Decision Limit Research Articles

A-014 Analytical Outlier Occurrence and False Positivity Rate of the Beckman Coulter Access High-sensitivity Cardiac Troponin I Assay

Abstract Background Cardiac troponin (cTn) is the preferred biomarker for the diagnosis of acute myocardial infarction due to its high sensitivity and specificity relative to other available cardiac markers. Accurate measurement is therefore paramount to proper risk stratification of patients. Analytical outliers and false positive results have been previously reported on various platforms though the exact cause remains unknown. These false positive test results may pose a risk for the misclassification of patients and lead to adverse clinical outcomes. We evaluated the performance of the Beckman Coulter Access high-sensitivity cTn I method by measuring the occurrence of analytical outliers between duplicate results and the false positivity rate (i.e., fliers). Methods We tested 2535 samples from 1879 patients in duplicate on the Beckman Coulter Access hs-cTn I assay. These specimens had initial troponin measurements above the gender-specific 99th percentile of the upper reference limit (URL) cutoff and were immediately aliquoted, re-centrifuged, and re-tested. Non-reproducible results (outliers) were defined as having a concentration difference between the replicates exceeding 10%. Critical outliers were defined as duplicate results with concentrations that differed by greater than 10% from the initial result and the second result was classified in a lower risk stratification category than the original result. Results Approximately 12% (305) of the positive samples tested in duplicate had a percent difference that was >10% and therefore did not meet the recommended precision requirements of the assay. Of these specimens, 33 (10.8%) were classified as critical outliers because the repeat test result crossed a critical decision limit threshold. For sixteen samples the risk classification for the patient was downgraded from high (>50 ng/L) to moderate risk (13–50 ng/L (F); 20–50 ng/L (M)). Seventeen false positive results were identified in which the patients were downgraded from the high to low risk category (<13 ng/L (F); <20 ng/L (M)). Nearly 85% of the samples identified as outliers were lithium heparinized plasma. Conclusion We evaluated the reproducibility of the Beckman Coulter Access hs-cTn I method across 11 hospitals in an integrated healthcare network to characterize the occurrence of analytical outliers and false positive results. Our data indicated an outlier rate of 12% (305/2535) for positive hs-cTn I results; however, the risk category only changed for 1.3% (33/2535) of positive troponin results upon repeat analysis. Overall the false positivity rate was determined to be <0.1% (n = 55 560).

A-010 Systemwide Implementation of High Sensitivity Troponin T in a Multi-center Hospital Set Up with Instrument Variability: The Baystate Health Experience

Abstract Background High-sensitivity cardiac troponin (hs-cTn) testing helps in rapid risk stratification in patients with acute coronary syndromes resulting in early identification of low-risk patients eligible for early discharge. A high sensitivity troponin test can measure the 99th percentile upper reference limit with an analytical imprecision ≤10% and it can measure cTn above the limit of detection in ≥50% of healthy subjects. We carried out method validation of the Elecsys Gen 5 hsTnT assay with variable instrument capacities like Roche cobas e801, e601, and e411 in our health system. The analytical variability of these instruments was studied and standardized to implement the Gen 5 hsTnT assay systemwide for clinical use in four hospitals. Methods The cTnT control samples with low, medium, and high concentrations were used for precision studies (N = 20). The calibration materials at five levels ranging from 8 ng/L to 10 000 ng/L were used for evaluating linearity, accuracy, and analytical measurement range. The observed and assigned values were compared using linear regression analysis. Heparinized plasma samples were tested using the Gen 5 hsTnT assay. Method comparisons were carried out on different Roche cobas analyzers in each hospital splitting remnant specimens. Analyzer e801 performance was compared with a reference laboratory. Simultaneously, the e601 and e411 analyzers were compared with the e801. Statistical analysis was carried out using EP Evaluator and GraphPad Prism. Results The Roche e801 demonstrated imprecision of 0.5%–2.2% (N = 20), the e601 had imprecision of 0.4%–1.4% (N = 20) and the e411 had 0.8%–4.8% (N = 20) across the three levels of control samples tested. These were well below the expected imprecision criteria of ≤10% for troponins. Deming regression analysis of the 5th GEN assays showed linear relationship across platforms: y (e801 5th GEN) = 1.007x (Reference Lab 5th GEN) + 50.65, R = 0.9899 with positive bias of 8% (N = 45); y (e601 5th GEN) = 0.929x(e801 5th GEN) + (−1.426), R = 0.9995 with negative bias of 7% (N = 48); y (e411 5th GEN) = 0.942x(e801 5th GEN) + (−7.044), R = 1.000 with negative bias of 7% (N = 49). The accuracy, reportable range, and linearity of Gen 5 hsTnT were tested on e801, e611 and e411 analyzers over a measured range of 8–10 000 ng/L. Allowable systematic error (SEa) was 1.5 ng/L (conc) or 5.0% (e801); 1.5 ng/L (conc) or 7.5% (e611 and e411). The maximum deviation for a mean recovery from 100% was 2.4% (e801), 2.7% (e601) and 6.2% (e411). The 5 of 5 mean recoveries were accurate within the SEa for all the three analyzers. The 15 out of 15 results were accurate within the allowable total error (TEa) of 3 ng/L (conc) or 10.0% (e801), 3 ng/L (conc) or 15.0% (e601 and e411). The three analyzers passed reportable range and accuracy tests, and the results were linear. Conclusion The TEa was determined from instrument specific imprecision, systemwide medical decision limits and parameters of rule out criteria. The standardization of analytical performance characteristics is important in a large health system with instrument variability and provision of transfer of care across health systems.

