Presentation and progression of MPO-ANCA interstitial lung disease.

The association between MPO-ANCA-associated vasculitis (AAV) and interstitial lung disease (ILD) has been well established. Pulmonary fibrosis may coexist with, follow, or even precede the diagnosis of AAV, and its presence adversely affects the prognosis. The optimal approach to investigating ANCA in patients with ILD remains a subject of ongoing debate. Here we aim to describe presentation and progression of MPO-ANCA ILD. We conducted a retrospective evaluation of a cohort of individuals diagnosed with MPO-ANCA ILD, with or without accompanying renal impairment, at the Immunology and Cell Therapy Unit, Careggi University Hospital, Florence, Italy, between June 2016 and June 2022. Clinical records, imaging studies, pathologic examinations, and laboratory test results were collected. Among the 14 patients identified with MPO-ANCA ILD, we observed a significant association between MPO-ANCA titers assessed at the time of ILD diagnosis and renal involvement. Renal impairment in these cases often manifested as subclinical or slowly progressive kidney damage. Interestingly, complement C3 deposits were consistently found in all renal biopsy specimens, thereby suggesting the potential for novel therapeutic targets in managing renal complications associated with MPO-ANCA ILD. The presentation of MPO-ANCA vasculitis as ILD can be the first and only clinical manifestation. MPO-ANCA levels at ILD diagnosis could warn on the progression to renal involvement in patients with MPO-ANCA ILD, hence caution is needed because renal disease can be subclinical or smoldering.

Thrombosis and Bleeding in Patients with Vaccine-Induced Immune Thrombotic Thrombocytopenia: A Systematic Review of Published Cases.

INTRODUCTION: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a highly prothrombotic reaction to COVID-19 (coronavirus disease 2019) adenoviral vector vaccines. Its distinct bleeding and thrombotic patterns compared with other platelet consumptive disorders remain unclear. METHODS: We performed a systematic review of the literature (PubMed and Embase) up to July 31, 2022, including case reports and case series providing nonaggregate data of VITT patients. Accurate VITT diagnosis required fulfillment of the following criteria: (1) endorsement by the authors, (2) consistent vaccine type and timing, (3) presence of thrombocytopenia and thrombosis, (4) detection of anti-platelet factor 4 antibodies. Data are presented as frequencies with 95% confidence intervals (CIs) calculated with the exact binomial method. RESULTS: We retrieved 143 eligible studies, describing 366 patients. Of 647 thrombotic events, 53% (95% CI: 49-56) were venous thromboses at unusual sites and 30% (95% CI: 27-34) were cerebral venous sinus thromboses (CVSTs). The ratio of venous-to-arterial events was 4.1. Thromboses in most sites were associated with at least another thrombotic event, with the exception of CVST and CNS arterial thrombosis (isolated in 49 and 39% of cases, respectively). Bleeding occurred in 36% (95% CI: 31-41) of patients; 68% (95% CI: 59-75) of bleeding events were intracranial hemorrhages (ICHs). Overall mortality was 24% (95% CI: 19-29), and 77% (95% CI: 58-90) in patients with isolated CVST complicated by ICH. CONCLUSION: VITT displays a venous-to-arterial thrombosis ratio comparable to heparin-induced thrombocytopenia. However, VITT is characterized by a higher prevalence of CVST and ICH, which contribute to the increased bleeding frequency and mortality.

Impairment of platelet function in both mild and severe COVID-19 patients.

