Assay Variability Research Articles

A Comprehensive Evaluation of Analytical Method Parameters Critical to the Reliable Assessment of Therapeutic mRNA Integrity by Capillary Gel Electrophoresis.

In recent years, messenger ribonucleic acid (mRNA)-lipid nanoparticle (LNP) biotherapeutics have demonstrated significant promise in disease treatment and prevention given their rapidly modifiable production processes and considerable capacity to adapt to complex or low-yielding proteins of interest. As a result, many products are currently being developed in this space. Critically, well-characterized and appropriately designed assays are required to monitor purity and integrity in order to maintain the efficacy and consistency of these novel products. Currently, capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) and ion-pair reversed-phase liquid chromatography (IP-RPLC) are techniques of choice for mRNA integrity analysis. However, most methods proposed for biotherapeutic analysis have been developed using naked mRNA without LNP components or proprietary buffer formulations, which can obscure undiscovered impurities or complex interactions between mRNA and the sample matrix. In this study, we addressed these methodological challenges by using a biotherapeutically relevant commercial mRNA-LNP sample (approx. 4200 b) to refine and optimize a customizable CGE-LIF method currently under consideration for mRNA-LNP biotherapeutic analysis. We systematically characterized how critical method parameters-such as denaturant type, concentration, and usage-and LNP disruption protocols can interfere with accurate mRNA integrity analysis in CGE-LIF and IP-RPLC. We found that optimal conditions for CGE-LIF assay sensitivity, variability, and resolution included sample precipitation by isopropanol, high urea concentrations, no formamide as a sample diluent, and high concentrations of dye. Finally, the advantages and disadvantages of both CGE-LIF and IP-RPLC are highlighted, and a discussion of key considerations when using or designing methods for mRNA integrity assessment is presented.

Preparation of robust synthetic control samples and their use in a metatranscriptomic clinical test

Metatranscriptomics (MT) has the potential to revolutionize the field of molecular diagnostics. Due to the complexity of MT diagnostic models, positive and negative control materials for specific disease indications can be difficult to obtain. Controls must often be sourced directly from patients. This introduces logistical burdens, assay variability, and limits high throughput clinical laboratory operations. To overcome this limitation, we developed a method for generating Synthetic Control (SC) samples, which duplicate the nucleic acid signature of complex clinical specimens and produce the desired test outcome. SCs can be easily and cost-effectively produced in large quantities (> 100,000 SCs per amplification cycle), enabling high throughput diagnostic testing. Here, we report the generation of Synthetic Positive Control (SPC) samples. SPCs were validated and implemented in a clinical laboratory. The SPCs produced robust positive signals (average OC risk score of 0.996) and high levels of reproducibility (%CV of 0.29%) in a high throughput automated CLIA laboratory. SCs are a novel and useful method for the generation of high quality controls for MT-based diagnostic tests, and their adoption could herald the widespread use of MT tests in molecular diagnostics.

A deep dive into four thyroglobulin immunoassays from analytical perspective

Backgrounds Serum thyroglobulin immunometric assays (sTg) are crucial for monitoring differentiated thyroid cancer (DTC) treatment. However, challenges such as anti-thyroglobulin autoantibodies (TgAb) and assay variability hinder evaluations. This study assessed four sTg methods—three second-generation (Architect, Access, Elecsys) and one first-generation (Immulite)—following Clinical and Laboratory Standards Institute (CLSI) and American Thyroid Association (ATA) guidelines. Methods The study compared sTg(Architect), sTg(Access), sTg(Elecsys), and sTg(Immulite). Precision was evaluated per CLSI EP05-A3, while the lower limits of detection (LLD) were assessed using EP17-A2. Passing–Bablok and Bland–Altman analyses were conducted as per EP09c, and semi-quantitative comparisons used Kappa statistics. Results The second-generation sTgs (Architect, Access, Elecsys) exhibited satisfactory precision (<7% coefficient of variation, CV%), unlike sTg(Immulite), which showed significant deviations and inadequate sensitivity for DTC recurrence (Limit of quantitation, LoQ = 4.59 μg/L). Second-generation sTgs had strong correlations (r > 0.884) across all concentration ranges (≤1, 1-10, >10 μg/L), with biases (slope: 1.131-2.027). sTg(Immulite) correlated well with second-generation methods for concentrations >10 μg/L (r > 0.945) but less so for <10 μg/L (r < 0.642). TgAb significantly impacted sTg(Immulite). Kappa statistics revealed strong agreement among second-generation methods (κ > 0.800) but lower concordance with sTg(Immulite), especially in TgAb(+) samples (κ: 0.562-0.653). Agreement ratios were high for second-generation methods (0.667-1.000) but variable for sTg(Immulite), particularly at lower concentrations and in TgAb(+) cases (0.097-0.727). Conclusions sTg(Immulite) did not meet LLD and precision criteria for DTC monitoring, facing issues with TgAb interference. Second-generation sTgs demonstrated consistent performance across all concentrations.