A-149 Performance Characteristics of VerifyNow PRU and ARU Tests During a Clinical Method Verification Exercise

Abstract Background Dual antiplatelet therapy with aspirin and a platelet P2Y12 receptor antagonist is indicated for the prevention of atherothrombotic events in patients with acute coronary syndromes and for those undergoing percutaneous coronary intervention procedure. Two different assays are available in the VerifyNow Test device for monitoring the anti-platelet therapy response. The aspirin assay (ARU Test) utilizes arachidonic acid as an agonist and is sensitive to acyl salicylic acid. The P2Y12 assay (PRU test) is sensitive to thienopyridines (such as clopidogrel, prasugrel, ticagrelor) and uses adenosine diphosphate (ADP) as an agonist. The PRU test is a non-waived test and ARU test is a waived test. A comprehensive method performance verification is required for the PRU test. We evaluated performance characteristics of the VerifyNow PRU Test and ARU Test for clinical use. Methods The standard precision and accuracy study using two levels (low and high) of quality control materials were performed (N = 10) for both the assays. Alternative qualitative method comparison was carried out (N = 49) using the VerifyNow PRU Test in a reference lab and in-house. The in-house inter-instrument comparisons were carried out (N = 42). The blood samples were obtained from patients taking P2Y12 inhibitors and from normal donors not on any drugs. The results of the PRU Test are expressed in P2YI2 reaction units (PRU). The VerifyNow-Aspirin assay results are expressed as aspirin reaction units (ARU). The statistical analysis to evaluate performance characteristics was carried out using the EP Evaluator. Results The VerifyNow device 1561 precision characteristics for low QC (Obs Mean = 1.5 PRU (N = 10), SD = 0.2; 95% CI for Obs Mean 1.0 to 2.0 PRU) and high QC (Obs Mean = 258.3 PRU (N = 10), SD = 8.2; 95% CI for Obs Mean 252.4 to 264.2 PRU) were within acceptable performance limits. The CV for high QC was 3.2%. The ARU test also demonstrated high precision for two levels (Low QC Obs Mean = 350ARU (N = 10), SD = 0.0); High QC Obs Mean = 583.3 (N = 10), SD = 7.2; 95% CI for Obs Mean = 578.1–588.5) with a CV of 1.2%. The PRU Test Method PRU 1561 In House vs Ref Method Verify Now PRU Test (N = 49) demonstrated overall agreement of 93.9% with positive agreement (intended use population on drugs, N = 27) of 92% and negative agreement (normal non-drug group, N = 22) of 95.8% (Cohen’s Kappa = 87.8%; Kappa >75% indicates “"high” agreement). The McNemar Test for Symmetry passed (P > 0.999, Fisher’s Exact test) in this qualitative method comparison. A value of P < 0.05 would suggest the methods are significantly different. The other in-house instrument 1562 also demonstrated comparable or better performance characteristics. Conclusion It is important to confirm the medical decision limit for the intended use population and determine acceptance criteria while considering biologic variability. However, the reference range verifications with drug responders and non-responders are not always feasible and cost-effective. Based on the qualitative method comparison in this study, we have transferred the medical decision limits from the peer group. The in-house multi-instrument comparison should be repeated every six months to monitor the performance of the instruments.

Measurement of sweat lactate levels in exercise and non-exercise activities using capillary electrophoresis system with contactless conductivity detection and cyclodextrin-modified buffer

