Identification of Qualitative Platelet Disorders in Adolescent Women with Heavy Menstrual Bleeding

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4922-4922
Author(s):  
Kristina M. Haley ◽  
Susan Lattimore ◽  
Cara McDavitt ◽  
Ayesha Khader ◽  
Colin Boehnlein ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Board Of Directors ◽  
Platelet Function ◽  
Research Funding ◽  
Blood Platelet ◽  
Bleeding Disorder ◽  
Advisory Committees ◽  
Adolescent Women ◽  
Platelet Disorder ◽  
Platelet Function Assays

Abstract Introduction: Nearly 40% of adolescent women experience heavy menstrual bleeding (HMB), and identifiable bleeding disorders are diagnosed in only 20-60% of these patients. We suspect that qualitative platelet disorders contribute to HMB, but are under-diagnosed. A pilot study was conducted to evaluate platelet function in adolescent women with HMB employing four novel, small-volume, whole blood platelet function assays. In addition, primary and secondary hemostasis, bleeding phenotype, and quality of life were assessed. Methods: Patients referred to the Young Women's Hematology Clinic at Oregon Health & Science University for evaluation of HMB were offered participation in the study. Participants underwent standard review of their medical and family history and physical exam. Standard lab evaluation included CBC, PT, PTT, fibrinogen, thrombin time, Von Willebrand Panel, PFA-100, and iron studies with platelet aggregation or phenotyping performed if clinically indicated. Using less than 0.5 mL of whole blood, platelet function was assessed with four novel platelet function assays: assessment of platelet activation, secretion, and aggregation was assessed by flow cytometry analysis, while platelet adhesion and aggregation was assessed under shear in a capillary tube. Quality of life (QOL) was assessed using the PedsQL tool. Bleeding phenotype was assessed with the ISTH Bleeding Assessment Tool (ISTH BAT). Menorrhagia was assessed with the Pictorial Bleeding Assessment Chart (PBAC), the Philipp Tool and the clinical history. Results: Nine participants have enrolled on study to date, with 2 completing the 3-month visit. The median age of the cohort was 16 years (14-18 years). Eight out of nine categorized their period as heavy, 6 also had epistaxis, and 7 reported excessive bruising. The median ISTH BAT score was 4 (3-7). Of the 7 patients who had a Philipp Score obtained, 5 were positive. Median PBAC score was 161 (64-196). Median ferritin was 13 ng/mL (4-65 ng/mL). Median QOL psychosocial score was 70 (68.36-88.25), comparable to that of pediatric patients with cancer. Of the 9 participants, 6 had platelet aggregation and phenotyping. Four participants did not receive a bleeding disorder diagnosis, 1 was diagnosed with Type 1 VWD, 1 was diagnosed with bleeding disorder, NOS, and 1 was diagnosed with Ehlers Danlos Syndrome. Two participants were diagnosed with a qualitative platelet disorder (QPD): one based on platelet aggregation and one based on thromboelastography. The four novel platelet function assays confirmed platelet function abnormalities in the participants diagnosed with QPD's (Figure 1&2). Impaired platelet response to agonist stimulation was also observed in participants with non-platelet disorder bleeding disorder diagnoses and in participants without a bleeding disorder diagnosis. Conclusions: In this pilot study, the etiology of HMB in adolescent women was evaluated with four novel platelet assays in addition to standard assays of hemostasis. A bleeding disorder diagnosis was not made with standard evaluations in 4 out of 9 participants. The novel assays detected platelet abnormalities not observed using currently available clinical labs, and confirmed the presence of abnormal platelet function in participants with abnormal platelet function testing. These assays require significantly less blood volume than currently available assays and expand investigation of platelet function to platelet adhesion and platelet interactions in whole and flowing blood. Further work is needed to determine the sensitivity and specificity of the novel assays in detecting platelet dysfunction. Continued investigation into the impact of HMB on the adolescent female population is needed. Disclosures Haley: CSL Behring: Honoraria; Baxalta: Membership on an entity's Board of Directors or advisory committees. Recht:Biogen: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Biogen: Research Funding; Genentech: Research Funding; Novo Nordisk: Research Funding; Baxalta: Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Chronic Lymphocytic Leukaemia Is Associated with a Broad Platelet-Function Defect Which Is Exacerbated By Ibrutinib and Acalabrutinib

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3756-3756
Author(s):  
Scott R McGregor ◽  
Grace Gilmore ◽  
Elizabeth E. Gardiner ◽  
Sarah Hicks ◽  
Shilpa Rakesh ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Platelet Function ◽  
Whole Blood ◽  
Research Funding ◽  
Blood Platelet ◽  
Advisory Committees ◽  
Function Defect ◽  
Btk Inhibitors ◽  
Platelet Function Assays

