scholarly journals Increased Platelet Force Is Associated with Decreased Bleeding Severity in Pediatric Immune Thrombocytopenia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1096-1096
Author(s):  
Oluwamayokun T. Oshinowo ◽  
Renee Copeland ◽  
Christina Caruso ◽  
Meredith E. Fay ◽  
Traci Leong ◽  
...  
Keyword(s):  
Platelet Count ◽  
Single Cell ◽  
Immune Thrombocytopenia ◽  
Stress Test ◽  
Severe Bleeding ◽  
Contraction Force ◽  
Platelet Counts ◽  
Asymptomatic Patients ◽  
Low Platelet Count ◽  
Bleeding Severity

Background: Immune Thrombocytopenia Purpura (ITP) can result in severely low platelet counts and while most of the 4,000 new cases of pediatric ITP diagnosed each year self-resolve, 10% of these patients have major bleeding episodes. The platelet count remains the mainstay method for predicting hemorrhage and unfortunately has only proven to be loosely correlated to bleeding severity. Consequently, there currently exists no biomarker that accurately and reliably predicts which patients need immediate medical treatment, all of which have side effects, and which patients only require monitoring. To that end, we leveraged our Platelet Contraction Cytometer (PCC), a versatile system that measures platelet contraction forces at the single-cell level and at high-throughput, to study platelets of patients with ITP prospectively. Buchanan bleeding scores were used to distinguish patients with severe symptoms from asymptomatic patients. With 49 patients, we observed two significant findings: 1) Tracking both single platelet force measurements and platelet count enables stratifying patients into having major, minor, or no risk for bleeding. Accordingly, patients in the major risk category have a combined low platelet force and low platelet count. 2) Longitudinal studies showed that when major risk patients had increased platelet force or higher platelet counts, this was associated with the alleviation of major symptoms. Thus, when utilized together, platelet force and platelet count can more accurately predict bleeding severity in pediatric ITP patients. Platelet Contraction Cytometry: Our PCC utilizes a large array of fibrinogen "microdot" pairs patterned on a flat hydrogel of known stiffness. Thrombin-activated platelets adhere to these microdots, spread to the neighboring microdot, and contract, pulling the microdots closer together. As platelet force is directly proportional to the microdot displacement, only a single microscopy measurement is necessary to determine the force applied by each single platelet (Fig 1). As such, hundreds of platelets are measured in a controlled mechanical and biochemical environment (Myers et al, 2017) Results: With our cohort of 49 ITP patients, we observed that low average single cell platelet contraction forces highly correlate with severe bleeding symptoms (Buchannan scores 2-4). Using a regression tree, we found that an average force of 26nN best separates symptomatic from asymptomatic patients with a diagnostic sensitivity of 100% and specificity of 86.8% (AUC 0.97, 95% CI: 0.9361-1). However, platelets from some asymptomatic patients exerted forces of less than 26nN as well. In a more thorough examination, we found that majority of those asymptomatic patients with low platelet forces had platelet counts > 40,000/uL. Although our data shows that platelet count alone is a poor predictor of severe bleeding symptoms (AUC 0.8, 95% CI: 0.6757-0.9189), when a platelet count <40k/uL is coupled alongside our criterion of average contraction force <26 nN, we find that the specificity of our system increases to 94.7% and thus platelet count and platelet force synergistically predict which patients are symptomatic with high accuracy. Using a logistic regression model, we also found that with each 1nN decrease in average contraction force, the odds of the patient bleeding increases 2.1 (95% CI: 1.1-4.0) times that of being asymptomatic and when this decrease in contraction force is increased 5nN, the odds of a patient bleeding is 44.4 (95% CI: 1.8-1066.5) times that of being asymptomatic (Fig 2A-B). Moreover, when tracking individual patients (n=4) over time, we demonstrate that whenever a patient's blood sample is associated with increased single platelet average contraction force >26 nN or platelet count >40k/uL, bleeding symptoms are alleviated. Conversely, whenever a patient exhibited high platelet contractile force and low platelet count, the onset of bleeding symptoms correlated with a decrease in platelet forces. Unlike bulk or micro-clot assays, our assay functions as a "stress test" of single platelets by placing them in conditions that maximize contractility, and in both cases, the reduction in the subpopulation of highly contractile platelets is correlated with bleeding severity. As such, our work suggests that single platelet forces could be used as a diagnostic biomarker to assess bleeding risk in patients with ITP regardless of count (Fig 2C-D). Disclosures Bennett: Novartis: Research Funding. Lam:Sanguina, LLC: Equity Ownership.

