Inter-assay Variability Research Articles

Evaluation of Biochemical Characteristics and Performance of the 3 Screen ICA ELISA Kit

We conducted a fundamental evaluation of the 3 Screen ICA ELISA kit, which can simultaneously measure three major anti-islet autoantibodies important in diagnosing and predicting type 1 diabetes, to assess its usefulness as a measuring reagent. In autoantibody-positive samples, the coefficient of variation for intra-assay variation ranged from 1.37% to 2.50%, inter-assay variation from 2.81% to 3.61%, and lot-to-lot variation from 2.01% to 8.61%, demonstrating good reproducibility. Additionally, interfering substances did not affect the autoantibody titers, and satisfying performance was observed in tests examining the sample freeze-thaw stability. Notably, even when the titer of GAD autoantibodies was below the cut-off value of the GAD autoantibody ELISA, the 3 Screen ICA signal was completely absorbed by recombinant GAD65 protein, indicating that the detection sensitivity for GAD autoantibody in the 3 Screen ICA ELISA is higher than that of the GAD autoantibody ELISA kit. Furthermore, in a study using IASP2020 samples from the Immunology and Diabetes Society, which aims to standardize anti-islet autoantibody assays, this kit achieved excellent results with a sensitivity of 96.0%, specificity of 100%, and accuracy of 98.57%. Measuring multiple anti-islet autoantibodies in combination is crucial for diagnosing and predicting type 1 diabetes. The ELISA kit used in this study is highly versatile and can be used in any measurement facility, making it extremely useful for routine testing.

Serum Transcobalamin Concentration in Cats—Method Validation and Evaluation in Chronic Enteropathies and Other Conditions

Hypocobalaminemia is common in cats with chronic enteropathy (FCE). However, the disruptions in cobalamin metabolism are not fully understood and may vary across species. Cobalamin is distributed to target tissues via binding to transcobalamin (TC) in blood, which has not been evaluated in cats. Thus, an in-house sandwich-ELISA was established to evaluate serum total TC concentrations in cats with FCE. Surplus sera served to analytically validate the assay, and serum TC concentrations were compared among cats with FCE and other diseases (gastrointestinal neoplasia, cholangiohepatopathy, and other neoplastic or non-neoplastic conditions) and healthy controls. Observed-to-expected ratios for serial dilutions ranged from 72.4 to 145.6% and were 75.1–126.7% for spiking-and-recovery. Intra- and inter-assay variability was <17.7% and <17.2% and the preliminary reference interval for feline serum TC was <160–2795 aU/L (lower detection limit: 160 aU/L). Serum TC levels were significantly decreased (p = 0.0067) but not correlated with paired cobalamin concentrations in FCE. Hypertranscobalaminemia predominated with hypercobalaminemia, reaching the highest levels in advanced-stage chronic kidney disease (CKD) cases. TC variations in cobalamin deficiency states with FCE may be linked to inflammation or autoantibodies. This and possible links between serum TC variation in FCE, intracellular cobalamin availability, response to supplementation, and concurrent CKD require further exploration.

Stability of the thromboxane B2 biomarker of aspirin pharmacodynamics in human whole blood and in long-term stored serum samples

BackgroundSerum thromboxane B2 (sTXB2) is a validated biomarker of low-dose aspirin pharmacodynamics. In the original method, non-anticoagulated blood samples must incubate at 37°C immediately after withdrawal, be centrifuged, serum should be frozen until assayed. Timely completion of all preanalytical steps may affect the feasibility and quality of sTXB2 measurements. The storage duration of frozen serum can also affect sTXB2 stability. AimsWe assessed the stability of sTXB2: in clotted blood samples stored at 4°C before further processing; in sera stored at -40°C for over a decade. MethodVenous whole blood withdrawn from individuals on chronic low-dose aspirin was dispensed in different tubes, immediately incubated at 37°C for 1 hour. The reference tube was promptly processed following the original protocol, the remaining tubes were stored at 4°C from 12 to 72 hours before further processing. Sera stored at controlled -40°C temperature for <1 to 15 years were re-assayed. Values within the inter-assay variation limits (±9%) versus baseline were considered acceptable. ResultsBaseline sTXB2 values (5.4[2.4-13.4] ng/ml, median [IQR], n=40) were comparable with those in samples at 4°C up to 48 hours (97[86-104]% of reference, n=26), but at 72 hours the variability exceeded the inter-assay variation. TXB2 levels were stable in frozen sera for up to 10 years (101[87-108]% of reference, n=32), but decreased significantly afterwards (87[74-109]% at 15 years, p=0.005, n=32). ConclusionsTXB2 is stable in clotted blood samples stored at 4°C for up to 48 hours before further processing and in serum samples stored at -40°C over 10 years.