Development and Validation of a Novel LC-MS/MS Based Proteomics Method for Quantitation of Retinol Binding Protein 4 (RBP4) and Transthyretin (TTR).

Retinol binding protein 4 (RBP4) is the plasma carrier of retinol that complexes with transthyretin (TTR). RBP4 is a potential biomarker of cardiometabolic disease. However, RBP4 quantitation has relied on immunoassays and western blots without concurrent measurement of retinol and TTR. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous absolute quantitation of serum and plasma RBP4 and TTR is needed to advance the understanding of RBP4 and TTR as biomarkers. Surrogate peptides with reproducible, linear LC-MS/MS response were selected for RBP4 and TTR quantitation. Purified proteins were used as quantitation standards and heavy labelled peptides as internal standards. Matrix effects were evaluated for quantitation. The method was validated using pooled human serum and applied to measure inter- and intra-individual variability in RBP4 and TTR concentrations in healthy individuals and in patients with diabetic kidney disease. The quantitation was linear for the clinically relevant concentration ranges of RBP4 (0.5 - 6 µM) and TTR (5.8 - 69 µM). The assay inter-day variability was <12% and precision within 5%. The inter-individual variability for RBP4 and TTR concentrations was 18 - 26%, while intra-individual variability was similar to assay variability. RBP4 and TTR quantitation correlated with commercially available ELISA assays. The developed LC-MS/MS method allows simultaneous absolute quantitation of RBP4 and TTR in human serum and plasma with reduced samples volumes. The method can be applied to clinical biomarker studies, for analysis of nutritional vitamin A status, and measurements of stoichiometry of RBP4, TTR and retinol in circulation.

Autoantibodies in Autoimmune Disorders: Mechanisms of Pathogenesis, Diagnostic Utility, and Therapeutic Implications

Autoantibodies, once considered mere biomarkers of autoimmune disorders, are now recognized as central players in disease pathogenesis, offering critical insights into the breakdown of immune tolerance. This review synthesizes current knowledge on the dual roles of autoantibodies—as diagnostic tools and direct mediators of tissue damage—across a spectrum of autoimmune diseases. We begin by exploring the mechanisms underlying autoantibody production, including genetic predispositions (e.g., HLA haplotypes), environmental triggers (infections, microbiome dysbiosis), and dysregulated B-cell/T-cell interactions. Pathogenic mechanisms are dissected, from complement activation and immune complex deposition to receptor blockade/activation (e.g., anti-TSH receptor in Graves’ disease) and molecular mimicry. Highlighting disease-specific autoantibodies, we detail their clinical implications in systemic lupus erythematosus (anti-dsDNA, anti-Smith), rheumatoid arthritis (anti-CCP), and neurological disorders (anti-NMDA receptor, anti-AQP4). The review underscores the diagnostic and prognostic utility of autoantibodies, while addressing challenges such as seronegative autoimmunity and assay variability. Current therapeutic strategies, including B-cell depletion (rituximab), complement inhibition (eculizumab), and cytokine modulation, are evaluated alongside emerging approaches like antigen-specific tolerance induction and CAR-T cell therapy. Unresolved questions—such as why some autoantibodies remain asymptomatic and their role in disease initiation—are discussed, emphasizing the need for interdisciplinary research. Advances in single-cell technologies, proteomics, and AI-driven diagnostics promise to revolutionize personalized medicine, enabling early intervention and tailored therapies. By bridging mechanistic insights with clinical applications, this review underscores the transformative potential of autoantibody research in redefining autoimmune disease management and moving toward curative strategies.

Bamboo-PCM: Comparative Analysis of Phase Change Material-Impregnated Dendrocalamus giganteus Culm Behavior Exposed to Thermal Variation in Wind Tunnel Assay.

