Clinical Chemistry and Laboratory Medicine (CCLM)

Objectives An isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (cRMP) was developed and validated to measure serum and plasma concentrations of the total and free form of valproic acid. Methods Quantitative nuclear magnetic resonance spectroscopic methodology was used to determine the absolute content (g/g) of the reference material, ensuring traceability to SI units. Separation of valproic acid from potential unknown interferences was achieved with reversed-phase chromatography. A protein precipitation protocol was established for sample preparation for total valproic acid, while the free form was separated by ultrafiltration. Assay validations and measurement uncertainties were aligned with guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement. Results The cRMPs were highly selective and specific with no evidence of matrix effects, allowing quantifying total and free valproic acid in a range of 2.40–145 μg/mL and 1.60–42.0 μg/mL, respectively. Intermediate precision was <4.0 % and repeatability CV ranged from 0.9 to 3.5% for all concentrations of free and total valproic acid. The relative mean bias ranged from −0.4 to 4.1 % for native serum and from −0.3 to 3.5 % for Li-heparin plasma levels for total valproic acid. Free valproic acid showed mean biases between −2.9 and 4.0 % for native serum and ultrafiltrates. Measurement uncertainties for single measurements and target value assignment ranged from 1.7 to 3.4 % and 0.9–1.3 %, respectively, for total valproic acid. Free valproic acid ranged from 2.0 to 4.1 % and from 0.8 to 1.5 % for single measurements and target value assignment, respectively. Conclusions We present novel ID-LC-MS/MS-based cRMPs for total and free valproic acid in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

Objectives The International Organisation for Standardisation (ISO) 15189 standard provides the requirements for quality and competence in medical laboratories. It aims to ensure that laboratories deliver accurate and reliable results for patient care. The first version of the same was published in 2003, followed by editions in 2007, 2012 and 2022. The objective of this article is to analyze the ethical commitments and requirements assumed to achieve reliable results in laboratory medicine incorporated in the ISO standard. Methods We compare the concepts and changes related to ethics introduced over time in the ISO 15189 versions. We also present, a comparative study about the evolution of ethical concepts in the clinical laboratory and how these changes have been reflected in ISO 15189 standards through 2003 to 2022. Results After a thorough comparative analysis, we observe that the ISO 15189:2022 version has strengthened the requirements for several key concepts of Ethics such as impartiality and confidentiality, and addressed the issues of laboratory users with regards to autonomy, beneficence and justice although these terminologies are not explicitly mentioned. However, several other key words and concepts have not been addressed with adequate emphasis. Conclusions We recommend that several key concepts related to ethics may need to be included in the future revisions, especially those related to new and upcoming technologies being used in laboratory diagnostics and research.

Objectives Ocular lymphomas are rare diseases that occur in ocular adnexa or specific intraocular regions. Although histology remains the gold standard, cytology and flow cytometry (FCM) are fundamental, mainly when a biopsy sampling is technically demanding. This study evaluated the effectiveness of FCM in diagnosing ocular diseases, especially hematologic malignancies, and compared its performance with the other techniques. Methods In this monocentric study we collected patient characteristics from 165 patients referred to Padua University Hospital. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV), accuracy and concordance rate were evaluated for cytologic, FCM, and/or histologic analyses performed. Results Patients were classified as having hematologic neoplasms (n=86), non-hematologic neoplasms (n=33) or non-neoplastic diseases (n=46). Hematologic neoplasms comprised 74 cases with ocular adnexal hematologic lesions and 12 cases with intraocular lymphomas and leukemias. FCM analysis of ocular specimens allowed valuable results in 71/86 hematologic cases, failure being mainly due to absence of cellularity (8/15). Histology showed 100 % PPV for both hematologic and non-hematologic neoplasms; FCM reached 97 and 66 %, and cytology 80 and 91 %, respectively. Concordance between FCM and cytology was lower (κ<0.61) than with histology (κ=0.677). FCM phenotype matched ocular lymphoma histotype in 95.8 % of cases. The lymphocytic infiltrate by FCM was 39.5 % in non-neoplastic, 64 % in non-hematologic and 89.5 % in hematologic diseases with B lymphocyte prevalence of 31.3 %, 21.0 % and 93.3 %, respectively. Clonality analysis remains essential regardless of these values. Conclusions FCM provides rapid results, studies numerous markers simultaneously, and analyzes ocular aspirates from minimally invasive samples. For this reason, it is desirable to permanently integrate this technique into the diagnostic process of ocular disorders, particularly when hematologic malignancies are suspected, as part of an integrated multidisciplinary pathway.

Electronic health records will increasingly aggregate longitudinal laboratory results from multiple providers, but availability alone does not guarantee safe interpretation. We present guidance, developed by laboratory professionals with the DGKL medical informatics division, for cumulative displays that are clinically meaningful. The core principle is to group medically comparable analyses while preserving laboratory provenance so that clinicians can follow true patient trends without conflating them with laboratory-induced variation. Comparability is defined algorithmically from Logical Observation Identifiers Names and Codes (LOINC) axis: analyses estimating the same patient property (allowing serum/plasma system equivalence and mathematically convertible properties such as substance vs. mass concentration) are grouped; coding of units is harmonized via Unified Code for Units of Measure (UCUM) with consistent conversion of numeric results and corresponding reference intervals, including inequality qualifiers. Analyte-specific conversion factors should come from authoritative sources; for poorly standardized measurands (e.g., tumor markers) or when conversions are inappropriate (e.g., Lp(a)), results remain separated. Methodological distinctions that affect interpretation – such as screening vs. confirmatory drug testing and point-of-care testing – are displayed independently to signal potential analytical discontinuities. A standardized, medically meaningful default result sequence – derived from LOINC metadata and clinical nomenclatures, with alphabetic naming as a pragmatic fallback – supports cross-laboratory aggregation; rare or novel tests lacking robust standardization remain as free text. The rules-based approach updates seamlessly with LOINC releases and remains compatible with the Nomenclature for Properties and Units (NPU), facilitating cross-border exchange within the European Health Data Space. While harmonized presentation improves trend analysis, true comparability ultimately requires measurement procedures traceable to reference methods and materials.

Saliva, a non-invasive biofluid with a variety of biomolecules, has been increasingly recognized as a reliable specimen in the diagnostics of viral infections. Compared to the standard specimens represented by blood and nasopharyngeal swab, saliva is advantageous in non-invasive, simple collection, convenient storage and compatibility with self-testing and large-scale screening. Saliva is an all-round specimen for viral detection as the shedding of viral nucleic acid fragments, antigens and the secretion of virus-specific antibodies are present in saliva. Accordingly, saliva has been widely used to detect a variety of viruses, including saliva-borne, air-borne and blood-borne transmitted viruses. However, challenges also arise as diverse methods during collection, storage and processing of the saliva could lead to discordant results among different studies. It is now aware that the methodology of saliva sampling is critical to the detection of viral biomarkers in the accuracy, repeatability and consistence with the data retrieved from the standard specimens. Therefore, establishing a standardized procedure for saliva sampling is crucial. Here we focus on a dozen factors that may affect the results of salivary diagnostic of viral infections and suggest a set of standardized procedures for saliva sampling, aiming to minimize result variation and benefit the application of saliva in salivary diagnostics of viral infections.

Objectives Measuring creatinine levels is essential for assessing kidney function. However, traditional venous sampling poses challenges in terms of sample collection, storage, and transportation. Recently, dried blood spot (DBS) analysis has emerged as a potential alternative for measuring creatinine levels, although its clinical application remains limited. Creatinine measurements from DBS could facilitate chronic kidney disease screening, medication management, and at-home patient monitoring. Methods Precision and hematocrit effect were assessed according to Clinical and Laboratory Standards Institute protocols to validate creatinine measurements in DBS. Subsequently, the protocol was applied in a method comparison involving 115 patients. Lastly, robustness was assessed by investigating creatinine stability over time and the influence of spotting effects. Results Creatinine measurements in DBS resulted in low bias and correcting for hematocrit further improved accuracy. A comparison of creatinine and estimated glomerular filtration rate (eGFR) between venous plasma and finger stick DBS showed an R 2 of 0.97 and 0.86, respectively. Moreover, sensitivity and specificity for detecting decreased eGFR (<60 mL/min/1.73 m 2 ) were 91 and 96 %, respectively. Creatinine remained stable in DBS for up to a month at −20 °C and 4 °C, up to six days at 20 °C, and one day at 37 °C. Finally, spotting effects such as double spotting had negligible effects. Conclusions In conclusion, creatinine from DBS can be analyzed using an automated high-throughput chemistry analyzer, enabling accurate eGFR classification. This method may support blood sampling at home for screening programs, patient follow-up, and medication management.

Objectives A candidate reference measurement procedure (RMP) based on isotope dilution (ID) liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated to accurately measure serum and plasma concentrations of mycophenolic acid (MPA). Methods Quantitative nuclear magnetic resonance (qNMR) spectroscopy was utilized for determining the absolute content of the reference material, guaranteeing its traceability to SI units. Separation of MPA from potential interferences, whether known or unknown, was accomplished by using a C18 column. For sample preparation, a protocol for protein precipitation followed by a high dilution step was established. Assay validation and measurement uncertainty determination were conducted following the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement guidelines. Results The RMP demonstrated high selectivity and specificity without any indication of a matrix effect, enabling the quantification of MPA in the range between 0.240 and 18.0 mg/L. The intermediate precision and repeatability (n=60, measurements) were found to be <3.7 % and <3.0 %, respectively, across all concentration levels and independent from the matrix. The relative mean bias ranged from −1.9 to 2.1 % for serum and from −0.5 to 1.8 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were found to be <6.4 % and <3.0 % (k=2), respectively. Conclusions We are pleased to present a new LC-MS/MS-based candidate RMP for MPA in human serum and plasma which offers a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

Objectives As (a lack of) vitamin D has been linked to a wide variety of chronic diseases, there is growing interest in generating robust epidemiological data. Consequently, the need for large-scale biological sample collection has increased. In this context, dried blood spot (DBS) microsampling offers a minimally invasive alternative to venous sampling. However, to allow interpretation of DBS-based 25-hydroxyvitamin D (25-(OH)D) results, the set-up of a clinical validation study is essential to assess the agreement with the reference matrix, plasma. Methods Venous plasma and whole blood, venous DBS (vDBS) and capillary DBS (cDBS) were collected from 44 healthy volunteers to evaluate the agreement between the different sample types for 25-(OH)D quantification. To transform cDBS-based results to plasma concentrations, a hematocrit (Hct)-dependent conversion factor was applied and evaluated using four different Hct determination approaches. All samples were analysed using previously described validated LC-MS/MS methods. Results No clinically relevant methodological (vDBS vs. whole blood) or sampling-site related (cDBS vs. vDBS) issues were observed. Following Hct-dependent conversion, good agreement between the cDBS-derived and actually measured plasma results was obtained, as 90 % of the results lay within 20 % of the plasma result, independent of the Hct approach used. Additionally, weighted Cohen’s kappa values of 0.83–0.85 were obtained across the different Hct approaches, indicating substantial to almost perfect agreement in vitamin D status classification. Conclusions Following Hct-dependent conversion, cDBS can be used as a reliable and practical alternative matrix to plasma for large-scale monitoring of vitamin D status in epidemiological and public health contexts.

Recreational nitrous oxide (N 2 O) use has emerged as a growing public health concern, with increasing reports of neurological, psychiatric, and thrombotic complications. This position paper from the EFLM Committee on Biological Markers of Nitrous Oxide Abuse highlights the urgent need for clinical and laboratory awareness, alongside coordinated action from healthcare systems and the scientific community. Short-term recommendations focus on early clinical recognition, prompt biological testing, and structured patient management. Key clinical symptoms include unexplained sensory disturbances, gait instability, bladder and bowel complaints, sexual dysfunction, mood disorders, and thromboembolic events in young individuals. Plasma homocysteine (HCY) and plasma methylmalonic acid (MMA) are central to the diagnostic approach, with HCY serving as a sensitive marker of recent exposure and MMA indicating clinical severity. Vitamin B12 and folate measurements are also essential, though B12 levels may be falsely reassuring (often in the low-normal range). Given the rapid normalization of biomarkers, testing should be performed on admission, and vitamin B12 supplementation should begin without delay, with caution regarding biomarker kinetics and possible self-supplementation by patients. In addition, this paper outlines long-term goals, including the harmonization of biomarker assays, creation of international patient registries, and development of interdisciplinary expert networks. Notably, the international network PROTOSIDE (www.protoside.com) plays a key role in facilitating case discussion, expert collaboration, and dissemination of best practices in the management of N 2 O-related complications. A continuous bibliographic watch and support for translational research are necessary to improve biomarker discovery and understanding of the underlying mechanisms of toxicity. By combining immediate clinical action with long-term scientific and policy development, this document lays the foundation for an international strategy to address the silent but expanding epidemic of nitrous oxide misuse.

