95% of samples were tested positive. A total of 230 contrived samples with concentrations lower than LoD were used to determine cut-off value. Inclusivity and exclusivity studies were conducted by in silico analysis of 80 sequences of MPXV (clade I), 3026 of MPXV (clade II), 10 of camelpox virus, 93 of cowpox virus, 6 of ectromelia virus, and 116 of vaccinia virus (NCBI and GISAID accessed on 10/03/22). Clinical evaluation of the assay was conducted using 80 contrived clinical samples in unique negative skin lesion matrix, including 40 positives (five samples at 5× LOD, five at 2.5×, ten at 1.5×, and twenty at 1×), along with 40 negatives. The positive (PPA) and negative percent agreement (NPA) of the assay were calculated. The study was approved by BRANY IRB. Results The qPCR assay had a cut-off Ct value of 40, and a LoD of 500 copies/mL for both MPXV and OPX with good inclusivity and exclusivity. Clinical evaluation for MPXV yielded a PPA and NPA of 94.9% (95% CI: 83.1%–98.6%) and 100% (89.9%–100%), respectively. For OPX, the PPA and NPA were 92.3% (79.7%–97.4%) and 100% (89.9%–100%), respectively. Conclusions Both analytical and clinical performances of the developed MPXV qPCR assay were good. Lyophilized reagents contain all required components, providing longer shelf-life and easier transportation. MPXV is the only member of the OPX genus known to be circulating among humans in the US currently. This assay as designed could potentially cross-detect different non-variola OPX, such as vaccinia virus and cowpox virus. Healthcare providers should always contact the local public health authorities for more guidance. Furthermore, this study was limited to lesion swab specimens and performance evaluation with additional specimen types is warranted.

A-349 Septec: A Microfluidic Platform for the Rapid Diagnosis of Bloodstream Infection Diagnosis and Associated Sepsis Management

Abstract Background Sepsis is a life-threatening disorder resulting from a dysregulated host response to infection. Globally, sepsis is responsible for 11 million deaths annually and is associated with high mortality and morbidity. Sepsis can arise from any infection by microbes or viruses; however, the most common cause is bloodstream infection (BSI) via bacterial pathogens. Rapid identification of organisms responsible for BSI may reduce time until the administration of suitable antibiotics and employment of rapid diagnostics at patient bedside further strengthens their value as informed clinical decisions can be made and acted upon within shorter timeframes, potentially enhancing patient outcomes and preventing progression to more critical sepsis or septic shock. This study reports on the analytical and clinical performance of the SepTec microfluidic assay, an in-vitro diagnostic device capable of rapid detection of bloodborne pathogens of gram-positive, gram-negative and fungal origin from whole blood within 15 minutes. Methods Analytical performance of the SepTec assay was studied as follows: limit of detection (LoD), reproducibility and impact of interfering substances. Sterile and infected blood samples were obtained either through the generation of contrived samples or through clinical collaboration. Clinical performance was determined retrospectively and prospectively on a total of 146 patients. For both the retrospective and prospective clinical studies, results were compared against blood culture. Results The LoD of the SepTec assay for S. Aureus, E. coli and C. albicans from contrived blood samples was 10 CFU/mL for each pathogen. CVs were calculated at 4% for organism detection in whole blood samples. From retrospective and prospective analysis, the SepTec assay showed 100% sensitivity and >96% specificity. Conclusion The SepTec assay can detect microbes from blood and differentiate between gram-positive, gram-negative or fungal pathogens with high sensitivity and specificity. Turnaround time for the test is estimated at 15minutes, rendering it with the potential to be a significantly more rapid approach than the gold-standard blood culture, or many molecular approaches. It is hoped that this work with yield a point-of-care diagnostic platform which will enhance sepsis management, overall improving patient outcomes.

