This paper is the tenth in a series of publications dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C and the certification of reference materials. In the former publications the general concept for reference procedures for enzymes (Part 1), the measurement of the catalytic activity concentrations of creatine kinase (Part 2), lactate dehydrogenase (Part 3), alanine aminotransferase (Part 4), aspartate aminotransferase (Part 5), γ-glutamyltransferase (Part 6), total α-amylase (Part 8), and alkaline phosphatase (Part 9) are described. Part 8 describes the measurement of the catalytic activity concentration of total amylase, comprising the isoenzymes salivary derived α-amylase and pancreas derived α-amylase. The catalytic activity concentrations of the pancreatic isoenzyme provides more diagnostic selectivity in cases of differential diagnosis of pancreatitis and its monitoring. The current article describes the IFCC primary reference measurement procedure for pancreatic α-amylase enabling metrological traceability to SI.

Objectives Developing procedures based on equilibrium dialysis (ED) that allow measuring the free drug concentration in plasma improves therapeutic drug monitoring (TDM) in those cases where its measurement is justified. However, this procedure requires specific sample preparation and presents different pitfalls, which are not error-free. As with any result provided by a clinical laboratory, this one should be as accurate as possible to allow a correct clinical interpretation. The measurement uncertainty (MU) is a parameter that enables the accuracy of results to be known, and that is mandated by ISO 15189. Herein, this study suggests how the MU for the results of the free drug concentrations in serum could be estimated when an ED is used. Methods A combination of the top-down and bottom-up approaches was used to estimate the MU based on the ISO/TS 20914:2019 and JCGM 100:2008 guidelines, including the concentration of free phenytoin in serum, as an example. Different scenarios were incorporated considering or not a significant bias related to the primary drawbacks of ED: the non-specific binding, the volume shift effect and the Gibbs-Donnan effect. Results The expanded uncertainties estimated ranged between 13.0 and 30.9 %. The highest MU corresponded to the free drug concentrations in serum results when significant biases related to the volume shift and Gibbs-Donnan effects exist. Conclusions A detailed estimation of MU for free drug concentrations is presented using ED, considering different scenarios. This study could stimulate clinical laboratories to perform MU studies and its application in TDM.

Objectives: Laboratory Medicine is a crucial discipline that contributes to the diagnosis, management and monitoring of patients. This branch of medicine faces two major challenges: New technologies and increased demand. There is limited information available of the state of affairs in Laboratory Medicine in Spain. This study provides a picture of clinical laboratories and clinical laboratory professionals.Methods: The Spanish Society of Laboratory Medicine distributed a questionnaire among the 250 most representative centers (the ones with the largest volume of determinations and training programs), of which 174 (69.6%) returned the questionnaire providing data for 2019.Results: Laboratories were classified according to the number of determinations. In total, 37% identified themselves as small (< 1 million determinations per year); 40% considered themselves medium-sized (1-5 million determinations per year) and 23% considered they were large laboratories (> 5 million determinations). The level of specialization of laboratory physicians and laboratory performance were higher in large laboratories. Most requests (87%) and determinations (93%) corresponded to biochemistry and hematology. As many as 63% of physicians had an indefinite contract, and 23% were older than 60 years.Conclusions: Laboratory medicine is a consolidated discipline that is gaining relevance in Spain. It adds value to the diagnosis, prognosis and follow-up of diseases, and to treatment response monitoring. The results of this study will help us address challenges such as the need for specialized training for laboratory professionals; the emergence of technological innovations; exploitation of Big Data; optimization of quality management systems and patient safety.

BACKGROUND:Good strategical programs are required for the early detection of disease even in the absence of evident clinical signs, which is crucial in satisfying animal welfare. Haptoglobin (Hp) and inter-α-trypsin inhibitor heavy chain H4 (ITIH4) are acute phase proteins and good biomarkers of early inflammation in cattle, with plasma levels that significantly increase after injury or infection.OBJECTIVES:We aimed to develop and validate two new immunoturbidimetric methods for Hp and ITIH4.METHODS:Species-specific antibodies were obtained and used to develop the immunoassays. For the Hp assay, antibodies were fixed to latex microparticles to enhance detection. The immunoassays were set up in an automated analyzer to carry out validation studies. Reference intervals were calculated using Reference Value Advisor.RESULTS:The Hp immunoturbidimetric method had a linear analytical range up to 0.40 mg/mL. The limit of detection (LoD) was 0.005 mg/mL, and the limit of quantification (LoQ) was 0.007 mg/mL. Total imprecision was less than 7%. Comparison with ELISA and single radial immunodiffusion (SRID) showed good correlation, whereas the comparison with the colorimetric method showed constant and proportional differences. The ITIH4 immunoassay showed linearity up to 5 mg/mL, and the LoD was 0.002 mg/mL. Total imprecision was less than 6%. Method comparison showed a good correlation with single radial immunodiffusion, both methods being equivalent. Bilirubin, triglycerides, and hemoglobin presented no interference in any of the assays. Reference intervals were 0.007-0.017 mg/mL for Hp and 0.2-0.7 mg/mL for ITIH4 in dairy cows 10 days before parturition.CONCLUSIONS:Immunoturbidimetric methods developed for Hp and ITIH4 can measure basal and increased levels of these proteins, showing adequate precision, accuracy, and robustness.

