Objectives To evaluate the diagnostic performance of chemiluminescent immunoassay (CLIA), latex immunoturbidimetric assay (LIA), and the combination of CLIA/LIA with respect to the functional heparin-induced platelet aggregation (HIPA) test.Methods An observational retrospective study was conducted on 100 patients. All samples were initially tested with CLIA on the ACL TOP AcuStar, and then we performed LIA and CLIA tests concurrently (on the same samples on the ACL TOP 970 CL) and the HIPA test.Results The CLIA test was performed on both the AcuStar and the ACL TOP 970 CL, and results were concordant: 68% of patients were negative, and 32% were positive. The HIPA test confirmed a diagnosis in 26 of 32 and identified 6 false-positive patients and 1 false-negative patient. The LIA test was performed on the ACL TOP 970 CL: 64% of patients were negative, and the remaining 36% were positive. The HIPA test confirmed a diagnosis in 24 patients and identified 12 false-positive and 3 false-negative patients. The combination of CLIA and LIA tests allowed us to categorize 27 true-positive, 13 false-positive, 0 false-negative, and 60 true-negative patients.Conclusions The combination of CLIA/LIA provides high sensitivity with a progressively greater probability of detecting platelet-activating antibodies with a higher assay reactivity, reaching 100% when both automated assays yield moderate or strong results.

BackgroundInclisiran is a subcutaneously administered synthetic small interfering RNA directed against proprotein convertase subtilisin–kexin type 9, leading to sustained low-density lipoprotein cholesterol (LDL-C) reduction with a twice-yearly dosing regimen. While its lipid-lowering efficacy is well established, its broader metabolic effects remain incompletely characterized. This study aimed to comprehensively evaluate serum metabolomic and lipoproteomic changes in patients with dyslipidemia before and after inclisiran treatment using an integrated nuclear magnetic resonance (NMR)-based approach.MethodsThis observational single-center study included 69 patients with dyslipidemia treated with inclisiran in routine clinical practice. Fasting serum samples were collected at baseline and 3 months after treatment initiation. A total of 30 metabolites and 112 lipoprotein-related parameters were quantified using NMR spectroscopy. Paired comparisons between time points were performed using the Wilcoxon signed-rank test with false discovery rate correction. Clustering based on lipoprotein changes was conducted using the KODAMA algorithm to identify distinct response patterns.ResultsInclisiran treatment was associated with extensive and coherent reductions in lipoprotein-related parameters. Seventy-four lipoprotein variables showed significant changes. Reductions were observed across LDL, IDL, and VLDL subclasses, while some HDL-related parameters were increased. In contrast, the global metabolomic profile remained largely unchanged; trimethylamine-N-oxide (TMAO) was the only metabolite significantly increased at follow-up. KODAMA clustering identified three patient groups with heterogeneous responses. Two groups demonstrated marked lipoprotein reductions, particularly those with higher baseline LDL-related parameters, whereas one group exhibited minimal changes. Baseline TMAO levels were lower in the group with the most pronounced lipid response and increased at follow-up, potentially reflecting changes in concomitant lipid-lowering therapy rather than a direct effect of inclisiran.ConclusionInclisiran induces a broad and consistent improvement in lipoprotein profiles, extending beyond LDL-C reduction, with minimal impact on the overall metabolome. These findings support the highly specific lipid-lowering action of inclisiran and highlight inter-individual variability in treatment response, especially in concomitant lipid-lowering therapies.

Ischemic stroke caused by the abrupt interruption of blood flow is one of the leading causes of death and disability worldwide. Despite expanding reperfusion treatment indications, a significant proportion of patients (54.5%) experiences poor outcomes, regardless of successful recanalization, emphasizing the need for clinically relevant preclinical stroke models to better understand the mechanisms associated with effective reperfusion. Here, we develop and characterize a novel mouse model of light-induced recanalization following the photothrombotic occlusion of the distal branch of the middle cerebral artery (MCA). The recanalization provides a meaningful reduction of the infarct volume compared to non-recanalized mice. Moreover, the generalized motor impairment is less severe, as measured by the Neuro Deficit score. Ex vivo investigation highlights that light-mediated recanalization mitigates astrocyte complexity in the periinfarct cortex of recanalized mice. Moreover, light-induced recanalization reduces cerebral edema occurrence. Finally, the investigation of circulating biomarkers shows that our model of occlusion and recanalization of the MCA recapitulates the neuroinflammatory cascade of the acute phase of ischemic stroke.