Abstract INTRODUCTION Bleeding from thrombocytopathy is a common complication of advanced chronic lymphocytic leukaemia (CLL). In addition to disease-related thrombocytopenia, the presence of the CLL clone and/or therapeutic interventions may further impair platelet function. In particular, the BTK inhibitors ibrutinib and acalabrutinib are known to inhibit platelet glycoprotein VI (GPVI)-mediated platelet aggregation. We compared platelet function and markers of GPVI activation between untreated CLL patients, ibrutinib-treated CLL patients and healthy controls, and studied the in vitro effects of ibrutinib and acalabrutinib on clinically utilised platelet function assays to assess their impact on GPVI-mediated as well as non-GPVI-mediated platelet activation pathways. METHODS Blood samples from 17 healthy volunteers and 8 untreated CLL patients were spiked with vehicle or comparable plasma concentrations of ibrutinib (0.3µM, 1.0µM) and acalabrutinib (1.8µM, 6.0µM) attainable during the treatment of CLL. Additional samples were obtained from 5 CLL patients undergoing ibrutinib treatment. Platelet function was evaluated using whole blood multiple electrode aggregometry (MEA - Multiplate®) and light transmission aggregometry (LTA - AggRAM®) in response to varying concentrations of aggregation-inducing reagents (collagen, CRP-XL, ADP, TRAP, ristocetin, arachidonic acid, and adrenaline). Shear-induced platelet adhesion was assessed using PFA-100®. Soluble GPVI plasma levels were assessed by ELISA. RESULTS In the absence of treatment, CLL patients exhibited significant platelet defects on whole-blood platelet function analyses in response to various agonists including ADP, ristocetin, TRAP and collagen (MEA) and prolongation of PFA-100® collagen/epinephrine closure time. This impairment was not replicated in assays using platelet-rich plasma (LTA). Ibrutinib-treated CLL patients demonstrated an additive impairment of platelet function, especially in regards to collagen-mediated activation by MEA or PFA-100®. There was no significant difference in soluble GPVI levels between normal, untreated or ibrutinib treated CLL patients. Addition of clinically-attainable concentrations of ibrutinib and acalabrutinib in vitro produced similar concentration-dependent inhibition of platelet function in healthy controls, with inhibition of aggregation evident in response to various agonists including collagen, CRP-XL, ristocetin and ADP but not arachidonic acid or TRAP. Ibrutinib also impaired aggregation in response to epinephrine, and caused selective prolongation of the PFA-100® collagen/epinephrine closure time, an effect not observed with acalabrutinib. MEA appears more sensitive and reproducible than LTA to describe the various inhibitory effects on platelet aggregation. Similar concentration-dependent inhibition of platelet function was observed by adding ibrutinib and acalabrutinib in vitro to blood samples from untreated CLL patients. CONCLUSIONS CLL is associated with a broad platelet function defect, which can be exacerbated by BTK inhibitors. Acalabrutinib induces a platelet function defect similar but less potent to that observed with ibrutinib, with the exception of shear-induced platelet adhesion (PFA-100®) which was only abnormal with ibrutinib. Routine platelet function assays are capable of quantifying BTK inhibitor-induced platelet dysfunction in CLL patients, with the most sensitive and reproducible measure being collagen-induced aggregation by MEA. There was no evidence for BTK-dependent platelet GPVI cleavage. Whole-blood platelet function assays may have utility in managing CLL patients presenting with bleeding or requiring urgent surgery during therapy with BTK inhibitors. Disclosures McGregor: Pfizer: Other: Conference travel support; Bristol-Myers Squibb: Other: Conference travel support; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria. Baker:CSL Behring: Research Funding; Biogen Idec: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Research Funding; Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support, Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support; Roche: Other: Conference travel support; Novo Nordisk: Other: Conference travel support.

Clot-On-A-Chip: A Microfluidic Device To Study Platelet Aggregation and Contractility Under Shear

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2363-2363 ◽  
Author(s):  
Lucas Ting ◽  
Shirin Feghhi ◽  
Ari Karchin ◽  
Wes Tooley ◽  
Nathan J White ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Board Of Directors ◽  
Microfluidic Device ◽  
Platelet Function ◽  
Research Funding ◽  
Force Generation ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Contractile Forces

Abstract Introduction In primary hemostasis, platelets adhere, activate, and aggregate at the wall of an injured vessel to form a hemostatic plug for the cessation of bleeding. After activation, platelets generate myosin-driven contractile forces to compact the size of the plug in order to reduce the space between platelets and prevent their disaggregation. Hemodynamic shear can be a major effector of platelet function in hemostasis, but its effect on the ability of platelets to produce contractile forces is an open question. Studying the dynamics of platelet aggregation and platelet force generation under hemodynamic shear can provide important insights into hemostasis and thrombosis. Method We have developed a microfluidic device that uses microscale blocks to induce platelet aggregation and microscale posts to measure platelet forces in a hemostatic plug. Whole human blood in heparin or citrate is pumped through a microfabricated chip containing microchannels with arrays of blocks and posts arranged along the bottom of a microchannel (Fig. 1). The surface of the blocks and posts are pre-coated with von Willebrand factor and type I collagen to allow for platelet adhesion. As blood is passes over a block, its rectangular shape induces a high shear rate that causes platelets to aggregate on its surface. A flexible micropost is situated behind each block. As platelets aggregate between the block and post, their contractile forces causes the post to bend toward the block. The deflection of the post is recorded under fluorescence microscopy and analyzed using quantitative image analysis of the videos. Since a microscale post bends like a cantilever beam, its deflection can be used to quantify the forces of platelets. Results Blebbistatin, a myosin inhibitor, was used to confirm that deflection of the posts by the platelets in heparinized blood was due to myosin activity. When blood was incubated with 2-MeSAMP, a P2Y12 antagonist, platelets were able to aggregate, but their ability to generate contractile forces was substantially reduced. This finding indicates that ADP activation is needed for platelet contractility under shear. The rate of hemodynamic shear was found to influence platelet function, for the rate of platelet aggregation and force generation were found to increase for blood sheared from 2000 to 12,000 s-1. Moreover, platelet aggregation and contractile forces were reduced when glycoprotein Ib-V-IX complex and integrin αIIbβ3 were inhibited with antibody AK2 and antibody fragment c7E3 Fab, respectively. When citrated blood was incubated with tissue plasminogen activator, platelets aggregate and produced contractile forces that increased steadily within the first ten minutes, but then the forces began to subside. Conclusions Our device can be used to study the role of hemodynamic shear in platelet function and gives insights into the role of platelet forces during hemostasis. Its microscale dimensions also allow us the study the biomechanics involved in the formation of a hemostatic plug during its early stages of growth and stability. Disclosures: White: Vidacare Corp: Honoraria; Stasys Medical Corp: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; NIH: Research Funding; Coulter Foundation: Research Funding; Washington State Life Sciences Discovery Fund: Research Funding. Sniadecki:Stasys Medical Corporation: Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees.

