Clinical Chemistry and Laboratory Medicine (CCLM)

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.

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 Lipoprotein(a) is an atherogenic particle causative of atherosclerotic cardiovascular disease. Novel treatments have been developed that lower lipoprotein(a) to unprecedented levels with cardiovascular outcomes trials ongoing. Many guidelines recommend testing once in the lifetime of everyone, but testing rates remain low. In this study we compare a lipoprotein(a) point of care testing device to laboratory analysers and assess its performance. Methods Lipoprotein(a) concentrations on residual patient samples measured on the Randox and Roche assays were compared to a novel point of care device, iProtin. Furthermore, assessment of iProtin performance characteristics were performed, including intra- and inter-assay coefficient of variation and dilutional studies. Results Lipoprotein(a) concentrations measured on the Randox and Roche assays showed strong correlation with iProtin. Regression analysis using Passing-Bablock showed the best fits for iProtin based on 58 serum samples were: 1.15 × Randox + 7.28 nmol/L and 1.02 × Roche + 17.54 nmol/L. The R 2 values for Randox/iProtin and Roche/iProtin were 0.906 and 0.912 respectively. Correlation between Roche and Randox showed Roche=1.15 × Randox − 13.33 nmol/L with an R 2 value of 0.973. Inter-assay coefficient of variation of the iProtin device showed a day-to-day imprecision over 5 days of 15.5 % (low concentration quality control) and 6.2 % (high concentration quality control). Within day imprecision was 13.2 % (lower concentration patient sample) and 14.3 % (higher concentration patient sample). Conclusions Point of care testing could be a complimentary option to laboratory testing of lipoprotein(a), especially in remote areas. It may help (re-)stratify cardiovascular risk and help tailor treatment decisions.

Laboratory medicine lies at the core of modern healthcare, enabling timely diagnosis, effective patient monitoring, and increasingly personalized therapeutic strategies. Over the past decades, automation has profoundly reshaped the role of clinical laboratories, substantially enhancing their contribution to clinical outcomes, operational efficiency, and the overall sustainability of healthcare systems. More recently, laboratory automation has emerged as a cornerstone of value-based laboratory medicine, representing not merely a technological upgrade but a strategic transformation of laboratory practice aimed at delivering measurable value to patients and healthcare stakeholders. Although automation has long been established in clinical chemistry and immunoassays, its scope is now expanding to molecular diagnostics and mass spectrometry – two disciplines that are central to precision medicine. Looking ahead, the convergence of automation, digitalization, and artificial intelligence is driving the emergence of hyperautomation in laboratory medicine. Within this paradigm, laboratories evolve from isolated testing units into integrated diagnostic hubs, in which results from multiple laboratory disciplines are harmonized and contextualized to effectively support clinical decision-making.

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.

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.

Objectives The Kappa Index has proven its diagnostic value for multiple sclerosis (MS), while its prognostic potential remains to be fully explored. The objective of this study is thus to investigate the value of the Kappa Index at disease onset in predicting disease activity and high-efficacy therapy (HET) initiation. Methods We enrolled MS patients with available Kappa Index values at disease onset and a follow-up of at least two years. Primary outcome was the time to loss of NEDA3 (no evidence of disease activity-3) defined as the absence of relapses, MRI activity, and disability progression. Secondary outcome was the time to HET initiation. Results Of 120 enrolled patients (36 M, 84 F, mean age: 35 ± 11 years), NEDA3 loss occurred in 89 (74 %) by the end of the follow-up period. A total of 98 (82 %) initiated a moderate efficacy therapy (MET); of these, 34 (28 %) transitioned to a HET during follow-up. Kappa Index values above the maximally selected log-rank statistic-derived cut-off of 38 were independent risk factors for NEDA3 loss (HR 1.75, 95 % CI: 1.09–2.80, p=0.021) and HET initiation (3.25, 95 % CI: 1.54–6.87, p=0.002) and also independently predicted HET following MET failure (2.54, 95 % CI: 1.17–5.51, p=0.018). Conclusions Elevated Kappa Index values at diagnosis predict disease activity, MET failure and HET initiation and may be a valuable adjunctive tool in identifying patients in need of prompt HET initiation.

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.

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.

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.

Objectives Evolution is difficult to predict for some patients at emergency department (ED). The soluble urokinase plasminogen activator receptor (suPAR ) is a non-specific prognostic inflammatory blood biomarker with a high negative predictive value for pejorative outcomes. The aim of this study was to investigate the relationship between low levels of suPAR at ED admission and patient discharge following hospitalization in a short stay unit. Methods We carried out a single-center prospective observational study in the acute-care hospital ward of a university hospital center, including patients over 18 years old with an intermediate triage score. Results Overall, 202 acute medical patients were included, exhibiting a mean suPAR level of 7.43 ± 3.36 μg/L. Of these patients, 25 (12.4 %) displayed a suPAR dosage below 4 μg/L and 177 (87.6 %) a dosage ≥4 μg/L. At 24 h, 55 patients (27.2 %) were discharged, 139 (68.8 %) were hospitalized, and five (2.5 %) were either admitted to intensive care or died. In contrast to group with a high suPAR rate, those with suPAR <4 μg/L benefited from secure ED discharge (OR=5.68; CI 95 %=2.6–12.4). For predicting hospital discharge, patients with a suPAR value <4 μg/L had an AUC-ROC of 0.75. (95% CI 0.67–0.83). Conclusions Our study revealed that in patients with a high triage scale level and requiring a monitoring period, SuPAR levels under 4 μg/L could have enabled five times more patients to return home compared with those exhibiting a level ≥4 μg/L at emergency visit.