This study presents the analysis of lactate in sweat using capillary electrophoresis with contactless conductivity detection. Two cyclodextrin (CD) compounds, carboxymethyl-β-cyclodextrin sodium salt (CM-β-CD) and heptakis (2,3,6-tri-O-benzoyl)-β-cyclodextrin (TRIME-β-CD), were evaluated as buffer modifiers for the separation of chloride, nitrate, sulfate, oxalate, maleate, acetate, lactate, and phosphate anions. The concentrations of these modifiers were tested at 0.05, 0.1, and 0.5 mM for eight anions separation. The buffer was 40.0 mM MES/L-His (pH 6.0) with 0.05 mM CTAB, resulting in anodic separation. Due to separation efficiency in resolving lactate and phosphate peaks, TRIME-β-CD at a concentration of 0.1 mM was selected as the suitable buffer additive for sweat lactate analysis. The calibration of the developed method, using maleate as an internal standard, resulted in the linear ranges of 0.1–5.0 mM, r2 of 0.9999, and an instrumental LOD of 0.042 mM. The intra-day and inter-day precisions of the relative migration time (RMT) were 0.3–0.4% and 3–4% RSD, respectively. Lactate levels in sweat samples from the same group of volunteers were measured after two distinct activities: exercise (after running) and non-exercise (after sauna). For non-exercise activities, lactate concentrations ranged from 15 ± 1 mM to 36 ± 2 mM. While, after exercise, the concentrations were between 26 ± 1 mM and 136 ± 1 mM. Further evaluation of the stability of sweat storage for monitoring lactate contents, it was found that the changes in lactate levels were insignificant when stored for up to 24 weeks at 4 °C. This was confirmed by paired t-tests (t-stat ragnes from -0.59 to 2.49; t-critical = 2.57, P = 0.05). The measured lactate concentrations from volunteers were significantly higher, approximately 2–6 times, in exercise activities compared to non-exercise activities. This substantial difference in lactate secretion indicates a clear distinction in the factors that stimulate sweat lactate production. To evaluate the intensity of exercise based on lactate levels in sweat, a proposed cut-off level of 36.0 mM, with a decision limit of 40.0 mM, can be proposed.

Identification of cortisol metabolites with LC-MS/MS in plasma, skin mucus, bile and faeces for stress evaluation of farmed Atlantic salmon

As a stress hormone, cortisol and more recently its metabolites are analysed when assessing fish stress and welfare status, although the exact identity of these metabolites is not clearly defined for the Atlantic salmon. LC-MS/MS techniques, owing to their specificity, sensitivity and ability to simultaneously identify and measure several relevant compounds, can be useful tools for this purpose. Using the guidelines provided by the European Decision no. 657/2002/EC for validation, the LC-MS/MS method presented here, can reliably identify and quantify cortisol and five of its metabolites (5β-THF, cortisone, 5β-DHE, 5β-THE and β-cortolone) in bile and faeces, and cortisol and cortisone in skin mucus and blood plasma of farmed Atlantic salmon within 15 min. Identified as the most predominant compound in faeces and bile, 5β-THE is proposed as a candidate stress biomarker when using these matrices. A decision limit (CCα) below 5 ng/mL, a detection capability (CCβ) and a limit of detection (LOD) below 10 ng/mL and a limit of quantitation (LOQ) below 30 ng/mL were typically obtained for most of the compounds. The concentrations of these compounds measured in either non-stressed or stressed fish were all above the CCα, CCβ, LOD and the LOQ of the method. The latter consequently demonstrated significant difference in cortisol metabolites concentrations between the two groups of fish. The present study further demonstrates that pooling of samples from several individuals could provide reliable results for farmed fish stress evaluation, when sample materials are insufficient in terms of quantity.

Multi-class/residue method for determination of veterinary drug residues, mycotoxins and pesticide in urine using LC-MS/MS technique

BackgroundVeterinary drugs are widely used in animals to prevent diseases and are a complex set of drugs with very different chemical properties. Multiclass and multi-residue methods for simultaneous detection of residues from veterinary drugs and contaminants in urine are very rare or non-existent. Therefore, the aim of this study was to develop and validate a sensitive and reliable quantitative LC-MS/MS method for simultaneous determination of a wide range of veterinary drug and pesticide residues and mycotoxins in bovine urine. This involved 42 veterinary drug residues (4 thyreostats, 6 anabolic hormones, 2 lactones, 10 beta agonists, 15 antibiotics, 5 sulphonamides), 28 pesticides and 2 mycotoxins. Stable isotopically labelled internal standards were used to facilitate effective quantification of the analytes. Analysis was performed in both positive and negative ionization modes with multiple reaction monitoring transitions over a period of 12 min.ResultsThe parameters validated included linearity, limit of detection (LOD), limit of quantification (LOQ), detection capability (CCβ), decision limit (CCα), stability, accuracy and precision. The process followed guidelines of the regulation 2021/808/EC. The calibration curves were linear with coefficient of correlation (R2) from 0.991 to 0.999. The LODs were from 0.01 to 2.71 µg/L, while the LOQs were from 0.05 to 7.52 µg/L. The CCα and CCβ were in range 0.05–12.11 µg/L and 0.08–15.16 µg/L. In addition, the average recoveries of the spiked urine samples were from 71.0 to 117.0% and coefficient of variation (CV) < 21.38% (intraday and interday).ConclusionA new isotopic LC-MS/MS method has been developed, validated and applied for identification and quantification of 72 residues of veterinary drugs and pesticides and other contaminants such as mycotoxins in bovine urine. The most appropriated sample preparation procedures involved sodium acetate buffer, enzymatic hydrolysis using β-glucuronidase and cleanup solid phase extraction with OASIS SPE cartridges. The parameters were satisfactorily validated fulfilling requirements under Regulation 2021/808/EC. Consequently, the method could be used in routine analysis of bovine urine samples for simultaneous detection of veterinary drug and pesticide residues as well as contaminants such as mycotoxins.

