Circulating Activated Platelets in Children With Long Covid: A Case-Controlled Preliminary Observation.

We investigated if children with Long Covid (n=14) have activated platelets compared with healthy controls (n=14). Platelet activation and secretion markers were investigated by flow cytometry using MoAbs directed against P-selectin, CD63, and PAC-1 in quiescent platelets and in platelets stimulated with 10-µM adenosine diphosphate and 25-µM protease activated receptor 1-activating peptide. Circulating platelets of patients with Long Covid had significantly increased expression of the activation marker cytometry using MoAbs directed against P-selectin (P = 0.019).

An integrated multi-tool analysis contributes elements to interpreting unclassified factor IX missense variants associated with haemophilia B.

BACKGROUND: Dissection of genotype-phenotype relationships in haemophilia B (HB) is particularly relevant for challenging (mild HB) or for HB-associated but unclassified factor IX (FIX) missense variants. Here, the identification of a FIX missense variant associated with mild HB, reported but unclassified, prompted a multiple-level approach to contribute elements to interpret unclassified HB-associated FIX missense variants. METHODS: Molecular modelling of wild-type (WT) and V92A FIX variants, expression studies in HEK293 cells with evaluation of protein (ELISA, Western blotting) and activity (aPTT-based/chromogenic assays) levels after recombinant expression, and multiple prediction tools. RESULTS: The F9(NM_000133.4):c.275T>C (p.V92A) variant was found in a mild HB patient (antigen, 45.4 U/dl; coagulant activity, 23.6 IU/dl; specific activity, 0.52). Newly generated molecular models showed alterations in Gla/EGF1-EGF2 domain conformation impacting on Ca++ affinity and protein-protein interactions with FXIa. Multi-tool analysis indicated a moderate impact on protein structure/function of the valine-to-alanine substitution, in accordance with patient's and modelling data. Expression studies on the V92A variant showed a specific activity (0.49±0.07; WT, 1.0±0.1) recapitulating that of the natural variant, and pointed toward a moderate activation impairment as the main determinant underlying the p.V92A defect. The validated multi-tool approach, integrated with evidence-based data, was challenged on a panel (n=9) of unclassified FIX missense variants, which resulted in inferred protein (secretion/function) outputs and HB severity. CONCLUSIONS: The rational integration of multi-tool and multi-parameter analyses contributed elements to interpret genotype/phenotype relationships of unclassified FIX missense variants, with implications for diagnosis, management and treatment of HB patients, and potentially translatable into other human disorders.

Safety and effectiveness of oral anticoagulants in patients with atrial fibrillation and stage 4 chronic kidney disease: a real-world experience.

It is still uncertain whether direct oral anticoagulants (DOACs) perform better than vitamin K antagonists (VKAs) in subjects with non-valvular atrial fibrillation (NVAF) and advanced chronic kidney disease (CKD). The aim of the study was to compare safety and effectiveness of DOACs and VKAs in patients with NVAF and stage 4 CKD (creatinine clearance 15-29 mL/min). We searched the hospital databases of two academic centers to retrospectively identify patients with stage 4 CKD who were on treatment with DOACs or VKAs for NVAF. Safety was the primary outcome of the study and was assessed in terms of incidence of major bleeding (MB). Secondary outcomes were clinically relevant non-major bleeding (CRNMB) and death for any cause. A total of 176 patients (102 on DOACs and 74 on VKAs) were found and included in the analysis. The incidence rate of MB was not statistically different between groups (8.6 per 100 patients-year in the DOAC group and 5.6 per 100 patients-year in the VKA group). Rates of IS/SSE and CRNMB were statistically similar in the two treatment groups, as well. There were less deaths for any cause in the DOAC group than in the VKA group (8.6 and 15.8 per 100 patients-year, respectively), but the difference was not statistically significant. This study found no difference in terms of safety and effectiveness between patients with NVAF and stage 4 CKD treated with DOACs and VKAs. Larger prospective or randomized studies are needed to confirm these findings.

Malnutrition as a Risk Factor in the Development of Oral Cancer: A Systematic Literature Review and Meta-Analyses.

