Background:VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a severe adult-onset autoinflammatory disease caused by somatic mutations in the X-linked UBA1 gene, most commonly affecting codon 41. Early molecular confirmation is essential, but sequencing-based methods may be limited by turnaround time, cost, and sensitivity for low-level somatic variants. We aimed to validate a rapid, accessible allele-specific real-time PCR assay for detection of the most frequent UBA1 hotspot mutations associated with VEXAS syndrome. Methods:In this prospective monocentric study conducted at the University Hospital "P. Giaccone" (Palermo, Italy), 17 adults were enrolled: six patients with high clinical suspicion of VEXAS syndrome and eleven healthy controls. UBA1 variants c.121A>G, c.121A>C, and c.122T>C were screened using a SYBR Green-based allele-specific real-time PCR kit with mutation-specific reaction mixes and an internal housekeeping control, followed by melting curve analysis for variant discrimination. All PCR-positive samples were confirmed by Sanger sequencing. Results:Allele-specific real-time PCR identified UBA1 mutations in 5/6 (83.3%) suspected VEXAS cases. Sanger sequencing confirmed all real-time PCR-positive results, demonstrating 100% concordance between methods. Conclusion:This allele-specific real-time PCR assay enables rapid and reliable detection of the most common UBA1 codon 41 mutations associated with VEXAS syndrome using standard real-time PCR platforms. The approach provides a practical, cost-effective screening strategy to support timely diagnosis in patients with high clinical suspicion.

BACKGROUND:In this study, we first evaluated the relationship between sTREM2 concentrations in CSF, serum, and plasma of Alzheimer's disease (AD) patients using the newly developed Lumipulse G sTREM2 assay. METHODS:sTREM2 was measured by the fully automated Lumipulse G1200 platform (Fujirebio). Associations and agreement between matrices were assessed using Passing-Bablok regression, Spearman correlation, and Bland-Altman analyses. Sub-analyses explored the influence of disease stage and tau pathology. RESULTS:Median sTREM2 concentrations were highest in CSF, followed by serum and plasma. Serum and CSF sTREM2 levels showed a moderate but significant correlation (ρ = 0.32; p = 0.0012), although regression analysis indicated poor linearity. In contrast, serum and plasma sTREM2 levels were strongly correlated (ρ = 0.74; p < 0.001). The association between CSF and serum sTREM2 levels was independent of total and phosphorylated tau. Notably, a strong CSF-serum correlation was observed in the MCI due to AD group (ρ = 0.74) but was completely lost in overt AD dementia, demonstrating a clear disease stage-dependent relationship. CONCLUSION:CSF and blood sTREM2 capture partly distinct biological processes and show limited overall agreement. While serum and plasma sTREM2 are closely related, they are not interchangeable.

BACKGROUND:Myasthenia gravis (MG) is an autoimmune disorder of the neuromuscular junction caused by antibodies against the acetylcholine receptor (AChR) or muscle-specific kinase (MuSK). Although these antibodies enable diagnosis, they correlate poorly with disease activity and prognosis. Emerging data suggest that MG involves broader neuroimmune mechanisms beyond the peripheral synapse. Glial fibrillary acidic protein (GFAP), a marker of astroglial activation, and neurofilament light chain (NfL), a marker of neuroaxonal injury, have been proposed as circulating biomarkers in several neurological diseases. This study aimed to evaluate serum GFAP and NfL concentrations in MG patients and to assess its potential diagnostic utility. METHODS:In this retrospective case-control study, 137 patients with confirmed MG and 338 healthy controls were enrolled. Clinical classification followed MGFA criteria. Anti-AChR and anti-MuSK antibodies were measured by ELISA. Serum GFAP and NfL concentrations were quantified using a fully automated chemiluminescent immunoassay (Lumipulse G1200). Group comparisons were performed using non-parametric tests, correlations using Spearman analysis, and independent determinants assessed with multivariable linear regression. Receiver operating characteristic (ROC) curve analysis was performed to evaluate diagnostic performance. RESULTS:Serum GFAP concentrations were higher in MG patients than controls (44.6 vs 29.6 pg/mL) and strongly correlated with age (r = 0.64, p < 0.0001), but not with antibody titers. In multivariable regression, age was the main independent determinant of GFAP (β = 0.00827, p < 0.0001), whereas MG status was not. GFAP showed moderate diagnostic performance (AUC = 0.704). In an age- and sex-matched analysis, NfL concentrations were modestly higher in MG patients compared with controls (30.2 vs 15.5 pg/mL; p = 0.047). A moderate positive correlation between GFAP and NfL was observed in both groups. CONCLUSIONS:Circulating GFAP levels are elevated in MG but are primarily driven by age and sex rather than disease-specific mechanisms, limiting their diagnostic utility. NfL shows a modest increase, suggesting subtle neuroaxonal involvement; however, its clinical relevance remains uncertain. Together, these findings indicate that GFAP and NfL reflect non-specific neurobiological processes rather than robust biomarkers of MG.