Development of an assay to quantify tranexamic acid levels in plasma

Dysregulations during blood clot breakdown (fibrinolysis) during vascular trauma can lead to excessive blood loss. Tranexamic acid (TXA) is an inhibitor of fibrinolysis that works by blocking the interaction between plasminogen and fibrin degradation products (FDPs) – a key step in fibrinolysis. Despite the widespread usage, there are no tests available in a clinical setting to monitor TXA levels. We developed a fluorescence resonance energy transfer (FRET)-based assay to quantify TXA concentrations in plasma by using 1) fluorescently labeled plasminogen, and 2) FDPs labelled with a fluorescence quencher. Once plasminogen binds the FDPs, the fluorescent signal is quenched. TXA causes plasminogen to dissociate from the FDPs, thus increasing fluorescence signal in a dose-dependent manner. The dose response was sensitive between 1 and 100 μM (0.16 and 15.7 mg/L). The intraassay and interassay variabilities were determined to be 5.7% and 3.0%, respectively. Limit of detection was estimated to be 0.28 μM (0.044 mg/L). When tested for measuring known levels of TXA added to plasma samples, the ratio between measured and expected TXA concentration was 1.0151. Our study demonstrates a novel assay that can rapidly quantify TXA concentrations in plasma samples, thus demonstrating its potential as an in-hospital tool.

Comparison of five tacrolimus measurement methods underscore the need for a standardized method to improve clinical outcomes

Abstract Tacrolimus is a widely used drug to prevent post-transplantation organ rejection by suppressing host immune response. While its efficacy is well established, tacrolimus is also known for narrow therapeutic index and debilitating adverse effects, rendering accurate measurement for therapeutic drug monitoring essential, to minimize drug toxicity and graft loss, and maximize organ rejection prevention. Tacrolimus can be quantitated by immunoassay (IA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, previous pairwise studies showed significant inter-assay variabilities between different IA methods, and between IA vs LC-MS/MS, which may be associated with the fact that IA measures parent drug and its metabolites, while LC-MS/MS measures only parent drug. No clear conclusion has been drawn about the optimal method. Transplant patients may also have their tacrolimus levels measured at different laboratories by different methods, causing inter-laboratory and intra-individual variabilities which may affect tacrolimus dose adjustment and monitoring and lead to adverse clinical outcomes. Here we performed a multi-platform comparison of tacrolimus quantification by two IA and 3 LC-MS/MS assays. A total of 216 specimens were analyzed for tacrolimus levels using chemiluminescence immunoassay (ChLIA, ARCHITECT, Abbott Diagnostic) and electrochemiluminescence immunoassay (ECLIA, Elecsys, Roche Diagnostic), and three LC-MS/MS assays (assigned names LC-MS/MS A (Agilent), Q (Waters), and W (Agilent)) in five different laboratories at four institutions. In one laboratory, the same analyses were performed on two different individual instruments (LC-MS/MS W1 and W2). Comparative results using LC-MS/MS A as reference were analyzed using Deming regression analysis, Pearson correlation, Bland Altman plots and bias calculation using GraphPad Prism version 10.1.1. While the correlations between each method with the LC-MS/MS A were good (r value: lowest 0.948 (LC-MS/MS Q), highest 0.988 (LC-MS/MS W2)), the ChLIA and ECLIA showed positive absolute bias of 2.1 and 1.2 ng/mL, respectively, and proportional bias of +26% and +17%, respectively. In comparison, the absolute bias of LC-MS/MS Q, W1 and W2 were -0.7, -0.02, and -0.1 ng/mL, respectively, with proportional bias of -11%, -0.2%, and -0.7%, respectively. The LC-MS/MS platforms showed tight agreement with one another, while IA methods were noticeably different, both from each other and from the LC-MS/MS, with a consistent positive bias, more pronounced with the ChLIA than the ECLIA. Here, our data-driven approach demonstrated that LC-MS/MS is superior to IA, and among IAs, ECLIA showed higher accuracy compared to ChLIA. However, inter-assay differences remain, emphasizing a need for tacrolimus assay standardization as well as prospective clinical trials to identify the most optimal method of tacrolimus measurement and improve patient outcomes.