Ristocetin-Induced Platelet Aggregation for Monitoring of Bleeding Tendency in Ibrutinib-Treated Patients with Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 718-718 ◽  
Author(s):  
Lukas Kazianka ◽  
Christa Drucker ◽  
Cathrin Skrabs ◽  
Philipp Bernhard Staber ◽  
Edit Anna Porpaczy ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Board Of Directors ◽  
Platelet Function ◽  
Research Funding ◽  
Bleeding Event ◽  
Normal Subjects ◽  
Lymphocytic Leukemia ◽  
Bleeding Tendency ◽  
Bleeding Events ◽  
Advisory Committees

Abstract Background. Inhibition of Bruton´s tyrosine kinase (BTK) with the small molecule ibrutinib has significantly improved the survival of patients with chronic lymphocytic leukemia (CLL). BTK is also expressed in platelets. Collagen- and von Willebrand Factor (vWF)-dependent (ristocetin-induced) impairment of platelet function has recently been described (Levade M et al., Blood 2014, 124:3991-5;Kamel S et al., Leukemia 2015, 29:783-787) . Bleeding events were observed in 61% of patients in a recently published 3 year follow-up (Byrd JC et al., Blood 2015, 125:2497-2506). Bleeding under ibrutinib is generally mild (CTC grade 1-2 corresponding to spontaneous bruising or petechiae), but grade 3 or 4 bleeding can be observed, particularly after trauma. We hypothesized that quantitative assessment of platelet aggregation in ibrutinib CLL patients could help (1) to predict bleeding tendency, and (2) to guide patients through invasive procedures. Patientsand Methods. Twenty-four adult patients with previously treated CLL (16 male/8 female, median age 67 years, range 55-84) received ibrutinib orally at a planned dose of 420mg/day and were regularly monitored and thoroughly investigated for bleeding tendency. The median time on ibrutinib was 7.5 months, (range 1-27). Bleeding events (any CTC grade) occurred in 13 (54%) and dose-reductions to 280 (N=12) or 140mg (N=3) (for bleeding, infections, or neutropenia) were made in 15 (63%) of patients during a median observation period of 5 months (range 1-12). Bleeding was observed in 4 of 6 patients with concomitant anticoagulation. Of note, only 1 of the 24 patients had a CTC grade 3 bleeding event, and no grade 4 or 5 events were observed. Ristocetin-induced platelet aggregation (RIPA, herein referred to as RCoF) was quantitatively measured in fresh hirudin-blood by whole blood aggregometry with a Multiplate® Analyzer (Roche Diagnostics). Platelet aggregation was expressed in AUC units (U) (normal range 98-180U). Controls included normal subjects (N=53). Consecutive samples before and during treatment were available in all patients. Statistical methods comprised t-Test and ANOVA using SAS. Results. Ristocetin-induced platelet aggregation was already diminished before ibrutinib treatment (median 51 RCoF U) when compared to normal controls (Table 1). This is likely due to lower platelet counts in CLL patients influencing overall platelet aggregability (Hanke AA et al., Eur J Med Res 2010, 15:214-219). During ibrutinib treatment, platelet aggregation was substantially impaired (median of 22U). A direct comparison of available paired samples in 5 patients showed a significant decrease after ibrutinib initiation (51 to 14.5U; p=0.0028). Of note, significantly lower values were measured at visits when bleeding events were documented (N=34) compared to patient visits without bleeding tendency (N=70) (median 13 vs. 42U; p<0.001). The median RCoF value was lower in patients with CTC grade > 2 (N=10) vs. <2 bleeding (11 vs. 14U). Similar results were obtained for collagen-dependent platelet function (bleeding vs. no bleeding: 17 vs. 19.5U; p=0.002). RCoF values were correlated with platelet count (r2 =0.34; p<0.0001) at median values of 103 vs. 138 G/L in patients with or without bleeding, respectively. There was also a significant difference between the lowest RCoF values in individual patients with or without bleeding (7.5 vs. 16.5U; p=0.027) (Figure 1). No bleeding event was observed in patients whose lowest RCoF value was greater than 25U. Long-term kinetics of vWF-dependent platelet function was assessed in 7 patients and corresponded with ibrutinib dose. When ibrutinib was stopped, recovery of RCoF to greater than 70U was observed in as little as 48hours, suggesting a short time to normalization of platelet function. Conclusion. These data indicate that quantitative assessment of vWF-dependent platelet function in ibrutinib treated patients may serve to monitor therapy particularly in the setting of bleeding tendency, anticoagulation, or planned invasive procedures. Further evaluation of platelet function as a pharmacodynamic marker seems warranted. Figure 1. VWF-dependent platelet function (RCoF) in normal subjects or CLL patients before and during ibrutinib treatment with or without bleeding. Figure 1. VWF-dependent platelet function (RCoF) in normal subjects or CLL patients before and during ibrutinib treatment with or without bleeding. Figure 2. Lowest RCoF values in individual patients with or without bleeding. Figure 2. Lowest RCoF values in individual patients with or without bleeding. Disclosures Staber: Genactis: Research Funding; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Takeda-Millenium: Research Funding; Karyopharm: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria. Pabinger:Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Baxter: Membership on an entity's Board of Directors or advisory committees. Jilma:True North Therapeutics, Inc.: Consultancy, Research Funding. Jaeger:Hoffmann La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; True North Therapeutics, Inc.: Research Funding.

scholarly journals Prospective Study Reveals Increased Platelet Function Associated with Multiple Myeloma and Its Treatment