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.

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 Serum protein electrophoresis is one of the keystone investigations for screening for monoclonal proteins and assessing the serum protein profile. The newly released Helena V8ultra capillary electrophoresis system has been evaluated. Methods In total, 164 serum samples were assessed on the Helena V8 UltraCE system and compared with the Sebia Capillarys™ instrument. Abnormalities suggestive of monoclonal proteins were confirmed by immunosubtraction. Imprecision studies intervals were determined. Special attention was paid to complement C3 polymorphism. Results The imprecision of the Helena V8ultra was inferior or equal from 0.73 % (albumin) to 4.32 % (alpha 2 globulin). The mean bias of Helena V8ultraCE vs. Sebia Capillarys was about −0.225 % for albumin; −0.791 % for alpha 1 globulins; 1.353 % for alpha 2 globulins; −2.317 % for beta globulins; 0.066 % for gamma globulins. Among the 6 samples with monoclonal proteins confirmed by immunofixation, all were seen on both methods, with only 1 discordant result. The theoretical plate number (albumin fraction) was 2,371 ± 718 (Helena V8UltraCE) vs. 445 ± 115 (Sebia Capillarys 2). The high resolution also allows to distinguish Complement C3 phenotypes. CV values for the various fractions were low, ranging from 0.47 % (albumin) to 0.89 % (gamma globulins). IgG, IgA and IgM M-proteins could be detected in the electropherogram with an excellent sensitivity (<0.1 g/L). Conclusions Our evaluation confirms the good analytical performance of the Helena V8 analyzer as a suitable alternative to the Sebia Capillarys instrument. The high resolution allows detailed analysis of individual protein fractions which is an excellent basis for studying microheterogeneity.

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 Prognostication in patients with severe acute brain injury (SABI) in the Intensive Care Unit (ICU) is complex, often requiring early decisions on life-sustaining therapy. Serum biomarkers such as neurofilament light (NFL), glial fibrillary acidic protein (GFAP) and neuron specific enolase (NSE) may aid in predicting outcomes. This study investigated the association between biomarker concentrations and hospital mortality in ICU patients with SABI. Methods In this prospective multi-center study, 103 adult ICU patients with SABI were enrolled within 24 h of injury. Serum concentrations (NFL, GFAP, NSE) were measured at 24 h, 72 h, 7 days, and 14 days post-injury. Biomarker concentrations between hospital survivors and non-survivors were compared using a linear mixed-effects model and ROC curves. Results GFAP concentrations peaked on day 1, while NSE and NFL concentrations peaked on day 14. Higher concentrations were observed in non-survivors for all biomarkers on day 3, a 1,4 fold increase for NSE (p=0.034) and approximately 2-fold increase for GFAP (p=0.041) and NFL (p=0.018). Distinct temporal patterns were observed for all biomarkers, although biomarker trajectories did not significantly differ between survivors and non-survivors. Prognostic accuracy was moderate when biomarkers were combined with established mortality predictors (AUC 0.749) on day 3. Conclusions NSE, GFAP and NFL show distinct temporal trajectories in SABI patients. Concentrations were higher in non-survivors during the early phase of admission. While individual biomarkers showed limited prognostic accuracy, in combination with other predictors mortality prediction improved. Serum biomarkers may still be valuable in a multimodal prognostication framework for SABI patients.

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 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.

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 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 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.

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 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.

Agentic artificial intelligence (AI) systems are distinguished by their ability to invoke multiple tools, compose command chains, and combine chain-of-thought reasoning with deep research to execute complex tasks and take actions. This represents a major evolution beyond machine learning and large language models (LLM), toward systems capable of planning, executing, and coordinating complex workflows. In contrast to traditional LLMs, which primarily focus on generating and classifying information, agentic AI introduces elements of autonomy, reasoning, and orchestration, while digital twins extend this concept to dynamic virtual representations of patients and laboratory processes, capable of continuous learning, simulation and adaptation. This transition has profound implications for laboratory medicine, a field characterized by high data complexity, multi-omics integration, and stringent operational demands. At the same time, laboratories face growing expectations regarding efficiency, resource stewardship, and value-based healthcare delivery. This article explores both the opportunities and limitations of agentic AI in laboratory medicine, highlighting its potential to move beyond static automation toward autonomous, outcome-driven innovation. It also examines the ethical, interpretability, and governance considerations that must accompany its implementation.

Objectives The rapid emergence of highly potent synthetic opioids represents a growing challenge for forensic and clinical laboratories. Methods We report the first identification in Europe of methiodone (IC-26) and the first detection in France of cychlorphine in drug samples collected by a drug-checking service in Paris. Results Preliminary screening by liquid chromatography with diode-array detection (LC-DAD) and gas chromatography coupled with mass spectrometry (GC-MS) revealed unknown peaks due to the lack of corresponding spectra in available spectral librairies. High-resolution mass spectrometry (LC-HRMS) provided accurate mass measurements consistent with the cychlorphine and Methiodone formulas. Structural confirmation was obtained by 1D/2D nuclear magnetic resonance (NMR) spectroscopy. Targeted LC-MS/MS quantified 5 % cychlorphine and 2 % methiodone, with no other psychoactive compounds detected. Conclusions These results highlight the limitations of library-based screening and the value of multi-platform strategies integrating LC-HRMS, LC-MS/MS, and NMR for the early detection of emerging drugs.

