Background:Standardization, machine learning techniques and comparison to normality are concepts that are changing the landscape of multiparameter flow cytometry data analysis in clinical hematology. We applied these concepts to the follow‐up of acute myeloid leukemia (AML), which remains challenging due to the heterogeneity of the disease.Aims:To evaluate a new flow cytometry data analysis approach for the follow‐up of measurable residual disease in AML.Methods:Based on retrospective flow cytometry data, we developed a strategy for personalized monitoring of AML. This strategy is an evolution of the “leukemia associated immunophenotype” or “LAIP”‐approach that has been used the last decades in specialized flow cytometry laboratories. It relies on new concepts, comparing simultaneously the patient's follow‐up and diagnosis results to control group samples. We named these concepts the “leukemic cloud” and the “abnormality ratio”. To evaluate this approach, flow cytometry data of 6 AML patients and 20 control patients was used. All samples were analyzed with the same 10‐parameter (FSC‐A, SSC‐A, CD45, CD34, CD117, CD13, CD33, CD19, CD3, HLA‐DR) FACS Canto II flow cytometry assay. The “abnormality ratio” results over time were compared with morphology and molecular biology results.Results:The “Abnormality Ratio” results over time were in line with the clinical evolution of the patients (see example on image below) and were overall more sensitive for detecting residual disease than morphology and molecular biology, excepted for NPM1‐mutated AML. The “Abnormality Ratio” did not consider phenotypic switches of the malignant cell population identified at diagnosis. However, the discrepancy between the “abnormality ratio” results and morphology or molecular biology results could be used to objectify these phenotypic switches.Summary/Conclusion:We developed a quasi‐fully automatable flow cytometry data analysis algorithm. It could be used as a basis for the development of “next generation” flow cytometry assays for monitoring of hematological malignancies such as AML. Prospective studies are needed to confirm the applicability and limitations of our concepts.image

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.

Objectives: Diffuse large B-cell lymphoma (DLBCL) is highly heterogeneous in terms of phenotype and treatment response in patients. These characteristics make the prognosis difficult to establish and hinder the use of new personalized treatments in clinical practice. In this context, there is currently a need to define new biomarkers enabling a better definition of DLBCL subtypes, prognosis evaluation, and an overview of the resistance to chemotherapeutics. The aim of this study was to evaluate the use of microRNAs found in plasma from patients with DLBCL as biomarkers of tumor evolution in these patients. Method: For this purpose, a plasma biobank was created with samples from patients with DLBCL. The evolution of the level of selected microRNAs during treatment has been studied. A total of 19 patients with DLBCL were included in this pilot mono-centered study and a total of 68 samples were analyzed. Results: The first step of this study was the selection of the microRNAs to be quantified in all the samples of the biobank and that could potentially be used as biomarkers. To this end, quantification of 377 microRNAs was performed on the plasma samples of 2 selected patients with DLBCL and 1 healthy donor with no history of cancer. Among the 377 microRNAs evaluated, 7 were selected and analyzed in the entire biobank. Conclusions: This study highlighted 5 circulating microRNAs whose plasma levels would be worth further investigating for the characterization of DLBCL evolution in patients. MiR-21 and miR-197 had a significant higher plasmatic level in patients with tumors unresponsive to treatment. With a higher plasma level in patients with complete remission, miR-19b, miR-20a, and miR-451 could enable to differentiate, at the remission review, patients with residual tumor, from patients with complete remission.