Abnormalities of platelet function were reported in patients with severe COVID-19 (severe-C), but few data are available in patients with mild COVID-19 (mild-C) and after COVID-19 recovery. The aim of this study was to investigate platelet parameters in mild-C patients (n = 51), with no evidence of pneumonia, and severe-C patients (n = 49), during the acute phase and after recovery, compared to 43 healthy controls. Both mild-C and severe-C patients displayed increased circulating activated platelets, low δ-granule content (ADP, serotonin), impaired platelet activation by collagen (light transmission aggregometry) and impaired platelet thrombus formation on collagen-coated surfaces under controlled flow conditions (300/s shear rate). The observed abnormalities were more marked in severe-C patients than in mild-C patients. Overall, 61% (30/49) of mild-C and 73% (33/45) of severe-C patients displayed at least one abnormal platelet parameter. In a subgroup of just 13 patients who showed no persisting signs/symptoms of COVID-19 and were re-evaluated at least 1 month after recovery, 11 of the 13 subjects exhibited normalization of platelet parameters. In conclusion, mild abnormalities of platelet parameters were present not only in severe-C but also, albeit to a lesser extent, in mild-C patients during the acute phase of COVID-19 and normalized in most tested patients after clinical recovery.

Production of anti-PF4 antibodies in antiphospholipid antibody-positive patients is not affected by COVID-19 vaccination.

BACKGROUND: Antibodies against cationic platelet chemokine, platelet factor 4 (PF4/CXCL4), have been described in heparin-induced thrombocytopenia (HIT), but also in patients positive for antiphospholipid antibodies (aPL) even in the absence of heparin treatment and HIT-related clinical manifestations. Anti-PF4 antibodies have been recently described also in subjects who developed thrombosis with thrombocytopenia syndrome (TTS) in association with adenoviral vector-based, but not with mRNA-based, COVID-19 vaccines. OBJECTIVE: To investigate whether COVID-19 vaccination affects the production of anti-PF4 antibodies in aPL-positive patients and in control groups. METHODS: Anti-PF4 immunoglobulins were detected in patients' and controls' serum samples by ELISA and their ability to activate normal platelets was assessed by the platelet aggregation test. RESULTS: Anti-PF4 were found in 9 of 126 aPL-positive patients, 4 of 50 patients with COVID-19, 9 of 49 with other infections, and 1 of 50 aPL-negative patients with systemic lupus erythematosus. Clinical manifestations of TTS were not observed in any aPL patient positive for anti-PF4, whose serum failed to cause platelet aggregation. The administration of COVID-19 vaccines did not affect the production of anti-PF4 immunoglobulins or their ability to cause platelet aggregation in 44 aPL-positive patients tested before and after vaccination. CONCLUSIONS: Heparin treatment-independent anti-PF4 antibodies can be found in aPL-positive patients and asymptomatic carriers, but their presence, titre as well as in vitro effect on platelet activation are not affected by COVID-19 vaccination.

The Development of Extra-Articular Manifestations in Children With Enthesitis-Related Arthritis: Natural Course or Different Disease Entity?

Introduction: Enthesitis-related Arthritis (ERA) is a specific category of juvenile idiopathic arthritis (JIA) characterized by axial and/or peripheral arthritis, and enthesitis, although other different extra-articular manifestations may encompass its clinical spectrum. Materials and Methods: In order to examine if ERA-JIA with extra-articular involvement may represent a different entity from ERA without extra-articular involvement, we performed a retrospective, observational, monocentric study, in a cohort of ERA patients followed between 2001 and September 2020 at the Pediatric Rheumatology Unit of Meyer Children Hospital of Florence. We analyzed the demographic, clinical, laboratory and imaging data at the disease onset, as well as after 3, 6, and 12 months follow up. Results: We have enrolled 53 patients, 33 males. At the time of diagnosis, average age was 10.9 years, 53 patients had active arthritis and 25 active enthesitis. The middle foot involvement was present in 20 patients. Twenty-five children achieved clinical remission on medication. Extra-articular manifestations were observed in 14 patients, of whom 3 had inflammatory bowel disease, 5 uveitis, one uveitis associated with Crohn disease, 4 SAPHO syndrome, one celiac disease. The cohort was stratified according to the presence/absence of extra-articular manifestations. It was observed that middle foot involvement was more frequent in patients with no extra-articular manifestations (18/39 vs. 2/14; χ2 = 4.45, p = 0.05). Additionally, patients presenting extra-articular manifestation needed more frequently (12/14 vs. 21/39, χ2= 4.45, p = 0.05), and preciously (months: 3.7 ± 5.4 vs. 16.7 ± 26.5, p = 0.02), treatment with biologic agents. Finally, these patients achieved belatedly (months: 31.6 ± 32.3 vs. 22.9 ± 18.3, p = 0.01) and less frequently (3/14 vs. 22/39; χ2= 5.50, p = 0.03) the clinical remission on medication. Eventually, extra-articular involvement inversely correlated with the middle-foot arthritis (ρs -0.29, p = 0.03), the chance to achieve remission on medication (ρs -0.31 e p = 0.02), as well as the chance to keep overall remission, with and without medication (ρs -0.28, p = 0.04). Conclusion: In our cohort, children diagnosed with ERA-JIA at the onset of disease and then developed extra-articular manifestations show the absence of middle foot involvement and worse prognosis with an early need for the use of biologic agents, and overall low chance to achieve remission.

