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Journal

Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Mario Plebani
Edited by: Philippe Gillery , Ronda Greaves , Karl J. Lackner , Giuseppe Lippi , Bohuslav Melichar , Peter Schlattmann and Shiyang Pan

Language: English

First published: January 1, 1963

Publication Frequency: 12 issues per year

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About this journal

Objective
Clinical Chemistry and Laboratory Medicine (CCLM) publishes articles on novel teaching and training methods applicable to laboratory medicine. It is focused on basic and applied research and cutting-edge clinical laboratory medicine. CCLM is one of the leading international journals in the field of clinical laboratory sciences and drives progress in the field. CCLM is led by a multi-institutional editorial board. All contributions submitted for publication in CCLM are single-blind peer reviewed by at least two experts in the field. CCLM is led by a multi-institutional editorial board. It is issued monthly, both in print and electronically. Letters to the Editor and Congress Abstracts are published online only.
CCLM is the official journal of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM).

Topics

clinical biochemistry
clinical genomics and molecular biology
clinical haematology and coagulation
clinical immunology and autoimmunity
clinical microbiology
drug monitoring and analysis
evaluation of diagnostic biomarkers
disease-oriented topics (cardiovascular disease, cancer diagnostics, diabetes)
new reagents, instrumentation and technologies
new methodologies
reference materials and methods
reference values and decision limits
quality and safety in laboratory medicine
translational laboratory medicine
clinical metrology

Article formats
Research Articles, Reviews, Mini Reviews and Opinion Papers on all aspects of clinical chemistry and laboratory medicine, Guidelines and Recommendations, Point/Counterpoint Articles, Letters to the Editor, Editorials

For EFLM Academy Members

To take advantage of all the opportunities offered to members of the EFLM Academy, please access your personal area in the EFLM Academy at the link https://academy.eflm.eu/secretariat/login (if you do not remember your login details, you can use the option FORGOT PASSWORD). Click on the section “International Journals” on the left-hand side where you find CCLM as the first listed journal.

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Latest developments in the clinical laboratory sciences
Fundamental and applied approach and novel teaching and training methods
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Authors have the option of publishing their articles under the Creative Commons Attribution (CC-BY-4.0) open access license. Open access allows authors to retain copyright and share their findings with the scientific community without restrictions. Authors who belong to an institution that has an agreement with the publisher can publish open access without paying an article processing charge (APC) or at a considerable discount

Previous Titles

Vol. 1 - Vol. 4, 1963-1966
Zeitschrift für Klinische Chemie
Vol. 5 - Vol. 9, 1967-1971
Zeitschrift für Klinische Chemie und Klinische Biochemie
Vol. 10 - Vol. 13, 1972-1975
Zeitschrift für Klinische Chemie und Klinische Biochemie Journal of Clinical Chemistry and Clinical Biochemistry
Vol. 14 - Vol. 26, 1976-1988
Journal of Clinical Chemistry and Clinical Biochemistry Zeitschrift für Klinische Chemie und Klinische Biochemie
Vol. 27 - Vol. 28, 1989-1990
Journal of Clinical Chemistry and Clinical Biochemistry
Vol. 29 - Vol. 35, 1991-1997
European Journal of Clinical Chemistry and Clinical Biochemistry

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Defining the analytical characteristics of a novel high-sensitivity point-of-care troponin I assay in its intended clinical environment

September 3, 2025

James Hatherley, Sarah Davies, Suzannah Phillips, Ahmed Dakshi, Guy Miller, Lisa Bailey, Paul Collinson, Aleem Khand

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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.
Open Access
Method comparison of plasma and CSF GFAP immunoassays across multiple platforms

September 2, 2025

Burak Arslan, Elzbieta Rembeza, Sofia Rasch, Ulf Andreasson, Kaj Blennow, Henrik Zetterberg, Hlin Kvartsberg

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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.
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Cerebrospinal fluid leptin in Alzheimer’s disease: relationship to plasma levels and to cerebrospinal amyloid

September 1, 2025

Elodie Bouaziz-Amar, Jeanne Neez, Fidaa Ibrahim, Matthieu Martinet, Claire Hourregue, Julien Dumurgier, Emmanuel Cognat, Francois Mouton-Liger, Agathe Vrillon, Jacques Hugon, Claire Paquet, Matthieu Lilamand

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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.
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Association of Clinical Biochemists in Ireland Annual Conference

September 1, 2025

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Open Access
Interference of therapeutic monoclonal antibodies with electrophoresis and immunofixation of serum proteins: state of knowledge and systematic review

September 1, 2025

Sacha Pelletier, Laetitia Florent, Philippe Gillery, Jean-Baptiste Oudart

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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.
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Fecal leukocyte esterase levels predict endoscopic severity as an alternative biomarker in inflammatory bowel disease

September 1, 2025

Feng-Pai Tsai, Meng-Tzu Weng, Hsin-Yun Wu, Zhi-Che Chen, Chien-Chih Tung, Chun-Ying Wang, Shu-Chen Wei

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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.
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Failure Mode and Effects Analysis (FMEA) integrating quality indicators for risk assessment of the total testing process in human papillomavirus genotyping testing: a proactive risk analysis model for molecular diagnostics

August 29, 2025

Tingting Li, Yuting He, Yuanhao Chen, Shunwang Cao, Yi Wang, Chunmin Kang, Hongmei Wang, Cheng Zhang, Chang Wen, Peifeng Ke

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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.
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Association of Clinical Biochemists in Ireland Annual Conference

August 29, 2025

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Are vitamins A and E results truly traceable and clinically useful? A practical and critical inquiry

August 28, 2025

Raúl Rigo-Bonnin, Estel Julià-Quiñones, Anna Escalante-Vilanova, Irene Aliart-Fernández

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Open Access
Quality assurance using patient split samples: recommendations for primary healthcare laboratories

August 26, 2025

Anne Stavelin, Eva Rønneseth, Anne Lise Fossum, Stein Binder, Camilla Aker, Maria Kim Nguyen, Sverre Sandberg

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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.
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Biological variation of serum Golgi protein 73 concentrations

August 26, 2025

Anna Stefanska, Joanna Siodmiak, Katarzyna Bergmann, Łukasz Szternel, Aleksandra Wolska, Aneta Klarkowska, Judyta Koneszko, Magdalena Krintus, Mauro Panteghini

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Age distorts the interpretation of FIB-4

August 26, 2025

Arne Åsberg, Gunhild Garmo Hov, Lena Løfblad

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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.
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An innovative immunoassay for accurate aldosterone quantification: overcoming low-level inaccuracy and renal dysfunction-associated interference

August 25, 2025

Kaijuan Wang, Hongying Cong, Zhangwei Gao, Xiaojing Gao, Wei Zhang, Xiaocui Shi, Zhou Zhou

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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.
Open Access
Method comparison of diabetes mellitus associated autoantibodies in serum specimens

August 25, 2025

Alexander Lenard, Andreas Rapp, Steffen Busch, Peter Findeisen

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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.
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Validation of a plasma GFAP immunoassay and establishment of age-related reference values: bridging analytical performance and routine implementation

August 25, 2025

Burak Arslan, Ulf Andreasson, Elzbieta Rembeza, Markus Axelsson, Lenka Novakova, Bjørn-Eivind Kirsebom, Tormod Fladby, Anna Dittrich, Silke Kern, Ingmar Skoog, Kaj Blennow, Henrik Zetterberg, Hlin Kvartsberg