Resumen Objetivos La medicina de laboratorio es una disciplina clave que contribuye al diagnóstico, terapia y seguimiento adecuado de los pacientes. Actualmente se enfrenta a grandes retos debido a la innovación tecnológica y al aumento de la demanda. Desafortunadamente hay una información limitada para comprender la situación actual en España. Este estudio pretende mostrar la situación de los laboratorios clínicos y de los profesionales de la medicina laboratorio. Métodos Desde la Sociedad Española de Medicina de Laboratorio se envió una encuesta a los 250 centros más representativos (mayor volumen y con programas formativos), de los cuales 174 (69,6%) respondieron con datos referidos a 2019. Resultados Los laboratorios se clasificaron en función del número de determinaciones. El 37% se consideraron pequeños (<1 millón de determinaciones al año), el 40% medianos (entre 1 y 5 millones de determinaciones al año) y el 23% grandes (>5 millones de determinaciones al año). El nivel de especialización de los facultativos y la eficiencia fueron mayores en los laboratorios grandes. La mayoría de las peticiones (87%) y determinaciones (93%) se concentraron en Bioquímica y Hematología. En relación con los facultativos, el 63% disponían de contrato fijo y el 23% superaban los 60 años. Conclusiones La medicina de laboratorio en España es una disciplina consolidada y en crecimiento, capaz de aportar valor al diagnóstico, pronóstico y seguimiento de enfermedades y sus tratamientos. El conocimiento del estado actual nos permitirá abordar retos tales como la necesidad de formación de profesionales, la innovación tecnológica, la aplicación del Big data, la optimización de los sistemas de gestión de calidad y la seguridad del paciente.

BACKGROUND:Hydroxychloroquine is an antimalarial drug that has been prescribed for the treatment of patients with COVID-19 infection. To assist in clinician decision-making, several clinical laboratories have developed and validated measurement procedures in-house based on HPLC or HPLC-MS/MS to measure the mass concentration of hydroxychloroquine in different biological fluids. In these cases, laboratories produce their calibration materials but rarely estimate the measurement uncertainty of their assigned values. Thus, we aimed to show how this uncertainty can be calculated, using the preparation of hydroxychloroquine calibrators in blood-hemolysate-based matrix as an example.METHODS:A bottom-up approach was used to estimate the uncertainty related to the values assigned to end-user calibration materials prepared in-house. First, a specification of the measurand and a measurement equation were proposed. Then, different sources of uncertainty related to the preparation of hydroxychloroquine calibration materials were identified and quantified. Afterwards, the combined uncertainty was calculated using the law for the propagation of uncertainty resulting in the final expanded uncertainty.RESULTS:In this study, the most significant source of uncertainty was that associated with the hydroxychloroquine's reference material mass obtained via balance, while the smallest contribution was from the uncertainty associated with the hydroxychloroquine reference material purity.CONCLUSIONS:A simple procedure to estimate the measurement uncertainty of values assigned to calibration materials is presented here, which would be easy to implement in clinical laboratories. Also, it could be put into practice for other pharmacological quantities measured by in-house HPLC or HPLC-MS/MS procedures commonly used in clinical laboratories.

