Volume 63, Issue 11

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Objectives 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.

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.

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.

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.

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.

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.

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.

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.

Objectives Glycosylation is a tightly controlled co-translational and post-translational enzymatic modification. Total N-glycome profiling in blood serum/plasma provides information on the most common serum/plasma glycosylation. Total plasma N-glycome from various population samples displayed predictive ability for type 2 diabetes incidence in two previous studies; we therefore explored the ability of total serum N-glycome to predict this disease in a general adult population. Methods This prospective cohort study included a random sample of 1516 adults from a single Spanish municipality. Participants’ glycemic status (non-diabetes, type 2 diabetes) was evaluated at baseline and at a mean follow-up of 7.4 years. Total serum N-glycome at baseline was also measured. Serum enzymatic N-glycan release was performed on a robotic platform followed by HILIC-UPLC glycan separation. Total serum N-glycans were quantified and employed alone, as well as in combination with classical risk factors, to construct type 2 diabetes prediction models. Results Total serum N-glycome peak 37, mainly composed of A3F1G3S3, predisposed participants to type 2 diabetes; however, total serum N-glycome peak 24, mainly composed of A2G2S2, was protective against type 2 diabetes. The interaction between total serum N-glycome peaks 37 and 24 predicted the incidence of type 2 diabetes over time (area under the curve 0.801 [0.750–0.853]). Their predictive power had an independent and additive effect on classical prediction factors. Conclusions The interaction between total serum N-glycome peaks 37 and 24 may constitute a promising predictive biomarker for type 2 diabetes improving the classical prediction tools.

Molecular ‘ in vitro diagnostic’ (IVD) tests are established for the diagnosis of tuberculosis (TB) and multi-drug resistant TB (MDR-TB). What is less clear is how the use of TB or MDR-TB molecular IVD results differ across regions, whether corroborative tests are conducted and what external quality assessment (EQA) infrastructure exists to underpin test confidence. This study investigated the current international status of molecular TB IVDs methods, application and quality assessment. An online survey was distributed by the IFCC’s Committee for molecular diagnostics to TB diagnostic laboratories worldwide. 118 laboratories from 41 nations indicated a range of IVDs were used. ∼75 % participated in EQA programs and 32 % reported this used the WHO International Standard. ∼65 % also delivered MDR-TB results the majority of which were used to change therapy; 1/6 of these do so without EQA evaluation of the MDR-TB result. The study demonstrates a range of IVD solutions in use for TB diagnosis along with a high uptake of EQA in support of this global uptake of this test modality. However, we also reveal gaps in quality assurance for MDR-TB testing with 10 % of the laboratories using resistant results alone without participating MDR-TB EQA. This suggest additional work is required to build on established use of EQA to better support MDR-TB testing and better ensure confident when results are used to guide antibiotic use. Addressing these gaps will ensure the accuracy of future MDR-TB results, which is critical for effective disease management and help combat TB on a global scale.