Ahead of Publication / Just Accepted

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

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

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

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

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

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

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.

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

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

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

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

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

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

Objectives Monocyte distribution width (MDW) is an early sepsis indicator measuring monocyte heterogeneity during massive infection. We compared MDW changes in Escherichia coli sepsis patients with the effects of living E. coli and lipopolysaccharide in an ex vivo sepsis model. We also investigated the dynamics of monocyte morpho-functional and inflammatory responses in the sepsis model. Methods Whole blood from healthy participants was in vitro stimulated with live E. coli (10 6 –10 10  CFU/mL) and LPS (0.1–10 μg/mL). Complete blood counts, including MDW, were evaluated at different time-points using DxH 690T Hematology Analyzer (Beckman Coulter). MDW values were compared with those retrospectively obtained from sepsis patients (n=23). May-Grunwald-Giemsa-stained blood smears were analyzed by digital cell morphology (CellaVision DM software). A panel of 27 inflammatory mediators was quantified in plasma (Bio-Plex 200). Results MDW values were early and significantly increased in a dose- and time-dependent manner by live E. coli and LPS treatments (p<0.01). MDW values were significantly higher in sepsis patients compared to controls and overlapped those observed in the ex vivo model. IL-1β, TNF-α, IL-8, MIP1-α, MIP-1β, Eotaxin, G-CSF, and PDGF-bb were significantly modulated after treatments. Conclusions Our findings confirm the clinical utility of MDW in sepsis diagnosis and sustain the reliability of the whole blood assay as ex vivo sepsis model. E. coli and LPS directly promote early monocyte morpho-functional modifications, mirrored by high MDW values and pro-inflammatory mediators. These results improve the knowledge on the biological basis of sepsis, providing novel evidence on the usefulness of MDW in septic conditions.

Objectives Ovarian cancer is the deadliest gynaecologic malignancy. Due to the lack of reliable biomarkers for the detection of the early disease, most patients are diagnosed at an advanced stage resulting in poor survival. We therefore aimed at establishing novel CA-125 glycovariant assays to improve the diagnostic sensitivity and specificity of ovarian cancer. Methods Blood samples of 184 patients with epithelial ovarian cancers (EOC), 127 benign ovarian tumors, and 115 healthy controls were measured using GLYVAR™ Ovarian I and II assays (Uniogen) and the conventional CA-125 protein assay (CanAg CA-125 EIA, Fujirebio). Results The two glycovariant assays differentiated benign and malignant ovarian masses with 88.0 % sensitivity at 99 % specificity, whereas CA-125 showed 72.8 % sensitivity. The improved performance was most evident in patients with borderline or moderately elevated CA-125 concentration at diagnosis, which is a challenging group for differential diagnostics. The CA-125 glycovariant assays showed 2.5 times higher sensitivity (33.3 % with CA-125 vs. 83.3 % with the CA-125 glycovariants) at 94 % specificity. CA-125 glycovariants corrected 82.4 % of false positive results given by CA-125 concentrations with the commonly used cutoff 35 U/mL. Importantly, the CA-125 glycovariant assays detected 63.6 % of early-stage serous carcinomas from benign and healthy controls with very high 99 % specificity, while CA-125 had a sensitivity of only 45.5 %, representing a 40 % increase. Conclusions This is the first study describing the clinical performance of GLYVAR Ovarian I and II assays in ovarian cancer diagnostics. The results indicate that the CA-125 glycovariant assays have remarkable potential to improve ovarian cancer diagnostics.

The integration of artificial intelligence (AI) and machine learning (ML) into laboratory medicine shows promise for advancing diagnostic, prognostic, and decision-support tools; however, routine clinical implementation remains limited and heterogeneous. Laboratory data presents unique methodological and semantic complexities – method dependency, analyte-specific variation, and contextual sensitivity-not adequately addressed by general-purpose AI reporting guidelines. To bridge this gap, the EFLM Committee on Digitalisation and Artificial Intelligence (C-AI) proposes an expanded checklist to support assessment of requirements and recommendations for the development of AI/ML models based on laboratory data. Building upon the widely adopted ChAMAI checklist (Checklist for assessment of medical AI), our proposal introduces six additional items, each grounded in the CRoss Industry Standard Process for Data Mining (CRISP-DM) framework and tailored to the specificities of laboratory workflows. These extensions address: (1) explicit documentation of laboratory data characteristics; (2) consideration of biological and analytical variability; (3) the role of metadata and peridata in contextualizing results; (4) analyte harmonization and standardization practices; (5) rigorous external validation with attention to dataset similarity; and (6) the implementation of FAIR data principles for transparency and reproducibility. Together, these recommendations aim to foster robust, interpretable, and generalizable AI systems that are fit for deployment in clinical laboratory settings. By incorporating these laboratory-aware considerations into model development pipelines, researchers and practitioners can enhance both the scientific rigor and practical applicability of AI tools. We advocate for the adoption of this extended checklist by developers, reviewers, and regulators to promote trustworthy and reproducible AI in laboratory medicine.

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

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

Objectives To investigate the relationship between uracilemia (U) and dihydropyrimidine dehydrogenase (DPD) activity in peripheral blood mononuclear cells (PBMC) and whether they are influenced by renal or hepatic impairment. Methods This retrospective study included 176 cancer patients with pre-treatment U (UPLC-MSMS assay) and PBMC-DPD (radioenzymatic assay) analyzed the same day (routine phenotyping). Blood renal (creatinine, BUN) and hepatic (ALT, AST, GGT, ALP, albumin, bilirubin) work-up was performed within 15 days before or up to 4 days after DPD phenotyping. Biochemical markers were categorized according to CTCAEv5.0 grade (G). Glomerular filtration rate (eGFR) was estimated (CKD-EPI and EKFC). Non-parametric statistical tests were used. Results Prevalence of partial deficiency was 3.4 % based on PBMC-DPD (i.e. ≤100 pmol/min/mg) and 6.3 % based on U (i.e. ≥16 μg/L). No complete deficiency was observed. Fifteen patients out of 176 (8.5 %) exhibited discordant DPD status between PBMC activity and U. The correlation between PBMC-DPD and U was significant but weak (r= −0.309, p<0.001). PBMC-DPD (mean 246, median 235, range 62–926 pmol/min/mg prot) was not influenced by renal or hepatic impairment. U (mean 9.6, median 8.5, range 1.7–57.8 μg/L) was significantly higher in patients with elevated BUN (normal vs. >1-UNL, p=0.009), GGT (G0 vs. G1 vs. G2 vs. G3, p<0.001), AST (G0 vs. G≥1, p=0.015), or with hypoalbuminemia (G0 vs. G ≥ 1, p=0.045). Categorized creatinine or eGFR did not influence U. Conclusions It remains unclear whether renal and/or hepatic impairment acts as a confounding factor affecting the accuracy of uracilemia testing, or whether truly impacts DPD activity, suggesting caution in U interpretation.

Objectives To evaluate the consistency of seven enzymatic glycated albumin (GA) assays in pregnant women based on a multicenter study. Methods Samples were collected from pregnant women at three different gestational stages: 4–13 weeks (n=150), 24–28 weeks (n=300, including 150 GDM subjects), and 29–40 weeks (n=300, including 150 GDM subjects), across three hospitals between July 2022 and December 2023 in China. These samples were analyzed using seven enzymatic GA methods (Lucica, Norudia, BSBE, Maccura, Meikang, Reebio, and Zybio assays). Spearman correlation analysis, Passing–Bablok regression, and Bland–Altman plots were used to evaluate the consistency between the Lucica used in our laboratory and the other selected assays. The effects of albumin concentration and gestational stage on the consistency of GA were evaluated through stratified analyses. Results The correlation coefficients between Lucica and the other six assays for GA% measurement ranged from 0.741 to 0.906 (p<0.0001), with the mean relative biases ranging from −15.5 to +6.7 %. In trimester-stratified analysis, the highest correlation coefficient was observed in the first trimester for all assays except Maccura, and the bias increased with advancing gestational age for all assays except BSBE. In albumin-stratified analysis (30–45 g/L), the correlation increased with increasing albumin concentration for all assays, while the bias decreased except for BSBE and Maccura assays. Conclusions Poor analytical consistency was observed in enzymatic GA assays for pregnant women, with discrepancies varying across gestational stages and albumin concentrations. Reference intervals for pregnant women should be established based on trimester-stratified and manufacturer-specific criteria.

