The impact of accuracy and precision of analytical test methods on the determination of withdrawal periods.

Treatment of food-producing animals with veterinary medicinal products (VMPs) can result in residues in foodstuffs (e.g. eggs, meat, milk, or honey) representing a potential consumer health risk. To ensure consumer safety, worldwide regulatory concepts for setting safe limits for residues of VMPs e.g. as tolerances (US) or maximum residue limits (MRLs, EU) are used. Based on these limits so-called withdrawal periods (WP) are determined. A WP represents the minimum period of time required between the last administration of the VMP and the marketing of foodstuff. Usually, WPs are estimated using regression analysis based on residue studies. With high statistical confidence (usually 95% in the EU and 99% in the US) the residues in almost all treated animals (usually 95%) have to be below MRL when edible produce is harvested. Here, uncertainties from both sampling and biological variability are taken into account but uncertainties of measurement associated with the analytical test methods are not systematically considered. This paper describes a simulation experiment to investigate the extent to which relevant sources of measurement uncertainty (accuracy and precision) can impact the length of WPs. A set of real residue depletion data was artificially 'contaminated' with measurement uncertainty related to permitted ranges for accuracy and precision. The results show that both accuracy and precision had a noticeable effect on the overall WP. Due consideration of sources of measurement uncertainty may improve the robustness, quality and reliability of calculations upon which regulatory decisions on consumer safety of residues are based.

Use of Uncertainty of Measurement for Traceability of Test Results and Setting up of own Quality Goal for Methods having Lower Stability- A Tertiary Care Hospital study.

Uncertainty of measurement (UM) provides a quantitative estimate for traceability of test results. The Nordtest guide was applied for calculating UM of 26 analytes. For this, internal and external quality control data from July 2019 to April 2020 was used. UM of test results were compared to %TEa values of CLIA '2019, RiliBÄK, and Ricos. It was observed that UM for all analytes were below %TEa values of RiliBÄK. UM value of Albumin, Calcium and Sodium could not meet CLIA '2019 and Ricos guidelines. For results of Albumin, Calcium and Sodium to be traceable, more frequent quality control protocols resulted in decrease in bias. Quality goals were set for these three parameters. This helped in reduction of quality control cycles and optimum utilization of resources.

Quality control of cytostatic drug preparations-comparison of workflow and performance of Raman/UV and high-performance liquid chromatography coupled with diode array detection (HPLC-DAD).

The drugs used for treatment during chemotherapy are manufactured individually for each patient in specialised pharmacies. Thorough quality control to confirm the identity of the delivered active pharmaceutical ingredient and the final concentration of the prepared application solution is not standardized yet except for optical or gravimetric testing. However, solution stability problems, counterfeit drugs, and erroneous or deliberate underdosage may occur and negatively influence the quality of the product and could cause severe health risks for the patient. To take a step towards analytical quality control, an on-site analytical instrument using Raman and UV absorption spectroscopy was employed and the results were compared to high-performance liquid chromatography coupled to diode array detection. Within the scope of the technology evaluation, the uncertainty of measurement was determined for the analysis of the five frequently used cytostatic drugs 5-fluorouracil, cyclophosphamide, gemcitabine, irinotecan and paclitaxel. The Raman/UV technique (2.0-3.2% uncertainty of measurement; level of confidence: 95%) achieves a combined uncertainty of measurement comparable to HPLC-DAD (1.7-3.2% uncertainty of measurement; level of confidence: 95%) for the substances 5-fluorouracil, cyclophosphamide and gemcitabine. However, the uncertainty of measurement for the substances irinotecan and paclitaxel is three times higher when the Raman/UV technique is used. This is due to the fact that the Raman/UV technique analyses the undiluted sample; therefore, the sample has a higher viscosity and tendency to foam. Out of 136 patient-specific preparations analysed within this study, 96% had a deviation of less than 10% from the target content.

Simplified approach to uncertainty of measurement in the clinical virology laboratory.

Medical laboratories are required to ensure the quality of their diagnostic results. Quality assurance procedures include quality assessments (internal and external), quality controls (negative, positive, or internal controls), equipment monitoring, and audits. Quality control data may be used to evaluate the uncertainty of measurement. All clinical virology laboratories require a standard operating procedure detailing their consideration of uncertainty of measurement, as this parameter may impact on the overall quality of diagnostic results as well as the clinical interpretation thereof. This review aims to provide a simplified approach to the concept of uncertainty of measurement, specific for clinical virology laboratories.

Uncertainty of measurement in andrology: UK best practice guideline from the Association of Biomedical Andrologists.