A-130 Analytical Performance Evaluation of Assays Measuring Acetaminophen and Ethanol on the Atellica CI 1900 Analyzer

Abstract Background The Atellica® CI 1900 Analyzer is an automated, mid-throughput, integrated chemistry and immunoassay analyzer employing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica CH Ethyl Alcohol (ETOH) and Acetaminophen (Acet) assays on the Atellica CI 1900 Analyzer. Methods The Atellica CI 1900 ETOH and Acet assays use the same reagents and calibrators as the comparable Atellica CH assays. Precision and method comparison (MC) were used as performance indicators for the Atellica CI 1900 Analyzer. Precision studies were performed according to CLSI EP05-A3. Native and contrived human serum samples were used. One aliquot of each sample pool was tested in duplicate in two runs per day >2 h apart on each analyzer for >20 days. MC studies were performed according to CLSI EP09-A3. Individual human serum samples were analyzed using the Atellica CH assays on both the Atellica CH and Atellica CI 1900 Analyzers. Precision and MC were evaluated with three reagent lots on one system. Results Representative precision and MC results observed for each assay are listed in the table. Over the two assays tested, repeatability and within-lab %CVs were <2.5% and <4.0%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1 for the ETOH and Acet assays. Conclusion Evaluation of the Atellica CH ETOH and Acet assays using the Atellica CI 1900 Analyzer demonstrated good precision and equivalent performance compared to the same assays on the Atellica CH Analyzer. *The products/features mentioned here are not commercially available in all countries. Their future availability cannot be guaranteed.

A-020 Evaluation of the Analytical Performance of BNP and NT-proBNP Assays on the Atellica CI 1900 Analyzer

Abstract Background The Atellica® CI 1900 Analyzer is an automated, high-throughput integrated chemistry and immunoassay analyzer employing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica IM B-type Natriuretic Peptide (BNP) and NT-proBNP (PBNP) assays* on the Atellica CI 1900 Analyzer. Methods The Atellica CI 1900 BNP and PBNP assays use the same reagents and calibrators as the Atellica IM assays. Precision and method comparison were used as performance indicators for the Atellica CI 1900 Analyzer. Precision studies were performed according to CLSI EP05-A3 and method comparison according to CLSI EP09-A3 using native and contrived human plasma (BNP) and serum (PBNP) samples. One aliquot of each sample pool was tested in duplicate in two runs per day >2 h apart on each analyzer for >20 days. BNP and PBNP were evaluated with one reagent lot on two systems. For the method comparison, individual native and contrived human plasma samples (BNP) or native human serum samples (PBNP) were analyzed using the Atellica IM BNP and PBNP assays on both the Atellica IM and Atellica CI 1900 Analyzers. Results Representative precision and MC results for each assay are listed in the table. For the two assays tested, repeatability and within-lab %CVs were <2.5% and <3.0%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1. Conclusion Evaluation of the Atellica IM BNP and PBNP assays using the Atellica CI 1900 Analyzer demonstrated acceptable precision and equivalent performance compared to the same assays on the Atellica IM Analyzer.

B-040 Performance Evaluation of Five Reproductive Endocrinology Assays on the Atellica CI 1900 Analyzer

Abstract Background The Atellica® CI 1900 Analyzer is an automated, mid-throughput, integrated chemistry and immunoassay analyzer utilizing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica IM Luteinizing Hormone (LH), Progesterone (PRGE), Follicle Stimulating Hormone (FSH), Prolactin (PRL), and Total hCG (ThCG) assays* on the Atellica CI 1900 Analyzer. Methods The Atellica CI 1900 LH, PRGE, FSH, PRL, and ThCG assays use the same reagents and calibrators as the comparable Atellica IM assays. Precision and method comparison (MC) studies were used as performance indicators for the Atellica CI 1900 Analyzer. Precision studies were performed according to CLSI EP05-A3 using quality control material and native and contrived human serum samples. One aliquot of each sample pool was tested in duplicate in two runs per day ≥2 hours apart on each analyzer for ≥20 days with one reagent lot on two systems. MC studies were performed according to CLSI EP09-A3. Individual native human serum samples were analyzed using the Atellica IM assays on both the Atellica IM and Atellica CI 1900 Analyzers. Results Representative precision and MC results are listed for each assay in the table. Over the five assays tested, repeatability and within-lab %CVs were <7% and <13.5%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1. Conclusion Evaluation of the Atellica IM LH, PRGE, FSH, PRL, and ThCG assays using the Atellica CI 1900 Analyzer demonstrated good precision and equivalent performance compared to the same assays on the Atellica IM Analyzer. *The products/features mentioned here are not commercially available in all countries. Their future availability cannot be guaranteed.

A capacitive laser-induced graphene based aptasensor for SARS-CoV-2 detection in human saliva.