Minimal residual disease (MRD) assessment has emerged as a key prognostic marker in multiple myeloma (MM), with MRD negativity (undetectable MRD, uMRD) correlating with improved progression-free and overall survival. The International Myeloma Working Group recommends standardized protocols for MRD detection, including the EuroFlow-based next-generation flow cytometry (NGF), although its implementation in routine care remains challenging. We evaluated the feasibility and clinical utility of two CE-IVD-approved EuroFlow tubes for MRD detection in a real-world clinical setting, and investigated the relationship between bone marrow (BM) immune profiles and disease outcomes. Between December 2021 and February 2024, 74 MM patients (58 of them classified as transplant-eligible) underwent 97 MRD evaluations after achieving at least a very good partial response (VGPR). Using ≥2 million events/sample, a median sensitivity of 10⁻⁵ was achieved; 45% of cases were uMRD. After a median follow-up of 19 months, one relapse occurred in the uMRD group, whereas 15 relapses and 3 deaths were observed among persistent MRD (pMRD) patients. Immunophenotypic profiling revealed a higher granulocyte-to-lymphocyte ratio and a lower proportion of CD56dim NK cells, possibly reflecting immune rebalancing after bone marrow recovery. Our findings confirm the feasibility and prognostic relevance of NGF-based MRD detection using standardized commercial panels, and highlight the potential of immune profiling to provide additional biological insights into MRD dynamics in multiple myeloma.

Alzheimer’s disease (AD) is increasingly recognized as a biological continuum characterized by early neuropathological and molecular changes that precede the onset of clinical symptoms. Fluid biomarkers have transformed the diagnostic landscape by enabling the in vivo detection of core AD pathologies, particularly amyloid-β deposition and tau-related neurodegeneration. Despite the rapid expansion of candidate biomarkers, however, only a limited number have successfully translated into clinical practice. Discovery-phase approaches, primarily driven by mass spectrometry-based proteomics, enable the unbiased identification of novel biomarker candidates across multiple biological pathways. Research-phase methods, including immunoassays such as enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassays (ECLIA), microfluidic platforms, and ultrasensitive technologies such as single-molecule array (SIMOA), support analytical and clinical validation in well-characterized cohorts. Clinical implementation has been advanced by fully automated platforms, including Lumipulse and Elecsys, which have obtained regulatory approval for cerebrospinal fluid biomarkers and, more recently, blood-based biomarkers. These developments represent a paradigm shift toward minimally invasive and scalable diagnostic strategies that may reduce dependence on neuroimaging techniques. Nevertheless, major challenges remain, including assay standardization, inter-platform variability, demonstration of clinical utility, and barriers to widespread clinical adoption. This review provides a comprehensive overview of analytical methods used to measure AD fluid biomarkers in cerebrospinal fluid and plasma, structured according to the biomarker development pipeline from discovery to clinical implementation. Overall, the review highlights a fit-for-purpose approach to biomarker development and emphasizes the complementary roles of diverse analytical technologies across the different phases of biomarker translation.

