Assay Performance Characteristics Research Articles

Clinical Significance of Positive Results of the BioFire Cerebrospinal Fluid FilmArray Meningitis/Encephalitis Panel at a Tertiary Medical Center in the United States.

The FilmArray Meningitis/Encephalitis (ME) panel is the first US Food and Drug Administration-cleared multiplex polymerase chain reaction panel for the detection of central nervous system infections. While the assay's performance characteristics have been described, the real-world significance of positive results has not been fully characterized. To evaluate the clinical significance of positive ME panel results in a tertiary care medical center in New York, New York. Four physicians independently performed retrospective clinical assessments of all positive ME panel results at Columbia University Irving Medical Center, including the Children's Hospital of New York, during an 18-month period. Each reviewer determined the likelihood of central nervous system infection for all cases and whether cases fit Brighton diagnostic criteria for meningitis, encephalitis, or meningoencephalitis. Among 119 cases, there was 75% positive agreement (95% CI, 54%-89%) between ME panel results and clinical consensus, which varied among panel targets. The ME panel showed good agreement with expert clinical consensus for patients presenting with acute meningitis/encephalitis. Factors contributing to clinically insignificant ME positive results included low pretest probability, traumatic lumbar puncture, specimen contamination, and detection of incidental viral targets such as human herpesvirus 6. Notably, the ME panel detected more than twice the number of cases of bacterial meningitis detected by culture alone, particularly among patients receiving empiric antimicrobial therapy before lumbar puncture. Appropriate test use and contextual interpretation of results are critical to leveraging the advantages of the platform while avoiding potential pitfalls.

Analytical validation of a real-time hydrolysis probe PCR assay for quantifying Plasmodium falciparum parasites in experimentally infected human adults

BackgroundVolunteer infection studies have become a standard model for evaluating drug efficacy against Plasmodium infections. Molecular techniques such as qPCR are used in these studies due to their ability to provide robust and accurate estimates of parasitaemia at increased sensitivity compared to microscopy. The validity and reliability of assays need to be ensured when used to evaluate the efficacy of candidate drugs in clinical trials.MethodsA previously described 18S rRNA gene qPCR assay for quantifying Plasmodium falciparum in blood samples was evaluated. Assay performance characteristics including analytical sensitivity, reportable range, precision, accuracy and specificity were assessed using experimental data and data compiled from phase 1 volunteer infection studies conducted between 2013 and 2019. Guidelines for validation of laboratory-developed molecular assays were followed.ResultsThe reportable range was 1.50 to 6.50 log10 parasites/mL with a limit of detection of 2.045 log10 parasites/mL of whole blood based on a parasite diluted standard series over this range. The assay was highly reproducible with minimal intra-assay (SD = 0.456 quantification cycle (Cq) units [0.137 log10 parasites/mL] over 21 replicates) and inter-assay (SD = 0.604 Cq units [0.182 log10 parasites/mL] over 786 qPCR runs) variability. Through an external quality assurance program, the QIMR assay was shown to generate accurate results (quantitative bias + 0.019 log10 parasites/mL against nominal values). Specificity was 100% after assessing 164 parasite-free human blood samples.ConclusionsThe 18S rRNA gene qPCR assay is specific and highly reproducible and can provide reliable and accurate parasite quantification. The assay is considered fit for use in evaluating drug efficacy in malaria clinical trials.

A robust reporter assay for the determination of the bioactivity of IL-4R-targeted therapeutic antibodies

Type 2 inflammatory cytokines, including IL-4, IL-5 and IL-13, contribute considerably to the pathogenesis of asthma. Anti-IL-4R monoclonal antibody (mAb) has been approved for the therapeutic treatment of asthma, and many mAbs with the same target are in the different stages of R&D and clinical trials. Bioactivity determination is required to ensure the quality control of mAbs. However, current ELISA and SPR assays or cell-based anti-proliferation assays for IL-4R mAbs are either not mechanism-of-action (MOA) representative or tedious and time consuming. Therefore, we developed a reporter gene assay (RGA) based on the HEK-293 cell line that stably expressed signal transducer and activator of transcription 6 (STAT6) and the luciferase reporter controlled by STAT6 binding elements. Anti-4R mAb could bind to IL-4R, and block the interaction between IL-4 and IL-4R, resulting in the reduction of IL-4 induced STAT6 controlled luciferase expression. After careful optimization of the experiment parameters, the RGA method demonstrated optimal dose-response curve between anti-IL-4R mAb concentration and luciferase expression level. Validation according ICH-Q2 proved the excellent assay performance characteristics of the established RGA, including specificity, accuracy, precision, linearity and range. The established transgenic cell line was stable for the bioactivity determination of anti-IL-4R mAb up to 46 generations, and the RGA was also suitable for the bioactivity determination of anti-IL-4 mAbs, and potentially of anti-IL-13 mAbs. The established RGA could be adopted to determine the bioactivity during the development, characterization, lot release, stability, and comparability studies of anti-IL-4R mAbs.