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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.
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An automatic chemiluminescence immunoassay for a novel biomarker NT-IGFBP-4: analytical performance and clinical relevance in heart failure

August 25, 2025

Shuzheng Cao, Jing Wang, Ling Li, Yu Wu, Juan Yang, Litao Zhang, Xin Shu, Hui Wang, Yisha Jing, Yi Zhang, Zhenlu Zhang

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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.
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Toxic beauty: acute kidney injury triggered by hair-straightening treatment

August 22, 2025

Oscar D. Pons-Belda, Paloma Livianos-Arias-Camison, Santiago Mariño-Lopez, Jorvi J. Aguilar-Valero, Cassandra E. Puig-Hooper

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Improvement in the turnaround time of PTH(1–84) as part of the intraoperative PTH monitoring for parathyroidectomy

August 22, 2025

Beverly Buffart, Dragos Barglazan

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Multivariate approaches to improve the interpretation of laboratory data

August 22, 2025

Mario Plebani

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Rethinking the use of “one-way ANOVA” in CLSI EP15-A3 – a call for terminological precision and methodological clarity

August 21, 2025

Chaochao Ma, Ling Qiu

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From assessment to action: experience from a quality improvement initiative integrating indicator evaluation and adverse event analysis in a clinical laboratory

August 21, 2025

Peirong Chen, Xing Chen, Yuanzhi Xia, Xiaoliang Liang, Qiuyi Hong

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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.
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Estimation of measurement uncertainty for free drug concentrations using ultrafiltration

August 21, 2025

Raúl Rigo-Bonnin, Virgínia Mas-Bosch, Francesca Canalias

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Evaluation of measurement uncertainty of 11 serum proteins measured by immunoturbidimetric methods according to ISO/TS 20914: a 1-year laboratory data analysis

August 21, 2025

Emine Feyza Yurt, Medine Alpdemir, Mehmet Şeneş

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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.
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Evaluating the performance of a multiparametric IgA assay for celiac disease diagnosis

August 20, 2025

Caterina Maria Gambino, Luisa Agnello, Fabio Del Ben, Anna Maria Ciaccio, Salvatore Milano, Roberta Vassallo, Francesco Cacciabaudo, Aurelio Seidita, Pasquale Mansueto, Antonio Carroccio, Marcello Ciaccio

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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.
Open Access
Urgent call to the European Commission to simplify and contextualize IVDR Article 5.5 for tailored and precision diagnostics

August 20, 2025

Christa Cobbaert, Christian Schweiger, Christoph Buchta, Thomas Streichert, Florent Vanstapel, Francois Mullier, Lucas Biaggini, Ettore Capoluongo, Patrick Bossuyt, Harjit Pal Bhattoa, Tom Melvin, Michael Neumaier, On behalf of the EFLM Committee on European Regulatory Affairs (EFLM C-ERA) and endorsed by the DGKL Board

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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.
Open Access
Clinical significance of anti-mitochondrial antibodies and PBC-specific anti-nuclear antibodies in evaluating atypical primary biliary cholangitis with normal alkaline phosphatase levels

August 20, 2025

Shasha Li, Kai Zhang, Hongli Liu, Miaoyang Chen, Jialuo Wang, Yuan Yang, Yuxiang Gong, Xing Liu, Yongfeng Yang

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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.
Open Access
Verification of the T50 Calciprotein Crystallization test: bias estimation and interferences

August 20, 2025

Joyce Y. Xu, Didier Falconnet, Tarah van den Berkmortel, Sandra de la Rosa, Vincent Linder, Robert de Jonge, Andreas Pasch, Marc G. Vervloet, Henrike M. Hamer

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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.
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Assessing the harmonization of current total vitamin B12 measurement methods: relevance and implications

August 20, 2025

Bruno Mario Cesana, Simona da Molin, Nuthar Jassam, Julian H. Barth, Sabrina Buoro, Martina Tosi, Gianvincenzo Zuccotti, Santica Marcovina, Simona Ferraro

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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.
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A multiplex allele specific PCR capillary electrophoresis (mASPCR-CE) assay for simultaneously analysis of SMN1/SMN2/NAIP copy number and SMN1 loss-of-function variants

August 19, 2025

Yunli Lai, Xu Yang, Shijie Wei, Yajun Chen, Yanjun Cai, Wenyu Wang, Zeyan Zhong, Xuexi Yang, Wanjun Zhou

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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.
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Kidney stones consisting of 1-methyluric acid

August 19, 2025

Søren Risom Kristensen, Kim Hovgaard Andreassen, Lene Hyldgaard Bigum, Charlotte H Graugaard-Jensen, Jørgen Bjerggaard Jensen, Mette Thrane Øhrstrøm, Claus Gyrup Nielsen

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Reference intervals in value-based laboratory medicine: a shift from single-point measurements to metabolic variation-based models

August 18, 2025

Abdurrahman Coskun, Mario Plebani

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Laboratory data can be meaningful only when compared with reliable reference data; therefore, the estimation of reliable reference data is just as important as the accurate measurement of measurands in patient samples. Since analyte concentrations in the human body are influenced by both random variations (such as biological fluctuations) and systematic variations (such as physiological rhythms and age-related changes), the conventional model for estimating reference data – based solely on the statistical distribution of single-sample measurements from reference individuals – may not provide sufficiently reliable information for interpreting patient results. Therefore, a paradigm shift from relying solely on single-sample measurement distributions to incorporating metabolic changes observed in the human body when estimating reference intervals may enhance the clinical value of laboratory data for an effective clinical decision making and patient care. This opinion paper aims to summarize how to facilitate this transition and to identify the most suitable model for estimating reference intervals that reflect underlying metabolic dynamics.
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Evaluation of stability and potential interference on the α-thalassaemia early eluting peak and immunochromatographic strip test for α-thalassaemia --^SEA carrier screening

August 18, 2025

Wing Kit Lam, Christina Pui Ying Fan, Winnie Yim Fong Law, Tsz Fung Wong, Darcy Lok Han Too, Lok Nga Ko, Anskar Yu Hung Leung, Sze Fai Yip

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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.
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From metabolic profiles to clinical interpretation: multivariate approaches to population-based and personalized reference intervals and reference change values

August 15, 2025

Abdurrahman Coskun, Jasmin Weninger, Ali Canbay, Mustafa Kemal Özçürümez

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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.
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The importance of laboratory medicine in the management of CKD-MBD: insights from the KDIGO 2023 controversies conference

August 15, 2025

Samuel D. Vasikaran, Konstantinos Makris, Harjit Pal Bhattoa, Tomáš Zima, Pierre Delanaye, Pieter Evenepoel, Hanne Skou Jørgensen, Mathias Haarhaus, Eugene McCloskey, Giovanni Lombardi, Etienne Cavalier, On behalf of the IFCC Committee on Bone Metabolism and EFLM Committee on Chronic Kidney Disease

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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.
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Analytical and clinical evaluation of an automated high-sensitivity cardiac troponin I assay for whole blood

August 15, 2025

Jin-xing Yu, Kai Cui, Yang Li, Fang-fang Yang, Fen Xu, Zhou Zhou, Ya-hui Lin

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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.
Open Access
Evaluating large language models as clinical laboratory test recommenders in primary and emergency care: a crucial step in clinical decision making