Background:A prior meta‐analysis demonstrated that 3 of the new generation BCR‐ABL tyrosine kinase inhibitors (TKIs) (i.e. dasatinib, nilotinib and ponatinib) are associated with an increased risk of vascular occlusive events compared with imatinib. Additional analyses demonstrate that this risk is mainly driven by the occurrence of arterial events. At the time of these analyses, few clinical data were available on bosutinib (only one study included with few events in each arms), inducing a low statistical power to compute the risk of vascular occlusive events with this treatment. Recent clinical data comparing bosutinib to imatinib have been published and need to be incorporated in the meta‐analysis.Aims:To update the meta‐analysis on the risk of arterial and venous occlusive events in patients with Ph+ CML treated with new generation BCR‐ABL TKIs in randomized clinical trials (RCTs), particularly to assess the cardiovascular safety of bosutinib.Methods:The literature search (data lock point: January, 25th, 2019) was conducted according to a registered protocol (PROSPERO 2014:CRD42014014147). All RCTs comparing a new generation BCR‐ABL TKI versus imatinib in patients with Ph+ CML were included. Two independent investigators screened the literature, reviewed and extracted the data using standard forms. The statistical analyses were performed using a random (REM) and a fixed (FEM) effect model according to the characteristics of the included studies. ORs with 95% CIs were computed using the Peto method. Statistical heterogeneity was quantified using the I2 value, and publication bias was assessed by funnel plots.Results:Among the 385 abstracts identified, 13 studies fulfilled the established criteria and were included in the statistical analysis. Overall, 7.93% (197/2,484) of patients treated with a new generation BCR‐ABL TKIs developed a vascular occlusive event compared with 2.61% (56/2,149) when treated with imatinib (REM ORPETO: 2.81; 95%CI: 2.09 to 3.80). The update does not change the increased risk of vascular occlusive events with ponatinib (REM ORPETO: 3.47; 95%CI: 1.23 to 9.78), nilotinib (REM ORPETO: 3.55; 95%CI: 2.29 to 5.51) and dasatinib (REM ORPETO: 2.87; 95%CI: 1.48 to 5.55) compared with imatinib. The addition of the results from the BFORE trial (NCT02130557) improve the statistical power of the analysis and demonstrate a non‐significant trend towards an increased risk of vascular events with this treatment (REM ORPETO: 1.67; 95%CI: 0.91 to 3.05). In accordance with the results on ponatinib, nilotinib and dasatinib, this risk is mainly driven by the occurrence of arterial occlusive events. Arterial occlusive events occur in 7.08% (176/2,487) of patients treated with bosutinib whereas only 0.85% (21/2,484) of patients treated with bosutinib report a venous occlusive event. Funnel plots demonstrate no evidences of publication bias, and the I2 statistic specifies no heterogeneity among studies.Limitations of this meta‐analysis include the absence of a time‐to‐event analysis and the inconsistent report of cardiovascular events in the literature. However, the use of a clinical trial register aimed to decrease this heterogeneity.Summary/Conclusion:This updated meta‐analysis confirmed the risk of arterial occlusive events with new generation BCR‐ABL TKIs. The addition of the results from the BFORE trial increases the statistical power of the bosutinib sub‐analysis and reduce the interval confidence of the analysis. Further investigations are needed to confirm the apparently safer profile of bosutinib in regard to cardiovascular occlusive events.

Background: Early diagnosis of immune heparin-induced thrombocytopenia (HIT) is challenging. HemosIL (R) AcuStar HIT and heparin-induced multiple electrode aggregometry (HIMEA) were recently proposed as rapid diagnostic methods.Objectives: We conducted a study to assess performances of AcuStar HIT-IgG (PF4-H) and AcuStar HIT-Ab (PF4-H). The secondary objective was to compare the performances of the combination of Acustar HIT and HIMEA with standardised clinical diagnosis.Methods: Sera of 104 suspected HIT patients were retrospectively tested with AcuStar HIT. HIMEA was performed on available sera (n = 81). The clinical diagnosis was established by analysing in a standardized manner the patient's medical records. These tests were also compared with PF4-Enhanced (R), LTA, and SRA in subsets of patients. Thresholds were determined using ROC curve analysis with clinical outcome as reference.Results: Using the recommended thresholds (1.00 AU), the negative predictive value (NPV) of HIT-IgG and HIT-Ab were 100.0% (95% CI: 95.9%-100.0% and 95.7%-100.0%). The positive predictive value (PPV) were 64.3% (95% CI: 35.1%-87.2.2%) and 45.0% (95% CI: 23.2%-68.6%), respectively. Using our thresholds (HIT-IgG: 2.89 AU, HIT-Ab: 9.41 AU), NPV of HIT-IgG and HIT-Ab were 100.0% (95% CI: 96.0%-100.0% and 96.1%-100.0%). PPV were 75.0% (95% CI: 42.7%-94.5%) and 81.8% (95% CI: 48.3%-97.7%), respectively. Of the 79 patients with a medium-high pretest probability score, 67 were negative using HIT-IgG (PF4-H) test at our thresholds. HIMEA was performed on HIT-IgG positive patients. Using this combination, only one patient on 79 was incorrectly diagnosed.Conclusion: Acustar HIT showed good performances to exclude the diagnosis of HIT. Combination with HIMEA improves PPV. (C) 2013 Elsevier Ltd. All rights reserved.