scholarly journals Management of Adult Patients with Primary Immune Thrombocytopenia (ITP) in Clinical Practice: A Consensus Approach of the Spanish ITP Expert Group

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
M. Eva Mingot-Castellano ◽  
M. Teresa Álvarez Román ◽  
Luis Fernando Fernández Fuertes ◽  
Tomás José González-López ◽  
José María Guinea de Castro ◽  
...  
Keyword(s):  
Platelet Count ◽  
Clinical Practice ◽  
Immune Thrombocytopenia ◽  
Adult Patients ◽  
Severe Bleeding ◽  
Clinical Practices ◽  
Questionnaire Response ◽  
Diagnosis And Management ◽  
Asymptomatic Patients ◽  
Primary Immune Thrombocytopenia

Background and Objective. Diagnosis and management of primary immune thrombocytopenia (ITP) have changed dramatically in the last decade. The aim of the study was to obtain information about the opinion of the Spanish ITP Group (GEPTI) members regarding the best clinical practices for diagnosis and management of adult patients with ITP. Materials and Methods. A two-round Delphi method was carried out by sending to 129 experts a 90-item questionnaire developed by 11 specialists, with a 4-point Likert scale (“never,” “sometimes,” “frequently,” and “always”) for the assessment of responses. Results. Forty out of the 129 experts participated in the survey (participation rate 30.2%) and 39 completed the questionnaire (response rate 97.5%). Salient consensus points included the following: the need to indicate workup studies from a sustained platelet count < 100 x 109/L in the absence of a clear etiology; bone marrow aspiration in elderly patients with suspected ITP; beginning treatment in asymptomatic patients with a platelet count < 20 x 109/L; not exceeding 6-7 weeks of corticosteroid therapy; switching from corticosteroids to one thrombopoietin receptor agonist (TRA); switching to other TRA or other options as combinations of them with immunosuppressive drugs in case of failure; how to reduce tapering TRA; treating patients with symptomatic persistent ITP and platelet count > 20 x 109/L; and considering mucosal or severe bleeding as a basic criterion for hospital admission. Conclusions. The present consensus document provides a reference framework for the management of patients with ITP in clinical practice.

scholarly journals Platelet Contraction Force As a Biophysical Biomarker for Bleeding Risk in Patients with Immune Thrombocytopenia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 517-517
Author(s):  
Oluwamayokun T. Oshinowo ◽  
Renee Copeland ◽  
Carolyn M. Bennett ◽  
Wilbur A Lam ◽  
David R. Myers
Keyword(s):  
Platelet Count ◽  
Single Cell ◽  
Platelet Function ◽  
High Throughput ◽  
Immune Thrombocytopenia ◽  
Conflicts Of Interest ◽  
Bleeding Risk ◽  
Contractile Force ◽  
Diagnostic Potential ◽  
Low Platelet Count

