Pathogenesis of immune thrombocytopenia in common variable immunodeficiency.

Immune Thrombocitopenic Purpura (ITP) is an autoimmune disease characterized by antibody-mediated platelet destruction and variable reduced platelet production. Besides antibody-mediated platelet destruction, new pathogenic mechanisms have been reported to be involved in reducing platelet count. Among these, desialylation is one of the most recent and innovative mechanisms that has been found to be implied, at least in part, in non-antibody mediated platelet clearance. Common Variable Immunodeficiency (CVID) is the most common Primary Immunodeficiency seen in clinical practice. About 25-30% of CVID patients are affected by autoimmune manifestation, among which ITP is the most common. Little is know about pathophysiological mechanisms that lead to ITP in CVID. Given the poor antibody production typical of CVID patients, we aimed at verifying whether platelet desialylation could be responsible for CVID associated thrombocytopenia. According to our results, we may suggest that in CVID patients, ITP is due to a decreased bone marrow platelets production, rather than an increased peripheral platelet destruction, which is more common in patients with primary ITP. An increased platelet desialylation does not appear to be implicated in the thrombocytopenia secondary to CVID, while it is implicated in the pathogenesis of primary ITP. Nevertheless an intriguing aspect has emerged from this study: regardless the presence of thrombocytopenia, the majority of CVID patients present a double platelet population as far as desialylation concerns, whilst no one of the healthy donors and of the patients with primary ITP shows a similar characteristic.

An unusual case of sodium citrate-dependent artifactual platelet count.

Background: Ethylenediaminetetraacetic acid (EDTA)-dependent pseudothrombocytopenia is a rare phenomenon. Spurious pseudothrombocytopenia has also been described in other circumstances, while artifactual platelet count in whole blood samples anticoagulated with sodium citrate is an exceptional occurrence. Case report: In this study, we describe the case of a 44-year-old ostensibly healthy woman who attended the local outpatient clinic for routine laboratory testing, including platelet count in EDTA and sodium citrate, for suspected artifactual pseudothrombocytopenia previously identified in another center. The results of hematological testing on both specimens were essentially normal, except for mild anemia. Nevertheless, the platelet number was 425 × 109/L in K2EDTA and 266 × 109/L (293 × 109/L after correcting for sample dilution) in sodium citrate, respectively. Microscopic revision of blood smears revealed the presence of platelet aggregates and satellitism only in the sodium citrate specimen. Conclusion: Unlike previous occasional reports of concomitant EDTA- and sodium citrate-dependent pseudothrombocytopenia, we first describe a paradigmatic case of artifactual platelet count attributable to platelet clumping and satellitism, exclusively developing in blood anticoagulated with sodium citrate.

Eltrombopag for the treatment of inherited thrombocytopenias: a phase II clinical trial.

Patients with inherited thrombocytopenias often require platelet transfusions to raise their platelet count before surgery or other invasive procedures; moreover, subjects with clinically significant spontaneous bleeding may benefit from an enduring improvement of thrombocytopenia. The hypothesis that thrombopoietin-mimetics can increase platelet count in inherited thrombocytopenias is appealing, but evidence is scarce. We conducted a prospective, phase II clinical trial to investigate the efficacy of the oral thrombopoietin-mimetic eltrombopag in different forms of inherited thrombocytopenia. We enrolled 24 patients affected by MYH9-related disease, ANKRD26-related thrombocytopenia, X-linked thrombocytopenia/ Wiskott-Aldrich syndrome, monoallelic Bernard-Soulier syndrome, or ITGB3-related thrombocytopenia. The average pre-treatment platelet count was 40.4 ×109/L. Patients received a 3- to 6-week course of eltrombopag in a dose-escalated manner. Of 23 patients evaluable for response, 11 (47.8%) achieved a major response (platelet count >100 ×109/L), ten (43.5%) had a minor response (platelet count at least twice the baseline value), and two patients (8.7%) did not respond. The average increase of platelet count compared to baseline was 64.5 ×109/L (P<0.001). Four patients with clinically significant spontaneous bleeding entered a program of long-term eltrombopag administration (16 additional weeks): all of them obtained remission of mucosal hemorrhages, with the remission persisting throughout the treatment period. Treatment was globally well tolerated: five patients reported mild adverse events and one patient a moderate adverse event. In conclusion, eltrombopag was safe and effective in increasing platelet count and reducing bleeding symptoms in different forms of inherited thrombocytopenia. Despite these encouraging results, caution is recommended when using thrombopoietinmimetics in inherited thrombocytopenias predisposing to leukemia. ClinicalTrials.gov identifier: NCT02422394.