Objectives Our study sought to determine the usefulness of biomarkers of systemic inflammation (C-reactive protein (CRP), procalcitonin (PCT) and proadrenomedullin (proADM)) on hospital admission, as compared to the CURB65 score, for predicting 30- and 90-day mortality in patients hospitalized for community acquired pneumonia (CAP). Methods Observational, prospective study of adults admitted for CAP in four Spanish teaching hospitals. Disease severity was determined within the first 24 h of diagnosis, using the CURB65 score. CRP, PCT and proADM levels were assessed from samples obtained in the Emergency Department (ED). We compared the capacity of the different biomarkers and the CURB65 score to predict pneumonia-related 30- and 90-day mortality. Results A total of 956 patients hospitalized with CAP were included, 462 in the internal and 494 in the external sample. Of the biomarkers, proADM showed the greatest AUC for predicting 30- and 90-day mortality (0.80 and 0.76 respectively). Mortality at 30 and 90 days increased as proADM levels rose. When proADM was used as a continuous variable, CURB65 showed a similar predictive capacity (AUC 0.80) to both crude and age-adjusted proADM (AUC 0.80 and 0.83 respectively) for 30-day mortality. The same was also true for 90-day mortality. However, proADM used as a categorical variable had a greater predictive capacity for 90-day mortality than the CURB65 score (<0.001). Conclusions Amongst patients admitted for CAP, the use of proADM obtained in the ED may be useful for identifying patients at greatest risk of mortality, with a similar predictive capacity to the CURB65 score.

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

Objectives In 2023, Croatia implemented a screening program for familial hypercholesterolemia (FH) targeting children entering the first grade of primary school. The program is based on total cholesterol (TC) concentration measurements. This pilot study aimed to assess and compare TC results obtained using different analytical platforms during the program’s initial phase (2023–2024). Methods Retrospective data from laboratories across Croatia were analyzed. Results covered the following analyzers: Beckman Coulter AU (BC), Abbott Alinity/Architect c (AA), Roche Cobas c (RC) and Siemens Atellica CH (SA). Statistical analyses included Kruskal–Wallis and chi-square tests using the program’s cut-off values. Bland–Altman and Passing-Bablok analyses were used to assess differences in a direct comparison study of 40 samples. To reduce variability, TC values were also expressed as multiples of the median (MoM), calculated separately for each platform. Results The study included 17,694 children. Median TC values (IQR) differed significantly across platforms: BC (4.3 [3.8–4.7] mmol/L), AA (4.1 [3.7–4.5]), RC (4.1 [3.7–4.5]), and SA (4.0 [3.6–4.4]); p<0.001. BC and SA results differed significantly from all other platforms, while no significant difference was found between RC and AA. The distribution of TC results also varied significantly (p<0.001). In the comparison study, all platforms showed statistically significant differences (p<0.001). In contrast, MoM values showed no statistically significant differences between analyzers (p=0.106) or in distribution when applying various MoM-based cut-offs. Conclusions Although variations in TC measurements across platforms are small, they are statistically significant and affect FH classification. MoM normalization may support harmonization in future screening protocols.

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

Objectives Pernicious anemia (PA) is a common cause of vitamin B12 deficiency and requires a robust diagnosis to guide lifelong treatment. Anti-parietal cell antibodies (APCA) are frequently used as a diagnostic marker, but their poor specificity raises concerns about overdiagnosis, particularly in patients lacking the more specific but less sensitive anti-intrinsic factor antibodies (IFA). We compared the diagnostic performance of two APCA detection methods: indirect immunofluorescence (IIF) and immunodot assay. Methods We prospectively enrolled patients with B12 deficiency and APCA positivity without IFA. PA diagnosis was adjudicated by blinded expert based on histology and response to oral B12. Patients were classified as true PA (APCA-PA), false positive (APCA-FP), or undetermined. Only APCA-PA and APCA-FP cases were analyzed. Results Among 56 included patients, 19 were classified as APCA-PA and compared to 24 APCA-FP. APCA immunodot assay was positive in 19/19 APCA-PA vs. 23/24 APCA-FP (p>0.99), while APCA IIF was positive in 13/19 APCA-PA vs. 2/24 APCA-FP (p<0.001), with higher IIF titers among APCA-PA (p<0.001). Only IIF positivity was significantly associated with PA (68.4 % vs. 8.3 %, p<0.001), with 92 % specificity. This association was confirmed in multivariate analysis (OR 37.1 [95 % CI: 6.1–439.4]). APCA IIF titer was also associated with PA diagnosis (AUC 0.82 [95 % CI: 0.68–0.96]), and titer ≥1:80 further improved specificity to 96 %. Conclusions IIF is more specific than immunodot for PA diagnosis, and its result should prevail over immunodot when deciding whether to perform EGD, when EGD is not feasible, and when establishing the diagnosis if biopsies are inconclusive.

Objectives To assess the imprecision and stability of the point-of-care troponin I assay in the Emergency Department and its correlation and bias to two central laboratory troponin I assays (Siemens Atellica and Abbott Alinity). Methods Imprecision and stability testing was performed on opportunistically selected samples using whole blood in the emergency department by non-laboratory trained personnel. Assay comparisons were undertaken on samples taken from participants of the Mersey Acute Coronary syndrome Rule Out Study. Results The coefficient of variation (95 % confidence interval), at the 99th percentile for the point-of-care assay, was 8.1 % (6.1–12.1 %) but with a wide confidence interval reflective of considerable scatter at values just below the 99th percentile. The 10 % limit of quantification was 7.5 ng/L (1.7–61.8 ng/L). All samples met the ≤2 ng/L stability criteria for a duration of 4 h and under. The point-of-care assay very strongly correlated and had a negative bias with the Siemens Atellica and Abbott Alinity assays, Pearson’s R=0.99 and 0.95, mean difference −29.7 ng/L and −13.3 ng/L respectively. Conclusions The Siemens VTLi point-of-care assay fulfils high-sensitivity criteria when operated by non-laboratory trained staff using whole blood in its intended environment. Lithium heparin samples are likely stable up to 4 h. Significant bias between the point-of-care and two central laboratory assays negates the use of these assays interchangeably.

Objectives Glial fibrillary acidic protein (GFAP) is a biomarker of astrocytic activation associated with neurodegenerative diseases, neuroinflammatory disorders, and traumatic brain injury. However, the lack of standardized methods for quantifying GFAP across different immunoassay platforms poses challenges for its clinical implementation. This study aimed to compare the analytical performance of multiple commercially available and in-house immunoassays for GFAP quantification in plasma and cerebrospinal fluid (CSF) to assess their agreement and potential interchangeability. Methods We conducted a method comparison using four plasma GFAP immunoassays (Simoa, Ella, Alinity, and MSD) and four CSF GFAP assays (ELISA, Ella, Alinity and MSD). Anonymized leftover plasma and CSF samples were analyzed across platforms. Sample sizes for the pairwise comparisons ranged from 23 to 52 for plasma and 34 to 51 for CSF. Pairwise comparisons were performed using Spearman correlation, Bland–Altman analysis, and Passing–Bablok regression to assess systematic and proportional biases. Outliers were identified and excluded to ensure robust statistical evaluation. Results Strong correlations were observed across all platforms (Spearman’s r=0.827–0.927 for plasma; r=0.937–0.958 for CSF). However, significant systematic and proportional biases were present in several comparisons, preventing direct interchangeability of results. In plasma, Simoa consistently reported higher GFAP concentrations compared with Ella and Alinity, while Alinity overestimated levels relative to Ella. Similarly, in CSF, ELISA tended to underestimate GFAP concentrations compared with Alinity, MSD, and Ella, with the largest discrepancy observed between ELISA and MSD. Conclusions Despite strong correlations, substantial method-dependent biases indicate that GFAP measurements across different immunoassay platforms need to be standardized to ensure harmonization and reliable clinical application of GFAP as a biomarker.

Objectives Dysregulation of brain leptin signaling contributes to Alzheimer’s disease (AD) pathophysiology, but plasma leptin may not accurately reflect central nervous system activity. This study examined the correlation between plasma and cerebrospinal fluid (CSF) leptin levels and their relationship with beta-amyloid (Aβ) and tau biomarkers. Methods Cross-sectional analysis of data from cognitively impaired patients from a tertiary memory clinic. All the patients were diagnosed using CSF AD biomarkers. CSF and plasma leptin were measured using standardized immunoassays. The correlation between plasma and CSF leptin levels was studied. CSF leptin levels were compared between patients with positive AD biomarkers (A+T+N+) and neurological controls (A−T−N−). Regression analyses were performed to study the relationship between CSF leptin and CSF amyloid and tau biomarkers, with adjustment for age, gender, body mass index (BMI) and estimated glomerular filtration rate (eGFR). Results Finally, 140 participants were included (age 69.8 [1.1], females=47.4 %). CSF and plasma leptin were highly correlated (r=0.79, p=<0.0001). Individuals with AD CSF profile (A+T+N+) showed lower CSF leptin concentrations than their A−T−N− counterparts (74.7 [4.7] vs. 120.9 [11.9] ng/L, p<0.01) and CSF leptin remained associated with ATN profile and more specifically with the amyloid beta ratio after adjusting for confounding factors (β=−0.60, [0.73], p<0.01). Conclusions CSF leptin levels strongly correlate with plasma leptin and are reduced in individuals with AD biomarker positivity. This suggests plasma leptin may reflect central leptin activity. Our findings support a link between leptin signaling and amyloid deposition in AD.