In-vitro and in-vivo metabolism of different aspirin formulations studied by a validated liquid chromatography tandem mass spectrometry method.

Low-dose aspirin (ASA) is used to prevent cardiovascular events. The most commonly used formulation is enteric-coated ASA (EC-ASA) that may be absorbed more slowly and less efficiently in some patients. To uncover these "non-responders" patients, the availability of proper analytical methods is pivotal in order to study the pharmacodynamics, the pharmacokinetics and the metabolic fate of ASA. We validated a high-throughput, isocratic reversed-phase, negative MRM, LC-MS/MS method useful for measuring circulating ASA and salicylic acid (SA) in blood and plasma. ASA-d4 and SA-d4 were used as internal standards. The method was applied to evaluate: (a) the "in vitro" ASA degradation by esterases in whole blood and plasma, as a function of time and concentration; (b) the "in vivo" kinetics of ASA and SA after 7 days of oral administration of EC-ASA or plain-ASA (100 mg) in healthy volunteers (three men and three women, 37-63 years). Parameters of esterases activity were Vmax 6.5 ± 1.9 and Km 147.5 ± 64.4 in plasma, and Vmax 108.1 ± 20.8 and Km 803.2 ± 170.7 in whole blood. After oral administration of the two formulations, tmax varied between 3 and 6 h for EC-ASA and between 0.5 and 1.0 h for plain-ASA. Higher between-subjects variability was seen after EC-ASA, and one subject had a delayed absorption over eight hours. Plasma AUC was 725.5 (89.8-1222) for EC-ASA, and 823.1(624-1196) ng h/mL (median, 25-75% CI) for plain ASA. After the weekly treatment, serum levels of TxB2 were very low (< 10 ng/mL at 24 h from the drug intake) in all the studied subjects, regardless of the formulation or the tmax. This method proved to be suitable for studies on aspirin responsiveness.

Patients with Essential Thrombocythemia may be Poor Responders to Enteric-Coated Aspirin, but not to Plain Aspirin.

Essential thrombocythemia (ET) patients are treated with aspirin (acetylsalicylic acid [ASA]) to prevent thrombosis. Previous studies showed that serum thromboxane (Tx) B2 was high 24 hours after enteric-coated (EC)-ASA in ET patients, due to increased number of noninhibited reticulated platelets (RPs), consequent to high platelet turnover, and that ASA should be given twice a day to ET patients. We studied ET patients (n = 17) and healthy subjects (n = 10) on 100 mg EC-ASA once daily; experiments were repeated after 14-day treatment with 100 mg plain-ASA once daily. Serum TxB2, plasma ASA, and salicylic acid (SA) were measured before the morning dose and up to 8 hours thereafter. Blood activity of ASA-deacethylating esterases, in vitro inhibition of collagen-induced TxB2 production by ASA (10-1,000 µM), and number of RP were measured. TxB2 inhibition by ASA in vitro and esterases activities were normal in all subjects. EC-ASA elicited highly variable responses; 6 ET patients were poor responders, as their serum TxB2 was high after EC-ASA; their plasma levels of ASA and SA were low/undetectable. In contrast to EC-ASA, plain ASA decreased serum TxB2 and increased plasma ASA and SA in all subjects. Serum TxB2 was high in ET patients at 24 hours and significantly correlated with RP count (but not RP percentage) and platelet count. Plain ASA should be used in ET patients to inhibit platelets efficiently. The identification of ET patients who might benefit from twice a day ASA could simply be based on their platelet count: since their platelet turnover is not increased, ET patients with normalized platelet count should not need twice a day ASA treatment.

