Antinuclear antibody (ANA)/extractable nuclear antigen (ENA) panels in connective tissue disease-associated interstitial lung disease (CTD-ILD) are usually interpreted antibody-by-antibody, leaving the joint mixture effect, statistically attributable burden, and operational stratification value unquantified. In 4771 ILD patients with a complete 12-target ENA panel at a tertiary respiratory center over 32 months, sequential logistic models with Benjamini-Hochberg adjustment, mixture analysis, dual-antibody mutual adjustment, E-values, and Greenland-Drescher adjusted population attributable fractions (PAF) were applied. Three antibodies were significant in the fully adjusted model at q < 0.05: anti-Ro52 (adjusted odds ratio [OR] 3.21, 95% CI 2.38-4.32), anti-SS-A (2.77, 1.98-3.86), and anti-Scl-70 (2.65, 1.67-4.22). Positivity for any one of these three antibodies captured 68.3% of CTD-ILD cases at a 31.2% cohort flag rate, with specificity 70.5%, negative predictive value 98.1% and positive likelihood ratio 2.31, providing an operational rule for prioritizing multidisciplinary discussion (MDD) referral. A 5-antibody mixture quintile test yielded a Q5 vs. Q1 OR of 2.54 (95% CI 1.68-3.83, P-trend = 6.6 × 10[-11]). The fully adjusted OR of anti-SS-A attenuated from 2.77 to 1.85 after additional adjustment for anti-Ro52, suggesting partial mediation of the SS-A signal through Ro52. Greenland-Drescher PAFs were 35.5% for anti-Ro52, 17.1% for anti-SS-A and 7.0% for anti-Scl-70. These figures are interpretable only under strong, unverifiable causal assumptions and are best read as a stratification yield. Among ENA-tested ILD patients, a simple any-positive rule comprising anti-Ro52, anti-SS-A, and anti-Scl-70 identifies two-thirds of CTD-ILD cases while flagging fewer than one-third of the cohort for prospective rheumatologic evaluation.

Coronary artery disease (CAD) is a complex disorder influenced by endothelial dysfunction, vascular inflammation, and thrombosis. This study evaluated selected genetic variants in endothelin signaling (EDN1, PHACTR1), nitric oxide metabolism (NOS3), and fibrinolysis (SERPINE1/PAI-1) in relation to CAD susceptibility, major clinical sub-phenotypes, and disease severity in a Turkish study population. A total of 1256 individuals who underwent coronary angiography were enrolled, including 748 CAD patients and 508 non-CAD controls. Genotyping of EDN1 rs3087459, PHACTR1 rs9349379, NOS3 rs1799983, and PAI-1 4G/5G variants were performed using quantitative real-time PCR. Logistic regression, haplotype, and gene-gene interaction analyses were conducted under multiple inheritance models. The EDN1 CC genotype was significantly associated with CAD (OR = 5.759, 95% CI: 1.319-25.149, p = 0.020), severe CAD (p = 0.012), and C allele with myocardial infarction (OR = 1.640, 95% CI: 1.091-2.465, p = 0.017) in males. Moreover, the PHACTR1 GG genotype was associated with premature CAD (OR = 1.721, 95%CI: 1.076-2.753, p = 0.024), and greater angiographic severity (p < 0.05). The NOS3 T allele carrier males had an increased risk for diabetes (OR = 1.449, 95%CI:1.057-1.985 p = 0.021), and CAD complexed with diabetes (CAD-DM) (OR = 1.619, 95%CI:1.004-2.610, p = 0.048). Notably, the interaction between NOS3 and PHACTR1 variants was consistently associated with CAD, severe CAD, premature CAD, and CAD-DM, and was further supported by higher stenosis severity indices and elevated troponin levels (p < 0.05). These findings demonstrate that variation within endothelial pathways extends beyond CAD susceptibility to shape angiographic complexity and clinical severity. Notably, a synergistic interaction between PHACTR1 and NOS3 modulates CAD related phenotypes, emphasizing that coordinated pathway disturbances contribute to CAD heterogeneity more than isolated variants.