Multiple Sclerosis (MS) is a neuroinflammatory disorder in which genetic and environmental factors contribute to disease onset. Evidence implicates the inflammasome pathway in MS pathophysiology. However, the interaction between inflammasome-related genetic variants and 25-OH-vitamin D3 (25(OH)D3) levels remains unclear. 105 MS patients and 109 healthy controls were enrolled. Genotyping of NLRP3 (rs10754558, rs3806265) and NLRC4 (rs479333) polymorphisms was performed using real-time PCR. Serum 25(OH)D3 levels were measured by high-performance liquid chromatography. Clinical severity was assessed using the Expanded Disability Status Scale (EDSS), Multiple Sclerosis Severity Score (MSSS), annualized relapse rate (ARR), and age at onset. MS patients showed significantly lower serum 25(OH)D3 levels than controls. Genotype distributions did not differ significantly under an additive model; however, the NLRP3 rs10754558 GG genotype was more frequent in MS patients under a recessive model and was significantly associated with disease status after adjustment for sex. Subjects carrying the GG genotype also had significantly lower serum 25(OH)D3 levels than CC/CG carriers, independently of sex. No significant associations were observed for NLRP3 rs3806265 or NLRC4 rs479333, and none of the investigated variants was associated with EDSS, MSSS, ARR, or age at onset. The NLRP3 rs10754558 polymorphism may be associated with MS susceptibility and reduced circulating vitamin D levels, suggesting a potential link between inflammasome-related genetic variability and immunometabolic regulation in MS.

OBJECTIVES:To establish the reference intervals (RIs) of serum glial fibrillary acid protein (GFAP) measured by the fully automated Lumipulse system. METHODS:The study population consisted of 340 healthy individuals, including 251 blood donors and 89 outpatients, with a median age of 56 years. Serum GFAP levels were measured by the Lumipulse G GFAP assay on the fully automated platform Lumipulse G1200 (FUJIREBIO Inc., Tokyo, Japan). GFAP RIs (2.5th and 97.5th percentiles) were calculated for the overall population and stratified by age and sex groups. For the overall population, males, and females partitions, we employed the nonparametric methods, while for the age-and-sex groups we employed the "robust" method, as recommended by CLSI. RESULTS:The RI in the whole population was 10.4-92.0 ng/L. When considering sex differences, females showed higher levels of serum GFAP than males across all age groups. A positive correlation was observed between age and GFAP (Spearman's rho=0.55, p<0.001). Specifically, the biomarker was stable until 60 years, while individuals aged>60 years demonstrated significantly and considerably higher levels than younger age groups. Additionally, in the 50-60 age group, we observed gender-related differences, with females having increased levels than males. CONCLUSIONS:GFAP levels are influenced by both age and sex. Accordingly, we established RIs of serum GFAP, taking into consideration age and sex-related differences.

BACKGROUND AND AIM:This study aims to explore the clinical significance of antinuclear antibodies (ANA) patterns in liver diseases. MATERIALS AND METHODS:We included 396 patients with a request for ANA testing for suspected autoimmune liver disease (AILD). For each patient, we collected demographical, clinical, and laboratory data. RESULTS:Among the patients, 33% had AILD, predominantly aiutoimmune hepatitis (AIH). The AC1 pattern was significantly more prevalent in AIH patients, while the AC21 pattern was strongly associated with primary biliary cholangitis (PBC). AC4-AC5 patterns were less frequent in AIH and PBC patients but more common in non-alcoholic hepatitis. Elevated alkaline phosphatase and gamma-glutamyl transferase levels were observed in AILD patients with AC11, AC12, and AC21 patterns. CONCLUSIONS:These findings highlight the different distribution of ANA patterns in liver diseases, with specific patterns showing strong associations with distinct liver conditions.

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder and represents a major public health challenge. With increasing life expectancy, the incidence of AD has also increased, highlighting the need for early diagnosis and improved monitoring. Traditionally, diagnosis has relied on clinical symptoms and neuroimaging; however, the introduction of biomarkers has revolutionized disease assessment. Traditional biomarkers, including the Aβ42/Aβ40 ratio, phosphorylated tau (p-Tau181, p-Tau217, and p-Tau231), total tau (t-tau), and neurofilament light chain (NfL), are fundamental for staging AD progression. Updated guidelines introduced the ATX(N) model, which extends biomarker classification to include additional promising biomarkers, such as SNAP-25, YKL-40, GAP-43, VILIP-1, progranulin (PGRN), TREM2, IGF-1, hFABP, MCP-1, TDP-43, and BDNF. Recent advancements have allowed for the detection of these biomarkers not only in CSF but also in plasma and neuron-derived exosomes, offering less invasive and more accessible diagnostic options. This review explores established and emerging biomarkers and emphasizes their roles in early diagnosis, patient stratification, and precision medicine.