Development of a robust reporter gene assay for measuring the bioactivity of OX40-targeted therapeutic antibodies.

OX40 plays a prominent role in the onset and development of solid tumors, and OX40-targeted monoclonal antibodies (mAbs) have entered clinical trials for various tumors. Bioactivity determination of therapeutic mAbs is of great significance in product quality, however, mechanism of action-based bioassays to determine the bioactivity of anti-OX40 mAbs is still lacking. Here, we established a reporter gene assay system based on two cell lines, namely Jurkat-OX40-NFκB-Luc which stably expresses NFκB-controlled luciferase, and Raji cells which inherently express FcγRs. In the model, FcγRs on Raji cells could crosslink the Fc of anti-OX40 mAbs, which leads to the further crosslinking between Fab of anti-OX40 mAbs and OX40 on Jurkat-OX40-NFκB-Luc cells. OX40 crosslinking could activate Jurkat-OX40-NFκB-Luc cells, and induce the expression of NFκB-controlled luciferase, the extent of which could reflect the bioactivity of anti-OX40 mAbs in a dose-dependent manner. After the optimization of various assay conditions, the validation of the cell-based bioassay showed good assay performance characteristics, including specificity, accuracy, precision, linearity, and stability. This innovative assay that is based on the OX40-NFκB pathway can be a powerful pool to measure the bioactivity of OX40-targeted mAbs.

Performance of BioFire array or QuickVue influenza A\u2009+\u2009B test versus a validation qPCR assay for detection of influenza A during a volunteer A/California/2009/H1N1 challenge study

BackgroundInfluenza places a significant burden on global health and economics. Individual case management and public health efforts to mitigate the spread of influenza are both strongly impacted by our ability to accurately and efficiently detect influenza viruses in clinical samples. Therefore, it is important to understand the performance characteristics of available assays to detect influenza in a variety of settings. We provide the first report of relative performance between two products marketed to streamline detection of influenza virus in the context of a highly controlled volunteer influenza challenge study.MethodsNasopharyngeal swab samples were collected during a controlled A/California/2009/H1N1 influenza challenge study and analyzed for detection of virus shedding using a validated qRT-PCR (qPCR) assay, a sample-to-answer qRT-PCR device (BioMerieux BioFire FilmArray RP), and an immunoassay based rapid test kit (Quidel QuickVue Influenza A + B Test).ResultsRelative to qPCR, the sensitivity and specificity of the BioFire assay was 72.1% [63.7–79.5%, 95% confidence interval (CI)] and 93.5% (89.3–96.4%, 95% CI) respectively. For the QuickVue rapid test the sensitivity was 8.5% (4.8–13.7%, 95% CI) and specificity was 99.2% (95.6–100%, 95% CI).ConclusionRelative to qPCR, the BioFire assay had superior performance compared to rapid test in the context of a controlled influenza challenge study.

Evaluation of the Analytical Performance Beckman Coulter Access Sensitive Estradiol Assay.

The study aimed to assess the analytical performance of the Access Sensitive Estradiol (SNSE2) Assay on a DxI800 (Beckman Coulter, Brea, CA, USA) and compared it with a Cobas E 601 (Roche Diagnostics, Penzberg, Germany). SNSE2 was assessed for imprecision, accuracy, limit of blank (LoB), limit of detection (LoD), limit of quantitation (LoQ), linearity, interference, and carryover. Two hundred and fourteen samples were run on both instruments. Bland-Altman plots, Passing-Bablok regression, and concordance correlation coefficient (CCC) graphs were used for comparisons. Access SNSE2 showed appropriate assay performance characteristics in terms of imprecision, LoB, LoD, LoQ, linearity, and interference. The Bland-Altman analysis of DxI 800 yielded negative bias from Cobas E 601 and the deviations for E2 ≤ 150 pmol/L, 150 - 500 pmol/L, and ≥ 500 pmol/L were found as 0.8%, -15%, and -8.9%, respectively. DxI 800 and E170 systems showed poor agreement for E2 levels ≤ 150 pmol/L and 150 - 500 pmol/L with CCC values of 0.7404 and 0.8342. For E2 levels ≤ 150 pmol/L there was a significant amount of both proportional and constant error with the highest slope of 1.518 (1.269 to 1.761) and an intercept of -45.08 (-66.09 to 18.78, respectively, according to the Passing-Bablok regression analysis). Analytical performance for SNSE2 assay was found appropriate. However, attempts to improve harmonization and standardization across assays do not seem to contribute much for E2 measurements. Results obtained with different systems cannot be used interchangeably and follow up of patients should be done with the same system.