Objectives To analyze the performance of laboratories in the China National External Quality Assessment Scheme (China NEQAS) for HbA 2 and HbF testing, and to propose strategies for quality improvement. Methods Data were obtained from China NEQAS from 2013 to 2024 using both commercially lyophilized controls and in-house whole blood controls. The distributions of methods and instruments were analyzed. Inter-laboratory coefficient variations (CVs) and target values were compared between two types of controls and between method-instrument platforms over 12 years. Results The in-house controls remained homogeneous and stable for almost one month at 2–8 °C and for one year at −80 °C. Capillary electrophoresis (CE) became the dominant method, adopted by 84.3 % of labs in 2024. For HbA 2 , two EQA controls had comparable concentration ranges and inter-laboratory CVs. HbF in-house controls covered broader pathological concentrations than commercial ones. CE demonstrated lower inter-laboratory CVs for both analytes: HbA 2 was 2.1 ± 1.8 % vs. 5.5 ± 4.8 % (high-performance liquid chromatography, HPLC) and 6.5 ± 4.1 % (agarose gel electrophoresis, AGE), while HbF was 3.2 ± 1.9 % vs. 5.0 ± 1.6 % (HPLC) and 8.6 ± 6.8 % (AGE). Significant discrepancies in maximum-to-minimum mean concentrations were observed among different method-instrument platforms when testing the same controls (up to 10 % for HbF). Conclusions In-house controls demonstrate homogeneity, stability and intrinsic commutability, and have an expanded concentration range, can serve as a reliable alternative to commercial controls for EQA. High-precision techniques such as CE should be favoured. Furthermore, the development of reference methods and commutable reference materials is essential for standardization.

New technologies and advances in human biology are paving the way for better patient management and outcomes in laboratory medicine. Precision diagnostics, as an essential component of P5 medicine, implies a shift towards selective testing of defined measurands, enabling a higher understanding of interindividual diversity and (patho-)biology at the molecular level. Precision diagnostics can potentially overcome some of the current inadequacies in diagnostic care pathways, caused by suboptimal tests leading to misclassifications and avoidable patient harm. Additionally, molecular methods have the potential to be standardizable upfront, allowing embedding of the metrological traceability concept. Over the last decade, especially mass spectrometry (MS) is gaining momentum and proving its potential as a selective measurement method for immunoassay replacement, add-on test and/or reference measurement procedure (RMP). In this context, the current place of MS in laboratory medicine is discussed, followed by cases from laboratory developed tests (LDTs), commercially available tests, highlighting also future MS applications. It is time for a wake-up call to promote a selective testing approach that accurately resembles individual biology, principally to be used when molecular definition is clinically relevant for classification ability and patient benefit. We provide a compilation for the laboratory specialist when to opt for molecularly defined tests. Ideally, the evolution to precision diagnostics is part of a changing healthcare landscape, where molecular fingerprints and patient diversity are inclusive. This will enable early detection of preclinical symptoms and start a shift from curative to preventive and personalized care.

External Quality Assessment (EQA) is central to quality assurance in laboratory medicine, providing independent measures of tests trueness, and inter-laboratory comparability. Traditional EQA schemes, designed around stable and well-characterised measurands, have been successful in routine analytes such as electrolytes and liver enzymes. However, the rise of precision medicine presents new challenges that extend beyond the scope of traditional EQA models. Advances in genomics, proteomics and metabolomics require quality systems capable of evaluating complex and context-specific biomarkers which may lack reference materials or established targets values. This creates a paradox: while EQA depends on standardisation, precision medicine is individualised. To remain relevant, EQA must evolve from static, one-size-fits-all models into adaptive, technology-driven, and clinically contextualised systems. In our view, the evolution of EQA must be immediate and decisive. Adaptive program design, cloud-based digital integration, AI-assisted analytics, and hybrid models provide a roadmap towards more flexible and clinically relevant systems. Together, these approaches offer a template for ensuring that EQA continues to safeguard quality while supporting innovation in personalised diagnostics.

Objectives Fecal immunochemical tests (FITs) from various manufacturers yield different results, leading to variable positivity rates and clinical performance. These differences can influence the healthcare costs of a colorectal cancer screening program (CRC-SP), specifically when switching among FIT manufacturers. The analytical and clinical performance of three FITs was investigated to determine adjusted thresholds using positivity rate harmonization. Methods A cohort of 6,600 participants from the Quebec CRC-SP received collection kits from three different manufacturers to sample the same fresh stool. Participants with positive results were referred for a colonoscopy which was considered positive if advanced neoplasia (AN) was detected. Positivity rates were determined for each FIT at an unadjusted threshold. Adjusted thresholds were determined using a positivity rate harmonization strategy. Clinical performance was then evaluated for each supplier at unadjusted and adjusted thresholds. Results Among 5,513 participants who fulfilled the inclusion criteria, 327 underwent colonoscopy. The FIT positivity rates at unadjusted thresholds differed between manufacturers. The adjusted thresholds determined to yield the same positivity rate were different for each FIT. A total of 69 participants were diagnosed with AN. Significant differences in concordance, discordance, sensitivity, and specificity were observed when using the unadjusted threshold. After applying adjusted thresholds, differences in clinical performance between manufacturers were no longer statistically significant. Conclusions Threshold adjustment using a positivity rate harmonization strategy render differences in clinical performance statistically nonsignificant and leads to a stable colonoscopy rate between manufacturers. CRC screening programs should determine adjusted thresholds when changing FIT suppliers or when using multiple assays.

The definition of analytical performance specifications (APS) by the Milan model 1b is based on indirect approaches investigating the impact of analytical performance of the laboratory test on clinical classification and thereby on the probability of patient outcomes. As direct diagnostic outcome studies (Milan model 1a) for defining APS are now considered very difficult and costly to be performed in practice, expert groups have gathered to reach consensus on how to use available information and apply Milan model 1b to the definition of APS. They have highlighted three major aspects: a) the definition of the clinically acceptable misclassification rate(s); b) the influence of the clinical pathway and patient population and setting (disease prevalence) when diagnostic thresholds are defined, e.g., in guidelines; and c) the intended use of the test. The basic question calling for an answer is how to move forward and provide specific APS for certain measurands that are key in clinical decision making. Here, cardiac troponin testing is used as a practical example for the application of model 1b-derived APS. Proposals are made for moving to practice with the application of this model to APS definition.

Recent debates have focused on the impact of analytical imprecision in high-sensitivity cardiac troponin (hs-cTnI and hs-cTnT) assays on the diagnosis of non-ST-elevation myocardial infarction (NSTEMI), based on the absolute delta thresholds for the rapid rule-in/rule-out algorithms in emergency department (ED) according to the 2020 ESC guidelines. Consensus guidelines by the AACC/IFCC and the Fourth Universal Definition of Myocardial Infarction emphasize precision at the 99th percentile upper reference limit and the clinical significance of biomarker changes. Evidence from the Italian Study Group on Cardiac Biomarkers demonstrates that hs-cTn assays generally achieve ≤10 % CV at concentrations relevant to diagnostic thresholds, and recommends a Reference Change Value (RCV) >30 % to define clinically meaningful variation. Conversely, van Schrojenstein Lantman et al. aimed to define the assay performance specifications (APS) required to reliably detect the recommended absolute delta thresholds and the consequences of failure to meet these APS. The aim of this opinion paper is to highlight the methodological and conceptual differences between these two approaches. More specifically, while APS are influenced by both assay characteristics and clinical context, the RCV approach is based on the intra-individual biological variation that remains consistently low (individual index ≈0.3), supporting the interpretation of circulating troponins as markers of physiological cardiomyocyte turnover. Ultimately, diagnosis of acute myocardial infarction requires integration of biomarker changes with clinical evidence of ischemia, ECG, and imaging findings. Emerging artificial intelligence–based models that combine hs-cTn with clinical variables further improve diagnostic accuracy, underscoring the importance of contextual interpretation and paving the way toward personalized medicine in the management of suspected NSTE-ACS.

Objectives The European Organization for External Quality Assurance Providers in Laboratory Medicine (EQALM) Haematology Working Group (WG) has previously provided two guidelines to support the standardization of haematology blood smear external quality assessment (EQA) (Vives CJL, Albarède S, Flandrin G, Heller S, Horvath K, Houwen B, et al. Guidelines for blood smear preparation and staining procedure for setting up an external quality assessment scheme for blood smear interpretation. Part I: control material. Clin Chem Lab Med 2004;42:922–6; Vives CJL, Van Blerk M, Albarède S, Gutierrez G, Heller S, Nordin G, et al. Guidelines for setting up an external quality assessment scheme for blood smear interpretation. Part II: survey preparation, statistical evaluation and reporting. Clin Chem Lab Med 2006;44:1039–43) but these recommendations did not include analytical performance specifications. In this paper the WG provides advice on the performance specifications for the evaluation of blood cell identification, comments on morphological characteristics and diagnostic hypothesis. Methods To develop these specifications, the WG made a survey of the practices in use by EQALM members and provided a standard set of EQA data, provided by one of the members, for performance evaluation. Results The results of this exercise show a variation in the performance assessment outcomes from one EQA provider to another, suggesting that a degree of standardisation would be of benefit to participating laboratories. Conclusions The WG has provided advice on common performance specifications, based on model 1 of the Milan Consensus (Sandberg S, Fraser CG, Horvath AR, Jansen R, Jones G, Oosterhuis W, et al. Defining analytical performance specifications: consensus statement from the 1st strategic conference of the European federation of clinical chemistry and laboratory medicine. Clin Chem Lab Med 2015;53:833–5).

Objectives To develop a proactive risk assessment model for human papillomavirus (HPV) genotyping testing by integrating Failure Mode and Effects Analysis (FMEA) with quality indicators (QIs), ensuring compliance with ISO 15189:2022 and improving diagnostic accuracy. Methods A multidisciplinary team designed and performed detailed FMEA across pre-analytical, analytical, and post-analytical phases of HPV genotyping testing. To improve objectivity, we integrated Sigma metrics into the FMEA framework through a molecular diagnostics-specific model of QIs (MQI). The FMEA model systematically identified testing phases, potential failure modes, their effects, root causes, and existing controls. Risk was quantified using Severity, Occurrence (from 1-year QI data), and Detection scores (1–5 scale). Risk Priority Numbers (RPNs) were calculated (Severity × Occurrence × Detection) to prioritize failure modes, with mandatory interventions implemented for high-risk items (RPN≥40). Results Five high-risk failure modes (e.g., sample misidentification, data analysis errors) were identified and successfully mitigated to acceptable levels (RPN<40) through process optimization and standardization, achieving RPN reductions of 20–80 %. We established a molecular diagnostics-specific MQI, comprising 14 pre-analytical, 25 analytical, and three post-analytical phase QIs. QI-based risk assessment of 35 evaluable QIs for HPV genotyping testing revealed one high-risk QI (“Incorrect results due to information system failures”) and three medium-risk QIs, all of which were addressed through corrective actions. Conclusions This study developed an integrated FMEA-QI model for HPV genotyping testing, establishing both a traditional FMEA framework and a molecular diagnostics-specific MQI. The combined approach improves risk assessment objectivity and enables multidimensional analysis compared to conventional methods.