Objectives Free triiodothyronine (FT3) and free thyroxine (FT4) are important diagnostic markers for assessing thyroid function. However, their accurate quantification remains challenging due to low serum concentrations. Significant variability exists among current assay methods for measuring FT3 and FT4. This study aims to establish a candidate Reference Measurement Procedure (cRMP) for simultaneous quantification of serum FT3 and FT4 based on isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) technology. Methods A convenient and reusable equilibrium dialysis (ED) device was utilized to separate free thyroid hormones from their protein-bound counterparts in serum. Key dialysis parameters, including temperature, pH, membrane type, and duration, were optimized to ensure consistent and reliable performance. The dialysate containing FT3 and FT4 was directly quantified by ID-LC-MS/MS. The method underwent systematic validation, comparative analysis with existing assays, and a comprehensive uncertainty assessment. Results The developed cRMP demonstrated limits of quantification (LoQ) of 1.54 pmol/L for FT3 and 3.22 pmol/L for FT4, and with an imprecision of less than 3 %. No interference from endogenous analogs was observed, and the method showed good consistency in interlaboratory comparison. In contrast, chemiluminescent immunoassay results exhibited poor agreement with and the cRMP. Conclusions This study developed a highly precise, accurate, specific, and sensitive ID-LC-MS/MS-based cRMP for the simultaneous measurement of FT3 and FT4 in human serum. This method provides a reliable tool for standardizing routine thyroid function tests.

The strategic direction of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) is guided by global healthcare trends, rapid technological advancements, and the evolving role of laboratory professionals in modern medicine. The aim is to position laboratory medicine as a leader in precision diagnostics, digital healthcare transformation and integrated diagnostics, ensuring timely, accurate, and cost-effective medical testing that improves patient outcomes. EFLM reaffirms that every innovation in laboratory medicine must ultimately serve the patient – ensuring equitable access to high-quality diagnostics, enhancing patient understanding, and strengthening trust in laboratory-generated data. In a world deeply reshaped by technological, economic, and geopolitical forces, EFLM must act as both a scientific authority and a societal compass, guiding the transformation of diagnostics into patient-centered, data-driven, and resilient care models. EFLM’s strategy focuses on integrating laboratory innovation and value-based laboratory medicine, strengthening workforce competencies, promoting sustainability and clinical outcomes improvement, and addressing key challenges in patient-centered healthcare. The following strategic priorities reflect the Federation’s commitment to improving the impact of laboratory medicine on clinical care, public health, and scientific advancement.

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 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.

The secondary scientific use of routine laboratory data will increasingly rely on LOINC as a semantic standard, particularly within the European Health Data Space (EHDS). LOINC enables the aggregation of large data sets from clinical care for research purposes, particularly in epidemiology. However, current approaches to semantic standardisation largely neglect metrological aspects – in particular, the considerable limitations of analytical standardisation for many fundamental analytes and the resulting scatter of result values across different assays that use common LOINC codes. This incomplete harmonisation leads to statistical uncertainty that must be taken into account when quantitative conclusions – such as diagnostic thresholds for analytes – are derived from aggregated, LOINC-derived data sets. In this opinion piece, we propose using the extensive global data pool generated by external quality assessment (EQA) programs to finally annotate LOINC codes with a sound and useful uncertainty metric. This represents secondary scientific use of EQA data that is analogous to and supports the secondary use of routine diagnostic data from patient care for research. With a proof-of-concept analysis, we demonstrate the feasibility of this approach, which offers a wide range of design options. We suggest that consortia of EQA providers, coding institutions, scientific societies, and the IVD industry could advance precision research through this concept. It is noteworthy that the proposed annotation strategy – linking semantic test codes to uncertainty metrics based on EQA data – is not limited to LOINC as a semantic coding system.

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 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 This study aimed to evaluate the feasibility of robotic venipuncture in clinical settings and compare its performance with manual venipuncture in terms of blood specimen quality and test results. Methods From March to April 2025, 63 participants (35 females, 28 males), aged 23–73 years, were recruited at Zoucheng People’s Hospital Medical Laboratory Center. Blood samples were collected using both robotic venipuncture and manual venipuncture on April 8, 2025. Specimen quality was assessed pre-testing, and coagulation/biochemical parameters were analyzed. Results Robotic venipuncture demonstrated advantages in reducing venipuncture pain and achieving more consistent blood sample mixing. Compared with manual venipuncture, the robotic system enables more precise control of blood collection volume and anticoagulant ratio. Statistically significant differences (p<0.05) were observed in fibrinogen (Fib), prothrombin time (PT), plasminogen activity (PTA), activated partial thromboplastin time (APTT), International Normalized Ratio (INR), thrombin time (TT), potassium (K + ), lactate dehydrogenase (LDH), α‐hydroxybutyrate dehydrogenase (HBDH) levels between the two methods. Conclusions Robotic venipuncture technology demonstrates clinical feasibility, offering more precise blood collection volumes and accurate anticoagulant-to-blood ratios compared to manual venipuncture, thereby enhancing specimen stability (Lippi G, Salvagno GL, Montagnana M, Lima-Oliveira G, Guidi GC, Favaloro EJ. Quality standards for sample collection in coagulation testing. Semin Thromb Hemost. 2012;38:565–75; Reneke J, Etzell J, Leslie S, Ng VL, Gottfried EL. Prolonged prothrombin time and activated partial thromboplastin time due to underfilled specimen tubes with 109 mmol/L (3.2 %) citrate anticoagulant. Am J Clin Pathol. 1998;109:754–7). However, challenges remain, including longer procedure times and initial user acceptance barriers.