Uncertainty of measurement has become a paramount factor to consider in pathology. In the UK, consideration of uncertainty of measurement is mandatory for medical laboratories who apply to be accredited against ISO15189:2012 via the United Kingdom Accreditation Service. This guideline intends to help those working within diagnostic andrology to better understand the concept of uncertainty, and how it can be applied to semen analysis and post-vasectomy semen analysis. The various areas where uncertainty may exist are identified, and guidance is provided to minimise this uncertainty. This guidance is produced by the Association of Biomedical Andrologists alongside experts in the field of andrology, in order to aid laboratory scientists in understanding and undertaking important tasks that will improve quality of their service.

Improving uncertainty in Widmark equation calculations: Alcohol volume, strength and density.

The Widmark equation is probably the most commonly used calculation for medicolegal purposes. Recently the National Research Council (USA) and the Forensic Science Regulator (UK) have called for the uncertainty of all results to be given with all forensic measurements and calculations. To improve the uncertainty of measurement of results from Widmark calculations we have concentrated on the uncertainties of measurement involved in the calculation of amount of alcohol, that of the volume of alcohol, the concentration of alcohol and the density of alcohol as previous studies have investigated some of the other factors involved. Using experimental studies, the scientific literature and legal statutes, we have determined revised and improved uncertainties of the concentration of ethanol for Widmark calculations for both the USA and UK. Based on the calculations that we have performed we recommend the use of Monte Carlo Simulation for the determination of uncertainty of measurement for Widmark Calculations.

The role of the uncertainty of measurement of serum creatinine concentrations in the diagnosis of acute kidney injury.

Uncertainty of measurement is the numeric expression of the errors associated with all measurements taken in clinical laboratories. Serum creatinine concentration is the most common diagnostic marker for acute kidney injury. The goal of this study was to determine the effect of the uncertainty of measurement of serum creatinine concentrations on the diagnosis of acute kidney injury. We calculated the uncertainty of measurement of serum creatinine according to the Nordtest Guide. Retrospectively, we identified 289 patients who were evaluated for acute kidney injury. Of the total patient pool, 233 were diagnosed with acute kidney injury using the AKIN classification scheme and then were compared using statistical analysis. We determined nine probabilities of the uncertainty of measurement of serum creatinine concentrations. There was a statistically significant difference in the number of patients diagnosed with acute kidney injury when uncertainty of measurement was taken into consideration (first probability compared to the fifth p = 0.023 and first probability compared to the ninth p = 0.012). We found that the uncertainty of measurement for serum creatinine concentrations was an important factor for correctly diagnosing acute kidney injury. In addition, based on the AKIN classification scheme, minimizing the total allowable error levels for serum creatinine concentrations is necessary for the accurate diagnosis of acute kidney injury by clinicians.

Determination of real-time polymerase chain reaction uncertainty of measurement using replicate analysis and a graphical user interface with Fieller's theorem.

BACKGROUND: Disease monitoring of viruses using real-time polymerase chain reaction (PCR) requires knowledge of the precision of the test to determine what constitutes a significant change. Calculation of quantitative PCR confidence limits requires bivariate statistical methods. OBJECTIVE: To develop a simple-to-use graphical user interface to determine the uncertainty of measurement (UOM) of BK virus, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) real-time PCR assays. METHODS: Thirty positive clinical samples for each of the three viral assays were repeated once. A graphical user interface was developed using a spreadsheet (Excel, Microsoft Corporation, USA) to enable data entry and calculation of the UOM (according to Fieller's theorem) and PCR efficiency. RESULTS: The confidence limits for the BK virus, CMV and EBV tests were ∼0.5 log, 0.5 log to 1.0 log, and 0.5 log to 1.0 log, respectively. The efficiencies of these assays, in the same order were 105%, 119% and 90%. The confidence limits remained stable over the linear range of all three tests. DISCUSSION: A >5 fold (0.7 log) and a >3-fold (0.5 log) change in viral load were significant for CMV and EBV when the results were ≤1000 copies/mL and >1000 copies/mL, respectively. A >3-fold (0.5 log) change in viral load was significant for BK virus over its entire linear range. PCR efficiency was ideal for BK virus and EBV but not CMV. Standardized international reference materials and shared reporting of UOM among laboratories are required for the development of treatment guidelines for BK virus, CMV and EBV in the context of changes in viral load.