The construction industry's pursuit of eco-friendly materials has sparked interest in bamboo, a renewable resource with exceptional physical and mechanical properties. This study analyzed the integration of Dendrocalamus giganteus bamboo with phase change materials (PCMs) to enhance thermal energy storage in building applications, aiming to improve temperature regulation and reduce energy consumption for climate control. The study compared the performance of bamboo impregnated with an industrial PCM or coconut oil, used in conjunction with a polyurethane resin (PU) coating treatment, assessing their thermal regulation performance against traditional building materials such as ceramic tiles, fiber cement, and metal sheets. From an anatomical perspective, the pores within bamboo culms offered ample space for PCM storage, resulting in a substantial heat storage capacity. Thermal behavior tests conducted in a wind tunnel revealed that the impregnated bamboo samples effectively mitigate temperature fluctuations by aligning them with the PCM's phase change temperature. Additionally, it was observed that air flow velocity had an impact on this phenomenon. The study concluded that bamboo culms impregnated with PCM hold promise for temperature regulation in construction applications, with variations in airflow exerting an impact on the outcomes obtained.

CHO-S expression of a novel human recombinant IgG1 of anti-ALD antibody isolated by phage mutation display.

CHO-S expression of a novel human recombinant IgG1 of anti-ALD antibody isolated by phage mutation display.

Comparison of Four Chimeric Antigens and Commercial Serological Assays for the Diagnosis of Trypanosoma cruzi Infection.

Chagas disease (CD), a neglected tropical disease caused by Trypanosoma cruzi, is a significant public health issue particularly in Latin America, affecting millions worldwide. Diagnosis is a challenge owing to the genetic diversity of T. cruzi and the complexities involved in selecting antigens for the detection of anti-T. cruzi antibodies. This study evaluated four chimeric recombinant antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) designed to enhance diagnostic accuracy by addressing assay variability. We compared the diagnostic performance of these chimeric antigens using indirect ELISA as a diagnostic platform, with three commercial serological assays in Brazil, analyzing 100 serum samples from individuals with confirmed CD and 86 from non-infected controls. The results revealed that all assays and antigens demonstrated an area under the receiver operating characteristic curve of 100%, signifying their exceptional ability to distinguish between CD-positive and CD-negative samples. Notably, the chimeric antigens achieved 100% sensitivity, specificity, accuracy, and kappa index, equaling or surpassing the commercial assays. This research highlights the efficacy of IBMP chimeric antigens as reliable diagnostic tools for CD, suggesting their potential integration into commercial diagnostic platforms to enhance the accuracy and reliability of CD detection.

Assessment of Inter-Laboratory Variability for Flow Cytometric Crossmatch Testing: Lessons Learned from Proficiency Surveys

Assessment of Inter-Laboratory Variability for Flow Cytometric Crossmatch Testing: Lessons Learned from Proficiency Surveys

Fast and accurate prediction of drug induced proarrhythmic risk with sex specific cardiac emulators

In silico trials for drug safety assessment require many high-fidelity 3D cardiac simulations to predict drug-induced QT interval prolongation, which is often computationally prohibitive. To streamline this process, we developed sex-specific emulators for a fast prediction of QT interval, trained on a dataset of 900 simulations. Our results show significant differences between 3D and 0D single-cell models as risk levels increase, underscoring the ability of 3D modeling to capture more complex cardiac responses. The emulators demonstrated an average error of 4% compared to simulations, allowing for efficient global sensitivity analysis and fast replication of in silico clinical trials. This approach enables rapid, multi-dose drug testing on standard hardware, addressing critical industry challenges around trial design, assay variability, and cost-effective safety evaluations. By integrating these emulators into drug development, we can improve preclinical reliability and advance the practical application of digital twins in biomedicine.