BACKGROUND:Pediatric sepsis remains a major global health challenge, complicated by age-related variability in presentation and diagnostic uncertainty. Monocyte Distribution Width (MDW), a measure of monocyte anisocytosis automatically reported with complete blood count, has shown promise as an early biomarker for sepsis in adults. However, its clinical utility in pediatric patients remains unclear. In this study, we explored the usefulness of MDW in pediatric patients presenting to the emergency department (ED). METHODS:We conducted a retrospective observational study of pediatric patients (<18 years) who presented to the ED for any cause. Patients were categorized into four groups: controls (no infection), infection, sepsis, and shock. MDW values were compared across groups and stratified by age (≤6 years and > 6 years). A receiver operating characteristic (ROC) curve analysis was performed to assess MDW diagnostic performance. RESULTS:A total of 393 patients were enrolled: 117 controls, 183 with infection, 88 with sepsis, and 5 with shock. Overall, MDW values increased with disease severity, peaking in patients with shock. However, significant overlap was observed between infection and sepsis groups, particularly in children ≤ 6 years, where MDW was elevated even in controls. In children > 6 years, MDW showed a clearer stepwise increase across disease categories. ROC analysis revealed an AUC of 0.73 for distinguishing infected from non-infected patients at a cutoff of 24. CONCLUSIONS:MDW is a readily accessible biomarker that may aid in identifying pediatric patients with infection in emergency settings.

BACKGROUND:Glial Fibrillary Acidic Protein (GFAP) is a well-established biomarker of astrocytes and astrogliosis, a pathological response observed in various neurological diseases. This study aimed to evaluate the diagnostic performance of serum GFAP in Alzheimer's disease (AD), multiple sclerosis (MS), and transthyretin amyloidosis (ATTR) polyneuropathy. METHODS:We performed a retrospective observational study, including 498 participants (337 healthy controls and 161 patients with AD, MS, or ATTR amyloidosis). Serum GFAP levels were measured using the Lumipulse G1200 platform, and statistical analyses were performed to compare levels across disease groups and assess their diagnostic accuracy. RESULTS:GFAP levels were significantly elevated in all neurological disease groups compared to age-matched controls, with the highest levels found in AD (79.4 pg/mL vs. 39.5 pg/mL, p = 2.55 × 10-12). ROC curve analysis revealed that GFAP had strong diagnostic performance for AD (AUC = 0.86), moderate performance for ATTR amyloidosis (AUC = 0.67), and poor performance for MS (AUC = 0.61). CONCLUSIONS:These findings suggest that GFAP is a promising biomarker for AD, reflecting astrocytic activation and neuroinflammatory processes. Its diagnostic utility in ATTR amyloidosis is moderate, while its role in MS remains limited.

Introduction: Neurofilament light chain (NfL) is one of the most important biomarkers in the field of clinical neurochemistry. Several analytical methods have been developed in the last decade. Recently, Fujirebio introduced a ready-to-use assay kit for measuring NfL levels in the cerebrospinal fluid (CSF) on the fully automated LUMIPULSE G System. In this study, we established the decisional cutoffs for CSF NfL. Materials and Methods: We performed a retrospective observational study including patients with cognitive decline. CSF NfL levels were measured by two analytical methods: the NF-light ELISA kit (UmanDiagnostics) and the Lumipulse G1200 fully automated system (Fujirebio). We calculated the cutoffs for the Lumipulse, starting from the consolidated cutoffs of the ELISA method for each age and using the equation obtained by the regression analysis. Results: The study population consisted of 100 patients with cognitive decline. The median levels of CSF NfL measured by Lumipulse and ELISA were 776.5 +/- 772.6 pg/mL and 473.5 +/- 443.5 pg/mL, respectively, significantly different (p < 0.001). The Spearman's rank correlation coefficient was 0.962, indicating a robust positive correlation between the two measurement methods. The equation derived from the Passing-Bablok regression analysis was CSF CLEIA = -61.16 + 1.83 x CSF ELISA. Based on this equation, we defined the decisional cutoff values. Conclusions: Decisional cutoffs are fundamental tools for guiding clinicians to use biomarkers' results and interpretation appropriately. This is the first study establishing the decisional cutoff value of NfL measured by Lumipulse, a fully automated platform widely used in clinical laboratories.