The concept of value, defined as health outcomes achieved per monetary unit spent, has profoundly reshaped modern healthcare delivery. While Value-Based Healthcare models have permeated many clinical disciplines, laboratory medicine has been slow to integrate this paradigm shift. In this opinion paper, we argue for a strategic repositioning of clinical laboratories as core enablers of value in healthcare systems. Laboratory diagnostics, long considered ancillary, should be reframed as pivotal tools that support outcome-based, cost-effective decision-making. We explore how laboratory parameters contribute to clinical value through predictive accuracy, diagnostic specificity, and operational appropriateness – factors that directly influence patient outcomes and resource allocation. Examples such as vitamin D testing, albumin as a biomarker of biological age, and NT-proBNP in heart failure demonstrate the potential and pitfalls of volume-driven laboratory utilization. Beyond technical excellence, we emphasize the importance of interpretive collaboration, health literacy, and ethical stewardship of diagnostic resources. Structural challenges, including commoditization, delocalization via point-of-care testing, and the limited use of patient-reported outcomes in laboratory settings, are critically examined. Finally, we highlight emerging policy frameworks across Europe that align reimbursement models with measurable outcomes, advocating for the integration of laboratories in clinical governance and value-based procurement. In this renewed perspective, laboratories are not merely data providers but agents of personalized, sustainable, and patient-centered care.

Objectives Accurate determination of blood potassium levels in neonates presents significant clinical challenges, primarily attributable to technical difficulties in phlebotomy procedures and the elevated susceptibility of neonatal blood samples to hemolysis. This study aimed to develop and validate a hemoglobin-based mathematical model to correct for hemolysis-induced pseudohyperkalemia in neonatal blood samples. Methods This prospective study analyzed 134 neonatal blood specimens (postnatal age ≤7 days). Controlled hemolysis induction established the potassium-hemoglobin relationship, with specimens allocated to model development (n=101) and validation (n=33) cohorts. The model utilized direct hemoglobin quantification and multivariate regression, with statistical analyses performed using SPSS 26.0. Results Analysis of the training cohort established a neonatal-specific potassium release coefficient (ΔK + /ΔHb=0.28 ± 0.03 mmol/L per g Hb) and revealed a strong linear correlation between hemoglobin concentration and potassium elevation (Y=0.2834X, r 2 =0.8606, p<0.0001). Method validation showed strong agreement between constant correction and model-predicted values (p=0.887). Independent validation confirmed clinical utility, demonstrating comparable corrected and baseline potassium concentrations (p=0.0693) with excellent correlation (r=0.8845, p<0.0001). The model improved hypokalemia detection from 8.9% to 26.7 %, effectively resolving pseudo-normalization artifacts. Conclusions The developed hemoglobin-driven correction model provides a validated and accurate method for hemolysis interference in neonatal potassium measurement, offering significant clinical value for managing irreplaceable specimens in neonatal intensive care settings.

Objectives Reference intervals (RIs) are essential for interpreting laboratory test results. It is recommended that each medical laboratory establishes and reviews its own RIs. The use of direct methods is often unfeasible for most laboratories, while indirect methods are a more viable alternative. However, these methods require not only medical, but also statistical and technical expertise, thereby limiting accessibility for many laboratories. To address this challenge, a web-based application was developed to facilitate the estimation and verification of RIs using real-world laboratory data. Methods The application was developed using R Studio and the Shiny web framework. The tool supports five indirect methods for reference interval estimation: refineR, TMC, TML, kosmic and reflimR. Furthermore, a Docker container was designed to enable a secure local deployment. Results Up to 200,000 laboratory test results can be included via a straightforward copy-and-paste input. The tool provides recommendations for sex-based stratification by performing statistical analysis. In addition, a drift-detection algorithm was developed to analyze whether age-based stratification is necessary. The results of RI estimation are displayed and visualized alongside the underlying data distribution. Existing RIs can be verified by comparing them against calculated intervals. Conclusions ReferenceRangeR is a user-friendly tool for estimating and verifying RIs using real-world laboratory data, eliminating the need for statistical or technological expertise, thereby supporting laboratory professionals in meeting the current regulatory standards.

Objectives Hemolysis is a common preanalytical interference in clinical biochemistry, affecting up to 3.3 % of routine samples and contributing to approximately 60 % of rejected specimens. It results from the release of intracellular constituents, such as hemoglobin and potassium, into the plasma due to cell membrane disruption. This interference is often evaluated through hemolysis index (HI) thresholds defined by in vitro diagnostic (IVD) analyzers, but these thresholds may not align with clinical needs or biological variation. Methods A descriptive study was conducted from February to April 2025, comparing the hemolysis interference data of 72 clinical chemistry analytes across five major manufacturers: Abbott, Beckman Coulter, BioSystems, Roche, and Siemens. Manufacturer package inserts were reviewed and compared to the CLSI C56-A guideline for hemolysis interference testing. Results The compliance of manufacturers with the CLSI guideline varied significantly. Siemens had the highest compliance, reporting hemolysis interference at two analyte levels in 46/71 of cases, while BioSystems did not report analyte concentrations for any tested parameters. Criteria for significant interference was often missing, and in many cases, manufacturers used arbitrary thresholds, such as 10 % bias. Additionally, acceptance criteria and reported bias values were inconsistent across manufacturers, with only Abbott and Siemens providing detailed bias data. Conclusions Manufacturer instructions often lack completeness and consistency, making it crucial for clinical laboratories to independently validate hemolysis thresholds. A harmonized approach, incorporating manufacturer data, research, and local validation, is essential to improve laboratory quality, minimize misinterpretations, and ensure compliance with regulatory standards.

Objectives Automated urine particle analyzers are increasingly employed in clinical laboratories to standardize urinalysis. These systems can reduce workload, turnaround time and the inter-observer variation. This study aimed to assess the performance of five automated urine particle analyzers in detecting and categorizing dysmorphic erythrocytes (isomorphic-, dysmorphic erythrocytes, acanthocytes) and pathological casts, in particular erythrocyte casts, for screening of glomerular vs. non-glomerular hematuria, compared to manual phase-contrast microscopy, as the reference method. Methods The analytical performance of five automated urine particle analyzers, Atellica UAS 800, FUS-3000Plus, iQ200, SediMAX conTRUST Pro, and UF-4000, for measuring and categorizing erythrocytes and pathological casts was evaluated. Urine samples spiked with erythrocytes were used to assess precision and performance characteristics, while 68 urine samples from patients with glomerular hematuria were analyzed for concordance. Results Automated analyzers demonstrated acceptable precision at middle and high erythrocyte concentrations. At low levels, the iQ200 performed relatively well and the SediMAX met the manufacturer’s precision criteria. Manual on-screen review was required for 90 % of samples analyzed to achieve acceptable results for dysmorphic erythrocytes and cast detection. None of the analyzers exhibited strong agreement with manual microscopy for identifying isomorphic-, dysmorphic erythrocytes or acanthocytes (concordance <0.80), whereas the FUS-3000 and UF-4000 had relatively higher sensitivities for pathological cast detection (58.6 and 69.6 %, respectively). Conclusions Current automated urine particle analyzers are limited in accurately identifying dysmorphic erythrocytes and pathological casts, necessitating manual microscopy for reliable assessment. While automation offers potential for standardization and efficiency, significant technological advancements are required to improve diagnostic accuracy, reliability, and clinical applicability.

Objectives MicroRNA-192-5p, a liver-enriched miRNA downregulated in hepatocellular carcinoma (HCC), is a promising biomarker, but its clinical use is limited by technical challenges in detecting low-abundance plasma miRNAs. This study innovatively uses droplet digital PCR (ddPCR) with locked nucleic acid (LNA)-modified probes to develop an ultrasensitive and standardized method for miRNA quantification in liquid biopsies. Methods Following Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines, seven primer-probe combinations were screened by qPCR, and one with the lowest Ct variability (Ct <35) was selected. LNA-modified Probe P-2 was designed to enhance target hybridization. Reaction conditions were optimized to 1 μM primers, 300 nM probes, and 55 cycles. Analytical validation included trueness, precision, sensitivity, linear range, and interference testing. Plasma from 87 HCC patients and 57 controls was analyzed, and a logistic model combining miR-192-5p, AFP, and AFU was evaluated. Results The LNA probe improved positive droplet counts by 32 %. The dPCR showed excellent precision (intra-batch CV 2.31–21.63 %, inter-batch 17.54 %) and trueness (R=0.92 vs. RT-qPCR). Sensitivity thresholds were LoB=1.75, LoD=3.33, LoQ=13.45 copies/μL, with linear range 13.45–129,693 copies/μL (R 2 =0.9965). It tolerated low hemoglobin and triglycerides but was affected by bilirubin. HCC patients had lower miR-192-5p (444.2 vs. 753.5 copies/μL, p<0.001), with AUC=0.70. The multi-marker model had AUC=0.88. Conclusions This LNA-optimized ddPCR assay resolves miRNA liquid biopsy barriers. The combinatorial model outperforms single biomarkers, offering a clinical tool for the precise quantification of HCC-specific miRNAs. Standardized workflows ensure reproducibility, and multicenter studies are needed for validation.

Objectives Regulatory guidelines recommend non-parametric Passing–Bablok regression for evaluating the agreement between two measurement methods in laboratory settings. However, concluding for the agreement if the 95 % CI of the slope and of the intercept include 1 and 0, respectively is incorrect since the agreement assessment must focus on a null hypothesis of not equivalence and an alternative hypothesis of equivalence. Methods We exhaustively simulated appropriate structural models with several values of slope, intercept and measurement error by keeping equal variances and means of the two methods. We calculated the slope and intercept bias of four regressions: non-parametric Passing–Bablok, Theil, Ordinary Least Squares and Deming. In addition, we calculated the percentages of the agreement according to the not shareable Passing–Bablok suggestion. Furthermore, we calculated the percentages of the 95 % CI of the slope and of the intercept included within sensible equivalence thresholds for assessing the agreement. Results Passing–Bablok procedure gives unbiased estimates, a little more and less biased than those from Deming’s regression. The percentages of rejecting the hypothesis of no-agreement, according to the wrong Passing–Bablok’s approach are correctly near to 0.05 Type I error under the agreement and also for 0.990≤slopes≤1.005. However, they are too low for slopes >1.05 and <0.950. Conclusions The Passing–Bablok 95 % CIs are too wide for being included in sensible agreement thresholds according to a population equivalence model and, finally, this approach cannot be considered under the best agreement model of the individual equivalence.

Accurate interpretation of laboratory data is a critical step in clinical decision-making. This requires the availability of reliable reference data for comparison. Reference data can be derived from various sources, including hospital or laboratory databases, groups of reference individuals, or an individual’s own data, and can be estimated using different statistical approaches. In addition to the possible lack of standardization of measurement methods this diversity results in the availability of multiple reference intervals for a given measurand. However, selecting the most appropriate reference data is challenging and requires a systematic approach to identify the best available option for each measurand. In this opinion paper, we aim to develop a systematic approach for constructing a hierarchical structure encompassing all known reference interval (RI) models, to discuss the advantages and disadvantages of each, and to provide a framework for selecting the most appropriate RI for routine clinical practice. To illustrate the model visually, we constructed a hierarchical pyramid with the less reliable reference intervals positioned at the base, gradually increasing in reliability toward the top. Based on the data sources and the statistical approaches used to estimate RIs, we conclude that, at least from a theoretical perspective, the currently widespread model – discrete population-based RIs derived from hospital or laboratory data – occupies the lowest level, that is, it represents the ground of the hierarchical pyramid, whereas multivariate continuous personalized RIs reside at the top.

Objectives To validate urine as a suitable matrix for copeptin measurements, derive a urinary copeptin/creatinine ratio in an apparently healthy sample of the population, and assess pre-analytical conditions such as collection type and storage temperatures that affect stability and recovery. Methods Matrix effect was determined by spiking the manufacturer’s calibrator F simultaneously into pooled serum and aliquots of spot urine sample donated by a single volunteer. Stability was assessed without preservatives as well as 0.0032× g of thymol or 1 mL of 5 M hydrochloric acid. The urinary copeptin-to-creatinine ratio reference limit was derived from random spot urines collected from 120 apparently healthy volunteers who met the selection criteria. Results Our in-house derived urinary copeptin-to-creatinine ratio was<2.5 pmol/mmol creatinine, and there was a statistically significant difference in urinary copeptin concentration between males and females, but this was eliminated when corrected for urine creatinine. 24-h and spot urine with and without preservatives were stable for 7 days at 4–8 °C, 23–25 °C, and −20 °C. Limits of blank, limits of detection, and limit of quantitation were 1.1 pmol/L, and 1.81 pmol/L, respectively. Conclusions Urine matrix does not distort the kinetics of the BRAHMS Thermo Fisher Copeptin assay and therefore could be measured with high accuracy. The clinical utility of urinary copeptin is still unknown, however, there is growing interest in this area in patient with autosomal polycystic kidney disease.