Lipoprotein(a) [Lp(a)] is a plasma lipoprotein that shares structural elements with low-density lipoprotein (LDL), such as apolipoprotein B-100 (apoB), but differs by the presence of apolipoprotein(a) [apo(a)], which is covalently linked to apoB via a disulphide bond. In recent years, interest in Lp(a) has increased considerably, as epidemiological, genetic and biological evidence supports its causal role in cardiovascular disease. Its heterogeneous structural features, metabolic peculiarities, and ability to transport biologically active and potentially pro- atherogenic, pro-inflammatory and pro-thrombotic molecules make Lp(a) a unique lipoprotein among the apoB-containing lipoproteins. Lp(a) is now recognised as an important risk factor in cardiovascular risk assessment, as it plays a causal and independent role in the development of both atherosclerotic disease and aortic valve stenosis. Measuring Lp(a), together with other determinants of cardiovascular risk, is now recognised as essential for appropriate clinical management and the identification of new therapeutic targets. Consequently, the need to include Lp(a) in global cardiovascular risk assessment has clearly emerged, especially in individuals with a personal history of early or recurrent events, familial hypercholesterolaemia, family history of early events, or family history of high Lp(a) levels. This document, produced through the collaboration of the main Italian scientific societies in the field of cardiovascular disease management and laboratory medicine (SISA, SIC, ANMCO and SIBioC), analyses the pathogenetic role of lipoprotein(a) [Lp(a)] and the clinical significance of its measurement.

Ischemic stroke is a leading cause of disability and mortality, particularly among the elderly. Recanalization therapies, including thrombolysis and thrombectomy, are essential for restoring blood flow and saving ischemic tissue. However, these interventions may trigger reperfusion injury, worsening inflammation and tissue damage, leading to blood-brain-barrier (BBB) disruption, cerebral edema (CE) and adverse functional outcomes. Here we propose a model integrating circulating inflammatory biomarkers with metabolomic and lipoproteomic data able to help clinicians in predicting BBB disruption, CE at 24 h post stroke onset and poor post-stroke functional outcome (Modified Rankin Scale (mRS > 2). Peripheral blood from 87 patients was collected at admission and 24 h after stroke onset. The logistic LASSO regression algorithm was employed to identify the optimal combination of metabolites, lipoprotein-related parameters and circulating biomarkers to discriminate the groups of interest at the two time-points. Multivariable logistic regression models included as covariates: age, sex, onset-to-treatment time, treatment with lipid-lowering medications before stroke, history of heart failure, history of atrial fibrillation and history of diabetes. The regression models showed that methionine, acetate, GlyA and MMP-2 were significant predictors of BBB disruption, methionine, acetate, TIMP-1 and CXCL-10 predicted 24-hours CE, whereas a poor functional outcome at three months was predicted by CXCL-10, IL-12 and LDL-5. As stroke has a heterogeneous pathophysiology, a personalized approach based on biomarkers, as presented in this study, shown to be effective in tackling patient individual risk and could help in developing novel diagnostic, prognostic, and therapeutic neuroprotective strategies for the management of stroke patients.

Background: Atrial fibrillation (AF) is one of the most common heart rhythm disorders encountered in clinical practice. Emerging evidence suggests a significant role of inflammation in the pathogenesis of AF, but certain questions still remain unanswered, in particular whether AF-related inflammation is a cause or a consequence of the arrhythmia, and whether inflammation reflects underlying disease or AF itself. At the current state of the art, scientific evidence on the role of oral anticoagulants (OAC) in modulating pro-inflammatory cytokines implicated in the pathogenesis of AF remains scarce. The aim of our study was to evaluate, in a population of AF patients undergoing OAC, the different roles of anticoagulant therapy [Vitamin K antagonists (VKAs) and direct oral anti-coagulants (DOACs)] in modulating the levels of inflammatory biomarkers in AF. Methods: The Strat-AF study is an observational, prospective, single center, hospital-based study enrolling elderly patients with AF. Results refer to 170 subjects with complete clinical and biohumoral assessment. Results: At multivariate logistic regression analysis, adjusted for several covariates, VKA treatment was an independent protective predictor for having a high grade of inflammation not balanced by anti-inflammatory cytokine levels [OR = 0.26 (0.10–0.69), p = 0.007]. Conclusions: These results from the Strat-AF study are “generators of hypotheses” and provide preliminary evidence for the differential effects of VKAs and DOACs on inflammatory biomarkers (e.g., IL-6, TNF-α) in AF patients. These findings suggest that inflammatory biomarkers could enhance stroke risk prediction models, potentially improving a tailored AF management.