Baseline Aβ and tau markers contribute to localized patterns of brain tissue atrophy across 1223 individuals from four harmonized longitudinal datasets

AbstractBackgroundAmyloid‐β (Aβ) plaques and tau pathogenesis in the brain precede cognitive decline in the progression of Alzheimer’s dementia, yet the extent to which these measures can predict localized brain tissue atrophy has not been studied in a large, diverse population. Multisite studies offer robust statistical power with larger sample sizes but are confounded by variations in biomarker quantification across studies, including variations in MRI scanners, PET tracers, and CSF assays. Longitudinal data from N=1223 individuals from four independent AD studies were harmonized to assess localized brain tissue atrophy over 2 to 5 years. We tested for associations between volumetric changes and baseline measures of Aβ, phosphorylated tau (p‐tau), and cognitive function. Associations between atrophy and ApoE4 and diagnostic conversion were also assessed.MethodBaseline (BL) and follow‐up (FU; mean=2.3+/‐0.5 years) 3T T1‐weighted brain MRI (T1w) from ADNI3 (N=373), Prevent‐AD (N=213), HABS‐MGH (N=213), and OASIS3 (N=424) were used along with corresponding PET measures obtained from FBB, FBP or PiB tracers, and CSF measures (Figure 1A). Subject‐wise maps of volumetric brain changes were created and warped to a common template for statistical analyses (Figure 1B). Voxel‐wise mixed effects linear regressions were run to test for associations between change in brain volume and Aβ‐42 positivity (CSF / PET), CSF p‐tau‐181 positivity, ApoE4 genotype, Clinical Dementia Rating (CDR), and diagnostic conversion (i.e., cognitively normal vs decline). Fixed effects included BL age, sex, scan time interval, and PET imaging tracer type or CSF processing batch; scan site was a random effect. FDR was used for voxel‐wise multiple comparisons correction.ResultAβ and p‐tau positivity at baseline, and the ApoE4 genotype, were associated with accelerated atrophy in the temporal lobe, medial‐parietal lobe and amygdala (Figure 2). CDR and conversion were associated with ventricular expansion and atrophy in the insula, temporal lobe, hippocampus and amygdala (Figure 3).ConclusionOur multi‐study brain mapping and harmonization approaches showed how baseline PET and CSF based amyloid and tau metrics are predictive of common and localized brain atrophy across multiple studies with diverse data collection protocols and study designs.

Variability of 7K and 11K SomaScan Plasma Proteomics Assays.

SomaScan is an aptamer-based proteomics assay designed for the simultaneous measurement of thousands of human proteins with a broad range of endogenous concentrations. The 7K SomaScan assay has recently been expanded into the new 11K version. Following up on our previous assessment of the 7K assay, here, we expand our work on technical replicates from donors enrolled in the Baltimore Longitudinal Study of Aging. By generating SomaScan data from a second batch of technical replicates in the 7K version as well as additional intra- and interplate replicate measurements in the new 11K version using the same donor samples, this work provides useful precision benchmarks for the SomaScan user community. Beyond updating our previous technical assessment of the 7K assay with increased statistics, here, we estimate interbatch variability, assess inter- and intraplate variability in the new 11K assay, compare the observed variability between the 7K and 11K assays (leveraging the use of overlapping pairs of technical replicates), and explore the potential effects of sample storage time (ranging from 2 to 30 years) in the assays' precision.

Assay variables and early clinical evaluation of low-angle light scattering for platelet function analysis.

The recently developed platelet aggregation technique based on low-angle light scattering (LaSca) in diluted platelet-rich plasma (PRP) requires only a small sample volume and provides information about platelet aggregation and shape change. This study aimed to investigate the influence of preanalytical and analytical variables and to validate the method in a real-life pediatric hematology hospital setting. Platelet aggregation was induced by ADP in diluted PRP in the presence of 2mM calcium at 23°C. The study included healthy adults (n = 30), healthy children (n = 20), and pediatric patients with suspected or diagnosed platelet function abnormalities (n = 25). The assay parameters were stable for at least 3h after isolation of PRP and were sensitive to plasma dilution in the range of 2-8%. The initial aggregation velocity was significantly reduced in pediatric patients compared with healthy children (p < 0.05). ADP-induced light transmission amplitude was moderately correlated with LaSca amplitude of aggregation in healthy children (p = 0.52, p < 0.05) but not in pediatric patients. We standardized the protocol for platelet aggregation assessment by LaSca and characterized the influence of preanalytical and analytical variables on it.