Objectives To compare the diagnostic performances of GFAP and UCH-L1 with S100B in detecting intracranial injury, while investigating the impact of confounding factors. Methods In a large prospective cohort of 1,010 patients with mild traumatic brain injury, we first evaluated the clinical performances of S100B and the GFAP/UCH-L1 combination. To explore the impact of pre-analytical interferences on GFAP and UCH-L1 levels, HIL indices (hemolysis, icterus, lipemia) were measured using the Atellica ® analyzer, and spiking experiments were performed with increasing concentrations of hemolysate, bilirubin, and Intralipid ® . We then assessed the influence of four confounders on biomarker specificity: age over 80 years, hemolysis, neurodegenerative diseases, and extracranial fractures. Finally, we evaluated the ability of the biomarkers to predict clinical outcomes at one month. Results S100B and the GFAP/UCH-L1 combination showed sensitivities of 96 and 100 %, and specificities of 25 and 27 %, respectively. False positives were significantly associated with age >80 and extracranial fractures for S100B; with age >80 and neurodegenerative diseases for GFAP; and with age >80, hemolysis, and extracranial fractures for UCH-L1. UCH-L1 levels were markedly increased by hemolysis, starting at 400 mg/L of hemoglobin. Age was the only confounding factor to significantly affect specificity. Using age-adjusted thresholds in patients over 80 increased specificity to 30 % for S100B and 33 % for GFAP/UCH-L1. Overall, the biomarkers exhibited limited predictive value and performed poorly for one-month clinical outcomes. Conclusions S100B and the GFAP/UCH-L1 combination demonstrated very high sensitivities, close to 100 %, with specificities of approximately 30 % for the diagnosis of intracranial lesions. Age-adjusted thresholds improve specificity in older patients, supporting their clinical implementation. This study also provides the first evidence that hemolysis significantly elevates UCH-L1 concentrations from 400 mg/L of hemoglobin.

Objectives Laboratories are required to routinely verify reported reference intervals (RIs), but common verification methods like the CLSI-EP28-A3c binomial test are often impractical due to sample collection requirements. Indirect verification methods like equivalence limits (ELs) use routine data from patient care but lack systematic evaluation. This study aimed to develop and evaluate a novel indirect verification method: verification of reference intervals based on the uncertainty of sampling (VeRUS). Methods VeRUS compares the to-be-verified candidate RI to an RI estimated from local routine data. Acceptable differences are based on the sampling uncertainty intrinsic to the nonparametric method for establishing RIs with n=120 samples. The three verification methods were systematically compared with simulated test sets resembling 10 differently distributed biomarkers and a wide range of plausible candidate RIs. Results The binomial test is inherently unable to reject too wide RIs; e.g. the 99.8 %-interval, for which ELs and VeRUS showed high rejection rates (mean 89.2 %, SD 31.5 % and mean 95.8 %, SD 2.3 %, respectively). Moreover, the binomial test incorrectly accepts 29.3 % of “too narrow” 80%-intervals, whereas the false acceptance rates of ELs and VeRUS were lower (mean 21.7 %, SD 40.9 % and mean 7.2 %, SD 4.7 %, respectively). Overall, both indirect verification methods demonstrated increased statistical power, while ELs were least consistent among different biomarker distributions. Conclusions Its robust performance without the need for sample collection makes VeRUS an attractive tool for RI verification. By enabling routine verification of previously practically unverifiable RIs (e.g., in pediatrics), VeRUS may enhance clinical decision-making and improve patient care.

Objectives To investigate the staining and serological characteristics of AC-30 pattern. Methods A total of 184 participants were recruited from patients who underwent routine antinuclear antibody testing between 2022 and 2023 at Peking Union Medical College Hospital. Cohort 1 (n=47) showed the AC-30 pattern on HEp-2 indirect immunofluorescence assay, and cohort 2 (n=137) showed AC-2 pattern as control. Anti-DFS70 antibody detection and DFS70 antigen adsorption assays were conducted. Pattern simulation assays were performed by combining serum samples exhibiting classic AC-2 pattern with other common HEp-2 IIFA patterns. Results Anti-DFS70 antibodies were detected in 97 % of patients in cohort one and in all patients in cohort 2. The titers of the IIFA pattern showed a weak correlation with anti-DFS70 antibody levels in cohort 1 (r=0.35, p=0.0331). In DFS70 antigen adsorption assays, a higher proportion of homogeneous nuclear staining was observed in cohort 1 (79 %) than in cohort 2 (62 %) (p=0.037). Simulated samples mixed classic AC-2 with homogeneous pattern resembled those of AC-30 pattern in both interphase and mitotic cells. Especially, the staining characteristics of AC-2 became increasingly indistinct when mixed with higher-titer homogeneous patterns. Among samples exhibiting homogeneous patterns post-DFS70 immunoadsorption, non-autoimmune conditions were more common in cohort one than cohort two. Conclusions The presence of relatively lower anti-DFS70 antibodies levels or coexisting high-titer homogeneous patterns may contribute to the development of the AC-30 pattern rather than AC-2. This finding needs to be further confirmed in larger-scale studies.

The College of American Pathologists (CAP) has maintained the highest standards for laboratory medicine through education, evaluation, and certification. One form of External Quality Assurance – proficiency testing (PT) – is the centerpiece of that mission. Over 500 medical and scientific experts oversee CAP PT programs which include more than 600 tests performed by 22,000 laboratories in over 100 countries. It is the most comprehensive laboratory peer-review comparison program in the world. The CAP offers four urine sediment PT products tailored to the needs of different laboratories. Each includes three or four digital images, shipped twice a year. The program is overseen by the Hematology and Clinical Microscopy Resource Committee. Images are graded if there is 80% or greater consensus of either referee or participant laboratories. Failing laboratories must analyze the reasons for the failure, report the results, and initiate corrective action. Over the years, there has been a progressive decline in the number of errors, demonstrating that education and regulatory oversight are major contributors to improved PT performance and, by extension, patient care. The PT urine sediment image databank is a unique resource, representing the consensus of many laboratories. Participant and referee responses identify which morphologic variants are unambiguous and which are more difficult to classify. The PT challenges include discussions of disease pathophysiology and key morphologic features. This teaching component is what helps to set the CAP’s program apart. The discussions formed the basis for the Color Atlas of Urinary Sediment published by the CAP in 2010.

Objectives Clinical laboratory operations consume large amounts of electricity and water and generate considerable quantities of solid waste. In this study, we quantified and compared the estimated solid waste produced from performing one million basic metabolic panels (BMPs) across five major high-throughput chemistry analyzer systems commonly used in the United States. Additionally, we estimated the carbon emissions associated with the electricity and water usage of each analyzer to better understand their environmental impact. Methods Five academic medical centers each using a different clinical chemistry analyzer system, collected their respective annual vendor purchase data (January 2022–December 2022). Consumables including reagent cartridges, packaging boxes, inserts associated with reagents, calibrators, and QC materials were categorized and weighed separately. Solid waste estimates for one million BMPs were calculated based on institutional testing volumes. Water and electricity usage for the analyzer systems was obtained and estimated CO 2 emissions (CO 2 e) were determined using standard emission factors and calculators. Results The study found that performing one million BMPs on wet chemistry analyzer platforms, Abbott, Beckman, Roche and Siemens generated 995 kg, 1,274 kg, 579 kg and 489 kg of solid waste respectively while the dry chemistry Ortho system produced 160,593 kg. The majority of waste across all platforms was plastic. Annualized CO 2 e from electricity and water use totaled to 69,665 kg CO 2 e. Conclusions Analysis of BMPs generates substantial amounts of solid waste and CO 2 e across major diagnostic platforms. These findings emphasize the urgent need for sustainability strategies in diagnostic laboratories to address their environmental footprint.

Objectives This study aimed to perform an extended analytical verification of the Immunoglobulin Isotypes (GAM) for the EXENT ® Analyzer (EXENT ® -GAM) assay, a MALDI-TOF mass spectrometry-based method for detecting and quantifying serum M-proteins in patients with plasma cell dyscrasias, and to compare it with conventional serum protein electrophoresis (SPEP), serum immunofixation electrophoresis (sIFE) and serum free light chains (sFLC) assays. Methods Imprecision, linearity, limit of quantification (LOQ), quantification comparison with SPEP, isotyping concordance with sIFE and sFLC, interference from therapeutic monoclonal antibodies (t-mAbs), sample stability, and reagent lot consistency were evaluated. Results EXENT ® -GAM demonstrated acceptable imprecision (CV ≤20 % for low and ≤15 % for high M-protein levels) and wide linear range (∼0.03–30 g/L). The polyclonal immunoglobulin background negatively influenced the assays LOQ. M-proteins with mass-shifted light chains (i.e., glycosylated light chains) are prone to non-linearity and inferior LOQ. M-protein quantification by EXENT ® differed systematically and proportionally from quantification by SPEP, highlighting non-interchangeability. EXENT ® demonstrated 97 % concordance with sIFE for M-protein isotyping and identified numerous additional (low-level) M-proteins. Some proved to be clinically relevant (residual disease in sIFE-negative samples); others lacked correlation with sFLC result or clinical diagnosis. EXENT ® reliably distinguished endogenous M-proteins from t-mAbs, except talquetamab, which interfered with quantification and was partially misclassified. Conclusions EXENT ® -GAM enables sensitive and reproducible quantification and isotyping of M-proteins below the detection limit of SPEP and sIFE. Its ability to resolve analytical challenges posed by SPEP and sIFE represents a significant advancement. Further clinical studies are needed to confirm its potential in residual disease detection.

Objectives The aim of this study was to evaluate the impact of introducing an autonomous courier in a clinical laboratory, focusing on specimen turnaround time (TAT). We assessed whether high-frequency robotic transport from the central accessioning area to analytical sections could reduce delays and variability compared with manual delivery. Methods We retrospectively analyzed routine lithium–heparin glucose specimens processed during the initial days of robot deployment (13–23 May 2025, weekdays, 08:00–16:00) and compared them with the same period in 2024 under manual transport. TAT was defined as the interval from check-in at accessioning to technical validation in the Laboratory Information System (LIS). Data were assessed globally and stratified by time of day, examining changes in central tendency, dispersion, and extreme delays. Results In total, 6,299 samples in 2024 and 5,759 in 2025 were analyzed, showing a leftward shift of the distribution with fewer delay. In 2025, the mean TAT decreased from 122.64 to 106.72 min, the median from 112 to 101 min, and dispersion tightened. Stratification by time of day also demonstrated consistent improvements. Conclusions Even in its earliest days of operation, the delivery robot reduced TAT and variability, converting specimen transport from batch runs into near-continuous flow. These findings highlight ease of adoption and the potential of robotic transport to improve speed, predictability, and safety in intralaboratory logistics. Further validation across longer periods and additional laboratory sections is warranted.

Objectives The potential relationship between cardiac troponin (cTn) composition and etiologies of myocardial injury may provide diagnostic insights for myocardial infarction (MI). We compared the performance of novel ternary cTnI–cTnT–TnC complex (ITC) assays with high-sensitivity (hs)-cTnI and hs-cTnT in differentiating MI patients at presentation. Methods Plasma samples from 1,210 patients with suspected MI were collected. Major cTn forms were quantified using long-cTnT ITC complex, hs-total ITC complex, hs-cTnI and hs-cTnT assay. We compared the assay performance based on the 99th percentile upper reference limits (URLs), evaluated their area under the receiver operating characteristic curve (AUC), and assessed their ability to differentiate MI using established cut-off values. Results A total of 138 (11.4 %) patients were diagnosed with MI. 86 % patients had long-cTnT ITC complex concentration below the 99th percentile URLs (sensitivity: 0.818, specificity: 0.943, NPV: 0.975, PPV: 0.649). AUC was 0.959 for long-cTnT ITC complex, comparable to hs-cTnI (0.953) and superior to hs-total ITC complex (0.889) and hs-cTnT (0.927). Using established cut-off values, the long-cTnT ITC complex assay classified 10 % of patients as rule-in and 52 % as rule-out (sensitivity: 0.993, NPV: 0.998, specificity: 0.972, PPV: 0.760), which was comparable to hs-cTnI and better than hs-total ITC complex and hs-cTnT. In early-presenting patients, the long-cTnT ITC complex demonstrated enhanced discrimination and improved diagnostic performance. Conclusions The long-cTnT ITC complex assay demonstrated performance comparable to hs-cTnI and superior to hs-total ITC complex and hs-cTnT in distinguishing MI using a single sample at presentation. These findings suggest its potential utility in improving risk stratification and triage strategies, particularly among early-presenting patients.