August 14, 2025

Ahmed Medhat Zayed, Glynis Frans, Nicolas Delvaux

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Objectives Large language models (LLMs), such as OpenAI’s GPT-4o, have demonstrated considerable promise in transforming clinical decision support systems. In this study, we focused on a single but crucial task of clinical decision-making: laboratory test ordering. Methods We evaluated the self-consistency and performance of GPT-4o as a laboratory test recommender for 15 simulated clinical cases of different complexities across primary and emergency care settings. Through two prompting strategies – zero-shot and chain-of-thought – the model’s recommendations were evaluated against expert consensus-derived gold-standard laboratory test orders categorized into essential and conditional test orders. Results We found that GPT-4o exhibited high self-consistency across repeated prompts, surpassing the consistency observed among individual expert orders in the earliest round of consensus. Precision was moderate to high for both prompting strategies (68–82 %), although relatively lower recall (41–51 %) highlighted a risk of underutilization. A detailed analysis of false negatives (FNs) and false positives (FPs) could explain some gaps in recall and precision. Notably, variability in recommendations centered primarily on conditional tests, reflecting the broader diagnostic uncertainty that can arise in diverse clinical contexts. Our analysis revealed that neither prompting strategy, case complexity, nor clinical context significantly affected GPT-4o′s performance. Conclusions This work underscores the promise of LLMs in optimizing laboratory test ordering while identifying gaps for enhancing their alignment with clinical practice. Future research should focus on real-world implementation, integrating clinician feedback, and ensuring alignment with local test menus and guidelines to improve both performance and trust in LLM-driven clinical decision support.
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Comparative analysis of population-based and personalized reference intervals for biochemical markers in peri-menopausal women: population from the PALM cohort study

August 14, 2025

Jiaming Wu, Penghui Feng, Jinming Zhang, Xingtong Chen, Rong Chen, Min Luo, Falin He

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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.
Open Access
Recent decline in patient serum folate test levels using Roche Diagnostics Folate III assay

August 14, 2025

Anders Larsson, Johan Saldeen, Frida Duell

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The current status of serum insulin measurements and the need for standardization

August 14, 2025

Curt Rohlfing, Gregory Petroski, Maya Hatten-Beck, Steven Hanson, Andrew N. Hoofnagle, Randie R. Little, Kuanysh Kabytaev

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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.
Open Access
Hemolysis detection using the GEM 7000 at the point of care in a pediatric hospital setting: does it affect outcomes?

August 13, 2025

Ridwan B. Ibrahim, Nazmin Bithi, Charlene Hernandez, Hanna Uhrova, Brandy D. Recio, Sridevi Devaraj

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Overview of laboratory diagnostics for immediate management of patients presenting to the emergency department with acute bleeding

August 13, 2025

Giuseppe Lippi, François Mullier, Henri Thonon, Emmanuel J. Favaloro

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Acute, life-threatening bleeding is a relatively common but critical presentation in the emergency department (ED), needing immediate assessment and intervention to reduce morbidity and mortality. Rapid identification of the bleeding source, evaluation of hemostatic function, and timely initiation of resuscitation are essential components of early management. Laboratory diagnostics plays a central role in this process, enabling clinicians to stratify the risk, guide therapeutic decisions, and predict outcomes. This opinion paper summarizes current evidence supporting the use of a core panel of laboratory tests in the initial evaluation of patients with emergency bleeding admitted to the ED. The leading characteristics of these initial tests encompass elevated diagnostic sensitivity, high precision and reproducibility, broad analytical measurement range, minimal turnaround time, low sample volume requirements, continuous availability, accessible measurement uncertainty, and proven clinical impact. The hypothetical core tests may include hemoglobin, blood lactate, prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, platelet count, viscoelastic assays, specific tests for measuring direct oral anticoagulants (DOACs), cardiac troponins and other organ-specific tests in patients with signs and symptoms of hypovolemic shock-induced organ failure. We believe that early implementation of a targeted, evidence-based initial laboratory diagnostic strategy in patients presenting to the ED with severely acute bleeding may support more effective resuscitation and transfusion protocols, reduce unnecessary interventions, and improve clinical outcomes.
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Biomarkers in body fluids and their detection techniques for human intestinal permeability assessment

August 13, 2025

Jian Zhang, Tianqi Wang, Xuanjiao Mao, Xinyu Hu, Li Lv, Haochen Qi, Lei Zheng

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Introduction Intestinal permeability (IP) is a crucial index for intestinal tract integrity, reflecting intestinal barrier function, injury, diseases, and related systemic conditions. However, current mainstream testing methods are invasive and qualitative. Biomarkers for IP in body fluids offer noninvasive and quantitative advantages for IP assessment while pose challenges for high-performance detection techniques. Content This review introduces the main types of biomarkers for IP, including sugar probes, endotoxin, D-lactic acid, intestinal fatty acid-binding protein, diamine oxidase, citrulline, zonulin and lipopolysaccharide-binding protein. Their sources, associated diseases and detection techniques are summarized, and the regulation of intestinal ecosystem and IP by diet are also briefly discussed. Summary By investigating existing studies, various biomarkers in body fluids and their detection techniques are introduced, and the effectiveness and realizability of IP assessment with body fluid markers are demonstrated. This review can be as a reference for gastroenterologists, laboratory technicists and researchers. Outlook Body fluid biomarker detection provides a promising proach for IP in vitro assessment. To improve the feasibility in clinics, more types of biomarkers, more accurate physiological and pathological mechanisms, and more innovative sensing technologies are expected to be explored and developed. It is foreseeable that fast, precise, and on-site IP assessment will contribute to the clinical diagnosis and monitoring of intestinal and secondary diseases.
Open Access
Cryoglobulin pre-analysis over the weekend

August 13, 2025

Corrie de Kat Angelino, Chantal P. Rovers, Joannes F.M. Jacobs

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Supplementation of pyridoxal-5′-phosphate in aminotransferase reagents: a matter of patient safety

August 12, 2025

Mauro Panteghini

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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.
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Reference ranges for ionized calcium in plasma in Danish children aged 0 days to 3 years using laboratory registry data

August 12, 2025

Anne-Sofie Allermann Faarvang, Thomas S. Rosengren, Lars E. Pedersen, Pia B. Larsen, Esther A. Jensen

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Objectives Measurement of ionized calcium is frequently used in the assessment of calcium metabolism. However, the utility of ionized calcium in young children can pose a challenge. In this study, we aimed at establishing novel accurate reference ranges for ionized calcium in plasma in a pediatric population. Methods All children aged 0–3 years who had ionized calcium measured in Region Zealand from 2012 to 2023 were included. Exclusion criteria included samples from intensive care units or nephrology departments and samples with pH under 7.2 or over 7.6. If a patient had repeated samples, only the last measurement per month for each child was used. In total, 20,516 ionized calcium measurements from 16,039 children were included. Based on these measurements, we established both pH corrected and non-corrected data sets. Results Data are described as median with 2.5th and 97.5th fractiles. Reference ranges were divided into age groups. We observed variation in reference ranges, especially in the first 10 days of life: 1.14–1.46 (pH corrected 1.13–1.43) mmol/L on day zero rising to 1.29–1.59 (pH corrected 1.27–1.55) mmol/L on day eight. After reaching peak levels on day eight, ionized calcium gradually decreased to 1.16–1.36 (pH corrected 1.17–1.35) mmol/L at 18–24 months of age. Conclusions We present novel reference ranges for ionized calcium in plasma. Results indicate a physiological rise in plasma ionized calcium after birth. After eight days, a steady decrease in plasma levels was seen before stabilizing at 18–24 months of age.
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Challenges of using natriuretic peptides to screen for the risk of developing heart failure in patients with diabetes: a report from the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Committee on Clinical Applications of Cardiac Bio-Markers (C-CB)