Abstract Background: Immune Thrombocytopenia Purpura (ITP) is defined by a low platelet count in the absence of any known causes and affects over 4,000 US children and 8,000 adults each year (Terrell et al, 2010). Deciding when and how to treat these patients remains difficult as there is no biomarker or diagnostic test that identifies which patients will self-resolve and which are at risk for major bleeding and life-threatening intracranial hemorrhage (~10%). In addition, the medications used to treat ITP all have significant side effects, forcing clinicians to balance risks associated with bleeding and medication. As such, an ongoing debate in the field of clinical hematology centers around which patients require therapy (Flores & Buchanan, 2013)(Cooper 2017)(Gralnek 2008). Here, we show that a new quantitative measurement of platelet function, which is independent of known platelet biomarkers (Myers et al, 2017), has diagnostic potential in identifying bleeding risk in ITP patients. Specifically, using high-throughput platelet contraction cytometry (PCC) measurements of individual platelet forces (Myers et al, 2017), we found that low platelet forces strongly correlate with bleeding symptoms. Unlike existing tests of platelet function that use bulk measurements, our test operates at the single cell level and therefore does not depend on the platelet count, enabling a direct comparison of individual platelet function in health versus disease states. In the broad context of overall function, our findings of impaired force generation agree with previous research demonstrating that impaired platelet function, and not low platelet count, correlates with bleeding in ITP patients (Frelinger et al, 2015). In addition, as the first demonstration of a correlation between single cell force and disease, our novel approach may represent an entirely new class of "physics-based" diagnostics. Platelet Contraction Cytometry (PCC): Within the PCC, a single platelet attaches, spreads, and applies contractile force to a pair of fibrinogen microdots that are attached to a moveable, spring-like, surface. Since the applied platelet contractile force is directly proportional to the microdot displacement, the force is calculated from a single fluorescence image of the platelet. Using microfabrication technology, thousands of microdots are created on a single device to enable high-throughput measurements in tightly controlled mechanical, biochemical, and shear microenvironments (Fig 1). Here, gel-filtered platelets from patients are activated (1U/mL thrombin), plated on the device with a moderately stiff surface (75 kPa), and measured as described previously (Myers et al, 2017). Results: In a cohort of ITP patients (n = 27), we observed that patients with bleeding and/or bruising symptoms exhibited significantly lower average platelet contraction forces than asymptomatic patients regardless of platelet count. Using an average force cutoff value of 26nN, we found that low forces identified bleeding in ITP with 100% sensitivity and 89.4% specificity. From a mechanistic perspective, our preliminary data also suggests that mean platelet volume does not correlate with platelet force or function, although further studies on a single platelet level are needed to confirm this result. While this data indicates that patients with bleeding symptoms have platelets with low forces, it is unclear whether low forces correlate with bleeding or whether the patient always has had low platelet forces that therefore render them susceptible to bleeding. Our preliminary prospective data suggests that low platelet forces correlate with the bleeding symptoms themselves and return to higher values when bleeding symptoms cease. Importantly, as our approach enables "single-cell" examinations of platelet forces, we identified different platelet subpopulations unique to ITP. Healthy individuals have a distribution of contractile forces with a single peak, while ITP patients tend to have two prominent peaks, one with a lower contractile force and one with a higher one. Surprisingly, patients with bleeding symptoms only have a low force peak, suggesting that the lack of highly contractile platelets may be a biophysical biomarker for bleeding (Fig 2). Ongoing studies are focused on using this finding to further improve specificity as well as elucidating the mechanistic underpinnings of low platelet contraction in ITP. Disclosures No relevant conflicts of interest to declare.

Functional platelet defects in children with severe chronic ITP as tested with 2 novel assays applicable for low platelet counts

Blood ◽  
2014 ◽  
Vol 123 (10) ◽  
pp. 1556-1563 ◽  
Author(s):  
Esther R. van Bladel ◽  
Annemieke G. Laarhoven ◽  
Laila B. van der Heijden ◽  
Katja M. Heitink-Pollé ◽  
Leendert Porcelijn ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Function ◽  
Platelet Reactivity ◽  
Severe Bleeding ◽  
Platelet Counts ◽  
Chronic Itp ◽  
Key Points ◽  
Low Platelet Count ◽  
Platelet Defects

Key Points Pediatric chronic ITP patients with a severe bleeding phenotype exhibit functional platelet defects. The platelet microaggregation test and the platelet reactivity assay are able to assess platelet function at extremely low platelet count.

Quality of Life Improvements Following Eltrombopag Treatment in Children with Chronic Immune Thrombocytopenia: Case Report

2021 ◽  
Vol 104 (4) ◽  
pp. 672-675
Keyword(s):  
Quality Of Life ◽  
Platelet Count ◽  
Receptor Agonist ◽  
Immune Thrombocytopenia ◽  
Case Series ◽  
Thrombopoietin Receptor ◽  
Thrombopoietin Receptor Agonist ◽  
Low Platelet Count ◽  
Chronic Immune Thrombocytopenia