HISTORIQUE: Pour surveiller les virus au moyen de la réaction en chaîne de la polymérase (PCR) en temps réel, il faut connaître la précision du test pour déterminer ce qui constitue un changement important. Il faut des méthodes statistiques bivariées pour calculer les limites de confiance de la PCR quantitative. OBJECTIF: Élaborer une interface utilisateur graphique facile à utiliser pour déterminer l'incertitude des mesures (IDM) du virus BK, du cytomégalovirus (CMV) et du virus d'Epstein-Barr (VEB) par PCR en temps réel. MÉTHODOLOGIE: Trente échantillons cliniques positifs de cha-cune des analyses virales ont été répétés une fois. Une interface utilisateur graphique a été élaborée au moyen d'un chiffrier (Excel, Microsoft Corporation, États-Unis) pour saisir les données et calculer l'IDM (selon le théorème de Fieller) et l'efficacité de la PCR. RÉSULTATS: Les limites de confiance des tests du virus BK, du CMV et du VEB étaient de ∼0,5 log, 0,5 log à 1,0 log, et 0,5 log à 1,0 log, respectivement. L'efficacité de ces analyses était de 105 %, de 119 % et de 90 %, respectivement. Les limites de confiance sont demeurées stables pendant la trajectoire linéaire de ces trois tests. EXPOSÉ: Un changement de la charge virale plus de cinq fois plus élevé (0,7 log) et plus de trois fois plus élevé (0,5 log) était important pour le CMV et le VEB lorsque les résultats étaient d'un maximum de 1 000 copies/mL et de plus de 1 000 copies/mL, respectivement. Un changement de la charge virale plus de trois fois élevé (0,5 log) était important pour toute la trajectoire linéaire du virus BK. La PCR avait une efficacité idéale pour le virus BK et le VEB, mais pas pour le CMV. Il faudra élaborer des normes de référence internationales standardisées et partager les signalements d'IDM entre laboratoires pour préparer des directives thérapeutiques relatives au virus BK, au CMV et au VEB dans le contexte des changements de la charge virale.

Determination of 73 multi-class pesticides in okra (Abelmoschus esculentus L.) fruits using LC-MS/MS and GC-MS/MS and estimation of analytical uncertainty of measurement.

This study developed a method to simultaneously determine 73 multi-class pesticides in okra fruit using LC-MS/MS and GC-MS/MS. The sample was extracted with acetonitrile and subsequent clean-up through dispersive-SPE method. The quantification level of the technique was 0.01 µg g-1 and compliance to the MRLs fixed by the regulatory bodies like EU and FSSAI. The recovery at 10, 50, and 100 µg kg-1 spiked levels; intra and inter-day precision at 50 µg kg-1 were found within 70-120% with RSD less than 15% with LC-MS/MS and GC-MS/MS. Measurement uncertainty was in the range of 1.81 to 12.91 µg kg-1 estimated at 50 µg kg-1. The matrix effects were slightly higher for LC than GC-compatible pesticides. Risk assessment for pesticides detected in the field and market samples found no hazardous to the consumers except profenofos. The proposed method is highly sensitive, reproducible for the complex matrix like okra, and meets the regulatory standards.

[Uncertainty of measurement and result from a complex calculation: about the risk of fetal Down's syndrome by maternal serum markers]. / Incertitude de mesure et résultat issu d'un calcul complexe : le cas du risque de trisomie 21 fœtale par les marqueurs sériques maternels.

Screening for fetal Down's syndrome has the peculiarity of combining the biochemical assay of 2 or 3 serum markers with the risk associated with maternal age. If the accuracy of measurement of each parameter is known by the biologist, the uncertainty of the ultimate risk to the patient is not. Indeed, the means of risk calculation involve numerous multi-parameter equations which are not practical for daily use. Defining a re-test limit on thresholds of 1/50 and 1/1,000 is therefore impossible. Since the use of an arbitrarily defined threshold is not being satisfactory, we propose, by default, a methodology based on the exploitation of patient files in the laboratory with risks close to the two decision thresholds. Modulations of the concentrations of all the markers according to their uncertainty allow new risks to be obtained, which can be averaged and framed by an interval of several standard deviations. Choosing the level of uncertainty, the number of files to include, the number of standard deviations framing the average risk, as well as the calculation software, are all choices available to the biologist. The proposed methodology is therefore highly empirical but open, and adaptable, to the specific environment and performance capabilities of each and every laboratory involved.

Circular dichroism of biopharmaceutical proteins in a quality-regulated environment.