August 12, 2025

Allan S. Jaffe, Yader Sandoval, Nicholas L. Mills, Torbjørn Omland, Kristin M. Aakre

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Background Several guidelines groups have made recommendations about how to use natriuretic peptides (NPs) to screen patients with diabetes for incipient heart failure. This group is at risk for undetected and stage B heart failure, where structural heart disease is present despite the absence of clinical signs and symptoms. These recommendations are based on trial data that suggest there are therapeutic options to benefit these patients. Content These guidelines do not adequately account for the marked analytic differences between NP assays nor their high degree of biological and analytical variability. Thus, without additional data and guidance, these recommendations could lead to excessive testing for some groups but might disadvantage others. In addition, these issues could lead to difficulties in the interpretation of values. Accordingly, the Committee on Clinical Applications of Cardiac Bio Markers of the International Federation of Clinical Chemistry has developed a consensus educational document to describe these difficulties and define areas where additional data are needed. Clinicians should be cognizant of differences in the assays for NPs, the high degree of variability of values, differences in sex and ethnicity and the need to factor in a variety of clinical and treatment variables in interpreting NP values. Summary These suggestions for diabetes eventually will include others at high risk. They will require close attention to the issues involved in measuring and interpreting NP values.
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HCV serology: an unfinished agenda

August 11, 2025

Claudio Galli, Mario Plebani

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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.
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A novel corrective model based on red blood cells indices and haemolysis index enables accurate unhaemolysed potassium determination in haemolysed samples – Hemokalc project

August 8, 2025

Charles R. Lefèvre, Bérénice Vigier, Mathilde Favalelli, Jordan Garnier, Alexandre Scanff, Maxime Pawlowski, Nicolas Collet, Claude Bendavid

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Objectives Haemolysis is a major preanalytical issue that affects potassium measurements, often leading to sample rejection and delayed clinical management. This study proposes a novel corrective model for accurate unhaemolysed potassium prediction. Methods Blood samples from 14 healthy volunteers were used to prepare a range of haemolysates via freeze-thaw method. First, the relationship between potassium variation and haemolysis variation (ΔK/ΔHI) was studied both individually and globally to assess inter-individual variability. Then, to achieve a more personalised unhaemolysed potassium prediction, a novel corrective model was developed based on: potassium levels in paired unhaemolysed and gradually haemolysed samples, measured haemolysis index, mean corpuscular haemoglobin concentration, mean corpuscular volume and intraerythrocytic potassium level. The bias between true and model-predicted unhaemolysed potassium values was calculated and compared to the reference change value (RCV%). Results Global data showed a strong correlation between ΔK and ΔHI (Pearson r=0.97, p<0.0001), following a linear relationship: ΔK=0.33*ΔHI (p<0.0001). However, individual data revealed substantial inter-individual variation (min ΔK=0.23*ΔHI and max ΔK=0.39*ΔHI). The correction model achieved 100 % accuracy for the 116 prepared samples, with predicted unhaemolysed potassium values falling within a ± 10 % bias range (mean ± standard deviation of bias = −0.5 ± 2.8 %). Conclusions We propose a novel, reliable, and cost-effective corrective model to predict unhaemolysed potassium from haemolysed samples. Compared with previously published models, the integration of red blood cells indices allows for a more personalised, patient-centred approach with high efficiency.
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Clinical validation of a liquid chromatography single quadrupole mass spectrometry (LC-MS) method using Waters Kairos™ Amino Acid Kit reagents

August 8, 2025

Rowan Hellier, Luke Griffiths, Chandra Sundas, Annabel Rodham, Stuart J. Moat

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Objectives Quantitation of plasma amino acids (AA) is critical for the diagnosis and monitoring of inherited disorders of AA metabolism. AA analysis using ion-exchange chromatography (IEC) with post-column ninhydrin derivatization is time consuming, with run times of ∼2 h, limiting sample throughput. Liquid chromatography mass-spectrometry can potentially address some of the current challenges. Methods Performance of components of the Waters Kairos Amino Acid Kit using liquid chromatography single quadrupole mass-spectrometry (LC-MS) following derivatization of samples with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ•Tag™ Ultra Derivatization Reagent) was evaluated. Results were compared with the Biochrom-IEC method using patient specimens (n=115), ClinChek ® control and external quality assessment (EQA) material. Results The kit reagents and our developed method had a 19-min analysis time, demonstrated acceptable inter-assay imprecision (CV<10 %) and bias vs. IEC-method (overall mean bias <2 %). Excellent correlation (concordance correlation coefficient (CCC) >0.99) with IEC was demonstrated for 10/23 analytes, good correlation (CCC >0.95) for 10/23, with the remaining three amino acids (aspartate, histidine and tryptophan) demonstrating moderate concordance (CCC ≥0.90 but <0.95). 1/23 AAs had a mean bias >10 % using EQA material. The method demonstrated a lower limit of quantitation of ≤2.5 μmol/L for all AA, making this assay suitable for CSF analysis. Calibration stability bias was <5 % over 12-weeks. Derivatized AAs were stable for ≤17 days. The analytical column supplied demonstrated good retention time stability (<0.4 %) and was capable of >2000 injections. Conclusions The tested methodology demonstrated good analytical performance and correlation with IEC. This approach confers practical advantages over IEC, including analytical selectivity and workflow time efficiency.
Open Access
Oral salt loading combined with postural stimulation tests for confirming and subtyping primary aldosteronism

August 8, 2025

Jenny Yeuk Ki Cheng, Wai Shan Clara Lo, Teresa Kam Chi Tsui, Wing Kar Edith Chow, Kitty Kit Ting Cheung, Ronald Ching Wan Ma, Risa Ozaki, Chung Shun Ho

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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.
Open Access
Total laboratory automation: fit for its intended purposes?

August 8, 2025

Mario Plebani

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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.
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Establishment of region-, age- and sex-specific reference intervals for aldosterone and renin with sandwich chemiluminescence immunoassays

August 8, 2025

Lin-Lin Cao, Bing Gu, Liqiong Yao, Gang Li, Chunling Wang, Huijun Cui, Ran Xu, Hui Wang

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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.
Open Access
Advancing diagnostic stewardship through claims-based utilization analysis: toward a system-wide vision of diagnostic excellence

August 8, 2025

Matthew L. Rubinstein, Mario Plebani

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Targeting low-value laboratory care

August 7, 2025

Michael Vogeser

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Investigation of the possible cause of over-estimation of human aldosterone in plasma, using a unique, non-synthetic human aldosterone-free matrix

August 1, 2025

Sheree Stoner, Sarah Mogambi, Lok Ko, Isabelle Ryan, Karen Young, Tamantha Harrower, Lee Price, Tony Badrick, Peter E. Hickman, Michael Stowasser, Greg Ward

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Objectives Aldosterone is over-estimated by direct immunoassay when compared to assay by LC-MS/MS. We have investigated the mechanism for this over-estimation, using dichloromethane (DCM) extraction and a unique matrix of non-synthetic aldosterone-free plasma (ALFP). Methods Samples used were from patients with normal estimated Glomerular Filtration Rate (eGFR). All samples were measured by direct DiaSorin immunoassay, followed by extraction using dichloromethane (DCM) and LC-MS/MS. Post-DCM samples were reconstituted in DiaSorin Liaison Endocrinology Diluent (DED), which is effectively a human serum albumin matrix, or in a unique matrix of non-synthetic human aldosterone-free plasma (ALFP). Patient plasma samples were selected to cover a wide range of aldosterone concentrations. Results Aldosterone direct immunoassay over-estimated aldosterone concentration in all samples, compared to LC-MS/MS. Post-DCM extraction and reconstitution with DED, aldosterone results matched well with LC-MS/MS results. Reconstitution with our unique ALFP plasma matrix rather than the DED, resulted in an over-estimation of aldosterone by approximately 50 pmol/L in all samples. Conclusions In this investigation, we have demonstrated that the most likely source of interference in the direct DiaSorin aldosterone immunoassay is an endogenous protein commonly found in patient samples. This protein probably interferes with the immunoassay by binding to the tracer.
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Is successfulness of platelet clump disaggregation by vortexing influenced by platelet measurement methods?