Is triple-positive serology for Epstein-Barr virus (VCA-IgG, VCA-IgM, EBNA-IgG) a specific feature of angioimmunoblastic T-cell lymphoma?

PURPOSE: We assessed the frequency of triple-positive serology (viral capsid antigen [VCA]-immunoglobulin G [IgG], VCA-immunoglobulin M, Epstein-Barr nuclear antigen-IgG) for Epstein-Barr virus (EBV) in a small number of patients with angioimmunoblastic T-cell lymphoma (AITL) at disease onset. METHODS: Nine patients with newly diagnosed AITL were retrospectively enrolled in the present study. For all of them, EBV serology data were available. RESULTS: Of 9 patients, 7 (77.7%) had a triple-positive serology (VCA-IgG, VCA-IgM, EBNA-IgG ) for EBV. These patients were characterized by bone marrow involvement, high incidence of thrombocytopenia, and poor prognosis according to Revised International Prognostic Index and Prognostic Index for Angioimmunoblastic T-cell Lymphoma scores. CONCLUSION: Assessment of both viremia and serology for EBV could be useful in patients with clinical and laboratory data suggesting lymphoma diagnosis; furthermore, although our data need to be validated in a larger cohort of patients, triple positivity for EBV serology might help to direct the diagnosis toward AITL.

Calcium-Dependent Src Phosphorylation and Reactive Oxygen Species Generation Are Implicated in the Activation of Human Platelet Induced by Thromboxane A2 Analogs.

The thromboxane (TX) A2 elicits TP-dependent different platelet responses. Low amounts activate Src kinases and the Rho-Rho kinase pathway independently of integrin αIIbß3 and ADP secretion and synergize with epinephrine to induce aggregation. Aim of the present study was to investigate the role Src kinases and the interplay with calcium signals in reactive oxygen species (ROS) generation in the activatory pathways engaged by TXA2 in human platelets. All the experiments were performed in vitro or ex vivo. Washed platelets were stimulated with 50-1000 nM U46619 and/or 10 µM epinephrine in the presence of acetylsalicylic acid and the ADP scavenger apyrase. The effects of the ROS scavenger EUK-134, NADPH oxidase (NOX) inhibitor apocynin, Src kinase inhibitor PP2 and calcium chelator BAPTA were tested. Intracellular calcium and ROS generation were measured. Platelet rich plasma from patients treated with dasatinib was used to confirm the data obtained in vitro. We observed that 50 nM U46619 plus epinephrine increase intracellular calcium similarly to 1000 nM U46619. ROS generation was blunted by the NOX inhibitor apocynin. BAPTA inhibited ROS generation in resting and activated platelets. Phosphorylation of Src and MLC proteins were not significantly affected by antioxidants agents. BAPTA and antioxidants reduced P-Selectin expression, activation of integrin αIIbß3and platelet aggregation. TXA2-induced increase in intracellular calcium is required for Src phosphorylation and ROS generation. NADPH oxidase is the source of ROS in TX stimulated platelets. The proposed model helps explain why an incomplete inhibition of TP receptor results in residual platelet activation, and define new targets for antiplatelet treatment.

Primary sphenoid lymphoma: Focus on imaging.

Primary lymphoma of the sphenoid is an extremely rare pathology, therefore it is difficult to hypothesize and the imaging characteristics are not well-known. Here we report the imaging features in computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET) scan of a 44-year-old patient who presented with severe headache. CT and MRI showed a sphenoid sinus mass that suggested rhinopharyngeal lesion or a chordoma. However, biopsy from the mass histologically proved it to be Diffuse large B-cell lymphoma and PET examinations revealed increased fluorodeoxyglucose uptake around the sphenoid bone and multiple spinal lesions.

CT imaging of primary pancreatic lymphoma: experience from three referral centres for pancreatic diseases.