Background Despite that measurement uncertainty data should facilitate an appropriate interpretation of measured values, there are actually few reported by clinical laboratories. We aimed to estimate the measurement uncertainty of some β-lactam antibiotics (β-LA), and to evaluate the impact of reporting the measurement uncertainty on clinicians’ decisions while guiding antibiotic therapy. Methods Measurement uncertainty of β-LA (aztreonam [ATM], cefepime [FEP], ceftazidime [CAZ], and piperacillin [PIP]) values, obtained by an UHPLC-MS/MS based-method, was estimated using the top-down approach called the single laboratory validation approach (EUROLAB guidelines). Main uncertainty sources considered were related to calibrators’ assigned values, the intermediate precision, and the bias. As part of an institutional program, patients with osteoarticular infections are treated with β-LA in continuous infusion and monitored to assure values at least 4 times over the minimal inhibitory concentration (4×MIC). We retrospectively evaluated the impact of two scenarios of laboratory reports on clinicians’ expected decisions while monitoring the treatment: reports containing only the β-LA values, or including the β-LA coverage intervals (β-LA values and their expanded measurement uncertainties). Results The relative expanded uncertainties for ATM, FEP, CAZ and PIP were lower than 26.7%, 26.4%, 28.8%, and 25.5%, respectively. Reporting the measurement uncertainty, we identified that clinicians may modify their decision especially in cases where 4×MIC values were within the β-LA coverage intervals. Conclusions This study provides a simple method to estimate the measurement uncertainty of β-LA values that can be easily applied in clinical laboratories. Further studies should confirm the potential impact of reporting measurement uncertainty on clinicians’ decision-making while guiding antibiotic therapy.

Clinicians request a large part of measurements of biological quantities that clinical laboratories perform for diagnostic, prognostic or diseases monitoring purposes. Thus, laboratories need to provide patient’s results as reliable as possible. Metrological concepts like measurement uncertainty and metrological traceability allow to know the accuracy of these results and guarantee their comparability over time and space. Such is the importance of these two parameters that the estimation of measurement uncertainty and the knowledge of metrological traceability is required for clinical laboratories accredited by ISO 15189:2012. Despite there are many publications or guidelines to estimate the measurement uncertainty in clinical laboratories, it is not entirely clear what information and which formulae they should use to calculate it. On the other hand, unfortunately, there are a small number of clinical laboratories that know and describe the metrological traceability of their results, even though they are aware of the lack of comparability that currently exists for patient’s results. Thus, to try to facilitate the task of clinical laboratories, this review aims to provide a proposal to estimate the measurement uncertainty. Also, different suggestions are shown to describe the metrological traceability. Measurement uncertainty estimation is partially based on the ISO/TS 20914:2019 guideline, and the metrological traceability described using the ISO 17511:2020. Different biological quantities routinely measured in clinical laboratories are used to exemplify the proposal and suggestions.

En infeccions osteoarticulars, per subministrar el tractament antibiotic adequat, evitant de retruc la resistencia a aquests, es precisa obtenir resultats de mesura dels antibiotics el mes exactes possible. Entre les estrategies, el Laboratori de Referencia d'Enzimologia Clinica (LREC) de la UAB i el Grup d'Infeccions de dificil tractament i us d'antimicrobians de l'Institut d'Investigacio Biomedica de Bellvitge (IDIBELL), proposen un model per a calcular la incertesa de mesura i avaluar l'impacte del seu coneixement sobre les decisions cliniques.

Background: Different reference change values approaches are widely accepted to interpret the change between two consecutive measured values from the same biological quantity in an individual. Nevertheless, we propose two uncertainty-based models to estimate reference change values which will include all possible variation sources. Methods: The models consisted in 1) estimation of the uncertainty related to each measured value, 2) calculation of the change between these values and its uncertainty, and 3) performing a compatibility study to know if the values are of the same degree of equivalence. Also, results obtained using the proposed models and the classical approaches are shown. Results: The primary uncertainty sources corresponded to the within-subject biological, followed by those related to the analytical or pre-analytical phase, and post-analytical, respectively. We observed higher reference change values when our models were applied than those obtained using the classical approaches. Conclusions: The estimation of reference change values using our models could be more realistic than the classical approaches because more identified variation sources were considered. We hope that this study will help and stimulate clinical laboratories to perform uncertainty and reference change values studies, and it allows a greater understanding of these concepts and its application in disease monitoring.

Reference intervals are commonly used as a decision-making tool. In this review, we provide an overview on "big data" and reference intervals, describing the rationale, current practices including statistical methods, essential prerequisites concerning data quality, including harmonization and standardization, and future perspectives of the indirect determination of reference intervals using routine laboratory data.

Resumen Los intervalos de referencia son habitualmente empleados como herramienta de apoyo a las decisiones clínicas. En esta revisión se resumen los aspectos relacionados con el big data y los intervalos de referencia, las prácticas actuales, incluyendo los métodos estadísticos, los requisitos de calidad de los datos, incluyendo la armonización y la normalización, y las perspectivas de futuro para la determinación indirecta de intervalos de referencia mediante datos de laboratorio de rutina.

