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Immune thrombocytopenia (ITP) is an autoimmune disease with a complex heterogeneous pathogenesis and a bleeding phenotype that is not necessarily correlated to platelet count. In this study, the platelet function was assessed in a well-defined cohort of 33 pediatric chronic ITP patients. Because regular platelet function test cannot be performed in patients with low platelet counts, 2 new assays were developed to determine platelet function: first, the microaggregation test, measuring in platelets isolated from 10 mL of whole blood the platelet potential to form microaggregates in response to an agonist; second, the platelet reactivity assay, measuring platelet reactivity to adenosine diphosphate (ADP), convulxin (CVX), and thrombin receptor activator peptide in only 150 mu L of unprocessed whole blood. Patients with a severe bleeding phenotype demonstrated a decreased aggregation potential upon phorbol myristate acetate stimulation, decreased platelet degranulation following ADP stimulation, and a higher concentration of ADP and CVX needed to activate the glycoprotein IIbIIIa complex compared with patients with a mild bleeding phenotype. In conclusion, here we have established 2 functional tests that allow for evaluation of platelet function in patients with extremely low platelet counts (

Background: Light transmission aggregometry (LTA) is still the most used test for the identification and diagnosis of platelet function defects (PFD). Disadvantages of LTA include its laborious nature, the large volumes of blood required and the relative insensitivity to small changes in platelet function. We investigated if the flow cytometry-based whole blood platelet activation test (WB-PACT) correlates with LTA or could be used as a complimentary test. Methods: WB-PACT quantifies αIIbβ3 activation and P-selectin expression in response to 3 agonists and VWF binding to platelets in response to ristocetin. In total, 212 patients (51 on dual antiplatelet therapy (acetylsalicylic acid and ADP receptor antagonist), 161 suspected for bleeding diathesis) were tested. For WB-PACT parameters, the 2.5th, 10th and 25th percentiles were determined in 56 healthy donors to score the patient samples. For LTA, maximal aggregation, disaggregation and prolongation of the lagtime in response to 4 agonists and ristocetin was scored. Patients with at least one parameter lower than the 10th percentile measured with WB-PACT in healthy donors or at least one value deviating from the normal range measured with LTA were diagnosed with PFD. A bleeding score (BS) was calculated with the ISTH/SSC bleeding assessment tool (Rodeghiero et al., JTH, 2010). Results: A moderate correlation between LTA versus WB-PACT was found with a R of 0.60 (Figure 1). In total, 95 patients were diagnosed with PFD by both tests (κ=0.40, Table 1) and BS were recorded for 28/95 patients. Of these 28 patients, 25% had an elevated BS (adapted according to gender and age: >5 in women, >3 in men and >2 in children) and 36% had a BS>3. BS were recorded for 30/38 and 22/24 patients who were diagnosed with PFD according to WB-PACT only and LTA only, respectively. Interestingly, 27% of patients with PFD according to WB-PACT had an elevated BS and 40% had a BS>3. For LTA, only 13% of patients with PFD had an elevated BS and 13% had a BS>3. Conclusions: Flow cytometric analysis of platelet function by WB-PACT gives additional value to LTA to determine PFD. Disclosures No relevant conflicts of interest to declare.

The antiphospholipid syndrome is characterized by the presence of antiphospholipid antibodies in plasma of patients with thromboembolic complications. A major problem in defining the syndrome is that serologic assays to detect antiphospholipid antibodies have a low specificity. We recently published a method that specifically detects lupus anticoagulant (LAC) caused by anti-beta(2)-glycoprotein I antibodies. Here, we studied the clinical relevance of detecting beta(2)-glycoprotein I-dependent LAC. Plasma samples were collected from 198 patients with autoimmune diseases. In those samples with a positive partial thromboplastin time-lupus anticoagulant (PTT-LA), a modified activated partial thromboplastin time (aPTT)-based LAC test was performed with cardiolipin as confirming agent. Twenty-five of 58 patients with an aPTT-based LAC were dependent on the presence of anti-beta(2)-glycoprotein I antibodies. Presence of beta(2)-glycoprotein I-dependent LAC was almost completely associated with a history of thromboembolic complications (odds ratio, 42.3; 95% confidence interval, 194.3-9.9). An increased frequency of thrombosis was not found in 33 patients with LAC independent of anti-beta(2)-glycoprotein I antibodies (odds ratio, 1.6; 95% confidence interval, 3.9-0.8). The use of an LAC assay with cardiolipin as confirming agent strongly improves the detection of patients at risk of thrombosis. Our findings suggest that anti-beta(2)-glycoprotein I antibodies with LAC activity are antibodies that are responsible for the thromboembolic complications in the antiphospholipid syndrome.

Introduction In hemophilia, unravelling the mechanisms behind interindividual variability in phenotype remains a big challenge. Objectives To study platelet activation and platelet responsiveness in relation to hemostatic phenotypes in hemophilia B by 1) comparing hemophilia B patients to healthy controls and 2) relating these to the clinical phenotype of the individual patients with severe hemophilia B. Methods Platelet reactivity was determined in whole blood measuring expression of P-selectin and binding of fibrinogen with FACS analysis after stimulating or inhibiting platelets with concentration series of several (ant-)agonists, including adenosine diphosphate (ADP), convulxin (CVX) and thrombin receptor associated protein (TRAP)and iloprost. Concentration of soluble platelet activation markers (soluble P-selectin, β-Thromboglobulin and RANTES) were measured in platelet poor plasma by ELISA. Markers of platelet activation and responsiveness were compared between healthy controls (n=12), patients with mild/moderate (n=12) or severe hemophilia B (n=19). In severe hemophilia patients, annual FIX consumption/kg was calculated over the last 6 years. Data were analysed with linear regression using SPSS 20. Results Levels of basal platelet activation (both P-selectin and fibrinogen) were equal between controls, mild/moderate and severe hemophilia B (Figure 1). For basal P-selectin expression, the mean area under the curves for were 28.5 (95% CI 23.3-33.6), 33.0(95% CI 24.4-41.6) and 28.4(95% CI 23.9-32.9) respectively (p=0.4). After stimulation with ADP, CVX and TRAP (with and without iloprost), platelet responsiveness was similar in all groups. There was no relation between annual FIX consumption and any marker of platelet activity. For basal P-selectin expression, the beta coefficient was -0.212 (p=0.38)(Figure 2). Conclusion In hemophilia B, platelet activity and responsiveness was similar to healthy controls. In severe hemophilia patients, there was no relation between FIX consumption and platelet activity. Figure 1. Basal P-selectin activity between healthy controls and hemophilia patients Figure 1. Basal P-selectin activity between healthy controls and hemophilia patients Figure 2. Basal P-selectin activity according to FIX consumption in severe hemophilia B Figure 2. Basal P-selectin activity according to FIX consumption in severe hemophilia B Disclosures Schutgens: Pfizer: Research Funding.