The alcohol biomarker phosphatidylethanol (PEth) – test performance and experiences from routine analysis and external quality assessment

Phosphatidylethanol (PEth) are membrane molecules formed from phosphatidylcholine and ethanol through transphosphatidylation catalyzed by phospholipase D. Measurement of the main PEth form 16:0/18:1 is used as a specific and sensitive alcohol biomarker, since its formation requires ethanol, it accumulates in the blood upon repeated ethanol exposure, and it is only slowly eliminated during abstinence. PEth formation correlates with alcohol intake at the population level, albeit with considerable inter-individual variation as for the half-life during withdrawal. Over the past decade, the use of PEth has increased significantly and the applications have broadened. In Sweden, routine decision limits and the interpretation of test results for PEth were harmonized in 2013, using < 0.05 µmol/L (∼35 µg/L) as the recommended lower reporting limit and values > 0.30 µmol/L (∼210 µg/L) to indicate regular high alcohol intake. Routine test results show a large variation with about half being < 0.05 µmol/L and some even exceeding 10 µmol/L. In 2013, an external quality assessment (EQA) scheme for PEth 16:0/18:1 measurement in whole blood was also started (Equalis, Uppsala, Sweden), presently involving 56 laboratories from 13 countries. The agreement of PEth results between the laboratories has gradually improved to a CV < 15%. The current clinical and scientific information suggests that PEth values below the lower reporting limit (typically ∼0.03–0.05 µmol/L, or ∼20–35 µg/L) indicates sobriety or only low or occasional alcohol consumption, while regular high alcohol intake at levels corresponding to harmful drinking is required in most cases to reach PEth values > 0.30 µmol/L.

Reference Values for Videofluoroscopic Measures of Swallowing: An Update.

It is essential that clinicians have evidence-based benchmarks to support accurate diagnosis and clinical decision making. Recent studies report poor reliability for diagnostic judgments and identifying mechanisms of impairment from videofluoroscopy (VFSS). Establishing VFSS reference values for healthy swallowing would help resolve such discrepancies. Steele et al. (2019) released preliminary reference data for quantitative VFSS measures in healthy adults aged < 60 years. Here, we extend that work to provide reference percentiles for VFSS measures across a larger age span. Data for 16 VFSS parameters were collected from 78 healthy adults aged 21-82 years (39 male). Participants swallowed three comfortable sips each of thin, slightly, mildly, moderately, and extremely thick barium (20% w/v). VFSS recordings were analyzed in duplicate by trained raters, blind to participant and task, using the Analysis of Swallowing Physiology: Events, Kinematics and Timing (ASPEKT) Method. Reference percentiles (p2.5, 5, 25, 50, 75, 95, and 97.5) were determined as per Clinical and Laboratory Standards Institute EP28-A3c guidelines. We present VFSS reference percentile tables, by consistency, for (a) timing parameters (swallow reaction time; the hyoid burst-to-upper esophageal sphincter (UES)-opening interval; UES opening duration; time-to-laryngeal vestibule closure (LVC); and LVC duration) and (b) anatomically scaled pixel-based measures of maximum UES diameter, pharyngeal area at maximum pharyngeal constriction and rest, residue (vallecular, pyriform, other pharyngeal locations, total), and hyoid kinematics (X, Y, XY coordinates of peak position; speed). Clinical decision limits are proposed to demarcate atypical values of potential clinical concern. These updated reference percentiles and proposed clinical decision limits are intended to support interpretation and reliability for VFSS assessment data. https://doi.org/10.23641/asha.24043041.

Simulated effects of ferritin screening on C-reactive protein levels in recruited blood donors.

Ferritin is commonly measured to evaluate iron stores in the body. Some countries have added or considered adding ferritin lower bounds to donor eligibility criteria. Ferritin is also elevated by inflammation. The main goal of this study is to estimate how different ferritin cut-offs would affect the proportion of donors with a C-reactive protein (CRP) level over 3 mg/L, which is the decision limit of the highest chronic cardiovascular risk. To simulate recruitment of new blood donors, we selected participants from two Finnish general population cohorts, namely FINRISK 1997 (n = 5369) and Health 2000 (n = 3278), that would likely fulfil the selection criteria of blood donation. We then calculated the proportion of individuals with high-sensitivity CRP values above 3 mg/L, over a range of ferritin values. We found that for several ferritin cut-offs the proportion of potential donors with CRP > 3 mg/L would rise by a statistically significant amount. The trend was significant and similar for all subgroups but weaker for non-menstruating women as well as men. Our results show that screening a population of potential blood donors with ferritin cut-offs raises the number of people with CRP > 3 mg/L within the blood donor population.

