Summary Background Microparticles are extracellular vesicles resulting from the budding of cellular membranes that have a high potential as emergent biomarkers; however, their clinical relevance is hampered by methodological enumeration concerns and a lack of standardization. Flow cytometry (FCM) remains the most commonly used technique with the best capability to determine the cellular origin of single MPs. However, instruments behave variably depending on which scatter parameter, (Forward (FSC) or Side scatter (SSC)), provides the best resolution to discriminate submicron particles. To overcome this problem, a new approach, based on two sets of selected beads adapted to FSC or SSC optimized instruments, was recently proposed to reproducibly enumerate platelet-derived MP counts among instruments with different optical systems. Objective The objective was to evaluate this strategy in an international workshop that included 44 laboratories accounting for 52 cytometers of 14 types. Methods/Results Using resolution capability and background noise level as criteria to qualify the instruments, the standardization strategy proved to be compatible with 85% (44/52) instruments. All instruments correctly ranked the PMP levels of two platelet-free plasma samples. The inter-laboratory variability of PMP counts was 37% and 28% for each sample. No difference was found between instruments using forward or side scattered light as the relative sizing parameter. Conclusions Despite remaining limitations, this study is the first to demonstrate a real potential of bead-based strategies for standardization of MP enumeration across different FCM platforms. Additional standardization efforts are still mandatory to evaluate MP clinical relevance at a multicenter level.

ObjectiveTo compare response to rituximab (RTX) between adult patients positive for myelin oligodendrocyte glycoprotein (MOG) and aquaporin‐4 (AQP4) antibodies.MethodsWe prospectively studied adult patients with MOG or AQP4 antibodies who received RTX under an individualized dosing schedule adapted to the biological effect of RTX monitored by memory B‐cell measurement. Memory B cells were counted monthly and when relapse occurred. The biological effect of RTX was considered significant with <0.05% memory B cells in peripheral blood lymphocytes.ResultsIn 16 patients with MOG antibodies and 29 with AQP4 antibodies, mean follow‐up was 19 (range = 9–38) and 38 (13–79) months. Under RTX, 10 relapses occurred in 6 of 16 (37.5%) patients with MOG antibodies, and 13 occurred in 7 of 29 (24%) with AQP4 antibodies. The median time of relapse after the most recent infusion was 2.6 (0.6–5.8) and 7 (0.8–13) months, respectively (p < 0.001). Memory B cells had reemerged in 2 of 10 (20%) relapses in patients with MOG antibodies and 12 of 13 (92.5%) with AQP4 antibodies (p < 0.001).InterpretationIn AQP4 antibody–associated disorder, relapse mostly occurs when the biological effect of RTX decreases, which argues for treatment efficacy. In MOG antibody–associated disorder, the efficacy of RTX is not constant, because one‐third of patients showed relapse despite an effective biological effect of RTX. In this subpopulation, memory B‐cell depletion was unable to prevent relapse, which was probably caused by different immunological mechanisms. These findings should be used to improve treatment strategies for MOG antibody–associated disorder. ANN NEUROL 2020;87:256–266

Platelets promote metastasis, however, their role in tumor growth remains controversial. Here, we investigated the effect of platelet interactions with colorectal tumor cells. Platelets extravasated into the tumor microenvironment and interacted with tumor cells in a cadherin-6–dependent manner. The interaction induced platelet spreading, release of their granule content, and the generation of three types of microparticles (iMP) that expressed platelet markers, tumor markers, or both. The presence of iMPs was confirmed in colorectal cancer tissue specimens. Platelets significantly reduced tumor growth and increased intratumoral macrophages. This was mediated by iMP recruitment of macrophages via the chemoattractants RANTES, MIF, CCL2, and CXCL12 and activation of their tumor cell killing capacity through IFNγ and IL4, which led to cell-cycle arrest of tumor cells in a p21-dependent manner. In contrast, in the bloodstream, iMPs activated endothelial cells and platelets and induced epithelial-to-mesenchymal transition of tumor cells, promoting metastasis. Altogether, these results indicate that depending on the environment, local or bloodstream, the consequences of the interactions between platelets and a tumor may promote or prevent cancer progression. Significance: Tumor cell interaction with platelets produces chimeric extracellular vesicles that suppress primary tumor growth by activating tumor-eliminating macrophages, while promoting metastasis through EMT and endothelial activation.