The MICELI (MICrofluidic, ELectrical, Impedance): Prototyping a Point-of-Care Impedance Platelet Aggregometer.

As key cellular elements of hemostasis, platelets represent a primary target for thrombosis and bleeding management. Currently, therapeutic manipulations of platelet function (antithrombotic drugs) and count (platelet transfusion) are performed with limited or no real-time monitoring of the desired outcome at the point-of-care. To address the need, we have designed and fabricated an easy-to-use, accurate, and portable impedance aggregometer called "MICELI" (MICrofluidic, ELectrical, Impedance). It improves on current platelet aggregation technology by decreasing footprint, assay complexity, and time to obtain results. The current study aimed to optimize the MICELI protocol; validate sensitivity to aggregation agonists and key blood parameters, i.e., platelet count and hematocrit; and verify the MICELI operational performance as compared to commercial impedance aggregometry. We demonstrated that the MICELI aggregometer could detect platelet aggregation in 250 µL of whole blood or platelet-rich plasma, stimulated by ADP, TRAP-6, collagen, epinephrine, and calcium ionophore. Using hirudin as blood anticoagulant allowed higher aggregation values. Aggregation values obtained by the MICELI strongly correlated with platelet count and were not affected by hematocrit. The operational performance comparison of the MICELI and the Multiplate® Analyzer demonstrated strong correlation and similar interdonor distribution of aggregation values obtained between these devices. With the proven reliability of the data obtained by the MICELI aggregometer, it can be further translated into a point-of-care diagnostic device aimed at monitoring platelet function in order to guide pharmacological hemostasis management and platelet transfusions.

Platelet Adhesion and Thrombus Formation in Microchannels: The Effect of Assay-Dependent Variables.

Microfluidic flow chambers (MFCs) allow the study of platelet adhesion and thrombus formation under flow, which may be influenced by several variables. We developed a new MFC, with which we tested the effects of different variables on the results of platelet deposition and thrombus formation on a collagen-coated surface. METHODS: Whole blood was perfused in the MFC over collagen Type I for 4 min at different wall shear rates (WSR) and different concentrations of collagen-coating solutions, keeping blood samples at room temperature or 37 °C before starting the experiments. In addition, we tested the effects of the antiplatelet agent acetylsalicylic acid (ASA) (antagonist of cyclooxygenase-1, 100 µM) and cangrelor (antagonist of P2Y12, 1 µM). RESULTS: Platelet deposition on collagen (I) was not affected by the storage temperature of the blood before perfusion (room temperature vs. 37 °C); (II) was dependent on a shear rate in the range between 300/s and 1700/s; and (III) was influenced by the collagen concentration used to coat the microchannels up to a value of 10 µg/mL. ASA and cangrelor did not cause statistically significant inhibition of platelet accumulation, except for ASA at low collagen concentrations. CONCLUSIONS: Platelet deposition on collagen-coated surfaces is a shear-dependent process, not influenced by the collagen concentration beyond a value of 10 µg/mL. However, the inhibitory effect of antiplatelet drugs is better observed using low concentrations of collagen.

