Introduction: Classical Hodgkin Lymphoma (cHL) has an incredibly high survival rate, exceeding 80% at 10 years. Continuous improvement in treatment strategies led to an increased number of survivors in the community. Most common complications encountered are secondary malignant neoplasms (SMN) and cardiovascular (CV) toxicity. Methods: We conducted a single-centre retrospective study including 157 patients diagnosed and treated for cHL between 1974 and 2016. Results: Median age at diagnosis in our cohort is 30 years old. Ninety-six patients (61%) had a stage 1 or 2 disease. Front line treatment consisted of radiotherapy alone for 3 patients (2%), chemotherapy alone for 74 patients (47%) and combined modalities for 80 patients (51%). Our median follow-up time is 11 years. We reported 96 cardiovascular events in 75 patients (48%). Median time to CV event was 10 years. Most of the events were graded 1 or 2 (71%) and were related to valvular heart disease. Grade 3 and 4 CV events (n = 21) were mainly due to coronary heart disease (43%) while most of the deaths related to CV toxicity were consequent to heart failure (57%). Twenty-seven patients (17%) were diagnosed with SMN, with a median time to SMN of 10 years. Most of them were solid malignancies (71%), including 6 lung cancers and 2 breast cancers (both BRCA2+). We encountered 2 secondary leukaemia, 3 myelodysplastic syndromes and 2 Non-Hodgkin Lymphoma. Overall survival rate in our cohort is 76% at 10 years. We observed 38 deaths, at a median age of 60 years old. Six patients (16%) died from cHL, and main causes of non-lymphoma deaths were secondary malignant neoplasms (10 pts, 28%) and cardiovascular events (7 pts, 18%). Discussion: Long-term complications in Hodgkin Lymphoma survivors are well-known and focus on how to decrease treatment related toxicity is constantly growing. Special attention is brought to the use of anthracyclines and mediastinal radiotherapy. Regarding CV toxicity, we tend to review our patients annually and to offer periodic screening with heart imaging. Our study showed that we detected CV events in almost half of our patients. Those events were mostly low-grade valvular disease not requiring treatment. However, more than 1 out of 4 CV event was graded 3 or more. The impact of the doses of anthracycline and/or radiotherapy received in our population still need to be determined. With a median time to CV event and to SMN of 10 years, we can confirm that long-term surveillance is recommended for the survivors. Conclusion: Cure is no longer enough when managing cHL. Our past and previous treatments led to an increasing number of survivors who are at higher risk of long-term complications. Recommendations for long-term surveillance programmes are emerging and are mostly experts-based opinions. More data are needed to identify high-risk patients and to provide them with an adequate monitoring. Keywords: Hodgkin lymphoma, late effects in lymphoma survivors, prevention and cancer interception No conflicts of interests pertinent to the abstract.

Background Thrombin generation testing has been used to provide information on the coagulation phenotype of patients. The most used technique is the calibrated automated thrombogram (CAT) but it suffers from a lack of standardization, preventing its implementation in routine. The ST Genesia is a new analyzer designed to assess thrombin generation based on the same principle as the CAT. Unlike the CAT system, the ST Genesia is a benchtop, fully automated analyzer, able to perform the analyses individually and not by batch, with strict control of variables such as temperature and volumes, ensuring, theoretically, maximal reproducibility. Objectives This study aimed at assessing the performance of the STG-DrugScreen application on the ST Genesia analyzer. We also aimed at exploring stability of plasma samples after freezing and defining a reference normal range. Results Results demonstrated the excellent interexperiment precision of the ST Genesia and confirmed that the use of a reference plasma helps reducing the inter-experiments variability. Stability revealed that plasma samples are stable for at least 11 months at -70 degrees C or lower, except for those containing low molecular weight heparins which have to be tested within 6 months. Freezing had no effect on the majority of thrombin generation parameters except on time to peak. Conclusions Our results suggest an easy implementation of thrombin generation with the use of ST Genesia in the routine laboratory. This will facilitate the design of multicentric studies and enable the establishment of reliable and evidence-based thresholds, which may improve the management of patients treated with anticoagulants.

