ASPYRE-Lung: validation of a simple, fast, robust and novel method for multi-variant genomic analysis of actionable NSCLC variants in FFPE tissue.

Introduction: Genomic variant testing of tumors is a critical gateway for patients to access the full potential of personalized oncology therapeutics. Current methods such as next-generation sequencing are costly and challenging to interpret, while PCR assays are limited in the number of variants they can cover. We developed ASPYRE® (Allele-Specific PYrophosphorolysis REaction) technology to address the urgent need for rapid, accessible and affordable diagnostics informing actionable genomic target variants of a given cancer. The targeted ASPYRE-Lung panel for non-small cell carcinoma covers 114 variants in 11 genes (ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET & NTRK1/2/3) to robustly inform clinical management. The assay detects single nucleotide variants, insertions, deletions, and gene fusions from tissue-derived DNA and RNA simultaneously. Methods: We tested the limit of detection, specificity, analytical accuracy and analytical precision of ASPYRE-Lung using FFPE lung tissue samples from patients with non-small cell lung carcinoma, variant-negative FFPE tissue from healthy donors, and FFPE-based contrived samples with controllable variant allele fractions. Results: The sensitivity of ASPYRE-Lung was determined to be ≤ 3% variant allele fraction for single nucleotide variants and insertions or deletions, 100 copies for fusions, and 200 copies for MET exon 14 skipping. The specificity was 100% with no false positive results. The analytical accuracy test yielded no discordant calls between ASPYRE-Lung and expected results for clinical samples (via orthogonal testing) or contrived samples, and results were replicable across operators, reagent lots, runs, and real-time PCR instruments with a high degree of precision. Conclusions: The technology is simple and fast, requiring only four reagent transfer steps using standard laboratory equipment (PCR and qPCR instruments) with analysis via a cloud-based algorithm. The ASPYRE-Lung assay has the potential to be transformative in facilitating access to rapid, actionable molecular profiling of tissue for patients with non-small cell carcinoma.

Potential and challenges of clinical high-dimensional flow cytometry: A call to action.

Clinical biomarker strategies increasingly integrate translational research to gain new insights into disease mechanisms or to define better biomarkers in clinical trials. High-dimensional flow cytometry (HDFCM) holds the promise to enhance the exploratory potential beyond traditional, targeted biomarker strategies. However, the increased complexity of HDFCM poses several challenges, which need to be addressed in order to fully leverage its potential and to align with current regulatory requirements in clinical flow cytometry. These challenges include among others extended timelines for assay development and validation, the necessity for extensive knowledge in HDFCM, and sophisticated data analysis strategies. However, no guidelines exist on how to manage such challenges in adopting clinical HDFCM. Our CYTO 2024 workshop "Potential and challenges of clinical high-dimensional flow cytometry" aimed to find consensus across the pharmaceutical industry and broader scientific community on the overall benefits and most urgent challenges of HDFCM in clinical trials. Here, we summarize the insights we gained from our workshop. While this report does not provide a blueprint, it is a first step in defining and summarizing the most pressing challenges in implementing HDFCM in clinical trials. Furthermore, we compile current efforts with the goal to overcome some of these challenges. As such we bring the scientific community and health authorities together to build solutions, which will accelerate and simplify the full adoption of HDFCM in clinical trials.

An immunoassay for the quantification of phosphorylated α-synuclein at serine 129 in human cerebrospinal fluid.

The link between alpha Synuclein (α-Syn) phosphorylation and Parkinson's disease pathogenesis has not been fully elucidated, in part due to analytical methods with finite specificity and sensitivity, resulting in conflicting data on pathophysiological levels of the protein.One factor hindering the assessment of the role of pSer129 α-Syn is the lack of a fit for purpose assay. Antibodies were assessed for quantification of pSer129 α-Syn, resulting in a sensitive and specific assay suitable for use in Parkinson's disease and control CSF, with no significant difference found between the two populations. Total α-Syn was measured using a commercial kit, demonstrating a positive correlation between total and pSer129 α-Syn.This adds to available methods for pSer129 α-Syn in support of α-synucleinopathy research.

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GoEnrich: creating high quality genomic DNA resources from limited voucher specimen tissues or museum specimens of at-risk species for conservation-friendly use in the validation of environmental DNA assays.