Rapid testing for respiratory syncytial virus in a resource-limited paediatric intensive care setting.

We analysed the performance characteristics of the respiratory syncytial virus lateral flow rapid antigen assay in use when compared to a multiplex polymerase chain reaction for detection of respiratory viruses. The study was conducted at a tertiary paediatric hospital in Port Elizabeth, South Africa, from 01 January 2017 to 31 December 2018. We found the clinical sensitivity (36.8%) of the rapid test to be too low for routine diagnostic use. Knowledge of assay performance characteristics of rapid tests are important for appropriate interpretation of rapid test results.

Best Practices for Validation of Measurable Residual Disease Assessments By Multiparameter Flow Cytometry in Emerging Clinical Trials of Acute Myeloid Leukemia

Background: Measurable Residual Disease (MRD) assessments are gaining increasing acceptance as a prognostic factor for tailoring treatment in hematological malignancies. Acute Myeloid Leukemia (AML) is a heterogeneous disease with high relapse rates and presents a high unmet need for effective treatment options. Measurement of residual disease after therapy reflects a combination of all resistance mechanisms and is currently used for guiding treatment options. Study Design: In this study, we aimed to validate an AML-MRD assay by multiparameter flow cytometry (MFC) methodology. This is a 4-tube, 8-parameter assay designed to incorporate cell differentiation (CD) markers for identification of a diverse group (covering roughly 90% of patients, Cloos et al, 2018) of Leukemia Associated Immunophenotypes (LAIPs) to accurately identify both native phenotypes and phenotype shifts after drug treatment. These CD markers were selected based on extensive investigation of many markers and in line with the consensus recommendations from European Leukemia Network AML working party (Schuurhuis et al, 2018), while specimen testing and interpretation principles were performed in accordance with Cloos et al, 2018. The assay validation focused on evaluation of sensitivity (MRD cut point and LOD), precision and accuracy as key criteria for evaluating assay performance utilizing primary patient specimens and AML cell lines representing different LAIPs. The results were orthogonally verified in a blinded manner by morphologic assessment at Navigate and by the MRD-team at VUMC Amsterdam. Results: Two experimental approaches were adopted to evaluate analytical and functional sensitivity (clinical applicability) of the assay. Results indicated analytical sensitivity (LOD) as low as 0.01% LAIPs of total WBC and functional sensitivity (LOQ) of 0.1% (MRD cut point). Excellent repeatability and reproducibility (less than 20% CV) was observed across instruments, operators and independent measurements (n = 75). The frequencies of AML blasts detected by MFC and morphological examination were highly concordant (Spearman r = 0.95, P value < 0.001, n = 24). LAIPs deduced across nine patient specimens by the Navigate laboratory were independently confirmed by the MRD-team at VUMC Amsterdam. Conclusion: In summary, based on the use of consensus markers recommended by ELN for reliable capture of a broad group of LAIPs in AML patients and verification of key assay performance characteristics, we believe this comprehensive MFC based AML MRD assay is fit-for-purpose for accurately assessing measurable residual disease. Following clinical trial validation, MRD might be used as a surrogate endpoint for approval of emerging agents. Disclosures Marques Ramos: Novartis: Current Employment. Larson:BMS, Bioline, Celgene, Juno, Janssen: Research Funding; TORL Biotherapeutics: Current equity holder in private company. Sarikonda:Novartis: Current Employment.

A Novel Bead-Based Immunoassay for the Measurement of Heat Shock Proteins 27 and 70.