Objectives Complement inhibitor pegcetacoplan binds to C3 and its activation product C3b. Pegcetacoplan has been approved for the treatment of paroxysmal nocturnal hemoglobinuria. Because pegcetacoplan exerts broad inhibition of the complement cascade its efficacy is also investigated in numerous other diseases caused by complement dysregulation, such as C3 glomerulopathy. Pegcetacoplan causes a number of counterintuitive changes in laboratory results. Methods In-depth complement analysis in two patients with PNH and three patients with C3 glomerulopathy, all treated with pegcetacoplan. Results C3 levels increase up to 300 % above reference levels. In vitro testing showed that this is not a turbidimetric artifact in the C3 immunoassay due to pegcetacoplan-C3 complex formation but appears to be caused by increased half-life of C3 bound to pegcetacoplan. Unbiased mass spectrometric plasma proteome analysis confirmed the dramatic pegcetacoplan-induced increase in circulating C3. Surprisingly, also a three-fold increase of properdin was observed during pegcetacoplan treatment. Serum protein electrophoresis showed an additional band in all patients after pegcetacoplan exposure. This C3-band does not migrate at its expected position because of changes in the mass and charge of C3 bound to pegcetacoplan and should therefore not be misinterpreted as an M-protein. Both in vitro experiments and real clinical practice laboratory results demonstrated that pegcetacoplan completely blocked the alternative complement pathway while the classical pathway is affected but remains largely intact. Conclusions Because of the increasing use of pegcetacoplan in routine clinical practice, it is important that both clinicians and laboratory specialists are aware of these unexpected therapy-induced laboratory findings.

Objectives Plasma renin activity (PRA) measures the rate of renin converting angiotensinogen to angiotensin I in circulation and reflects the function of renin-angiotensin-aldosterone system (RAAS). The study aimed to demonstrate the performance of the MAGLUMI Angiotensin I assay for the measurement of PRA. Methods The MAGLUMI Angiotensin I assay was evaluated for precision, linearity, limit of blank (LoB), limit of detection (LoD), limit of quantification (LoQ), interferences, cross-reactivity and reference intervals. It was compared to liquid chromatography tandem mass spectrometry (LC-MS/MS) using 54 samples, and impact of clinical factors was assessed in 181 samples. Results The assay demonstrated repeatability of 1.5 %, 1.85%, and 1.6 % across three different pool concentrations, with corresponding within-laboratory imprecision values of 2.0 %, 2.5%, and 1.9 %. The linear range was [0.10, 24.0] ng/mL. The LoB, the LoD, and the LoQ were 0.03 ng/mL, 0.07 ng/mL, and 0.10 ng/mL, respectively. The interferences and cross-reactivity met the criterion for acceptable cross-reactivity (CR) with <±10 %. Reference interval validation shows <10 % of a total of 40 healthy individuals’ plasma samples falling outside the established interval for standing and lying position. The MAGLUMI assay showed excellent agreement with LC-MS/MS for both angiotensin I (slope=1.076, intercept=0.109 ng/mL/h, r=0.987, p<0.001) and plasma renin activity (slope=1.131, intercept=−0.386 ng/mL/h, r=0.973, p<0.001). Conclusions The MAGLUMI Angiotensin I chemiluminescence immunoassay is a sensitive and reliable commercial kit for detecting plasma renin activity. The assay exhibited robust analytical performance and strong agreement with LC-MS/MS.

Objectives The performance of a novel urine particle analyzer, fluidlab 2 (Anvajo GmbH, Dresden, Germany), was evaluated against phase-contrast visual microscopy according to the most recent EFLM European Urinalysis Guideline. Methods The fluidlab 2 device combines digital holographic microscopy with neural network-based object detection for particle classification. Its compact benchtop design is suitable for bedside use, reducing turnaround times. The analytical performance (imprecision, linearity, LoQ) was evaluated according to the 2023 EFLM Urinalysis Guideline. Method comparison involved the analysis of 450 urine samples, assessing RBC, WBC, and SEC counts against visual microscopy using Passing-Bablok regression and Spearman’s correlation. Bland-Altman plots were used to evaluate the agreement with clinical performance standards, while weighted Cohen’s kappa was used to measure diagnostic agreement on an ordinal scale. Results By applying Dahlberg’s procedure, a desirable relative coefficient of variation R(CV) ≤2.0 was obtained for RBC and WBC. Linearity of up to 7 × 10 6 /L and 6 × 10 6 /L was achieved. The estimated LoQ at CV=30 % reached 20 × 10 6 /L for RBC and 5 × 10 6 /L for WBC. Spearman’s correlation coefficient against visual microscopy was 0.86, 0.92 and 0.94 for RBC, WBC and SEC, respectively. Agreement with visual microscopy (Cohen’s weighted kappa) was 0.92 for RBC, 0.93 for WBC, 0.96 for SEC, 0.86 for casts, 0.82 for non-SEC, 0.33 for crystals and 0.51 for bacterial counts. Conclusions Fluidlab 2 provides desirable imprecision for RBC and WBC, and meets the criteria for linearity and LoQ. Cohen’s weighted kappa coefficients show an optimal comparison to visual microscopy for RBC, WBC and SEC and a minimum comparison for casts and non-SEC. This evaluation demonstrated promising results for the use of the fluidlab 2 analyzer in a clinical setting to detect kidney-related diseases based on urine particle analysis.