The present case series described six chronic immune thrombocytopenia patients (cITP), with a median age of 7.7 (7.0 to 13.0) years and low platelet count at 15,500 (7,000 to 20,000)/uL. They were suffering from bleeding symptoms and side effects of treatment. After enrollment, they were treated with thrombopoietin receptor agonist (eltrombopag). Five patients responded positively, showing a median platelet count of 115,000 (39,000 to 433,000)/uL. The median dose of eltrombopag used was 1.3 (0.8 to 2.2) mg/kg/day. The quality of life (QoL) improved for all patients, with their median overall score using a Pediatric QoL questionnaire showing 25.0% improvement. Median scores also showed improvements in each sphere of life functioning as physical (30.8%), emotional (26.4%), social (16.4%), and school (21.4%). The present report demonstrated that a select group of cITP patients, with low platelet count and bleeding symptoms, benefitted from treatment with eltrombopag, as shown by increased platelet counts and improved QoL. Keywords: Chronic ITP, Thrombopoietin receptor agonist, Children

scholarly journals Long Term Discontinuation of Eltrombopag after Remission in Primary Immune Thrombocytopenia: 8-Year Follow-up Data from 15 Spanish Centers

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2352-2352
Author(s):  
Tomas Jose Gonzalez-Lopez ◽  
Fernando Fernandez-Fuertes ◽  
Maria Cristina Pascual Izquierdo ◽  
Isabel Caparros ◽  
Silvia Bernat ◽  
...  
Keyword(s):  
Platelet Count ◽  
Median Time ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Previous Treatment ◽  
Complete Response ◽  
Platelet Response ◽  
Platelet Counts

Background: Successful discontinuation of eltrombopag in certain immune thrombocytopenia (ITP) patients after complete response has already been demonstrated. However, the frequency of this phenomenon and type of candidate patients are still matter of discussion. Moreover, possibility of long term discontinuation responses is not clearly established. Methods: Here we retrospectively evaluated our whole cohort of 508 adult patients (aged 18 years or more) with primary ITP treated with eltrombopag included in the Spanish Eltrombopag Registry with a focus on the patients who achieved a durable (at least six months) platelet response after stopping eltrombopag. Successful discontinuation of eltrombopag (SDOE) was defined as those patients who reached remission and maintained platelet counts ≥ 50x109/l for at least 6 months in absence of eltrombopag or any rescue therapies administered. Long term discontinuation of eltrombopag (LTDOE) was defined as those patients who reached remission and maintained platelet counts ≥ 50x109/l for at least 36 months in the absence of eltrombopag or any rescue therapies administered. The study was approved by the Hospital Universitario de Burgos Ethics Committee and fulfilled Helsinki declaration standards. Results: While 37.4% of our patients relapsed of ITP with subsequent platelet count drop sometime during first six months of discontinuation of eltrombopag, a total of 74 patients (14.6%) were able to achieve SDOE. The median age of SDOE patients was 62 [range, 47-79] years. There were 47 women and 27 men. According to the standard definition, patients were allocated to newly diagnosed (n=17), persistent (n=15) and chronic (n=42) ITP groups. The median time from diagnosis to eltrombopag initiation was 31 [range, 4-104] months. The median number of previous therapies was 2 [range, 1-2], including splenectomy (14%), rituximab (18%) and romiplostim (12%). As expected, all patients but 1 achieved a complete response (platelet count ≥100 x 109/L) prior to eltrombopag discontinuation The median duration of eltrombopag treatment was 7 [range, 2-19] months. Reasons for eltrombopag discontinuation were: persistent response despite a reduction in dose over time (n=43), platelet count >400x109/L (n=16), aspartate aminotransferase elevation (n=5), diarrhea (n=4), thrombosis (n=3), patient's request (n=2) and other reasons (n=1). Analysis of these SDOE discontinued patients show that with a median follow-up of 55 [range, 29-79] months, 38 patients (51.3%) maintained treatment-free response 36 months after stopping eltrombopag with no need of additional ITP therapies (median time of eltrombopag discontinuation was 70 [range, 50-77] months).This condition is what we define now as LTDOE. Nevertheless, 36 patients relapsed beyond 6 months but before 36 months of eltrombopag discontinuation (median time of eltrombopag discontinuation was 10 [range,7 -22] months). Characteristics of LTDOE population were a median time since ITP diagnosis of 32 [range, 5-88] months with 15/38 patients having ITP <1 year. 9 patients (24%) were male and their median age was 50 [range, 37-64] years. They had received a median of only two previous treatment lines [range: 1-2 lines]. The median platelet count before starting eltrombopag was 19 x 109/L [range, 8-40]. Meanwhile, platelet count before eltrombopag stop was 218 x 109/L [range, 123-356]. The main characteristics (age, gender, duration of ITP, prior ITP lines, platelet count before starting eltrombopag, duration of eltrombopag treatment, and platelet count before eltrombopag withdrawal) of the 38 patients with LTDOE were compared with those of the SDOE cohort who did not achieve a LTDOE. Unfortunately, no predictive factors of LTDOE could be identified. Conclusion: Durable platelet response following eltrombopag cessation may be observed in only 15% of primary ITP patients treated with this drug. On the contrary, half of patients who achieve a sustained response after eltrombopag withdrawal will get a long term discontinuation. However, we are lacking predictor factors for successful and long-term discontinuation of eltrombopag in primary ITP. Disclosures Gonzalez-Lopez: Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau. Pascual Izquierdo:Novartis: Consultancy; Sanofi: Consultancy. Sánchez-González:Amgen: Consultancy, Speakers Bureau; Gilead: Speakers Bureau; Navartis: Consultancy, Speakers Bureau; Shire: Speakers Bureau; Takeda: Consultancy, Speakers Bureau. Jarque:Takeda: Consultancy, Speakers Bureau; Shire: Consultancy, Speakers Bureau; Shionogi: Consultancy, Speakers Bureau; Servier: Speakers Bureau; Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Grifols: Consultancy; Gilead: Consultancy, Speakers Bureau; CellTrion: Consultancy; Celgene: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Abbie: Consultancy, Speakers Bureau; Alexion: Consultancy, Speakers Bureau.