This systematic review and meta-analyses aimed to assess whether malnutrition may increase the incidence of oral cancer. Following the PRISMA statement, the research was conducted on PubMed, Scopus, and MEDLINE via OVID without any time restrictions. The risk of bias was assessed, and the quality of evidence for each performed meta-analysis was evaluated using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) framework. Sixty-one articles met the inclusion criteria and seven studies underwent quantitative evaluation. For our meta-analysis on hypovitaminosis B, three studies with a total of 90,011 patients were included. An odds ratio of 2.22 was found. Our meta-analysis on the correlation between vitamin C and oral cancer included one study with a total of 866 patients and the derived odds ratio was 1.06. Our meta-analysis on the relationship between vitamin D deficiency and the incidence of oral cancer included three studies with a total of 12,087 patients and the odds ratio was -2.58. The GRADE system showed a moderate strength of evidence due to the presence of studies with a high risk of bias and high indirectness of the data given. The present findings suggest that an inadequate intake of vitamins, particularly vitamin D, poses a risk for the onset of oral cancer.

Extended coagulation profile of children with Long Covid: a prospective study.

Emerging data suggests that endotheliopathy changes can be associated with post covid condition (PCC) in adults. Research on the matter in children is lacking. We analyzed an extended coagulation profile including biomarkers of endothelial damage in children with PCC and compared it with a control group of children that fully recovered post- SARS-CoV-2 infection. A case-control study enrolling children below 18 years of age with previous microbiologically confirmed SARS-CoV-2 infection in a pediatric post-covid unit in Italy ≥ 8 weeks after the initial infection. Samples were taken at 8 and 12 weeks after the SARS-CoV-2 diagnosis and analyzed for coagulation profiling (fibrinogen, prothrombin time, international normalized ratio, activated partial thromboplastin time, d-dimers, factor VIII coagulant activity, plasma von Willebrand factor (VWF) antigen and VWF ristocetin cofactor (RC)). We compared coagulation profiles in samples from children identified with PCC (at least one, or three or more symptoms, which could not be explained by an alternative diagnosis, at the 8- and 12-week follow-up assessment using the pediatric Long Covid International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC) survey. Seventy-five children were enrolled, 49.3% were females, the median age was 10.2 (IQR 4.9) years. Forty-six (61%) of the children had at least one persisting symptom at the eight weeks post-onset, (PCC8); 39/75 (52%) had persistent symptoms for more than 12 weeks (PCC12) and 15/75(32%) had at least three persisting symptoms (PCC ≥ 3) at 12 weeks. Children with PCC presented more frequently with abnormal D-Dimer levels above the reference range compared to children that had fully recovered at the 8-12 weeks (39.1% vs. 17.2%, p = 0.04), and 12 week follow up or more (41% vs. 17.2%, p = 0.05), and in children with three or more symptoms at 12 weeks follow up compared to those that had recovered (64.3% vs. 22.2%, p = 0.002). For the other coagulation profiles, there were abnormal values detected for VWF, FVIII, RC and Fibrinogen but no significant differences between children with PCC compared to controls. Although the majority of children in our cohort showed coagulation profile within or close to normal ranges, we found that a higher proportion of children with PCC, and specifically those with a more severe spectrum characterized with three or more persisting symptoms, had abnormal D-dimer levels compared to other children that fully recovered from an acute SARS-CoV-2 infection.

Oral Squamous Cell Carcinoma-Associated Thrombosis: What Evidence?

Venous thromboembolism (VTE) disease is the second leading cause of mortality in cancer patients. In the general population, the annual incidence of a thromboembolic event is about 117 cases per 100,000 persons, but cancer increases this risk about fourfold, while in patients receiving chemotherapy and surgical treatment, it is about sevenfold. Oral squamous cell carcinoma (OSCC) is the most common form of oral cancer and represents a multistep process in which environmental factors and genetic alterations are implicated. Thrombotic risk is considered empirically low in OSCC patients, although few data are available. Having limited information available may result in poor awareness of VTE prevention in OSCC, risking jeopardising the oncologic treatment and increasing the morbidity and mortality among these patients. In this paper, the topic of OSCC-associated thrombosis will be discussed.

Long-term outcomes of pediatric infections: from traditional infectious diseases to long Covid.

There is limited evidence available on the long-term impact of SARS-CoV-2 infection in children. In this article, the authors analyze the recent evidence on pediatric long Covid and lessons learnt from a pediatric post-Covid unit in Rome, Italy. To gain a better understanding of the concerns raised by parents and physicians in relation to the potential long-term consequences of this novel infection, it is important to recognize that long-term effect of a post-infectious disease is not a new phenomenon.

The authors analyze the recent evidence on pediatric long Covid and lessons learnt from a pediatric post-Covid unit in Rome, Italy. Also, we analyze the long-term effects of other infectious diseases.

Direct oral anticoagulants and therapeutic adherence: do not let your guard down.

