Update on Mechanisms, Pathogenicity, Heterogeneity of Presentation, and Laboratory Diagnosis of Heparin-Induced Thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is the most life-threatening adverse effect of heparin therapy and is provoked by the development of drug-dependent antibodies. It occurs more frequently in patients with cardiac or orthopedic surgery or severe circulatory diseases, and the risk depends on the patient pathological status. As heparin is an anticoagulant used for treating thrombotic events or their risk, this iatrogenic complication has a paradoxal effect as it can induce thromboembolic diseases, frequently associated to severe morbidity or fatal outcomes. Diagnosis involves clinical evaluation of disease probability and laboratory tools for testing the presence of heparin-dependent antibodies with immunoassays or their capability to activate platelets with functional assays. Antibodies developed when stoichiometric complexes of platelet factor 4 (PF4) with heparin are formed during therapy. In few cases non-platelet factor 4 antigens can be involved. Antibodies can remain asymptomatic, but pathogenicity occurs in the presence of high concentrations of IgG isotype antibodies, with high avidity: they target and activate platelets or endothelial cells exposing heparin-PF4 (HPF4) complexes and produce thrombocytopenia and sometimes thrombosis. Risk factors which favor the development of antibodies and their pathological effect are discussed. The present understanding of mechanisms underlying disease development and diagnostic strategies of this heparin adverse effect is presented

Optimization of Heparin Monitoring with Anti-FXa Assays and the Impact of Dextran Sulfate for Measuring All Drug Activity

Heparins, unfractionated or low molecular weight, are permanently in the spotlight of both clinical indications and laboratory monitoring. An accurate drug dosage is necessary for an efficient and safe therapy. The one-stage kinetic anti-FXa assays are the most widely and universally used with full automation for large series, without needing exogenous antithrombin. The WHO International Standards are available for UFH and LMWH, but external quality assessment surveys still report a high inter-assay variability. This heterogeneity results from the following: assay formulation, designed without or with dextran sulfate to measure all heparin in blood circulation; calibrators for testing UFH or LMWH with the same curve; and automation parameters. In this study, various factors which impact heparin measurements are reviewed, and we share our experience to optimize assays for testing all heparin anticoagulant activities in plasma. Evidence is provided on the usefulness of low molecular weight dextran sulfate to completely mobilize all of the drug present in blood circulation. Other key factors concern the adjustment of assay conditions to obtain fully superimposable calibration curves for UFH and LMWH, calibrators’ formulations, and automation parameters. In this study, we illustrate the performances of different anti-FXa assays used for testing heparin on UFH or LMWH treated patients’ plasmas and obtained using citrate or CTAD anticoagulants. Comparable results are obtained only when the CTAD anticoagulant is used. Using citrate as an anticoagulant, UFH is underestimated in the absence of dextran sulfate. Heparin calibrators, adjustment of automation parameters, and data treatment contribute to other smaller differences

Editorial for the Special Issue of Monitoring Anticoagulants

This Special Issue focuses on monitoring anticoagulant therapies and presents all the most recent updates introduced for laboratory practice, which benefit anticoagulated patients [...

Updates on Anticoagulation and Laboratory Tools for Therapy Monitoring of Heparin, Vitamin K Antagonists and Direct Oral Anticoagulants

Anticoagulant drugs have been used to prevent and treat thrombosis. However, they are associated with risk of hemorrhage. Therefore, prior to their clinical use, it is important to assess the risk of bleeding and thrombosis. In case of older anticoagulant drugs like heparin and warfarin, dose adjustment is required owing to narrow therapeutic ranges. The established monitoring methods for heparin and warfarin are activated partial thromboplastin time (APTT)/anti-Xa assay and prothrombin time – international normalized ratio (PT-INR), respectively. Since 2008, new generation anticoagulant drugs, called direct oral anticoagulants (DOACs), have been widely prescribed to prevent and treat several thromboembolic diseases. Although the use of DOACs without routine monitoring and frequent dose adjustment has been shown to be safe and effective, there may be clinical circumstances in specific patients when measurement of the anticoagulant effects of DOACs is required. Recently, anticoagulation therapy has received attention when treating patients with coronavirus disease 2019 (COVID-19). In this review, we discuss the mechanisms of anticoagulant drugs—heparin, warfarin, and DOACs and describe the methods used for the measurement of their effects. In addition, we discuss the latest findings on thrombosis mechanism in patients with COVID-19 with respect to biological chemistry