Introduction The increasing use of therapeutic monoclonal antibodies (t-mAbs) has improved cancer and autoimmune disorder treatment. These therapeutics can interfere with serum protein electrophoresis (SPEP) and immunofixation (IF), potentially leading to the appearance of monoclonal bands that may be misinterpreted as monoclonal gammopathies. Identifying the migration patterns and detection thresholds of t-mAbs is crucial to avoid misinterpretation in clinical laboratories. Content A systematic review following PRISMA guidelines was conducted using Pubmed and ScienceDirect databases, with algorithm-based searches and double-blind article selection. Data on the matrix used, separation methods and type of interference were collected into an extraction table. Summary A total of 30 articles were included and 30 t-mAbs were described. 11 t-mAbs migrated at the end of the gamma region, 12 in the mid-gamma region, 5 in the early gamma region, one in the beta-2 globulin region and one in the alpha-2 globulin region. Most t-mAbs were detectable by SPEP and IF at concentrations above 100 mg/L. Outlook Caution is needed when a new peak appears on SPEP, as it may be mistaken for a monoclonal spike leading to misdiagnosis. Therefore, understanding the migration profiles of these t-mAbs is essential. Different methods are available to remove t-mAbs interference and could be used in daily practice.

Objectives Our previous study revealed a correlation between fecal leukocyte esterase and fecal calprotectin levels. This study assessed the predictive value of fecal leukocyte esterase compared with fecal calprotectin and C-reactive protein of inflammatory bowel disease. Methods Patients with inflammatory bowel disease who underwent ileocolonoscopy at National Taiwan University Hospital from March 2022 to March 2024 were included. Fecal leukocyte esterase and fecal calprotectin levels from stool samples collected within one month of endoscopy were analyzed. Active ulcerative colitis and Crohn’s disease were defined as Mayo endoscopic score ≥2 or simple endoscopic score for Crohn’s disease ≥7, respectively. Sensitivity, specificity, predictive values, and areas under the receiver operating characteristic curve were calculated using IBM SPSS Statistics 29. Results Of the 203 patients (100 with ulcerative colitis and 103 with Crohn’s disease), fecal leukocyte esterase levels were significantly correlated with fecal calprotectin levels (r=0.425, p<0.001) and endoscopic severity in ulcerative colitis (r=0.432, p<0.001) and Crohn’s disease (r=0.311, p=0.001). For predicting Mayo endoscopic scores ≥2 in ulcerative colitis using fecal leukocyte esterase, fecal calprotectin, and C-reactive protein, areas under the curve were 0.731, 0.785, and 0.558, respectively. For predicting simple endoscopic scores for Crohn’s disease ≥7, areas under the curve were 0.706, 0.800, and 0.770, respectively. No significant difference was observed between fecal leukocyte esterase and fecal calprotectin. Conclusions Fecal leukocyte esterase correlates with fecal calprotectin and predicts endoscopic severity in inflammatory bowel disease.

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

Objectives The increasing adoption of point-of-care testing (POCT) in primary healthcare highlights the need for robust quality assurance (QA) procedures to ensure result reliability. The patient split sample approach, i.e. comparing POCT and central laboratory results from the same patient, can be a valuable QA tool, though practical guidance for its implementation remains scarce. This study aimed to develop clear, user-friendly recommendations for non-laboratory personnel in primary healthcare laboratories on when and how to perform such comparisons and to recommend acceptable limits for the compared results. Methods In 2023, an expert group was established, composed of medical specialists in laboratory medicine, researchers, and laboratory advisors. The recommendations were formulated based on relevant literature, the professional experience of the group members, and Noklus’ educational framework for primary care laboratories. Pragmatic acceptability limits were established taking several approaches into account. The draft recommendations were audited among more than 120 Noklus employees and final consensus was reached in 2024. Results Comparing POCT results with central laboratory results is recommended when: (A) no suitable EQA program exists for a given POCT; (B) appropriate internal quality control materials are lacking or inadequate; or (C) POCT results contradict clinical expectations. Acceptable limits for the compared results were set at 15 or 20 %, depending on the measurand. Results outside these limits should be reviewed using a structured checklist. Conclusions These are the first published recommendations for using patient split samples in primary healthcare that are designed to be simple and user-friendly for non-laboratory personnel, facilitating widespread adoption.

Objectives To determine whether the diagnostic accuracy of the liver fibrosis marker FIB-4 and the likelihood ratios (LRs) of specific FIB-4 values vary with age. Methods We used a published dataset of 540 patients diagnosed with non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis. Liver biopsy showed no or early fibrosis in 391 patients, and advanced fibrosis in 149. For each group we established the relation between the mean of the natural logarithm of FIB-4 (log(FIB-4)) and age, and the standard deviation (SD) of log(FIB-4) and age. Using a parametric method, we calculated the area under the ROC curve of FIB-4 as a function of the difference in mean values of log(FIB-4) between the two groups, and the ratio of the SDs of log(FIB-4). LRs were calculated as functions of age at 35, 50 and 65 years, using a parametric method. Results The mean log(FIB-4) increased with age in both groups. The SDs of log(FIB-4) did not change with age. There was a trend towards decreasing diagnostic accuracy with age, but this finding did not reach statistical significance. The area under the ROC curve was 0.73 using a parametric method that accounted for age, and 0.79 using a traditional non-parametric method that did not account for age. The LRs of specific FIB-4 values decreased with age. Conclusions Age distorts the estimation of both diagnostic accuracy and LRs.

Objectives Accurate quantification of aldosterone is critical for screening and diagnosing primary aldosteronism (PA). Current competitive chemiluminescence immunoassays (cCLIA) overestimate plasma aldosterone concentration (PAC) compared to liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, LC-MS/MS is technically demanding and time-consuming, limiting its widespread clinical utility. Therefore, a novel two-step sandwich chemiluminescence immunoassay (sCLIA) for accurate quantification of PAC was systematically evaluated. Methods Precision, trueness, linear range, and maximum dilution factor of the new immunoassay were comprehensively validated. In a multicenter study involving 2,696 samples from seven Chinese centers, PAC measurements were performed in parallel using sCLIA, cCLIA, and LC-MS/MS. The study specifically focused on evaluating the assay’s performance at low aldosterone concentrations and in patients with chronic kidney disease (CKD), investigating potential interference from renal impairment by comparing the consistency between immunoassays and LC-MS/MS results across different CKD stages. Results The sCLIA exhibited excellent analytical performance for PAC measurement, with intra-assay imprecision <4.64 % and bias <5.71 % against certificated reference materials. The assay exhibited a wide reportable range (30–100,000 ng/L) with a limit of quantification at 30 ng/L and dilution capability ≥50-fold. Compared to cCLIA, sCLIA showed superior agreement with LC-MS/MS, particularly at low PAC concentrations (<110 ng/L) and in subjects with reduced renal function (eGFR<60 mL/min/1.73 m 2 ). Conclusions This novel sCLIA method exhibited excellent analytical performance, combining the practical advantages of immunoassays with LC-MS/MS accuracy, thereby offering an ideal solution for large-scale primary aldosteronism screening in clinical practice.

Objectives Autoantibodies against structure elements of the pancreas are an essential part of laboratory diagnosis of diabetes mellitus. The heterogeneity of current methods and difficult inter-assay comparability of results poses challenges to both clinical interpretation as well as research integrity. Methods We conducted a method validation study comparing the measurement of autoantibodies against glutamic acid decarboxylase (GADA), islet antigen 2 (IA-2A), and zinc transporter 8 (ZnT8A) on three commercially available platforms (EUROLabWorkstation analyzer, enzyme-linked immunosorbent assay (ELISA); iFlash 1800 (chemiluminescence immunoassay (CLIA); Maglumi 800 (CLIA)). Results Compared to the results from EUROLabWorkstation analyzer that was routinely used in our laboratory, the Maglumi demonstrated an acceptable agreement and deviation for GADA (75.4−35.8 %) and IA-2A (71.7−50.3 %). The iFlash displayed less favorable results with agreement and deviation for GADA (51.5−108.8 %) and IA-2A (68−32.5 %). While the iFlash showed excellent agreement for ZnT8A, the Maglumi produced almost exclusively negative results below a 200 U/mL threshold which results in severe underestimation of results on the Maglumi. Overall, no consistent agreement was observed across all three parameters among the CLIA devices tested. Conclusions Our study reveals significant and clinically relevant discrepancies in the detection of GADA and IA-2A when comparing CLIA devices to the ELISA method routinely used in our laboratory. For individual antibodies up to 65 % of patients who tested positive using ELISA showed negative or borderline results on at least one CLIA device. These deviations are unpredictable and cannot be detected through standard calibration and internal control procedures.