Objectives Hereditary transthyretin amyloidosis with polyneuropathy (ATTRv-PN) is a rare, progressive neurodegenerative disorder caused by mutations in the transthyretin (TTR) gene. The disease leads to systemic amyloid deposition, primarily affecting the nervous system and, in some cases, the heart. Early diagnosis and monitoring are critical for effective management, yet reliable biomarkers remain limited. This study aimed to investigate the role of serum glial fibrillary acidic protein (GFAP) and neurofilament light chain (NfL) as biomarkers in ATTRv-PN. Methods A retrospective observational study was conducted at the University Hospital Paolo Giaccone, enrolling ATTRv-PN patients, asymptomatic TTR mutation carriers, and healthy blood donors. Serum GFAP and NfL levels were measured using a fully automated immunoassay (Lumipulse G1200). Results A total of 119 participants were included: ATTRv-PN (n=23), carriers (n=27), and healthy controls (n=69). GFAP levels were significantly elevated in ATTRv-PN patients compared to carriers and healthy controls (p<0.001), with the highest levels observed in individuals with the V122I mutation. The median NfL levels were also significantly elevated in ATTRv-PN patients (30.74 pg/mL) compared to carriers (11.59 pg/mL) and healthy controls (12.86 pg/mL) (p<0.001). Additionally, a significant negative correlation was observed between NfL levels and clinical severity scores, indicating its association with disease severity. Conclusions These findings support the usefulness of serum NfL as a prognostic tool in ATTRv-PN and highlight the potential involvement of astrocyte activation in disease pathology. Further longitudinal studies are needed to validate these biomarkers for clinical application.

BackgroundIn this study, we explored the accuracy of two new sepsis biomarkers, monocyte distribution width (MDW) and presepsin (PSP), compared to traditional ones, C-reactive protein (CRP) and Procalcitonin (PCT), to identify sepsis and predict intra-hospital mortality by analyzing their kinetic at different time points during hospitalization stay.MethodsWe enrolled 104 patients admitted to the intensive care unit (ICU) of University Hospital “Paolo Giaccone”, Palermo. Among these, 30 (29%) had a clinical diagnosis of sepsis. MDW, PCT, CRP, and PSP were evaluated at admission (T0), after 24 h (T24), 48 h (T48), 72 h (T72), at day 5 (T5), and at discharge (TD).ResultsPatients with sepsis displayed higher levels of PCT and PSP than patients without sepsis at each timepoint; differently, CRP displayed statistically significant differences only at T0, while MDW only at T0 and T24. Patients with increasing levels of PSP displayed lower median survival time than patients with decreasing levels; differences reached statistical significance only at 48 h (20 vs. 29 days, log rank test, p = 0.046). Interestingly, PSP was an independent predictor of ICU mortality at 48 and 72 h after hospital admission. Also, the kinetic of PSP had prognostic value, with increased values at 48 h after admission being associated with reduced survival.ConclusionOur findings support the role of PSP and its kinetic as a predictor of ICU mortality.

Cerebrospinal fluid (CSF) core biomarkers of Alzheimer’s disease (AD), including amyloid peptide beta-42 (Aβ42), Aβ42/40 ratio, and phosphorylated tau (pTau), are precious tools for supporting AD diagnosis. However, their use in clinical practice is limited due to the invasiveness of CSF collection. Thus, there is intensive research to find alternative, noninvasive, and widely accessible biological matrices to measure AD core biomarkers. In this study, we measured AD core biomarkers in saliva and plasma by a fully automated platform. We enrolled all consecutive patients with cognitive decline. For each patient, we measured Aβ42, Aβ40, and pTau levels in CSF, saliva, and plasma by Lumipulse G1200 (Fujirebio). We included forty-two patients, of whom 27 had AD. Levels of all biomarkers significantly differed in the three biofluids, with saliva having the lowest and CSF the highest levels of Aβ42, Aβ40, and pTau. A positive correlation of pTau, Aβ42/40 ratio, and pTau/Aβ42 ratio levels in CSF and plasma was detected, while no correlation between any biomarker in CSF and saliva was found. Our findings suggest that plasma but not saliva could represent a surrogate biofluid for measuring core AD biomarkers. Specifically, plasma Aβ42/40 ratio, pTau/Aβ42 ratio, and pTau could serve as surrogates of the corresponding CSF biomarkers.