Heat shock proteins (HSPs) play an essential role in protecting proteins from denaturation and are implicated in diverse pathophysiological conditions like cardiovascular diseases, cancer, infections, and neurodegenerative diseases. Scientific evidence indicates that if HSP expression falls below a certain level, cells become sensitive to oxidative damage that accelerates protein aggregation diseases. On the other hand, persistently enhanced levels of HSP can lead to inflammatory and oncogenic changes. To date, although techniques for measuring HSPs exist, these assays are limited for use in specific sample types or are time consuming. Therefore, in the present study, we developed a single-molecule assay digital ELISA technology (Single Molecule Array—SIMOA) for the measurement of HSPs, which is time effective and can be adapted to measure multiple analytes simultaneously from a single sample. This technique combines two distinct HSP-specific antibodies that recognize different epitopes on the HSP molecule. A recombinant human HSP protein was used as the standard material. The assay performance characteristics were evaluated by repeated testing of samples spiked with HSP peptide at different levels. The limit of detection was 0.16 and 2 ng/mL for HSP27 and HSP70, respectively. The inter- and intra-assay coefficients of variation were less than 20% in all tested conditions for both HSPs. The HSP levels assayed after serial dilution of samples portrayed dilutional linearity (on average 109%, R2 = 0.998, p < 0.001, for HSP27 and 93%, R2 = 0.994, p < 0.001, for HSP70). A high linear response was also demonstrated with admixtures of plasma exhibiting relatively very low and high levels of HSP70 (R2 = 0.982, p < 0.001). Analyte spike recovery varied between 57% and 95%. Moreover, the relative HSP values obtained using Western blotting correlated significantly with HSP values obtained with the newly developed SIMOA assay (r = 0.815, p < 0.001 and r = 0.895, p < 0.001 for HSP70 and HSP27, respectively), indicating that our method is reliable. In conclusion, the assay demonstrates analytical performance for the accurate assessment of HSPs in various sample types and offers the advantage of a huge range of dilution linearity, indicating that samples with HSP concentration highly above the calibration range can be diluted into range without affecting the precision of the assay.

The tumor genetics of acral melanoma: What should a dermatologist know?

Dermatologists stand at the gateway of individualization of classification, treatment, and outcomes of acral melanoma patients. The acral melanoma genetic landscape differs in vital ways from that of other cutaneous melanomas. These differences have important implications in understanding pathogenesis, treatment, and prognosis. The selection of molecularly targeted therapy must be adapted for acral melanoma. It is also critical to recognize that tumor development is far more complex than an isolated event, reliably treated by a medication acting on a single target. Tumors exhibit intratumor genetic heterogeneity, metastasis may have different genetic or epigenetic features than primary tumors, and tumor resistance may develop because of the activation of alternative genetic pathways. Microenvironmental, immune, and epigenetic events contribute and sustain tumors in complex ways. Treatment strategies with multiple targets are required to effectively disrupt the tumor ecosystem. This review attempts to translate the current molecular understanding of acral melanoma into digestible concepts relevant to the practice of dermatology. The focus is tumor genetics defining potentially treatable cancer pathways, contextualized within the relevant pathologic and molecular features.

Evaluation of Serological Tests for SARS-CoV-2: Implications for Serology Testing in a Low-Prevalence Setting.

BackgroundRobust serological assays are essential for long-term control of the COVID-19 pandemic. Many recently released point-of-care (PoCT) serological assays have been distributed with little premarket validation.MethodsPerformance characteristics for 5 PoCT lateral flow devices approved for use in Australia were compared to a commercial enzyme immunoassay (ELISA) and a recently described novel surrogate virus neutralization test (sVNT).ResultsSensitivities for PoCT ranged from 51.8% (95% confidence interval [CI], 43.1%–60.4%) to 67.9% (95% CI, 59.4%–75.6%), and specificities from 95.6% (95% CI, 89.2%–98.8%) to 100.0% (95% CI, 96.1%–100.0%). ELISA sensitivity for IgA or IgG detection was 67.9% (95% CI, 59.4%–75.6%), increasing to 93.8% (95% CI, 85.0%–98.3%) for samples >14 days post symptom onset. sVNT sensitivity was 60.9% (95% CI, 53.2%–68.4%), rising to 91.2% (95% CI, 81.8%–96.7%) for samples >14 days post symptom onset, with specificity 94.4% (95% CI, 89.2%–97.5%).ConclusionsPerformance characteristics for COVID-19 serological assays were generally lower than those reported by manufacturers. Timing of specimen collection relative to onset of illness or infection is crucial in reporting of performance characteristics for COVID-19 serological assays. The optimal algorithm for implementing serological testing for COVID-19 remains to be determined, particularly in low-prevalence settings.