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Dalia Khan ◽  
Joanne Mitchell ◽  
Rekha Rana ◽  
Neline Kriek ◽  
Amanda Unsworth ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Platelet Function ◽  
Research Funding ◽  
Platelet Reactivity ◽  
Immunoblot Analysis ◽  
Healthy Controls ◽  
Advisory Committees ◽  
Fibrinogen Binding ◽  
The Impact

Background: Multiple Myeloma (MM) is a rare incurable bone marrow cancer characterised by a malignant proliferation of plasma cells. MM is usually preceded by a premalignant and benign Monoclonal Gammopathy of Undetermined Significance (MGUS). The incidence of arterial and venous thrombosis in MM is substantially higher than in the normal population, however the cause of this increased thrombosis risk and the impact of MM on platelet function is unclear. Treatments for both newly diagnosed and relapsed/refractory patients with MM include Immunomodulatory drugs (IMiDs) such as thalidomide/lenalidomide-based combinations. These treatments improve considerably patient outcomes, however iMiD treatment also increases the risk of thrombotic complications in these patients. Aims: In this prospective study we explored the impact of MM and its treatment on platelet function. Methods: High throughput functional analysis was performed using platelets from normal healthy controls (n=31) and patients with MGUS (n=18), smouldering multiple myeloma (SMM, n= 20), and MM (26). The MM group was further divided into 3 treatment cohorts; (1) no treatment, (2) treatment with proteasome inhibitor (PI) and dexamethasone (Dex), and (3) treatment with PI, Dex, immunomodulatory drug (iMiD) and direct oral anticoagulant. Platelet aggregation and activation (fibrinogen binding and P-selectin exposure) were measured in response to a concentration range of agonists including ADP, the thrombin receptor agonist TRAP-6, collagen, collagen-related peptide (CRP), a thromboxane receptor agonist U46619 and epinephrine. Cereblon protein was detected in platelet protein extracts by immunoblot analysis. Results: Consistent with previous reports, modestly increased VWF and factor VIII levels were detected in MM patients, but no additional differences in coagulation parameters were detected in patient groups compared to normal healthy controls (other than expected due to anticoagulant usage). Platelet aggregation in response to each agonist was increased significantly in the MM patient group compared to the normal healthy controls, suggesting that platelet reactivity is elevated in MM patients through a common mechanism that is shared by different activation pathways or the involvement of multiple mechanisms. P-selectin exposure on platelets from MM patients was not significantly different from normal healthy donors, indicating that enhanced platelet reactivity in MM is specifically through modulation of integrin αIIbβ3 activation, fibrinogen binding and therefore enhanced aggregation. The effects of treatment on platelet function in patients on iMiD vs. non iMiD treatment were assessed. In the iMiD treatment group, patient platelets aggregated in response to lower concentrations of ADP, collagen, epinephrine and CRP in samples taken post-treatment compared to those taken before and during treatment. This demonstrates an increased sensitivity to platelet activation in these patients induced by treatment. Immunoblot analysis revealed that platelets contain cereblon, a therapeutic target of lenalidomide. The potential direct effects of iMiDs on platelets in vitro was therefore explored. Lenalidomide treatment (10mM) increased the ability of platelets to aggregate in response to low concentrations of each agonist tested when compared to normal controls. Conclusions: Platelet reactivity is increased in multiple myeloma and increased further upon iMiD treatment. The presence of the key therapeutic target for iMiDs in platelets and the ability of lenalidomide to modulate platelet function directly, reveals new avenues for investigation to determine the underlying mechanism of action. Disclosures Laffan: CSL: Consultancy; Pfizer: Consultancy; Sobi: Consultancy; Roche: Consultancy; LFB: Consultancy; Shire: Consultancy; Octapharma: Consultancy; Bayer: Speakers Bureau; Roche-Chugai: Speakers Bureau; Takeda: Speakers Bureau; Leo-Pharma: Speakers Bureau; Pfizer: Speakers Bureau. Shapiro:Bayer: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; NovoNordisk: Consultancy, Speakers Bureau; Sobi: Consultancy, Speakers Bureau; Chugai/Roche: Consultancy, Speakers Bureau; Shire/Takeda: Consultancy, Speakers Bureau. Thakurta:Oxford University: Other: visiting professor; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Ramasamy:Takeda: Research Funding; Janssen: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding; Amgen: Research Funding; Amgen: Honoraria; Takeda: Honoraria; Sanofi: Honoraria; Oncopeptides: Honoraria; Takeda: Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Bristol Myers Squibb: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers squibb: Membership on an entity's Board of Directors or advisory committees. Gibbins:Bristol Myers Squibb: Research Funding; Arena Pharmaceuticals: Research Funding.

scholarly journals Idelalisib Rapidly Improves Platelets Function in Patients with Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5566-5566
Author(s):  
Gianluigi Reda ◽  
Ramona Cassin ◽  
Andrea Artoni ◽  
Anna Lecchi ◽  
Bruno Fattizzo ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Pilot Study ◽  
Board Of Directors ◽  
Research Funding ◽  
Bleeding Risk ◽  
Bleeding Tendency ◽  
Advisory Committees ◽  
Coagulation Tests ◽  
Haematological Remission