With growing regulatory interest in ensuring the integrity and consistency of the higher order structure of biopharmaceutical and vaccine products, validated methods will be required that operate in quality-regulated environments and in compliance with guidelines such as those published by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use [ICH]. Circular dichroism [CD] is a widely available, rapid and sensitive method to compare the secondary and tertiary structures of samples. For use in quality-regulated analyses multiple factors need to be considered. Firstly, a thorough understanding of the sources of experimental error is required, alongside protocols and reference standards for sample preparation, instrument calibration and performance monitoring. Secondly, validated algorithms to objectively compare spectra are required, with statistical evaluation to assess when values are significantly different and to define compliance criteria. Thirdly, regulatory authorities mandate the process of method assessment and validation to be acceptable for use in regulated environments. In this review I discuss regulatory expectation, the sources and magnitudes of instrument- and sample-derived uncertainties, the use of reference standards to define and correct instrument performance, and the algorithms used to objectively compare spectra. The ideas discussed here support the use of CD not only in quality-regulated environments but also best practice when used in a research environment.

Disregarded Measurement Uncertainty Contributions and Their Magnitude in Measuring Plasma Glucose.

BACKGROUND: Each measurement is subject to measurement uncertainty (MU). Consequently, each measurement of plasma glucose concentration used for diagnosis and monitoring of diabetes mellitus (DM) is affected. Although concepts and methods of MU are well established in many fields of science and technology, they are presently only incompletely implemented by medical laboratories, neglecting MU of target values of internal quality control (IQC) materials. METHODS: An empirical and practical approach for the estimation of MU based on the analysis of routine IQC using control samples with assigned target values is presented. Its feasibility is demonstrated exemplarily by analyzing IQC data from one year obtained for glucose employing the hexokinase method with IQC of two different concentrations. RESULTS: Combined relative extended (k = 2) MU comprising bias, coefficient of variation (CV), and MU of the target values assigned to control materials were about 9% with a lower (~ 56 mg/dL; ~3.1 mmol/L) and 8% with a higher (~ 346 mg/dL; ~19.2 mmol/L) concentration sample, analyzing IQC of one year from three different devices. CONCLUSIONS: Estimation of MU in this study is quite reliable due to the large number of IQC data from one year. The MU of the target values of the commercial control material in this study was considerably larger than other MU contributions, ie, standard deviation and bias. In the future, the contribution of MU of commercial IQC should be addressed more carefully and technologies to measure glucose should be geared toward smaller MU possible, as needed, especially for glucose concentration measurements in diagnosis and management of DM.

Qualitative threshold method validation and uncertainty evaluation: A theoretical framework and application to a 40 analytes liquid chromatography-tandem mass spectrometry method.

Qualitative methods hold an important place in drug testing, filling central needs in screening and analyses, among others, linked to per se legislation. Nevertheless, the bioanalytical method validation guidelines do not discuss this type of method or describe method validation procedures ill-adapted to qualitative methods. The output of qualitative methods are typically categorical, binary results, such as presence/absence or above cut-off/below cut-off. As the goal of any method validation is to demonstrate fitness for use under production conditions, qualitative validation guidelines should evaluate performance based on discrete, binary results instead of the continuous measurements obtained from the instrument (e.g. area). A tentative validation guideline for threshold qualitative methods was developed by in silico modelling of measurements and derived binary results. This preliminary guideline was applied to a liquid chromatography-tandem mass spectrometry method for 40 analytes, each with a defined threshold concentration. Validation parameters calculated from the analysis of 30 samples spiked above and below the threshold concentration (false negative rate, false positive rate, selectivity rate, sensitivity rate and reliability rate) showed a surprisingly high failure rate. Overall, 13 out of the 40 analytes were not considered validated. A subsequent examination found that this was attributable to an appreciable shift in the standard deviation of the area ratio on a day-to-day basis, a previously undescribed and unaccounted-for behaviour in the qualitative threshold method validation literature. Consequently, the developed guideline was modified and used to validate a qualitative threshold method, based on the binary results for performance evaluation and incorporating measurement uncertainty.

Development of an analytical method for simultaneously determining TBBPA and HBCDs in various foods.

Tetrabromobisphenol A (TBBPA) and Hexabromocyclododecanes (HBCDs) are commonly used as brominated flame retardants in large volumes, and accumulate in plants and animals in the environment, and people are exposed to them when consuming food. As many countries are monitoring them in food, it is necessary to develop a method to analyze them simultaneously for cost efficiency. A method was developed and optimized under different conditions using accelerated solvent extraction to extract the lipids from the samples, acid silica column to clean the samples and liquid chromatography coupled with tandem mass spectrometry to determine TBBPA and HBCDs. The method was validated in different kinds of food. Uncertainty of measurement was calculated by combining all uncertainties of contributors. Intermediate precision (reproducibility) was the most influential contributor to uncertainty. 5 food categories with 115 samples were analyzed with the method, and mackerels containing high level of fat were highly contaminated by TBBPA and HBCDs.