Abstract Thrombosis are severe complications of paroxysmal nocturnal hemoglobinuria (PNH), effectively reduced by eculizumab. Extracellular vesicles (EVs) may play a central role. The objective of this study was to assess the procoagulant activity of plasma isolated from PNH patients (treated or not by eculizumab) and to quantify their circulating EVs. We iteratively collected the platelet-free-plasma of 17 PNH patients and 16 matched healthy volunteers, quantified their circulating EVs by flow cytometry and evaluated their procoagulant activity by thrombin generation and STA-Procoag-procoagulant phospholipid (PPL) assays. A significant decrease of EVs from platelets (P = .024) and an increase of the STA-Procoag-PPL clotting time (P = .049) was observed after initiation of eculizumab and up to 11 weeks after. This reduction of prothrombotic biomarkers was not observed with the thrombin generation test due to a lack of sensitivity of this assay. Active hemolysis was observed in 90% of patients and elevated D-dimers in 41% of them. However, no significant difference was observed between patients and control subjects regarding the procoagulant activity, the EVs quantity, or the cellular origin. Lactate dehydrogenase (LDH) levels were lower in eculizumab-treated patients compared to nontreated patients (441 vs 2448 IU/L). D-dimers and LDH decreased after administration of eculizumab (mean decrease of 1307 ng/mL and 4159 IU/L, respectively). These observations suggest a decrease of the phospholipid-dependent procoagulant potential of EVs after eculizumab therapy in PNH patients. Trial registration: NUB: B039201214365

Red blood cell (RBC) concentrates may be stored for up to 42 days before transfusion to a patient. During storage extracellular vesicles (EVs) develop and can be detected in significant amounts in RBC concentrates. The concentration of EVs is affected by component preparation methods, storage solutions, and inter-donor variation. Laboratory investigations have focused on the effect of EVs on in vitro assays of thrombin generation and immune responses. Assays for EVs in RBC concentrates are not standardized. The aims of this review are to describe the factors that determine the presence of erythrocyte-EVs in RBC concentrates, the current techniques used to characterize them, and the potential role of EV analysis as a quality control maker for RBC storage.

Despite its considerable morbidity and mortality, paroxysmal nocturnal haemoglobinuria (PNH) is still underdiagnosed. Patients with PNH can suffer from cardiovascular, gastrointestinal, neurological or haematological symptoms and refer to several specialists. The aim of this paper is to review the diagnosis and the management of PNH patients, with the primary focus on identifying high-risk groups. Additionally, the implementation and prognostic value of the defined high-risk groups will be commented on and the management of PNH patients is discussed from a Belgian perspective. Finally, based on the available data, recommendations are provided. Eculizumab is a potent C5 complement inhibitor and reduces intravascular haemolysis and thrombosis in PNH patients and improves their quality of life. As thrombosis is the main cause of death in PNH patients, identifying high-risk PNH patients in need of therapy is essential. Currently, novel complement inhibitors are in development and the first data seem promising. Another challenge in PNH is to identify new markers to assess the thrombotic risk to achieve a better risk-based prophylactic anti-thrombotic management. Finally, because of the low prevalence of the disease, PNH patients should be included in the prospective PNH registry, which will offer new insights on the natural course of the disease and the impact of treatment of PNH.

Abstract The impact of direct oral anticoagulants (DOACs) on laboratory assays used for thrombophilia testing (e.g., antithrombin, protein S, protein C, lupus anticoagulant and activated protein-C resistance) is a well-known issue and may cause false-positive and -negative results. Therefore, the correct interpretation of tests that are performed in patients taking DOACs is mandatory to prevent misclassification and the subsequent clinical consequences. We aimed at evaluating the efficiency of a new and simple procedure (DOAC-Stop®; Haematex Research, Hornsby, Australia) to overcome the effect of all DOACs in real-life settings and to assess the percentage of erroneous results due to the presence of DOACs on thrombophilia screening tests. For this purpose, 135 DOAC-treated patients (38 apixaban, 40 dabigatran, 15 edoxaban, and 42 rivaroxaban) and 20 control patients were enrolled. A significant drop in apixaban, dabigatran, edoxaban, and rivaroxaban plasma concentrations following the DOAC-Stop® treatment was observed (74.8–8.2 ng/mL [p < 0.0001], 95.9–4.7 ng/mL [p < 0.0001], 102.1–8.8 ng/mL [p = 0.001], and 111.3–7.0 ng/mL [p < 0.0001], respectively). The DOAC-Stop® treatment was mostly effective to overcome the effect of DOACs on PTT-LA, dilute Russell's viper venom time (dRVVT) screen, and dRVVT confirm tests. Using our procedures, false-positive results due to DOACs were observed only with lupus anticoagulant tests (up to 75%) and fell to zero after the DOAC-Stop® procedure, regardless of the DOAC considered. In conclusion, the DOAC-Stop® adsorbent procedure appeared to be an effective and simple way to overcome the interference of DOAC on coagulation tests and should facilitate the interpretation of thrombophilia screening tests in patients taking DOACs.