Abstract INTRODUCTION Platelet function has never been studied systematically in patients with CLL. Novel drugs are now available for CLL treatment that may impact on platelet functions. Fifty per cent of patients treated with Ibrutinib suffered from minor bleedings and only 5% from major bleedings, partly caused by the drug driven inhibition of platelet glycoprotein VI signaling. No data on bleeding tendency has yet emerged on patient treated with Idelalisib, the first specific inhibitor of PI3K δ p110 approved for the treatment of relapsed/refractory CLL or for patients with del17p or TP53 as first line therapy. In animal models a reduction of p110δ on platelets (PLT) does not increase bleeding, causing only a slight reduction of platelet aggregation and activation. Knowledge about potential bleeding complications associated with the use of small molecules may be relevant in older patients and those at increased bleeding risk due to concomitant therapies. PATIENTS AND METHODS Ten patients with CLL (M/F: 6/4; median age: 71 years, range 47-82) who started therapy with Idelalisib were enrolled in a prospective observational pilot study. The Bleeding Severity Score (BSS), a validated questionnaire, was administrated to patients to estimate bleeding before and during idelalisib therapy. All patients underwent coagulation tests and platelet aggregation/secretion studies with different aggregating agents before starting therapy with Idelalisib, after 28 + 7 days and after 3 months. Patients with a platelet count less than 80.000/mm3, in antiplatelet or anticoagulant therapy, with recent use (within 7 days) of NSAIDs and a diagnosis of hereditary thrombocytopenia/pathy were excluded. We defined complete haematological remission (CHR) as Hb more than 11g/dl, PLT more than 100.000/mm3, lymphocyte less than 5.000/mm3 and partial haematological remission (PHR) as a response not fulfilling criteria for CHR. RESULTS No cases of hemorrhagic complications or increased bleeding tendency were observed in patients with CLL and no patients had a pathologic BSS (>5) at enrolment. All patients had coagulation tests within normal limits at baseline and after 28 days. Platelet count was below 100.000/mm3 in 5 patients. In 9 out of 10 patients platelet aggregation was pathological with at least 2 of the 4 aggregating agents tested. Platelet secretion before initiation of treatment with Idelalisib was particularly impaired with ADP (8/10 patients), while was pathological with collagen, a strong agonist, in only 2 patients. In 8 patients intraplatelet ATP/ADP ratio was pathological, as observed in delta storage pool disease. After 28 days of treatment 4 of 10 patients were in CHR and 3 in PHR. Platelets count was still below 100.000/mm3 in 2 subjects. At 28 days in 5 out of the 9 patients with pathological baseline test, platelet aggregation improved, while 3 remained unchanged and in one worsened. Even ADP secretion normalized in 4 patients. ATP/ADP ratio did not significantly change. At three months 7 patients reached CR and 2 reached PR. At three months platelets count was still below 100000/mm3 in 2 patients. In 3 patients platelet aggregation further ameliorated. CONCLUSIONS In this pilot study, treatment with idelalisib improved platelet aggregation tests in most of the CLL patients who presented a pathological test before starting therapy. It's unlikely that the drug has a direct effect on platelets, given their low expression of PI3Kδ; therefore our results are probably due to the rapid idealisib effect on CLL clone. Based on these preliminary data, Idelalisib seems to be safe in patients with an increased bleeding risk. Disclosures Reda: Roche: Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding. Peyvandi:Alexion: Other: research funding paid to Luigi Villa Foundation, Research Funding; Ablynx: Membership on an entity's Board of Directors or advisory committees, Other: research funding paid to Luigi Villa Foundation, Research Funding; CSL Behring: Speakers Bureau; Biotest: Other: research funding paid to Luigi Villa Foundation, Research Funding, Speakers Bureau; Octapharma: Consultancy; Kedrion Biopharma: Consultancy, Other: research funding paid to Luigi Villa Foundation, Research Funding; LFB: Consultancy; Grifols: Speakers Bureau; Novo Nordisk: Other: research funding paid to Luigi Villa Foundation, Research Funding, Speakers Bureau; SOBI: Speakers Bureau; Bayer: Speakers Bureau.

scholarly journals Initial Evaluation of Adolescent Females Hospitalized with Heavy Menstrual Bleeding

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1216-1216
Author(s):  
Lauren E Amos ◽  
Shannon L Carpenter
Keyword(s):  
Board Of Directors ◽  
Platelet Function ◽  
Adolescent Females ◽  
Bleeding Disorder ◽  
Diagnostic Evaluation ◽  
Heavy Menstrual Bleeding ◽  
Bleeding Disorders ◽  
Advisory Committees ◽  
Platelet Function Disorders ◽  
Von Willebrand