12426 Performance Of Dehydroepiandrosterone-Sulphate In Diagnosing Mild Autonomous Cortisol Secretion: A Systematic Review And Meta-Analysis

Abstract Disclosure: J. Saini: None. B. Salama: None. S.R. Chacko: None. K. Yu: None. I. Bancos: None. Objective: Measurement of baseline dehydroepiandrosterone sulfate (DHEAS) is suggested by the guidelines in support of diagnosing mild autonomous cortisol secretion (MACS). However, evidence on the diagnostic accuracy of DHEAS in MACS is difficult to interpret due to differences in the diagnostic cutoffs, small sample size, and heterogeneous reference standards. We aimed to conduct a systematic review and meta-analysis of published literature to assess the performance of DHEAS in diagnosing MACS, as defined in the 2023 guidelines. Methods: A comprehensive search was conducted using several databases from each database’s inception to January 8th, 2024. Databases include Ovid MEDLINE (R) and Epub Ahead of Print, In-Process &amp; Other Non-Indexed Citations, and Daily, Ovid EMBASE, Ovid Cochrane Central Register of Controlled Trials, Ovid Cochrane Databases of Systematic Reviews, and Scopus. Only original studies of at least 20 participants were included. MACS was defined as post-dexamethasone cortisol &amp;gt;1.8 mcg/dL. QUADAS-2 was used to assess the risk of bias. Bivariate and random effects meta-analysis was used to generate pooled diagnostic accuracy estimates. Results: The initial search yielded 285 articles, and only 7 studies met the inclusion criteria. In 7 studies (1 from North America, 3 from Asia, and 3 from Europe), a total of 574 patients with MACS and 830 referent subjects (patients with adrenal tumors without MACS) were included. The mean age was specified only in 5 studies and was similar in patients with MACS (55.5 years) and referent group (53.3 years). Patients with MACS had a higher number of women (323/472, 68.4%) as compared to the referent group (352/658, 53.4%). DHEAS cutoffs studied included: lower cutoff of age-and sex-specific ranges (3 studies), 40 mcg/dL (3 studies), 60 mcg/dL (1 study), and 67.5 mcg/dL (1 study). With increasing DHEAS cutoff, sensitivity for MACS diagnosis increased, and specificity decreased. Most studies (4 studies) used the chemiluminescent immunoassay to measure DHEAS. A meta-analysis of studies using DHEAS cut-off between 40-70 mcg/dL was performed, with a pooled sensitivity of 68% (95% CI: 51-81) and a pooled specificity of 85% (78-89). The positive likelihood ratio was 4 (95% CI: 3-6; I2=36.94, P=0.175) and the negative likelihood ratio was 0.4 (0.3-0.6; I2=49.97, P=0.092). Most studies were at a high risk of bias for selection of patients, index, or reference standard. Conclusion: Based on limited heterogeneous evidence, measurement of DHEAS provides additional value in diagnosing MACS. Further larger diagnostic accuracy studies should explore various DHEAS cutoffs and consider assay variability. Presentation: 6/3/2024

7560 Using a Single Pre-test Cortisol Level To Reduce The Need For Dynamic Testing For Adrenal Insufficiency In A Southeast Asian Population

Abstract Disclosure: V. Ramadoss: None. L. Tan: None. A.E. Teo: None. K. Lazarus: None. K. Meeran: None. D. Deepak: None. L. Ong: None. C. Khoo: None. P. Eng: None. Introduction: The Short Synacthen Test (SST) is widely used in clinical practice to assess Adrenal Insufficiency (AI). Despite its relative ease of administration, Synacthen use is limited by its availability and its administration demands supervision by experienced staff, resulting in increased testing cost. A single measurement of serum cortisol has been shown to predict AI, but the suggested threshold varies according to the patient cohort studied, assay variation and the prevalence of the condition relative to the size of population tested. This study aims to (a) identify a threshold cortisol level to screen for AI in a South-East Asian population and (b) to determine the utility of a stimulated 60-minute cortisol level in the diagnosis of AI. Methods: This is a retrospective analysis of SSTs (250 micrograms) performed in our institution between 28th February 2022 to 28th February 2023. Serum cortisol in our institution was measured by Abbott Alinity analysers. We defined a normal response as a stimulated cortisol value of ≥ 420nmol/L at either 30-minutes, 60-minutes, or at both time points. Logistic regression analysis was performed to predict normal responses based on baseline cortisol level. Results: Median cortisol level at baseline, 30-minutes, and 60-minutes after Synacthen stimulation were 254 (IQR 191-320) nmol/L, 499 (IQR 415 -585) nmol/L, 574 (IQR 476 -662) nmol/L respectively. A total of 785 SSTs were performed, out of which 83.1% were normal and 16.9% were abnormal. Of the 785 SSTs, 55 (7.0%) would have failed if only the 30-minute cortisol was assessed without the 60-minute value, whereas 10 (1.4%) would have failed if only the 60-minute cortisol was assessed without the 30-minutes value. An early morning basal cortisol (0800am to 1200pm) cut-off of &amp;lt;300nmol/L identified subnormal cortisol with 95.0% sensitivity and an afternoon cortisol (1200pm to 1700) of &amp;lt; 312nmol/L achieved 95.2% sensitivity. A basal cortisol level of &amp;lt; 100nmol/L will confirm AI with 97.3% specificity. Using a basal cortisol level of ≥ 300nmol/L, 94.8% of individuals go on to pass the SST. Use of this basal cortisol value would avoid 252 (32.1%) SSTs. Conclusion: A single measurement of basal cortisol of ≥ 300nmol/L has the potential to exclude AI with 95% sensitivity on Abbott Alinity platform in our cohort of patients. Using a threshold cortisol level of 300nmol/L, at least 32% of the SSTs could be avoided. This would have improved patients experience with cost savings implications. Furthermore, a stimulated 60-minute cortisol level identifies 55 (7%) of individuals who would otherwise be misclassified as AI, thereby avoiding unnecessary long-term glucocorticoid replacement. Presentation: 6/1/2024