BACKGROUND: Direct oral anticoagulants (DOAC) and vitamin K antagonist drugs (VKA) are recommended for stroke prevention in atrial fibrillation and for treatment of venous thromboembolism. Undoubtedly, DOAC have contributed to improve quality of life of these patients, but unfortunately, available 'real world' data show a very high variable compliance to DOAC. AIMS AND OBJECTIVES: to evaluate predictors that adversely affect therapeutic adherence in patients naive naïve  to DOAC. METHODS AND POPULATION: this study was conducted on an outpatient population in oral anticoagulant therapy in a period between January 2019 and February 2020. Patients naiveto DOAC and treated for at least 6 months were enrolled. Non-Italian-speaking patients, cognitive or psychiatric disorders, refusal to participate or non-consent to the interview were exclusion criteria. A socio-demographic scale and the 8-item Morisky scale (MMAS-8) questionnaire assessed therapeutic adherence. RESULTS: One hundred two DOAC-naïve patients were selected from a population of 407 patients on the first visit at our centre. The population was homogeneously represented for gender (males 48%). The mean age was 79.5 years. Atrial fibrillation (65.7%) resulted the main reason for DOAC prescription and a polypharmacy was detected in 47.1% of the patients. Moreover, an optimal adherence to DOAC therapy was assessed in less than 30% of patients. CONCLUSIONS: Polypharmacy, patient's isolation, such as a low education level were statistically associated with a low therapeutic adherence. Therapeutic adherence remains an unsolved problem for anticoagulated patient. To identify patients at higher risk of poor compliance and therapeutic failure and establish targeted care pathways is a priority.

Direct oral anticoagulants in cancer patients: which help from the specialist hemostasis and thrombosis laboratory?

Oncologists continue to prefer traditional low molecular weight heparins over direct oral anticoagulants (DOAC) for the treatment of venous thromboembolism (VTE) cancer-related. Recently published studies supporting the use DOACs in this patient setting confirm the safety and efficacy of the treatment, but this still does not seem to be enough. The major obstacles to their use are interaction with chemotherapeutic agents, fear of haemorrhage, poor habit of using DOACs for VTE prophylaxis. But what if the haemostasis and thrombosis laboratory reassured oncologists? In this short report we talk about this topic in order to stimulate a fruitful discussion between expert.

Noncanonical type 2B von Willebrand disease associated with mutations in the VWF D'D3 and D4 domains.

We observed a 55-year-old Italian man who presented with mucosal and cutaneous bleeding. Results of his blood analysis showed low levels of von Willebrand factor (VWF) antigen and VWF activity (both VWF ristocetin cofactor and VWF collagen binding), mild thrombocytopenia, increased ristocetin-induced platelet aggregation, and a deficiency of high-molecular-weight multimers, all typical phenotypic hallmarks of type 2B von Willebrand disease (VWD). The analysis of the VWF gene sequence revealed heterozygous in cis mutations: (1) c.2771G>A and (2) c.6532G>T substitutions in the exons 21 and 37, respectively. The first mutation causes the substitution of an Arg residue with a Gln at position 924, in the D'D3 domain. The second mutation causes an Ala to Ser substitution at position 2178 in the D4 domain. The patient's daughter did not present the same fatherly mutations but showed only the heterozygous polymorphic c.3379C>T mutation in exon 25 of the VWF gene causing the p.P1127S substitution, inherited from her mother. The in vitro expression of the heterozygous in cis VWF mutant rVWFWT/rVWF924Q-2178S confirmed and recapitulated the ex vivo VWF findings. Molecular modeling showed that these in cis mutations stabilize a partially stretched and open conformation of the VWF monomer. Transmission electron microscopy and atomic force microscopy showed in the heterozygous recombinant form rVWFWT/rVWF924Q-2178S a stretched conformation, forming strings even under static conditions. Thus, the heterozygous in cis mutations 924Q/2178S promote conformational transitions in the VWF molecule, causing a type 2B-like VWD phenotype, despite the absence of typical mutations in the A1 domain of VWF.

Hemophilia A and von Willebrand deficiency: therapeutic implications.

: Hemophilia A is an X-linked bleeding disorder caused by a deficiency of factor VIII. Depending on the factor VIII activity in patient's plasma, we can have three different forms of hemophilia A: mild (5-40 IU/dl), moderate (1-5 IU/dl) and severe (<1 IU/dl). The most common symptoms include recurrent bleeding episodes of soft tissues and joints. The treatment is based on the prophylactic use of clotting factor concentrates to prevent bleeding episodes. We describe three cases of patients with initially diagnosis of hemophilia A that show different clinical severity, undergoing prophylactic therapies with low benefit. In these patients, the dosage of von Willebrand antigen revealed either low level or absence of this factor, which in one case was caused by the occurrence of a type III form of von Willebrand disease.