Abstract Background: Heavy menstrual bleeding (HMB) in adolescents can be severe and life-threatening. Up to 30% of young women who are hospitalized with anemia due to HMB have a bleeding disorder. Guidelines from the American College of Obstetrics and Gynecology (ACOG) and the National Heart, Lung, and Blood Institute (NHLBI) recommend evaluation for bleeding disorders in such patients. ACOG recommendations include testing for von Willebrand disease (VWD) and specify that consultation with a hematologist may help in interpreting results. NHLBI recommends testing for vWD be done in conjunction with a hematologist. As von Willebrand factor is an acute phase reactant, testing when patients are severely anemic and bleeding may not provide accurate results. ACOG guidelines do not include testing for platelet function disorders (PFD), though PFD may be as prevalent as VWD in females with HMB. Early and accurate diagnosis of bleeding disorders is important for health and quality of life, yet limited data exists on the diagnostic evaluation for bleeding disorders in adolescent females hospitalized for HMB. Objectives: To evaluate the diagnostic evaluation of bleeding disorders in adolescent females hospitalized for HMB. Methods: A retrospective, single center chart review of female patients aged 9-21 years hospitalized for HMB and anemia at a tertiary care children's hospital from January 1, 2000 until December 31, 2017 was done. HMB was defined as menses ≥7 days in length, use of 8 or more pads or tampons per day during menses, pictorial bleeding assessment chart (PBAC) score greater than 100, or symptomatic anemia. Patients were identified from our Hemophilia Treatment Center (HTC) registry, review of patients seen at a comprehensive clinic staffed by pediatric hematologists and gynecologists for adolescent females with HMB and bleeding disorders, and by an Electronic Medical Record (EMR) query of admission and discharge diagnoses of HMB and anemia. Data obtained included clinical features, diagnostic evaluation, and laboratory results. Results: 118 patients hospitalized for HMB and anemia were included. Inpatient Hematology consult or outpatient referral occurred in 68 (58%) of the patients; 60/68 (88%) had a bleeding disorder evaluation completed. 34 patients had a hematologic disorder. PFD was the most common (15/34; 44%) followed by VWD (9/34; 26%). 42% (50/118) of the patients did not have a Hematology consult or outpatient referral (Table 1). While hospitalized for HMB and anemia, 29 of the 50 patients had testing for vWD performed and only 4/29 (14%) had testing repeated as an outpatient once hemoglobin normalized. No patients tested for VWD while inpatient had results consistent with the diagnosis. Platelet function testing was performed in 10/50 patients using the platelet function analyzer (PFA-100) in 8 patients and platelet aggregometry in 2 patients. Conclusions: Despite national guidelines and the presence of known risk factors such as HMB since menarche and HMB causing severe anemia, the hematology service was not involved in the diagnostic process for a significant number of adolescent females. In these patients, testing often occurred while patients were hospitalized and was not repeated. Testing for platelet function disorders occurred infrequently and mainly consisted of the PFA-100 which lacks sensitivity and specificity. When patients were evaluated by Hematology and tested for bleeding disorders, a large proportion had a bleeding disorder, of which PFD were most common. This study demonstrates the need for standardization of the evaluation of adolescent females hospitalized for HMB. Guidelines should be updated to include testing for PFD. Hematologists should be involved when females are hospitalized for HMB and anemia. Disclosures Carpenter: Genentech Incorporated: Membership on an entity's Board of Directors or advisory committees; Nationwide Children's Hospital: Speakers Bureau; Bayer: Honoraria; Kedrion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk Pharmaceuticals, Inc: Consultancy; HEMA Biologics: Consultancy; American Academy of Pediatrics: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Consultancy; National Hemophilia Foundation (Impact Education): Speakers Bureau; Kane County State's Attorney: Consultancy; CSL Behring: Speakers Bureau; 4th Judicial District Attorney's Office- Colorado: Consultancy; Kedrion Biopharmaceuticals: Consultancy.

scholarly journals The Secreted Tyrosine Kinase Vlk Is Essential for Normal Platelet Activation and Thrombus Formation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-11
Author(s):  
Leila Revollo ◽  
Glenn Merrill-Skoloff ◽  
Karen De Ceunynck ◽  
James Dilks ◽  
Mattia Bordoli ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Kinase ◽  
Board Of Directors ◽  
Platelet Function ◽  
Secretory Pathway ◽  
Thrombus Formation ◽  
Extracellular Proteins ◽  
Advisory Committees ◽  
Extracellular Domains

Tyrosine phosphorylation of proteins secreted into the extracellular space has been observed in cell cultures and in vivo, yet little is known about the role that phosphorylation of extracellular proteins serves in modulating cell function. An important reason for the gap in our knowledge of the functional significance of extracellular protein phosphorylation has been the delay in identifying extracellular kinases. Within the last decade, however, bioinformatic strategies to identify kinases with signal peptides, coupled with biochemical approaches to characterize kinases in the secretory pathway, have described several kinases that phosphorylate secretory pathway and extracellular substrates. Of the known kinases containing signal sequences, Fam20B and VLK have been identified in platelets. VLK has been identified as a broadly expressed secretory pathway tyrosine kinase secreted from platelets in an activation dependent manner. Its role in platelet function, however, has not been previously studied. To understand the contribution of tyrosine phosphorylation of secreted factors and extracellular domains of transmembrane proteins in platelet function and thrombus formation, we generated mice whose platelets lacked VLK. Mice with megakaryocyte/platelet-specific VLK deficiency (Vlk-cKO) exhibited normal platelet abundance, volume and morphology, and tail clip bleeding times, but showed dramatic changes in platelet function in vitro and in vivo. In vivo, platelet accumulation was reduced by 90% in Vlk-cKO mice compared to control (Vlkf/f) littermates (P = 0.02) following laser-induced injury of cremaster arterioles (Figure). Likewise, fibrin generation was reduced in mice lacking platelet VLK by 62% (P = 0.009). In vitro, evaluation of resting and thrombin-stimulated VLK-deficient platelets demonstrated a significant decrease of several tyrosine phosphobands compared to control. Platelet function testing of VLK-deficient platelets (Figure) showed decreased platelet aggregation in response to stimulation with 100 µM AYPGKF, a PAR4 agonist, (Vlkf/f: 70+5.1%; Vlk-cKO: 23+8.0%) or 4 µg/mL collagen (Vlkf/f: 53+2.5%; Vlk-cKO: 27.5+2.9%). Dense and α-granule release in response to AYPGKF were also significantly decreased in platelets in which VLK had been silenced. In contrast, Vlk-cKO platelets aggregated normally in response to either 10 µM, 40 µM, or 100 µM ADP, and the aggregation defect in response to low doses of AYPGKF was reversed by subthreshold concentrations (2.5 µM) of ADP. Furthermore, stimulation with high-dose 150 µM AYPGFK or 5 U/ml thrombin resulted in comparable platelet function and ATP secretion in control and Vlk-cKO platelets respectively, ruling out a storage pool defect. Taken together, these results suggest that a dense granule secretion defect contributes to the decrease in platelet aggregation observed in platelets in which VLK is absent. In human platelets, tyrosines phosphorylated in secreted and extracellular domains of transmembrane proteins implicated in the regulation of platelet function were identified by mass spectroscopy analysis. Extracellular proteins or proteins with phosphosites that mapped to extracellular domains included ectonucleoside triphosphate diphosphodydrolase 6 [ENTPD6], platelet basic protein, integrin αIIß, and multimerin-1. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus dependent platelet aggregation and thrombus formation, and provide the first evidence that secreted kinases contribute to platelet function. Disclosures De Ceunynck: Sanofi: Current Employment. Dilks:PlateletBiogenesis: Current Employment. Peters:PlateletBiogenesis: Current Employment. Noetzli:Anylam: Current Employment. Rosen:Keros Therapeutics: Membership on an entity's Board of Directors or advisory committees. Italiano:PlateletBioGenesis: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Flaumenhaft:STRM.Bio: Membership on an entity's Board of Directors or advisory committees; PlateletDiagnostics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; QuercisPharma: Membership on an entity's Board of Directors or advisory committees.