How I Manage cyclic thrombocytopenia

Blood ◽  
2020 ◽  
Author(s):  
Paul A Kyrle ◽  
Sabine Eichinger
Keyword(s):  
Platelet Count ◽  
Immune Thrombocytopenia ◽  
Hematological Malignancies ◽  
Severe Bleeding ◽  
Typical Pattern ◽  
Periodic Fluctuation ◽  
Thrombopoietin Receptor ◽  
Primary Immune Thrombocytopenia ◽  
Platelet Count Monitoring ◽  
Mucocutaneous Bleeding

Cyclic thrombocytopenia (CTP) is a rare disease, which is characterized by periodic fluctuation of the platelet count. The pathogenesis of CTP is unknown and most likely heterogeneous. Patients with CTP are almost always misdiagnosed as having primary immune thrombocytopenia (ITP). The interval between ITP and CTP diagnosis can be many years. CTP patients often receive ITP-specific therapies including corticosteroids, thrombopoietin receptor agonists, rituximab and splenectomy which are followed by a transient increase in platelet count that is wrongly attributed to treatment effect with inevitable "relapse". CTP can be diagnosed by frequent platelet count monitoring which reveals a typical pattern of periodic platelet cycling. An early diagnosis of CTP will prevent these patients from being exposed to possibly harmful therapies. The bleeding phenotype is usually mild and consists of mucocutaneous bleeding at the time when the platelet count is at its nadir. Severe bleeding from other sites can occur but is rare. Some patients respond to cyclosporine A or to danazol, but most patients do not respond to any therapy. CTP can be associated with hematological malignancies or disorders of the thyroid gland. Nevertheless, spontaneous remissions can occur, even after many years.

Plerixafor (Mozobil ®)Plus G-CSF Is Effective in Mobilizing Hematopoietic Stem Cells in Patients with Concurrent Thrombocytopenia Undergoing Autologous Hematopoietic Stem Cell Transplantation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3229-3229 ◽  
Author(s):  
Ivana N Micallef ◽  
Eric Jacobsen ◽  
Paul Shaughnessy ◽  
Sachin Marulkar ◽  
Purvi Mody ◽  
...  
Keyword(s):  
Stem Cell ◽  
Platelet Count ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Platelet Counts ◽  
Cd34 Cells ◽  
Low Platelet Count ◽  
Equity Ownership