Neurodegenerative disorders (NDs) represent a group of different diseases characterized by the progressive degeneration and death of the nervous system’s cells. The diagnosis is challenging, especially in the early stages, due to no specific clinical signs and symptoms. In this context, laboratory medicine could support clinicians in detecting and differentiating NDs. Indeed, biomarkers could indicate the pathological mechanisms underpinning NDs. The ideal biofluid for detecting the biomarkers of NDs is cerebrospinal fluid (CSF), which has limitations, hampering its widespread use in clinical practice. However, intensive efforts are underway to introduce high-sensitivity analytical methods to detect ND biomarkers in alternative nonivasive biofluid, such as blood or saliva. This study presents an overview of the ND molecular biomarkers currently used in clinical practice. For some diseases, such as Alzheimer’s disease or multiple sclerosis, biomarkers are well established and recommended by guidelines. However, for most NDs, intensive research is ongoing to identify reliable and specific biomarkers, and no consensus has yet been achieved.

The aim of this systematic review was to evaluate the diagnostic accuracy of molecular-based LTPs serum sIgE for the diagnosis of food allergies in patients with suspected allergy to one of the LTPs-containing foods. Cohort, prospective or retrospective cross-sectional studies were considered for inclusion in this review. Oral food challenge (both open and double-blind placebo-controlled) was the reference standard for the diagnosis. PubMed (MEDLINE), Web of Science, Scopus, and ClinicalTrial.org were searched for relevant papers. The risk of bias was assigned by the QUADAS-2 tool. Data were reported as the sensitivity and specificity. The study protocol was registered in the PROSPERO database (CRD42022321985). Fifteen articles, including 2395 individuals, were included. The sensitivity of Ara h 9 for peanut allergy diagnosis ranged from 6 to 61%; the specificity ranged from 57 to 100%. The Cor a 8 sensitivity ranged from 11 to 43%, with the specificity ranging from 59 to 94%. Ara h 9 and Cor a 8 serum sIgE may be useful for confirming the diagnosis of peanut and hazelnut allergy in symptomatic patients, although the diagnostic accuracy is limited by the low sensitivity. More investigation on other LTPs and in adult populations may be important to define the clinical role of this test in food allergy diagnostics.

Objectives Non-celiac wheat sensitivity (NCWS) is an emerging clinical condition characterized by gastrointestinal and extraintestinal symptoms following the ingestion of gluten-containing foods in patients without celiac disease (CD) or wheat allergy. Despite the great interest for NCWS, the genetic risk factors still need to be fully clarified. In this study, we first assessed the possible contribution of KIR genes and KIR haplotypes on the genetic predisposition to NCWS. Methods Fifty patients with NCWS, 50 patients with CD, and 50 healthy controls (HC) were included in this study. KIR genes and KIR genotyping were investigated in all subjects by polymerase chain reaction with the sequence oligonucleotide probe (PCR-SSOP) method using Luminex technology. Results We found a statistically different distribution of some KIR genes among NCWS, CD, and HC. Specifically, NCWS showed a decreased frequency of KIR2DL1, -2DL3, -2DL5, -2DS2, -2DS3, -2DS4, -2DS5, and -3DS1 genes, and an increased frequency of -3DL1 gene respect to both CD and HC. No difference was detected in the KIR haplotype expression. At the multivariate analysis, KIR2DL5, -2DS4, and -2DS5 were independent predictors of NCWS. Conclusions Our findings suggest a role of KIR genes in NCWS susceptibility, with KIR2DL5, -2DS4, and -2DS5 having a protective effect. Further large-scale multicentric studies are required to validate these preliminary findings.