Objectives To investigate the feasibility of at-home collection of liquid capillary blood samples for clinical chemistry analyses. Methods Kidney transplant recipients performed a fingerprick to collect capillary blood samples. Samples were kept at room temperature for 24 h before further processing. Results were compared to results obtained from venipuncture samples using Passing-Bablok analysis and by calculating mean relative differences, which were compared to CLIA and TEa acceptance limits. In addition, transport times/temperatures and effects of postal handling were examined. Results Of 38 patients that performed a fingerprick, 29 properly filled 2 microtainers, 4 filled 1 microtainer and 5 were unable to fill any microtainer. Capillary samples were generally more hemolytic, but we found mean differences within CLIA/TEa for hemoglobin, leukocytes, hemoglobinA1c, creatinine, alanine transaminase, C-reactive protein, albumin, calcium and gamma-glutamyl transferase. For alkaline phosphatase and thrombocytes, we found a negative bias, but mean differences within CLIA/TEa. Mean differences for potassium, aspartate transaminase, hematocrit and mean corpuscular volume exceeded CLIA and/or total allowable error (TEa). For phosphate, individual samples showed large variation. Transport times did not exceed 24 h, but temperatures up to 10 °C above outside temperatures were observed. Transport of samples resulted in slight increases in H-index, lactate dehydrogenase (LDH) and aspartate transaminase (ASAT). Conclusions At-home capillary blood collection is feasible for many patients and samples are suitable for a subset of clinical chemistry analyses. Instruction, monitoring of transport time/temperature and knowledge of the pitfalls when interpreting results are key elements upon implementation in clinical practice.

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 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).

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.

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.

Objectives Despite standardization efforts, significant inter-assay variability persists among prostate-specific antigen (PSA) tests, impacting prostate cancer (PCa) diagnosis and monitoring. We aimed to evaluate the analytical and clinical performance of the newly developed Atellica IM high-sensitivity PSA (hsPSA Atellica) assay compared with established PSA assays. Methods A total of 236 serum samples from healthy individuals and patients with or without PCa were analyzed using the hsPSA Atellica assay and four FDA-approved PSA assays: Hybritech Access, Architect, Atellica, and Cobas. Analytical performance included limit of detection (LOD), limit of quantification (LOQ), linearity, inter-assay precision, and hook effect. Method comparison was performed using Passing-Bablok regression, Bland-Altman analysis, and kappa index concordance. Results The hsPSA Atellica assay demonstrated a LOD of 0.01 μg/L and LOQ of 0.028 μg/L (CV: 3.2 %, accuracy: 115 %). Precision was maintained across concentrations, with CVs of 4.2 %, 3.8 %, and 2.4 % at low, medium, and high levels of PSA. Strong agreement was observed with the compared tests, particularly with Cobas and Hybritech PSA assays. Diagnostic sensitivity and specificity at the 4 μg/L clinical decision threshold were 98 % and 35 %, respectively. In 87 samples between 3 and 10 μg/L, concordance between hsPSA Atellica and Hybritech reached 96.6 % (κ=0.82). The assay remained accurate up to PSA concentrations of 13,311 μg/L, showing minimal hook effect. Conclusions The hsPSA Atellica assay shows excellent analytical sensitivity and strong agreement with established assays. To our knowledge, this is the first published evaluation of this assay, supporting its clinical utility in both PCa diagnosis and follow-up.

Background Method-comparison studies in laboratory medicine are routinely interpreted using regression-based or Bland–Altman analyses. Although useful descriptively, these statistical procedures are frequently misapplied to infer “agreement”, “equivalence”, or “interchangeability”. Such interpretations overlook essential metrological conditions – including the definition of the measurand, the traceability chain, and measurement uncertainty – leading to misconceptions with potential clinical consequences. Content This Opinion Paper clarifies the distinct meanings of four metrological concepts that are often treated as synonyms: comparability , compatibility , equivalence , and interchangeability . We explain why numerical similarity or statistical association does not establish metrological relatedness, and outline the specific requirements for each concept. Comparability requires a shared measurand and calibration hierarchy; compatibility requires differences smaller than the combined uncertainty; equivalence requires clinically irrelevant residual differences; and interchangeability requires stability of clinical decisions when substituting one measuring system for another. We also discuss familiar sources of misinterpretation, including ambiguous definitions of the measurand, incomplete traceability chains, and uncritical reliance on regression- or bias-based summaries. Summary and outlook Distinguishing among comparability, compatibility, equivalence, and interchangeability is essential for the metrological interpretation of method-comparison studies and for ensuring safe analytical and clinical decision-making. Integrating these concepts explicitly into study design, harmonisation strategies, and reporting practice will strengthen traceability implementation, prevent erroneous claims of “agreement”, and support more reliable patient care.