8008 Insulin-like Growth Factor-1 (IGF-1) Level Fluctuations and Patient Reported Outcomes (PROMS) During Treatment with Long-acting Somatostatin Receptor Ligands (SRLs) for Acromegaly: a Prospective Study

Abstract Disclosure: I. Remba-Shapiro: None. J.R. Schweizer: None. K.J. Liebert: Consulting Fee; Self; Crinetics, Recordati Rare Diseases, Novo Nordisk. K. Schilbach: None. M. Adams: Consulting Fee; Self; Chiesi. N.A. Tritos: None. M. Bidlingmaier: Consulting Fee; Self; Ipsen, Ionis Pharmaceuticals Inc., Novartis Pharmaceuticals, Novo Nordisk, Pfizer, Inc., Roche Pharmaceuticals. L.B. Nachtigall: Consulting Fee; Self; Crinetics. Grant Recipient; Self; Amryt, Recordati Rare Diseases. Background: SRLs are the most used medical therapy for acromegaly. Some patients report symptoms of acromegaly towards the end of the injection cycle even when IGF-1 levels are controlled. IGF-1 fluctuations within an injection cycle have been observed. In this study we investigated the variability of biochemical markers and PROMS in patients receiving long-acting SRLs. Methods: Subjects with acromegaly controlled with monthly lanreotide depot or octreotide LAR were recruited. Blood samples at 1 and 4 weeks after SLR injection were collected in 2 consecutive cycles. Clinical characteristics were evaluated. Quality of life (QoL) surveys included: Acromegaly Quality of Life Questionnaire (AcroQoL) (higher scores = better QoL) and Patient Assessed Acromegaly Symptom Questionnaire (PASQ) (higher scores = worse QoL). Week 1 vs. week 4 values were compared. Quest Diagnostics LC-MS assay was used to measure IGF-1. Coefficient of variation (CV = (IGF-1 SD)/(IGF-1 mean)%) of IGF-1 was calculated. Values ≤5% were interpreted as being a consequence of assay variability. Variables are presented as mean (SD) and compared using t-test; p &amp;lt;0.05 was considered significant. Results: Patients (6 women, 4 men) had a mean age of 56.1 (15.4) years. Eight underwent TSS. One had diabetes mellitus, 3 had hypertension. Mean IGF-1 index (IGF-1 x upper limit of normal) at screening was 0.63 (0.23). BMI was 27.7 (5.8) kg/m2. IGF-1 index at week 1 was 0.57 [0.16] vs. 0.61 [0.23] at week 4, p=0.271. Insulin levels were lower in week 1 vs. week 4 (7.3 [4.4] vs. 9.7 [6.0]mIU/mL p=0.041). There were no significant differences in GH, glucose, weight, blood pressure and ring size in week 1 vs. week 4. PASQ varied significantly within the injection cycle with higher scores in week 4 (18.1 [10.8]) compared to week 2 (16.1 [10.4] p=0.032) and week 3 (14.2 [8.8] p= 0.002). ACROQoL scores did not differ by injection phase. IGF-1 increased more than assay CV from week 1 to week 4 in 8/20 (40%) determinations. An ROC curve identified an IGF-1 index cutoff of 0.57 at week 4 (AUC: 0.812, p=0.021) as a predictor of IGF-1 index increase within the injection cycle (CV &amp;gt; 5%): sensitivity 88%, specificity 75%. Five patients had IGF-1 index &amp;gt; 0.57 at week 4 during both cycles (range 0.60-1.2). In this group, mean IGF-1 index was higher at week 4 vs. week 1 (0.77 [0.21] vs. 0.66 [0.14] p=0.026). The 5 patients with IGF-1 index &amp;lt; 0.57 at week 4 had no difference in IGF-1 index between week 4 and week 1 (0.48 [0.14] vs. 0.44 [0.09] p=0.144). Conclusions: This prospective study of patients with acromegaly controlled with long-acting SRLs demonstrates injection phase variability in self-reported symptoms by PASQ. An IGF-1 index &amp;gt; 0.57 at the end of the cycle may warrant injection phase-dependent monitoring. Larger studies to confirm the IGF-1 cutoff above which patients should be monitored in a cycle-dependent manner will allow an individualized approach to optimize management. Presentation: 6/2/2024

