Analytical Imprecision Research Articles

Simultaneous Quantification of Serum Symmetric Dimethylarginine, Asymmetric Dimethylarginine and Creatinine for Use in a Routine Clinical Laboratory.

Background Symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA) are naturally occurring amino acids classed as uremic toxins by the EUTOX working group. SDMA is principally excreted through the kidneys and is a well-known renal function marker, ADMA is a potent inhibitor of nitric oxide production. Here, we describe the development of a rapid and sensitive liquid chromatography tandem mass spectrometry method for simultaneous measurement of SDMA, ADMA and creatinine. Method Serum samples were prepared by protein precipitation and dilution with acetonitrile prior to injection onto a Waters TQS-Micro. Multiple reaction monitoring was used to detect SDMA, ADMA, creatinine, and their corresponding internal standard transitions after separation with a HILIC analytical column. Sample stability and intra-individual variation studies were also assessed following ethical approval. Results The retention time for creatinine was 0.43, SDMA 1.10 and ADMA 1.14 minutes. Mean recovery for creatinine was 103%, SDMA was 100% and ADMA was 103%, matrix effects were minimal (<6%). Lower limit of quantitation for creatinine and SDMA/ADMA was 17.5 µmol/L and 0.1 µmol/L respectively. Analytical imprecision showed a coefficient of variation <10% for all analytes across the working range of the assays. Intra-individual variation for creatinine was 4.7%, SDMA 7.5% and ADMA 7.6%. Discussion We have developed a rapid assay for LC-MS/MS measurement of SDMA, ADMA and creatinine in a routine clinical laboratory. It is simple, reproducible, and easy to perform. The stability of SDMA and ADMA pre- and post-centrifugation allows for their routine use without any special sample handling requirements.

An accurate measurement method for serum homocysteine measurement by ID-LC/MS/MS and the application of external quality assessment

BackgroundSerum homocysteine (Hcy) measurement accuracy is essential for detecting and diagnosing cardio-cerebrovascular illnesses. Although many different methods have been developed to determine the concentration of Hcy, those different assays showed nonequivalent results. This study aimed to develop an accurate and precise isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC/MS/MS) method for serum homocysteine (Hcy) and assign values for reference materials used in external quality assessment (EQA) programs. MethodsDifferent concentrations of homocysteine calibration solutions were prepared with d4-Hcy as the internal standard. This method employed DTT to reduce protein-bound Hcy, followed by protein precipitation, and gradient elution on a Supelcosil LC-CN column for chromatographic separation, and multiple reaction monitor (MRM) mode with electrospray ionization (ESI) for mass spectrometric detection. After optimized ID-LC/MS/MS parameters, imprecision, trueness, the limit of quantification (LoQ), the limit of detection (LoD), and measurement uncertainty were evaluated to check the methodological performance. The established method was employed in assigning values to EQA samples, which were sent to 63 clinical laboratories in Beijing to measure Hcy. ResultsAt 10.69,15.99, and 37.80 μmol/L levels, the present method demonstrated analytical imprecision of 0.57 %, 0.65 %, and 0.57 %, and recoveries of 99.67 % to 100.21 %, respectively. The bias between the target values of GBW (Guojia Biaozhun Wuzhi) were − 0.79 % to 0.62 %. The LoQ and LoD for Hcy were 0.36 nmol/L and 0.27 nmol/L. The method had an uncertainty (U 95 %) of 1.34 % to 1.48 %. For the three levels of EQA samples, the percentage of laboratories meeting the trueness evaluation criteria (±10 %) was 81.67 %, 83.33 %, and 71.67 % respectively. ConclusionsWith optimal method precision and trueness, the ID-LC/MS/MS method to measure serum Hcy can be used for value assignment of EQA samples, which can provide reliable data for monitoring the accuracy of clinical laboratory for Hcy measurement.

Toward Analytical Performance Specifications for Immunosuppressive Drug Quantification in Transplantation: An Opinion Article.