PURPOSE: To describe CT characteristics of primary pancreatic lymphoma (PPL), a rare disease with features in common with adenocarcinoma. MATERIALS AND METHODS: Fourteen patients were enrolled. CT: unenhanced scan, contrast-enhanced pancreatic and venous phases. Image analysis: tumour location; peri-pancreatic vessel encasement; necrosis; enlarged lymph nodes; fat stranding; enlarged bile duct and pancreatic duct; neoplasm longest dimension, volume and density. RESULTS: Histopathological diagnoses: follicular non-Hodgkin lymphoma (5/14), diffuse large B-cell lymphoma (6/14) and high-grade B-cell lymphoma not otherwise specified (3/14). Six of 14 PPLs were located in the pancreatic head and 7/14 in the body-tail; 1/14 involved the whole gland. In 5/14 cases the superior mesenteric artery and vein were encased; splenic vein and artery encasement was depicted in 2 PPLs. Necrosis was present in 2/14. Enlarged retroperitoneal lymph nodes were found in 11 cases and fat stranding in all patients. The bile duct was dilated in six cases and the pancreatic duct in five. Mean neoplasm longest diameter and volume were 8.05 cm and 210.8 cm3. Mean tumour attenuation values were 39.1 HU at baseline, 60.6 HU in the pancreatic phase and 71.4 HU in the venous phase. CONCLUSIONS: PPL presents as a large mass lesion with delayed homogeneous enhancement; peri-pancreatic fat stranding and vessel encasement are present, without vascular infiltration. Pancreatic duct dilatation is rare. KEY POINTS: • Primary pancreatic lymphoma (PPL) is a rare haematological disease • PPL presents imaging features in common with pancreatic carcinoma but also some distinctive findings • The majority of PPLs are large lesions with delayed homogeneous enhancement • Peri-pancreatic fat stranding and vessel encasement are common in PPL • Vascular infiltration and pancreatic duct dilatation are rare in PPL.

Observational retrospective study of vascular modulator changes during treatment in essential thrombocythemia.

Essential thrombocythemia (ET) patients are at risk of developing thrombotic events. Qualitative platelet (PLT) abnormalities and activation of endothelial cells (ECs) and PLTs are thought to be involved. Microparticles (MPs) can originate from PLTs (PMPs), ECs (EMPs), or red cells (RMPs). Previous studies have indicated that MPs contribute to ET pathophysiology. Endothelial modulators (eg, nitric oxide [NO], adrenomedullin [ADM], and endothelin-1 [ET-1]) are also involved in the pathophysiology of this condition. We hypothesized that treatments for reducing PLT count might also indirectly affect MP generation and endothelial activity by altering endothelial modulator production. The rationale of this study was that hydroxyurea (HU), a cytostatic drug largely used in ET, induces the production of a potent vasoactive agent NO in ECs. An observational retrospective study was designed to investigate the relationship between MPs, NO, ADM, and ET-1 in ET patients on treatment with HU, anagrelide (ANA), aspirin (ASA), and a group of patients before treatment. A total of 63 patients with ET diagnosis: 18 on HU + ASA, 15 on ANA + ASA, 19 on ASA only, and 11 untreated patients, and 18 healthy controls were included in this study. Blood samples were analyzed for MP (absolute total values) and functional markers (percentage values) by flow cytometry. PLT-derived MPs were studied using CD61, CD62P, CD36, and CD63, whereas endothelial-derived MPs were studied using CD105, CD62E, and CD144. Endothelial modulator markers (NO, ADM, and ET-1) were measured by ELISA. Total MP count was higher in the group treated with ANA + ASA (P < 0.01). MP markers modified in ET patients returned to levels of healthy controls following treatment, in particular, in patients on ANA treatment. NO and ADM values were higher in the HU group (P < 0.001). HU and ANA treatment also affected MP production in a cell origin-specific manner. HU and ANA, although acting via different pathways, have similar final effects. For instance, HU causes vasodilatation by increasing NO and ADM levels, whereas ANA impairs vasoconstriction by reducing ET-1. In conclusion, therapy with HU cytostatic drugs and ANA can reduce PLT count in ET, and also affect endothelial modulatory agents, with HU sustaining vasodilation and prothrombotic MP concentration, whereas ANA decreases vasoconstriction.

Thrombocytopenia and infections.

INTRODUCTION: Acquired thrombocytopenia recognizes a myriad of causes. Among these, infectious diseases play a relevant role since a low platelet count is commonplace along with other abnormal laboratory data. Areas covered: This narrative review, after a brief presentation of the possible pathogenic mechanisms, is focused on the most prevalent infections associated with thrombocytopenia, namely those attributable to hepatitis C virus (HCV), human immunodeficiency virus (HIV) and Helicobacter pylori. Expert commentary: An underlying HCV or HIV infection should always be suspected in patients at risk who present with isolated thrombocytopenia. The eradication of Helicobacter pylori is advisable in infected patients with secondary immune thrombocytopenia, because this will increase the platelet count in a substantial number of cases, thus avoiding more aggressive and prolonged treatments.