7938 Validation of Inflammatory Cytokine Multiplex Array With Variations in Target Panel

Abstract Disclosure: W.M. Sanders: None. C.A. Karvonen-Gutierrez: None. D.S. McConnell: None. Background: Multiplex arrays are an invaluable tool for conducting large-scale biomarker research as they provide a high-throughput, cost-effective method for quantifying a panel of endocrine markers. However, the shared reaction environment between antibodies of differing reactivities creates the potential for inducing a technical difference in the assay methods due to any variation in panel selection. Methods: We evaluated the R&amp;D Systems Luminex HS Cytokine Panel A (Catalog # FCSTM09) for systematic differences when measuring a “panel” of IL-1β, TNF-α, and IL-10 vs. measuring “IL-10 only.” We selected 72 serum samples and assayed them in singleton 4 times: 2 separate “panel” batches, and 2 separate “IL-10 only” batches. Differences in log-transformed IL-10 results for sample replicates within and between the “panel” and “IL-10 only” batches were assessed for significance with paired t-tests (α=.01). We also assessed the differences graphically with Bland-Altman plots of differences and absolute percent differences.All batches included control materials from R&amp;D Systems and internal lab serum pools (male and female). To retroactively assess the test batches, we graphically compared their quality control results to those of the subsequent, larger assay project we were piloting. Results: The “IL-10 only” assay yielded an average result of .879 pg/mL (SD .57) on batch 1 and .712 pg/mL (SD .58) on batch 2, with an average paired sample difference of .167 pg/mL, and an average absolute percent difference of 28.8%. The “panel” assay produced an average IL-10 result of .645 pg/mL (SD .50) on batch 1 and .720 pg/mL (SD .52) on batch 2, with an average paired sample difference of .075 pg/mL, and an average absolute percent difference of 15.5%. Comparing pooled IL-10 results from the “IL-10 only” batches with the “panel” batches, there was an average paired difference of .113 pg/mL and absolute percent difference of 16.2%. Paired t-testing of log-transformed results for within “panel,” within “IL-10 only,” and between pooled results from the two methods demonstrated that each had significant differences (p&amp;lt;.0001). The Bland-Altman plots further revealed that these differences were ubiquitously large for samples with low concentrations of IL-10.Test batch quality control results were consistent with those in the primary assay project. The serum pool QCs exhibited significant inter-assay variation – the male pool average result was .41 pg/mL (SD .10) vs. .359 in the test batches, and the female pool average was .57 pg/mL (SD .10) vs. .548 in the test batches. Conclusion: The multiplex assay had considerable inter-assay variation at low concentrations of IL-10, and low results were prevalent. Due to this, we determined that any methodological differences between the “panel” and “IL-10 only” methods were undetectable compared to ambient variation, and irrelevant given the sample characteristics. Presentation: 6/3/2024

Microarray-based evaluation of selected recombinant timothy grass allergens expressed in E. Coli and N. Benthamiana

BackgroundTimothy grass (Phleum pratense) is a significant source of allergens, and recombinant allergens are increasingly used for diagnostic purposes. However, the performance of different recombinant allergen production systems in diagnostic assays needs further investigation to optimize their use in clinical settings.ObjectiveThe main objective of this study was to analyze and compare the diagnostic performance of recombinant timothy grass allergens produced in E. coli and N. benthamiana using a custom-made microarray chip.MethodsRecombinant timothy grass allergens Phl p 1, Phl p 2, Phl p 5, Phl p 6, Phl p 11, and Phl p 12 were produced in E. coli and/or N. benthamiana. A total of 113 patient serum samples were tested to evaluate the diagnostic sensitivity, specificity, inter-assay variability, and correlation of allergen-specific IgE detection compared to commercial multiplex tests (ALEX and ISAC). Additionally, the prevalence of sIgE to these allergens was assessed.ResultsPhl p 1, Phl p 2, Phl p 5, Phl p 6 and Phl p 11 showed high or very high positive correlation in immunoreactivity with other commercial multiplex tests. Notably, Phl p 11 fused with maltose-binding protein (MBP) demonstrated high diagnostic specificity and sensitivity, with a 0.3 arbitrary cut-off value. However, a high intra-assay variation was observed. The study also assessed specific IgE prevalence to timothy grass allergens within the tested patient cohort.ConclusionsRecombinant allergens from both E. coli and N. benthamiana demonstrated strong diagnostic potential on the microarray platform, with Phl p 11 (MBP-fused) showing particularly high performance. High intra-assay variation highlights the need for further optimization in allergen formulation and microarray storage conditions. These results highlight the potential of recombinant allergens for diagnostic applications, despite challenges with allergen stability in microarray formats. Specific IgE prevalence to timothy allergens revealed a sensitization profile consistent with findings from multiple studies.Graphical

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.