Analytical methods require performance that meets the clinical needs. Different approaches for setting up permissible analytical imprecision goals (pCVA%) for drug analyses have been reported. The aim of this study was to calculate the pCVA% for cyclosporine, tacrolimus, everolimus, sirolimus, and mycophenolic acid using 4 alternative approaches, to compare the results and to critically discuss advantages and disadvantages of each model. The approaches to evaluate pCVA% were (A) based on biological variation observed in routine measurement results between 2022 and 2023 in the authors' laboratory, (B) derived from the terminal elimination half-life and dosing interval of the drugs, and (C and D) explored from the width of the therapeutic ranges (TR) by the 2 methods. For approach A, routine measurement data for cyclosporine and tacrolimus, obtained through liquid chromatography-tandem mass spectrometry and electrochemiluminescence immunoassays, were evaluated separately. The 4 alternative approaches for deriving pCVA% yielded similar results, for cyclosporine and tacrolimus in an analytical method dependent manner. The average pCVA% was 5.2%, 5.6%, 5.1%, 4.8%, and 7.7% for cyclosporine, tacrolimus, everolimus, sirolimus, and mycophenolic acid, respectively. The most challenging goals were those using TR-related approaches, while those using the biological variation approach were most easily achievable. Approach B resulted in more stringent goals for drugs with longer elimination half-lives (eg, everolimus and sirolimus). There is no single ideal approach for setting goals of drug analysis. However, the pCVA% values derived from the various approaches are similar and confirm that a <6% target proposed by the International Association of Therapeutic Drug Monitoring and Clinical Toxicology is adequate and realistic in combination with state-of-the-art measurement technologies. In the authors' opinion, approaches based on the width of the TR are preferable, as they represent a common basis for clinical decisions and reflect elements of biological variation and analytics used to establish the TR.

PrecivityAD2™ Blood Test: Analytical Validation of an LC-MS/MS Assay for Quantifying Plasma Phospho-tau217 and Non-Phospho-tau217 Peptide Concentrations That Are Used with Plasma Amyloid-β42/40 in a Multianalyte Assay with Algorithmic Analysis for Detecting Brain Amyloid Pathology.

Alzheimer's disease (AD) is a progressive irreversible neurodegenerative disorder that represents a major global public health concern. Traditionally, AD is diagnosed using cerebrospinal fluid biomarker analysis or brain imaging modalities. Recently, less burdensome, more widely available blood biomarker (BBM) assays for amyloid-beta (Aβ42/40) and phosphorylated-tau concentrations have been found to accurately identify the presence/absence of brain amyloid plaques and tau tangles and have helped to streamline AD diagnosis. However, few BBMs have been rigorously analytically validated. Herein, we report the analytical validation of a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) multiplex method for quantifying plasma phosphorylated-tau217 (p-tau217) and non-phosphorylated-tau217 (np-tau217) peptide concentrations. We combined the p-tau217/np-tau217 concentrations ratio (%p-tau217) and the previously validated LC-MS/MS multiplex assay for plasma Aβ42/40 into a new multianalyte assay with algorithmic analysis (MAAA; PrecivityAD2™ test) that identifies brain amyloid status based on brain amyloid positron emission tomography. We found (a) the %p-tau217 assay is precise, accurate, sensitive, and linear over a wide analytical measurement range, and free from carryover and interference; (b) the pre-analytical specimen collection, processing, storage, and shipping conditions that maintain plasma tau peptide stability; and (c) using the measured analytical imprecision for plasma Aβ42/40 and p-tau217/np-tau217 levels in a worst-case scenario model, the PrecivityAD2 test algorithm for amyloid pathology classification changed for only 3.5% of participants from brain amyloid positive to negative, or from negative to positive. The plasma sample preparation and LC-MS/MS methods underlying the PrecivityAD2 test are suitable for use in the clinical laboratory and valid for the test's intended purpose: to aid in the diagnostic evaluation of individuals aged 55 and older with signs or symptoms of mild cognitive impairment or dementia.

LC-MS-MS quantification of Δ8-THC, Δ9-THC, THCV isomers and their main metabolites in human plasma.