Abstract Abstract 3229 Poster Board III-166 Introduction Low platelet count prior to mobilization is a significant predictive factor for mobilization failure in patients with non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD) undergoing autologous hematopoietic stem cell (HSC) transplantation (auto-HSCT; Hosing C, et al, Am J Hematol. 2009). The purpose of this study is to assess the efficacy of HSC mobilization with plerixafor plus G-CSF in patients with concomitant thrombocytopenia undergoing auto-HSCT. Methods Patients who had failed successful HSC collection with any mobilization regimen were remobilized with plerixafor plus G-CSF as part of a compassionate use program (CUP). Mobilization failure was defined as the inability to collect 2 ×106 CD34+ cells/kg or inability to achieve a peripheral blood count of ≥10 CD34+ cells/μl without having undergone apheresis. As part of the CUP, G-CSF (10μg/kg) was administered subcutaneously (SC) every morning for 4 days. Plerixafor (0.24 mg/kg SC) was administered in the evening on Day 4, approximately 11 hours prior to the initiation of apheresis the following day. On Day 5, G-CSF was administered and apheresis was initiated. Plerixafor, G-CSF and apheresis were repeated daily until patients collected the minimum of 2 × 106 CD34+ cells/kg for auto-HSCT. Patients in the CUP with available data on pre-mobilization platelet counts were included in this analysis. While patients with a platelet count <85 × 109/L were excluded from the CUP, some patients received waivers and were included in this analysis. Efficacy of remobilization with plerixafor + G-CSF was evaluated in patients with platelet counts ≤ 100 × 109/L or ≤ 150 × 109/L. Results Of the 833 patients in the plerixafor CUP database, pre-mobilization platelet counts were available for 219 patients (NHL=115, MM=66, HD=20 and other=18.). Of these, 92 patients (NHL=49, MM=25, HD=8 and other=10) had pre-mobilization platelet counts ≤ 150 × 109/L; the median platelet count was 115 × 109/L (range, 50-150). The median age was 60 years (range 20-76) and 60.4% of the patients were male. Fifty-nine patients (64.1%) collected ≥2 × 109 CD34+ cells/kg and 13 patients (14.1%) achieved ≥5 × 106 CD34+ cells/kg. The median CD34+ cell yield was 2.56 × 106 CD34+ cells/kg. The proportion of patients proceeding to transplant was 68.5%. The median time to neutrophil and platelet engraftment was 12 days and 22 days, respectively. Similar results were obtained when efficacy of plerixafor + G-CSF was evaluated in 29 patients with platelet counts ≤ 100 × 109/L (NHL=12, MM=10, HD=3 and other=4). The median platelet count in these patients was 83 × 109/L (range, 50-100). The median age was 59 years (range 23-73) and 60.4% of the patients were male. The minimal and optimal cell dose was achieved in 19(65.5%) and 3(10.3%) patients, respectively. The median CD34+ cell yield was 2.92 × 106 CD34+ cells/kg. The proportion of patients proceeding to transplant was 62.1%. The median time to neutrophil and platelet engraftment was 12 days and 23 days, respectively. Conclusions For patients mobilized with G-CSF alone or chemotherapy ±G-CSF, a low platelet count prior to mobilization is a significant predictor of mobilization failure. These data demonstrate that in patients with thrombocytopenia who have failed prior mobilization attempts, remobilization with plerixafor plus G-CSF allows ∼65% of the patients to collect the minimal cell dose to proceed to transplantation. Thus, in patients predicted or proven to be poor mobilizers, addition of plerixafor may increase stem cell yields. Future studies should investigate the efficacy of plerixafor + G-CSF in front line mobilization in patients with low platelet counts prior to mobilization. Disclosures Micallef: Genzyme Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding. Jacobsen:Genzyme Corporation: Research Funding. Shaughnessy:Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Marulkar:Genzyme Corporation: Employment, Equity Ownership. Mody:Genzyme Corporation: Employment, Equity Ownership. van Rhee:Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

A Novel Canine Model of Immune Thrombocytopenia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3326-3326
Author(s):  
Dana N LeVine ◽  
Adam J Birkenheuer ◽  
Marjory B Brooks ◽  
Shila K Nordone ◽  
Dwight A Bellinger ◽  
...  
Keyword(s):  
Platelet Count ◽  
Immune Thrombocytopenia ◽  
Canine Model ◽  
Target Range ◽  
Dose Titration ◽  
Platelet Glycoprotein ◽  
Control Group ◽  
Large Animal ◽  
Isotype Control ◽  
Platelet Counts