Carbamazepine interaction with direct oral anticoagulants: help from the laboratory for the personalized management of oral anticoagulant therapy.

Current guidelines recommend caution in prescribing concomitant use of direct-acting oral anticoagulants (DOACs) and antiepileptic drugs due to drug-drug interactions leading to potential risk of DOACs subtherapeutic concentration and treatment failure. Herein we report a significant interaction between carbamazepine (CZP) and apixaban, causing subtherapeutic concentration of the drug in a patient with atrial fibrillation who had a transient ischemic attack (TIA) episode. Another anti-Xa DOAC, edoxaban, administered to the patient after TIA occurrence did not show significant interaction with CZP. In addition to confirm that cautions should be used when antiepileptic and DOACs are concomitantly prescribed, the present case also demonstrates that, in the management of certain subsets of patients who need anticoagulant treatment, measurement of DOAC plasma concentration can help guide a personalized management and avoid adverse clinical outcomes.

Leukocyte Activation in Obese Patients: Effect of Bariatric Surgery.

The rising prevalence of obesity is a major global health problem. In severe obesity, bariatric surgery (BS) allows to obtain a significant weight loss and comorbidities improvement, among them one of the factors is the thrombotic risk. In this observational study, we measured indices of leukocyte activation in severely obese patients as markers of increased thrombotic risk in relation with serum markers of inflammation before and after BS. Frequency of polymorphonuclear neutrophil-platelet (PLT) and monocyte (MONO)-PLT aggregates as well as of tissue factor (TF) expressing MONOs was measured in the peripheral blood of 58 consecutive obese patients and 30 healthy controls. In 31 of the 58 obese patients, data obtained at the enrollment were compared with those obtained at 3, 6, and 12 months after BS. Compared with healthy controls, obese patients showed a higher frequency of polymorphonuclear leukocyte (PMNL)-PLT aggregates (7.47 ± 2.45 [6.82-8.11]% vs 5.85 ± 1.89 [5.14-6.55]%, P = 0.001), MONO-PLT aggregates (12.31 ± 7.33 [10.38-14.24]% vs 8.14 ± 2.22 [7.31-8.97]%, P < 0.001), and TF expressing MONOs (4.01 ± 2.11 [3.45-4.56]% vs 2.64 ± 1.65 [2.02-3.25]%, P = 0.002). PMNL-PLT and MONO-PLT aggregate frequency was positively correlated with TF expressing MONOs (R2 = 0.260, P = 0.049 and R2 = 0.318, P = 0.015, respectively). BS was performed in 31 patients and induced a significant reduction of the body mass index, and waist and hip circumferences. These effects were associated with a significant decrease of PMNL-PLT aggregates at 12 months (7.58 ± 2.27 [6.75-8.42]% vs 4.47 ± 1.11 [3.93-5.01]%, P < 0.001), and a reduction of TF expressing MONOs at 6 (3.82 ± 2.04 [3.07-4.57]% vs 1.60 ± 1.69 [0.30-2.90]%, P = 0.008) and 12 months (3.82 ± 2.04 [3.07-4.57]% vs 1.71 ± 0.54 [1.45-1.97]%, P = 0.001) after BS.These data suggest that leukocyte-PLT aggregate formation and MONO activation represent an important mechanism underlying the increased thrombotic risk of obese patients. We also show that BS is effective in normalizing these inflammatory indices.

Differential diagnosis of pulmonary embolism in outpatients with non-specific cardiopulmonary symptoms.

Most cardiopulmonary diseases share at least one symptom with pulmonary embolism (PE). The aim of this study was to identify the most common acute causes of dyspnea, chest pain, fainting or palpitations, which diagnostic procedures were performed and whether clinicians investigate them appropriately. An Italian multicenter collaboration gathered 17,497 Emergency Department (ED) records of patients admitted from January 2007 to June 2007 in six hospitals. A block random sampling procedure was applied to select 800 hospitalised patients. Results of the overall 17,497 patients were obtained by weighting sampled cases according to the probability of the randomisation block variables in the whole population. The case-mix of enrolled patients was assessed in terms of cardiopulmonary symptoms, and the prevalence of acute disorders. The actual performance of procedures was compared with a measure of their accuracy as expected in the most common clinical presentations. PE occurred in less than 4% of patients with cardiopulmonary symptoms. Acute heart failure, pneumonia and chronic obstructive pulmonary disease exacerbation were the most likely diagnoses in patients with dyspnea. Acute myocardial infarction was present in roughly 10% of patients with chest pain. Atrial fibrillation was the prevalent diagnosis in patients with palpitations. Echocardiography, computed tomographic pulmonary angiography, perfusion lung scan, D-dimer test and B-type natriuretic peptide were performed less than expected from their accuracy. Diagnostic strategies, starting from non-specific symptoms and coping with the eventuality of PE, are likely to benefit from an increased awareness of the examination's accuracy in discriminating among several competing hypotheses, rather than in testing the single PE suspicion.