In recent years, potential therapeutic applications of several different cannabinoids, such as Δ9-tetrahydrocannabinol (Δ9-THC), its isomer Δ8-THC and Δ9-tetrahydrocannabivarin (Δ9-THCV), have been investigated. Nevertheless, to establish dose-effect relationship and to gain knowledge of their pharmacokinetics and metabolism, sensitive and specific analytical assays are needed to measure these compounds in patients. For this reason, we developed and validated an online extraction high-performance liquid/liquid chromatography-tandem mass spectrometry (LC/LC-MS-MS) method for the simultaneous quantification of 13 cannabinoids and metabolites including the Δ8 and Δ9 isomers of THC, THCV and those of their major metabolites in human plasma. Plasma was fortified with cannabinoids at varying concentrations within the working range of the respective compound and 200 µL was extracted using a simple one-step protein precipitation procedure. The extracts were analyzed using online trapping LC/LC-atmospheric pressure chemical ionization-MS-MS running in the positive multiple reaction monitoring mode. The lower limit of quantification ranged from 0.5 to 2.5 ng/mL, and the upper limit of quantification was 400 ng/mL for all analytes. Inter-day analytical accuracy and imprecision ranged from 82.9% to 109% and 4.3% to 20.3% (coefficient of variance), respectively. Of 534 plasma samples following controlled oral administration of Δ8-THCV, 236 were positive for Δ8-THCV (median; interquartile ranges: 3.5 ng/mL; 1.8-11.9 ng/mL), 383 for the major metabolite (-)-11-nor-9-carboxy-Δ8-tetrahydrocannabivarin (Δ8-THCV-COOH) (95.4 ng/mL; 20.7-328 ng/mL), 260 for (-)-11-nor-9-carboxy-Δ9-tetrahydrocannabivarin (Δ9-THCV-COOH) (5.8 ng/mL; 2.5-16.1 ng/mL), 157 for (-)-11-hydroxy-Δ8-tetrahydrocannabivarin (11-OH-Δ8-THCV) (1.7 ng/mL; 1.0-3.7 ng/mL), 49 for Δ8-THC-COOH (1.7 ng/mL; 1.4-2.3 ng/mL) and 42 for Δ9-THCV (1.3 ng/mL; 0.8-1.6 ng/mL). We developed and validated the first LC/LC-MS-MS assay for the specific quantification of Δ8-THC, Δ9-THC and THCV isomers and their respective metabolites in human plasma. Δ8-THCV-COOH, 11-hydroxy-Δ8-THCV and Δ9-THCV-COOH were the major Δ8-THCV metabolites in human plasma after oral administration of 98.6% pure Δ8-THCV.

CytogeneticsComparability of Beckman Coulter Access Cardiac Troponin I Assays

Abstract Cardiac troponins (cTn) are the most sensitive and specific biomarkers of myocardial injury. The rise and/or fall of cardiac troponins above the 99th percentile of the upper reference limit (URL) is required in the assessment of acute myocardial infarction. In our institution, high sensitivity troponin I (hsTnI) is performed on four Access 2 Immunoassay Systems and three DXI 800 Access Immunoassay Systems. As a regional medical center, we accept patients from outside hospitals where contemporary cardiac troponin I (cTnI) were performed. The results from different assays and platforms may disagree and lead to incorrect explanation of the results as false rise and/or fall of cTn. To harmonize results among these assays, we analyzed the variation between the two assays of cTnI and hsTnI, and the concordance of same assay hsTnI on different instruments using patient specimens. To investigate the between-method variance, 424 patient specimens with troponin levels ranging from 0-23370 ng/L were used to measure both cTnI and hsTnI on DXI 800 instruments. To investigate the within-method agreement, 43 patient specimens with troponin levels ranging from 2-114394 ng/L were used to measure hsTnI on Access 2 and DXI instruments. The between-method comparison of cTnI and hsTnI on DXI showed a slope of 1.003 with intercept of -43.31 and correlation coefficient (CC) of 0.9590. The mean bias was -5.1%, which was within the analytical imprecision range determined in our laboratory (2.2%-10.7%). However, comparison of cTnI and hsTnI in patients with troponin &amp;lt;20ng/L (n=103), the 99th percentile of the URL for males, showed the slope was 1.325 with intercept of -0.97 and CC of 0.7462. The mean bias was 1.91 (21.53%), which was much larger than the analytical imprecision. This is consistent with the fact that cTnI results are not reliable at the lower level due to the imprecision problem. Comparison of cTnI and hsTnI in patients with troponin &amp;gt;20ng/L (n=321) showed the slope was 1.006 with intercept of –60.09 and CC of 0.9576. The mean bias was -54.30 (-5.23%), which was within the analytical imprecision range. The within-method comparison of hsTnI on Access 2 and DXI 800 showed the slope was 1.138 with intercept of -633.0 and CC of 0.9888. The mean bias was 2126.4 (10.6%), within the analytical imprecision range. These data indicate that cTnI results above the 99th percentile URL are comparable with hsTnI results if both are tested on DXI 800, and hsTnI results tested on Access 2 and DXI 800 are comparable. Thus, to use the cardiac troponins assay properly, we suggest the laboratory report cardiac troponins with the assay and instrument names to reduce confusion during results comparison.

Impact of analytical imprecision and bias on patient classification.