Background Direct oral anticoagulants (DOACs) have been developed to address some of the limitations of Vitamin-K antagonists. Deviations from the recommended use have been reported, sometimes leading to serious adverse events. Purpose Our objective was to evaluate the appropriateness of prescribing DOACs in real-life clinical practice. Material and methods We conducted a prospective study including patients admitted to a 450-bed teaching hospital from April to June 2013 and taking rivaroxaban or dabigatran in prevention of stroke or systemic embolism in non-valvular atrial fibrillation. A clinical pharmacist collected clinical and pharmaceutical data from the electronic medical record and patient interview. Appropriateness of prescribing was evaluated using 9 criteria of the Medication Appropriateness Index.2,3 Explicit instructions specific to the appropriate use of DOACs were added based on EU summary of the product characteristics and (inter)national guidelines. The primary outcome measure was the prevalence of patients with ≥1 inappropriate criterion. Results Fifty-two patients were evaluated (median age 74 years; 29 and 23 taking Xarelto® and Pradaxa®, respectively). Twenty-eight (53.8%) patients had at least one inappropriate rating: 1 inappropriate criterion in 26.9% and >1 in 26.9% of patients. The most frequent inappropriate criteria were: wrong dosage (32.7%, e.g. dose not adapted to renal function); inappropriate choice and modalities of administration (28.8%, e.g. prescription of a DOAC in a VKA-naive patient with extreme body weight, once daily administration of Pradaxa®); and unpractical modalities of administration (25.0%, e.g. Pradaxa® in non-adherent patients). Twenty-four patients (47.0%) had experienced one adverse event. The clinical pharmacist made 29 interventions during the study period; e.g. 11 request for specific coagulation assay, 8 switches to another oral anticoagulant. Conclusion The quality of prescribing DOACs was suboptimal. Off-label use was frequent and suggests that reinforcing education of patient and health care professionals is needed. References 1. Larock AS, et al . Appropriateness of prescribing dabigatran etexilate and rivaroxaban in patients with nonvalvular atrial fibrillation: a prospective study. Ann Pharmacother 2014;48(10):1258–68 2. Hanlon JT, et al . A method for assessing drug therapy appropriateness. J Clin Epidemiol 1992;45(10):1045–51 3. Samsa GP, et al . A summated score for the medication appropriateness index: development and assessment of clinimetric properties includingcontent validity. J Clin Epidemiol 1994;47(8):891–6 References No conflict of interest.

Background Early diagnosis of immune heparin-induced thrombocytopenia (HIT) is essential to improve clinical outcome but remains challenging. The release of platelet microparticles (PMPs) is considered of major pathophysiological significance. Objectives The aim of this study was to evaluate performances of PMP generation assay (PMPGA) compared to clinical outcome to diagnose HIT. The second objective was to compare PMPGA with performances of 14C-serotonin release assay (SRA) on the same series of patients. Methods Sera of 53 HIT-suspected patients were retrospectively incubated with citrated-whole blood from healthy donors with 1 IU and 500 IU/ml of unfractionated heparin (UH). PMPGA was performed using FACSAria® flow cytometer. The clinical diagnosis was established by two blinded independent investigators analysing in a standardized manner the patient's medical records. Performances of PMPGA and SRA (n = 53) were evaluated using ROC curve analysis with clinical outcome as reference. Results In positive HIT patients, PMPs expressing phosphatidylserine are generated with low UH concentration whereas PMP rate decreases significantly in presence of high UH concentration. Using clinical outcome as reference, sensitivity and specificity of PMPGA reached 88.9% (95% CI: 50.7-99.4) and 100.0% (95% CI: 90.0-100.0). Sensitivity and specificity of 14C-SRA were 88.9% (95% CI: 50.7-99.4) and 95.5% (95% CI: 83.3-99.2). Conclusions PMPGA is a rapid and reliable assay for HIT diagnosis. PMPGA showed good correlation with 14C-SRA performances and predominately with clinical outcome.

Microvesicles (MVs) play a major role in cellular crosstalk, inflammation, infection, immunity, thrombosis and angiogenesis [1]. They are not only defined by size but also by concentration, morphology, biochemical composition, cellular origin, physical properties and activity [2]. Today, the MV nomenclature is still a matter of debate [3]. This lack of scientific consensus is mainly due to inaccuracy of size measurement [2]. Notably, the detection and quantification of MVs in blood are hampered by their methods of isolation, their nanometric size, and the relatively low abundance of some specific MV subpopulations such as endothelial MVs [4,5]. In this issue, Mobarrez and colleagues report on the influence of factor (F) VIII injection on MP count and activity in hemophilia A patients [6]. The authors nicely showed that plasma MP count (except leucocyte MPs) decreased after on-demand treatment with FVIII concentrate in hemophilia A patients and that the decrease in circulating MPs correlated with hemostatic activation. This is an additional piece of evidence that suggests that plasmatic MP measurement and characterization in biological fluids could reveal new diagnostic and/or prognostic information in human diseases [1]. As a limitation, the authors recognized that pre-analytical variables such as transportation of the samples by flight and storage may affect the results. Actually, despite the ongoing efforts of the Scientific Subcommittee on Vascular Biology of the International Society of Thrombosis and Haemostasis (ISTH), one must admit that there is still a need for guidelines on standardized and harmonized pre-analytical protocols. Some key principles and guidance on pre-analytical issues related to blood sampling, sample handling, plasma generation and plasma freezing/storing are discussed here and summarized in Table 1. They are based on our experience in the field and an up-to-date review of the literature. Very few pre-analytical data exist on exosomes (30– 100 nm) for clinical studies. Therefore, only MPs from 100 nm to 1 lm will be discussed.

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