Thrombosis in myeloproliferative and myelodysplastic syndromes.

Myeloproliferative neoplasms (MPNs) and myelodisplastic syndromes (MDs) are clonal disorders caused by mutations of myeloid stem cells. Among MPNs, polycythemia vera and essential thrombocythemia are relatively benign disorders in which arterial and venous thromboses represent the main cause of morbidity and mortality. The natural history of MDs is often complicated by both thromboses and haemorrhages, mainly due to platelet quantitative and quantitative anomalies, as well as to treatment complications. In this short review, we focus the attention on the main aspects of thrombophilia in both disorders.

Incidence of thromboembolic complications in patients with atrial fibrillation or mechanical heart valves with a subtherapeutic international normalized ratio: a prospective multicenter cohort study.

Subtherapeutic international normalized ratio (INR) is frequently encountered in clinical practice, and patients with high-risk atrial fibrillation (AF) and with mechanical heart valve (MHV) with inadequate anticoagulation may be exposed to an increased risk of thromboembolic events (TE). However, there are no prospective data evaluating this risk. Consecutive patients with a history of stable anticoagulation, but with a subtherapeutic INR, were prospectively included. Data on use and dose of low-molecular weight heparin (LMWH) bridging therapy were collected. The incidence of objectively confirmed TE and of major bleeding events within 90 days after the index INR was assessed. Five hundred and one patients with INR value 0.5-1 INR units below the lower limit of the patient-specific target INR were included in the study (280 with MHV and 221 with AF and CHADS2 score ≥3). LMWH was prescribed for 64 patients (12.8%). During follow-up, seven patients had a TE (1.40%; 95% confidence interval 0.68, 2.86%; 5.58 events for 100 patients year). All the events occurred within 14 days after the index INR. When we consider only patients who did not receive bridging therapy, the incidence of TE was 1.14% (5 of 437 patients; 95% confidence interval 0.49, 2.64%; 4.58 events for 100 patients year). There were no major bleeding events. The risk of TE in this population was not negligible. Given the frequent observation of subtherapeutic INR levels when monitoring vitamin K antagonists, this finding warrants additional investigation to improve the management of these patients.

Polycythemia vera: gender-related phenotypic differences.

In polycythemia vera, gender has recently been shown to influence the JAK2(V617F) allele burden, but its effect on the disease phenotype is unknown. This issue was investigated using the database of the European Collaboration on Low-dose Aspirin in Polycythemia Vera (ECLAP) Study. The ECLAP Study recruited 1,638 polycythemic subjects and followed for 2.7 ± 1.3 years. At study entry, men, compared to women, had a higher prevalence of myocardial infarction (11.3 vs. 5.8%; P < 0.0001) and peripheral arterial disease (6.1 vs. 2.9%; P < 0.05) while a history of venous thrombosis was more common in women (11.4 vs. 7.9%, P = 0.016). Among 234 venous thrombosis, there were 39 splanchnic vein thromboses (33 extra-hepatic portal vein thromboses and 6 Budd-Chiari syndromes). Most of these events occurred as an early disease presentation in young female subjects. Women, compared to men, had higher platelet counts (average value 430 ± 213 vs. 375 ± 201 × 10(9)/L; P < 0.0001) and lower hematocrits (0.46 ± 0.06 vs. 0.48 ± 0.06 l/l; P < 0.0001). Cholesterol plasma level, available in 995 subjects (61%), was lower in male patients (180.8 ± 43.1 vs. 196 ± 46.6 mg/dl; P < 0.0001). During follow-up there were 205 major thromboses confirming an high incidence of myocardial infarction in men although not statistically significant (1.2 vs. 0.6 cases per 100 person-years; P > 0.05). These data show several gender-related differences both in the thrombotic diathesis and in the prevalence of vascular risk factors of PV patients.