A History of Abnormal Bleeding Correlates with Platelet Dysfunction in Aggregation Studies but Not PFA-100 Analyses

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3447-3447 ◽  
Author(s):  
Dharmesh Gopalakrishnan ◽  
Heesun J Rogers ◽  
Paul Elson ◽  
Keith R. McCrae
Keyword(s):  
Flow Cytometry ◽  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Board Of Directors ◽  
Platelet Function ◽  
Bleeding Event ◽  
Advisory Committees ◽  
History Of ◽  
Abnormal Bleeding ◽  
Platelet Release

Abstract Introduction:In this retrospective medical records-based study we analyzed the association between abnormal spontaneous or procedure-related bleeding and patterns of abnormality in tests of platelet function. Commonly used platelet function tests include Platelet Function Analyzer -100 Closure Times (PFA-100 CT), platelet aggregation testing using Light Transmission Aggregometry (LTA), and platelet dense granule release assay (by lumi-aggregometry). Other related tests include Von Willebrand factor (VWF) analysis, platelet flow-cytometry for cell surface glycoprotein expression, and electron microscopy. LTA in our center uses five different agonists - ADP, arachidonic acid, collagen, epinephrine and ristocetin, while lumi-aggregometry is performed using four agonists - ADP, arachidonic acid, collagen and epinephrine. Methods:This study included 497 patients who had platelet aggregation testing done using LTA between August 2008 and August 2013. Sixty-nine percent (n = 354) of these patients had a history of abnormal bleeding. Since the data were not normally distributed, Wilcoxon rank-sum test (for continuous variables) and Fisher's exact test (for categorical variables) were used wherever appropriate. P value of < 0.05 was considered as significant. Results:Of the patients with a history of abnormal bleeding, 81% had spontaneous bleeding, 29% had surgery/procedure-related bleeding, and 13% had history of both types of abnormal bleeding. Three hundred nine of these patients had a recent (< 4 weeks) bleeding event. Abnormal bleeding, recent or historical, was found to associate significantly with impaired platelet aggregation, as well as platelet release in response to ADP, arachidonic acid, collagen or epinephrine (P<0.001 for all). Abnormal aggregation and release in response to ≥2 different agonists was also significantly associated with abnormal bleeding, as was the total number of abnormalities on the aggregation and release panel (P<0.001). A history of a recent bleeding event (<4 weeks) was found to be associated with reduced aggregation in response to arachidonic acid (P = 0.001), impaired aggregation and release in response to collagen (P =0.04 and <0.001 respectively), and reduced platelet release in response to epinephrine (P = 0.005). Recent bleeding also correlated with the total number of abnormalities in the aggregation (P = 0.002) and release panels (p =0.03). No significant association was found between a history of abnormal bleeding (either recent or historical) and prolonged PFA-100 closure times (collagen/ADP or collagen/epinephrine), or any abnormality in VWF analyses or platelet flow-cytometry studies. Conclusions:While PFA-100 closure times, VWF analyses and platelet flow-cytometry panel failed to show an association with abnormal bleeding (historical or recent), impaired platelet aggregation or release in response to several agonists was found to correlate with abnormal spontaneous and/or procedure-related bleeding, recent as well as historical, suggesting that platelet aggregation and release assays may be useful in diagnosis of a bleeding diathesis in patients with an appropriate clinical history Disclosures McCrae: Halozyme: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Syntimmune: Consultancy; Momenta: Consultancy.

Platelet Function Disorders in Bleeding Patients without Other Coagulopathies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4650-4650
Author(s):  
Francine R. Dembitzer ◽  
Ellinor I.B. Peerschke ◽  
Caroline Cromwell ◽  
Xiaofei Zhang ◽  
Louis M. Aledort
Keyword(s):  
Platelet Aggregation ◽  
Board Of Directors ◽  
Platelet Function ◽  
Coagulation Factor ◽  
Clinical History ◽  
Study Cohort ◽  
Advisory Committees ◽  
Normal Platelet ◽  
Platelet Function Disorders ◽  
Age Range