Development, validation and application of a high-performance liquid chromatography method for the detection of ibuprofen in elephant plasma

A simple and accurate method for the determination of ibuprofen concentrations in elephant plasma was developed and validated using reverse phase high-performance liquid chromatography (HPLC). Utilizing a liquid-liquid extraction with ethyl acetate, methanol, and phosphoric acid, samples were separated on an XBridge C18 column. The mobile phase consisted of 0.005 M ammonium phosphate and acetonitrile (40:60, v/v). Both ibuprofen and the internal standard flurbiprofen were quantitated using an ultraviolet (UV) detector set at 214 nm. The lower limit of quantification (LLOQ) was 0.05 μg/mL in 100 μL of plasma. The average recovery was over 95% while the intra-assay variability ranged from 0.8 to 9.9% and the inter-assay variability ranged from 2.7 to 9.8%. The method was used successfully in a therapeutic drug monitoring program for elephants and could be used in future pharmacokinetic studies.

An Automated pre-Dilution Setup for Von Willebrand Factor Activity Assays.

Accurate quantification of von Willebrand factor ristocetin cofactor activity (VWF:RCo) is critical for the diagnosis and classification of von Willebrand disease, the most common hereditary and acquired bleeding disorder in humans. Moreover, it is important to accurately assess the function of von Willebrand factor (VWF) concentrates within the pharmaceutical industry to provide consistent and high-quality biopharmaceuticals. Although the performance of VWF:RCo assay has been improved by using coagulation analyzers, which are specialized devices for blood and blood plasma samples, scientists still report a high degree of intra- and inter-assay variation in clinical laboratories. Moreover, high, manual sample dilutions are required for VWF:RCo determination of VWF concentrates within the pharmaceutical industry, which are a major source for assay imprecision. For the first time, we present a precise and accurate method to determine VWF:RCo, where all critical pipetting and mixing steps are automated. A pre-dilution setup was established on CyBio FeliX (Analytik-Jena) liquid handling system, and an adapted VWF:RCo method on BCS-XP analyzer (Siemens) is used. The automated pre-dilution method was executed on three different, most frequently used coagulation analyzers and compared to manual pre-dilutions performed by an experienced operator. Comparative sample testing revealed a similar assay precision (coefficient of variation = 5.9% automated, 3.1% manual pre-dilution) and no significant differences between the automated approach and manual dilutions of an expert in this method. While no outliers were generated with the automated procedure, the manual pre-dilution resulted in an error rate of 8.3%. Overall, this operator-independent protocol enables standardization and offers an efficient way of fully automating VWF activity assays, while maintaining the precision and accuracy of an expert analyst. Key features • Automated pre-dilution setup for von Willebrand factor concentrates of various natures. • Combination of a liquid handling system (CyBio FeliX) with a coagulation analyzer (BCS-XP). • Simplifies method transfer to other laboratories. • Basic training for CyBio FeliX and BCS-XP is required. Graphical overview VWF:RCo assay principle and measurement setup. Platelets (yellow ellipsoids) with negative surface charge (- - -) are treated with formaldehyde, which partly denatures the cell surface and thus stabilizes platelets for use as assay reagents. Stabilized platelets (dark-yellow-framed yellow ellipsoids) are then brought in contact with ristocetin A (chemical structure shown; black dots), which binds to the platelet surface and facilitates binding of VWF (green circles). The graphs show an example of quantitative determination of platelet agglutination by measurement of light transmission, where increasing amounts of VWF increase light transmission over time. The photo in the left-bottom corner shows the CyBio FeliX setup for VWF sample dilution and the photo in the right-bottom corner displays the BCS-XP system, which is used for VWF:RCo measurements.

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.

To optimize and evaluate methods for the detection of the inflammatory biomarkers myeloperoxidase (MPO) and calprotectin (CP) in equine feces by ELISA. Healthy horses (n = 28) and horses with intestinal inflammation (n = 10). 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. 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. 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.