Objectives Dried blood spot (DBS) sampling enables convenient at-home blood collection, suitable for patients needing regular check-ups, such as those receiving testosterone replacement therapy (TRT). This study assessed the feasibility and reliability of DBS sampling for monitoring testosterone concentrations in patients undergoing TRT, aiming to provide a convenient at-home alternative to venipuncture. Methods Twenty healthy volunteers (12F/8M) and 56 male patients (n=59 samples) receiving TRT were included. Blood was collected parallel by venipuncture (reference) and patient-performed finger prick (DBS) following instruction materials and additional verbal guidance from the andrology consultant. Feasibility and patients’ experience were evaluated through questionnaires. Testosterone concentrations in both serum and DBS were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results In healthy volunteers, serum and DBS testosterone concentrations were highly correlated (y=−0.021 + 0.66x; r=0.99). For patients receiving intramuscular testosterone undecanoate, this correlation was strong (y=1.3 + 0.54x; r=0.87). However, DBS samples from patients using testosterone gel showed extremely elevated testosterone levels compared to serum, likely due to local contamination. Most healthy volunteers and in general all patients preferred at-home DBS-sampling over venipuncture at the hospital when regular check-ups are necessary, citing convenience and reduced travel time. Conclusions At-home DBS sampling is a feasible and reliable method for monitoring testosterone in patients treated with intramuscular testosterone undecanoate, improving patient comfort and convenience. However, DBS sampling at-home is currently unsuitable for patients using testosterone gel, likely due to contamination issues. Comprehensive patient education on collection of DBS samples is crucial to ensure high-quality DBS and accurate testosterone quantification.

Objectives Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne pathogen that can cause a fatality rate as high as 12–50 %, posing a significant threat to public health. SFTSV is prevalent in mountainous and hilly regions with relatively poor medical conditions. Therefore, there is an urgent need to develop a new convenient, rapid and sensitive method for SFTSV detection in low-resource environments. Methods We developed a one-pot and visualized method for SFTSV detection using loop-mediated isothermal amplification assisted by CRISPR/Cas12b with G478A/K396A double mutations (LAC12b-2M). The specificity, sensitivity, accuracy and limit of detection (LOD) of LAC12b-2M were evaluated using clinical reverse transcription-quantitative PCR (RT-qPCR) as the reference method, with gradient dilutions of strong positive SFTSV RNA samples and 215 clinical serum samples from two cohorts. Results LAC12b-2M is sensitive to detect SFTSV with a LOD of 1 copy/μL at 61 °C within 30 min. Compared to clinical RT-qPCR, LAC12b-2M demonstrated a sensitivity of 98.8 % (82/83), a specificity of 100.0 % (96/96), and an accuracy of 99.4 % (178/179) in cohort 1 (n=179), and an accuracy of 100.0 % in cohort 2 (n=36). Conclusions Our LAC12b-2M method holds promise for point-of-care SFTSV testing in different healthcare settings, particularly in low-resource region where SFTSV is prevalent.

Objectives Multiplex arrays offer a high-throughput, cost-effective means for quantifying multiple analytes simultaneously, essential for large-scale biomarker research. However, the shared reactive environment in multiplex assays could lead to variations in results compared to single plex assays, potentially impacting outcomes. This study aimed to explore these differences by examining a “3-plex” cytokine panel vs. single plex assays for tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10). Methods Using the R&D Systems Luminex HS Cytokine Panel A, 72 serum samples were tested across several batches, including three “3-plex” and two “1-plex” batches each for IL-10 and TNF-α. We evaluated differences in cytokine values using paired t-tests, comparing within 1-plex, between 3-plex, and between 1-plex and 3-plex assays. Bland-Altman plots visually assessed absolute and percentage differences. Results IL-10 values were similar between 1-plex and 3-plex assays, showing an average difference of 4.2 fmol/L (10.7 %), which was less than the within plex differences. Conversely, TNF-α showed a 16.7 % difference in the between plex comparison, compared to a 12 % difference in the within plex comparisons. Statistically significant differences emerged mainly for IL-10 across all comparisons. Bland-Altman analyses indicated pronounced variability at low analyte concentrations. Conclusions While the multiplex assays demonstrated variation at low analyte levels, especially for IL-10, such differences might not substantially affect comparability when mixed with single plex assays, particularly in datasets dominated by low concentrations.

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 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 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.