Abstract Introduction: We present a series of 105 patients referred for hematologic consultation to evaluate a clinical bleeding disorder. Little is known regarding the prevalence of non-genetic qualitative platelet disorders and which of these might respond to desmopressin. Methods: Patients were assessed over a two-year period, from December 2011 through December 2013. Patients, who were found to have neither von Willebrand's disease nor coagulation factor deficiencies, nor quantitative platelet defects, were tested for platelet function abnormalities by PFA-100 (Dade Behring, Marburg, Germany) and platelet aggregation and secretion studies using lumi aggregation (Chrono-Log Corp., Havertown, PA, USA). Selected patients with abnormal platelet function were given a trial of intravenous desmopressin to determine efficacy, and platelet function studies were repeated 2 hours later. Results: Of the 105 referred patients (26 males, age range 15-83 years; 79 females, age range 21-86 years), 67 with either normal (n=43) or abnormal (n=24) PFA-100 results were not evaluated further based on their clinical history and presentation. 18 patients with abnormal PFA-100 results, consisting of Collagen/ADP, Collagen/Epinephrine, or both, had platelet aggregation and secretion studies performed. 16 of these patients had abnormal platelet aggregation and secretion studies, while 2 patients had normal studies. In addition, 20 patients with normal PFA-100 results underwent platelet aggregation and secretion studies. Of these, 17 had abnormal platelet aggregation and secretion predominantly in response to two or more weak agonists, including ADP, epinephrine and/or arachidonic acid. Only 3 of these 20 patients had normal platelet aggregation and secretion. Of the 16 patients with both abnormal PFA-100 and abnormal platelet aggregation and secretion tests, 11 underwent subsequent pre- and post- desmopressin platelet function testing. 7 of the 11 patients showed improvement or complete normalization of at least one PFA-100 parameter, i.e., either Collagen/ADP, Collagen/Epinephrine, or both. However, results of platelet aggregation and secretion tests remained unchanged, irrespective of PFA-100 results. Conclusions: In the present study, platelet aggregation and secretion studies in patients with no other coagulopathies revealed platelet function defects in approximately 30% of the study cohort. Platelet function defects were in response to weak agonists, including ADP, epinephrine, and/or arachidonic acid.Our data support the recent SCC ISTH recommendations against the use of PFA-100 for platelet function screening.We recommend that the hematologist carefully assess the bleeding history, and, if significant, pursue platelet aggregation and secretion studies to identify potential platelet function defects. Disclosures Aledort: Baxter Healthcare: Membership on an entity's Board of Directors or advisory committees, Other: DSMB Participation; Kedrion BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees.

scholarly journals A Novel Method for Flow Cytometric Quantification of Human Platelet Membrane Glycoproteins in Citrated Whole Blood

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2367-2367
Author(s):  
Christina Berens ◽  
Jens Müller ◽  
Heiko Rühl ◽  
Johannes Oldenburg ◽  
Bernd Pötzsch
Keyword(s):  
Flow Cytometry ◽  
Board Of Directors ◽  
Whole Blood ◽  
Research Funding ◽  
Platelet Membrane ◽  
Percentage Increase ◽  
Advisory Committees ◽  
Flow Cytometric ◽  
Platelet Disorder ◽  
Novel Method

Introduction: Diagnosis of platelet receptor defects can be confirmed by flow cytometry using a panel of monoclonal antibodies. The conventional method utilizing platelet rich plasma (PRP) has many limitations such as time-consuming preparation, requirement of a large sample volume or limitations in samples from patients with thrombocytopenia or abnormally large platelets. In order to overcome these difficulties a novel method was developed using a second antibody directed against an erythrocyte protein which allows differentiation between platelets and erythrocytes in citrated whole blood (CWB). Methods: CWB or PRP from healthy blood donors and outclinic patients without platelet disorder (n= 18) as well as from two patients with Glanzmann thrombasthenia (GT) was stained with fluorescein isothiocyanate (FITC)-conjugated antibodies against CD42b (GP Ib), CD41a (GP IIb/IIIa), or CD62. CWB was stained in addition with a phycoerythrin (PE)-conjugated antibody against CD235a, a sialoglycoprotein expressed by erythroid precursors and erythrocytes. Platelet activation was induced by addition of 2 µM phorbol 12-myristate 13-acetat (PMA). The expression of CD42b, CD41a and CD62 on CD235a-negative cells was quantified by flow cytometry with and without PMA stimulation. Results: CWB and PRP samples showed no differences of mean (± standard deviation) fluorescence intensity (MFI)(GP Ib 74.78 ± 21.39 vs. 85.67 ± 26.03, GP IIb/IIIa 250.33 ± 59.08 vs. 234.50 ± 56.96, GP IIb/IIIa with PMA 349.50 ± 76.87 vs. 328.67 ± 72.92, GP IIb 26.10 ± 6.17 vs. 29.15 ± 8.29, GP IIb with PMA 35.58 ± 8.86 vs. 37.50 ± 8.98, CD62 1.91 ± 0.49 vs. 2.49 ± 1.14, CD62 with PMA 43.88 ± 10.89 vs. 37.78 ± 7.20, respectively). The increase of the fluorescence signal with PMA vs. without PMA was similar in CWB and PRP with a percentage increase of 141.17 ± 10.97 vs. 142.74 ± 15.08 % for GP IIb/IIIa, 136.76 ± 16.65 vs. 131.83 ± 16.64 % for GP IIb and 2432.14 ± 785.49 vs. 1701.37 ± 474.72 % for CD62. Patients with GT showed MFI values for GP IIb/IIIa and GPIIb expression less than 5 % in comparison to that of a healthy control. Conclusion: Flow cytometric analysis of platelet membrane proteins utilizing CWB is comparable to the conventional PRP method. Using an antibody against CD235a allows distinct differentiation between platelets and erythrocytes in CWB. This new method requires smaller amounts of blood, spares time-consuming preparation of PRP and is feasible to identify rare inherited platelet membrane disorders. Disclosures Berens: Baxter: Honoraria; Novo Nordisk: Honoraria; CSL Behring: Honoraria; Biotest: Honoraria; Shire: Honoraria; Pfizer: Honoraria. Müller:Roche: Membership on an entity's Board of Directors or advisory committees; Siemens: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; NovoNordisk: Membership on an entity's Board of Directors or advisory committees. Rühl:Sanofi Genzyme: Honoraria; SOBI: Honoraria; Shire: Honoraria; Bayer: Honoraria; CSL Behring: Honoraria; Grifols: Honoraria. Oldenburg:Swedish Orphan Biovitrum: Consultancy, Speakers Bureau; Grifols: Consultancy, Speakers Bureau; NovoNordisk: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Speakers Bureau; Takeda (Shire): Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Speakers Bureau; Bayer: Consultancy, Research Funding, Speakers Bureau; Biotest: Consultancy, Research Funding, Speakers Bureau; CSL Behring: Consultancy, Research Funding, Speakers Bureau; Octapharma: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau.

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