OBJECTIVE: Environmental DNA (eDNA) methods are crucial for monitoring populations, particularly rare and cryptic species. For confident eDNA application, rigorous assay validation is required including specificity testing with genomic DNA (gDNA). However, this critical step is often difficult to achieve as obtaining fresh tissue samples from at-risk species can be difficult, highly limited, or impossible. Natural history museum collections could serve as a valuable and ethical voucher specimen resource for eDNA assay validation. The present study demonstrates the effectiveness of whole genome amplification (WGA) in providing enough gDNA to assemble high quality mitogenomes from which robust targeted eDNA assays can be designed. RESULTS: Using fresh and historical museum tissue samples from six species spanning fish, birds, and mammals, we successfully developed a WGA method with an average yield of 380 to 1,268 ng gDNA per 20 µL reaction. This gDNA was used for whole genome shotgun sequencing and subsequent assembly of high quality mitogenomes using mtGrasp. These mitogenomes were then used to develop six new robust, targeted quantitative real time polymerase chain reaction-based eDNA assays and 200 ng WGA-enriched yielded satisfactory Cq values and near 100% detection frequencies for all assays tested. This approach offers a cost-effective and non-invasive alternative, streamlining eDNA research processes and aiding in conservation efforts.

A European Bioanalysis Forum recommendation for requiring a context-of-use statement for successful development and validation of biomarker assays.

The European Bioanalysis Forum, alongside key industry stakeholders, has been driving the discussions around the implementation of context-of use for biomarker assays to ensure that these assays are validated appropriately depending on their purpose. Insights into understanding why the implementation of context-of-use in assay strategies has also shown that the key stakeholder, or requester for the biomarker data, is responsible for providing the context-of-use statement for all biomarker assay requests. Experts from across the industry haves repeatedly sought a cross-industry recommended format in which the context-of-use statement could be provided. In this manuscript, the European Bioanalysis Forum suggests a format for this.

Non-specific markers of inflammation in bare-nosed wombats (Vombatus ursinus) with sarcoptic mange.

Sarcoptic mange, caused by epidermal infection with Sarcoptes scabiei, negatively impacts the health, welfare, and local abundance of bare-nosed wombats (Vombatus ursinus) in Australia. Improved understanding of the host immune response to disease and its contribution to pathophysiology could be used to inform management actions for this species in and ex situ. To evaluate the immune response of bare-nosed wombats to sarcoptic mange, we validated three assays (haptoglobin, agarose gel electrophoresis, and micro-erythrocyte sedimentation rate) measuring non-specific markers of inflammation using serum samples from free-living wombats from Tasmania (n = 33). We then analysed correlations between the assay results for each non-specific marker of inflammation and wombat's sarcoptic mange scores, and performed histopathological examinations to investigate association of the acute phase response with systemic amyloidosis. We present evidence that haptoglobin and erythrocyte sedimentation rate increased, and albumin decreased, in association with sarcoptic mange scores. This research demonstrates links between the acute phase response and sarcoptic mange severity in bare-nosed wombats, highlighting the utility of non-specific markers of inflammation for aiding assessment of the systemic effects of mange. Showing the value of agarose gel electrophoresis, we also identified specific acute phase proteins warranting future evaluation and found evidence of an immunoglobulin response in mange-affected wombats, revealed by increasing γ-globulins in association with apparent disease severity. Meanwhile, owing to its relatively low resource requirements and rapidity, the erythrocyte sedimentation rate assay may be useful as a point-of-care test to support therapeutic decisions in the field. Our methods and findings are likely to be applicable to a range of other clinical and population health scenarios in captive and free-living wombats, and species impacted by sarcoptic mange globally.

Development and validation of an enzyme-linked immunosorbent assay for the quantification of a recombinant humanized anti-IL-4Rα monoclonal antibody CM310 in serum and its application to pharmacokinetic study in Sprague-Dawley Rats.

CM310 is a recombinant humanized monoclonal antibody targeting Interleukin (IL)-4 receptor alpha (IL-4Rα). IL-4Rα blockade prevents IL-4 and IL-13 from binding to their receptor, thereby inhibiting downstream signaling pathways that drive Type 2 helper T-cell (Th2) inflammation. CM310 holds potential for treating Th2-related inflammatory diseases, such as asthma, atopic dermatitis and chronic sinusitis with nasal polyposis. In this study, a direct enzyme-linked immunosorbent assay (ELISA) was developed to measure the concentrations of CM310 in rat serum. Seven calibration standards (ranging from 25 to 1600 ng/mL) and three quality controls (70, 500 and 1250 ng/mL) were defined. The limit of detection (LOD), lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) were 13, 25 and 1600 ng/mL, respectively. The method exhibited excellent precision and accuracy and successfully applied to in vitro serum stability and pharmacokinetic (PK) studies. In conclusion, we have developed and validated a highly sensitive and selective method for measuring CM310 in Sprague-Dawley rats. The development and validation ELISA method met the acceptable criteria, which suggested that these can be applied to quantify CM310, as well as in PK studies.