The purpose of the present study is to establish the influence of age on serum biochemistry reference intervals (RIs) for unweaned calves and recently-weaned piglets using large number of animals sampled at different ages from populations under different season trials. Specifically, milk replacer (MR)-fed calves from April–July 2017 (n = 60); from December 2016–March 2017 (n = 76) and from April–August 2018 (n = 57) and one group of healthy weaned piglets (n = 72) were subjected to the study. Serum enzymes and metabolites of calves at age of 24 h (24 h after colostrum intake), 2, 5, and 7 weeks from merged trials and piglets at 0, 7, and 14 days post-weaning (at 21, 28, and 35 days of age) were studied. The main variable is age whereas no major trial- or sex-biased differences were noticed. In calves, ALT, AST, GGT, GPx, SOD, NEFAs, triglycerides, glucose, creatinine, total protein, and urea were greatly elevated (p < 0.001) at 24 h compared with other ages; glucose, creatinine, total protein, and urea constantly decreased through the age; cholesterol's lowest level (p < 0.001) was found in 24 h compared with other ages and the levels of haptoglobin remained unchanged (p > 0.1) during the study. In comparison with the adult RIs, creatinine from 24 h, NEFAs from 2 w, GGT from 5 w, and urea from 7 w are fully comparable with RIs or lie within RIs determined for adult. In piglets, no changes were noticed on glucose (p > 0.1) and haptoglobin (p > 0.1) and there were no major changes on hepatic enzymes (ALT, AST, and GGT), total protein, creatinine and urea even though several statistical differences were noticed on 7 days post-weaning. Cholesterol, triglycerides, NEFAs, cortisol and PigMAP were found increased (p < 0.05) while TNF-alpha was found less concentrated (p < 0.001) at 0 days post-weaning compared with other times. Moreover, the RIs of creatinine and GGT are fully comparable with RIs or lie within RIs determined for adult. In conclusion, clinical biochemistry analytes RIs were established for unweaned calves and recently-weaned piglets and among them some can vary at different ages.

BACKGROUND In dogs, as in humans, C-reactive protein (CRP) is a major acute phase protein that is rapidly and prominently increased after exposure to inflammatory stimuli. CRP measurements are used in the diagnosis and monitoring of infectious and inflammatory diseases. OBJECTIVES The study aim was to develop and validate a turbidimetric immunoassay for the quantification of canine CRP (cCRP), using canine-specific reagents and standards. METHODS A particle-enhanced turbidimetric immunoassay was developed. The assay was set up in a fully automated analyzer, and studies of imprecision, limits of linearity, limits of detection, prozone effects, and interferences were carried out. The new method was compared with 2 other commercially available automated immunoassays for cCRP: one turbidimetric immunoassay (Gentian CRP) and one point-of-care assay based on magnetic permeability (Life Assays CRP). RESULTS The within-run and between-day imprecision were <1.7% and 4.2%, respectively. The assay quantified CRP proportionally in an analytic range up to 150 mg/L, with a prozone effect appearing at cCRP concentrations >320 mg/L. No interference from hemoglobin (20 g/L), triglycerides (10 g/L), or bilirubin (150 mg/L) was detected. Good agreement was observed between the results obtained with the new method and the Gentian cCRP turbidimetric immunoassay. CONCLUSIONS The new turbidimetric immunoassay (Turbovet canine CRP, Acuvet Biotech) is a rapid, robust, precise, and accurate method for the quantification of cCRP. The method can be easily set up in automated analyzers, providing a suitable tool for routine clinical use.

Background: Due to the high inter-variability in immunosuppressants pharmacokinetics, therapeutic drug monitoring of these drugs is essential in order to minimize the risk of rejection after organ transplantation. Thus, results facilitated to clinicians by clinical laboratories for these drugs should be as reliable as possible. The knowledge of metrological traceability and performing compatibility studies can allow ensuring reliability of these results, mainly, when laboratories introduce substantial changes in their measurement procedures, e.g., when they change the calibration materials. The aim of this study was to investigate, as an example, the traceability of the calibrator's assigned values from two manufacturers (Recipe and Chromsystems) and to perform compatibility studies for sirolimus and tacrolimus. Material and methods: Traceability was described according to ISO 17511. Compatibility studies, based on the CLSI EP029-A guideline, were performed verifying the trueness processing the ERM (R)-DA111a and ERM (R)-DA110a reference materials. Results: Traceability studies reveled that sirolimus and tacrolimus results were traceable to SI, except for sirolimus if Chromsystems' calibrators was used. Using Recipe's calibrators, the absolute relative biases and their expanded uncertainties were 1.23% and +/- 6.10% for sirolimus, and 1.41% and +/- 3.02% for tacrolimus. Furthermore, when Chromsystems' calibrators were used, these values were 12.2% and +/- 6.02% for sirolimus, and 2.64% and +/- 2.94% for tacrolimus. Conclusions: Results reported a lack of traceability and compatibility of sirolimus' results when Chromsystems' calibrators was used. In order to avoid it, laboratories should only use calibrators with assigned values traceable to highest reference materials or, alternatively, perform a compatibility study and apply a bias correction factor.