BACKGROUND:Thrombotic effects are possible complications of red blood cell transfusion. The generation and accumulation of procoagulant red blood cell extracellular vesicles during storage may play an important role in these thrombotic effects. The objective of this study was to assess the value of a simple phospholipid-dependent clot-based assay (STA®-Procoag-PPL) to estimate the procoagulant activity of stored red blood cells and changes in this activity during storage of the blood component.MATERIALS AND METHODS:Extracellular vesicles from 12 red blood cell concentrates were isolated at 13 storage time-points and characterised by quantitative and functional methods: the degree of haemolysis (direct spectrophotometry), the quantification and determination of cellular origin (flow cytometry) and the procoagulant activity (thrombin generation and STA®-Procoag-PPL assays) were assessed.RESULTS:The mean clotting time of extracellular vesicles isolated from red blood cell concentrates decreased from 117.2±3.6 sec on the day of collection to 33.8±1.3 sec at the end of the storage period. This illustrates the phospholipid-dependent procoagulant activity of these extracellular vesicles, as confirmed by thrombin generation. Results of the peak of thrombin and the STA®-Procoag-PPL were well correlated (partial r=-0.41. p<0.001). In parallel, an exponential increase of the number of red blood cell-derived extracellular vesicles from 1,779/μL to 218,451/μL was observed.DISCUSSION:The STA®-Procoag-PPL is a potentially useful technique for assessing the procoagulant activity of a red blood cell concentrate.

Aims To determine the preventability of serious adverse drug reactions (ADRs) related to the use of direct oral anticoagulants (DOACs), and to explore contributing factors to preventable ADRs. Results were compared with vitamin K antagonists (VKAs). Methods We conducted a prospective observational study in the emergency departments of two teaching hospitals from July 2015 to January 2016. Patients admitted with a thrombotic or bleeding event while under DOAC or VKA were included. Four independent reviewers assessed causality, seriousness and preventability of ADRs using pilot‐tested scales. For cases of serious and potentially preventable ADRs, we performed semi‐structured interviews with general practitioners to identify contributing factors to ADRs. The primary outcome was the proportion of serious ADRs that were potentially preventable. Results The analysis included 46 DOAC and 43 VKA patients (median age 79 years). Gastrointestinal ( n = 34) and intracranial ( n = 16) bleedings were the most frequent ADRs. Results were that 53% of DOAC‐ and 61% of VKA‐related serious ADRs were deemed potentially preventable. Prescribing issues and inadequate monitoring were frequent for DOAC and VKA respectively. We identified many causes of preventable ADRs that applied to all oral anticoagulants, such as pharmacodynamic drug interactions and lack of communication. Conclusions More than half of serious ADRs were potentially preventable for both DOACs and VKAs. Interventions focusing on prescribing, patient education and continuity of care should help improve the use of DOACs in practice.

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a disease characterized by the susceptibility of blood cells to attack by the complement system, inducing extracellular vesicle (EV) production. Thromboembolism is the leading cause of death in this condition. Eculizumab, a humanized monoclonal antibody which inhibits the C5 protein of the complement, reduces the thrombotic risk in PNH.Materials and method: We conducted a pilot, prospective, open-label, longitudinal clinical study with six PNH patients treated with eculizumab. The aim was to measure, by flow cytometry, the EVs' production in the patients' platelet-free plasma (PFP) before and during the treatment. We also assessed the procoagulant activity in PFP using STA (R)-Procoag-PPL and thrombin generation assays (TGA). A high-sensitive version of TGA was also used to study the procoagulant profile induced by the EVs using EVs pelleted from PFP.Results: We observed a decrease in platelet EV count with eculizumab treatment (p < 0.05). STA (R)-Procoag-PPL assay showed a decrease of the procoagulant profile induced by procoagulant phospholipids (PL) during treatment. These results were not confirmed by TGA on PFP, due to a lack of sensitivity. Thus, we used a high-sensitive version of TGA that enabled us to observe variation in the procoagulant profile induced by the EVs with eculizumab (p < 0.05).Conclusions: Eculizumab has an impact on the extent of EV production and on the procoagulant profile induced by the procoagulant PL and the EVs. One factor in the antithrombotic action of eculizumab is its ability to decrease EV production and the procoagulant profile induced by PL and EVs. (C) 2017 Elsevier Ltd. All rights reserved.

Submicron-sized extra-cellular vesicles generated by budding from the external cell membranes, microparticles (MPs) are important actors in transfusion as well as in other medical specialties. After briefly positioning their role in the characterization of labile blood products, this technically oriented chapter aims to review practical points that need to be considered when trying to use flow cytometry for the analysis, characterization and absolute counting of MP subsets. Subjects of active discussions relative to instrumentation will include the choice of the trigger parameter, possible standardization approaches requiring instrument quality-control, origin and control of non-specific background and of coincidence artifacts, choice of the type of electronic signals, optimal sheath fluid and sample speed. Questions related to reagents will cover target antigens and receptors, multi-color reagents, negative controls, enumeration of MPs and limiting artifacts due to unexpected (micro-) coagulation of plasma samples. Newly detected problems are generating innovative solutions and flow cytometry will continue to remain the technology of choice for the analysis of MPs, in the domain of transfusion as well as in many diverse specialties.