Systematic evaluation supports the use of ELISA for quantification of calprotectin in equine feces, a first step toward noninvasive quantification of intestinal inflammation in horses.

OBJECTIVE: To optimize and evaluate methods for the detection of the inflammatory biomarkers myeloperoxidase (MPO) and calprotectin (CP) in equine feces by ELISA. ANIMALS: Healthy horses (n = 28) and horses with intestinal inflammation (n = 10). METHODS: Feces were suspended in buffer to create fecal supernatant. Serum and fecal supernatant were analyzed using ELISA kits validated for the detection of MPO and CP in equine serum. Assay validation steps included intra- and interassay variability (coefficient of variation [CV]), dilution linearity, spike recovery, and sample type correlation. Variations in sample handling protocols (centrifugation speed, extraction buffer, and filtration) were evaluated. RESULTS: 17 paired fecal and serum samples were used for initial analysis (10 healthy horses, 7 colitis). Previously reported sample handling protocols resulted in detectable MPO and CP but poor CV, linearity, and spike recovery. There was a linear correlation between serum and fecal samples for CP but not MPO. There was a significant difference between the concentration and CV of alternative sample handling protocols for CP and MPO, with improved CV for CP (2.1% to 18.6%) but not MPO (14.4% to 53.4%). Processing fresh feces with a fecal extraction buffer and filtration of supernatant resulted in the best CV (0.5% to 3.8%) and recovery (45% to 64%) for CP. Detection of MPO was inconsistent regardless of method. CLINICAL RELEVANCE: There are few reliable diagnostic modalities for inflammation of the equine large colon. Findings support quantification of CP in equine feces using the described ELISA kit and protocol. With additional study to establish reference interval and clinical utility, the fecal inflammatory biomarker CP may allow for noninvasive quantification of intestinal inflammation in horses.

Identifying MAGE-A4-positive tumors for TCR T cell therapies in HLA-A∗02-eligible patients.

T cell receptor (TCR) T cell therapies target tumor antigens in a human leukocyte antigen (HLA)-restricted manner. Biomarker-defined therapies require validation of assays suitable for determination of patient eligibility. For clinical trials evaluating TCR T cell therapies targeting melanoma-associated antigen A4 (MAGE-A4), screening in studies NCT02636855 and NCT04044768 assesses patient eligibility based on: (1) high-resolution HLA typing and (2) tumor MAGE-A4 testing via an immunohistochemical assay in HLA-eligible patients. The HLA/MAGE-A4 assays validation, biomarker data, and their relationship to covariates (demographics, cancer type, histopathology, tissue location) are reported here. HLA-A∗02 eligibility was 44.8% (2,959/6,606) in patients from 43 sites across North America and Europe. While HLA-A∗02:01 was the most frequent HLA-A∗02 allele, others (A∗02:02, A∗02:03, A∗02:06) considerably increased HLA eligibility in Hispanic, Black, and Asian populations. Overall, MAGE-A4 prevalence based on clinical trial enrollment was 26% (447/1,750) across 10 solid tumor types, and was highest in synovial sarcoma (70%) and lowest in gastric cancer (9%). The covariates were generally not associated with MAGE-A4 expression, except for patient age in ovarian cancer and histology in non-small cell lung cancer. This report shows the eligibility rate from biomarker screening for TCR T cell therapies and provides epidemiological data for future clinical development of MAGE-A4-targeted therapies.

Pig Acute Phase Proteins as Non-Antibody Systemic Biomarkers of Intracellular Infections.

As an alternative to traditional serological markers, that is, antibodies, for serum-based specific diagnosis of infections, circulating non-antibody markers may be used to monitor active disease. Acute phase proteins (APPs) are a prominent class of such markers widely used for diagnosing ongoing inflammation and infection. In this chapter, basic theoretical and practical considerations on developing APP assays and using APPs as markers of ongoing infection are presented with a specific focus on intracellular infections in pigs. Examples on APP-based monitoring of infection in pigs with viruses such as porcine respiratory and reproductive syndrome virus (PRRSV), porcine endemic diarrhea virus (PEDV), and influenza A virus (IAV), as well as intracellular bacteria (Lawsonia intracellularis) and the protozoan intracellular parasites Toxoplasma gondii and Cryptosporidium parvum are presented, with an emphasis on major pig APPs C-reactive protein (CRP), haptoglobin, serum amyloid A (SAA), and pig major acute phase protein (pig-MAP). The performance of these APPs as biomarkers in a range of experimental infection studies in pigs is described as examples on their use for estimating the severity of infection, vaccine efficacy, herd health characterization, and differential diagnosis.