July 31, 2025

Vanja Radišić Biljak, Brankica Šimac, Branka Krešić, Ana Nikler

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Open Access
Response to the editorial by Karl Lackner

July 30, 2025

Marith van Schrojenstein Lantman, Marc Thelen

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Accelerating time from result to clinical action: impact of an automated critical results reporting system

July 29, 2025

Elisa M. Aponte, Amos J. Shemesh, Taylor Kalomeris, Bulent Oral, Stanley Diaz, Yaxin Li, He S. Yang, Melissa M. Cushing, Zhen Zhao

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Robustness of steroidomics-based machine learning for diagnosis of primary aldosteronism: a laboratory medicine perspective

July 25, 2025

Graeme Eisenhofer, Mirko Peitzsch, Kevin Mantik, Manuel Schulze, Georgiana Constantinescu, Zhong Lu, Hanna Remde, Carmina T. Fuss, Tracy Ann Williams, Sven Gruber, Jacques W. M. Lenders, Andrea Horvath, Christina Pamporaki

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Objectives Use of machine learning (ML) in diagnostics offers promise to optimise interpretation of laboratory data and guide clinical decision-making. For this, ML-based outputs should provide robustly reproducible results at least as good as the underlying laboratory data. The objective of this study was to assess robustness of ML-based steroid-probability-scores for diagnosis of primary aldosteronism (PA). Methods Reproducibility of ML-based steroid-probability-scores was assessed from coefficients of variation (CVs) for pools of quality control plasma from selected groups of patients with and without PA. Intra-patient measurement variability was assessed from CVs of three consecutive plasma specimens obtained on different days from 77 patients. Inter-laboratory reproducibility was assessed from 47 duplicate plasma specimens analysed in two different laboratories. Results Support vector machine-derived steroid-probability-scores for diagnosis of PA for seven sets of quality control plasma pools yielded an averaged CV (2.5 % CI 0.4–4.4 %) that was lower (p=0.0078) than the averaged CV for seven steroids employed in that model (12.0 % CI 7.4–16.6). Using three sets of plasma samples from 77 patients, CVs for intra-patient measurement variability of steroid-probability-scores were 7 % (CI 5–9 %) and lower (p<0.0001) than CVs for measurements of aldosterone (38 % CI 32–42 %), 18-oxocortisol (36 % CI 29–43 %), 18-hydroxycortisol (25 % CI 21–28 %) and the aldosterone:renin ratio (46 % CI 38–55 %). ML-derived probability scores for 47 duplicate plasma samples analysed at two separate laboratories displayed excellent agreement and negligible bias. Conclusions ML-based steroid-probability-scores for diagnosis of PA display remarkably high robustness according to reproducibility of measurements within and between laboratories as well as within patients.
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A step forward in pediatric hemophagocytic lymphohistiocytosis and autoimmune disease: pediatric reference interval for serum soluble IL-2 receptor and soluble CD163

July 25, 2025

Victor Bruun, Tina Parkner, Anne Winther-Larsen, Mia Elbek Schjørring, Holger Jon Møller, Cindy Soendersoe Knudsen

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Objectives Hemophagocytic lymphohistiocytosis (HLH) is a severe condition requiring timely diagnosis to initiate immunomodulatory therapy. Soluble interleukin-2 receptor (sIL-2R) and soluble CD163 (sCD163) are biomarkers crucial for making this diagnosis. This study aims to establish pediatric reference intervals (RIs) for serum concentrations of sIL-2R and sCD163, and to evaluate the pre-analytic stability of sIL-2R. Methods Serum samples from 243 healthy children (aged 0–18 years) were analyzed to determine RIs for sIL-2R and sCD163. Samples were processed according to stringent pre-analytic protocols, with additional stability studies evaluating sIL-2R under various conditions, including temperature fluctuations and freeze-thaw cycles. The concentration of sCD163 was additionally determined in a protein reference material. RIs were calculated using non-parametric quantile regression, and subgroups were analyzed for potential partitioning by age, sex, and inflammation status (C-reactive protein (CRP) >8 mg/L). Results Discrete and continuous RIs were established for serum sIL-2R and sCD163 in healthy children, with distinct age-dependent variations observed for sIL-2R. Stability studies confirmed sIL-2R’s robustness under common pre-analytic conditions. Results were consistent across samples obtained from general practitioners and hospital clinics, enhancing their transferability. Conclusions This study provides comprehensive pediatric RIs for serum sIL-2R and sCD163 to support clinical decision-making in HLH and related inflammatory disorders. The stability of both sIL-2R and sCD163 under diverse conditions supports their reliable use in clinical settings. These findings may facilitate broader clinical implementation, pending on local validation and standardization.
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MAGLUMI^® Tacrolimus (CLIA) assay: analytical performances and comparison with LC-MS/MS and ARCHITECT Tacrolimus (CMIA) assay

July 16, 2025

Mingpeng Fu, Shanchun Chen, Xianliang Zheng, Xuehong Li, Honggao Sun, Jin Chen, Hua Pei

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Objectives Tacrolimus has been a cornerstone of immunosuppressive therapy over the past two decades. Due to its narrow therapeutic window and pharmacokinetic variability, drug monitoring is vital for enhancing the efficacy and safety during therapy. In the present study, we evaluated the analytical performances of the MAGLUMI ® Tacrolimus assay based on chemiluminescent immunoassay (CLIA), and compared with LC-MS/MS and the previously validated ARCHITECT Tacrolimus assay based on chemiluminescent microparticle immunoassay (CMIA). Methods We assessed the precision, limit of blank (LoB), limit of quantification (LoQ), limit of detection (LoD) and linearity of the MAGLUMI ® Tacrolimus assay using patient whole blood samples. Interference was assessed by introducing potential interferents into clinical samples. We also analyzed the correlation and agreement with the gold standard method (LC-MS/MS) and another previously validated high-performing ARCHITECT Tacrolimus (CMIA) assay by including 125 whole blood samples from patients and 44 spiked samples. Results MAGLUMI ® Tacrolimus (CLIA) assay exhibits superior precision, as coefficients of variation (CVs) for reproducibility and between-run precision were 0.55–3.63 % and 2.18–5.14 %, respectively. The LoB and LoQ were 0.1 μg/L and 0.5 μg/L. All samples in LoD verification had tacrolimus concentrations above LoB. The assay exhibited excellent linearity ( r =0.99990, 0.5–50 μg/L) with no interference. Additionally, the results of the MAGLUMI ® Tacrolimus (CLIA) assay showed strong correlation and concordance with LC-MS/MS and the CMIA assay. Conclusions The MAGLUMI ® Tacrolimus (CLIA) assay has excellent performance and strong concordance with LC-MS/MS and the ARCHITECT assay, making it a good alternative for tacrolimus measurement.
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Is time a significant factor in the release of potassium from lithium heparin plasma and serum?