Using targeted proteomics-based detection of collagen propeptides to quantify fibrillar collagen biogenesis in vitro

The collagen superfamily, as the major structural component of the extracellular matrix, encompasses 28 distinct subtypes, with type-I and –III forming fibrils crucial for the matrix scaffold. During collagen biogenesis, trimers of type-I and –III procollagen are secreted into the extracellular matrix. The N- and C-terminal propeptides of these trimers are proteolytically cleaved from procollagen during secretion, initiating collagen fibril formation. The propeptides are released into extracellular space and, therefore, have been used to quantify collagen biogenesis. But high-throughput methods for the quantification of these biomarkers are still lacking. This study presents a state-of-the-art multiplexed approach for the simultaneous quantification of PINP, PICP, PIIINP and PIIICP from cell culture supernatants. The ability of targeted proteomics to quantify these propeptides from cell culture samples was assessed in this study. Using tryptic digestion and solid phase extraction, we were able to accurately quantify precollagen propeptides in a range of 3–1000 ng/mL. The assay showed an average inter-assay variance of 6.86 % with an overall recovery ranging from 92 to 98 %. The assay was validated using recombinant protein standards diluted in surrogate matrix and tested using transforming growth factor β1 mediated induction of normal human dermal fibroblasts. In summary, the assay presented in this paper offers a novel, robust, and precise high-throughput method for measuring human collagen propeptides in cell culture supernatants, empowering researchers to assess collagen biogenesis effectively in in vitro experiments.

Establishment and application of quadruple fluorescence quantitative RT-PCR method for the identification of waterfowl astrovirus.

Avian astrovirus can infect a variety of poultry species and cause viral diarrhea, with a wide epidemic range strong pathogenicity and a high incidence. Among them, Duck astrovirus 3(DAstV-3), Duck astrovirus 4(DAstV-4), Goose astrovirus 1(GoAstV-1) and Goose astrovirus 2(GoAstV-2) are four types of astroviruses newly discovered in waterfowl in recent years. In order to realize the rapid detection of these four kinds of waterfowl stellate viruses, specific primers and probes were engineered to target a highly conserved region of ORF1b gene of DAstV-3, GoAstV-1 and GoAstV-2 and the ORF2 gene of DAstV-4, and a quadruple fluorescence quantitative RT-PCR method was developed. The results indicate that the method established in this study has good specificity and no cross reactivity with other pathogens. This method can detect viruses with a minimum concentration of 1 × 101 copies/μL for DAstV-4, GoAstV-1 and GoAstV-2, respectively, while the minimum concentration for DAstV-3 is 1 × 102 copies/μL. Compared with the routinely used RT-PCR method, the limit of detection by the multiplex RT-PCR lower. Both intra- and inter-assay variability tests revealed excellent reproducibility. This method was then used to analyze 269 field samples, and the results were verified by genome sequencing. In conclusion, this study presents a sensitive, accurate, and specific method for detecting DAstV-3, DAstV-4, GoAstV-1, and GoAstV-2 in a single reaction, enabling the monitoring and differential diagnosis of these four types of waterfowl astroviruses.

P-646 Prognostic statistical model of age-specific anti-Müllerian hormone (AMH) decrease based on hormone values of 23,528 women