How to Accelerate Early Stage of Malaria Vaccine Development by Optimizing Functional Assays.

While two Plasmodium falciparum circumsporozoite protein-based pre-erythrocytic vaccines (PEV), RTS,S and R21, have been approved by the WHO, no blood-stage vaccine (BSV) or transmission-blocking vaccine (TBV) has reached a phase 3 trial. One of the major obstacles that slows down malaria vaccine development is the shortage (or lack) of in vitro assays or animal models by which investigators can reasonably select the best vaccine formulation (e.g., antigen, adjuvant, or platform) and/or immunization strategy (e.g., interval of inoculation or route of immunization) before a human phase 2 trial. In the case of PEV, RTS,S and R21 have set a benchmark, and a new vaccine can be compared with (one of) the approved PEV directly in preclinical or early clinical studies. However, such an approach cannot be utilized for BSV or TBV development at this moment. The focus of this review is in vitro assays or in vivo models that can be used for P. falciparum BSV or TBV development, and I discuss important considerations during assay selection, standardization, qualification, validation, and interpretation of the assay results. Establishment of a robust assay/model with proper interpretation of the results is the one of key elements to accelerate future vaccine development.

Specimen type validation and establishment of normal cytokine reference intervals in cerebrospinal fluid.

Cerebrospinal fluid (CSF) is a critical body fluid to examine in attempts to discover potential biomarkers for neuroinflammatory and other disorders of the central nervous system (CNS). Serum and/or plasma cytokine levels have been associated with a variety of inflammatory conditions, and some have been shown to be actionable therapeutic targets. Less is known, however, about cytokine levels in CSF. Serum and plasma cytokine testing is widely available in clinical and research laboratories, but cytokine testing in CSF is extremely limited and if performed, accompanied by a disclaimer that it is an unvalidated specimen type. In this study, we validate CSF as a suitable specimen type and determine normal reference intervals for multiple cytokines as well as a soluble cytokine receptor. CSF was validated as a specimen type for testing using a laboratory developed multiplexed cytokine assay previously validated to measure 13 cytokines/markers in serum and plasma. Performance parameters including specimen dilution, specimen interference, linearity and precision were examined. Reference intervals were established using 197 normal and control CSF specimens by non-parametric quantile-based methods. CSF cytokine analysis demonstrated within and between run precision of <10% and < 20% CV, respectively and linearity of ±15% for all analytes throughout the analytical measurement range of the assay. Reference intervals for the 13 cytokines/markers were established from 197 normal and control CSF specimens (78 Male; mean 44.8 y ± 21.7 SD, 119 Female; mean 42.8 y ± 20.3 SD). Cytokine concentrations in CSF from normal donors and controls were less than the lower limit of quantitation of our assay for 6 of the 13 measured cytokines/markers. The chemokine IL8 demonstrated the highest concentration of all analytes measured. CSF demonstrated acceptable performance as a specimen type in our multiplexed cytokine assay. By validating CSF as a specimen type and establishing normal reference intervals for cytokine concentrations in CSF, their potential as biomarkers for infectious, autoimmune and other inflammatory CNS disorders can be more appropriately investigated.

Development and validation of ELISA method for quantification of Q-1802 in serum and its application to pharmacokinetic study in ICR Mouse.

Q-1802 is a humanized bispecific antibody targeting programmed death-ligand 1 (PD-L1) and Claudin 18.2 (CLDN18.2). It can bind to CLDN18.2 and mediate antibody-dependent cell-mediated cytotoxicity against tumor cells. The Fc segment of the antibody recognizing PD-L1 blocks PD-1 signaling and activates innate immunity and adaptive immunity. In this study, we report the development, validation, and application of sensitive and high-throughput enzyme-linked immunosorbent assays (ELISA) to measure the concentrations of Q-1802 in ICR mouse serum. The assay is sensitive, with a lower limit of quantification of 50 ng/mL, has a broad dynamic range of 50-3200 ng/mL, and exhibits excellent precision and accuracy. These assays were successfully applied to in vitro serum stability and pharmacokinetic (PK) studies. In conclusion, we have developed and validated a highly sensitive and selective method for measuring Q-1802 in ICR mouse serum. The development and validation steps of assays met the required criteria for validation, which suggested that these can be applied to quantify Q-1802, as well as in PK studies.