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 Our aim is to determine whether drone transport introduces relevant variability in analytical results and to assess its suitability for integration into routine hospital laboratory operations. Methods A two-phase study was conducted. The stability phase involved three sets of samples from 31 healthy volunteers transported by drone, by road, and processed without transport. The pilot phase included paired samples from 104 primary care patients transported by drone and by road. A broad panel of 53 biochemical, hematological and urinary analytes was assessed. Analytical performance was evaluated using mean bias, comparison with reference change values (RCVs) and hemolysis grading. Results Most analytes showed minimal differences between transport methods. In the stability study, glucose and MCH in road-transported samples, and lactate dehydrogenase (LDH) and mean corpuscular hemoglobin (MCH) in drone-transported samples, exceeded their respective RCVs, with LDH likely affected by mild hemolysis. In the pilot study, statistically significant differences were observed between transport methods for potassium, exhibiting a bias of −1.7 % (−2.6 to −0.8 %). Hemolysis was slightly more frequent in drone samples during the stability study but comparable in the pilot study. Conclusions Drone transport preserved the analytical integrity of a comprehensive test panel and has the potential to reduce logistical constraints. These findings support its implementation as a reliable and sustainable alternative to conventional transport in clinical laboratory settings.

Objectives To establish evidence-based analytical performance specifications (APS), specifically allowable total error (TE a ), for five fundamental semen analysis parameters (sperm concentration, progressive motility, total motility, morphology, and vitality) in China utilizing the state-of-the-art model, and to compare these specifications with existing international benchmarks. Methods We examined national external quality assessment (EQA) data from 2019 to 2025, encompassing 315 laboratories across 30 provinces in China. TE a was calculated using the state-of-the-art model as half the 10th–90th percentile range of result deviations from assigned values. Given that the participant count was below 100 from 2019 to 2022, this study eventually utilized data from 2023 to 2025 for the calculation of TE a . The pass rate of participating laboratories was evaluated against the established TE a criteria. Results The established TE a values as follows: sperm concentration, 34.3 %; progressive motility, 26.9 % (for assigned value ≤40 %) and 13.5 % (>40 %); total motility, 26.3 % (≤50 %) and 10.5 % (>50 %); morphology, 67.7 %; vitality, 24.6 % (≤60 %) and 7.9 % (>60 %). The pass rates for all parameters in recent EQA cycles were approximately 80 %, supporting the practical achievability of the proposed specifications. Comparisons showed that the derived TE a values are generally consistent with or more refined than previous state-of-the-art and biological variation-based standards. Conclusions This study provides the first large-scale, evidence-based TE a for semen analysis in China. The stratified TE a for motility and vitality based on clinical thresholds, improves diagnostic accuracy. The proposed specifications are practical, achievable, and clinically relevant, providing a consistent foundation for quality assurance in reproductive medicine laboratories.

Objectives Neurofilament light chain (NfL) is a biomarker of neuroaxonal damage in various neurological conditions, including multiple sclerosis (MS). With new analytical platforms entering the market, standardization of serum NfL (sNfL) measurement is essential. This study compared sNfL levels across three assays – NF-light Advantage V2/Plus (Quanterix), Lumipulse G NfL blood (FujiRebio), and Elecsys NfL (Roche Diagnostics) – and derived conversion formulas for harmonization. Methods Serum samples from three cohorts including healthy donors (n=303) and MS patients (n=181) were analyzed across three platforms. Passing-Bablok regression and correlation analyses assessed inter-platform agreement and potential systematic differences. Assays were further evaluated for their ability to detect MS disease activity. Results All platforms showed strong correlations (Pearson>0.98; Spearman>0.90, p<0.001). Absolute values from Elecsys were ∼6-fold lower than the other assays. Fourteen of 484 samples (2.90 %) showed substantial, retest-stable deviations (>4 SD) on at least one platform. After excluding outliers, conversion formulas were derived to transform the values to the NF-light Advantage V2/Plus scale: NfL=6.31 × [Elecsys NfL] + 2.33 ng/L; NfL=0.94 × [Lumipulse G NfL] + 1.30 ng/L. In MS patients, all platforms detected significant differences between relapse and remission (p<0.001). Age-adjusted Z scores from converted values yielded the highest effect sizes (Cohen’s d: 0.84 for NF-light Advantage, 1.11 for Elecsys, 0.93 for Lumipulse). Conclusions The three platforms demonstrated high correlation and comparable performance in sNfL quantification. About 3 % of samples showed strong deviations, highlighting the need for vigilance and further harmonization. Age-adjusted Z scores may enhance clinical and research utility.

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 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.

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.

This scoping review followed the PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews) guidelines to systematically map the current landscape of artificial intelligence (AI) and machine learning (ML) applications in the field of thrombosis and hemostasis (T&H), specifically targeting diagnostic enhancements in clinical and laboratory settings. Utilizing comprehensive searches across MEDLINE, EMBASE, Web of Science, and Scopus (2020–2025), 107 original studies met inclusion criteria and were analyzed. Clinical applications predominantly focused on predictive modelling for venous thromboembolism (VTE), pulmonary embolism (PE), deep vein thrombosis (DVT), anticoagulant management, and disease risk stratification, employing algorithms including neural networks, random forests, and gradient boosting. Laboratory-based AI implementations, though fewer, provided automated quality control, clot detection, and assay interpretation enhancements for potential better decision-making. Significant limitations addressed by the include studies include reliance on retrospective, single-center, small-sample datasets, limited external validation, model interpretability concerns, and integration challenges into clinical workflows. Persistent interdisciplinary disconnect between hemostasis domain experts and AI-ML specialists, compounded by regulatory hurdles, fragmented data, and labor-intensive data labelling processes, was highlighted as a major barrier to broader adoption. Recommendations for future research include developing large, externally validated multicenter datasets, transparent and interpretable ML models, prospective clinical validations, and user-centered integration strategies. Enhancing collaboration between laboratory scientists and AI-ML experts, establishing structured education programs, and creating regulatory frameworks are essential next steps to fully realize the potential of AI for significantly improving diagnostic accuracy, clinical decision-making, and patient management in T&H.