Objectives High-sensitivity cardiac troponin T (hs-cTnT) is used in pediatric healthcare to diagnose and assess the prognosis of various cardiac diseases, such as acute myocarditis, congenital heart disease, dilated cardiomyopathy, and heart failure. However, the standardized age-related reference intervals, 99th percentile cut-offs, and clinical guidelines are currently unavailable, especially in newborns and infants, making interpreting this biomarker challenging. Methods Residual serum samples were collected from 1,047 pediatric subjects with no cardiovascular disease, including 145 neonates and 902 infants. The concentrations of hs-cTnT were analyzed using Roche’s Elecsys Troponin T hs assay. The age-related 99th percentile cut-offs with 90 % confidence intervals (CIs) of hs-cTnT were established. Results Subjects were divided into age subgroups as follows: newborns (0–30 days, n=142), infants aged 1 month (31–60 days, n=223), 2 months (61–90 days, n=152), 3–4 months (91–150 days, n=211), 5–6 months (151–210 days, n=149), and 7 months-1 year (211–365 days, n=149). Serum hs-cTnT concentrations were highest during the first month of life and progressively declined in a year. The 99th percentile cut-offs of hs-cTnT concentration in each group were as follows: 114 (90 % CI: 114-114), 65.6 (63.7–66.1), 55.2 (54.4–55.2), 30.4 (29.1–30.9), 23.5 (22.1–23.5), 12.7 (12.4–12.7) ng/L. The 97.5th percentile cut-offs for each group were 109.4 (105.2–114), 62.5 (60.5–63.9), 52.3 (50.3–54.4), 29 (28.5–29.1), 20.5 (19.2–23.4), and 12 (12–12.1) ng/L. Conclusions This study aimed to provide reliable pediatric reference values for hs-cTnT based on a population of well-child children. These reference intervals and 99th percentile cut-offs will inform clinical decisions in the pediatric cardiology setting.

Objectives Procalcitonin (PCT) is increasingly used to support sepsis diagnosis, but its role in predicting outcomes remains uncertain, particularly in low-resource settings. We evaluated whether single and serial PCT measurements were associated with 28-day mortality in critically ill adults with suspected sepsis. Methods We conducted a retrospective study of adult intensive care unit (ICU) patients with suspected sepsis and at least one PCT measurement at a tertiary hospital in South Africa (August 2022-July 2023). Baseline PCT was analysed using multivariable logistic regression. Among patients with ≥2 PCT values, we examined the association between PCT slope (from patient-level linear regression) and mortality. Latent class mixed models (LCMM) were used to identify PCT trajectory subgroups. Results Of 371 patients, 119 (32 %) died within 28 days. Higher baseline log-PCT was independently associated with increased mortality (adjusted odds ratio [aOR] 1.58; 95 % CI 1.01–2.50). A rising PCT slope trended toward higher mortality (aOR 3.56 per unit/day; p=0.06). LCMM identified three trajectory classes with distinct mortality risks (class 2: aOR 4.53; class 3: aOR 4.35, vs. reference). These models were based entirely on laboratory data and did not assess clinical scoring systems. Conclusions Both baseline and serial PCT measurements predicted mortality in ICU patients with suspected sepsis. Modelling approaches based on routine laboratory data may offer scalable tools for early risk stratification in resource-limited settings.

Objectives Liquid chromatography-tandem mass spectrometry (LC–MS/MS) is increasingly used in clinical laboratories due to its high analytical specificity and multiplexing capabilities. While the pre-analytical and analytical phases have seen significant advances in automation, the post-analytical phase remains a bottleneck with limited standardisation. This survey aimed to identify gaps, benchmark practices against guidelines, and inform recommendations for improving consistency and quality in post-analytical data handling. Methods A descriptive electronic survey with 53 questions was distributed via the IFCC mailing list and collaborating organisations from May to July 2024. Questions covered participant characteristics, post-analytical quality indicators (e.g., chromatograms, calibration curves, ion ratios), quality assurance, and data management. Responses were benchmarked against three LC-MS/MS guidelines. Results Of 311 initial responses, 203 valid submissions from 57 countries were analysed. Laboratories reported diverse throughput: low (<100 samples/week, n=50), medium (100–2,000 samples/week, n=125), and high (>2,000 samples/week, n=28). Key findings included inconsistent acceptance criteria (e.g., variable ion ratios and signal-to-noise thresholds), reliance on manual data transcription (38 % of laboratories), and continued use of in-house spreadsheets. Practices often deviated from guidelines, with opportunities identified for harmonisation and automation. Conclusions In collaboration with the clinical mass spectrometry community, the IFCC-ETD developed six key recommendations from this survey to address challenges and enhance consistency, quality, and efficiency in post-analytical data handling.

Objectives Dehydroepiandrosterone sulfate (DHEAS), the sulfate ester of dehydroepiandrosterone, is one of the most common steroid hormones in the human body and the precursor of several other androgens. It is primarily used as a diagnostic or prognostic indicator in adrenal and reproductive disorders. Present immunoassays for DHEAS lack sensitivity and specificity, being vulnerable to cross-reactivity with endogenous interferences. Therefore, an isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) was developed to quantify DHEAS in human serum/plasma. Methods We ensured traceability to the International System of Units by using quantitative nuclear magnetic resonance to characterize a commercially available DHEAS reference material used for assay calibration. To mitigate matrix effects and prevent interference co-elution, a two-dimensional heart-cut LC method was employed for LC-MS/MS, in combination with a solid phase extraction sample preparation protocol. Selectivity was determined by spiking the prepared internal standard with the interferences testosterone, epi-testosterone, dehydroepiandrosterone, 5α-dihydrotestosterone, and estrone, in analyte free matrix. A post-column infusion experiment and comparison of standard line slopes were performed to evaluate matrix effects. Precision and accuracy were assessed via a multi-day validation experiment, and variability components estimated using analysis of variance-based variance-components analysis. Measurement uncertainty (MU) was evaluated in compliance with current guidelines. Results This RMP was suitable for analyzing DHEAS within the 0.800 to 8,400 ng/mL (2.17–22,800 nmol/L) range, demonstrating selectivity, sensitivity, and matrix-independence. Trueness and accuracy assessment revealed a relative bias (n=6) between −1.9 and 0.3 % for surrogate matrix samples (except for 5.9 % at the lowest level), −2.3 to 3.6 % for Li-heparin plasma samples and sample dilutions, and an overall bias between 0.7 and 1.8 % (n=60), indicating no statistically significant bias. The measurement process resulted in standard measurement uncertainties (MUs) ranging from 4.0 to 5.6 % for the low range and 3.5–4.2 % for the high range. At a 95 % confidence level (k=2), these uncertainties expanded to 7.9–11.1 % and 7.1–8.3 %, respectively. Reference values, determined from six measurements over multiple days (n=6), had standard MUs between 1.6 and 2.1 % for the low range and 0.9–1.7 % for the high range, with expanded MUs of 3.2–4.3 % and 1.9–3.5 %. Conclusions This RMP exhibited high analytical performance for DHEAS quantification and met requirements for measurement uncertainty. Additionally, it enabled differentiation between the DHEAS and other androgens. Consequently, this RMP is suitable for routine assay standardization and clinical sample evaluation.

Objectives Oral squamous cell carcinoma (OSCC) is the most prevalent form of squamous cell carcinomas of the head and neck (SCCHN), accounting for over 90 % of all oral cavity malignancies (approximately 275,000 new cases are worldwide diagnosed annually). Early-stage oral squamous cell carcinoma (T1 and T2) has a 5-year survival rate of up to 80 %. Survival rates decrease to 20–30 % at later stages (T3-T4). Each year, there are between 275,000 and 300,000 new cases of OSCC, and over 150,000 deaths worldwide. OSCC are usually non-hereditary tumors, although familial epidemiology has been recently reported. Methods From 2022 to 2024, we enrolled 56 patients from the complex structure of maxillofacial surgery and ORL, National Cancer Institute – IRCCS – Fondazione G. Pascale. The individuals enrolled underwent molecular testing via a multigene panel of 56 genes related to cancer predisposition customized in our laboratory. The panel included BRCA1 and BRCA2 . Results We identified a total of 7 pathogenic mutations annotated in clinical databases as ClinVar, in BRCA2 (two different variants), BRCA1 , MUTYH , BRIP1 , FANCM and FANCC genes (approximately 12.5 % of our patients). The results show a frequent predisposition to head and neck tumors similar to or even greater than that observed in other types of neoplasia, such as breast and ovarian cancers or colon cancer), with a predisposition of approximately 10 %. Conclusions Our results confirm that, similarly to other more studied tumors, predictive genomic medicine can play a crucial role in the early identification of germline mutations in head and neck cancers. This approach should be considered for the early detection of OSCC particularly for individuals at increased risk, e.g., those with a family history of the disease, who may also be candidates for targeted molecular therapies based on their genetic profile.

Objectives Dental amalgam (50 % mercury (Hg) by weight) is a commonly used material to restore a tooth damaged by decay. In recent years, amalgam safety has become a matter of ongoing controversy. This hypothesis-testing epidemiological study evaluated the relationship between blood Hg concentrations and amalgams in American adults. Methods Examination of the 2015–2016 National Health and Nutrition Examination Survey (NHANES) was undertaken using SAS, version 9.4 (Cary, NC, USA), survey regression statistical modeling (with adjustments for covariates). A total of 180,811,187 weighted-Americans (n=1,377) between the ages of 18–70 years-old, with known: dental filing surface status; urinary Hg concentrations; total and blood Hg species (inorganic and methyl-Hg) concentrations; bodyweight; and urine flow rates were examined. Results Significant increases were found in the blood concentrations of total and inorganic Hg, when comparing adults exposed to amalgams as compared to adults not exposed to amalgams. Amalgam surfaces significantly correlated with blood inorganic Hg concentrations, and estimated daily Hg vapor doses from amalgams significantly correlated with blood total and inorganic Hg concentrations. This study supports the importance of blood as an important transport avenue for Hg, which is dose-dependently released by amalgams, to accumulate in tissues and cells throughout the body. Conclusions Persons with amalgams, desiring to lower their blood Hg concentrations, should consult with a dentist trained and certified in safe amalgam removal. Also, pharmaceutical treatments to reduce/render non-toxic the blood Hg concentrations from amalgams should be considered. Efforts should be made to reduce/eliminate the continued use of amalgams.

Objectives DNA methylation in short homeobox gene 2 ( SHOX2 ) and RAS association domain family protein 1 ( RASSF1A ) shows significant auxiliary diagnostic value for early-stage lung cancer. However, the detection accuracy is frequently compromised because of the complexity of the analytical procedures. This pilot external quality assessment (EQA) study evaluated the performance of methylated SHOX2 ( mSHOX2 ) and methylated RASSF1A ( mRASSF1A ) detection across clinical laboratories in China. Methods A sample panel was prepared containing two mSHOX2 -positive samples, one mRASSF1A -positive sample, one mSHOX2 and mRASSF1A double-positive sample, and one negative sample. The panel was randomly coded and distributed to clinical laboratories for mSHOX2 and mRASSF1A detection in 2023 and 2024. The returned results were compared and scored to evaluate laboratory performance. Results The sample panel demonstrated sufficient stability and applicability. Results were collected from 21 laboratories in 2023 and 29 laboratories in 2024. Approximately 90.5 % (19/21) and 75.9 % (22/29) laboratories achieved correct results in 2023 and 2024, respectively. Out of the 105 results in 2023 and 145 results in 2024, 1 false negative and 3 false positives were observed in 2023 and 6 false negatives and 12 false positives in 2024. Conclusions This two-round EQA study in China emphasizes the need for improvement in the performance of mSHOX2 and mRASSF1A methylation detection. These results underscore the critical role of EQA schemes in monitoring and improving the detection quality in clinical laboratories. To address the challenges in mSHOX2 and mRASSF1A detection, targeted recommendations are proposed to enhance detection accuracy and reliability.