Background: Bicuspid aortic valve (BAV) is the most common congenital heart defect, and its complications (namely, dilatation of the thoracic ascending aorta) raise concerns regarding the proper timing of aortic surgery. The study aim is to unravel the genetic basis of BAV and its complications through a high-throughput sequencing (HTS) approach and segregation analysis if family members were available. Methods: Fifty-two Italian BAV patients were analyzed by HTS using the Illumina MiSeq platform. Targeted sequencing of 97 genes known to be or plausibly associated with connective tissue disorders or aorthopathy was performed. Thirty-five first-degree relatives of N = 10 probands underwent mutational screening for variants identified in the index cases. Results: HTS identified 194 rare (MAF < 0.01) variants in 63 genes. Regarding previously reported genes, five NOTCH1 variants in four BAV patients, four FBN1 variants in two patients and one GATA5 variant in one patient were identified. Interestingly, among further loci, the possible contribution of PDIA2, LRP1 and CAPN2 was suggested by (a) the increased prevalence of rare genetic variants, independently from their ACMG classification in the whole BAV cohort, and (b) segregation analyses of variants identified in family members. Moreover, the present data also suggest the possible contribution of rare variants to BAV complications, specifically MYLK in aortic dilatation, CAPN2 in BAV calcification and VHL and AGGF1 in valve stenosis. Conclusions: Our results underline clinical and genetic diagnosis complexity in traits considered monogenic, such as BAV, but characterized by variability in disease phenotypic expression (incomplete penetrance), as well as the contribution of different major and modifier genes to the development of complications.

BACKGROUND:Direct oral anticoagulants (DOACs) have simplified anticoagulant therapy, but plasma level testing (i.e., anti-Xa and anti-IIa activities) remains important in specific clinical situations (e.g., emergency surgery, bleeding, renal impairment, or suspected non-adherence). However, the impact of sample storage on assay reliability is not well defined. This study evaluated the stability and potential systematic errors in DOACs (apixaban, rivaroxaban, edoxaban, dabigatran) plasma levels after 24 h storage under different conditions. METHODS:We enrolled 182 patients on one of the four DOACs. Blood samples were collected in duplicate citrated tubes. The first tube was processed within 4 h to obtain baseline values, with plasma successively stored at room temperature, 4°C, and -20°C for 24 h. The second tube was stored as whole blood at room temperature before measuring DOACs concentrations. DOACs were measured using dedicated clotting or chromogenic assays. Stability was assessed using non-parametric statistics, Passing-Bablok regression, and Bland-Altman analysis, with Acceptable Change Limits based on assay variability. RESULTS:All DOACs showed good stability across conditions, with median recoveries ranging from 93% to 102%. No significant proportional errors were observed. Minor constant biases were observed for apixaban (at 4°C and -20°C), and more consistently for rivaroxaban and edoxaban. Dabigatran showed no significant bias. Variability was generally low (< 7%), and most measurements near clinical thresholds remained accurate. CONCLUSION:DOACs plasma levels remain stable after 24 h storage under various conditions. While minor biases exist, particularly for rivaroxaban and edoxaban, they are unlikely to affect clinical interpretation in most cases. Whenever needed, DOACs measurement can be deferred after blood drawing without jeopardizing results interpretation.

