Volume 63, Issue 12

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.

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.

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.

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

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

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.

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

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

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

Objectives 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 (1,791 determinations) based on human subjects-pool materials and 97 serum samples were retrieved to characterize the agreement between Alinity and Architect (Abbott Diagnostics), Access DXi (Beckman Coulter Diagnostics), Atellica and ADVIA Centaur (Siemens Healthcare Solution) compared to Cobas (Roche Diagnostics), considered as comparator because its calibrator traceability to the World Health Organisation (WHO) International Standard (IS) code 03/178. Ordinary least squares and Bland-Altman were used for this purpose. Results Abbott and Beckman methods overestimated and underestimated, respectively, B12 concentrations vs. Roche and the other methods. We reported similar systematic or proportional error patterns between EQA and serum samples. Only Beckman was affected by both errors. Due to the wide Limit of Agreement Interval, we cannot confidently conclude on the agreement between Roche and the other methods. However, the inter-method bias was well lower than the desirable goal of 9.4 % for Abbott Architect and also lower for Siemens ADVIA Centaur. Conclusions The recommended thresholds for serum total B12 should not be assumed applicable to all assays, due to the poor agreement among the currently available methods, a limitation that persists despite the release of the WHO IS 03/178.

Objectives 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.

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

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

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

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

Objectives 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.

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.

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

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

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

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

Objectives 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.

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

Objectives 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.