Characterization of a reliable cell-based reporter gene assay for measuring bioactivities of therapeutic anti-interleukin-23 monoclonal antibodies

Environmental disturbances may result in dysregulation of interleukin-23 (IL-23), which is a crucial modulator of immunity. Several therapeutic monoclonal antibodies (mAbs) have been developed for treating IL-23-related autoimmune inflammation, such as ustekinumab, guselkumab, tildrakizumab, and risankizumab. Accurate bioactivity determination of therapeutic mAbs is essential for their quality control and clinical application. However, the current methods are tedious and complicated. In the present study, we employed low-background lentivirus carrying sis-inducible element (SIE)-driven firefly luciferase to generate a stable DB-SIE-Luc cell line that expresses endogenous IL-23 receptors and developed a sensitive and straightforward reporter gene assay (RGA) based on DB-SIE-Luc cells. After the optimization of various assay parameters, we set up a bioassay with the best fit of a four-parameter model and an appropriate signal-to-noise ratio (SNR) for bioactivity determination of guselkumab. We further verified the excellent assay performance characteristics of our RGA, including specificity, linearity, accuracy, precision, and stability, according to ICH-Q2. Taken together, we established a reliable and robust cell-based RGA, which potentially serves as a valubale alternative bioactivity determination assay for the release control and stability study of anti-IL-23 mAbs.

Performance characteristics of thromboelastometry assays using incompletely filled and prolonged stored samples.

Thromboelastometry (TEM) is often used to guide transfusion therapy in patients with massive bleeding. The effect of testing incompletely filled samples and those stored for a prolonged time at 4°C was investigated. Whole blood samples were collected from 15 healthy blood donors and were pooled according to ABO group. From these pools, aliquots were taken and diluted to produce final whole blood:citrate buffer ratios ranging from 90:10 (fully filled sample) to 40:60 (extremely under filled samples). These samples were then tested by EXTEM, INTEM, and FIBTEM on calibrated ROTEM delta machines. Separately, the four samples at 90:10 dilution were kept at 4°C for 16-20 hours and then retested on the ROTEM machines. All of the samples at the 90:10 and 80:20 (half-filled sample) whole blood:citrate buffer dilutions demonstrated ROTEM parameters within their respective reference ranges, although the samples from the 80:20 dilution tended to demonstrate slightly longer or slower times, depending on each ROTEM parameter, compared to the completely filled samples. All of the samples with more dilute whole blood to citrate buffer ratios (i.e., 70:30 to 40:60) yielded abnormal TEM results. The TEM results for the 90:10 dilution samples exposed to 16-20 hours of storage at 4°C were within the reference intervals. Completely and half-filled samples, and completely filled samples after prolonged cold storage, produced normal ROTEM results. Tubes that are less than half-filled should not be used for ROTEM testing.

Streamlining basophil activation testing to enable assay miniaturization and automation of sample preparation

BackgroundNumerous studies have demonstrated the capabilities of the basophil activation test (BAT) but various parameters such as a lack of standardization and a time consuming and labor intensive workflow continue to hinder the field to fully leverage the capabilities of this technique. When pediatric patients have to be considered, an additional limitation is related to blood volume consumption. ObjectivesThis work aimed at developing and characterizing a simplified and standardized whole-blood based BAT prototype procedure and at further assessing the feasibility of automating and miniaturizing the developed assay into a 96 well plate format. MethodsA dry and room temperature stable reagent technology was used to simplify and standardize BAT. Under optimized conditions, EDTA anticoagulated whole blood samples of non-allergic and allergic donors (<24 h old) together with calcium containing buffer were added to ready-to-use dry reagent tubes or 96 well plates (negative controls, positive controls and allergen tests) containing a 5 color compensation-free antibody panel (CD45-KrO/CD3-PC7/CRTH2-A647/CD203c-PE/CD63-PB). Upon mixing and incubation at 37 °C for 15 min, erythrocytes were lysed and samples were analyzed by flow cytometry without further washing steps. While it is important to precisely control the incubation time to minimize the assay variability, herein, a 15 min incubation time was chosen as it provides a suitable compromise for both the magnitude of basophil activation and the quality of the staining. A Biomek NXP robotic platform (Beckman Coulter) was used for automation and both CD203c and CD63 levels were monitored to characterize basophil reactivity. ResultsThis streamlined BAT protocol is no-wash, compensation free and only requires 4 pipetting steps to be completed. The assessment of assay performance characteristics showed wide applicability, satisfactory repeatability and a high degree of standardization as demonstrated by very low intra-assay and inter-operator variabilities (CVs < 10%). Leveraging these technical foundations, it was then proven that this new BAT procedure can easily be transposed into the 96 well plate format, thereby benefiting from a miniaturized format and full automation capabilities. When considering 8 dilution points to characterize the ex vivo basophil reactivity of a given whole blood sample, we found that as little as 5 μL of blood per point could be used. ConclusionsA whole blood based and simplified procedure for BAT is proposed. It relies on a dry antibody formulation technology and requires only a few manual steps to be completed. This procedure can also be transposed in a 96 well plate format, fully automated and miniaturized, when sample volume reduction, throughput increase or unattended sample preparation is required.