An increase in analytical imprecision and/or the introduction of bias can affect the interpretation of quantitative laboratory results. In this study, we explore the impact of varying assay imprecision and bias introduction on the classification of patients based on fixed thresholds. Simple spreadsheets (Microsoft Excel) were constructed to simulate conditions of assay deterioration, expressed as coefficient of variation and bias (in percentages). The impact on patient classification was explored based on fixed interpretative limits. A combined matrix of imprecision and bias of 0%, 2%, 4%, 6%, 8%, and 10% (tool 1) as well as 0%, 2%, 5%, 10%, 15%, and 20% (tool 2) was simulated, respectively. The percentage of patients who were reclassified following the addition of simulated imprecision and bias was summarized and presented in tables and graphs. The percentage of patients who were reclassified increased with increasing/decreasing magnitude of imprecision and bias. The impact of imprecision lessens with increasing bias such that at high biases, the bias becomes the dominant cause for reclassification. The spreadsheet tools, available as Supplemental Material, allow laboratories to visualize the impact of additional analytical imprecision and bias on the classification of their patients when applied to locally extracted historical results.

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-161 Analytical Performance of Reagents for Determination of Activated Partial Thromboplastin Time: A Sigma Metric Perspective

Abstract Background Sigma Metric (SM) assesses the degree of deviation of lab. tests from established specifications, combining Total Acceptable Error (ETa), imprecision and analytical bias. Quality criteria, defined by models of desirable specification, are used to determine the analytical performance of lab. tests. The concepts of biological variation are used as the preferred approach for establishing specifications. Reagents (RE) for the determination of activated partial thromboplastin time (APTT) use thromboplastin obtained from different tissue extracts and activation factors, being standardized to reduce the variability of the tests, and ensure equivalence of clinical diagnosis. This work evaluated three RE for APTT and the fulfillment of the desirable specifications of analytical quality, using the SM as an analysis tool. Material &amp; methods APTT determinations were performed on the Sysmex®CS-2500 (Siemens Healthineers) with Actin®FS (AFS), Actin®FSL (FSL), and Pathromtin®SL (PSL) RE and bi-level Control Plasma N (CN) and Ci-Trol®2 Control (CI). Two different lots were used for each RE and control (CT), and the results were analyzed in RE-CT combination. We determined (a)analytical bias (Bias), from intra-assay imprecision determined by 30 repetitions in a single analytical run and by comparison with manufacturer's data, and (b)analytical coefficient of variation (CVa), from inter-assay imprecision, by triplicate in 3 periods each 3 h, for the 2 CT levels. Analytical quality specifications for APTT were selected in the literature, adopting desirable ETa values of 4.5% and minimum of 6.7%, obtained from a database for biological variation and acceptable limit of 15%, based on CLIA-CAP. SM was calculated with formula: Sigma = ETa–Bias/CVa, with graphic representation adapted from the Westgard’s model. Results For CVa, AFS and FSL results with CN and CI were &amp;lt;1.0%. PSL presented results above 1.0% for CN and CI. All RE presented results lower than those recommended by the manufacturer, respectively 4.0% for AFS; 3.0% for FSL; and 2.8% for PSL. For bias, a greater variation was observed for the combinations of RE and CT, ranging from 1.16% to 4.07%, being related to the differences between the averages obtained and the values defined in the leaflets. For the desirable specification of ETa (4.5%), 3 combinations of RE and CT level AFS-CN, FSL-CN and PSL-CI presented Sigma values &amp;gt;3 (respectively, 3.61, 3.87 and 3.33). When using the minimum specification (6.7%), the PSL-CN combination presented a Sigma = 2.06. With the CLIA-CAP specification, all RE and CT combinations had Sigma &amp;gt;6. Discussion All RE showed similar analytical performance when compared by SM, demonstrating that different commercial formulations for APTT determination do not show differences in analytical Bias and CVa determinations. The graphical representation by SM allows you to visually compare analytical imprecision and inaccuracy values. Conclusion All RE showed no differences in analytical performance, with SM being a visual tool for analyzing and comparing RE performance.

Reducing Lipid Panel Error Allowances to Improve the Accuracy of Cardiovascular Risk Stratification.