July 15, 2025

Astrid Petersmann, Uta Ceglarek, Karen Friederike Gauß, Winfried März, Alexander von Meyer, Matthias Nauck

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Utilization analysis of laboratory tests using health insurance claims data: advancing nationwide diagnostic stewardship monitoring systems

July 15, 2025

Se-eun Koo, Jiyeon Kim, Kuenyoul Park

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Objectives Diagnostic stewardship aims to optimize laboratory testing practices by ensuring appropriate test utilization while minimizing unnecessary testing. However, the nationwide monitoring systems for laboratory test utilization remain underdeveloped. In this study, we analyzed nationwide health insurance claims data to identify inappropriate test utilization patterns and inform diagnostic stewardship interventions. Methods We analyzed the health insurance claims data from the Health Insurance Review and Assessment Service of Korea from January 2018 to December 2023. Three laboratory tests with concerns were evaluated. Inappropriate test utilization was classified based on adherence to recommended methodologies and institutional testing practices. Results The proportion of tuberculosis cultures performed using both liquid and solid media increased from 84.6 % in 2018 to 91.4 % in 2023; however, some institutions continued to perform >90 % of tests using only a single medium. Regional disparities were observed, with nonmetropolitan areas exhibiting higher rates of inappropriate tuberculosis testing. Stool ova and parasite tests showed a persistent reliance on direct smear-only methods. The correlation between endemic parasitic regions and the high proportions of direct smear-only tests suggests an urgent need for improved diagnostic methodologies. Despite longstanding recommendations against their use, bleeding time tests remained frequently ordered. The total number of bleeding time test claims exceeded 400,000 annually, with a limited decline over time. Conclusions Systematic monitoring frameworks based on nationwide claims data can identify inappropriate test utilization and support diagnostic stewardship efforts by informing targeted interventions, thereby optimizing laboratory resource allocation and enhancing patient safety.
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Assessment of 2023 ACR/EULAR antiphospholipid syndrome classification criteria in a Spanish cohort

July 14, 2025

Rodrigo Cantera Estefanía, Rafael Gálvez Sánchez, María Abando Casuso, José Antonio Flores García, Raquel García Ruiz, Irene Gorostidi Álvarez, Héctor Cruz Barquín, María Oviedo Madrid, Marina Herrero López, Ligia Gabriela Gabrie Rodríguez, Juan José Domínguez García, Ariadna García Ascacíbar, José Luis Hernández Hernández, Lucrecia Yáñez San Segundo, Belén González-Mesones Galán, María Luisa González Ponte, Marcos López Hoyos, Gala Aglaia Méndez Navarro, Sara del Barrio Longarela, Víctor Manuel Martínez Taboada

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Objectives The 2023 ACR/EULAR criteria aim to improve the classification of antiphospholipid syndrome (APS). This study aims to validate these criteria and compare clinical and laboratory domains between classified and non-classified patients. Methods A retrospective cohort study was conducted at the Hospital Universitario Marqués de Valdecilla, a tertiary referral center in Cantabria, Spain. Data were retrieved from the laboratory information system, identifying patients with at least one positive determination of lupus anticoagulant (LA), anticardiolipin (aCL), or anti-β2 glycoprotein I (anti-β2GPI) between January 2018 and March 2024. Patients were classified according to the 2006 Sydney and 2023 ACR/EULAR criteria. Results Among 375 patients meeting the Sydney criteria, 152 (40.6 %) fulfilled the 2023 ACR/EULAR criteria. The sensitivity and specificity of the 2023 ACR/EULAR criteria were 30.2 and 97.7 % respectively, with a positive predictive value of 0.84, and a negative predictive value of 0.77. Area under the ROC curve was 0.639 (95 % CI: 0.605–0.673). The exclusion of thrombosis with high-risk thrombotic profiles and recurrent pregnancy loss or fetal death as individual events on obstetric APS accounted for most declassified patients. Additionally, patients meeting the 2023 ACR/EULAR criteria had a higher prevalence of arterial thrombosis without cardiovascular risk factors. In laboratory domains, isolated IgM aPL positivity was a major exclusion factor due to its lower weight in the new criteria. Conclusions The 2023 ACR/EULAR criteria enhance specificity but significantly reduce sensitivity, excluding many APS patients, particularly those with obstetric APS or IgM aPL. This raises concerns about clinical trial eligibility and applicability in diverse populations.
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What matters most: an age-friendly approach to pathology and laboratory medicine

July 14, 2025

Melody Boudreaux Nelson, Candice Coffey

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Under the Age-Friendly Health System initiative, “What Matters” is defined as knowing the older adult’s specific health outcome goals and care preferences. This involves multiple settings of care (e.g., hospital, skilled nursing facility, outpatient visits) and can include end-of-life care. However, the establishment of testing frequency criterion and the development of wide-scale diagnostic algorithms are often left undefined in older adults with poor prognosis and/or shortened life expectancy. Thus, multidisciplinary development of Geriatric 5M-informed optimization plans at the institution level and quality improvement strategies within the laboratory community may further the successful implementation of age-friendly efforts. While patients, end-users, and systems can attribute to implementation barriers, the development of an evidence-base wherein laboratory expertise is directly associated with patient outcomes is vital. Thus, a concentrated, cooperative age-friendly approach centered on What Matters may present a sustainable strategy for early transformation. Future research centered on piloted interventions on the laboratory’s role in older adult care and end of life diagnostic management is needed.
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Performance of afternoon (16:00 h) serum cortisol for the diagnosis of Cushing’s syndrome

July 11, 2025

Hippolyte Dupuis, Emilie Merlen, Julien Elices-Diez, Pierre Balayé, Christine Cortet, Arnaud Jannin, Christine Do Cao, Claire Douillard, Benoit Soulez, Nassima Ramdane, Benoit Soudan, Marie-Christine Vantyghem, Stéphanie Espiard

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Introduction Late-night salivary cortisol (LNSC), which assesses the loss of the circadian rhythm of cortisol, is one of the first-line tests performed to diagnose Cushing’s syndrome (CS). Unfortunately, access to LNSC is still limited in some institutions. Alternatively, midnight serum cortisol can be measured, often combined with additional cortisol sampling over 24 h. This study investigates the performance of afternoon serum cortisol (F16h) for the positive diagnosis of hypercortisolism. Methods Retrospective study including consecutive patients evaluated for suspicion of CS by at least two tests among urine-free cortisol (UFC), overnight 1-mg dexamethasone suppression test (DST), and midnight serum cortisol (F00h). Patients assessed for adrenal incidentalomas or Cushing’s disease recurrence were excluded. Results Of the 589 included patients (41.3 % women, mean age 50.7 ± 16.3 years), 49 (8.3 %) were diagnosed with CS. F16h demonstrated significant correlations with DST, UFC, and F00h (r=0.24, p<0.001, r=0.41, p<0.001 and r=0.42, p<0.001, respectively). The optimal cut-off of the F16h was 218 nmol/L, achieving 83.7 % sensitivity, 67.4 % specificity, and a 97.8 % negative predictive value. The area under the ROC curve (AUC) for the F16h did not differ from UFC (0.83 vs. 0.79, p=0.3), yet its sensitivity was higher using the optimal diagnostic threshold. The AUC for the F16h was significantly lower than that for F00h and DST. Conclusions Given the limited diagnostic accuracy of both F16h and UFC, particularly in mild to moderate CS, efforts should prioritize expanding access to reliable LNSC assays for circadian rhythm assessment. Meanwhile, F16h may help rule out CS when LNSC is unavailable.
Open Access
Acoustophoresis-based blood sampling and plasma separation for potentially minimizing sampling-related blood loss