Objectives Glial fibrillary acidic protein (GFAP) is a well-established biomarker of astrocytic activation associated with neurodegenerative diseases, neuroinflammatory disorders, and traumatic brain injury. With increasing interest in blood-based biomarkers, the need for analytically validated assays and reliable reference intervals is critical for routine clinical implementation. This study aimed to analytically validate the MSD S-Plex ® GFAP immunoassay for plasma and to establish age-stratified reference intervals in an apparently healthy population. Methods This study was conducted in two phases. First, key analytical validation parameters – including repeatability, intermediate precision, measurement range, interferences, and sample stability – were evaluated following Clinical and Laboratory Standards Institute (CLSI) and published protocol guidelines. Second, reference intervals were derived from 579 apparently healthy individuals aged 17–91 years using a right-sided non-parametric percentile method. Age-specific upper reference limits were calculated for three predefined age groups, and a continuous age-dependent centile model was applied. Results MSD S-Plex ® GFAP assay demonstrated strong analytical performance, with coefficients of variation for repeatability and intermediate precision below 12 %. After accounting for the 1:2 dilution ratio, the validated measurement range was 0.425–1760 ng/L, with all calibration residuals remaining within ±15 %. GFAP concentrations were unaffected by hemolysis (p=0.85) and remained stable for up to 7 days at 4 °C and under frozen storage conditions. Age-stratified upper reference limits for plasma GFAP were established as 38 pg/mL (18–<50 years), 73 pg/mL (≥50–<70 years), and 156 pg/mL (≥70 years). Additionally, sex-related differences were observed after age 50, with females showing higher absolute GFAP levels than males. A strong positive correlation between age and plasma GFAP levels was observed (Spearman’s r=0.832, p<0.0001). Conclusions This study demonstrates the robust analytical performance of the MSD S-Plex ® GFAP assay and establishes age-related reference values for plasma GFAP. These findings support its suitability for routine clinical use and enhance its applicability in the diagnosis and monitoring of central nervous system (CNS) pathologies, such as neurodegenerative diseases, neuroinflammatory disorders, and acute brain injuries, within biomarker-supported clinical algorithms.

Objectives Insulin-like growth factor binding protein-4 (IGFBP-4) fragments are reported as emerging biomarkers for cardiovascular disease (CVD) risk assessment. To ensure data reliability and improve clinical application, the first automatic chemiluminescent immunoassay (CLIA) for NT-IGFBP-4 was developed and its distribution across CVDs was evaluated in this study. Methods Fragment-specific monoclonal antibodies were used to develop immunoassay, followed by comprehensive analytical validation, including limit of blank (LoB), limit of detection (LoD), limit of quantification (LoQ), linearity, specificity, precision, sample type compatibility, and stability. Reference intervals for NT-IGFBP-4 were established in healthy individuals, with variations analyzed based on gender, age, body mass index (BMI), and renal function. Additionally, NT-IGFBP-4 distribution was explored in CVD patients. Results The newly developed chemiluminescence assay demonstrated high specificity for NT-IGFBP-4, with excellent sensitivity (LoQ=1.0 ng/mL), broad linearity (1.0–1,000.0 ng/mL), and strong precision (CV≤3.0 %). It showed no interference from common endogenous substances, maintained compatible with various sample types, and remained stable under different storage conditions. Reference intervals showed slight variations by gender and age, with levels being independent of BMI but influenced by renal function. NT-IGFBP-4 levels were significantly elevated in CVDs, especially in heart failure, correlating with NYHA classification and LVEF (%), with higher levels indicating worse cardiac function. Conclusions The new automatic NT-IGFBP-4 (CLIA) assay offers a highly specific, sensitive and precise method for quantifying IGFBP-4 fragments. Its validated performance and disease association findings would enhance its diagnostic and prognostic potential in CVD research, particularly in heart failure.

Objectives ISO 15189:2022 recommends the use of quality indicators (QIs) to monitor laboratory performance and focuses on risk management and continual improvement. This study aims to compare hospital laboratory data with national and international quality specifications and to share experiences in quality improvement and adverse event management. Methods We evaluated 15 QIs from 2023 by comparing their percentages and sigma levels with those of national benchmarks to identify performance gaps. On the basis of this analysis, a series of data-driven continuous improvement projects were implemented in 2024. Adverse events were closely monitored and corrective actions were taken accordingly. Data from 2023 to 2024 were compared with national and international specifications, and statistical analysis was conducted using R Studio. Results Excluding internal quality control and interlaboratory comparison indicators, the remaining 13 QIs generally exhibited acceptable performance (σ>3.5). Statistically significant improvements (p<0.05) were observed in seven indicators. Overall, laboratory performance was ranked relatively low at the national level but favourably at the international level. The analysis of 15 adverse events revealed that errors occurred across all testing phases and involved multiple departments. Conclusions The quality improvement initiatives implemented in 2024 yielded measurable improvements. Effective communication and interdisciplinary collaboration are essential for reducing errors and enhancing laboratory performance.

Objectives Measurement uncertainty (MU) plays an important role in the interpretation of laboratory results, but data on serum proteins analyzed by immunoturbidimetry according to ISO/TS 20914 are limited. Methods MU of 11 serum proteins, including CRP, RF, ASO, IgG, IgA, IgM, C3, C4, ceruloplasmin, transferrin, and β2-microglobulin, were estimated using 1-year internal quality control (IQC) data obtained from Roche Cobas analyzers. MU was calculated using uncertainty and calibrator uncertainty according to ISO/TS 20914, assuming negligible deviation from external quality assessment data. Analytical performance specification (APS) models were selected according to the EFLM APS selection criteria, and maximum allowable uncertainty (MAU) values were determined based on sources such as EFLM models and literature. Results IgA and RF were the only two analytes that met the required and minimum MAU values, respectively, at both IQC levels. MU values for CRP, ceruloplasmin, transferrin, and β2-microglobulin exceeded targets at both levels. MU for C3, C4, IgG, and IgM exceeded the minimum MAU at IQC1 but remained acceptable at IQC2. MU values for ASO were calculated as 10.01 and 7.22 % but could not be evaluated due to a lack of reference data. Assay precision should be improved for CRP, IgG, IgM, ceruloplasmin, transferrin, and β2-microglobulin. Use of updated calibration materials for CRP may help reduce MU. Conclusions Maintaining acceptable precision over a long period remains a challenge for serum proteins analyzed by immunoturbidimetry, highlighting the need for methodological improvements and stricter quality monitoring. In this context, MU assessment is crucial.

Objectives Celiac disease (CD) is a systemic autoimmune disorder triggered by gluten in genetically predisposed individuals. Accurate diagnosis remains challenging due to clinical heterogeneity and reliance on invasive biopsy. This study aimed to evaluate the diagnostic performance of a novel multiparametric membrane-based enzyme immunoassay (AESKUBLOTS ® ) for the simultaneous detection of IgA antibodies targeting eight CD-related antigens. Methods A retrospective, single-centre study was conducted on 180 participants: 80 with CD (30 untreated, 50 on gluten-free diet, GFD), 50 with non-celiac wheat sensitivity (NCWS), and 50 healthy controls (HC). Serum samples were analysed using the AESKU assay. Diagnostic accuracy was assessed via ROC curve analysis and 5-fold cross-validation, examining individual markers and a composite antibody score. Results The assay demonstrated high diagnostic performance, particularly in untreated CD patients. Anti-tTG neo IgA showed the highest accuracy (AUC=0.93), followed by anti-tTG IgA (AUC=0.92). A composite score of ≥4 positive markers yielded an AUC of 0.99, while ≥6 positive markers achieved 100 % specificity and PPV, with 76.7 % sensitivity. Notably, anti-mTG IgA levels were elevated in all CD patients regardless of diet, suggesting potential utility in monitoring or identifying ongoing mucosal immune activity. Conclusions This multiparametric IgA assay offers a sensitive, specific, and non-invasive diagnostic tool for CD. Larger, prospective studies are warranted to confirm the clinical utility and expand the applicability to broader populations.

The European Commission (EC) started targeted evaluations and public consultations regarding the EU IVDR 2017/746 to assess its effectiveness, efficiency, relevance, and EU-added value. The goal is to identify implementation challenges and unintended consequences, experienced by either IVD-manufacturers that put CE-IVDs on the EU market or by medical laboratories that establish and operate in-house-IVDs (IH-IVDs) in their healthcare institution (network). Based on stakeholder feedback the EC aims to be informed about potential regulatory revisions by late 2025. As IH-IVDs, used in different modes, have a vital role in the EU healthcare system, the authors make the statement that Article 5.5 of the IVDR should be systemically amended, removing conditions (a) and (d) through (i) for in-house tests, while retaining conditions (b) and (c). Having the prime objectives of the IVDR in mind, i.e. patient safety and clinical utility, this opinion paper urges the EC to revise IVDR Article 5.5 to prevent disservices to patients and caregivers by taking into account available quality management infrastructure in ISO 15189:2022 accredited medical laboratories and guaranteed professionality of registered laboratory specialists, both already monitored at the level of the EU member states. By abandoning unnecessary, non-valuable requirements from Article 5.5 overregulation is prevented and the sustainability of important specialty and orphan tests, essential for patients with rare diseases, is guaranteed.