Blood Cell-Bound C4d as a Marker of Complement Activation in Patients With the Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is a chronic and disabling condition characterized by recurrent thrombosis and miscarriages mediated by antibodies against phospholipid-binding proteins (aPL), such as beta2glycoprotein I (ß2GPI). Complement is involved in APS animal models and complement deposits have been documented in placenta and thrombotic vessels despite normal serum levels. Analysis of circulating blood cells coated with C4d displays higher sensitivity than the conventional assays that measure soluble native complement components and their unstable activation products in systemic lupus erythematosus (SLE). As C4d-coated blood cell count has been reported to be more sensitive than serum levels of complement components and their activation products in systemic lupus erythematosus (SLE) patients, we decided to evaluate the percentage of C4d positive B lymphocytes (BC4d), erythrocytes (EC4d), and platelets (PC4d) in primary APS patients and asymptomatic aPL positive carriers as marker of complement activation in APS. We assessed by flow cytometry the percentages of BC4d, EC4d, and PC4d in primary APS (PAPS; n. 23), 8 asymptomatic aPL positive carriers, 11 APS-associated SLE (SAPS), 17 aPL positive SLE, 16 aPL negative SLE, 8 aPL negative patients with previous thrombosis, 11 immune thrombocytopenia (ITP) patients, and 26 healthy subjects. In addition, we used an in vitro model to evaluate the ability of a monoclonal anti-ß2GPI antibody (MBB2) to bind to normal resting or activated platelets and fix complement. EC4d and PC4d percentages were significantly higher in PAPS and aPL carriers as well as aPL positive SLE and SAPS than in aPL negative controls. The highest values were found in PAPS and in SAPS. The EC4d and PC4d percentages were significantly correlated with serum C3/C4 and anti-ß2GPI/anti-cardiolipin IgG. In vitro studies showed that MBB2 bound to activated platelets only and induced C4d deposition. The detection of the activation product C4d on circulating erythrocytes and platelets supports the role of complement activation in APS. Complement may represent a new therapeutic target for better treatment and prevention of disability of APS patients.

Aggregometry in the settings of thrombocytopenia, thrombocytosis and antiplatelet therapy.

A variety of laboratory tests have been developed, which can diagnose a number of both congenital and acquired disorders of platelet function. Many tests of platelet function measure the ability of platelets to adhere to each other, forming platelet aggregates, which represent the major constituents of hemostatic plugs and of arterial thrombi. Light transmission aggregometry (LTA) is still considered the gold standard of platelet aggregation tests, but other platelet aggregation-based tests are also available. Among them, the flow cytometry-based methods may be more convenient than LTA for the study of patients with very low or very high platelet counts. The use of platelet aggregation tests has also been advocated to monitor the treatment with antiplatelet agents (mostly the P2Y12 antagonist clopidogrel) of patients with thrombotic arterial occlusions, with the aim of improving their efficacy and safety. However, randomized clinical trials failed to show any advantage of this strategy; as a consequence, international guidelines now recommend against laboratory monitoring of antiplatelet therapy.

Reduced platelet count, but no major platelet function abnormalities, are associated with loss-of-function ATP-binding cassette-1 gene mutations.

Loss-of-function mutations of the the ATP-binding cassette-1 (ABCA1) gene are the cause of Tangier disease (TD) in homozygous subjects and familial HDL deficiency (FHD) in heterozygous subjects. These disorders are characterized by reduced plasma HDL-cholesterol (HDL-C) and altered efflux of cholesterol from cells. Previous studies in TD patients and ABCA1-/- murine models reported defects in platelet count, morphology, and function, but the issue is still controversial. We analyzed three subjects with low to very low HDL-C levels due to the loss-of-function mutations of the ABCA1 gene. Two related patients with FHD were heterozygous carriers of two mutations on the same ABCA1 allele; one, with TD, was homozygous for a different mutation. Mild to moderate thrombocytopenia was observed in all the patients. No morphological platelet abnormalities were detected under optical or EM. History of moderate bleeding tendency was recorded only in one of the FHD patients. Only limited alterations in platelet aggregation and activation of the integrin αIIbß3 were observed in one FHD patient. While α-granule secretion (P-selectin), content, and secretion of platelet δ-granules (serotonin, ATP, and ADP) and thromboxane (TX) A2 synthesis were normal in all the patients, the expression of lysosomal CD63, in response to some agonists, was reduced in TD patients. In conclusion, three patients carrying ABCA1 genetic variants had low platelet count, with the lowest values observed in TD, not associated with major alterations in platelet morphology and response to agonists or bleeding.