The standard lipid panel forms the backbone of atherosclerotic cardiovascular disease risk assessment. Suboptimal analytical performance, along with biological variability, could lead to erroneous risk assessment and management decisions. The current National Cholesterol Education Program (NCEP) performance recommendations have remained unchanged for almost 3 decades despite improvements in assay technology. We investigated the potential extent of risk misclassification when the current recommendations are met and explored the impact of improving analytical performance goals. We extracted lipid panel data for 8506 individuals from the NHANES database and used these to classify subjects into 4 risk groups as recommended by the 2018 US Multisociety guidelines. Analytical bias and imprecision, at the allowable limits, as well as biological variability, were introduced to the measured values to determine the impact on misclassification. Bias and imprecision were systematically reduced to determine the degree of improvement that may be achieved. Using the current performance recommendations, up to 10% of individuals were misclassified into a different risk group. Improving proportional bias by 1%, and fixing imprecision to 3% across all assays reduced misclassifications by up to 10%. The effect of biological variability can be reduced by taking the average of serial sample measurements. The current NCEP recommendations for analytical performance of lipid panel assays allow for an unacceptable degree of misclassification, leading to possible mismanagement of cardiovascular disease risk. Iteratively reducing allowable error can improve this.

The risk of clinical misinterpretation of HbA1c: Modelling the impact of biological variation and analytical performance on HbA1c used for diagnosis and monitoring of diabetes

BackgroundThe validity of clinical interpretation of HbA1c depends on the analytical performance of the method and the biological variation of HbA1c in patients. The contribution of non-glucose related factors to the biological variation of HbA1c (NGBVA1c) is not known. This paper explores the cumulative impact of analytical errors and NGBVA1c on the risk of misinterpretation. MethodsA model has been developed to predict the risk of misinterpretation of HbA1c for diagnosis and monitoring with variables for analytical performance and levels of NGBVA1c. ResultsThe model results in probabilities of misinterpretation for a given HbA1c. Example: for an HbA1c 43 mmol/mol (6.1%), bias 1 mmol/mol (0.09%), CV 3% (2%) used for diagnosis, the probabilities of misinterpretation range from 1 to 19% depending on the contribution of NGBVA1c to the biological variation of HbA1c. ConclusionsIn addition to analytical bias and imprecision, NGBVA1c contributes to the risk of misinterpretation, but the relative impact is different per clinical application of HbA1c. For monitoring, imprecision is the predominating factor, for diagnosis both biological variation and analytical bias. Given the increasing use of HbA1c for diagnosis, increase of knowledge on NGBVA1c, decrease of analytical bias, and awareness of the risk of misinterpretation are required.

Raising the Dead Volume: Analysis of Microsamples Diluted and Corrected with Near Infrared Tracer.

Sample processing robotics require large liquid volumes to operate efficiently. Robotics are impractical in settings that deal in small specimen volumes such as pediatric laboratories. Short of manual sample handling, remedies for the current state include a redesign of current hardware or specialized adaptation for submilliliter specimens. We blindly increased the volume of plasma specimens with diluent containing a near infrared dye, IR820, to assess the change to the original specimen volume. Diluted specimens were analyzed using a variety of assay formats/wavelengths (sodium, calcium, alanine aminotransferase, creatine kinase, cholesterol, HDL cholesterol, triglyceride, glucose, total protein, creatinine), and results were compared to neat specimens. Recovery of analyte in the diluted specimens vs neat was the primary outcome measure. Mean analytic recovery from the diluted specimens across all assays ranged from 93% to 110% after correction using IR820 absorbance. Absorbance correction compared favorably to mathematical correction using known volumes of specimens and diluents (93%-107%). Pooled mean analytic imprecision across all assays ranged from 2% using the neat specimen pool to 8% when plasma pool was diluted to 30% of its original concentration. No interference from dye addition was noted, indicating the diluent was broadly applicable and chemically inert. The greatest variability in recovery was observed when respective analyte concentrations were present near the lower limits of assay detectability. Addition of a chemically inert diluent containing a near-infrared tracer is a feasible way to raise specimen dead volume and potentially automate processing and measurement of clinical analytes in microsamples.

Biological Variation and Reference Change Value of Routine Hematology Measurands in a Population of Managed Bottlenose Dolphins (Tursiops truncatus).