BACKGROUND:Values of mass concentration of tacrolimus in whole blood are commonly used by the clinicians for monitoring the status of a transplant patient and for checking whether the administered dose of tacrolimus is effective. So, clinical laboratories must provide results as accurately as possible. Measurement uncertainty can allow ensuring reliability of these results. The aim of this study was to estimate measurement uncertainty of whole blood mass concentration tacrolimus values obtained by UHPLC-MS/MS using two top-down approaches: the single laboratory validation approach and the proficiency testing approach.MATERIAL AND METHODS:For the single laboratory validation approach, we estimated the uncertainties associated to the intermediate imprecision (using long-term internal quality control data) and the bias (utilizing a certified reference material). Next, we combined them together with the uncertainties related to the calibrators-assigned values to obtain a combined uncertainty for, finally, to calculate the expanded uncertainty. For the proficiency testing approach, the uncertainty was estimated in a similar way that the single laboratory validation approach but considering data from internal and external quality control schemes to estimate the uncertainty related to the bias.RESULTS:The estimated expanded uncertainty for single laboratory validation, proficiency testing using internal and external quality control schemes were 11.8%, 13.2%, and 13.0%, respectively.CONCLUSIONS:After performing the two top-down approaches, we observed that their uncertainty results were quite similar. This fact would confirm that either two approaches could be used to estimate the measurement uncertainty of whole blood mass concentration tacrolimus values in clinical laboratories.

Background: Traceable and accurate results of cyclosporine A (CsA) mass concentrations in whole blood are required to ensure the monitoring of immunosuppressive therapy in transplant recipients. Metrological traceability and measurement uncertainty can allow ensuring reliability and comparability of these results over time and space. In this study, we provide a practical and detailed example of how the traceability and uncertainty of mass concentration of CsA results, obtained using an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) procedure, can be described and estimated. Methods: Traceability was described mainly according to ISO 17511 and information obtained from certificates facilitated with the manufacturer's calibrators. Uncertainty estimation was performed using the bottom-up and top-down approaches. For the bottom-up approach, the most relevant sources of uncertainty were identified and later used to estimate the standard, combined and expanded uncertainties. For the top-down approach, expanded uncertainty was estimated directly using intralab quality control data mainly. Results: Mass concentration of CsA results was traceable to the manufacturer's product calibrators used to calibrate the UHPLC-MS/MS procedure. The expanded uncertainties estimated by the bottom-up and top-down approaches were 7.4% and 7.2%, respectively. Conclusions: After performing the bottom-up and top-down approaches, we observed that their results were quite similar. This fact would confirm that the top-down approach could be sufficient for estimating uncertainty of CsA mass concentrations in whole blood results in clinical laboratories. Finally, we hope that this study can help and motivate clinical laboratories to describe metrological traceability and to perform measurement uncertainty studies based on the simpler top-down approach.

BACKGROUND:The availability of a species-specific reference material is essential for the harmonization of results obtained in different laboratories by different methods.OBJECTIVES:We describe the preparation of a canine C-reactive protein (cCRP) serum reference material containing purified cCRP stabilized in a serum matrix. The material can be used by manufacturers to assign values to their calibrator and control materials.METHODS:The serum matrix was obtained using blood collected from healthy dogs, stabilized and submitted for a delipidation process. The reference material was prepared by diluting purified cCRP in the serum matrix containing 1.0 mol/L HEPES buffer, 3.0 mmol/L calcium chloride, 80,000 kUI/L aprotinin, and 1.0 mmol/L benzamidine hydrochloride monohydrate at a pH of 7.2, and dispensing (0.5 mL) the matrix into vials that were then frozen.RESULTS:The pilot batch of 200 vials was shown to be homogeneous and stable after a stability study at various temperatures and over a total time of 110 days. The prepared material was submitted to an assignment value study. Eight laboratories from different European countries participated by using the same reagents for an immunoturbidimetric method adapted for different analyzers. The obtained cCRP concentration in the reference material was 78.5 mg/L with an expanded uncertainty (k = 2) of 4.2 mg/L.CONCLUSIONS:Canine C-reactive protein serum reference material has been produced that allows harmonization of results obtained by different methods and different laboratories, thus reducing the possibility of errors and misunderstandings.