Objectives Insulin-like growth factor-1 (IGF-1) is a peptide hormone currently lacking data surrounding its pre-analytical stability. This study aimed to investigate the impact of delaying centrifugation and analysis at different storage temperatures on the in vitro stability of IGF-1. Methods The study followed the Checklist for Reporting Stability Studies (CRESS). Ten healthy, adult participants were recruited to donate 40 mL of blood each. Blood was collected into serum separator tubes. Samples were aliquoted and split into two main categories i.e., immediate centrifugation (timepoint zero) and delayed centrifugation (by 8 and 24 h). These categories were further subdivided into different storage temperatures i.e., −20 °C, 4 °C, 20–25 °C and 30 °C. All measurements were conducted in duplicate on a DiaSorin LIAISON ® XL (Via Crescentino snc, Saluggia, Italy). Analysis occurred immediately after centrifugation (timepoint zero), after 8, 24, 48, 72, 168 and 672 h in the respective categories. The percentage deviation (PD) from baseline was calculated and a maximum permissible difference of ≥10.7 % (based on biological variation) was considered significant. Results In our study, IGF-1 was stable for at least 72 h regardless of delay in centrifugation or storage temperature. Samples that were refrigerated were stable until 168 h (PD≤5.5 %) regardless of a delay in centrifugation. Samples that were frozen at −20 °C were stable up to 672 h (PD −1.5 %). Conclusions IGF-1 is stable up to 72 h after sampling regardless of delay in centrifugation or storage temperature. Stability may be lengthened to 7 days by refrigerating at 4 °C.

Objectives Detection of paraneoplastic anti-neuronal antibodies (PNS autoantibodies) aids in diagnosing the particular syndrome and guides the search for an underlying tumor. To identify opportunities for harmonization that could enhance diagnostic value, we assessed variability in autoimmune serological testing for PNS across laboratories. Methods The European Autoimmunity Standardisation Initiative (EASI) developed a questionnaire addressing testing methods, digital tools, interpretation, clinical context, and quality assurance. This was distributed through two external quality assessment (EQA) providers (UK NEQAS and IfQ Lübeck) to clinical labs routinely performing PNS autoantibody assays. Results Among 139 responding laboratories, 78 % perform both cerebellum indirect immunofluorescence (IIF) and line/dot blot assays on serum and cerebrospinal fluid (76 %). Alignment on sample dilution (61 %) and conjugate type (70 %) is variable. Differences in antigen composition and reporting strategies contribute to variability in line/dot blot testing. Digitization is widespread for line/dot blot data (87 %) but limited for cerebellum IIF (31 %). About 53 % use testing algorithms that vary by sample type and requested antibodies. Internal quality control is performed by 89 % (IIF) and 48 % (line/dot blot), mainly using commercial controls. Nearly half (43 %) participate in multiple EQA programs; 46 % (IIF) and 42 % (line/dot blot) have ISO15189 certification, with 20 % planning certification. Positivity rates are low (<6 % in 73 % of labs), indicating low pre-test probability. While only 46 % restrict testing by discipline, most labs access clinical context (84 %) and interact with clinicians (63 %). Conclusions Considerable variability exists in PNS autoantibody assay execution, reporting, and quality control. Introducing lab-specific recommendations may harmonize practices and improve diagnostic quality.

Objectives To prevent intoxication with 5-fluorouracil (5-FU) during chemotherapy, the initial measurement of plasma uracil levels has recently been recommended by the European Medicines Agency. Here we present and compare two new chromatographic methods for analysis of uracil in plasma. Two independent laboratories analyzed the same set of samples using in-house-developed analytical methods. The aim of this comparison is to demonstrate that our methods are harmonized, providing comparable concentrations and consistent clinical interpretations. Methods The approaches presented employ mass spectroscopic detection in negative ion mode but differ in sample preparation and chromatographic techniques. Method 1: Protein precipitation using acetonitrile, followed by injection of a small sample volume for hydrophilic interaction liquid chromatography (HILIC). Method 2: Protein precipitation with acetonitrile combined with filtration through a 96-well filter plate, followed by evaporation and reconstitution in the chromatographic mobile phase for C18 reverse-phase chromatography. Results Both methods are demonstrated robust and accurate quantification of uracil. Results obtained from patient samples (n=161) show a strong correlation between the two methods, with a relative mean deviation of 2 %. The same clinical interpretation is obtained for the majority of samples (150/161). The remaining samples (11/161) were close to the lower clinical decision limit (16 μg/L). Conclusions For the analysis of uracil, both presented methods, which are based on simplified sample preparation procedures, provide accurate results without significantly impacting the clinical interpretation. However, the results indicate a potential gray area of analytical imprecision from 14 to 20 μg/L, encompassing the lower clinical decision limit (16 μg/L).

Objectives Precise and accurate determination of urinary cortisol is important for diagnosis and monitoring of cortisol excess and deficiency. To achieve this, a selective and specific, isotope-dilution two-dimensional heart-cut liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method was developed. Methods A novel, two-dimensional heart-cut LC-MS/MS method was developed using a Waters Acquity BEH C18 column and a Raptor Biphenyl column, with water (A1) and with 0.2 mM ammonium fluoride in water (A2) and methanol (B1+B2) as mobile phases. This approach was complemented by a supported liquid extraction (SLE) sample preparation protocol. Matrix effects were assessed through post-column infusion experiments and comparisons of standard line slopes across matrices. A multi-day validation study evaluated accuracy and precision of the candidate reference measurement procedure (RMP) and measurement uncertainty (MU) was estimated in compliance with current guidelines. Reproducibility was assessed by performing a method comparison study between two independent laboratories. Results In the first LC column dimension, cortisol and prednisolone were separated from cortisone, prednisone, and 20-beta-dihydrocortisone. Baseline separation of cortisol from prednisolone was achieved in the second dimension. No interfering signals were detected at the retention time for quantifier and internal standard transitions in any urine samples analysed. No significant matrix effect, ion suppression, or enhancement was observed. Linearity was successfully demonstrated (r≥0.999); residuals were randomly distributed in a quadratic model via linear regression with an 1/x weighting. The lower limit of the measuring interval was determined at 2.00 ng/mL (5.52 nmol/L), displaying a relative deviation of −0.1 %, a coefficient of variation (CV) of 2.9 %, and recoveries of 96–103 %. Intermediate precision ranged from 1.9–3.2 % and repeatability CV from 1.6–2.7 %. Relative mean bias ranged from −3.5–1.2 %, indicating method accuracy was unaffected by matrix variance. Measurement uncertainties (k=1) for cortisol were ≤3.4 % across different concentrations and sample types. The expanded uncertainty at a 95 % confidence level (k=2) was ≤6.8 %. An increased number of measurements (n=6) for target value assignment reduced the uncertainty to 0.8–1.3 % (k=1), yielding an expanded uncertainty (k=2) of 1.7–2.5 %. Conclusions The validation results confirm the robustness, accuracy, and reliability of this RMP for determination of cortisol in human urine within a working range of 2.00–220 ng/mL (5.52–552 nmol/L).

Capillary blood sampling is gaining recognition as a patient-centered alternative to traditional venipuncture. Its main advantages are that it is less invasive, reduces patient discomfort, and potentially can be performed without the direct supervision of a healthcare professional. These features could facilitate access to healthcare, particularly for patients with limited access to clinical facilities or those requiring frequent testing, thereby supporting telemedicine, and improving participation in screening programs and clinical trials. However, its integration into routine clinical practice requires the development of standardized traceable processes and user-friendly collection kits that preserve sample quality and have preanalytical error rates comparable to those of venipuncture. Compliance with ISO 15189 and integration into existing laboratory workflows are essential. Moreover, device affordability and compatibility with analyzers will determine accessibility and scalability. Addressing logistical, regulatory, and technical challenges is key to fully realizing its potential as a patient-centric solution in modern healthcare, where the robustness of analytical data derived from capillary blood could be comparable to that obtained from venous blood. In this review, we analyze the potential role of capillary blood in routine clinical practice, highlighting its benefits and challenges and determining the conditions necessary for its successful implementation.

Objectives Identifying the potential sources of bias in the direct measurement of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) is important. In this study, we aimed to investigate the effect of remnant cholesterol on LDL-C and HDL-C levels measured using homogenous methods. Methods We obtained 41 commutable frozen serum samples and measured LDL-C and HDL-C levels. Eight measurement systems were used, and the degree of bias was obtained by comparing with the values obtained using the reference methods. Correlations among remnant cholesterol/LDL-C, remnant cholesterol/HDL-C, and bias were analyzed using Spearman’s analysis. Results In all eight systems, samples with a positive bias >4 % had lower LDL-C levels and higher remnant cholesterol levels, as measured by the reference methods, compared to those with a bias ≤4 %. A significant correlation between remnant cholesterol/LDL-C and a positive bias of LDL-C was observed in six of the eight systems evaluated. For HDL-C bias, three systems showed a positive correlation, and three systems showed a negative correlation. In some systems, LDL-C bias was higher in samples with remnant cholesterol/LDL-C ≥0.25 than in those with remnant cholesterol/LDL-C <0.25. Conclusions Remnant cholesterol has a potential effect on direct LDL-C and HDL-C measurements, which has been observed when several measurement systems are used. For these systems, manufacturers should improve the methods to reduce the interference of remnant cholesterol.

With the advent of flow cytometry and dedicated fluorescent dyes in the 1980s, automated reticulocyte enumeration has effectively superseded conventional manual microscopy, dramatically boosting throughput and precision while, through metrics including the immature reticulocyte fraction (IRF) and reticulocyte production index (RPI), delivering previously unattainable diagnostic resolution for anemia phenotyping, longitudinal tracking of marrow erythropoiesis, and evaluation of functional iron deficiency. Yet appreciable heterogeneity in analytical principles, reagent chemistry, and calibration frameworks persists across platforms, constraining result comparability and uniform interpretation across systems. The International Council for Standardization in Haematology (ICSH) is advancing commutable reference materials built on stable cell lines or synthetic microspheres, together with globally harmonized reference intervals, to achieve interoperable performance among instruments. Concurrently, the capital expense and servicing requirements of high-end fully automated analyzers continue to be a major impediment to clinical access to automated reticulocyte testing. This narrative review focuses on the “standardization and clinical accessibility of automated reticulocyte counting,” systematically elaborating the core technical principles and characteristics of mainstream instruments, analyzing advances and challenges in standardization, and envisioning its application prospects in global clinical practice.

Objectives To develop and validate a machine-learning model based on routinely available biochemical and hematological parameters for detecting wrong blood in tube (WBIT) errors in hospitalized patients. Methods A retrospective multicenter study including one internal cohort (IC) and two external validation cohorts (EVC, EVC2). The IC was balanced (50 % correct, 50 % WBIT; 25 % real, 25 % simulated), while EVC (n=800) and EVC2 (n=460) represented more realistic scenarios (95 % correct, 5 % WBIT; equally distributed between real and simulated). Parameters present in ≥ 95 % of requests were selected, and their normalized variation from the immediately preceding result was calculated. The IC was divided into a training set (IC-TS, n=324) and an internal validation set (IC-VS, n=108). Feature selection was refined with Elastic Net before training an XGBoost model. Performance was assessed in IC-VS, EVC, and EVC2. For benchmarking, the model’s discriminative ability was also compared with a multivariate Mahalanobis-based approach and with univariate delta checks within IC-TS/IC-VS. Results Sixteen of 25 candidate variables were retained. The model achieved ROC-AUC values of 0.98–0.99 and PR-AUC values of 0.93–0.99 across all validation cohorts. Recalibration improved positive predictive value and net benefit by reducing false positives, with a slight decrease in sensitivity, although all values remained ≥90 %. Specificities ranged from 98 to 99 %. The model consistently outperformed both the multivariate Mahalanobis approach and univariate delta checks within the internal cohort. Conclusions This machine-learning model, leveraging widely available routine laboratory parameters, shows strong potential for integration into clinical workflows, enhancing WBIT detection and improving patient safety.