Introduction:Heparin-induced thrombocytopenia (HIT) is a relatively rare condition burdened by a high rate of complications and mortality. Cardiac surgery is a high risk setting for this condition. But in this area, some particularities can make both diagnosis and treatment difficult. Warfarin is often needed after valvular surgery but may be dangerous if HIT is ongoing. Danaparoid is the only anticoagulant whose mechanism of action has been demonstrated to counteract the pathogenesis of HIT. However, the 24-hour half-life challenges its use after surgery. Case Presentation:Here, we present a case in which HIT occurred six days after cardiac surgery. Warfarin was initiated two days after surgery but was stopped five days later, given the high risk of bleeding due to concomitant thrombocytopenia. HIT probability was initially underestimated, because a misleading diagnosis of endocarditis was made. When redo surgery was performed, no infectious masses were found, but a large thrombus was removed from the left atrium. Bivalirudin and danaparoid were used as alternative anticoagulants during the subsequent postoperative course. Conclusion:HIT should always be kept in mind after cardiac surgery, even if a more plausible cause of thrombocytopenia is present. Discontinuation of warfarin could lead to catastrophic consequences if an unrecognized HIT is ongoing, and an alternative anticoagulant is not started. Bivalirudin and danaparoid were used after the diagnosis of HIT, adapting anticoagulant therapy to the needs of recent surgery.

Background: Atrial fibrillation (AF) is the most common supraventricular arrythmia and one of the most commonly encountered heart conditions in clinical practice. Emerging evidence suggests a significant role of inflammation in the pathogenesis of AF. Population studies have also suggested an association between AF and cognitive impairment and dementia. The aim of this study is therefore to assess, in a population of AF patients on oral anticoagulant therapy, the association between circulating biomarkers involved in the pathogenesis of AF and the cognitive and motor performances of the enrolled patients. Methods: The Strat-AF study is an observational, prospective, single-center, hospital-based study enrolling elderly patients with AF. Results refer to 180 subjects who underwent a complete clinical, biohumoral, cognitive, and functional evaluation. Results: At multivariate logistic regression, Clot Lysis Time (CLT) and circulating levels of von Willebrand Factor (vWF) remained significantly associated with pathological performances at the Stroop test (expressed as execution time) [OR 95% CI 1.54 (1.02–2.35), p = 0.042 and 1.75 (1.08–2.82), p = 0.023, respectively]. With regard to the Short Physical Performance Battery (SPPB), the circulating levels of IL-8 remained significantly associated with the clinical endpoint [OR 95% CI 2.19 (1.13–4.25), p = 0.020]. Conclusions: Our results suggest a potential innovative tool able to identify AF patients at risk of worse prognosis in terms of cognitive and motor performances. The clinical relevance of these results is due to the fact that we have no efficient methods to predict a deterioration in the cognitive performance and, consequently, the possible onset of dementia in AF patients undergoing oral anticoagulant therapy.

Background: Atherosclerotic cardiovascular disease (ASCVD) is often perceived as a male-dominant condition, yet recent European data show that more women live with and die from it. Gender disparities have been reported in the management of dyslipidemia, with women less likely to receive high-intensity lipid-lowering therapy and to reach low-density lipoprotein cholesterol (LDL-C) goals. This study aimed to assess sex-specific differences in response to and tolerance of PCSK9-targeted therapies—monoclonal antibodies (evolocumab, alirocumab) and small interfering RNA (inclisiran)—as well as LDL-C goal attainment according to current ESC guidelines. Methods: We conducted a prospective registry of patients initiating PCSK9-targeted therapy at a specialized lipid center between April 2018 and June 2024. Baseline lipid profiles were recorded and monitored over follow-up. Results: Of the 341 patients, 122 (35.8%) were women and 219 (64.2%) were men, with a mean age of 66.4 ± 12.6 years for the women and 63.9 ± 11.8 years for the men. The women more frequently had heterozygous familial hypercholesterolemia (HeFH) (61.5% vs. 38.4%, p < 0.001) and a lower prevalence of previous cardiovascular events compared to the men (62.3% vs. 84.5%, p < 0.001). A higher proportion of the women were classified as high cardiovascular risk compared to the men (37.7% vs. 15.5%, p < 0.001). Risk categories were assigned according to ESC guidelines, with LDL-C targets of <70 mg/dL for high-risk patients and <55 mg/dL for very high risk patients, along with a ≥50% LDL-C reduction for both categories. In the very high risk group, fewer women achieved LDL-C targets at the first two follow-up visits (first follow-up: 50.0% vs. 76.6%, p = 0.008; second follow-up: 55.3% vs. 68.1%, p = 0.049). Although treatment prescription and tolerance were similar between sexes, women showed smaller LDL-C reductions at the first follow-up (51.7 ± 23.9% vs. 57.3 ± 24.9%, p = 0.044). Conclusions: PCSK9-targeted therapies were effective in both sexes at third follow-up, although women showed a tendency toward a delayed response and lower target attainment, indicating the potential need for more personalized management strategies.