Abstract Abstract 3326 Immune thrombocytopenia (ITP) is a relatively prevalent disease in dogs with significant morbidity and mortality. Canine ITP is clinically analogous to human ITP, with heterogeneity in bleeding manifestations in individuals with similar platelet counts. With a view to ultimately investigate this bleeding heterogeneity, we set out to develop a canine model of ITP. There are currently no existing large animal models of ITP. An induced canine ITP model would be representative of ITP without the confounding co-morbidities seen in clinical cases. Since spontaneous ITP occurs in both dogs and humans, the dog is an ideal translational model. We hypothesized that 2F9, a murine IgG2a monoclonal antibody to the canine platelet glycoprotein GPIIb (a common target of autoantibodies in ITP), would induce predictable dose-dependent thrombocytopenia (TCP) in healthy dogs. 2F9 had not been previously administered in vivo. We produced highly purified 2F9 and αYFA antibodies from the 2F9 hybridoma (gift of David Wilcox, Blood Research Institute, Wisconsin) and an isotype control murine anti-yellow fever antibody (αYFA) hybridoma. A dose titration (2 dogs) and a dose repeatability study (3 dogs) were performed in healthy adult research dogs by repeated intravenous infusion (≤ 6 doses) of 2F9 antibody until a target nadir of 5–30 × 103 platelets/μl was reached. Platelet counts were performed hourly until the platelet count reached the desired nadir range (t=0 hrs), after which complete blood counts were performed at 2, 4, 6, 8, 12, 24 hours, then q 24 hours for 10 days. The following were evaluated throughout the study: physical examination, buccal mucosal bleeding time (BMBT, baseline and t=0 only), serum cytokines and chemokines (INFγ, Interleukin (IL) 2, 6, 7, 8, 10, 15, 18, KC, IP-10, MCP-1, GM-CSF, TNFα; Milliplex CCYTOMAG-90K), fibrinogen, and D-dimers. Specificity of the 2F9 effect was confirmed by IV infusion of the isotype control (αYFA) to 3 dogs at the highest cumulative effective dose of 2F9 (167 μg/kg); all parameters were measured as above (t=0 hrs was one hour after αYFA dosing). Within 2 hours of a median cumulative 2F9 administration of 63 μg/kg (range 50.0–166.6 μg/kg), all dogs developed profound TCP (range 11–28 × 103/μl). Compared to the control group, platelet nadir was significantly lower (median (range): 6 (4–11) × 103/μl vs. 200 (179–209) × 103/μl; p= 0.036) and change in platelet count from baseline to nadir was significantly greater in the 2F9-treated group (median (range): 238 (179–325) × 103/μl vs. 4 (0–10) × 103/μl; p=0.036) (Fig 1); p-values were calculated using the exact Wilcoxon rank-sum test. Platelet nadir was in our target range and platelet count remained < 40 × 103/μl in all 2F9-treated dogs for 24 hours. Dosing was predictable: in each dog, after an initial dose of 50 μg/kg 2F9, the second dose needed to reach the target nadir could be accurately calculated from the initial platelet decrease. 2F9-treated dogs developed a range of clinical bleeding from none to petechiae, ecchymoses, melena, and hematuria. At t=0 hrs, BMBT increased 3–8 fold in treated dogs, compared to < 2 fold in control dogs. Dogs had no changes in vital signs or demeanor and did not require any transfusion support. The model does not appear pro-thrombotic as fibrinogen and D-dimers were similar over time in 2F9-treated vs. control dogs. 2F9 infusion also generated negligible systemic inflammation, as assessed by white blood cell count and serum cytokine measurement. Unexpectedly, however, serum IL8 tracked faithfully with platelet count, demonstrating that platelets are a major source of serum IL8 in dogs (Fig 2). Although α granules are known to contain IL8, platelets have not been previously described as a significant serum IL8 source. Since IL8 is an important neutrophil chemokine, our finding may illuminate a novel mechanism of platelet-neutrophil cross-talk. In summary, we have developed a novel large animal ITP model that is highly representative of the spontaneous disease. Like naturally-occurring ITP, dogs demonstrate bleeding heterogeneity despite similar platelet counts (data not shown). We expect our model to lead to further insights into bleeding mechanisms in ITP. Ultimately, understanding what factors predispose certain patients to bleed will allow us to exploit these factors therapeutically as novel ITP treatments. Disclosures: No relevant conflicts of interest to declare.