A Pseudovirus-Based Neutralization Assay for SARS-CoV-2 Variants: A Rapid, Cost-Effective, BSL-2-Based High-Throughput Assay Useful for Vaccine Immunogenicity Evaluation.

Neutralizing antibody responses from COVID-19 vaccines are pivotal in conferring protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Effective COVID-19 vaccines and assays measuring neutralizing antibodies against emerging variants (i.e., XBB.1.5, XBB.1.16, and XBB.2.3) are needed. The use of biosafety level (BSL)-3 laboratories for live virus assays results in higher costs and a longer turnaround time; therefore, a BSL-2-based pseudovirus neutralization assay (PNT) was developed. The pseudoviruses were produced by cotransfecting cells with plasmids encoding a lentiviral backbone-expressing luciferase reporter; non-surface proteins for lentiviral production; and ancestral or Omicron (BA.1 and BA.5) SARS-CoV-2 spike (S) proteins. The PNT was developed and optimized in dose and kinetics experiments. The representative serum samples (COVID-19-convalescent or NVX-CoV2373-vaccinated participants enrolled in the 2019nCoV-101 trial) demonstrated a wide dynamic range. The neutralization data showed robust correlation with validated anti-recombinant spike IgG levels and angiotensin-converting enzyme 2 inhibition titers (ancestral). This assay is suitable for measurement of the neutralization ability in clinical samples from individuals infected with SARS-CoV-2 or immunized with a COVID-19 vaccine. The results suggest that this PNT provides a lower cost, high-throughput, rapid turnaround alternative to BSL-3-based microneutralization assays and enables the discovery and development of effective vaccines against emerging variants.

Determination of minimum inhibitory concentrations using machine-learning-assisted agar dilution.

Effective policy to address the global threat of antimicrobial resistance requires robust antimicrobial susceptibility data. Traditional methods for measuring minimum inhibitory concentration (MIC) are resource intensive, subject to human error, and require considerable infrastructure. AIgarMIC streamlines and standardizes MIC measurement and is especially valuable for large-scale surveillance activities. MICs were measured using agar dilution for n = 10 antibiotics against clinical Enterobacterales isolates (n = 1,086) obtained from a large tertiary hospital microbiology laboratory. Escherichia coli (n = 827, 76%) was the most common organism. Photographs of agar plates were divided into smaller images covering one inoculation site. A labeled data set of colony images was created and used to train a convolutional neural network to classify images based on whether a bacterial colony was present (first-step model). If growth was present, a second-step model determined whether colony morphology suggested antimicrobial growth inhibition. The ability of the AI to determine MIC was then compared with standard visual determination. The first-step model classified bacterial growth as present/absent with 94.3% accuracy. The second-step model classified colonies as "inhibited" or "good growth" with 88.6% accuracy. For the determination of MIC, the rate of essential agreement was 98.9% (644/651), with a bias of -7.8%, compared with manual annotation. AIgarMIC uses artificial intelligence to automate endpoint assessments for agar dilution and potentially increases throughput without bespoke equipment. AIgarMIC reduces laboratory barriers to generating high-quality MIC data that can be used for large-scale surveillance programs. IMPORTANCE: This research uses modern artificial intelligence and machine-learning approaches to standardize and automate the interpretation of agar dilution minimum inhibitory concentration testing. Artificial intelligence is currently of significant topical interest to researchers and clinicians. In our manuscript, we demonstrate a use-case in the microbiology laboratory and present validation data for the model's performance against manual interpretation.

Correlation between variant call accuracy and quality parameters in comprehensive cancer genomic profiling tests.

Background: Comprehensive genomic profiling (CGP) tests have been widely utilized in clinical practice. In this test, the variant list automatically output from the data analysis pipeline often contains false-positive variants, although the correlation between the quality parameters and prevalence of false-positive variants remains unclear. Methods: We analyzed 125 CGP tests performed in our laboratory. False-positive variants were manually detected via visual inspection. The quality parameters of both wet and dry processes were also analyzed. Results: Among the 125 tests, 52 (41.6%) required more than one correction of the called variants, and 21 (16.8%) required multiple corrections. A significant correlation was detected between somatic false-positive variants and quality parameters in the wet (ΔΔCq, pre-capture library peak size, pre-capture library DNA amount, capture library peak size, and capture library concentration) and dry processes (total reads, mapping rates, duplication rates, mean depth, and depth coverage). Capture library concentration and mean depth were strong independent predictors of somatic false-positive variants. Conclusions: We demonstrated a correlation between somatic false-positive variants and quality parameters in the CGP test. This study facilitates gaining a better understanding of CGP test quality management.