Reference measurement procedure for total bilirubin in serum re-evaluated and measurement uncertainty determined.

BACKGROUND: For the determination of total bilirubin in serum the candidate reference method developed by Doumas et al. has international recognition. The primary standard SRM 916a (NIST) was recommended for use as the primary reference material for calibration. Nowadays, no primary standard is anymore commercially available. Further, a description of uncertainty components was missing. METHODS: Two reference laboratories have re-investigated the candidate reference measurement procedure. Beside minor modifications, mainly the use of a molar absorption coefficient instead of calibration by use of bilirubin standard solutions has facilitated the operating, and improved the analytical performance. All relevant sources of measurement uncertainty were investigated. RESULTS: A measurement range of 5-525 µmol/L and a CV of 0.5% to 1.4% (long term imprecision) were determined. Excellent agreement was obtained comparing to Doumas procedure (r = 0.9999) and during a two laboratory comparison participating at IFCC RELA ring trials (mean deviation: 0.6%). The combined expanded measurement uncertainty (probability 95%) for bilirubin concentrations >30 µmol/L was estimated as 2.2%. CONCLUSION: A reference system for total bilirubin based on the described reference procedure shall enable metrological traceability and optimized standardization of the values obtained in clinical routine laboratories.

A new approach in evaluating the surface beta contamination using the direct method of measurement.

This paper describes a new approach in evaluating the surface beta contamination using the direct method of measurement. It makes use of previous results obtained in numerical modeling of electron transport in planar geometry and is mainly based on the estimation of the efficiency of contamination sources for beta radiation and its standard uncertainty using the available information concerning the component materials of the sources and their main parameters. Experimental results illustrate the appropriateness of the new approach for surface beta contamination measurements.

Design, development and metrological characterization of a low capacity precision industrial force transducer.

The paper discusses the development of the ring shaped force transducers for measurement of force in lower capacity to meet the industrial requirements with the increasing technological developments. A 50 N ring shaped force transducer for tension mode has been developed by studying the analytical and computational methods. The force transducer developed has been metrologically studied according to the calibration procedure based on the standard ISO 376 and uncertainty of measurement of the force transducer is found to be±0.10% (k=2), while taking into account the relative uncertainty contribution due to necessary factors like repeatability, reproducibility, zero offset, interpolation, resolution and reversibility. The force transducer developed may further be studied for improvement of metrological performance and may suitably be developed for other lower capacities like 10 N, 20 N etc. The force transducer developed offers very economical alternative of complex shaped force transducers with simple design and manufacturing features. The force transducer developed may be proved very helpful in providing traceability to the user industries and calibration laboratories in the lower range of force measurement and serve as force transfer standard.

Use of Combined Uncertainty of Pesticide Residue Results for Testing Compliance with Maximum Residue Limits (MRLs).

The uncertainty of pesticide residue levels in crops due to sampling, estimated for 106 individual crops and 24 crop groups from residue data obtained from supervised trials, was adjusted with a factor of 1.3 to accommodate the larger variability of residues under normal field conditions. Further adjustment may be necessary in the case of mixed lots. The combined uncertainty of residue data including the contribution of sampling is used for calculation of an action limit, which should not be exceeded when compliance with maximum residue limits is certified as part of premarketing self-control programs. On the contrary, for testing compliance of marketed commodities the residues measured in composite samples should be greater than or equal to the decision limit calculated only from the combined uncertainty of the laboratory phase of the residue determination. The options of minimizing the combined uncertainty of measured residues are discussed. The principles described are also applicable to other chemical contaminants.

A series of strategies for solving the shortage of reference standards for multi-components determination of traditional Chinese medicine, Mahoniae Caulis as a case.

In order to solve the bottleneck of reference standards shortage for comprehensive quality control of traditional Chinese medicines (TCMs), a series of strategies, including one single reference standard to determine multi-compounds (SSDMC), quantitative analysis by standardized reference extract (QASRE), and quantitative nuclear magnetic resonance spectroscopy (qNMR) were proposed, and Mahoniae Caulis was selected as an example to develop and validate these methods for simultaneous determination of four alkaloids, columbamine, jatrorrhizine, palmatine, and berberine. Comprehensive comparisons among these methods and with the conventional external standard method (ESM) were carried out. The relative expanded uncertainty of measurement was firstly used to compare their credibility. The results showed that all these three new developed methods can accurately accomplish the quantification by using only one purified reference standard, but each of them has its own advantages and disadvantages as well as the specific application scope, which were also discussed in detail in this paper.