Quantification of Urine and Plasma Porphyrin Precursors Using LC-MS in Acute Hepatic Porphyrias: Improvement in Routine Diagnosis and in the Monitoring of Kidney Failure Patients.

The quantification of delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) in urine are the first-line tests for diagnosis and monitoring of acute hepatic porphyrias (AHP). Ion-exchange chromatography (IEC), which is time- and staff-consuming and limited to urine, is still the preferred method in many specialized laboratories, despite the development of mass spectrometry-based methods. We describe a new LC-MS method that allows for rapid and simple quantification of ALA and PBG in urine and plasma with an affordable instrument that was used to analyze 2260 urine samples and 309 blood samples collected in 2 years of routine activity. The results were compared to those obtained with IEC, and urine reference ranges and concentrations in asymptomatic carriers were determined. Plasma concentrations were measured in healthy subjects and subgroups of symptomatic and asymptomatic AHP carriers. In urine, the clinical decision limits were not impacted by the change of method despite discrepancies in low absolute concentrations, leading to lower normal values. Two-thirds of asymptomatic AHP carriers (with the exception of coproporphyria carriers) showed an increased urine PBG concentration. Urine and plasma levels showed a good correlation except in patients with kidney disease in whom the urine/plasma ratio was relatively low. We described an LC-MS based method for the routine diagnosis and monitoring of AHP that allows for the detection of more asymptomatic carriers than the historical method. Blood analysis appears to be particularly relevant for patients with kidney disease, where urine measurement underestimates the increase in ALA and PBG levels.

Residue analysis of nitrofuran metabolites in five food commodities from Croatia using ultra-high performance liquid chromatography–tandem mass spectrometry

The aim of the current study was to validate a screening and confirmatory method for the determination of nitrofuran residues: 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 1-aminohydantoin (AHD), semicarbazide (SEM), 3,5-dinitrosalicylic acid hydrazine (DNSAH), nifurpirinol (NPIR), nifuroxazide (metabolite 4-HBH), and nitrovin (metabolite AMG) in muscle, milk, eggs, honey, and casings, in accordance with the legislation 2002/657/EC and 2021/808/EC. Sample preparation included hydrolysis and derivatization, followed by ethyl acetate extraction and measurement by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Validation parameters specified in this method are specificity/selectivity, trueness, precision, decision limit (CCα), and detection capability (CCβ). In the screening method, the CCβ value ranged from 0.275 to 0.582 µg kg-1, while in the confirmatory method, the CCα value ranged from 0.264 to 0.407 µg kg-1. The recovery values ranged from 93.5% to 127.5%. A total of 497 routine samples from the National Residue Monitoring Control Plan (NRCP) and border inspection were analysed from 2021 to 2022. SEM was detected in a total of 11 samples. Only four samples had a concentration above the Reference Point for Action (RPA = 0.5 µg kg-1). The validated method is suitable for monitoring nitrofuran residues at levels below the recently established RPA.

Ionic liquid-based dispersive liquid-liquid microextraction of anthelmintic drug residues in small-stock meat followed by LC-ESI-MS/MS detection.

An ionic liquid-based dispersive liquid-liquid microextraction (IL-DLLME) of 20 anthelmintic drugs followed and detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed, optimized, and validated. The parameters affecting the anthelmintic extraction efficiencies such as selection of extraction solvent (ionic liquids), selection of disperser solvent, volume of extraction solvent, volume of disperser solvent, pH of the aqueous phase, extraction time, salt addition, and centrifugation time were optimized. Validation was conducted according to ISO/IEC 17025:2017 and Commission Implementing Regulation (EU) 2021/808 of 22 March 2021. Validation parameters such as calibration function, matrix effect, limit of detection (LOD), limit of quantification (LOQ), decision limit (CCα), accuracy, and precision were established. Coefficient of determination (R 2) values ranging from .99938 to .99995 were obtained using the matrix calibration curve spiked at 0, 0.25, 1.0, 1.5, and 2.0 times MRL. The LODs and LOQs were calculated using the standard deviation of the response and the slopes of the calibration curves ranged from 0.35 to 26.1 μg/kg and from 1.2 to 87.0 μg/kg, respectively, and were dependent on calibration range. The CCα values ranged from 23 to 1022.0 μg/kg and are also dependent on the MRL concentration levels. The coefficient of variation (CV) values calculated are within the reproducibility range of 16%-30% adapted from the Horwitz Equation CV = 2(1-0.5 log C) and ranged from 1.7% to 16.9%. The developed and validated and the standard QuEChERS method were compared. The IL-DLLME LC-MS/MS method was applied to 32 small stock (18 caprine [goat] and 14 ovine [sheep]) liver samples received from municipal abattoirs at Botswana National Veterinary Laboratory for the analysis of anthelmintic drug residues. The results obtained indicated that the anthelmintic drug residues were all below the detection capability, and therefore, the samples were passed as fit for human consumption.