BackgroundThe activation of complement during platelet activation is incompletely understood. Objectives: We sought to explore the formation of C5b-9 and anaphylatoxins binding to collagen-activated platelets.MethodsC5b-9, anaphylatoxins C3a, C4a and C5a, and anaphylatoxin receptors C3aR1 and C5aR were measured by flow cytometry and/or confocal microscopy. Platelet microparticles were quantified by flow cytometry, and their C5b-9 content was determined by western blot analyses. In all experiments, sodium citrate was used for blood anticoagulation.ResultsC5b-9 rapidly formed on the platelet surface following activation with collagen, TRAP, ADP or A23187, but was surprisingly restricted to a subset of platelets (1 to 15%) independently of P-selectin or phosphatidylserine exposure. Following collagen activation, C5b-9-positive platelets in thrombi were found associated with collagen fibres. C5b-9 formation was obliterated by Mg2+-EGTA and significantly reduced by the thrombin inhibitor hirudin (−37%, p<0.05), but was unaffected by chondroitinase, compstatin, SCH79797 (PAR-1 inhibitor), or in the PRP of a MBL-deficient donor. Compstatin and Mg2+-EGTA, but not hirudin, SCH79797 or chondroitinase, inhibited the formation of collagen-induced microparticles (−71% and −44%, respectively, p<0.04). These microparticles contained greater amounts of C5b-9 compared with the other agonists. Platelet activation by collagen or convulxin resulted in the strong binding of anaphylatoxins and the exposure of receptors C3aR1 and C5aR (CD88) on their surface.ConclusionsC5b-9 formation on collagen-activated platelets is i) partially controlled by thrombin, ii) restricted to a subset of platelets, and iii) can occur without P-selectin expression or phosphatidylserine exposure. Activated platelets bind anaphylatoxins on their surface and express C3a and C5a receptors, which may contribute to the localization of inflammatory processes during thrombosis.

Microparticles (MPs) are submicronic vesicles which are formed by budding of the cellular membrane of virtually any cell type in response to cell activation or apoptosis. Both circulating MPs and MPs generated within tissues harbor molecules with a large repertoire of biological activities and transfer material to target cells. Depending on their cellular origin, the stimuli triggering their formation, or their localization, they may participate in the maintenance of organ or vascular homeostasis as well as inducing dysfunction. MPs have mostly been described as having procoagulant properties. However, the fact that some MP subsets are able to efficiently generate plasmin suggests that the role of MPs in hemostasis is more complex than initially thought. In this review, we summarize key findings showing that MPs provide a heterogeneous catalytic surface for plasmin generation, according to their cellular origin. We further address the specific features of the MP-dependent fibrinolytic system. Potential consequences of this MP-associated fibrinolytic activity in pathology are illustrated in cancer.

SummaryThe level of circulating platelet-, erythrocyte-, leucocyte-and endothelialderived microparticles detected by high-sensitivity flow cytometry was investigated in 37 b-thalassaemia major patients receiving a regular transfusion regimen. The phospholipid procoagulant potential of the circulating microparticles and the microparticle-dependent tissue factor activity were evaluated. A high level of circulating erythrocyte-and platelet-microparticles was found. In contrast, the number of endothelial microparticles was within the normal range. Platelet microparticles were significantly higher in splenectomized than in non-splenectomized patients, independent of platelet count (P < 0Á001). Multivariate analysis indicated that phospholipid-dependent procoagulant activity was influenced by both splenectomy (P = 0Á001) and platelet microparticle level (P < 0Á001). Erythrocyte microparticles were not related to splenectomy, appear to be devoid of proper procoagulant activity and no relationship between their production and haemolysis, dyserythropoiesis or oxidative stress markers could be established. Intra-microparticle labelling with anti-HbF antibodies showed that they originate only partially (median of 28%) from thalassaemic erythropoiesis. In conclusion, when b-thalassaemia major patients are intensively transfused, the procoagulant activity associated with thalassaemic erythrocyte microparticles is probably diluted by transfusions. In contrast, platelet microparticles, being both more elevated and more procoagulant, especially after splenectomy, may contribute to the residual thrombotic risk reported in splenectomized multi-transfused b-thalassaemia major patients.

No abstract

Summary Introduction In daily practice in haematology laboratories, spurious increased MCHC induces an analytical alarm and needs prompt corrective action to ensure delivery of the right results to the clinicians. The aim of this study was to establish a ‘decision tree’ using the new parameters red blood cells (RBC‐O) and haemoglobin (HGB‐O) from the Sysmex XN‐10 RET obtained by flow cytometry to deliver appropriate results. Methods From 128 unknown patients with MCHC > 365 g/L, all erythrocyte parameters including reticulocyte parameters were measured and analysed in parallel with blood smears, chemistry index and osmolarity. Differences between optical parameters (RBC‐O, HGB‐O) and usual parameters (RBC, HGB) obtained by impedance and photometry were reported also. Results Four groups were defined from observations: ‐RBC agglutination (n = 22); ‐optical interference (n = 17); ‐RBC disease (n = 18); and ‐others (n = 71). The use of RBC‐O and HGB‐O permitted efficient correction of the abnormalities when RBC agglutination and/or optical interference were present in 36 of 39 patients. Reticulocyte parameters permitted to elaborate an RBC score that allowed a highly sensitive detection of RBC disease patients (17/18). Conclusion Based on new parameters, we propose a ‘decision tree’ that delivers time savings and supports biological interpretation in case of elevated MCHC.