A-038 Interlaboratory Comparison of Parathyroid Hormone Analytical Results Across Clinical Assays from 8 Manufacturers - Steps Towards Standardization

Abstract Background Parathyroid hormone (PTH) is an 84 amino acid peptide produced by the parathyroid gland and is a key biomarker used for the diagnosis and treatment of Chronic Kidney Disease-Mineral and Bone Disorder, as well as hypo- and hyperparathyroidism. PTH clinical assays were previously reported to exhibit significant inter-assay measurement variability, which limits the utility of PTH as a diagnostic biomarker and can result in the misclassification of patients. Thus, there is a need for PTH clinical assay standardization to improve clinical assay measurement agreement. The Centers for Disease Control and Prevention’s Clinical Standardization Program (CDC-CSP) will launch a PTH standardization program, which is in support of and coordinated with the IFCC Committee on Bone Metabolism. In preparation, the CDC-CSP conducted an Inter-Laboratory Comparison Study to evaluate the current status of PTH inter-assay variability and to investigate potential sources of variability. Methods Eight PTH assay manufacturer’s laboratories measured PTH in 42 deidentified, fresh-frozen single donor serum samples in duplicate over 2 independent runs. Sample PTH concentrations ranged from 1.3 - 324 pg/mL, based on initial screening values. Five manufacturers submitted data for 2 different assays, resulting in PTH measurements from 13 assays total. Data were submitted to the CDC CSP for analysis. Results Assay precision was &amp;lt;5% for 10/13 measurement procedures (MP) assessed and 3 MPs exhibited precisions between 5 - 18%. Assay-specific PTH measurements demonstrated good linear correlation compared to the all-assay mean concentration. Interestingly, inter-assay variability increased with increasing PTH concentrations, with individual sample standard deviations increasing from 2.9 - 123, with a mean SD of 24.1 and individual sample CVs increasing from 18.9 - 44.5%, with a mean CV of 24.2%. Conclusions This study provides new insights into PTH inter-assay variability and assay performance for assays commonly used in the clinical setting. The findings of this PTH Interlaboratory Comparison Study establish a baseline for PTH inter-assay variability and will guide future PTH standardization efforts.

Interpretation of TSH results can be improved by reference values fluctuating in time.

The secretion of thyroid stimulating hormone (thyrotropin, TSH), is characterized by a marked circadian rhythm. Plasma or serum TSH values are significantly lower in the afternoon and in the evening as compared to the early morning. As in clinical practice, blood sampling time shows an important variation, a reliable assessment of thyroid status is often not an easy task for the clinician. The biological variation of TSH plays a major role in the intra-individual variability of TSH results in serum or plasma. The observed intra-day variation largely exceeds the reported inter-vendor variation and the coefficient of variation of clinical TSH assays. Therefore, a mathematical solution was sought for correcting interpretation of TSH results for sampling time. We have developed a cosinor model which allows to compensate TSH decision values for the fluctuating serum or plasma TSH concentrations throughout the day. The following mathematical function could be derived: corrected TSH cutoff_value (mIU/L)=0.40+0.24*cos(((π/12) *T)+6) in which T represents the time (hours). This mathematical function can be easily implemented into a laboratory's informatics system and furthermore allows a better tailored diagnosis of (subclinical) hyperthyroidism, regardless the blood sampling time. Implementing the corrected cut-off values result in a marked reduction of apparent (false positive) hyperthyroidism diagnosis, in particular in the afternoon.