Hematological analyses are particularly useful in assessing a dolphin's health status. However, the creation of appropriate reference intervals for this species is difficult due to the low number of reference individuals. The implementation of individual reference intervals (iRIs) allows researchers to overcome this limitation and, moreover, also consider the within-individual variability. The aims of this study were (1) to evaluate the biological variations in some hematological measurands, including erythrocytes (RBC), hematocrit (Hct), mean cellular volume and hemoglobin content (MCV and MCHC, respectively), RBC distribution width (RDW), leukocytes (WBC), and platelets (PLT); and (2) to calculate the index of individuality (IoI) and reference change value (RCV), which enable the production of iRIs, in healthy managed bottlenose dolphins. Seven dolphins were included, and the results of six hematological exams were analyzed for each animal. Analytical imprecision (CVa), within-dolphin variation (CVi), and between-dolphins variations (CVg) were calculated, and the IoI and RCV were derived for each measurand. All the hematological measurands had intermediate IoI except WBC, for which Iol was low. The calculated RCV ranged from 10.33% (MCV) to 186.51% (WBC). The results reveal that the majority of hematological measurands have an intermediate level of individuality in dolphins, and thus the application of iRIs is appropriate. The calculated RCV can also be applied to other managed dolphins and could be useful in interpreting serial CBC exams.

An Approach for Evaluating Potential Screening Thresholds Using Biomarker Population Distribution and Analytical Imprecision.

A common approach in laboratory medicine is to use a simple but sensitive test to screen samples to identify those that require additional investigation with a more complex and informative method. Selection of screening thresholds can be guided by biomarker distribution in the tested population and the analytical imprecision of the method. A simulation using joint probabilities derived from the population distribution for galactose-1-phosphate uridylyltransferase (GALT) activity and the analytical imprecision for the GALT assay was used to estimate the number of samples that would require repeat analysis and the number of samples with possibly false-negative screening determinations due to analytical imprecision. In the case of GALT activity, screening a conservative initial threshold 6 standard deviations from the confirmation threshold can essentially eliminate the chance of a false-negative screening determination due to analytical imprecision. The trade-off is a greater number of samples requiring follow-up testing (n = 222, equivalent to 0.15% of samples annually). Selection of thresholds in a screening algorithm is informed by estimates of the number of samples that would require repeat testing and the number that could be false negative due to analytical imprecision.

TDFCAM: A method for estimating stable isotope trophic discrimination in wild populations.

Stable isotope mixing models (SIMMs) are widely used for characterizing wild animal diets. Such models rely upon using accurate trophic discrimination factors (TDFs) to account for the digestion, incorporation, and assimilation of food. Existing methods to calculate TDFs rely on controlled feeding trials that are time-consuming, often impractical for the study taxon, and may not reflect natural variability of TDFs present in wild populations.We present TDFCAM as an alternative approach to estimating TDFs in wild populations, by using high-precision diet estimates from a secondary methodological source-in this case nest cameras-in lieu of controlled feeding trials, and provide a framework for how and when it should be applied.In this study, we evaluate the TDFCAM approach in three datasets gathered on wild raptor nestlings (gyrfalcons Falco rusticolus; peregrine falcons Falco perigrinus; common buzzards Buteo buteo) comprising contemporaneous δ13C & δ15N stable isotope data and high-quality nest camera dietary data. We formulate Bayesian SIMMs (BSIMMs) incorporating TDFs from TDFCAM and analyze their agreement with nest camera data, comparing model performance with those based on other relevant TDFs. Additionally, we perform sensitivity analyses to characterize TDFCAM variability, and identify ecological and physiological factors contributing to that variability in wild populations.Across species and tissue types, BSIMMs incorporating a TDFCAM outperformed any other TDF tested, producing reliable population-level estimates of diet composition. We demonstrate that applying this approach even with a relatively low sample size (n < 10 individuals) produced more accurate estimates of trophic discrimination than a controlled feeding study conducted on the same species. Between-individual variability in TDFCAM estimates for ∆13C & ∆15 N increased with analytical imprecision in the source dietary data (nest cameras) but was also explained by natural variables in the study population (e.g., nestling nutritional/growth status and dietary composition).TDFCAM is an effective method of estimating trophic discrimination in wild animal populations. Here, we use nest cameras as source dietary data, but this approach is applicable to any high-accuracy method of measuring diet, so long as diet can be monitored over an interval contemporaneous with a tissue's isotopic turnover rate.