Objectives The clinical implications of positive anti-mitochondrial antibodies (AMAs) and/or primary biliary cholangitis (PBC)-specific anti-nuclear antibodies (ANAs) in patients with normal alkaline phosphatase (ALP) remain uncertain. Methods Patients at The Second Hospital of Nanjing with positive AMAs/PBC-ANAs and normal ALP levels from January 2016 to July 2024 were categorized into three groups based on autoantibody profiles: [AMA/AMA-M2] + [gp210/sp100] + , [AMA/AMA-M2] + [gp210/sp100] − , and [AMA/AMA-M2] − [gp210/sp100] + . The study compared PBC diagnostic rates, clinical symptoms, laboratory results, and pathology across these groups. Results A total of 53.3 % (88/165) of the patients showed cholangitis activity diagnosed of PBC. The PBC diagnosis rate in the three groups was 82.1 % (23/28), 48.4 % (46/95), and 45.2 % (19/42) respectively. Multivariate analysis indicated a significant association between the absence of liver disease-related etiology (p<0.001), baseline serum immunoglobulin M (IgM) exceeding 0.796 times the upper limit of normal (ULN) (p<0.001), and the diagnosis of PBC as determined by liver biopsy. In the three groups of non-PBC patients, the major pathological injury patterns were minor nonspecific reactive changes (40.0 %/28.6 %/39.1 %), inflammation (40.0 %/24.5 %/47.8 %), and fatty changes (20 %/20.4 %/4.3 %). The diagnosis included viral hepatitis, autoimmune hepatitis, fatty liver disease, vascular liver disorders, drug-induced liver injury, congenital hepatic fibrosis, and porphyria. Conclusions Over 50 % of patients with positive PBC-specific antibodies and normal ALP have PBC. Liver biopsy is recommended when AMAs and/or PBC-specific ANAs are positive, especially when baseline serum IgM exceeds 0.796 × ULN, and when other etiologies related to liver disease are absent. The area under the curve (AUC) for these three indicators reaches 0.84.

Objectives The T50 Calciprotein Crystallization test (T50 test) is a novel blood-based in vitro diagnostic assay that determines the calciprotein crystallization time in patients. It is based on the one-half maximum transition time of calciprotein particle 1 (CPP1) to calciprotein particle 2 (CPP2) in serum, as detected by nephelometry. To date, the T50 test has only been performed at Calciscon AG, where the assay has been developed and is manufactured. The aim of this study was to compare the agreement and precision of the T50 test in a routine clinical laboratory. Additionally, the interference of free hemoglobin, bilirubin, lipid and low molecular weight heparin (LMWH) was analyzed in the T50 test. Methods Serum samples were measured at both laboratory sites to determine the agreement. The CLSI EP15-A3 protocol was used to evaluate the precision. Interference was analyzed by spiking pooled serum samples with interfering analytes. Results Both laboratories showed excellent agreement in the T50 values (y=1.002x-4). Furthermore, high precision was observed for the clinically relevant lower range of T50 with a total variation coefficient of 6.4 %. Serum samples with mid and higher ranges of T50 failed the CLSI precision criteria with a total variance of 10.1 % and 6.2 %, respectively. Lastly, no interferences were observed within the normally observed clinical serum concentrations of free hemoglobin, bilirubin, lipid, and LMWH. Conclusions The T50 test was successfully implemented in a routine laboratory setting. Additionally, the precision and interference observed in this study largely agreed with the manufacturer’s claims.

Objectives The UK National Institute for Health and Care Excellence has recommended thresholds for total vitamin B12 (B12) assays with interchangeable results for identifying B12 deficiency. We assessed the agreement between B12 methods, to evaluate whether the thresholds may be assumed applicable to all assays. Methods A total of 19 External Quality Assessment (EQA) exercises (1791 determinations) based on human subjects-pool materials and 97 serum samples were retrieved to characterize the agreement between Alinity and Architect (Abbott Diagnostics), Access DXi (Beckman Coulter Diagnostics), Atellica and ADVIA Centaur (Siemens Healthcare Solution) compared to Cobas (Roche Diagnostics), considered as comparator because its calibrator traceability to the World Health Organisation (WHO) International Standard (IS) code 03/178. Ordinary least squares and Bland-Altman were used for this purpose. Results Abbott and Beckman methods overestimated and underestimated, respectively, B12 concentrations vs. Roche and the other methods. We reported similar systematic or proportional error patterns between EQA and serum samples. Only Beckman was affected by both errors. Due to the wide Limit of Agreement Interval, we cannot confidently conclude on the agreement between Roche and the other methods. However, the inter-method bias was well lower than the desirable goal of 9.4 % for Abbott Architect and also lower for Siemens ADVIA Centaur. Conclusions The recommended thresholds for serum total B12 should not be assumed applicable to all assays, due to the poor agreement among the currently available methods, a limitation that persists despite the release of the WHO IS 03/178.

Objectives Spinal muscular atrophy (SMA) is a severe inherited neuromuscular disorder with a high carrier frequency and incidence rate. An accurate molecular method for SMA genes is crucial in carrier screening, clinical diagnosis, outcome assessment and precision therapies. Methods Comprehensively using the multiplex allele specific PCR (mASPCR) and capillary electrophoresis (CE), a novel single tube assay was developed to simultaneously determine the copy number of SMN1 / SMN2 / NAIP genes and five common loss-of-function variants in SMN1 . A total of 283 genotype known subjects were detected to evaluate the accuracy, while 564 clinical random samples were double-blind detected with this assay and MLPA to assess the specificity and sensitivity. Results This assay had high accuracy of 100 % consistency with the predetermined values in 283 genotype known subjects. Among 564 clinical random samples, the correlation between this assay and comparative method was 100 %, which showing high specificity and sensitivity. Conclusions This mASPCR-CE assay is easy to use and cost-effective, making it suitable for routine use in molecular screening and clinical diagnosis of SMA.

Objectives α-Thalassaemia screening is crucial for identifying carriers at risk of having offspring with haemoglobin (Hb) Bart’s hydrops fetalis syndrome. This study evaluated the performance of two potential screening methods: the α-thalassaemia early eluting peak (αEEP) identified by high-performance liquid chromatography (HPLC) and an immunochromatographic strip test (ICT), focusing on stability and potential interferences. Methods Ninety-two peripheral blood samples were used. Thirty were for assessment of αEEP and ICT stability and interference by icterus/lipaemia, and saline washing for interference removal. Diagnostic performance of αEEP and ICT were evaluated in 40 samples with glycated Hb (P2) ≥6.0 % and 22 with Hb F≥2.0 % on HPLC, using α-globin genotyping as the gold standard. Results Both αEEP and ICT results remained stable for 14 days. Neither test was significantly affected by icterus or lipaemia, though 3 of 20 ICT results (15 %) showed discordance after saline washing. Elevated HbA 1c and Hb F altered early eluting peak patterns but did not affect αEEP interpretation. For detecting -- SEA mutation, αEEP showed 100 % sensitivity and 100 % specificity when P2≥6.0 % or Hb F≥2.0 %, while ICT a low specificity (45 %) when Hb F≥2.0 %. Conclusions αEEP showed reliable performance in detecting α 0 -thalassaemia with -- SEA mutation in all conditions tested, while ICT showed low specificity when Hb F≥2.0 %. These findings support αEEP as a reliable test for routine clinical laboratory use, while cautions should be made for ICT in case of elevated Hb F levels.

Interpretation of laboratory test results is a comparative process that requires reference data. Such data are derived for each analyte separately, without accounting for, the interrelationships among analytes. Physicians use test panels containing multiple analytes to enhance clinical significance and improve the accuracy of decision-making. However, current interpretation practices apply reference intervals and reference change values in a univariate manner – that is, each analyte in the panel is interpreted independently and no reference data are available to interpret the panel as a whole. Yet, metabolism is a network of biomolecules, each of which is related to others. Therefore, a multivariate approach – based on the correlations among biomolecules – can provide a more informative reference than univariate approaches and can be used more effectively in the interpretation of laboratory data. This concept can be summarized by a motto: Combine single tests into meaningful groups, but interpret the group as a single clinical entity. In this opinion paper, we present a practical approach for obtaining reference data for both reference intervals and reference change values to interpret laboratory test panels composed of related analytes.