Renal function, sex and age influence purines and pyrimidines in urine and could lead to diagnostic misinterpretation

Glomerular filtration rate (GFR) is commonly used in clinical practice for the diagnosis and follow-up of chronic kidney disease. Screening for inborn errors of metabolism (IEM) is based on analysis of biomarkers in urine, reported by their ratio to urinary creatinine (crn). Impaired renal function may complicate the interpretation of several biomarkers used for screening of IEM. Our goal was to investigate the influence of kidney function, in terms of measured GFR (mGFR) on purines and pyrimidines in urine, in addition to the relationship to sex, age, pH and ketosis. Children (n = 96) with chronic kidney disease (CKD), in different CKD stages, were included. Urine samples were obtained prior to the injection of iohexol. Serum samples at 7 time-points were used to calculate mGFR based on iohexol plasma clearance. The association with sex, age, ketosis and pH was examined in samples of the laboratory production from 2015 to 2021 (n = 8192). Age was a highly significant covariate for all markers. GFR correlated positively to several purines and pyrimidines; the ratios hypoxanthine/crn, xanthine/crn and urate/crn (p = 2.0 × 10−14, < 3 × 10−15 and 7.2 × 10−4, respectively), and the ratios orotic acid/crn, uracil/crn, and carbamyl-β-alanine/crn (p = 0.03, 1.4 × 10−6 and 0.003, respectively). The values of urate/crn, xanthine/crn, uracil/crn, and carbamyl-β-alanine/crn were higher in females above 16 years of age. Ketosis and pH influenced some markers. In conclusion, decreased renal function interferes with the excretion of urinary purines and pyrimidines, and this could change decision limits substantially, e.g. result in false negative results in Lesch-Nyhan syndrome. SynopsisGFR influences purines and pyrimidines in urine.Clinical Trial Registration: ClinicalTrials.gov, Identifier NCT01092260, https://clinicaltrials.gov/ct2/show/NCT01092260?term=tondel&rank=2

A rapid and simple multi-residue methodology for quantification of anthelmintic macrocyclic lactones in cow, sheep and goat milk by HPLC: development, validation and application for the real samples

Anthelmintic macrocyclic lactones (MLs) [ivermectin (IVM), abamectin (ABA), doramectin (DRM), eprinomectin (EPR) and moxidectin (MXD)] are used worldwide for the treatment of parasitic infections in large and small ruminants because of their low mammalian toxicity and higher efficacy against internal and external parasites at low dose levels. A rapid, simple and sensitive HPLC and a modified QuEChERS extraction method were developed and validated for the analysis of five anthelmintic MLs residues in cow, sheep and goat milk. The analytes were extracted from milk samples by acetonitrile deproteinization, followed by the addition of sodium sulphate and sodium chloride to generate a phase partition. HPLC separation was conducted at 50⁰C using a C18 analytical column (Zorbax, Eclipse Plus, 250 mm × 4,6 mm, 5µ). The mobile phase consisted of a mixture of acetonitrile: methanol: water (58:38:4, v/v/v), delivered by an isocratic program at the flow rate of 1.3 ml/min. The elution of five analytes was completed within 11 min. The fluorescence detector (FLD) was set at an excitation wavelength of 375 nm and an emission wavelength of 470 nm. Recoveries ranged between 78.44% and 92.66%. The quantification limits (LOQ) of anthelmintic MLs in the milk samples from 3 different ruminant species ranged between 2.01 and 3.10 ng/g. The developed method was validated in accordance with the criteria of Commission Decision 2002/657/EC. Linearity, decision limit, detection and quantification limits, recovery, accuracy, precision, sensitivity and selectivity were determined, and satisfactory results were obtained. This developed and validated method is rapid, simple, sensitive and environmentally friendly, and can be used for multiple residue analyses of anthelmintic MLs in cow, sheep and goat milk for human consumption. The quantification limits for all drugs were below the maximum residue limits (MRLs), making the method appropriate for routine testing. Finally, the application of this method was assessed using real samples of cow, sheep and goat milk. The presence of MLs was confirmed in 8 out of 70 tested milk samples, which shows the method can be successfully used to determine the residue of anthelmintic MLs in three types of milk.

O-285 The 6th edition of the WHO manual for the examination and processing of human semen: How should it affect our practice?