Laboratory-related issues in the measurement of cardiac troponins with highly sensitive assays.

A number of assay-related issues can affect the performance of cardiac troponin (cTn) measurement in everyday practice. In this respect, it is vital that all information on cTn assays is known and that the performance characteristics of assays are objectively assessed and adequately described. The advent of the latest generation of more sensitive cTn assays has heralded a new wave of information about low concentrations of cTn in blood. These recent generation assays have improved analytical sensitivity and corresponding performance at low cTn concentrations when compared to their predecessors, providing a convincing goal for laboratory medicine in helping clinicians in the diagnosis of acute myocardial infarction. Crucial to the clinical utility of highly sensitive cTn assays is the laboratorians' role in closely scrutinizing proposed assays and defining their value in relation to available evidence. Analytical, as well as pre-analytical and post-analytical, aspects must be documented. In this review, we describe what laboratory professionals should know about their cTn assay performance characteristics and the pre-analytical prerequisites for robustness to ensure optimal post-analytical reporting.

Proficiency Testing of Standardized Samples Shows High Interlaboratory Agreement for Clinical Next Generation Sequencing-Based Hematologic Malignancy Assays With Survey Material-Specific Differences in Variant Frequencies.

As laboratories increasingly turn from single-analyte testing in hematologic malignancies to next-generation sequencing-based panel testing, there is a corresponding need for proficiency testing to ensure adequate performance of these next-generation sequencing assays for optimal patient care. To report the performance of laboratories on proficiency testing from the first 4 College of American Pathologists Next-Generation Sequencing Hematologic Malignancy surveys. College of American Pathologists proficiency testing results for 36 different engineered variants and/or allele fractions as well as a sample with no pathogenic variants were analyzed for accuracy and associated assay performance characteristics. The overall sensitivity observed for all variants was 93.5% (2190 of 2341) with 99.8% specificity (22 800 of 22 840). The false-negative rate was 6.5% (151 of 2341), and the largest single cause of these errors was difficulty in identifying variants in the sequence of CEBPA that is rich in cytosines and guanines. False-positive results (0.18%; 40 of 22 840) were most likely the result of preanalytic or postanalytic errors. Interestingly, the variant allele fractions were almost uniformly lower than the engineered fraction (as measured by digital polymerase chain reaction). Extensive troubleshooting identified a multifactorial cause for the low variant allele fractions, a result of an interaction between the linearized nature of the plasmid and the Illumina TruSeq chemistry. Laboratories demonstrated an overall accuracy of 99.2% (24 990 of 25 181) with 99.8% specificity and 93.5% sensitivity when examining 36 clinically relevant somatic single-nucleotide variants with a variant allele fraction of 10% or greater. The data also highlight an issue with artificial linearized plasmids as survey material for next-generation sequencing.

Development and Validation of a Free Carbamazepine Assay on an Automated Chemistry Analyzer.

Carbamazepine is an effective drug for treating seizures and trigeminal neuralgia. Therapeutic drug monitoring of free carbamazepine in serum can be useful in situations that drug--protein binding is altered to guide regimen adjustment and to aid in the diagnosis of clinical toxicity. Separation of the nonprotein bound carbamazepine was achieved via ultrafiltration through a molecular weight cut-off filter. A method for free carbamazepine measurement was developed on the automated cobas chemistry analyzers (Roche Diagnostics) by modifying the Carbamazepine Gen 4 assay (Roche Diagnostics). Assay performance characteristics were established including precision, accuracy, reportable range, analytical specificity, and stability. The intra- and inter-assay imprecision was 0%-1.4% and 2.4%-5.1%, respectively. The lower limit of quantitation was 0.3 µg/mL, and the assay was linear up to 10.0 µg/mL. A spike recovery study, using reference standard material, showed recovery was 93.5%--101.3% across the analytical measurement range. Method comparison with a reference laboratory method demonstrated equivalent performance with a slope of 1.01, intercept of 0.09, and correlation coefficient of 0.9948. This assay provides a simple and accurate method for monitoring free carbamazepine with a fast turnaround time.