Abstract Study question The analysis of anti-Müllerian (AMH) levels, can a useful statistical model be constructed to predict the rate of decrease of AMH levels at different ages? Summary answer A determination of AMH levels can be an objective marker of a ovarian age and a prognostic marker of the rate of ovarian function decline. What is known already The anti-Müllerian hormone is a dimeric glycoprotein produced by granulosa cells of the antral follicles in the ovaries, and AMH participates in the formation of the dominant follicle during folliculogenesis. Serum AMH levels are relatively constant and show little variability between and within the menstrual cycle. The level of serum AMH is proportional to the number of antral follicles and therefore most suitable for determining ovarian reserve. For this reason, AMH is a convenient marker for the biological age of the ovary and for the response to controlled ovarian stimulation (COS). Study design, size, duration A nationwide cross-sectional retrospective study was conducted between 2017 and 2023 and included 23,528 women the ages of 20 - 50, in which AMH levels were measured once. We performed an analysis of the different AMH levels at different ages and constructed a predictive statistical model for the rate of AMH decline. Results were analyzed in subgroups with AMH values &amp;lt; 1 ng/mL, between 1 and 3,5 ng/mL, and &amp;gt; 3,5 ng/mL, at one-year intervals. Participants/materials, setting, methods All women who visited the laboratory for AMH testing were included in the analysis. The samples were collected in 38 points throughout the country and the analysis was carried out in one centralized laboratory using the Ansh Labs (Webster, TX, USA) manual assays, including the ultra-sensitive AMH enzyme-linked immunosorbent assay (ELISA), with a management range of 0,08 - 14,2 ng/ml. The intra-assay variations were 1,97% and 4,00%. The inter-assay variations were 4,63% and 1,98%, respectively. Main results and the role of chance When analyzing all samples in each consecutive year, we found that the rate of AMH decline was approximately linear and inversely proportional to the woman’s age. However, when analyzing the results for the rate of AMH reduction in the different subgroups, we found out that: In women with AMH &amp;lt; 1 ng/ml, the rate of decline with advancing age changes as follows: until age 34, the decline in AMH is very slow, between ages 35 and 45 there is a rapid acceleration of the process.. In women with AMH between 1 and 3,5 ng/ml, the rate of decline with age was approximately constant. In women with AMH &amp;gt; 3,5 ng/ml, the rate of hormone decline is approximately constant but the year-on-year decline rate is decreasing. Based on our analysis and clinical experience, it is our opinion that the rate of AMH decline for the low AMH group has a non-linear characteristic, while ingeneral different groups exhibit different dynamics for the rate of decline. On this basis, we created a statistical model to predict the decrease in AMH levels at different ages. Limitations, reasons for caution Women of only one ethnicity were included in the study. The studied group is heterogeneous, which does not exclude the inclusion of women with a certain pathology and infertility. In some cases, a single determination of AMH could not predict individual differences in rates of hormone reduction. Wider implications of the findings Determination of reference values for AMH at different ages, based on the large volume of examined samples may facilitate the application of AMH in clinical practice. Based on our statistical analysis, creating a prognostic model for decreasing ovarian function and time to menopause appears feasible. Trial registration number NA

Performance of an automated immunoturbidimetric assay for bovine serum amyloid A.

Acute phase proteins are a group of vital constituents of the innate immune system, which may also serve as circulatory biomarkers of inflammation. The major acute phase protein serum amyloid A (SAA) is a reliable and sensitive biomarker in cows, allowing for rapid detection of inflammatory disease. A multispecies automated immunoturbidimetric assay (VET-SAA, Eiken) has been validated for horses, dogs, and cats, and it has been used to measure SAA concentrations in bovine samples. The aim of the present study was to perform an analytical validation of the VET-SAA immunoturbidometric assay based on monoclonal antihuman SAA antibodies for the measurement of SAA in clinical samples from cows. The validation included an assessment of imprecision, inaccuracy, and detection limit, as well as an evaluation of the overlap performance, using banked serum from healthy and sick cows with or without inflammatory disease. Intra- and interassay variation ranged from 0.91% to 2.9% and 2.5% to 5.8%, respectively. The assay was performed with acceptable accuracy within a clinically relevant range of SAA, although minor signs of inaccuracy were detected. Overlap performance was acceptable, with the VET-SAA assay able to differentiate between healthy cows and cows with inflammatory and noninflammatory conditions. The automated VET-SAA assay is considered acceptable for the measurement of SAA in cows.

A Quadruplex Reverse Transcription Quantitative Polymerase Chain Reaction for Detecting Canine Coronavirus, Canine Rotavirus, Canine Parvovirus, and Canine Distemper Virus