Laboratory investigations are important in the clinical management and the study of chronic kidney disease–mineral and bone disorder (CKD-MBD)- including CKD-associated osteoporosis. Parathyroid hormone (PTH) is the major hormone in the regulation of bone and calcium balance but is significantly affected in advanced CKD. Knowledge of PTH concentration is important in the assessment of osteoporosis including CKD-associated osteoporosis; however, measurement of PTH in the laboratory is bedevilled by interferences and inter-method differences compounded by lack of standardisation of commonly used immunoassays. Vitamin D is important for bone health and its deficiency contributes to the development of osteoporosis. Vitamin D metabolism is impaired in advanced CKD, augmenting the effects of its deficiency on bone health. Lack of consensus on optimal serum 25-hydroxyvitamin D (25-(OH)D) concentrations for bone health, including in the various CKD stages, is compounded by lack of analytical specificity of immunoassays. Liquid chromatography tandem mass spectrometry (LC-MS/MS) assays would help overcome these issues. Ionised calcium measurement is recommended for assessment of serum calcium, especially in CKD. Fibroblast growth factor 23 (FGF23) is important in the homeostasis of phosphate that accumulates in CKD, though this marker is not yet utilized in clinical context. Calculated maximal tubular reabsorption of phosphate normalized to glomerular filtration rate (TmP/GFR) may help in assessment of phosphate homeostasis. Bone specific alkaline phosphatase (BALP) and tartrate-resistant acid phosphatase isoform 5b (TRACP5b), the reference markers of bone formation and resorption for CKD-associated osteoporosis, have been shown to reflect bone turnover by histomorphometry in patients with advanced CKD.

Objectives To evaluate the performance of the iStar high-sensitivity cardiac troponin I (hs-cTnI) assay, focusing on its sensitivity, precision, linearity, and consistency with plasma samples, and to establish sex-specific 99th percentile upper reference limits. Methods The iStar hs-cTnI assay was assessed using the Drawray iStar 500 analyzer. Key performance metrics such as the limits of blank (LoB), detection (LoD) and quantitation (LoQ), precision, linearity, and agreement between sample types were evaluated according to Clinical and Laboratory Standards Institute (CLSI) guidelines. A methodological comparison was performed with the Abbott ARCHITECT hs-cTnI assay, and cross-reactivity with others troponins was assessed. Results The iStar hs-cTnI assay demonstrated robust sensitivity with a LoB of 0.09 ng/L and LoD of 0.31 ng/L. The LoQ was 0.79 ng/L for 20 % coefficient of variation (CV) and 1.85 ng/L for 10 % CV. Precision testing revealed CVs of 1.4–4.8 % near the 99th percentile upper reference limit (URL). The assay exhibited excellent linearity (r=1.00) and high agreement between whole blood and plasma samples (slope=0.936). Methodological comparison with the Abbott ARCHITECT hs-cTnI showed a high correlation coefficient of 0.983. Cross-reactivity with skeletal muscle troponin I, cardiac troponin C, and cardiac troponin T was negligible. In healthy individuals, the overall 99th percentile URL was 16 ng/L, with sex-specific values of 18 ng/L for males and 14 ng/L for females. Conclusions The iStar hs-cTnI assay demonstrates high sensitivity and precision, supporting it suitable for the rapid diagnosis of acute myocardial infarction using whole blood samples. Its high agreement with plasma and established sex-specific URL support its potential for clinical use in acute coronary syndrome management.

Objectives Significant changes in clinical biochemical markers occur during the peri-menopausal period. Traditional population-based reference intervals (popRIs) may not reflect individual physiological variability, limiting clinical interpretation. This study aimed to establish personalized reference intervals (prRIs) for menopausal women and compare them with popRIs. Methods We analyzed 899 healthy women aged 35–64 from the Peking Union Medical College Hospital Aging Longitudinal Cohort of Women in Midlife (PALM) cohort. 13 biochemical markers were evaluated across reproductive, menopausal transition, and postmenopausal stages. Six key biomarkers were selected through Kruskal–Wallis tests and ranked by their importance in menopausal status classification using a Random Forest model. Biological variation (BV) data were used to calculate total variation (TV) and index of individuality (II). The prRIs were constructed based on BV estimates, and the reference interval index (RII) was applied to compare popRIs and prRIs. Results ALT, TG, and FSH showed significant differences across menopausal stages and ranked highly in the Random Forest model. These markers also had large BV and differed across three menopausal stages. Most II values ranged from 0.6 to 1.4, and all median RII values were below 1.0, suggesting limited utility of popRIs. Crea in reproductive women had the highest proportion of RII>1.0, while FSH showed RII<0.5 in over 90 % of women in the menopausal transition. Conclusions For women in the menopausal transition with high BV estimates, combining popRIs with prRIs improves interpretation. Larger, more diverse cohorts are needed to validate and optimize prRIs for clinical application.

Objectives Insulin assays are used to assess insulin resistance and to aid in the diagnosis of conditions such as insulinoma and various forms of hypoglycemia. However, discrepancies among commercial assays limit their clinical and research utility. This study evaluates the current comparability of the most widely used insulin assays. Methods Forty serum samples, including those from healthy individuals and patients with type 1 or type 2 diabetes, were analyzed by nine manufacturers using 12 commercial immunoassays. Results from each assay were compared both to the isotope dilution mass spectrometry (IDMS) method and across the different immunoassays. Results Intra-assay repeatability was excellent (ICCs>0.99), but substantial inter-assay variability was observed. Differences relative to LC-MS ranged from −298.2 to +302.6 pmol/L. Several assays overestimated insulin concentrations at low levels and underestimated them at higher levels. Only one assay method showed full agreement with the IDMS method. Conclusions Despite all methods claiming traceability to the WHO 66/304 standard, significant variability persists among insulin assays. These findings highlight the urgent need for insulin assay standardization using commutable certified reference materials.

As aminotransferases represent the first-level tests to detect individuals with hepatocellular damage, standardization of their measurements is a priority need for patient care. The obtaining of the harmonization in aminotransferase results is however strongly dependent on supplementation of pyridoxal-5′-phosphate (P-5-P) in aminotransferase reagents as this exclusively enables reporting of measured values on clinical samples that are traceable to the IFCC reference measurements procedures, representing the highest metrological level defining the respective measurable enzyme quantity. The lack of P-5-P addition in the employed commercial reagents is the most frequent cause of unacceptably biased results of aminotransferases, which opposes to consistency of results across laboratories and commercial assays. Nevertheless, only about one-third of medical laboratories include P-5-P in their aminotransferase measurements. This demonstrates a lack of awareness of the clinical benefits of P-5-P supplementation, which is not just an analytical preference, but it represents a laboratory priority for providing a high-quality service in pertinent clinical situations.

Over 50 years have elapsed since the clinical definition of non-A, non-B hepatitis and 36 years since the unveiling of hepatitis C virus (HCV) and the availability of specific serological assays, but few advances in the serological diagnosis of HCV infection have occurred. Testing for anti-HCV is still based on the detection of reactivity towards the structural Core region of HCV, which appears to be dominant throughout the different phases of infection, plus antibodies towards antigens expressed by several non-structural regions. Unlike testing for other viral diseases, antibodies towards the envelope region are not detectable by the first line assays employed for screening or diagnosis and are scarcely represented in the supplementary assays employed to confirm the reactivity by screening assays. Clinical laboratories are often confronting issues on samples that give discrepant results among assays and are not confirmed by supplemental testing. Results obtained on those samples are usually labelled as ‘indeterminate’ and are often considered as false positive – though a true reference to define anti-HCV positivity is still lacking. The diagnostic answer is then deprived of clinical significance and affects patient management and counselling. The only advance, though not recent, has been the availability of assays detecting the HCV core antigen, which is considered as a surrogate of HCV RNA, with lesser sensitivity but bearing some operational and economic advantages in diagnosis and population screening, and lately of assays combining HCV antigen and anti-HCV detection. This paper summarizes the history of HCV serology and provides some insights on its limitations and potential developments.

Objectives Primary aldosteronism (PA) is an underdiagnosed yet important condition. This study aimed to evaluate the diagnostic performance of a combined protocol using oral salt loading and postural stimulation tests (PST), named the balance study, to confirm and subtype PA. Methods We retrospectively analyzed the data of 402 patients with either confirmed PA or essential hypertension who underwent the balance study. Biochemical markers were measured using liquid chromatography-tandem mass spectrometry. Receiver operating characteristic curves were used to assess the diagnostic performance. The cut-off values for biochemical markers were determined by Youden J’s statistics. Results For PA confirmation, urinary aldosterone (UALD; area under the curve [AUC] 0.894), urinary tetrahydroaldosterone (UTHA; AUC 0.875), the sum of UALD and UTHA (SUM; AUC 0.884), and supine plasma aldosterone (PALD; AUC 0.902) showed comparable performance. Corresponding cut-offs were: UALD>38 nmol/day, UTHA>160 nmol/day, SUM>259 nmol/day, and supine PALD>273 pmol/L. For PA subtyping, the difference or ratio between erect and supine PALD in PST achieved an AUC of 0.90. An increase of less than 19 pmol/L or less than 5 % from a supine to an erect position suggested an aldosterone-producing adenoma. Conclusions This combined protocol using oral salt loading and PST could help confirm a diagnosis of PA and predict the subtype. UTHA and supine PALD may add value to UALD for PA confirmation, whereas the erect/supine PALD ratio from PST could assist in PA subtyping. Further studies are required to validate these findings.