Automated Clinical Chemistry Platform Performance Across Two Academic Medical Centers

Abstract Our objective was to define the performance of automated chemistry platforms at two large academic medical centers by calculating and comparing sigma metrics for 28 analytes. Performance characteristics of chemistry assays on two Roche Cobas analyzers and four Abbott Architect analyzers were estimated using 12 months of Bio-Rad quality control (QC) data at two concentrations. Method imprecision was calculated as the cumulative QC coefficient of variation (CV) across analyzers at each QC concentration and percent bias was calculated by comparison of analyzer QC mean to peer group means. Sigma values were calculated for each method as [(TEa – Bias%)/CV%] using allowable total error (TEa) from two sources: the CLIA evaluation limits and desirable biological variation. Average sigma values were generated for each site and graded as optimal, &gt;6 sigma; good, 5 to 6 sigma; marginal, 3 to 5 sigma; or poor, &lt;3 sigma. Analysis of NIST SRM1950 standards for a subset of analytes allowed an estimation of absolute bias. Sigma metrics were highly comparable across both study sites. Considering CLIA TEa, just over half (Roche 57%; Abbott 54%) of the 28 analytes met the six-sigma standard of performance. The highest performing groups to meet or exceed the six-sigma standard were lipids (cholesterol, LDL, HDL, and triglycerides) and enzymes (ALP, ALT, AST, CK, GGT, LDH, and lipase). Electrolytes (Na, K, Cl, Mg) and metabolites (total bilirubin, BUN, CO2) failed to meet six-sigma. Notably, there were dramatic differences in sigma values calculated using CLIA and Ricos TEa criteria. Almost 40% of the analytes had at least one QC that performed poorly using Ricos criteria. Only 4 of the 28 assays (CK, GGT, lipase, and triglycerides) demonstrated optimal performance at both study sites using Ricos and CLIA criteria. Overall, 11 analytes at each institution exhibited unacceptable performance using Ricos criteria as opposed to only two analytes with the CLIA TEa limits. Analysis of NIST SRM1950 at both study sites gave comparable sigmas for all analytes except total bilirubin, cholesterol, Mg, and total protein. Neither Abbott nor Roche analyzers met six-sigma quality standards for all analytes tested. Overall assay performance across these two platforms at two major academic medical centers was almost identical. CLIA TEa and RICOS TEa criteria are significantly different, with wider limits of acceptability for CLIA. Variations between individual analyzers and manufacturers and limitations in automation would make tailored QC rules based on sigma metrics difficult to implement in a high-volume laboratory.

Abstract 3976: “Fit-for-purpose” PIK3CA assays for patient stratification in breast cancer clinical trials