Barometers Behaving Badly I: Assessing the Influence of Analytical and Experimental Uncertainty on Clinopyroxene Thermobarometry Calculations at Crustal Conditions

Abstract The composition of clinopyroxene and clinopyroxene-liquid (Cpx-Liq) pairs are frequently used to calculate crystallization/equilibration pressures in igneous systems. While canonical uncertainties are often assigned to calculated pressures based on fits to calibration or test datasets, the sources of these uncertainties (and thus ways to reduce them) have not been rigorously assessed. We show that considerable uncertainties in calculated pressures arise from analytical error associated with Electron Probe Microanalyser (EPMA) measurements of Cpx. Specifically, low X-ray counts during analysis of elements with concentrations &amp;lt;1 wt% resulting from insufficient count times and/or low beam currents yield highly imprecise measurements (1σ errors of 10–40% for Na2O). Low analytical precision propagates into the calculation of pressure-sensitive mineral components such as jadeite. Using Monte Carlo approaches, we demonstrate that elemental variation resulting from analytical precision alone generates pressures spanning ~4 kbar (~15 km) for a single Cpx and ~6 kbar for a single Cpx-Liq pair using popular barometry expressions. In addition, analytical uncertainties in mineral compositions produce highly correlated arrays between pressure and temperature that have been previously attributed to transcrustal magma storage. Before invoking such geological interpretations, a more mundane origin from analytical imprecision must be ruled out. Most importantly, low analytical precision does not just affect the application of barometers to natural systems; it has also affected characterization of Cpx in experimental products used to calibrate and test barometers. The impact of poor precision on each individual measurement is often magnified by the small number of measurements made within experimental charges, meaning that low analytical precision and true variability in mineral compositions have not been sufficiently mediated by averaging multiple EPMA analyses. We compile the number of Cpx measurements performed in N = 307 experiments used to calibrate existing barometers, and N = 490 new experiments, finding ~45% of experiment charges were characterized by ≤5 individual Cpx analyses. Insufficient characterization of the true composition of experimental phases likely accounts for the fact that all Cpx-based barometers exhibit large errors (± 3 kbar) when tested using global experimental datasets. We suggest specific changes to analytical and experimental protocols, such as increased count times and/or higher beam currents when measuring low concentration elements in relatively beam resistant Cpx in experiments and natural samples. We also advocate for increasing the number of analyses per experimental charge, resolving interlaboratory analytical offsets and improving data reporting. Implementing these changes is essential to produce a more robust dataset to calibrate and test the next generation of more precise and accurate Cpx-based barometers. In turn, this will enable more rigorous investigation of magma storage geometries in a variety of tectonic settings (e.g. distinguishing true transcrustal storage vs. storage in discrete reservoirs).

Plasma Kallikrein Cleaved H-kininogen: An End-Point Marker for Contact Activation in vitro and ex vivo.

ObjectivesThe contact system consists of coagulation factor XII (FXII), prekallikrein, and H-kininogen (HK) and plays important roles in many diseases. Plasma kallikrein (PKa) cleaved HK (cHK) is a marker of contact activation. Presently, we developed a specific and precise enzyme-linked immunosorbent assay (ELISA) for determination of cHK in vitro and ex vivo.MethodsCleaved HK specific mouse monoclonal antibodies (mAbs) were generated using a peptide corresponding to the PKa cleavage site on HK as immunogen. ELISA, surface plasmon resonance analysis, and immunoprecipitation established the specificity of the antibody, which subsequently was used in a sandwich ELISA. The analytical imprecision and the concentration of cHK in a reference population and in women receiving oral contraceptives (OC) were determined. cHK was assessed in vitro in plasma exposed to polytetrafluoroethylene, silicone, and glass tubes.ResultsThe selected mAb showed excellent specificity towards cHK. The intra-assay and inter-assay CV of the ELISA were 3.6 and 6.0%, respectively. The reference population (60 women, 60 men) displayed a median cHK plasma concentration of 1.38 μg/mL and a reference interval of 0.82 – 2.56 μg/mL. Women receiving OC had significantly higher concentrations, p < 0.001. cHK was significantly elevated in plasma exposed to polytetrafluoroethylene, p = 0.001, and glass, p < 0.0001.ConclusionThe ELISA showed excellent precision and specificity. cHK assessment ex vivo demonstrated ongoing contact activation in healthy individuals, augmented by OC. The cHK antibody and the ELISA could be promising tools in contact activation related diseases and in vitro investigations of the plasma compatibility of blood contacting biomaterials.

