Leveraging platelet contraction cytometry for immune thrombocytopenia

项目摘要/摘要 免疫性血小板减少性紫癜(ITP)在没有任何其他原因的情况下，以低血小板计数为特征 每年影响超过4,000名美国儿童和8,000名成年人。虽然大多数ITP案件都能解决 ITP患者本身出血的风险增加，10%的患者发生大出血，以及 有0.5%的患者出现危及生命的颅内出血。目前还没有ITP的生物标志物，甚至更少， 有出血风险，而且所有的治疗都有明显的副作用。这给临床医生留下了一个重要的 在决定是否治疗方面左右为难。最终处于高危状态的患者可能得不到治疗 直到发生严重出血，低风险患者可能会暴露于不必要的治疗副作用。 这一建议的研究目的是调查一个新的假设，即，收缩的力量 单个血小板与ITP的出血表型相关，与传统使用的生物学无关 血液学功能的标志物或化验。使用一种新开发的高通量血小板收缩 细胞仪(PCC)平行测量单个血小板的收缩力，我们的最新研究结果是 儿童原发性ITP患者的血小板力明显不同于健康对照组， 2)与出血密切相关(n=49)；3)同一患者随时间变化(n=7)。使用 平均用力截断值为26nN，我们发现低力识别ITP出血的敏感性为100%。 89.4%的特异度，仅考虑有血小板的患者时特异度提高到94% 计数：40,000 ul。目标1建立在这些初步数据的基础上，并建议对 血管紧张力、血小板特征、临床特征与预后的关系 研究一组新入选的ITP患者(n=100)，在单个时间点进行前瞻性研究，为期12个月。 我们将具体观察血小板收缩力是否与出血评分、未成熟血小板分数、 血小板活化、血小板形态、患者人口统计、治疗和解决的时间。 PCC还提供了一个独特的机会，从患者那里获得对血小板功能的新见解 ITP和低力的机械基础。以前的研究被认为是血小板的传统工具 功能，如聚集测量，血小板功能分析仪，或血栓弹性图，是混淆的 ITP患者的血小板计数偏低。我们将使用PCC来测试我们的假设，即固有的血小板变化和 外源性血浆因子改变了血小板的收缩能力。从外在的角度来看，我们的数据显示 这种免疫球蛋白与更严重的出血和更低的收缩力相关，而且孵化 非自体血浆中的血小板导致较低的收缩力。从内在的角度来看，我们有 研究发现，平均血小板体积低的患者，血小板作用力较低，出血增多。作为 机械论的基础尚不清楚，目标2寻求对 可能调节血小板功能的内在和外在因素。

PROJECT SUMMARY/ABSTRACT Defined by low platelet count in the absence of any other cause, immune thrombocytopenic purpura (ITP) affects over 4,000 US children and 8,000 adults each year. While the majority of ITP cases resolve themselves, patients with ITP have an enhanced risk of bleeding, with 10% experiencing major bleeding, and 0.5% of experiencing life-threatening intracranial hemorrhage. There is no biomarker for ITP or much less, bleeding risk, and all treatments involve significant side effects. This leaves clinicians with a significant dilemma in deciding whether or not to treat. Patients who are ultimately at high risk may not receive treatment until serious bleeding occurs, and low risk patients may be exposed to unnecessary treatment side effects. The research objective of this proposal is to investigate a novel hypothesis, namely, that the contractile force of individual platelets correlates with bleeding phenotype in ITP, independent of traditionally used biological markers or assays of hematological function. Using a newly developed high-throughput platelet contraction cytometer (PCC) to measure single platelet contractile forces in parallel, our latest results of a study of pediatric patients with primary ITP suggests that platelet forces 1) vary significantly from healthy controls, 2) strongly correlate with bleeding (n=49 patients) and 3) change over time in the same patient (n=7). Using an average force cutoff value of 26nN, we found that low forces identified bleeding in ITP with 100% sensitivity and 89.4% specificity, with a specificity improvement to 94% when only considering patients with a platelet count <40,000 uL. Aim 1 builds on this preliminary data and proposes a rigorous investigation into the relationship between contractile force, platelet characteristics, clinical characteristics, and outcomes by studying a cohort of newly enrolled ITP patients (n=100) at a single time point and prospectively for 12 months. We will specifically see if platelet contractile force correlates with bleeding score, immature platelet fraction, platelet activation, platelet morphology, patient demographics, treatments, and time to resolution. The PCC also offers a unique opportunity to gain new insights into the function of platelets from patients with ITP and mechanistic underpinnings of low force. Previous studies were hindered as traditional tools of platelet function such as aggregometry, platelet functional analyzers, or thromboelastography are confounded by the low platelet count in ITP. We will use the PCC to test our hypothesis that both intrinsic platelet changes and extrinsic plasma factors modify platelet contractile force. From an extrinsic perspective, our data has shown that IgG correlates with more severe bleeding and lower contractile forces, and furthermore that incubating platelets in non-autologous plasma leads to lower contractile forces. From an intrinsic perspective, we have found that patients with low mean platelet volume have lower platelet force and increased bleeding. As the mechanistic underpinnings are unclear, Aim 2 seeks to perform systemic, unbiased investigation into both the intrinsic and extrinsic factors that may modulate platelet function.