Background: Canine coronavirus (CCoV), canine rotavirus (CRV), canine parvovirus (CPV), and canine distemper virus (CDV) cause gastroenteritis in dogs, and co-infections of these pathogens are common in China. In particular, CCoV and CRV are confirmed to have important zoonotic potential and cause public health issues. It is difficult to diagnose these diseases based only on clinical manifestations and pathological damage. Methods: In this study, four pairs of specific primers and probes targeting the CCoV M, CRV VP7, CPV VP2, and CDV N genes were designed. The reaction conditions, including the primer and probe concentrations, annealing temperatures, and reaction cycles, were optimized for the development of a quadruplex RT-qPCR for the detection of CCoV, CRV, CPV, and CDV. The assay was used to test 1028 clinical samples to validate its application. Results: A quadruplex RT-qPCR was successfully established for the differential detection of CCoV, CRV, CPV, and CDV, with good specificity, high sensitivity, and excellent repeatability. The assay could specifically detect CCoV, CRV, CPV, and CDV without cross-reactivity with the other canine viruses tested. It showed high sensitivity with limits of detection (LOD) of 1.1 × 102 copies/reaction for all four plasmid constructs. It showed excellent repeatability, with 0.05–0.90% intra-assay variation and 0.02–0.94% inter-assay variation. The 1028 clinical samples were tested using the quadruplex RT-qPCR and a reported reference RT-qPCR. The positivity rates of CCoV, CRV, CPV, and CDV were 9.53%, 0.97%, 25.68%, and 5.06% using the developed assay, and 9.05%, 0.88%, 25.68%, and 4.86% using the reference assay, with agreements higher than 99.32%. Conclusion: The results indicated that a rapid and accurate quadruplex RT-qPCR was developed for the detection and differentiation of CCoV, CRV, CPV, and CDV.

Development and Validation of Multiplex Assays for Mouse and Human IgG and IgA to Neisseria gonorrhoeae Antigens.

There is an urgent need for vaccines against Neisseria gonorrhoeae, the causative agent of gonorrhea. Vaccination with an outer membrane vesicle-based Neisseria meningitidis vaccine provides some protection from N. gonorrhoeae; however, the mechanisms underlying this cross-protection are unknown. To address this need, we developed multiplexed bead-based assays for the relative quantification of human and mouse IgG and IgA against N gonorrhoeae antigens. The assays were evaluated for analyte independence, dilutional linearity, specificity, sensitivity, intra- and interassay variability, and robustness to sample storage conditions. The assay was then used to test samples from mice and humans immunized with an N meningitidis outer membrane vesicle vaccine.

Cryopreservation of cerebrospinal fluid cells preserves the transcriptional landscape for single-cell analysis

Cerebrospinal fluid (CSF) matrix biomarkers have become increasingly valuable surrogate markers of neuropsychiatric diseases in research and clinical practice. In contrast, CSF cells have been rarely investigated due to their relative scarcity and fragility, and lack of common collection and cryopreservation protocols, with limited exceptions for neurooncology and primary immune-based diseases like multiple sclerosis. The advent of a microfluidics-based multi-omics approach to studying individual cells has allowed for the study of cellular phenotyping, intracellular dynamics, and intercellular relationships that provide multidimensionality unable to be obtained through acellular fluid-phase analyses. Challenges to cell-based research include site-to-site differences in handling, storage, and thawing methods, which can lead to inaccuracy and inter-assay variability. In the present study, we performed single-cell RNA sequencing (10x Genomics) on fresh or previously cryopreserved human CSF samples from three alternative cryopreservation methods: Fetal Bovine Serum with Dimethyl sulfoxide (FBS/DMSO), FBS/DMSO after a DNase step (a step often included in epigenetic studies), and cryopreservation using commercially available Recovery© media. In comparing relative differences between fresh and cryopreserved samples, we found little effect of the cryopreservation method on being able to resolve donor-linked cell type proportions, markers of cellular stress, and overall gene expression at the single-cell level, whereas donor-specific differences were readily discernable. We further demonstrate the compatibility of fresh and cryopreserved CSF immune cell sequencing using biologically relevant sexually dimorphic gene expression differences by donor. Our findings support the utility and interchangeability of FBS/DMSO and Recovery cryopreservation with fresh sample analysis, providing a methodological grounding that will enable researchers to further expand our understanding of the CSF immune cell contributions to neurological and psychiatric disease.

Interassay Variability and Clinical Implications of Five Different Prostate-specific Antigen Assays