Since the 1980s, automation has profoundly transformed clinical laboratory operations, enhancing efficiency, standardization, and throughput. This technological evolution has enabled laboratories to meet rising testing demands, address persistent workforce shortages, and control operational costs. Beyond analytical consolidation, significant progress has been made through the integration of pre-analytical and post-analytical phases, thereby improving the overall quality of the Total Testing Process (TTP). Despite the well-recognized benefits of Total Laboratory Automation (TLA) – notably improved worker safety and faster turnaround times – a number of limitations have emerged, particularly concerning operational challenges and the lack of evidence for clinical effectiveness. Future improvements should focus on the integration of pre-pre-analytical processes, miniaturization of testing platforms, consolidation of all laboratory medicine subspecialties, and enhanced data management. However, the most critical issue remains the limited evidence supporting the impact of TLA on key clinical outcomes – such as reduced length of stay in emergency departments and hospital wards, optimized diagnostic-therapeutic pathways, improved quality of care, and reduced morbidity and mortality.

Objectives Accurate measurement of plasma aldosterone (ALD) and renin is important in primary aldosteronism screening and diagnosis. The high variability of ALD and renin due to salt intake, age, and sex, as well as the lack of immunoassay standardization necessitates the establishment of region-, age-, sex- and assay-specific reference intervals (RIs). This study established RIs for ALD and renin using novel sandwich chemiluminescent immunoassays. Methods ALD and renin were measured on a fully automated chemiluminescence platform (Maglumi X8, Snibe, China). The analytic performances were estimated following CLSI guidelines. A total of 2,281 healthy participants from Beijing, Jiangsu, Henan, Gansu and Guangdong were recruited. Results Total imprecision were 1.42–5.09 % for ALD and 1.07–4.11 % for renin. The maximum dilution rates were 50× and 10× for ALD and renin. Interferences of 22 and six interferents for ALD and renin were acceptable. The sandwich ALD immunoassay results highly correlated with LC-MS/MS ( r =0.994). The RI of ALD was partitioned based on salt intake, with central 95 % RIs for males of 16.9–185 ng/L (46.8–513 pmol/L) in Guangdong and 26.5–242 ng/L (73.5–671 pmol/L) in other four regions. Females showed an age-dependent decline in ALD levels (RIs in Guangdong: 26.9–442 ng/L [74.6–1,226 pmol/L, 18–40 years], 32.4–326 ng/L [89.9–904 pmol/L, 41–60 years], 21.2–199 ng/L [58.8–552 pmol/L, >60 years], whereas males exhibited no significant change. Renin RIs: females: 3.43–89.2 mU/L (18–40 years), 0.92–67.1 mU/L (41–60 years), 0.66–55 mU/L (>60 years); males: 2.23–95.1 mU/L (≤60 years), 1.71–68.9 mU/L (>60 years). Conclusions This is the first study to establish accurate ALD and renin RIs for sandwich immunoassays. Region-, age- and sex-specific RIs were recommended, which will enhance the accuracy of clinical screening and diagnosis of primary aldosteronism.

Objectives An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) was developed and validated to accurately measure serum and plasma concentrations of total and free phenytoin. Methods Quantitative nuclear magnetic resonance spectroscopy (qNMR) was used to determine the absolute content of the reference material, ensuring its traceability to SI units. The separation of phenytoin from potential unknown interferences was achieved with reversed-phase chromatography, utilizing a C8 column. A protein precipitation protocol was established for preparation of total phenytoin samples, while free phenytoin samples were prepared by membrane separation utilizing a commercially available ultrafiltration device. Assay validation and determination of measurement uncertainties was performed according to the guidelines of the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement. Results These RMPs demonstrated high selectivity and specificity, with no evidence of matrix effects, allowing quantification of total and free phenytoin in ranges of 0.640–48.0 μg/mL and 0.0800–4.80 μg/mL, respectively. Intermediate precision was <3.8 %, and repeatability was 1.4–3.8 %, over all concentration levels, for both forms of phenytoin. For total phenytoin, relative mean bias ranged from −2.7–0.3 % in native serum and from 0.0–1.1 % in lithium heparin plasma. Relative mean biases for free phenytoin were 3.5–4.1 % for both native serum and ultrafiltrates. Measurement uncertainties for single measurements and target value assignment were 1.8–2.5 % and 0.9–1.7 %, respectively, for total phenytoin. For free phenytoin, these measurement uncertainties were 2.0–3.9 % and 0.9–1.4 % for single measurements and target value assignment, respectively. Conclusions We present a novel LC-MS/MS-based RMP for phenytoin in human serum and plasma that provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

Objectives An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in human serum and plasma is presented. Methods Quantitative Nuclear Magnetic Resonance (qNMR) spectroscopic methodology has been utilized to assign absolute content (g/g) and International System of Units (SI)-traceability to the reference materials used as primary calibrators. This RMP was developed for the simultaneous quantification of 25OHD2 and 25OHD3 in human samples, utilizing supported liquid extraction (SLE) clean-up and a two-dimensional heart-cut ID-LC-MS/MS method to minimize matrix effects and prevent the co-elution of 3-Epi-25OHD3 and 3-Epi-25OHD2. The method underwent validation in accordance with current guidelines. Selectivity was assessed using spiked samples. To evaluate potential matrix effects, a post-column infusion experiment and a comparison of standard line slopes were performed. A 5-day validation study was conducted to determine precision, accuracy and trueness of the method. Measurement uncertainty for reference value assignment was evaluated in line with the Guide to the Expression of Uncertainty in Measurement (GUM). Equivalence to Joint Committee on Traceability in Laboratory Medicine (JCTLM) listed RMPs was demonstrated through the participation in the CDC Vitamin D Standardization-Certification Program (VDSCP) as well as the RELA scheme. Results The RMP enabled the quantification of 25OHD2 and 25OHD3 within the range of 1.50 ng/mL–180 ng/mL (3.64–436 nmol/L for 25OHD2 and 3.74–449 nmol/L for 25OHD3), without interference from their respective epimer and no evidence of matrix effects. Intermediate precision was determined to be ≤4.0 % for 25OHD2 and ≤3.6 % for 25OHD3, while repeatability was ≤3.3 % for 25OHD2 and ≤2.9 % for 25OHD3 across all concentration levels. The relative mean bias for the secondary reference materials varied from −1.0 to 1.1 %, regardless of the analyte. For the spiked samples, the relative mean bias ranged from −4.2 to 1.0 % for 25OHD2 and from −3.9 to 0.9 % for 25OHD3, irrespective of all levels and matrices. Expanded measurement uncertainties (k=2) for target value assignment (n=6) were ≤3.9 % for 25OHD2 and ≤3.2 % for 25OHD3. Participation in the VDSCP and the RELA scheme showed a good agreement with results from the JCTLM listed RMPs and laboratories. Conclusions The RMP enables the accurate, precise and consistent determination of 25OHD3 and 25OHD2. The robust performance of this method supports standardization of routine assays and guarantees traceability in the measurement of individual patient samples.