Abstract The WHO laboratory manual (World Health Organization 2021) has widened the scope of semen examination from primarily a prognostic tool for Medically Assisted Reproduction (MAR) by indicating the importance of evaluating men with possible disorders in male reproductive functions. Practical instructions for basic examination have been clarified and focused to measures of sperm production and motility as judged from sperm number, motility (vitality if poor motility) and sperm morphology. In addition to the recommendations by the WHO, an international standard based on the same laboratory science as the WHO manual has been published (International Organization for Standardization 2021) to facilitate for laboratories to provide reliable results of semen examination. Each ejaculate must be handled with great care before analysis begins. Time and temperature between sample collection and start of assessments is crucial. To reduce assessment variability, it is essential to ascertain that the examined aliquots are representative for the entire ejaculate and that the number of observations is sufficient to reduce the risk for significant influence of random factors. The former is handled both by aliquot volume (at least 50 µL for sperm concentration) and replicate assessment and comparison that the two assessments don’t differ too much. Regarding number of observations, 400 sperm counted in sperm concentration and in sperm motility assessments is required to reduce this source of errors to ± 10%. For assessment of sperm morphology and vitality a minimum number of observations is 200 spermatozoa. To achieve acceptable performance, thorough in-house training is necessary. There are protocols how to set up and run such schemes (Mortimer 1994, 1994). Basic medical laboratory standards also require that the laboratory regularly performs internal quality control to monitor that inter- and intra-personal variability is under control. Each laboratory should also participate in an external scheme for quality assessment (EQA) (International Organization for Standardization 2012). For the proper interpretation of semen examination results reference limits are important. However, the reference limits suggested by the WHO (World Health Organization 2021) come from a very mixed group of men and should therefore not be mistaken for true limits between fertility and infertility. The new WHO manual therefore argues for development of decision limits (when is it reasonable to act?) that are much more important than limits from a mixed population. It has been argued that semen examination must be better standardized (Björndahl et al. 2016), but the compliance has been almost non-existent (Vasconcelos et al. 2022) leading to even stronger appeals for stronger improvement of basic laboratory investigations of male reproductive functions (Björndahl et al. 2022) References Björndahl L, Barratt CL, Mortimer D, and Jouannet P. ‘How to count sperm properly': checklist for acceptability of studies based on human semen analysis. Hum Reprod 2016: 31; 227-232. Björndahl L, Barratt CLR, Mortimer D, Agarwal A, Aitken RJ, Alvarez JG, Aneck-Hahn N, Arver S, Baldi E, Bassas L, et al. Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology. Hum Reprod 2022. International Organization for Standardization. ISO 15189:2012 Medical Laboratories – Requirements for Quality and Competence 2012. Geneva. International Organization for Standardization. ISO 23162:2021 Basic semen examination — Specification and test methods 2021. ISO, Geneva. Mortimer D. Laboratory standards in routine clinical andrology. Reproductive Medicine Review 1994: 3; 97-111. Mortimer D. Practical Laboratory Andrology 1994. Oxford University Press, Oxford. Vasconcelos AL, Campbell MJ, Barratt CLR, and Gellatly SA. Do studies published in two leading reproduction journals between 2011 and 2020 demonstrate that they followed WHO5 recommendations for basic semen analysis? Hum Reprod 2022. World Health Organization. WHO laboratory manual for the examination and processing of human semen. 6th edn, 2021. World Health Organization, Geneva.

The Change in tPSA, fPSA and f/tPSA Levels in Men Undergoing Hemodialysis Effect of Hemodialysis on Serum PSA Levels

Purpose: This study aimed to assess the impact of hemodialysis treatment and different dialysis membranes with varying surface areas on serum levels of total prostate-specific antigen (tPSA), free prostate-specific antigen (fPSA), and the fPSA/tPSA ratio in patients undergoing hemodialysis treatment.&#x0D; Material and Methods: The study was conducted at the Central Laboratory of....Hospital in May, June, and July 2020. tPSA, fPSA, and fPSA/tPSA ratios measured in pre-dialysis and post-dialysis samples were determined. Correlation between pre-dialysis and post-dialysis of fPSA and tPSA levels and patients' ultrafiltrates were evaluated. The fPSA, tPSA, and fPSA/tPSA measured in pre-dialysis and post-dialysis samples grouped according to membrane type were compared.&#x0D; Results: The fPSA levels and fPSA/tPSA ratios of pre-dialysis samples were significantly lower than post-dialysis samples. tPSA values were not significantly different in pre-dialysis and post-dialysis samples. According to membrane types, it was found that pre-dialysis and post-dialysis tPSA, fPSA, and fPSA/tPSA were not significantly different. There was a positive correlation between difference in fPSA concentrations measured in pre-dialysis and post-dialysis samples and ultrafiltrate (rho=0.380). A positive correlation was found between difference in tPSA concentrations measured in pre-dialysis and post-dialysis samples and ultrafiltrate (rho=0.562).&#x0D; Conclusion: Post-dialysis fPSA values and fPSA/tPSA ratios were found to be elevated in patients receiving hemodialysis treatment due to hemoconcentration and other potential effects of dialysis. Therefore, interpreting fPSA values and fPSA/tPSA ratios in patients undergoing hemodialysis treatment according to current clinical decision limits may lead to misinterpretation. In this group of patients, the use of tPSA testing is safe as it remains unaffected by dialysis treatment.