July 7, 2025

Amal Nath, Sara Marie Larsson, Andreas Lenshof, Wei Qiu, Thierry Baasch, Linda Nilsson, Magnus Gram, David Ley, Thomas Laurell

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Objectives Frequent blood sampling in vulnerable patient groups, such as prematurely born infants, can lead to significant blood loss and increased transfusion needs. Current pre-analytical technology requires comparably large blood volumes and leads to discarding of cells. This study investigates a device prototype enabling in-line sampling where cell-reduced plasma for clinical chemistry analyses is generated through acoustophoresis. Methods Blood samples were collected from healthy adult donors in lithium-heparin tubes without gel. Plasma separated via acoustophoresis was compared with centrifuged plasma (2000 g × 10 min) for cell counts (n=14), cell-free hemoglobin (n=21), and 12 routine clinical chemistry analyte tests (n=21). Wilcoxon signed-rank tests and Bland Altman analysis were used for statistical comparison. Results Both acoustophoresis (AF) and centrifugation (CEN) generated cell-reduced plasma with<0.01 % of cells remaining after separation. However, compared to CEN plasma, more cells (median count per μL 642 vs. 205, p<0.01) and platelets (median count per μL 20,477 vs. 1,537, p<0.0001) remained in AF plasma. Cell-free hemoglobin (fHb) in AF plasma samples (range 0.0–0.2 g/L) was lower (p<0.01) than in CEN plasma samples (range 0.1–0.3 g/L). Statistically significant relative mean differences in test results ranging from 0.84 % (95 % CI 0.48–1.19) for sodium to 10.50 % (95 % CI 5.02–15.99) for AST were found. Conclusions This proof-of-concept study demonstrates that acoustophoresis has the potential to produce sufficiently cell-free plasma for several commonly performed clinical chemistry analyses. Further studies should assess pathological samples, platelet activation, and improve the design for more efficient removal of platelets.
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External quality assessment in resource-constrained laboratories: a survey of practices and perceptions in Nepal

July 7, 2025

Vivek Pant, Santosh Pradhan, Tony Badrick

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Cellular expression of PD-1, PD-L1 and CTLA-4 in patients with JAK2^V617F mutated myeloproliferative disorders

July 3, 2025

Christoph Winkler, Markus Anliker, Stefan Schmidt, Clemens Feistritzer, Britta Höchsmann, Hubert Schrezenmeier, Anita Siller, Andrea Griesmacher, Lorin Loacker

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Objectives The acquired, somatic JAK2 V617F mutation is the most common molecular aberration in patients with myeloproliferative neoplasms (MPN) and also significantly involved in the regulation of T cell immunity. PD-1, PD-L1 and CTLA-4 are key immune checkpoint regulators that are elevated in patients with solid tumors, infectious diseases and chronic inflammation. We aimed further investigating the significance of immune checkpoint expression in JAK2 V617F positive MPN. Methods The surface expression of PD-L1, PD-1 and CTLA-4 on peripheral blood leukocytes was determined by flow cytometry in 27 patients with JAK2 V617F positive MPN and in a control group of 26 healthy individuals and analyzed by immune checkpoint and leukocyte subpopulation. In addition, the concentration of soluble PD-L1 (sPD-L1) in plasma was examined by ELISA. Results PD-1, PD-L1 and CTLA-4 are significantly overexpressed on the surface of granulocytes in JAK2 positive patients compared to the control group. Soluble PD-L1 (sPD-L1) is elevated in the plasma of JAK2 positive patients and increases with decreased renal function. In CD8+ T-cells and CD4+ T-cells there is a significant negative correlation between PD-1 expression or sPD-L1 concentration and their corresponding cell count. Conclusions Our study shows a significant increase of immune checkpoint regulators on the cellular surface as well as soluble PD-L1 in JAK2 mutated patients compared to healthy individuals. Increased activation of the JAK2/STAT signaling pathway by JAK2 V617F appears to be a mechanism of reduced immune activation in patients with MPN. Immune checkpoint inhibition might therefore represent a potential additional therapeutic target in this disease group.
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Comprehensive evaluation of antiphospholipid antibody testing methodologies in APS diagnosis: performance comparisons across assay systems and clinical subtypes

June 27, 2025

Yun Wang, Xu Yuan, Ting Wang, Wei Wei, Rujia Chen, Renren Ouyang, Feng Wang, Hongyan Hou, Shiji Wu

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Objectives Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by thrombosis and obstetric complications associated with antiphospholipid antibodies (aPLs). This study aimed to compare the diagnostic performance of six commercial assay systems for detecting aCL and aβ2GPI antibodies. Methods Sixty-three APS patients, 50 SLE patients, 67 disease controls, and 62 healthy controls were enrolled. aCL and aβ2GPI antibodies of IgA, IgG, and IgM isotypes were measured using six commercial platforms, including three ELISA-based systems and three CLIA-based systems. Inter-assay concordance was compared across all detection platforms, and ROC curve analysis was performed to evaluate and compare their diagnostic performance in APS. Results Inter-assay concordance varied across platforms, with IgG isotypes showing the highest consistency and IgA exhibiting the lowest agreement. Overall, CLIA-based systems demonstrated superior classification performance compared to ELISA-based methods. The highest area under the curve (AUC) reached 0.811, with sensitivity and specificity up to 0.730 and 0.891, respectively. IgG isotypes demonstrated the best overall performance, while IgA and IgM showed greater variability. The inclusion of IgA modestly improved sensitivity in some systems, although this was sometimes accompanied by decreased specificity. LA-positive patients had higher aPL positivity rates than LA-negative ones, and aPL levels were higher in thrombotic vs. obstetric APS. Conclusions Significant variability exists among commercial aPL detection systems. CLIA-based methods provided better consistency and diagnostic accuracy than ELISA. The inclusion of IgA provided additional diagnostic value in identifying APS patients who tested negative for aCL and aβ2GPI of the IgG and IgM isotypes.
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Oligoclonal banding analysis: assessing plasma use and time interval requirements for paired CSF and blood

June 26, 2025

Lily Olayinka, Yu Chen, Karina Rodriguez-Capote, Jessica L. Gifford, Caitlin Buch, Spencer Weber, Michelle L. Parker, Natalia Volodko, Mathew P. Estey, Dustin Proctor, Ashley Newbigging, Pierre Bordeleau, Maggie Powell, Daniel R. Beriault, Joseph Macri, Victoria Higgins

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Exploring commutable materials for serum folate measurement: challenges in cross-method harmonization

June 26, 2025

Xiaerbanu Nizhamuding, Qian Zhang, Lizi Jin, Rui Wu, Youli Lu, Xilian Yi, Meiwei Zhang, Jiangtao Zhang, Weiyan Zhou, Jie Zeng, Tianjiao Zhang, Chuanbao Zhang