Objectives A new candidate isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based reference measurement procedure (RMP) has been developed for the accurate and precise quantification of 17β-estradiol (E2) in human serum and plasma covering a measurement range from 0.400 to 5,000 pg/mL (1.47–18,357 pmol/L). To address this broad range, two separate methods were created: a high sensitivity (HS) method for concentrations between 0.400 and 5.00 pg/mL (1.47–18.4 pmol/L) and a standard range (SR) method for concentrations between 5.00 and 5,000 pg/mL (18.4–18,357 pmol/L). Methods As the primary reference material, E2 (CRM 6004-a) from the National Metrology Institute of Japan was used to ensure traceability to the international system (SI). A two-dimensional heart-cut LC approach was utilized for LC-MS/MS analysis, employing a supported liquid extraction sample preparation protocol for the SR method and a liquid-liquid extraction protocol followed by derivatization for the HS method. Assay validation was conducted following current guidelines. Selectivity and specificity were assessed using spiked serum samples, while potential matrix effects were evaluated through a post-column infusion experiment and comparison of standard line slopes. Precision, accuracy, and trueness were determined using an extensive 5-day protocol. Standard measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM), with three individual sample preparations performed on at least two different days. Equivalence with higher-order RMPs was demonstrated through participation in the CDC Steroid Hormones Standardization (HoSt) program. Results The RMP enabled the quantification of E2 within the range of 0.400–5,000 pg/mL (1.47–18,357 pmol/L), demonstrating no interference from structurally related compounds and no evidence of matrix effects. The relative mean bias of the SR method ranged from −2.4 to 1.9 % across all levels, including secondary reference materials and spiked samples, whereas the HS method exhibited a mean bias ranging from −3.0 to 2.9 %. Expanded measurement uncertainties (k=2) for target value assignment ranged from 1.7 to 4.4 % for the SR method and were found to be ≤6.1 % for the HS method. The method’s transferability was demonstrated in a comparison study at a second laboratory. Additionally, the candidate RMP exhibited excellent correlation and equivalence to JCTLM-listed RMPs through the CDC HoSt program. Conclusions In summary, the ID-LC-MS/MS-based RMP accurately quantifies E2. Its robust performance makes it suitable for standardizing routine assays and measuring individual patient samples, ensuring traceability.

Objectives Androstenedione (ASD) is a biomarker used in the diagnosis of hyperandrogenism and adrenal hyperplasia. Therefore, accurate measurement of serum ASD is pivotal in clinical settings. We aimed to develop a novel highly selective reference measurement procedure (RMP) based on isotope dilution-two dimensional-liquid chromatography-tandem mass spectrometry (ID-2D-LC-MS/MS) for the quantification of ASD in human serum/plasma. Methods To achieve high selectivity and sensitivity, a two-dimensional heart-cut LC approach for LC-MS/MS and a supported liquid extraction sample preparation protocol were employed. Matrix effects were evaluated through a post-column infusion experiment and comparison of standard line slopes. Precision and accuracy were determined via a multi-day validation experiment. Reproducibility was assessed by comparing results from two independent laboratories, and measurement uncertainty (MU) was evaluated in compliance with current guidelines. Results Our novel RMP proved effective for measuring ASD concentrations ranging from 0.00800 ng/mL to 12.0 ng/mL (0.0279 nmol/L to 41.9 nmol/L) and demonstrated matrix-independence. The relative mean bias varied between 0.6 and 2.2 % across different matrices and concentration levels. Intermediate precision was observed to be between 1.2 and 1.9 %. The expanded measurement uncertainty for ASD target value assignment ranged from 1.7 to 2.5 %, irrespective of sample concentration. Equivalence to the JCTLM-listed RMP was evaluated through a method comparison study, revealing a high degree of agreement (r≥0.997). Additionally, by comparing results from two independent laboratories, the transferability and reproducibility of the RMP were confirmed. Conclusions This isotope dilution two-dimensional LC-MS/MS method can be used for the evaluation and standardization of routine assays and for measuring individual patient samples, ensuring traceability.

Objectives Cortisone is an inert precursor/metabolite of the potent steroid hormone cortisol. Measurement of serum cortisone levels and the cortisol-cortisone ratio can be useful for the diagnosis of dysfunction in the regulation of cortisol levels (i.e., severe and subtle apparent mineralocorticoid excess, low-renin primary aldosteronism). Therefore, an isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedure (RMP) to quantify cortisone in human serum/plasma was developed and validated. Methods Quantitative nuclear magnetic resonance (qNMR) was utilized to assign absolute content (g/g) and SI-traceability to reference materials used as primary calibrators. A supported liquid extraction sample preparation protocol as well as a two-dimensional heart-cut LC approach for LC-MS/MS analysis were employed to mitigate matrix effects and prevent co-elution of interferences. Selectivity was determined by analyzing a matrix sample containing the analyte, the internal standard and six potential interferents. A post-column infusion experiment and a comparison of standard line slopes were performed to evaluate matrix effects. An extensive protocol over five days was applied to determine precision, accuracy and trueness. Measurement uncertainty (MU) was evaluated in compliance with current guidelines. Results This RMP is suitable for analyzing cortisone within the 0.0800–120 ng/mL (0.222–333 nmol/L) range, demonstrating selectivity, sensitivity and matrix independence. Intermediate precision was ≤3.4 %, repeatability was ≤2.9 % across all concentration levels and relative mean bias ranged from −3.7 to 2.8 % across all tested matrices and concentrations. Expanded MU (k=2) for target value assignment (n=6) ranged from 2.1 to 5.5 %, irrespective of concentration or sample type. Conclusions This RMP allows for accurate and reproducible determination of cortisone in human serum and plasma. Implementation of this method supports routine assay standardization and patient sample measurement with confirmed traceability.

Objectives Isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedures (RMPs) for the quantification of 24,25(OH) 2 D2 and 24,25(OH) 2 D3 in human serum and plasma are presented. Methods Quantitative nuclear magnetic resonance (qNMR) spectroscopic methodology was utilized to assign absolute content (g/g) and SI-traceability to reference materials used as primary calibrators. For liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis a two-dimensional heart cut LC approach, in combination with a supported liquid extraction protocol, was established to mitigate matrix effects and prevent co-elution of interferences. Selectivity was determined by spiking the internal standards and similar compounds, in human serum. A post-column infusion experiment and comparison of standard line slopes was performed to evaluate matrix effects. Precision and accuracy were assessed via a multi-day validation experiment, utilizing certified secondary reference materials from the National Institute of Standards and Technology (NIST). Measurement uncertainty (MU) was evaluated per the Guide to the Expression of Uncertainty in Measurement (GUM). To demonstrate equivalence with the JCTLM-listed RMP, certified secondary reference materials were utilized. Additionally, a method comparison study was conducted with the 24,25(OH) 2 D3 method used by the CDC Vitamin D Reference Laboratory. Results The RMP allowed quantification of 24,25(OH)2D2 and 24,25(OH)2D3 within the range of 0.150–18.0 ng/mL (0.350–42.0 nmol/L 24,25(OH) 2 D2 and 0.360–43.2 nmol/L 24,25(OH) 2 D3) without interference from structurally-related compounds and no evidence of matrix effects. Intermediate precision was ≤2.3 % for 24,25(OH) 2 D2 and ≤2.9 % for 24,25(OH) 2 D3; repeatability was ≤1.4 % for 24,25(OH) 2 D2 and ≤2.1 % for 24,25(OH) 2 D3, across all concentration levels. The relative mean bias was −4.5 to 2.9 % for 24,25(OH) 2 D2, and −3.7 to 3.6 % for 24,25(OH) 2 D3. Expanded MU for reference value assignment for 24,25(OH) 2 D2 and 24,25(OH) 2 D3 for reference value assignment was ≤2.5 %, regardless of concentration level and sample type. Passing-Bablok regression revealed strong agreement between the 24,25(OH) 2 D3 results from the candidate RMPs and those provided by the CDC Vitamin D Reference Laboratory. Conclusions These RMPs permit accurate and reproducible determination of 24,25(OH) 2 D2 and 24,25(OH) 2 D3. Implementation of these methods supports routine assay standardization and patient sample measurement with confirmed traceability.

Objectives Accurate measurement of serum cortisol is crucial for the diagnosis and management of adrenal disorders. Thus, we have developed a novel isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedure (RMP) to quantify cortisol in human serum/plasma, offering higher sensitivity and reliability compared to existing RMPs. Methods Quantitative Nuclear Magnetic Resonance spectroscopic (qNMR) methodology has been utilized to assign the absolute content (g/g) and SI-traceability to the reference materials. A novel two-dimensional heart-cut liquid chromatography (LC) approach was implemented for the LC-MS/MS, combined with a supported liquid extraction (SLE) sample preparation protocol. A multi-day validation experiment assessed precision and accuracy. Reproducibility was assessed by comparing procedure results between two independent laboratories, and measurement uncertainty (MU) was evaluated in compliance with current guidelines. Results The established RMP exhibited high sensitivity, with a quantification range of 0.800–600 ng/mL (2.21–1,655 nmol/L), exceeding the ranges of existing JCTLM-listed RMPs. Intermediate precision was ≤2.6 %, and repeatability ranged from 0.9 to 1.9 % across all concentration levels. The relative mean bias ranged from −1.3 to 1.4 % for all matrices and concentration levels. Measurement uncertainties (MU) for cortisol in single measurements were ≤2.8 % regardless of the concentration level and sample type. Using the certified International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference panel, the equivalence between the candidate RMP and the Joint Committee on Traceability in Laboratory Medicine (JCTLM) listed RMPs (NRMeth 57 and NRMeth 8) was assessed, revealing excellent agreement. Conclusions This RMP allows for highly sensitive and reproducible determination of cortisol. The performance of the RMP facilitates the standardization of routine assays and ensures traceability in the measurement of individual patient samples.

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.