Italian daily platelet transfusion practice for haematological patients undergoing high dose chemotherapy with or without stem cell transplantation: a survey by the GIMEMA Haemostasis and Thrombosis Working Party.

BACKGROUND: Following high-dose chemotherapy/bone marrow transplantation, patients are routinely, prophylactically transfused with platelet concentrates (PC) if they have a platelet count ≤10×109/L or higher in the presence of risk factors for bleeding. However, whether such transfusions are necessary in clinically stable patients with no bleeding, or whether a therapeutic transfusion strategy could be sufficient and safe, is still debated. MATERIALS AND METHODS: The GIMEMA Haemostasis and Thrombosis Working Party sent a questionnaire to Italian haematology departments to survey several aspects of daily platelet transfusion practice, such as the cut-off platelet count for transfusion, the evaluation of refractoriness and the type of PC administered. RESULTS: The questionnaire was answered by 18 out of 31 centres (58%). A total of 23,162 PC were transfused in 2,396 patients in 2013. The vast majority of centres (95%) transfused PC according to Italian and international guidelines; only a few transfused always at platelet counts ≤20×109/L. The broad agreement on platelet count cut-off for transfusion (≤10×109/L) was not confirmed when the World Health Organization (WHO) bleeding score was considered: only a third of centres (33%) used transfusions as recommended when the bleeding grade was ≥2. Platelet refractoriness was poorly monitored and most centres (89%) evaluated, mostly empirically (67%), response to transfusion only 24 hours later. Thirty percent of centres transfused platelets in asymptomatic refractory patients. DISCUSSION: Although most Italian haematology departments transfuse PC according to Italian and international guidelines, our survey shows that in routine daily practice physicians do not comply closely with the WHO recommendations on platelet transfusions and monitoring platelet refractoriness. This causes excessive platelet transfusions, with a resulting increase of costs and waste of public health resources.

Autoimmunity and infection in common variable immunodeficiency (CVID).

Common variable immunodeficiency (CVID) is a heterogeneous group of diseases, characterized by primary hypogammaglobulinemia. B and T cell abnormalities have been described in CVID. Typical clinical features of CVID are recurrent airway infections; lymphoproliferative, autoinflammatory, or neoplastic disorders; and autoimmune diseases among which autoimmune thrombocytopenia (ITP) is the most common. The coexistence of immunodeficiency and autoimmunity appears paradoxical, since one represents a hypoimmune state and the other a hyperimmune state. Considering both innate and adaptive immune response abnormalities in CVID, it is easier to understand the mechanisms that lead to a breakdown of self-tolerance. CD21(low) B cells derive from mature B cells that have undergone chronic immune stimulation; they are increased in CVID patients. The expansion of CD21(low) B cells is also observed in certain autoimmune diseases. We have studied CD21(low) B cells in patients with CVID, CVID, and ITP and with ITP only. We observed a statistically significant increase in the CD21(low) population in the three pathological groups. Moreover, we found statistical differences between the two groups of CVID patients: patients with ITP had a higher percentage of CD21(low) cells. Our data suggest that CD21(low) cells are related to autoimmunity and may represent a link between infection and autoimmunity.

Thrombopoietin receptor agonists for preparing adult patients with immune thrombocytopenia to splenectomy: results of a retrospective, observational GIMEMA study.

In patients with immune thrombocytopenia (ITP) refractory to corticosteroids and intravenous immunoglobulins (IVIG), splenectomy may result at higher risk of peri-operative complications and, for this reason, potentially contraindicated. The thrombopoietin receptor agonists (TPO-RAs) romiplostim and eltrombopag have shown high therapeutic activity in primary ITP, but data of efficacy and safety regarding their use in preparation for splenectomy are missing. Thirty-one adult patients, median age 50 years, with corticosteroids and/or IVIG refractory persistent and chronic ITP who were treated with TPO-RAs (romiplostim= 24; eltrombopag= 7) with the aim to increase platelet count and allow a safer execution of splenectomy were retrospectively evaluated. Twenty-four patients (77%) responded to the use of TPO-RAs with a median platelet count that increased from 11 × 10(9) /L before starting TPO-RAs to 114 × 10(9) /L pre-splenectomy, but a concomitant treatment with corticosteroids and/or IVIG was required in 19 patients. Twenty-nine patients underwent splenectomy while two patients who responded to TPO-RAs subsequently refused surgery. Post-splenectomy complications were characterized by two Grade 3 thrombotic events (1 portal vein thrombosis in the patient with previous history of HCV hepatitis and 1 pulmonary embolism), with a platelet count at the time of thrombosis of 260 and 167 × 10(9) /L, respectively and one Grade 3 infectious event. TPO-RAs may represent a therapeutic option to improve platelet count and reduce the risk of peri-operative complications in ITP candidates to splenectomy. An increased risk of post-splenectomy thromboembolic events cannot be ruled out and thromboprophylaxis with low-molecular weight heparin is generally recommended.