Transmission-blocking activities of artesunate, chloroquine, and methylene blue on Plasmodium vivax gametocytes.

Plasmodium vivax is now the main cause of malaria outside Africa. The gametocytocidal effects of antimalarial drugs are important to reduce malaria transmissibility, particularly in low-transmission settings, but they are not well characterized for P. vivax. The transmission-blocking effects of chloroquine, artesunate, and methylene blue on P. vivax gametocytes were assessed. Blood specimens were collected from patients presenting with vivax malaria, incubated with or without the tested drugs, and then fed to mosquitos from a laboratory-adapted colony of Anopheles dirus (a major malaria vector in Southeast Asia). The effects on oocyst and sporozoite development were analyzed under a multi-level Bayesian model accounting for assay variability and the heterogeneity of mosquito Plasmodium infection. Artesunate and methylene blue, but not chloroquine, exhibited potent transmission-blocking effects. Gametocyte exposures to artesunate and methylene blue reduced the mean oocyst count 469-fold (95% CI: 345 to 650) and 1,438-fold (95% CI: 970 to 2,064), respectively. The corresponding estimates for the sporozoite stage were a 148-fold reduction (95% CI: 61 to 470) and a 536-fold reduction (95% CI: 246 to 1,311) in the mean counts, respectively. In contrast, high chloroquine exposures reduced the mean oocyst count only 1.40-fold (95% CI: 1.20 to 1.64) and the mean sporozoite count 1.34-fold (95% CI: 1.12 to 1.66). This suggests that patients with vivax malaria often remain infectious to anopheline mosquitos after treatment with chloroquine. Use of artemisinin combination therapies or immediate initiation of primaquine radical cure should reduce the transmissibility of P. vivax infections.

Performance of three multiplex real-time PCR assays for simultaneous detection of 12 infectious pathogens in mice affected with respiratory and digestive diseases.

Research quality can be improved with reliable and reproducible experimental results when animal experiments are conducted using laboratory animals with guaranteed microbiological and genetic quality through health monitoring. Therefore, health monitoring requires the rapid and accurate diagnosis of infectious diseases in laboratory animals. This study presents a performance evaluation of a commercially available multiplex real-time PCR (mRT-PCR) assay for the rapid detection of 12 infectious pathogens (Set 1: Sendai virus [SeV, formally murine respirovirus], Mycoplasma spp., Rodentibacter pneumotropicus, and Rodentibacter heylii; Set 2: Helicobacter spp., Murine norovirus [MNV], Murine hepatitis virus [MHV], and Salmonella spp.; Set 3: Staphylococcus aureus, Streptobacillus moniliformis, Corynebacterium kutscheri, and Pseudomonas aeruginosa). To evaluate the efficacy of the mRT-PCR assay, 102 clinical samples encompassing fecal and cecal specimens were analyzed. The resulting data were then compared with the findings from sequence analysis for validation. The assay's detection limit ranged from 1 to 100 copies per reaction. Specificity testing involving various viruses and bacteria indicated no cross-reactivity between strains. Additionally, the assay exhibited good reproducibility, with mean coefficients of variation for inter- and intra assay variation below 3%. The overall positive rate was 52.9% (n = 54), with the mRT-PCR assay findings matching sequence analysis results (κ = 1). MHV (n = 29, 28.4%) was the most prevalent pathogen, followed by Helicobacter spp. (n = 28, 27.5%), R. heylii (n = 18, 17.6%), Mycoplasma spp. (n = 14, 13.7%), MNV (n = 12, 11.8%), S. aureus (n = 9, 8.8%), P. aeruginosa (n = 4, 3.9%), and R. pneumotropicus (n = 1, 0.9%). This assay offers a rapid turnaround time of 100 min, including 30 min for DNA preparation and 70 min for target DNA/RNA amplification. It ensures accuracy, minimizing false positives or negatives, making it a convenient tool for the simultaneous detection of infectious diseases in many samples. Overall, the propose‑d assay holds promise for the effective detection of the most important pathogens in laboratory animal health monitoring.