Analysis of Aminoglycoside Antibiotics: A Challenge in Food Control.

Aminoglycosides are a widely used group of antibiotics in veterinary medicine. However, misuse and abuse of these drugs can lead to residues in the edible tissues of animals. Due to the toxicity of aminoglycosides and the exposure of consumers to the emergence of drug resistance, new methods are being sought to determine aminoglycosides in food. The method presented in this manuscript describes the determination of twelve aminoglycosides (streptomycin, dihydrostreptomycin, spectinomycin, neomycin, gentamicin, hygromycin, paromomycin, kanamycin, tobramycin, amikacin, apramycin, and sisomycin) in thirteen matrices (muscle, kidney, liver, fat, sausages, shrimps, fish honey, milk, eggs, whey powder, sour cream, and curd). Aminoglycosides were isolated from samples with extraction buffer (10 mM NH4OOCH3, 0.4 mM Na2EDTA, 1% NaCl, 2% TCA). For the clean-up purpose, HLB cartridges were used. Analysis was performed using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) with a Poroshell analytical column and a mobile phase of acetonitrile and heptafluorobutyric acid. The method was validated according to Commission Regulation (EU) 2021/808 requirements. Good performance characteristics were obtained for recovery, linearity, precision, specificity, and decision limits (CCα). This simple and high-sensitivity method can determine multi-aminoglycosides in various food samples for confirmatory analysis.

NT-proBNP Reference Intervals in Healthy U.S. Children, Adolescents, and Adults.

N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a cardiac biomarker used in the clinical management of heart failure. We sought to create updated reference intervals for NT-proBNP for healthy US children, adolescents, and adults. We identified a population of healthy individuals using the 1999 to 2004 cycles of the National Health and Nutrition Examination Survey (NHANES). We measured serum NT-proBNP in 12 346 adults and 15 752 children and adolescents with the Elecsys NT-proBNP assay on the Roche e601 autoanalyzer. We compared 4 methods for reference interval calculation, and presented the final reference intervals using the robust method partitioned by age and sex categories. NT-proBNP values were available for 1949 healthy adults and 5250 healthy children and adolescents. NT-proBNP concentrations in males and females varied according to age, being higher in early childhood, relatively lower in late adolescence, and highest through middle age and older age. Females tended to have higher NT-proBNP concentrations compared to men from late adolescence until middle age. The upper reference limit, or 97.5th percentile, for 50 to 59 year-old men was 225 ng/L (90% CI: 158 to 236), and for 50 to 59 year-old women, 292 ng/L (90% CI: 242 to 348). Among healthy individuals, NT-proBNP concentrations varied greatly according age and sex. The reference intervals presented here should inform future clinical decision limits and suggest that age- and sex-specific intervals may be necessary to more precisely characterize risk.

The regulation of threshold levels for prohibited substances in the world anti-doping program

Technological advancements in the equipment and laboratories used by anti-doping bodies means that minute levels of prohibited substances can be detected in an athlete's blood or urine. This had led to an increase in athletes testing positive for prohibited substances where the quantity of that substance in the athlete's sample is very low. This article will consider the role that decision limits and minimum reporting levels play with respect to prohibited substances identified in the World Anti-Doping Program. Recent CAS awards are analyzed to determine whether, and how, the issue of threshold requirements for prohibited substances should be further regulated.

Determination of quinolones, macrolides, sulfonamides and tetracyclines in honey using QuEChERS sample preparation and UHPLC-MS/MS analysis

Monitoring beehive health and timely prevention of possible infections by bacteria, mould, viruses or parasites is exceptionally important in beekeeping. Antibiotics originating from the environment can be found in honey and due to improper beekeeping practices. Enabling the detection of antibiotic residues in honey and suppressing the development of antibiotic resistance require the development of a sensitive multi-class method for the determination of antibiotics. For the purpose of analysing honey, a screening and confirmatory method for the determination of 36 antibiotics was developed. The QuEChERS procedure and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was selected to achieve high sensitivity and selectivity. Validation according to the new Commission Implementing Regulation (EU) 2021/808 included the following performance characteristics: selectivity, trueness, precision, decision limit (CCα), detection capability (CCβ) and relative matrix effect. The method was validated in the measurement range from 1 to 10 μg/kg, where maximum trueness and acceptable coefficients of variation were achieved. The detection capability (CCβ) equals the lowest concentration level for all analytes, ranging from 0.1 μg/kg to 2.5 μg/kg, as the false compliant rate less than 5% was confirmed at this level of interest. For unauthorised pharmacologically active substances, the CCα was determined and ranged from 0.16 μg/kg for sulfaquinoxaline to 3.67 μg/kg for difloxacin. The high matrix influence of floral and chestnut honey indicated the need for quantitative analysis using the matrix calibration curve.