AIMS:Long-term oral anticoagulation is the primary therapy for preventing ischemic stroke in patients with atrial fibrillation (AF). Different types of oral anticoagulant drugs can have specific effects on the metabolism of patients. Here we characterize, for the first time, the serum metabolomic and lipoproteomic profiles of AF patients treated with anticoagulants: vitamin K antagonists (VKAs) or direct oral anticoagulants (DOACs). MATERIALS AND METHODS:Serum samples of 167 AF patients (median age 78 years, 62 % males, 70 % on DOACs treatment) were analyzed via high resolution 1H nuclear magnetic resonance (NMR) spectroscopy. Data on 25 metabolites and 112 lipoprotein-related fractions were quantified and analyzed with multivariate and univariate statistical approaches. KEY FINDINGS:Our data provide evidence that patients treated with VKAs and DOACs present significant differences in their profiles: lower levels of alanine and lactate (odds ratio: 1.72 and 1.84), free cholesterol VLDL-4 subfraction (OR: 1.75), triglycerides LDL-1 subfraction (OR: 1.80) and 4 IDL cholesterol fractions (ORs ∼ 1.80), as well as higher levels of HDL cholesterol (OR: 0.48), apolipoprotein A1 (OR: 0.42) and 7 HDL cholesterol fractions/subfractions (ORs: 0.40-0.51) are characteristic of serum profile of patients on DOACs' therapy. SIGNIFICANCE:Our results support the usefulness of NMR-based metabolomics for the description of the effects of oral anticoagulants on AF patient circulating metabolites and lipoproteins. The higher serum levels of HDL cholesterol observed in patients on DOACs could contribute to explaining their reduced cardiovascular risk, suggesting the need of further studies in this direction to fully understand possible clinical implications.

Background: The impact of non-vitamin K antagonist oral anticoagulants (NOACs) on platelet function is still unclear. We conducted a comprehensive ex vivo study aimed at assessing the effect of the four currently marketed NOACs on platelet function. Methods: We incubated blood samples from healthy donors with concentrations of NOACs (50, 150 and 250 ng/mL), in the range of those achieved in the plasma of patients during therapy. We evaluated generation of thrombin; light transmittance platelet aggregation (LTA) in response to adenosine diphosphate (ADP), thrombin receptor-activating peptide (TRAP), human γ-thrombin (THR) and tissue factor (TF); generation of thromboxane (TX)B2; and expression of protease-activated receptor (PAR)-1 and P-selectin on the platelet surface. Results: All NOACs concentration-dependently reduced thrombin generation compared with control. THR-induced LTA was suppressed by the addition of dabigatran at any concentration, while TF-induced LTA was reduced by factor-Xa inhibitors. ADP- and TRAP-induced LTA was not modified by NOACs. TXB2 generation was reduced by all NOACs, particularly at the highest concentrations. We found a concentration-dependent increase in PAR-1 expression after incubation with dabigatran, mainly at the highest concentrations, but not with FXa inhibitors; P-selectin expression was not changed by any drugs. Conclusions: Treatment with the NOACs is associated with measurable ex vivo changes in platelet function, arguing for antiplatelet effects beyond the well-known anticoagulant activities of these drugs. There are differences, however, among the NOACs, especially between dabigatran and the FXa inhibitors.