SARS-CoV-2 virus induced CoVID-19 pandemic has accelerated the development of diagnostic tools. Devices integrated with electrochemical biosensors may be an interesting alternative to respond to the high demand for testing, particularly in contexts where access to standard detection technologies is lacking. Aptamers as recognition elements are useful due to their stability, specificity, and sensitivity to binding target molecules. We have developed a non-invasive electrochemical aptamer-based biosensor targeting SARS-CoV-2 in human saliva. The aptamer is expected to detect the Spike protein of SARS-CoV-2 wildtype and its variants. Laser-induced graphene (LIG) electrodes coated with platinum nanoparticles were biofunctionalized with a biotin-tagged aptamer. Electrochemical Impedance Spectroscopy (EIS) for BA.1 sensing was conducted in sodium chloride/sodium bicarbonate solution supplemented with pooled saliva. To estimate sensing performance, the aptasensor was tested with contrived samples of UV-attenuated virions from 10 to 10,000 copies/ml. Selectivity was assessed by exposing the aptasensor to non-targeted viruses (hCoV-OC43, Influenza A, and RSV-A). EIS data outputs were further used to select a suitable response variable and cutoff frequency. Capacitance increases in response to the gradual loading of the attenuated BA.1. The aptasensor was sensitive and specific for BA.1 at a lower viral load (10-100 copies/ml) and was capable of discriminating between negative and positive contrived samples (with strain specificity against other viruses: OC43, Influenza A, and RSV-A). The aptasensor detected SARS-CoV-2 with an estimated LOD of 1790 copies/ml in contrived samples. In human clinical samples, the aptasensor presents an accuracy of 72%, with 75% of positive percent of agreement and 67% of negative percent of agreement. Our results show that the aptasensor is a promising candidate to detect SARS-CoV-2 during early stages of infection when virion concentrations are low, which may be useful for preventing the asymptomatic spread of CoVID-19.

Capillary flow-driven immunoassay platform for COVID-19 antigen diagnostics

Over the last few years, the SARS-CoV-2 pandemic has made the need for rapid, affordable diagnostics more compelling than ever. While traditional laboratory diagnostics like PCR and well-plate ELISA are sensitive and specific, they can be costly and take hours to complete. Diagnostic tests that can be used at the point-of-care or at home, like lateral flow assays (LFAs) are a simple, rapid alternative, but many commercially available LFAs have been criticized for their lack of sensitivity compared to laboratory methods like well-plate ELISAs. The Capillary-Driven Immunoassay (CaDI) device described in this work uses microfluidic channels and capillary action to passively automate the steps of a traditional well-plate ELISA for visual read out. This work builds on prior capillary-flow devices by further simplifying operation and use of colorimetric detection. Upon adding sample, an enzyme-conjugated secondary antibody, wash steps, and substrate are sequentially delivered to test and control lines on a nitrocellulose strip generating a colorimetric response. The end user can visually detect SARS-CoV-2 antigen in 15–20 min by naked eye, or results can be quantified using a smartphone and software such as ImageJ. An analytical detection limit of 83 PFU/mL for SARS-CoV-2 was determined for virus in buffer, and 222 PFU/mL for virus spiked into nasal swabs using image analysis, similar to the LODs determined by traditional well-plate ELISA. Additionally, a visual detection limit of 100 PFU/mL was determined in contrived nasal swab samples by polling 20 untrained end-users. While the CaDI device was used for detecting clinically relevant levels of SARS-CoV-2 in this study, the CaDI device can be easily adapted to other immunoassay applications by changing the reagents and antibodies.

MATRIX plasma-in-plasma contrived samples assessment and detection of MRD using the Personalis NeXT Personal assay.