Role of blood cells dynamism on hemostatic complications in low-risk patients with essential thrombocythemia.

Patients with essential thrombocythemia (ET) aged less than 60 years, who have not suffered a previous vascular event (low-risk patients), may develop thrombotic or hemorrhagic events. So far, it has not been possible to identify useful markers capable of predicting which of these patients are more likely to develop an event and therefore who needs to be treated. In the present study, we analysed the relationship between vascular complications and longitudinal blood counts of 136 low-risk ET patients taken over a sustained period of time (blood cells dynamism). After a median follow-up of 60 months, 45 out of 136 patients (33%) suffered 40 major thrombotic and 5 severe hemorrhagic complications. A total number of 5,781 blood counts were collected longitudinally. Thrombotic and hemorrhagic events were studied together (primary endpoint) but also separately (thrombotic alone = secondary endpoint; hemorrhagic alone = tertiary endpoint). The primary endpoint showed no significant association between platelet and WBC count at diagnosis and risk of any event (platelet, p = 0.797; WBC, p = 0.178), while Hb at baseline did show an association (p = 0.024). In the dynamic analysis with Cox regression model, where the blood count values were studied by time of follow-up, we observed that the risk for Hb was 1.49 (95% CI 1.13-1.97) for every increase of 1 g/dL, and that this risk then marginally decreased during follow-up. WBC was associated with an increased risk at baseline for every increase of 1 × 10(9)/L (hazard ratio (HR) 1.07, 95% CI 1.01-1.13, p = 0.034), the risk was stable during follow-up (HR 0.95, p = 0.187 at 60 months). Also, for each increment at baseline of 100 × 10(9) platelets/L, HR was increased by 1.08 (95% CI 0.97-1.22, p = 0.159) and decreases during follow-up. In conclusion, this study is the first to evaluate in ET low-risk patients, the risk of developing a thrombotic/hemorrhagic event considering blood counts over time. Overall our study shows that the risk changes over time. For example, the risk associated with WCC is not linear as previously reported. An interesting new finding is that PLT and even Hb contribute to the risk of developing vascular events. Future treatments should take into consideration these findings and aim to control all parameters over time. We believe this early study may help develop a dynamic analysis model to predict thrombosis in the single patient. Further studies are now warranted to further validate our findings.

Role of JAK2 V617F mutation and aberrant expression of microRNA-143 in myeloproliferative neoplasms.

BACKGROUND: Myeloproliferative neoplasms (MPNs) are clonal myeloid disorders characterized by the overproduction of mature blood cells. The pathogenetic hallmark of MPNs is the dysregulation of JAK-STAT signaling, usually associated with the JAK2 V617F mutation. Multiple additional genetic and epigenetic alterations that constitutively activate the JAK-STAT signaling pathway have been described, including the modulation of the microRNAs (miRs) expression levels. The aims of our study were to investigate JAK2 V617F mutation allele burden and miR-143 expression levels in MPNs patients and to investigate the correlation between these genetic signatures and hematological parameters. METHODS: In total 78 patients with a clinical diagnosis of polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IM), made according to the WHO 2008 criteria, were included in the study. Twenty healthy subjects were checked as controls. Quantification of JAK2 V617F mutation and miR-143 expression levels were determined by real-time quantitative polymerase chain reaction. RESULTS: The miR-143 expression in MPNs patients was 2.97-fold higher than in controls. JAK2 V617F mutation allele burden and miR-143 expression level resulted higher in PV and IM respect to ET patients. Patients who had V617F allele burden >50% displayed a higher miRNA-143 expression level than patients with allele burden <50%. In MPNs patients, a statistically significant positive correlation was observed between JAK2 V617F mutation allele burden and hemoglobin and hematocrit values and between miR-143 expression levels and platelet count. CONCLUSIONS: Our findings of aberrant miR-143 expression support the concept that factors other than JAK2 V617F mutation may contribute to the pathogenesis and some clinical signs of MPNs.