Objectives Vitamin K homologues are essential to human health, and their concentrations in biological samples serve as valuable diagnostic biomarkers. This study was aimed to develop a method for determining vitamins K1 (phylloquinone, VK1) and K2 (menaquinone, MK-4) in human serum. The proposed method was validated and applied to the serum of a cohort of 20 Russian individuals. Methods High-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was used to analyse the content of VK1 and MK-4 in serum. Atmospheric pressure chemical ionisation (APCI) in negative mode was applied to ionise VK1 and MK-4. Protein precipitation and solid-phase extraction (SPE) on polystyrene-divinylbenzene resin were combined to isolate and preconcentrate the analytes from serum. Results The HPLC-MSMS method was developed and validated for the determination of vitamins VK1 and MK-4 in human serum. The method demonstrated a lower limit of quantification (LLOQ) of 0.05 μg/L, with more than 71 % recoveries and precision within 17 %. To demonstrate the applicability of the method to real samples, serum from 20 healthy adults was analyzed. VK1 was detected in four individuals (0.094–0.96 μg/L), whereas MK-4 concentrations were below 0.22 μg/L in all cases. Conclusions The validated HPLC-MS/MS workflow provides a reliable and sensitive approach for the quantification of VK1 and MK-4 in minimal serum volumes. The method demonstrates robustness, reproducibility, and suitability for large-scale analytical applications. The proposed LC-MS/MS protocol successfully applied to native human serum samples, illustrating its applicability for future clinical and biochemical studies involving vitamin K.

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.

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 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.

Objectives Compare Elecsys (Roche) and Simoa (Quanterix) immunoassays for serum glial fibrillary acidic protein (GFAP) using our reference database and Z scores, and evaluate their prognostic value for progression independent of relapse activity (PIRA) in multiple sclerosis (MS). Methods Platform correlation was assessed in 612 samples from healthy controls (n=188; median [interquartile range, IQR] age 45.1 [36.4–61.7] years) and people with MS (n=424; 45.3 [35.2–53.9] years). Elecsys values were converted to Z scores via Passing-Bablok-derived regression and validated in fingolimod (n=414), and B-cell depleting therapy (BCDT; n=353) cohorts. Z scores and hazard ratios (HRs) for time-to-PIRA were compared using Cox regression. Results GFAP Simoa and GFAP Elecsys measurements were correlated (r=0.94), with Elecsys values ∼54 % lower (GFAP Elecsys , ng/L=2.847 [95 % confidence interval, CI: 1.335 – 4.98] + 0.457 [0.434 – 0.478] * GFAP Simoa , ng/L). In univariable Cox models, GFAP Simoa and GFAP Elecsys Z scores were associated with time-to-PIRA in both validation cohorts. In multivariable Cox models, higher GFAP Simoa Z scores were associated with shorter time-to-PIRA in fingolimod cohort (HR: 1.27 [95 % CI 1.08 – 1.50], p=0.0031) and trended toward significance in BCDT (1.18 [0.99 – 1.41, p=0.0693). In contrast, GFAP Elecsys Z scores were associated with time-to-PIRA in both cohorts (fingolimod: 1.27 [1.09 – 1.48], p=0.0023; BCDT: (1.19 [1.00 – 1.40], p=0.0487). Conclusions Serum GFAP measured by Elecsys shows a comparable association with time-to-PIRA as Simoa, and GFAP Simoa Z scores can be successfully bridged to GFAP Elecsys Z scores, supporting Elecsys`s potential for clinical implementation.

Nowadays, we are facing major changes in the landscape of test harmonization through metrological traceability approach, all expected to promote equivalence of reported laboratory results. However, having traceability tools in place does not automatically mean that metrological traceability is correctly implemented. As concepts that are poorly implemented do not produce expected benefits, it is time to move from just discussing the effectiveness of traceability theory to how correctly implement it and effectively monitor its effects. In particular, considering the implementation outcomes, there is still a limited knowledge about the actual harmonization status of laboratory test results. We need to increase the information about the expected outcome by assessing result uniformity across different in vitro diagnostic medical devices (IVD-MDs) through an approach providing an ongoing robust evidence of standardization effectiveness. External Quality Assessment (EQA) programs have an irreplaceable role in this IVD traceability surveillance providing that they fulfill specific requirements. i.e., the use of commutable materials with target values assigned by reference measurement procedure (RMP) or strictly controlled procedures, if RMP does not exist, and with validation of results in relation to fit-for-purpose analytical performance specifications. If adequately designed, EQA programs can create evidence about intrinsic standardization status and equivalence of the examined IVD-MDs, serving as management tool for the medical laboratory and IVD manufacturers, and forcing them to investigate and eventually fix the identified problems. In this way, major stumbling blocks opposing the correct implementation of metrological traceability and the obtaining of harmonized laboratory results can be identified and removed.

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.