e15056 Background: Detection of circulating tumor DNA (ctDNA) has been shown to correlate with clinical outcome of oncology patients. Molecular residual disease (MRD) profiling by ctDNA is being rapidly deployed. Current tumor-informed MRD tests assess a relatively small number of personalised variants, placing limits on their detection sensitivity and ultimately leading to false-negative results due to insufficient assay limit of detection (LOD). Here we report a performance evaluation of the Personalis NeXT Personal tumor-informed MRD assay, using a novel design for contrived samples that are highly commutable to clinical samples, enabling robust assessment of these emerging MRD assays. Methods: Our novel samples were built from commercially acquired matched tumor and plasma samples, across different indications. The cancer plasma was diluted into a background of healthy donor plasma to create the 72 plasma-in-plasma samples in each study (total of 144). Personalis performed WGS on the matched solid tumor and normal samples from each patient. ~1800 somatic variants were selected for each patient. A personalized panel was designed and used to enrich for the selected targets in the individual plasma samples. An aggregated positive variant score was evaluated to determine the presence of ctDNA in each plasma sample. Results: MATRIX 1 panels averaged 1863 variants (range: 1818 to 1888) selected for MRD tracking. With 152 clinically relevant variants and additional assay components, an average of 2206 variants were assessed per patient. MATRIX 2 panels averaged 1836 variants (range: 1819 to 1867) selected for MRD tracking. With 2130 clinically relevant variants they averaged 4094 variants assessed per patient. In the MATRIX 1 study, tumor signal was detected in all analyzed samples, including 9 samples at 0.002% tumor plasma dilution, with an assay sensitivity of 100%. The MATRIX 2 study showed tumor signal detected in 14 of 16 samples at 0.001% tumor plasma dilution. The 2 false negatives were reported at the 0.001% tumor plasma dilution. Thus, the assay sensitivity was 87.5% at the lowest dilution (0.001%) and 96.8% overall. Conclusions: The MATRIX plasma-in-plasma approach is a robust option for assessing the sensitivity of MRD assays to low levels of ctDNA. This approach addresses all parts of an MRD assay for both the plasma and tumor/normal tissues, and is the closest thing to real clinical samples while allowing direct interrogation of sensitivity. Additionally, it allows assessment of any genomic or epigenomic biological feature. The Personalis NeXT Personal assay shows ultra-high sensitivity with reproducible data down to the 1-3 PPM range, and expansion of the personalized panel fixed clinical content allows tracking of more driver and resistance mutations with no impact to the MRD sensitivity.

Analytical performance assessment of ASPYRE-Lung: A new assay for rapid detection of actionable variants from tissue and plasma in NSCLC patients.

e15038 Background: We developed ASPYRE technology to make detection of genomic variants in oncology patients more accessible. Although ASPYRE is not traditional PCR, it can be performed using standard qPCR instruments on nucleic acid extracted from FFPE tissue or plasma and is therefore easily adoptable by clinical laboratories. Using ASPYRE, we designed a targeted panel called ASPYRE-LungTM consisting of 114 variants in 11 genes ( ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET & NTRK1/2/3) to inform the clinical management of patients with NSCLC. We have previously published general assay descriptions of ASPYRE-Lung (Silva et al., 2021, Scientific Reports and Gray et al., 2022, BMC Medical Genomics). Here we extend our prior studies with a detailed analytic performance assessment across all variants using precisely controlled synthetic samples. We determined assay-wide performance using samples with low variant allele fractions (VAFs) near the limit of detection (LoD) of the assay and compared results across 2 separate laboratory settings. Methods: Nine independent batches of reagents were split between 2 laboratories. Contrived test samples were prepared with known VAFs (DNA variants) or copy numbers (RNA variants). Each sample contained a single variant with VAFs of 0.1-0.5% for DNA variants, and 6-150 copies for RNA variants. Multiple replicates were performed at both testing sites using 20ng DNA/6ng RNA. Data was processed using an internally designed algorithm and analyzed using a machine learning classifier which determined the presence or absence of each individual variant. Results: Study data resulted in a total of 133,083 variant calls from 2,319 samples. For each variant at each VAF or copy number, the detection rate was calculated, and the results for each variant were used to calculate an LoD95 using probit regression. Target assay specifications and the assay-wide analytical performance data are shown in the table below. Conclusions: The data show that ASPYRE-Lung meets or exceeds target specifications for the detection of single nucleotide variants (SNVs), insertions/deletions (indels), and gene fusions. Estimates of LoD for key variants fall within target specifications, and assay-wide performance demonstrates highly sensitive detection of each class of variant. Performance was similar at the two laboratory sites further supporting the clinical potential of the assay. Moreover, the assay only requires 20ng DNA and as little as 6ng RNA, depending on sample type, so it is highly suitable for use with typical NSCLC patient samples such as needle core biopsies or plasma where available nucleic acid may be limited. This data supports further clinical development of ASPYRE-Lung. [Table: see text]