Objectives The aim of this study is to develop a core competency-based EPAs framework for undergraduate interns in the clinical laboratory. Methods The EPAs framework was developed in three stages: formation of a research team, drafting of an initial EPAs framework, and reviewing EPAs framework. This study employed a literature review and a modified Delphi method to construct EPAs. Furthermore, a preliminary validation of established EPAs framework was conducted among 23 interns and their supervisors. Results A framework encompassing 10 EPAs was developed, including: occupational exposure and protection, collection of various types of clinical specimens, reception and preprocessing of various types of clinical specimens, basic manual operations for laboratory tests, basic instrument operations for laboratory tests, judgment on the releasability of test reports, notification of critical values, laboratory quality control, interpretation of test reports, communication with clinicians and patients. Experts in the two rounds of Delphi showed high enthusiasm, authority, coordination, and concentration. Scores for each EPA indicator’s importance, familiarity, observability, universality, and accuracy were all exceeded 3.5 points, with a coefficient of variation below 0.25. Furthermore, expected confidence levels for each indicator were established at four key internship milestones. In pilot study, significant discrepancy were found between self- and director-assessments across most EPAs (p<0.05), with interns consistently rating themselves higher, particularly in complex tasks. Conclusions With its robust validity and systematic structure, this EPAs framework provides a scalable model for assessing clinical laboratory interns. It effectively enhances internship competency and promotes global standardization in clinical laboratory education.

Objectives Progesterone regulates reproductive processes and is used clinically to monitor ovarian function in people experiencing fertility problems. Measuring serum progesterone is challenging as it is highly protein-bound and exists at very low physiological levels. An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate RMP to quantify progesterone in human serum/plasma has been developed. Methods To ensure traceability to the SI Units, this RMP utilized primary reference material from the NMIJ. For the determination of progesterone, two-dimensional heart-cut chromatography, in combination with a straightforward protein precipitation protocol, was employed to minimize matrix effects and the coelution of isobaric interferences. Accuracy and precision of the candidate RMP was assessed in a multi-day validation experiment using certified secondary reference materials, spiked serum and plasma samples; measurement uncertainty was evaluated according to the GUM. Equivalence to JCTLM-listed RMPs was determined using leftover samples from the RELA scheme. Results The candidate RMP was highly selective for progesterone within a measurement range of 0.0400–72.5 ng/mL (0.127–231 nmol/L) and matrix independent. Intermediate precision was ≤3.3 %, and repeatability ranged from 1.4 to 2.7 % across all concentration levels. The mean bias ranged from 0.1 to 0.7 % for secondary certified reference materials, from −1.6 % to −0.2 % for serum samples, and from −2.3 to 4.0 % for plasma samples. Expanded measurement uncertainty (k=2) for target value assignment (n=6) was found to be ≤3.7 %. Equivalence to JCTLM-listed RMPs demonstrated a relative bias of −2.4 to 2.2 %, all within the measurement uncertainty of the respective RMP. Conclusions A candidate RMP based on ID-LC-MS/MS for progesterone quantification is presented, providing metrological traceability, target value assignment, routine test standardization, and the analysis of clinical samples comprising human serum and plasma to ensure the accuracy and traceability of individual patient results.

Monoclonal gammopathies (MGs) represent a diverse group of plasma cell disorders that range from asymptomatic premalignant conditions to aggressive malignancies such as multiple myeloma (MM). Mass spectrometry (MS) based serum analysis offers a non-invasive, highly sensitive alternative to conventional techniques for identifying and quantifying M-proteins. It enables more precise assessment of treatment response and residual disease, particularly in patients with suspected complete response (CR), those not eligible for bone marrow procedures, or those treated with monoclonal antibodies that may interfere with standard assays. This guideline document, endorsed by the Spanish Society of Laboratory Medicine (SEMEDLAB) and the Spanish Society of Hematology (SEHH), provides comprehensive recommendations for the clinical and analytical application of MS in the detection and monitoring of M-proteins in patients with MGs. The document outlines optimal use of MS at diagnosis, during follow-up, and in reporting practices, with a focus on standardized implementation, interpretative criteria, and clinical decision-making support.

Objectives Calcitonin (CT) is the established biomarker for medullary thyroid carcinoma (MTC), but its measurement is hampered by analytical limitations. Procalcitonin (PCT) and pro-gastrin-releasing peptide (ProGRP) have been proposed as alternative or complementary markers, yet the diagnostic value of ProGRP remains uncertain. We conducted a systematic review and meta-analysis to evaluate the diagnostic accuracy of ProGRP for MTC and compare its performance with CT. Methods This study followed PRISMA-DTA and SEDATE guidelines. PubMed, Embase, Cochrane Library, and Open Grey were searched through June 2025 without language or date restrictions. Eligible studies assessed serum ProGRP for diagnosis or follow-up of MTC and reported or allowed reconstruction of 2 × 2 contingency data. Two reviewers independently screened, extracted data, and assessed study quality using QUADAS-2. A Bayesian bivariate random-effects meta-analysis estimated pooled sensitivity, specificity, likelihood ratios, and diagnostic odds ratios. Comparative analyses were performed for ProGRP studies also reporting CT. Results Eight studies (n=4,080) were included; four reported both ProGRP and CT (n=1,064). ProGRP showed pooled sensitivity of 74 % and specificity of 95 % (AUC 0.82), while CT achieved 94 % sensitivity and 91 % specificity (AUC 0.89). Subgroup analyses confirmed consistent ProGRP performance across diagnostic and follow-up settings. No publication bias was observed. Conclusions ProGRP demonstrates high specificity but lower sensitivity than CT for MTC diagnosis and surveillance. Although not a replacement for CT, ProGRP may serve as a valuable complementary biomarker, particularly in inconclusive cases. Harmonization of assays and prospective validation are required to define universal thresholds and integrate ProGRP into multimarker diagnostic algorithms.

Objectives Neuron-specific enolase (NSE) is a clinically relevant biomarker used in the assessment of neuronal damage and in the diagnosis and monitoring of certain cancers. Despite its diagnostic importance, paediatric-specific reference intervals (RIs) for NSE are currently lacking. This study aimed to establish paediatric RIs for NSE in serum and to evaluate the influence of preanalytical factors on NSE measurements. Methods Residual serum samples from routine allergy testing in 242 Danish children (aged 0.1–17.9 years) were analysed using the Roche Elecsys ® NSE assay on a Cobas platform. Both traditional non-parametric, age-partitioned RIs and continuous RIs derived via quantile regression were established. In addition, we assessed the impact of preanalytical variables, including haemolysis, and evaluated a previously proposed correction method for haemolysed samples within this cohort. Results Both the non-parametric and continuous approaches yielded consistent RIs, showing an age-dependent decline in serum NSE concentrations irrespective of sex. The traditional age-partitioned RI (95th percentile, one-sided) indicated upper limits of 36.9 μg/L and 32.0 μg/L for the age groups 0–5 and 6–17 years of age, based on samples with haemolysis <10 mg/dL haemoglobin. Conclusions This study defines age-specific paediatric RIs for serum NSE, demonstrating a physiological decline with age and highlighting higher NSE levels in healthy children compared to adults. Furthermore, within a limited range, a previously proposed simple linear correction method was validated for adjusting NSE values in mildly haemolysed samples using the newly established RIs.

Objectives To maximize participation in the international standardization effort, this recommendation aims to update the international guidance of the Working Group on Laboratory Errors and Patient Safety of the International Federation of Clinical Chemistry and Laboratory Medicine by identifying a limited and globally applicable panel of Essential Quality Indicators (QIs) focused on patient safety, clinical outcomes, and harmonization across medical laboratories. Methods Through a consensus meeting, experts from multiple countries reviewed the IFCC Model of Quality Indicators (MQI) to identify high-priority indicators. Selection prioritized the probability of patient harm, ease of detection, and feasibility of data collection and implementation within national contexts. Results Six essential QIs covering the total testing process were identified and ratified: (1) rate of misidentified requests (Pre-MisR) and misidentified samples (Pre-MisS); (2) rate of sample rejections (Pre-RejS); (3) rate of hemolysis detected either by automated hemolysis index (Pre-HemI) or visual inspection (Pre-HemV); (4) rate of unacceptable results in External Quality Assessment/Proficiency Testing (Intra-Unac); (5) turnaround time of cardiac troponin at the 90th percentile for the emergency room (Post-TnTAT, Post-TnTAT clin); and (6) rate of incorrect laboratory reports (Post-RectRep). Recommendations on calculation, reporting frequency, and integration into IFCC and national comparison programs are provided. Conclusions The proposed essential QI panel provides a standardized and feasible framework to support its integration into national comparison programs and the IFCC MQI platform. Its implementation will facilitate data consolidation, strengthen the development of national and global quality specifications, and contribute to continuous improvement in patient safety.

EU laws are usually evaluated after 10 years; in the case of the IVDR, the targeted evaluation was brought forward and stakeholders in laboratory diagnostics were invited to comment. The Association of the Scientific Medical Societies in Germany (AWMF; Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften e.V.), recognises a relevant need to amend the IVDR with regard to in-vitro -diagnostic medical devices that are manufactured and used in healthcare facilities but are not placed on the EU market. Inadequate overregulation is recognised and should be corrected as part of the generally required reduction of bureaucracy.

Objectives This study aims to determine reliable reference intervals (RIs) for total cortisol (TC) in adults considering the effects of both age and blood collection time, using indirect methods and machine learning approaches. Methods Serum TC results from blood samples collected between 08:00 and 10:00 am at the first outpatient visit were included in the study. Serum TC were measured using a Roche Elecsys Cortisol II kit. Estimated RIs by indirect methods with the support of R packages (refineR and reflimR) for the implementation of machine learning algorithms (mclust and rpart) were compared with the manufacturer’s reference interval (RI) (48–195 μg/L). Results Estimated RIs by refineR and reflimR (57–256 μg/L and 62–271 μg/L, respectively) were wider than the manufacturer’s RI. When reflimR was applied to Box-Cox-transformed data with the lambda value of 0.284 suggested by refineR, an RI of 57–251 μg/L was obtained, which was like that obtained with refineR. An even better match with the manufacturer’s RI was achieved using Gaussian mixture modelling with the mclust, which suggested one out of four clusters with an RI of 55.8–187 μg/L. Clustering the data with rpart suggested stratification into two age groups (≤35 and >35 years) and three blood collection periods (08:00–08:45, 08:45–09:35, and 09:35–10:00). The TC levels demonstrated the highest concentrations in the early morning (8:00–08:45) and in young adults (18–35 years). Conclusions This study highlights the necessity of considering both age and blood collection time in clinical interpretation and demonstrates the effectiveness of indirect methods and machine learning approaches in the verification of RIs for hormones with known heterogeneity.

Objectives The Fourth Universal Definition of Myocardial Infarction recommends high-sensitivity cardiac troponin (hs-cTn) as the preferred biomarker for diagnosing myocardial infarction. This study evaluated the analytical and clinical performance of a novel hs-cTnT chemiluminescence immunoassay (CLIA) developed by Mindray. Methods Analytical performance characteristics including precision, sensitivity (limit of detection [LoD] and limit of quantification [LoQ]), imprecision profile, linearity, sample type comparison, method comparison, and potential interferences were assessed. Sex-specific 99th percentile upper reference limits (URLs) were established in 895 healthy individuals. Clinical diagnostic performance was evaluated in 559 patients with suspected acute myocardial infarction and compared with the Roche hs-cTnT assay. Results The Mindray assay showed excellent precision (repeatability CV≤1.99 %, within-laboratory precision CV≤6.11 %), superior sensitivity (LoD: 1.4 ng/L; LoQ: 2.4 ng/L), and linearity (r=0.9978), along with robust performance against common and immunological interferences. Using the manufacturer-claimed LoD of 2.0 ng/L, measurable values were found in 99.36 % of males, 83.26 % of females, and 91.48 % overall, meeting criteria for high-sensitivity assays. The derived sex-specific 99th percentiles were 18.6 ng/L (males), 9.6 ng/L (females), and 16.0 ng/L (overall). In the clinical cohort, the assay showed 100.0 % sensitivity, 83.3 % specificity, 80.0 % PPV, 100.0 % NPV, and an AUC of 0.988, comparable to the Roche Gen 5 hs-cTnT assay. Conclusions The Mindray hs-cTnT (CLIA) assay meets high-sensitivity assay criteria and demonstrates robust analytical and diagnostic performance, offering a reliable alternative to the Roche Gen 5 assay for clinical evaluation of myocardial injury.