Background Apolipoproteins have been recently proposed as novel markers of cardiovascular disease (CVD) risk. However, evidence regarding effects of diet on apolipoproteins is limited. Aim To compare the effects of Mediterranean diet (MD) and lacto-ovo vegetarian diet (VD) on apolipoproteins and traditional CVD risk factors in participants with low-to-moderate CVD risk. Methods Fifty-two participants (39 women; 49.1 ± 12.4 years), followed MD and VD for 3 months each. Medical and dietary information was collected at the baseline. Anthropometric parameters and blood samples were obtained at the beginning and the end of interventions. Results MD and VD resulted in significant improvement in anthropometric and lipid profiles. Both diets led to a reduction in most of the inflammatory parameters. As for apolipoproteins, a significant change was observed for ApoC-I after VD (+ 24.4%; p = 0.020). MD led to a negative correlation between ApoC-III and carbohydrates (R = − 0.29; p = 0.039) whereas VD between ApoD and saturated fats (R = − 0.38; p = 0.006). A positive correlation emerged after VD between HDL and ApoD (R = 0.33; p = 0.017) and after MD between plasma triglycerides and ApoC-I (R = 0.32; p = 0.020) and ApoD (R = 0.30; p = 0.031). IL-17 resulted to be positively correlated with ApoB after MD (R = 0.31; p = 0.028) and with ApoC-III after VD (R = 0.32; p = 0.019). Subgroup analysis revealed positive effects on apolipoproteins from both diets, especially in women, individuals older than 50 years-old or with < 3 CVD risk factors. Conclusions Both diets seem to improve CVD risk, however, MD showed a greater positive effect on apolipoproteins in some subgroups, thus suggesting how diet may influence new potential markers of CVD risk. Trial registration : registered at clinicaltrials.gov (identifier: NCT02641834) on December 2015.

Background: In February 2021, a few cases of unusual, severe thrombotic events associated with thrombocytopenia reported after vaccination with ChAdOx1 nCoV-19 (Vaxzevria) or with Johnson & Johnson's Janssen vaccine raise concern about safety. The vaccine-induced thrombotic thrombocytopenia (VITT) has been related to the presence of platelet-activating antibodies directed against platelet Factor 4. Objectives: We investigated VITT subject genetic background by a high-throughput whole exome sequencing (WES) approach in order to investigate VITT genetic predisposition. Methods: Six consecutive patients (females of Caucasian origin with a mean age of 64 years) were referred to the Atherothrombotic Diseases Center (Department of Experimental and Clinical Medicine, Azienda Ospedaliero-Universitaria Careggi, Florence) with a diagnosis of definite VITT underwent WES analysis. WES analysis was performed on the Illumina NextSeq500 platform. Results:WES analysis revealed a total of 140,563 genetic variants. Due to VITT's rare occurrence, we focused attention on rare variants. The global analysis of all high-quality rare variants did not reveal a significant enrichment of mutated genes in biological/functional pathways common to patients analyzed. Afterwards, we focused on rare variants in genes associated with blood coagulation and fibrinolysis, platelet activation and aggregation, integrin-mediated signaling pathway, and inflammation with particular attention to those involved in vascular damage, as well as autoimmune thrombocytopenia. According to ACMG criteria, 47/194 (24.2%) rare variants were classified as uncertain significance variants (VUS), whereas the remaining were likely benign/benign. Conclusion: WES analysis identifies rare variants possibly favoring the prothrombotic state triggered by the exposure to the vaccine. Functional studies and/or extensions to a larger number of patients might allow a more comprehensive definition of these molecular pathways.

Background:Cardiac surgery is a high-risk setting for heparin-induced thrombocytopenia (HIT). However, large differences in its incidence, rate of thrombotic complications, and mortality have been reported in this context. Few studies address the pharmacologic management of HIT specifically in this setting. Objectives:To describe the incidence, outcomes, and management of patients with HIT in our cohort and to compare them with patients presenting platelet factor 4/heparin antibodies but without platelet-activating capacity. Methods:A retrospective observational study was conducted over a period of 10 years and 6 months on 13,178 cardiac operations in a single high-volume cardiac surgery center. Results:HIT was diagnosed in 0.22% of patients. HIT with associated thromboembolic complications occurred in 0.04% of cases. Two deaths at 30 days were registered, both in patients with associated thrombosis. The 4T score showed a 99.9% negative predictive value. The immunoglobulin G-specific chemiluminescence test positivity rate was highly predictive of HIT. Warfarin was often started early after surgery, and although it was rarely stopped when the diagnosis of HIT was made, no new thromboembolic complications subsequently occurred. Thrombocytopenia appeared to be a poor prognostic sign, whatever the cause. Conclusion:Although rare, HIT is characterized by high mortality in this setting, especially if thrombotic complications occur. Large multicentric studies or an international registry should be created to enhance the scientific evidence on HIT diagnosis and management in this context.