Association Of Platelet Function Markers, Independent Of Platelet Count, With Bleeding Score In Patients With Immune Thrombocytopenia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3534-3534
Author(s):  
Andrew L. Frelinger ◽  
Anja J Gerrits ◽  
Michelle A. Berny-Lang ◽  
Travis Brown ◽  
Sabrina L. Carmichael ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Activation ◽  
Platelet Function ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Univariate Analysis ◽  
Blood Draw ◽  
Platelet Surface ◽  
Platelet Counts ◽  
Bleeding Score

Abstract Background Immune thrombocytopenia (ITP) patients with similarly low platelet counts differ in their tendency to bleed. Aim To determine if differences in platelet function in ITP patients with similarly low platelet counts partly account for the variation in bleeding tendency. Methods The relationship between bleeding scores and platelet function markers was investigated in a single center cross-sectional study of pediatric patients with ITP. Following informed consent, blood was collected from ITP patients and bleeding was graded using the Buchanan and Adix Score (J Pediatr 2002) at routine clinic visits or while admitted to the hospital. Bleeding scores were obtained by one of three hematologists blinded to platelet function results, and investigators performing platelet function tests were blinded to clinical results. Platelet function was assessed by whole blood flow cytometric measurement of unstimulated, ADP- or TRAP-stimulated platelet surface activated GPIIb-IIIa (as measured by PAC1 binding), P-selectin, and GPIb and by unstimulated, convulxin-, or ADP plus TRAP-stimulated platelet surface phosphatidylserine expression (as determined by annexin V binding). Platelet count, immature platelet fraction (IPF) and mean platelet volume (MPV) were determined by a Sysmex XE-2100, and platelet forward angle light scatter (FSC) was measured by flow cytometry. Results Platelet function and bleeding scores were evaluated in 34 consecutive consenting pediatric ITP patients (16 female, 18 male, age 9.7 ± 5.7 years [mean ± SD]). ITP was newly diagnosed (< 3 months) in 10 patients, persistent (3 -- 12 months) in 7 patients, and chronic (>12 months) in 17 patients. Platelet count at the time of the blood draw was 47 ± 55 x 109/L. The median bleeding score on day of blood draw was 1 (range 0 to 4). By univariate analysis, higher IPF, and lower platelet count were significantly associated with a higher bleeding score (odds ratio [OR] >1, p<0.05) but MPV was not. Multiple measures of platelet function were associated with bleeding scores by univariate analysis: higher levels of platelet FSC (a measure affected by multiple variables including size) surface GPIb on unstimulated, ADP- or TRAP-stimulated platelets, surface P-selectin on unstimulated platelets, and platelet FSC were associated with increased odds for higher bleeding scores (ORs each >1, p<0.05), while higher ADP- and TRAP-stimulated platelet surface activated GPIIb-IIIa and P-selectin were associated with reduced odds of higher bleeding scores (ORs each <1, p<0.05). After adjustment for platelet count, higher levels of platelet surface P-selectin on unstimulated platelets, GPIb on TRAP-stimulated platelets, and FSC remained significantly associated with increased odds for higher bleeding scores (Figure), but IPF did not. Similarly, after adjustment for platelet count, higher TRAP-stimulated percentage of P-selectin and activated GPIIb-IIIa positive platelets remained significantly associated with reduced odds of higher bleeding scores (Figure). These findings were independent of recent ITP-related treatment. Conclusions In this study of pediatric ITP patients, we identified selected platelet function markers which, independent of platelet count, are associated with increased (platelet FSC, platelet surface P-selectin on unstimulated platelets, and GPIb on TRAP-stimulated platelets) or decreased (TRAP-stimulated percent P-selectin and GPIIb-IIIa positive platelets) odds of high bleeding scores. Possible hypotheses to explain these associations are as follows: 1) Increased P-selectin on unstimulated platelets demonstrates in vivo platelet activation, possibly as a consequence of the recent bleeding. 2) Because platelet activation results in a reduction in platelet surface GPIb and increases in platelet surface activated GPIIb-IIIa and P-selectin, the ORs associated with all of these markers could be explained by reduced ability of platelets in patients with higher bleeding scores to respond to agonists. 3) While platelet FSC is partly related to size, the finding that MPV and IPF, adjusted for platelet count, were not associated with bleeding score suggests that factors other than size account for the association of platelet FSC with higher bleeding scores. Further study is required to validate these findings and determine if differences in platelet function are associated with future risk for bleeding. Disclosures: Off Label Use: Eltrombopag was given to WAS/XLT patients for treatment of thrombocytopenia. Neufeld:Shire: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Apopharma: Consultancy. Michelson:Sysmex: Honoraria.

Sign in / Sign up

Export Citation Format

Share Document