Evaluation of extracellular vesicle isolation and detection repeatability from cell cultures, biofluids, and mammalian tissues

Unaccounted variations in biological marker sample handling, preparation, and data collection can result in inaccurate or unreliable data sets. Herein, we assess the repeatability (test‐retest reliability) of extracellular vesicle (EV) size and concentration measurements by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) methods. We evaluate the contribution of pre‐analytical variation and analytical imprecision factors toward the total experimental variability in routine preparation and quantitative analysis of crude large (microvesicle, MV) or small (exosome, EXO) EV particle‐enriched fractions following differential velocity centrifugation. This is performed on EV‐enriched cell culture media, human biofluids, and mammalian tissues, which represent an increasing degree of analyte complexity and heterogeneity. Incremental increase in NTA camera level (CL) setting (shutter and gain) was associated with a proportional detection of larger small‐sized particle counts. There was a strong linear relationship between the raw sample volume or wet tissue weight input, and EV output. At optimal non‐oversaturating particle detection conditions, NTA and DLS techniques delivered low (&lt; 10%) inter‐sample coefficient of variability (CV%) when estimating EV size or number in technical replicates (TRs) prepared by using a 2‐step analyte dilution and sampling strategy. However, according to Bland‐Altman analysis, the repeatability of intra‐sample EV concentration measurements, but not size measurements by NTA, was prone to a significant run order bias within the first data capture cycle (DCC). Although analyte origin (random anatomical sub‐locations or different batches) did not represent a substantial source of error in EV analysis, a parallel EV isolation in TRs derived from a single supply, and a successive sample pooling with a potential preparation of second‐tier serially diluted TRs, is advised. To reduce the amount of analytical error in NTA analytes with the particle per frame (PPF) number between 40 and 100, and the polydispersity index (PDI) of &lt; 30%, no less than three 60 sec‐long or six 30 sec‐long repeated intra‐sample measurements by at least two successive DCCs should be recorded. In DLS, ten 30 sec‐long repeated measurements should be captured in each of three independent runs.

Analytical variability and uncertainty in canine leukocyte ratios obtained with manual counts

This study aimed to determine the analytical imprecision of calculated neutrophil to lymphocyte (NLR) and lymphocyte to monocyte ratios generated from manual differential white blood cell (WBC) counts in peripheral blood smears, and to describe how to report the uncertainty around a single WBC ratio result. No information on the analytical imprecision of WBC ratios in dogs is available. Coefficient of variations (CVs) of paired readings of one operator on 105 smears (intraoperator variability) and of three operators on 301 smears (interoperator variability) were calculated. The interoperator agreement was examined with the Fleiss' kappa coefficient (κ). Observed total errors (TEos), expanded measurement of uncertainty (EMU) and reporting intervals (RIs) were also calculated. Median CVs ranged from 3.14 to 28.28 (intraoperator) and from 5.39 to 53.85 (interoperator). No agreement among operators was found around the cut-offs. TEos were higher than allowable total errors in 32%-88% of smears. EMU ranged from 0.10 to 1.13. According to the RI, the calculated WBC ratios should be rounded to the nearest 10. WBC ratios should be interpreted cautiously in dogs. The EMU should be reported to make the clinician aware of the uncertainty of these parameters. For example, an NLR result of 17 is needed to have high confidence that the result is above a cut-off of 6.

Analytical and clinical evaluation of DiaSorin Liaison® Calprotectin fecal assay adapted for serum samples.

BackgroundCalprotectin is a calcium‐binding protein that can be measured in serum, plasma, and feces. Increased serum and plasma calprotectin concentrations have been found in chronic inflammatory rheumatic disorders. An analytical and clinical evaluation of the DiaSorin Liaison® fecal Calprotectin assay using LIAISON® XL was performed.MethodsThe protocol included an analytical and clinical evaluation in which imprecision, the linearity of dilution, differences between serum and plasma samples and method comparison with CalproLab™ ELISA kit were assessed. Serum calprotectin concentrations in active (n = 26) and remission (n = 23) rheumatoid arthritis (RA) patients were compared.ResultsThe intra‐day and inter‐day analytical imprecision CVs ranged from 2.9% to 4.0% and 2.7% to 10.4%, respectively. Correlation between measured and expected values was high (R > 0.99), indicating good linearity. The Wilcoxon signed‐rank test showed that serum and plasma matched samples presented statistically significant differences (p < 0.001) being the highest concentrations of calprotectin observed in serum samples. Deming regression equation was as follows: Diasorin calprotectin (μg/ml) = −0.32 (95% CI: −0.65 ‐ −0.05) +1.58 (95% CI: 1.42–1.79).* Calprolab calprotectin (μg/ml). Significantly higher serum calprotectin levels were found in RA patients with active disease when compared to patients with low disease activity or in clinical remission (mean ± SD) [(3.35 μg/ml ± 1.55) vs. (1.63 μg/ml ± 0.52), p < 0.001] and these levels correlated well with all disease activity indices.ConclusionsThe DiaSorin Liaison® fecal Calprotectin assay adapted for serum samples showed adequate technical performances and the clinical performances were similar to other assays.