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder. Although the quantification of very long-chain fatty acids (VLCFA) is an old technique implemented to diagnose and monitor, the limited number of experienced clinical biochemists in this field and also the limited availability of technical resources have resulted in scarce reports on the prevalence of X-ALD in developing countries like Brazil. This retrospective cross-sectional study analyzed all records of Brazilian patients diagnosed with X-ALD in LAM/SGM/HCPA from November 1992 to June 2026. Plasma VLCFA levels were analyzed by GC at diagnosis, along with patients' symptoms and their country's geographical origin. This is the largest reported Brazilian cohort (N = 275), comprising the three clinical forms of the disease: Addison disease, adrenomyeloneuropathy and childhood cerebral. Plasma VLCFA levels were consistently elevated across all phenotypes, characterizing the diagnosis together with the clinical manifestations and age. CCALD was primarily characterized by neurodevelopmental regression, seizures, behavioral disturbances, and visual impairment, whereas AMN was mainly associated with progressive myelopathy, and adrenocortical insufficiency was the predominant clinical manifestation in AD. Family history was identified in 43.3% of patients, and the mean diagnostic delay was 2.4 years for CCALD and 7.7 years for AMN, highlighting the importance of early recognition and family screening. Therefore, a detailed description of the patient's clinical presentation is essential for the correct diagnosis and interpretation of biochemical tests. Professionals need to be alert to this disease, given that multidisciplinary care is required.

Interpretation of pediatric coagulation results depends on the reference interval (RI) applied, yet the extent to which RI source affects result classification is rarely quantified. This retrospective single-center study determined how RI source affects out-of-RI flagging in pediatric coagulation testing. Analyzer-specific RIs for prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen in children aged 1 to <18 years were derived from 37,167 routine results measured on Roche Cobas t511/t711 analyzers (PT, n = 17,000; aPTT, n = 17,001; fibrinogen, n = 3166). Indirect RIs were estimated using refineR, continuous RIs using generalized additive models for location, scale and shape (GAMLSS), and direct a posteriori RIs from CLSI EP28-A3c healthy subsets. Out-of-RI flagging rates were compared across manufacturer, locally derived indirect, continuous, direct and published Turkish pediatric RIs, and transferability of published Turkish PT and aPTT intervals was assessed using a bootstrap analogue of the CLSI EP28-A3c 20-sample procedure. Indirect, continuous, and direct RIs were broadly concordant. Manufacturer aPTT limits flagged 32.3% of results outside reference limits (95% CI, 31.6-33.0), compared with 10.7% (10.2-11.2) using continuous RIs. Published same-platform Turkish PT and aPTT intervals did not meet transferability criteria in any single-year age group. Local pediatric coagulation RIs markedly reduced excess abnormal flagging, particularly for aPTT. RI source can materially alter pediatric coagulation result classification, supporting local verification and analyzer-specific implementation before adopting external or manufacturer-supplied limits.

Acute myeloid leukemia (AML) is a genetically heterogeneous and dynamically evolving malignancy in which morphologic remission does not necessarily indicate eradication of disease. Bone marrow examination remains central to diagnosis and response assessment, yet repeated aspiration is invasive, may be affected by hemodilution and spatial sampling, and provides only intermittent snapshots of an evolving clonal process. Liquid biopsy offers a complementary strategy by interrogating circulating cell-free DNA (cfDNA), the leukemia-derived fraction termed circulating tumor DNA (ctDNA), extracellular vesicles, exosomal nucleic acids, and circulating leukemic cells. Among the analytical platforms applicable to these materials, droplet digital polymerase chain reaction (ddPCR) is attractive because it partitions a specimen into thousands of reactions and enables highly precise absolute quantification without a calibration curve. This narrative review critically examines the biological rationale, analytical performance, and clinical evidence supporting ddPCR-based liquid biopsy in AML. The strongest current evidence concerns molecular measurable residual disease (MRD), particularly for NPM1 mutations and selected patient-specific variants, with additional applications in IDH1/2, FLT3-TKD, KIT, CEBPA, DNMT3A, and fusion-transcript monitoring. We discuss the roles of peripheral blood and plasma cfDNA at diagnosis, during induction and consolidation, before and after allogeneic hematopoietic stem-cell transplantation, and at suspected molecular relapse. We also compare ddPCR with multiparameter flow cytometry, reverse-transcription quantitative PCR, and error-corrected next-generation sequencing, emphasizing that analytical sensitivity alone does not establish clinical validity. Major barriers include pre-analytical variation, low and fluctuating ctDNA abundance, assay-specific false-positive droplets, incomplete target coverage, clonal hematopoiesis, and the absence of harmonized thresholds. Emerging multi-analyte strategies that combine mutation tracking, methylation, exosomal RNA, chimerism, and computational modeling may improve robustness. At present, ddPCR is best positioned as a rapid, targeted, and complementary tool embedded in a multimodal MRD framework rather than as a universal replacement for bone marrow or broad genomic profiling.

Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer-related mortality worldwide, largely due to delayed diagnosis, high recurrence rates, therapeutic resistance, and marked molecular heterogeneity. The identification of reliable biomarkers for early detection, prognostic stratification, and therapeutic monitoring is therefore of considerable clinical importance. Circular RNAs (circRNAs), a class of highly stable covalently closed non-coding RNAs, have emerged as promising molecular biomarkers owing to their tissue specificity, evolutionary conservation, and detectability in body fluids and extracellular vesicles. Accumulating evidence indicates that circRNAs play pivotal roles in hepatocarcinogenesis by regulating key biological processes, including metabolic reprogramming, immune evasion, and ferroptosis. Through ceRNA networks, epigenetic modulation, and interactions with RNA-binding proteins, circRNAs orchestrate glycolytic metabolism, lipid metabolic pathways, hypoxia adaptation, immune checkpoint signaling, tumor-associated macrophage polarization, and ferroptosis-associated antioxidant defenses. These regulatory functions not only contribute to tumor progression but also influence sensitivity to systemic therapies and immunotherapeutic interventions. Beyond their mechanistic roles, numerous circRNAs have demonstrated significant diagnostic and prognostic potential in HCC. Circulating and exosomal circRNAs are increasingly recognized as attractive liquid biopsy candidates because of their remarkable stability and accessibility in clinical specimens. Furthermore, specific circRNA signatures have been associated with tumor aggressiveness, recurrence risk, immune landscape remodeling, and treatment response, highlighting their utility as predictive biomarkers and potential therapeutic targets. In this review, we comprehensively summarize the current understanding of circRNA-mediated regulation of metabolic reprogramming, immune escape, and ferroptosis in HCC, with particular emphasis on their clinical and translational relevance. We further discuss the emerging role of circRNAs as diagnostic, prognostic, and predictive biomarkers, as well as the challenges that must be addressed before their integration into routine clinical practice. Collectively, circRNAs represent a promising class of biomolecules with substantial potential to improve precision diagnostics and personalized management of hepatocellular carcinoma.

ISO/TS 20914:2019 yields total expanded uncertainty (U%) without decomposing its components. Grey zone requires the full budget, including systematic uncertainty; RCVincrease requires imprecision alone, presupposing knowledge of budget composition. The MU budget of anti-TPO antibody testing across four Atellica IM modules was quantified over two assay generations (aTPOI and aTPOII) using an extended approach that supplements ISO/TS 20914:2019 with an EQA-derived systematic uncertainty component (usys) per Nordtest TR 537. uRw, EQA-derived usys, and ucal were combined in quadrature; within-IQC-lot CVs pooled by root-mean-square (2 independent cycles; aTPOI: 15 samples, aTPOII 8 samples; 8 months IQC). Grey zones derived using the overlap-based approach (C1+U%/100 and C1-U%/100,k = 2) and usys-exclusive U%. Upward logarithmic reference change value (RCVincrease) (Z = 1.645) from uRw and CVI. aTPOII lower combined uRw at Lv1 (8.27% vs 10.30%) but higher at Lv2 (6.86% vs 4.82%). RMSdev increased (3.78% → 7.04%), and uref increased (0.74% → 2.99%), making usys the co-dominant component in aTPOII (Lv1: 40.2%; Lv2: 43.4%). U% was comparable at Lv1 (24.96% vs 24.11%) but diverged at Lv2 (13.23% vs 23.21%). Excluding usys narrowed grey zone widths by 5.5% and 24.5% (normalized: 53.2% and 51.2%); RCVincrease: 37.9-42.7% and 33.4-38.5%. Budget composition differed between generations despite comparable U% at Lv1. Knowledge of composition is a prerequisite for correct grey zone and RCVincrease derivation. Nordtest TR 537 supplementation addresses this gap. Uncertainty budget decomposition into systematic and random components is a practical step during assay generation transitions; EQA-based peer-group deviation estimation may further characterize usys where material commutability can be established.