Background and objectiveProstate-specific antigen (PSA) remains a critical marker for prostate cancer (PCa) detection and monitoring. Recognising historical variability in PSA assays and the evolution of assay technology and calibration, this study aims to reassess interassay variability using the latest generation of five assays in a contemporary cohort of men undergoing prostate biopsy. MethodsFive different commercially available PSA assays were tested in a blood sample of 76 men before undergoing a prostate biopsy. Total PSA (tPSA) and free-to-total PSA ratio (%fPSA) were compared across assays, using Roche (Basel, Switzerland) as the benchmark, and correlated with biopsy outcome to analyse the impact on PCa diagnosis. The statistical analysis included Passing-Bablok regression and Bland-Altman plots, with a p value threshold of <0.05 for significance. Key findings and limitationsAmong the 76 men, 28 (36.8%) were diagnosed with significant PCa (defined as International Society of Urological Pathology grade ≥2). A high correlation was observed between tPSA and %fPSA values among the different PSA assays tested (r2 ≥ 0.9). The Passing-Bablok analysis showed that tPSA results varied substantially among the assays, with slopes ranging between 0.78 and 1.04. Compared with the tPSA of Roche, tPSA values were on average 20.7% lower by Beckman (Oststeinbeck, Germany), 15.2% lower by Abbott (Chicago, IL, USA), 6.1% lower by Diasorin (Saluggia, Italy), and 9.6% higher by Brahms (Hennigsdorf, Germany; p < 0.001 for all). The %fPSA values by Abbott and Brahms were higher at 15.7% and 10.6%, respectively (p < 0.001), while the Beckman and Diasorin values had minimal differences of -0.3% and 2.3%, respectively (p > 0.05). The variability across assays would have resulted in discrepancies in both the sensitivity and the specificity for tPSA and %fPSA by at least 14%, depending on the cut-offs applied. Conclusions and clinical implicationsDespite the use of the latest PSA assays, relevant variability of tPSA and %fPSA results can be observed among different assays. There is an urgent need for standardised calibration methods and greater awareness among practitioners concerning interassay variability. Clinicians should acknowledge that clinically relevant thresholds may depend on the specific PSA assay and that ideally the same assay is applied over time for better clinical decision-making. Patient summaryProstate-specific antigen (PSA) is a critical marker for prostate cancer (PCa) detection and monitoring. However, significant variations were observed in the results of the latest PSA assays. Thus, standardised calibration methods and greater awareness among practitioners concerning interassay variability are needed.

Performance evaluation of the SMG HHV-6 Q Real-Time PCR Kit for quantitative detection and differentiation of human herpesvirus 6A and 6B.

The aim of this study was to compare the performance of the newly developed SMG HHV-6 Q Real-Time PCR Kit (SMG assay) with the RealStar HHV-6 PCR Kit (RealStar assay). The analytical sensitivity and specificity, linearity, and precision of the SMG assay were evaluated. The clinical performance of the SMG assay was assessed and compared with that of the RealStar assay using 207 clinical specimens (HHV-6A positive, n = 51; HHV-6B positive, n = 64; HHV-6A/B negative, n = 92). The limit of detection of the SMG assay was 2.92 log10 copies/mL for HHV-6A DNA and 2.88 log10 copies/mL for HHV-6B DNA. The linear range was determined to be 3.40-9.00 log10 copies/mL for both viruses. Intra- and inter-assay variability were below 5% at concentrations ranging from 4 to 9 log10 copies/mL. No cross-reactivity was observed with the 25 microorganisms included in the specificity panel. The clinical sensitivity and specificity of the SMG and RealStar assays compared to in-house polymerase chain reaction and sequencing were as follows: SMG assay, 98.0% and 100% for HHV-6A DNA, respectively, and 96.9% and 100% for HHV-6B DNA, respectively; RealStar assay, 98.0% and 100% for HHV-6A DNA, respectively, and 90.6% and 100% for HHV-6B DNA, respectively. The correlation coefficients between viral loads measured by the two assays were 0.948 and 0.975, with mean differences of 0.62 and 0.32 log10 copies/mL for HHV-6A and HHV-6B DNA, respectively. These results demonstrate that the SMG assay is a sensitive and reliable tool for the quantitative detection and differentiation of HHV-6A and HHV-6B DNA.IMPORTANCEQuantitative real-time PCR (qPCR) that can distinguish between HHV-6A and HHV-6B DNA is recommended for diagnosis of active infection. The SMG HHV-6 Q Real-Time PCR Kit (SMG assay) is a newly developed qPCR assay that can differentiate between HHV-6A and HHV-6B DNA; however, little is known about its performance. In this study, we assessed the performance of the SMG assay and compared it with that of a commercially available qPCR assay, the RealStar HHV-6 PCR Kit (RealStar assay). The SMG assay demonstrated excellent analytical sensitivity and specificity, precision, and linearity. Furthermore, the viral loads measured by the SMG assay were highly correlated with those measured by the RealStar assay. Our results suggest that the SMG assay is a useful diagnostic tool for quantitative detection and differentiation of HHV-6A and HHV-6B DNA.