Objectives The soluble urokinase plasminogen activator receptor (suPAR) is a well-established biomarker of immune activation, reflecting the severity of systemic inflammation. Recent evidence showed increasing interest in suPAR as a prognostic marker, with elevated levels consistently associated with greater disease severity and mortality, in different clinical settings. Methods In this retrospective study, suPAR levels were assayed at Emergency Department admission in patients who received a diagnosis of sepsis or systemic infection during their initial clinical workup, using an automated turbidimetric assay (suPARnostic ViroGates kit on Atellica CH analyzer Siemens). The primary endpoint of this study was to evaluate the association between baseline suPAR levels and mortality, while the secondary endpoint aimed to explore their potential as indicators of clinical severity and predictors of patient outcomes. Results suPAR levels were elevated in all patients (median 6.99 μg/L) consistent with the severity of their clinical condition. A threshold of 10.2 μg/L was strongly associated with mortality, while a cut-off of 5.96 μg/L identified patients with severe disease and prolonged hospital stays. Conclusions suPAR seems to be a reliable, rapid, and clinically useful prognostic biomarker in the Emergency Department in patients with sepsis or systemic infections. Its early measurement by turbidimetric immunoassay in automation can support risk stratification, improve triage decisions, and enhance the management of these patients.

The discovery of circulating tumor DNA (ctDNA) prompted many scientists and companies to apply this new technology for cancer diagnostics. One valuable application of ctDNA is in the screening for cancer. This procedure has been coined “liquid biopsy” and unlike classical biopsy, is minimally invasive. This technology can be used to detect one, a few or several cancers, hopefully at an early, treatable stage. There is considerable debate on the ability of this technology to efficiently detect small, localized tumors since the amount of ctDNA in the circulation is miniscule, potentially leading to many false negatives. Additionally, the false positive rate is concerning, especially for low prevalence tumors. Here, we provide an update and underline important issues that need to be addressed before this technology enters the clinic. Due to substantial financial rewards of successful companies and the prospective large investment of public healthcare resources, scientists have the responsibility to thoroughly validate these technologies and make sure that these tests not only detect cancer, but they also trigger actionable interventions that improve patient survival and/or quality of life.

Laboratory errors are an important component of medical errors and are predominantly associated with the extra-analytical phases of the total testing process, particularly the pre-pre-analytical and post-post-analytical phases, which are largely dependent on clinical activities but also require laboratory support. The post-post-analytical phase is the stage in which clinicians interpret patients’ laboratory data using population-based reference data, such as population-based reference intervals (popRIs) and population-based decision limits (popDLs), typically provided by the laboratory. To minimize errors in this phase, it is essential that more accurate tools – such as personalized reference intervals (prRIs) and personalized decision limits (prDLs) – are made available to clinicians. However, population-based references are still widely used, whereas their personalized counterparts have not yet been implemented in routine practice. The discrepancy between inadequate population-based references and more appropriate personalized references can introduce systematic yet latent errors in the interpretation of patients’ laboratory data, potentially compromising patient safety even when clinicians are highly competent in data interpretation. In this opinion paper, we (1) summarize the limitations of popRIs and popDLs, (2) develop the concept of latent errors, and (3) discuss how personalized RIs and personalized DLs can be used to reduce latent errors and enable more accurate interpretation of patient laboratory data.

Objectives Neurofilament light (NfL) is an established biofluid marker of neuroaxonal injury for neurological diseases. Several high-throughput and sensitive immunoassays have been developed to quantify NfL in blood and cerebrospinal fluid (CSF), facilitating the use of NfL as a biomarker in research and clinical practice. However, because of the lack of rigorous comparisons of assays, it has been difficult to determine whether data are comparable and whether assay performance differs. Here, we compared the performance of five NfL immunoassays. Methods To assess the five NfL immunoassays (Fujirebio, ProteinSimple, Quanterix, Roche and Siemens), we used pooled plasma or pooled CSF, as well as unique samples from 20 healthy controls and 20 individuals with El Escorial defined probable or definite amyotrophic lateral sclerosis (ALS), to evaluate precision, parallelism and/or bias. We also examined correlations between plasma and CSF NfL concentrations within and across assays and evaluated their ability to differentiate healthy controls from individuals with ALS. Results Four of the five assays demonstrated exemplary performance based on our analyses of precision and parallelism. Across the five assays, NfL concentrations were lower in plasma than in CSF, although they displayed a high degree of correlation. We noted bias across assays; plasma NfL concentrations were lowest for the Roche assay and highest for the ProteinSimple assay. In addition, all assays reliably distinguished healthy controls from individuals with ALS using plasma or CSF NfL. Conclusions Four NfL assays demonstrated similar analytic performance. Alongside performance, other factors such as costs, accessibility, usability, footprint, and intended use, should be considered.

Currently, there is no accepted standard practice for determining the frequency of EQA challenge. The challenge frequency has evolved based on history and local requirements. However, EQA frequency should be based on identifying patient risks caused by poorly performing laboratories, methods, or processes in any phase of the total testing cycle. The role of IQC is to ensure result consistency from day to day and to halt reporting of results if there is an analytical failure. Historically, both activities have been based on synthetic control material. With the development of patient-based approaches to IQC and EQA, it is possible to continuously monitor analytical systems using the same patient parameter, usually the mean or median. These techniques can provide laboratories with additional information to reduce patient risk. There are limitations of Patient-Based Quality Assurance (PBQA), fundamentally the lack of middleware and connection of the analyzers to the EQA provider. It cannot be used to monitor the success of harmonization/standardization of assays to a reference measurement procedure. But the use of patient-based approaches offers an opportunity to reconsider how EQA can be undertaken and the relationship between IQC and EQA. If PBRTQC and PBQA could be implemented to provide daily peer group comparisons, then method-specific bias could be identified quickly by a laboratory. If this could be supplemented with a commutable, reference value assigned EQA program, then monitoring harmonization/standardization of assays to a reference measurement procedure could be achieved.

Objectives Transporting blood samples with pneumatic tube systems (PTSs) is routine in many hospitals. The influence of PTS transport on cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) analyses has not been comprehensively tested. Methods Paired blood samples from non-patient donors and cancer patients were transported manually or by PTS at two Danish hospitals using different PTS. A g-logger evaluated the forces applied to samples in each transport method. The concentration of short and long cfDNA fragments and ctDNA markers was analyzed using droplet digital PCR (ddPCR) and compared according to transportation. Different blood collection tubes (BCTs) were also investigated. Results All transportation methods subject samples to significantly different forces. The cfDNA concentration was slightly higher in samples transported with the Sumetzberger PTS than manually. There were no differences in cfDNA, whether using EDTA or Roche BCTs transported using Tempus600 or manually. Colorectal cancer patient samples showed insignificant changes in ratio of fragment lengths when transported by PTS compared to manual transport. In metastatic cancer patients, only a minor change in long/short fragment ratio was observed in PTS-transported samples. No discrepancy in ctDNA concentration/fraction was observed in cancer patient samples. Higher yields of cfDNA were obtained from EDTA samples, but no other differences were observed among the tested BCTs. Conclusions Although PTS transport introduced minor variations in cfDNA concentrations, these did not impact ctDNA interpretation. However, as PTSs vary in design, their pre-analytical effects should be carefully assessed, particularly as ctDNA analysis becomes more widely implemented in clinical practice.

Objectives The accurate and timely identification and notification of critical results (CR) are crucial to ensure patient safety and is an integral part of the process of medical validation of laboratory results. The objective of the study was to evaluate whether the implementation of an automated alert system care process based on CR from emergency department and inpatient units, could reduce the average length of stay and mortality. Methods A 2-year case-control study, where we compared the process of reporting CR by telephone with reporting via secure text messaging, in which the CR was directly communicated to the provider on their smartphone device. Results During the study, a total of 1,242 CR were included in the control group and 1,185 CR in the intervention group. Comparing pre- and post-implementation data, a significant improvement was observed in CR notification efficiency, with a marked reduction in turnaround time when using secure text messaging. Additionally, there was a significant decrease in the delay to first therapeutic intervention, average length of stay, and mortality. Conclusions Secure text messaging has facilitated the reporting of CR to targeted physicians to provide more effective care to their patients and to facilitate rapid and timely intervention.

Objectives With the development of genomic technologies, the isolation of genomic DNA (gDNA) from clinical samples has become more important for both clinical diagnostics and research studies. Blood samples collected in PAXgene Blood RNA Tubes, which are typically used for RNA extraction, can be used for gDNA extraction, particularly in clinical studies when only such blood samples are available. Methods We optimized the pre-treatment of blood samples collected in PAXgene Blood RNA Tubes. For the first time, three magnetic bead-based automated platforms (QIAsymphony SP, Maxwell RSC and KingFisher Apex) were compared for gDNA extraction from these blood samples. Additionally, the effects of storage at 4 °C or freeze-thaw cycles of the blood samples on gDNA yield were investigated. High-throughput extraction in 96-well format was evaluated. Results Systematic optimization of blood sample pre-treatment shows that prolonged incubation at room temperature and/or increased centrifugation speed and time improved gDNA yield from blood samples collected in PAXgene Blood RNA Tubes. QIAsymphony SP (4.27 ± 2.19 µg) and Maxwell RSC (4.82 ± 2.96 µg) produced significantly higher gDNA yields than KingFisher Apex (1.09 ± 0.61 µg). Higher gDNA yields were obtained with shorter storage time at 4 °C or fewer freeze-thaw cycles of the blood samples. In the 96-well format extraction, gDNA yields ranged from 0.24 to 13.46 µg. Conclusions Not all magnetic bead-based automated platforms are suitable for gDNA extraction from blood samples collected in PAXgene Blood RNA Tubes. Systematic pre-treatments optimization provides guidance for routine and high-throughput workflows, and storage conditions and freeze-thaw cycles offer practical reference for biobanking.

Objectives A candidate reference measurement procedure (RMP) based on isotope dilution (ID) liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated to accurately measure serum and plasma concentrations of mycophenolic acid glucuronide (MPAG). Methods Quantitative nuclear magnetic resonance (qNMR) spectroscopy was utilized for determining the absolute content (mass fraction; g/g) of the reference material, thereby, establishing the traceability to SI units. Separation of MPAG from potential interferences, whether known or unknown, was accomplished by using a Phenomenex Luna C18(2) column. For sample preparation, a protocol based on protein precipitation followed by a high-dilution step was established. A multi-day validation experiment evaluated precision and accuracy. Reproducibility was determined by comparing the results of the procedure between two independent laboratories. Measurement uncertainty (MU) was assessed in accordance with current guidelines. Results The RMP demonstrated high selectivity and specificity enabling the quantification of MPAG in the range between 0.750 and 600 μg/mL. The intermediate precision and repeatability (n=60, measurements) were found to be in the range from 0.9 to 3.7 % for serum samples and from 1.2 to 4.6 % for plasma samples. The repeatability was less than 3.5 % for serum samples and less than 4.0 % for plasma samples. The relative mean bias ranged from −0.9 to 3.2 % for serum samples and from −0.3 to 2.9 % for plasma samples. The expanded measurement uncertainties (k=2) for single measurements ranged between 2.4 and 7.7 % and were further reduced performing a target value assignment (n=6) resulting in expanded measurement uncertainties between 1.8 and 3.3 % (k=2), respectively. Conclusions We herein present a new LC-MS/MS-based candidate RMP for MPAG in human serum and plasma which offers a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

Background Investigations into human performance and health status during long-duration spaceflights are ongoing aboard the International Space Station (ISS) and are critical for planning missions beyond Low Earth Orbit (LEO). This review evaluates the current evidence on point-of-care-testing (POCT) in space, discussing requirements for POCT to support astronaut health during extended deep space missions and the potential for technology transfer to terrestrial healthcare. Contents Microgravity disrupts biochemical and hormonal regulation, leading to reversible homeostatic dysregulation across multiple organ systems in astronauts during spaceflight in LEO. Missions beyond LEO may significantly increase risks of morbidity and mortality in particular for cardiovascular disease. The ability to assess key organ functions by expanding the range of detectable biomarkers on POCTs is crucial for ongoing health monitoring, urgent clinical decisions, and future mission planning. The recent implementation of high-sensitivity cardiac troponin I on POCT will be essential for early identifying myocardial injury and facilitating telemedicine support. Furthermore, the development and use of reference change values (RCV) in space environment, derived from biomarkers measured longitudinally in real-time onboard, will be crucial to differentiate clinically relevant changes in biomarkers levels from alterations induced by microgravity. This approach may overcome the use of traditional cut-off values which are assessed and generally applied under stable terrestrial conditions. Summary Clinical laboratory capabilities on the ISS are currently minimal. As missions lengthen, and extend beyond LEO, enhanced POCT is needed. Limited data on POCT performance in space highlight the importance of laboratory professionals’ involvement to ensure high-quality testing and accurate interpretation.