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Objectives Commutability is a critical attribute for reference materials (RMs) employed in standardization and calibration activities, it ensures the accuracy and equivalence of analytical results across heterogeneous measurement systems and laboratories. In the context of folate quantification, commutability holds particular significance, as it validates that RMs exhibit analytical behavior equivalent to native clinical specimens when subjected to diverse folate detection platforms. This study aims to identify and characterize commutable candidate RMs for serum folate assays, with the ultimate goal of enhancing the harmonization and metrological traceability of folate measurements in clinical diagnostics. Methods Thirty study materials and pooled clinical serum samples were measured by two isotope-dilution liquid chromatography tandem mass spectrometry methods and three immunoassays in four different laboratories. Six external quality assessment materials for endocrine, 6 trueness verification materials for vitamins, 13 processed materials and 5 candidate RMs (cRM-1,2,3,4,5) were assessed. Deming regression analysis and the difference-in-bias approach were employed to evaluate commutability. Results cRM-3 demonstrated commutability (C or +) across all assays, being the most ideal candidate reference material in this study. Seven materials (cRM-1, cRM-2, cRM-3, TV202111, TV202012, CS-L, and RS 2W-1) were commutable for two LC-MS/MS methods regardless of the statistical method. Conclusions The complexity of folate species in serum, differences in detection principles between immunoassays and isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method (immunoassays using folate-binding protein with variable affinities and LC-MS/MS quantifying specific vitamers), and variations in calibration substrates among immunoassays collectively challenge the harmonization of folate measurement and commutability assessment.
Open Access
Isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedures for the quantification of total and free phenytoin in human serum and plasma

June 23, 2025

Tobias Schierscher, Linda Salzmann, Neeraj Singh, Manuel Seitz, Janik Wild, Carina Schäfer, Friederike Bauland, Andrea Geistanger, Lorenz Risch, Christian Geletneky, Christoph Seger, Judith Taibon

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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.
Open Access
An isotope dilution-liquid chromatography-tandem mass spectrometry based candidate reference measurement procedure for the simultaneous quantification of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum and plasma

June 20, 2025

Kerstin Kandler, Neeraj Singh, Friederike Bauland, Elie Fux, Andrea Geistanger, Christian Geletneky, Judith Taibon

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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.
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Validation of (self-collected) capillary blood using a topper collection system as alternative for venous sampling for 15 common clinical chemistry analytes

June 2, 2025

Rümeysa Geujar, Dilana Treffers, Maaike Roelofs, Anna van Dongen, Kalpana Ramkisoensing, Rixt Even, Huub H. van Rossum

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Objectives Home blood sampling for clinical purposes has gained much interest. This study validates the recently developed Topper-based capillary blood collection procedure for 15 commonly used chemistry analytes. Methods A total of 120 study participants (21 healthy volunteers and 99 patients) were enrolled. A venous sample was obtained and then participants were asked to self-collect blood by the Topper system (SelfSafeSure Blood Collection Devices). Collected sera were analyzed for 15 common clinical chemistry analytes and the serum indices on a Cobas Pro (Roche) system. Spearman correlations, Passing-Bablok regression analysis, and Bland-Altman difference analysis were performed. Comparability was determined using allowable bias criteria based on biological variation (EFLM database). Results In 113 out of 120 (94 %) self-collections a sample was obtained that allowed for the analysis of at least one analyte. Bland-Altman difference analysis showed that glucose and uncorrected AST did not meet the minimum bias criterion, creatinine and albumin were within the minimum bias criterion and urea and calcium were within the desirable bias criterion. ALP, corrected AST, ALT, total- HDL- and LDL-cholesterol, CRP, GGT, total protein and triglycerides were all within the optimal bias criterion. Conclusions Our study demonstrates that self-collected capillary blood can be used as a reliable alternative to venous sampling for most, if not all, analytes studied. Based on the analyte stability prior to sample processing, home sampling appears to be a reliable sampling option for a selection of these analytes.
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No fault or negligence after an adverse analytical finding due to a contaminated supplement: mission impossible. Two examples involving trimetazidine

May 30, 2025

Pascal Kintz

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Since several years, sports authorities, including national anti-doping organisations and the World Anti-Doping Agency (WADA) have published that the consumption of food supplements can be at risk for athletes due to potential contamination by prohibited substances. Despite these warnings, supplements are largely used by elite athletes and doping violations involving supplements are weekly reported in the media. Anabolic steroids, selective androgen receptor modulators (SARMs), metabolic modulators, stimulants and diuretics are among the most frequently detected substances in contaminated supplements. The author was involved in 2 cases where trimetazidine was identified as the source of the doping violation but the sport authorities sentenced both athletes. Case 1: trimetazidine in urine at 0.1 ng/mL; trimetazidine negative in 3 × 2 cm hair segments (LOQ at 1 ng/g [pg/mg]); trimetazidine at 4 ng/tablet in the supplement. Case 2: trimetazidine in urine at 0.1 and 1.6 ng/mL on 2 occasions; trimetazidine negative in 6 × 1 cm hair segments (LOQ at 1 ng/g [pg/mg]); trimetazidine at 7.2 μg/tablet in the supplement. Despite all pharmacological parameters were consistent with minute amounts of trimetazidine intake during a scenario of proven contamination, the presence of trimetazidine in the urine samples was recognized as an anti-doping rule violation and the athletes were sanctioned with a period of ineligibility of 6 months. From a forensic perspective, contamination is not doping. To avoid these conflicting issues with contaminations, the debate should move to the interest of using hair test results and the need of implementing a threshold for reporting the presence of a drug in urine at very low concentration.
Open Access
An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of 17β-estradiol in human serum and plasma

May 26, 2025

Myriam Ott, Tobias Santner, Neeraj Singh, Friederike Bauland, Daniel Köppl, Alexander Gaudl, Andrea Geistanger, Uta Ceglarek, Manfred Rauh, Christian Geletneky, Judith Taibon

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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.
Open Access
Candidate reference measurement procedure based on isotope dilution-two dimensional-liquid chromatography–tandem mass spectrometry for the quantification of androstenedione in human serum and plasma

May 23, 2025

Myriam Ott, Friederike Bauland, Vanessa Fischer, Daniel Köppl, Alexander Gaudl, Andrea Geistanger, Uta Ceglarek, Manfred Rauh, Judith Taibon

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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.
Open Access
An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of cortisone in human serum and plasma

April 17, 2025

Myriam Ott, Neeraj Singh, Marie Kubicova, Friederike Bauland, Daniel Köppl, Alexander Gaudl, Andrea Geistanger, Uta Ceglarek, Manfred Rauh, Christian Geletneky, Judith Taibon

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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.
Open Access
Isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedures for the quantification of 24(R),25-dihydroxyvitamin D2 and 24(R),25-dihydroxyvitamin D3 in human serum and plasma

March 6, 2025

Kerstin Kandler, Michael Stadlmeier, Neeraj Singh, Friederike Bauland, Andrea Geistanger, Christian Geletneky, Judith Taibon

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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.
Open Access
An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of cortisol in human serum and plasma

February 10, 2025

Myriam Ott, Neeraj Singh, Friederike Bauland, Daniel Köppl, Kerstin Kandler, Alexander Gaudl, Andrea Geistanger, Uta Ceglarek, Manfred Rauh, Christian Geletneky, Judith Taibon

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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.
Publicly Available
Retraction of: External Quality Assessment Programs in the US with an emphasis on urinary sediment testing: the College of American Pathologists experience

May 23, 2018

Eric F. Glassy, David J. Blomberg

Article number: 20180453

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External Quality Assessment Programs in the US with an emphasis on urinary sediment testing: the College of American Pathologists experience

August 11, 2015

Eric F. Glassy, David J. Blomberg

Article number: 20150658

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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.

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Journal Impact Factor	3.7	2024, Journal Citation Reports (Clarivate, 2025)
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