Abstract Background: Prospective clinical trials stratifying by PIK3CA genotype is necessary to determine if the mutation predicts for increased sensitivity to agents targeting the phosphoinositide 3-kinase (PI3K) pathway. It is crucial to select appropriate assays to optimize operational, scientific and timeline requirements for patient enrollment in a real world setting. Here we report application of three different PIK3CA mutation test methods during the course of a PI3K TKI development. Methods: We customized Sanger sequencing, cobas® assay and a proprietary investigational use only (IUO) assay to detect point mutations in PIK3CA in order to accommodate the development needs at the different stages of clinical development of PIK3CA TKIs. We performed method comparison studies to determine the most cost effective and “fit-for-purpose” assay for clinical trial testing. Specimen requirements, assay performance characteristics, turnaround-time (TAT) and valid result rates were assessed to select the best test method for patient stratification to meet the requirements of each stage of drug development. Results: PIK3CA test results can be obtained from a single, 5μm FFPE section with ≥0.1% and ≥10% tumor in the IUO and cobas® tests respectively. Sanger Sequencing requires longer processing time and ≥30% tumor content. The cobas® assays were validated in FFPE samples and can reliably detect PIK3CA mutations in exons 9 and 20 with ≥5% mutation level. The method comparison study showed that the concordance rate between Sanger sequencing and cobas® PIK3CA assay is 86% among the 360 samples tested while it is 96% between the cobas® assay and the IUO assay among 395 samples tested. The IUO assay is slightly more sensitive, and calls out the exact mutation while the cobas® assay may not. The Sanger sequencing demonstrated the lowest sensitivity and required more DNA input, which resulted in more samples failing to yield a valid result. The cobas® assay is a more user-friendly assay allowing better laboratory efficiency and TAT. Patients selected using the IUO assay showed a median progression-free survival nearly twice as long among those receiving a PI3K TKI compared to placebo. Conclusions: The most important factor in selecting a testing platform for clinical trial patient stratification is the assay performance characteristics. We employed the Sanger sequencing assay in the initial trial testing, then switched to the cobas® assay, and finally to the IUO assay to meet the requirements as the drug progressed through different stages of clinical development. The IUO assay has the performance characteristics that meet the regulatory requirements and a validated clinical utility in selecting patients for PI3K TKI treatment, thus on its way to a companion diagnostic submission. Citation Format: Hua C. Gong, Sharmila Manjeshwar, Violet Abraham, Shawn Rivera. “Fit-for-purpose” PIK3CA assays for patient stratification in breast cancer clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3976.

Abstract LB-011: Fit-for-purpose quantitative programmed cell death ligand -1 (PD-L1) droplet digital PCR assay development using PAXgene RNA blood samples

Abstract Purpose Programmed death ligand receptor 1, (PD-L1) is a key immune checkpoint inhibitory receptor expressed on activated tumor specific immune cells. While immunohistochemistry assays have been developed for the detection of PD-L1 expression in several cancer types for patient treatment decision, a blood-based test for PD-L1 expression evaluation would be beneficial in providing complimentary evidence for potential clinical application. This study developed a fit-for-purpose clinical biomarker assay for the detection of PD-L1 mRNA expression in blood using droplet digital polymerase chain reaction (ddPCR) methods. Study Procedure Taqman primer/probe assays were selected based on coverage of PD-L1 gene and were tested for linearity and efficiency using a cDNA construct. Four housekeeping genes were analyzed in positive control cell line (A549 treated with interferon gamma), negative control cell line (A549 untreated) and PAXgene blood from healthy volunteers. Real-time quantitative PCR method was tested first, following initial experiments there were concerns with the performance of our qPCR assay prompting the switch to ddPCR which offers absolute quantification and a lower limit of detection. Assay performance characteristics of the ddPCR assay were evaluated including linearity, limit of blank (LOB), limit of detection (LOD), and precision. Thirty-five healthy volunteer samples were evaluated to establish a baseline level of PD-L1 expression. Data Summary Conducting linearity comparison between Taqman primer/probe kits using cDNA titrations showed no significant difference in linearity (r2=0.99) or efficiency (~95%) among the three primer/probe kits. Housekeeping genes B2M, GUSB, RPLP0, and TBP were compared and GUSB was most consistently expressed between IFN gamma treated and untreated cells; it was selected for use thereafter. In ddPCR, the LOB was determined to be 0 copies and the LOD was determined to be 19 copies of PD-L1. The average intra-run coefficient of variation (CV) was 7.44% and average inter-run CV was 7.70%. Analysis of healthy control samples yielded an average of 2415 PD-L1 copies with a range of 768-7510 copies. Treatment of A549 cells with IFN gamma resulted in a 6.7-fold increase in PD-L1 expression (21,580 copies in untreated cDNA versus 145,000 copies in treated cDNA). Conclusion Our fit-for purpose clinical biomarker assay development efforts have demonstrated that the ddPCR assay is capable of reliably and accurately detecting PD-L1 in PAXgene blood with low limit of detection and minimal inter-and-intra run variability. Compared to real-time PCR, ddPCR is a purely quantitative and offers a much broader dynamic range and provides more sensitive detection of PD-L1 in blood. Further evaluation and comparison of cancer to healthy volunteer samples is underway for more information on assay performance and disease biology. Citation Format: Dennis O'Rourke, Danyi Wang, Maria Cusano, Jorge F. Sanchez-Garcia, Ti Cai, Juergen Scheuenpflug, Zheng Feng. Fit-for-purpose quantitative programmed cell death ligand -1 (PD-L1) droplet digital PCR assay development using PAXgene RNA blood samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-011.