Introduction to qualification and validation of an immunoassay.

Immunoassays play an important role in drug development of products targeting the immune system. Consistent quality of the results from an immunoassay is essential to make unbiased and accurate claims about the drug product during preclinical and clinical development stages. Assay qualification and validation shed light on the performance of the assay. It is the first evaluation and the verification, respectively, of the assay's performance. This tutorial explains and illustrates the calculation methodology for important assay qualification parameters including precision, relative accuracy, linearity, the lower limit of quantification (LLOQ), the upper limit of quantification (ULOQ), the assay range and dilutability. This tutorial focuses on assays used for (pre-) clinical purposes, characterized by a lognormal distribution of the measurements on its original untransformed scale and by the lack of well characterized reference material. Statistical calculations are illustrated with qualification data from an enzyme-linked immunosorbent assay (ELISA) vaccine immunoassay.

Development and performance of a next generation sequencing (NGS) assay for monitoring of dd-cfDNA post solid organ transplantation.

The aim of this study was to evaluate the analytical performance of a novel NGS assay, intended for monitoring of donor-derived cell-free DNA (dd-cfDNA), and describe its validity in clinical plasma samples from kidney transplanted patients. Artificial and clinical samples with increasing amounts of patient DNA were evaluated using NGS analysis of indel markers. Monitoring of dd-cfDNA with the NGS assay presented herein demonstrated a sensitivity of ≥0.1% dd-cfDNA and excellent accuracy (R2 0.99) throughout an extensive range of dd-cfDNA (0.1-30%). The precision of the test was determined for two levels (0.1% (LoD) and 1%) of dd-cfDNA. The between run precision (CV%) for the respective level was 16% and 9% and the corresponding result for the within run precision was 19% and 7%. To evaluate performance of the assay in clinical samples, 507 retrospective monitoring samples from 21 patients transplanted either with kidneys from living or deceased donors were analyzed. Monitoring samples were sampled at multiple time points from 24 h up to 90 days post-transplantation. We show that in one patient, increase of dd-cfDNA preceded increase of creatinine caused by acute cellular rejection by several days. In conclusion, the NGS assay displayed a combination of high sensitivity with good accuracy and precision in both artificial and clinical dd-cfDNA samples.

Impact assessment of metabolite instability in the development and validation of LC-MS/MS bioanalytical assays for measurement of rosuvastatin in human plasma and urine samples.

During bioanalytical assay development and validation, maintaining the stability of the parent drug and metabolites of interest is critical. While stability of the parent drug has been thoroughly investigated, the stability of unanalyzed metabolites is often overlooked. When an unstable metabolite is known or suspected to interfere with measurement of the parent drug or other metabolites of interest through back-conversion or other routes, additional tests with these unstable metabolites should be conducted. Here, the development and validation of two assays for quantification of rosuvastatin, one in human plasma and one in human urine, was reported. To this end, additional sets of quality control samples were added during assay validation to ensure the reliability of the assays. Acid treatment of samples is shown to be necessary for rosuvastatin quantification. In this regard, stability issues caused by the metabolite, rosuvastatin lactone, may have been overlooked if assay development and validation had only considered the parent drug, rosuvastatin. These assays represent a case study for how to develop and validate assays with unstable metabolites. Taken together, unstable metabolites should be included in all applicable stability tests.

The calculation of historical control limits in toxicology: Do's, don'ts and open issues from a statistical perspective.

For reporting toxicology studies, the presentation of historical control data and the validation of the concurrent control group with respect to historical control limits have become requirements. However, many regulatory guidelines fail to define how such limits should be calculated and what kind of target value(s) they should cover. Hence, this manuscript is aimed to give a brief review on the methods for the calculation of historical control limits that are in use as well as on their theoretical background. Furthermore, this manuscript is aimed to identify open issues for the use of historical control limits that need to be discussed by the community. It seems that, even after 40 years of discussion, more issues remain open than solved, both, with regard to the available methodology as well as its implementation in user-friendly software. Since several of these topics equally apply to several research fields, this manuscript is addressed to all relevant stakeholders who deal with historical control data obtained from toxicological studies, regardless of their background or field of research.