An Optimized and Standardized Rapid Flow Cytometry Functional Method for Heparin-Induced Thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a thrombocytopenia caused by heparin and mediated by an atypical immune mechanism leading to a paradoxical high thrombotic risk, associated with severe morbidity or death. The diagnosis of HIT combines a clinical scoring of pretest probability and laboratory testing. First-line routine tests are antigen binding assays detecting specific antibodies. The most sensitive of these tests have a high HIT-negative predictive value enabling HIT diagnosis to be ruled out when negative. However, HIT-positive predictive value is low, and a functional assay evaluating the pathogenicity of the antibodies should be performed to exclude false-positive results. In contrast to screening assays, functional assays are highly specific but technically challenging, and are thus performed in referral laboratories, where platelet activation is detected using radioactive serotonin (serotonin release assay, SRA) or visually (heparin-induced platelet activation, HIPA). Flow cytometry is a possible alternative. It is, however, currently not widely used, mostly because of the lack of standardization of the published assays. This article describes and discusses the standardization of a HIT flow cytometry assay (HIT-FCA) method, which subsequently led to the development and commercialization of a CE-marked assay (HIT Confirm®, Emosis, France) as a suitable rapid HIT functional test

Thrombospondin (TSP1) mediates in vitro proliferation of human MG-63 osteoblastic cells induced by α-thrombin

AbstractThrombospondin (TSP) is a 450-kDa glycoprotein synthesized and secreted by human MG-63 osteoblastic cells. In this study, we have first studied the effect of α-thrombin on TSP expression by human MG-63 cells. In situ hybridization indicated that TSP mRNA level in thrombin-treated MG-63 cells was increased when compared to unstimulated cells. As judged by immunofluorescence, thrombin-treatment of MG-63 cells resulted in increased cell surface expression of TSP when compared to quiescent cells. Because thrombin stimulates proliferation of osteoblastic cells, the involvement of TSP in proliferation of thrombin-stimulated osteoblastic cells was then investigated using a serum-free mitogenesis assay. Both α-thrombin (0.01 to 0.15 Uml) and TSP (5 to 600 ngml) caused a dose-dependent increase in [3H]thymidine incorporation by MG-63 cells. Proliferation of osteoblastic cells induced by α-thrombin or TSP was specifically and totally inhibited by anti-TSP monoclonal antibodies (3–10 μgml) or by indomethacin (1 μM), an inhibitor of prostaglandin synthesis. Anti-TSP antibodies which inhibited cell proliferation also inhibit TSP expression to the surface of these cells. Our experiments support the existence of a mechanism whereby TSP bound to the cell surface of thrombin-treated MG-63 cells stimulates secretion of prostaglandins which, in turn, allow cell proliferation to proceed

Assessment of platelet function on the routine coagulation analyzer Sysmex CS-2000i

International audienceBackground: Light transmission aggregometry (LTA) is considered as the gold standard for testing platelet function in the setting of both platelet disorders suspicion and response to antiplatelet therapy evaluation. LTA requires however specialized equipment, substantial blood sample volumes, is technically challenging and time-consuming.Aim: To evaluate an automated platelet aggregation method performed on a routine coagulation analyzer Sysmex CS-2000i. Methods: 46 patients presenting a bleeding syndrome and 62 patients with acute coronary syndrome receiving dual antiplatelet therapy were studied in total. Platelet aggregations were performed on CS-2000i equipped with a dedicated software and on APACT-4004 (Elitech, France) as the reference instrument. Aggregation was measured by monitoring the changes in light absorbance occurring in response to ADP 2.5, 5 and 10µM, collagen 3.3 µg/mL, epinephrin 10µM, ristocetin 1.25 mg/mL and arachidonic acid 0.5 mg/mL in platelet rich plasma (PRP). PRP were tested simultaneously on both CS-2000i and APACT-4004 devices. Platelet stirred speed were 800 rpm for both instruments.Results: Significant correlations were observed between CS-2000i and LTA after all stimulations (p 0.70).Conclusion: Platelet aggregation on the routine coagulation analyzer CS-2000i is an easily accessible, handy, reliable, standardized, and rapid tool to assess platelet function which allows to skirt most of the LTA limitations