Diagnostic Assay for Thrombocytosis

血小板增多症的诊​​断测定

• 批准号: 7999771
• 负责人: Dmitri V GNATENKO
• 金额: $ 17.89万
• 依托单位: STONY BROOK BIOTECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-16 至 2012-03-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7999771
• 关键词: Address; Age; Algorithms; Arteries; Biological Assay; Biological Markers; Blood; Blood Platelets; Bypass; Cardiac; Classification; Clinical; Custom; Cytolysis; Data; Detection; Diagnosis; Diagnostic; Diagnostic tests; Disease; Early treatment; Etiology; Exclusion; Gender; Gene Expression; Generations; Genes; Genetic screening method; Goals; Hematological Disease; Hemorrhagic Thrombocythemia; Human; In Vitro; Individual; Laboratories; Left; Measurement; Measures; Megakaryocyte Proliferation; Messenger RNA; Microspheres; Molecular; Myeloproliferative disease; Myocardial Infarction; Neurologic; Patients; Peripheral; Phase; Platelet Count measurement; Procedures; Process; Protocols documentation; Publishing; RNA; Rare Diseases; Reagent; Reproducibility; Reverse Transcription; Role; Sample Size; Sampling; Series; Services; Signal Transduction; Specificity; Stroke; Surveys; Techniques; Technology; Testing; Time; Transcript; Validation; Venous blood sampling; Whole Blood; base; cohort; cost; experience; in vitro Assay; instrumentation; novel; prototype; public health relevance; research clinical testing; thrombocytosis; tool

DESCRIPTION (provided by applicant): Our goal is to develop an In Vitro Diagnostic Multivariate Assay (IVDMIA) to distinguish Essential Thrombocythemia (ET) from non-clonal reactive thrombocytosis (RT) etiologies. ET represents a distinct subtype of myeloproliferative disorders, thrombocytosis, characterized by increased proliferation of megakaryocytes and resultant elevated levels of circulating platelets. To date, hematologic criteria for distinguishing among the various causes of thrombocytosis remains limited in their capacity to delineate clonal ET from RT. Jak2 genetic testing offers some diagnostic value, but it does not have adequate specificity or selectivity. Therefore a long series of clinical testing is utilized to rule-out RT and other disease. Currently ET diagnosis is essentially by exclusion. Our PI and collaborators have pioneered studies of the human blood platelets transcriptome1. We discovered and were the first to publish on platelet and platelet-specific mRNAs using Affymetrix GeneChips, to create custom microarrays, survey, discover and validate, with quantitative real-time reverse-transcription PCR (Q-PCR), differences in gene expression between ET, RT and normal platelets2. A custom microarray study of 95 subjects (Cohort I: ET [N=24]; RT [N=23]; healthy controls [N=48]) identified an 11-gene biomarker subset that discriminated among the three groups with 86.3% accuracy. Two-way class prediction (ET vs. RT) was 93.6% accurate. Discriminant power was validated with an independent group of patients (Cohort II: ET [N=16]; RT [N=15]) using Q-PCR and gave accurate (87.1%) phenotypic classification of Cohort II, RT vs ET patients. A separate 4-biomarker gene subset predicted JAK2-wild type ET in >85% of patient samples using either microarray or Q-PCR profiling. For diagnostic testing, preliminary studies indicate that a novel microsphere-based, signal amplification platform (Panomics and Luminex, Inc.) would be preferred. Using simplified procedures, it allows simultaneous profiling of up to 33 individual transcripts. This novel platform also uses intact platelets lysed in vitro, thus skipping RNA manipulation and allowing accurate platelet transcript profiling from as few as 5 x 107 intact platelets4, equivalent to 0.1ml of whole blood (other mRNA profiling techniques require 20 ml of blood or much more depending upon platelet levels). We propose to develop a simplified, sensitive, and reliable diagnostic assay to discriminate ET from RT using routine phlebotomy followed by platelet transcript profiling. Phase I will focus on (i) generation and optimization of microsphere-based technology to measure expression of biomarkers in platelets, and (ii) comprehensive comparison of this technology to traditional Q-PCR. Phase II will focus on validation of the power of this assay (combined with class prediction algorithms) to discriminate a large sampling of platelets from ET and RT patients. Other factors, such gender and other disease etc, will be analyzed during this large study. If successful, this project will result in a diagnostic tool for ET based on platelet transcript profiling. PUBLIC HEALTH RELEVANCE: This project seeks to create a clinical laboratory diagnostic test for the blood/hematologic disease called essential thrombocytosis (ET). ET is an important subset of all thrombocytoses, high platelet levels in the blood. It is a relatively rare disorder (2-3 per 100,000 people per year), but if left untreated nearly 50% of patients will experience thrombohemorrhagic complications. These include severe neurological, cardiac or peripheral artery manifestations such as stroke or a heart attack. Early definitive, diagnosis will allow early treatment and we have data that supports the potential for a diagnostic test.

描述（由申请方提供）：我们的目标是开发一种体外诊断多变量试验（IVDMIA），以区分原发性血小板增多症（ET）和非克隆反应性血小板增多症（RT）病因。ET代表骨髓增生性疾病血小板增多症的一种独特亚型，其特征在于巨核细胞增殖增加和由此产生的循环血小板水平升高。迄今为止，血液学标准之间的血小板增多症的各种原因的区分仍然是有限的，在他们的能力来描绘克隆ET从RT。Jak 2基因检测提供了一些诊断价值，但它没有足够的特异性或选择性。因此，一系列的临床试验被用来排除RT和其他疾病。目前，ET诊断基本上是通过排除。我们的PI和合作者开创了人类血小板转录组1的研究。我们发现并首次发表了使用Affyscore GeneChips的血小板和血小板特异性mRNA，创建定制微阵列，使用定量实时逆转录PCR（Q-PCR）调查，发现和验证ET，RT和正常血小板之间的基因表达差异2。95名受试者的定制微阵列研究（队列I：ET [N=24]; RT [N=23];健康对照[N=48]）鉴定了11个基因生物标志物子集，其以86.3%的准确度区分三组。双向分类预测（ET vs. RT）的准确率为93.6%。使用Q-PCR对独立的患者组（队列II：ET [N=16]; RT [N=15]）验证判别能力，并对队列II、RT与ET患者进行准确（87.1%）的表型分类。使用微阵列或Q-PCR分析，单独的4-生物标志物基因子集预测>85%的患者样品中的JAK 2-野生型ET。对于诊断测试，初步研究表明，一种新的基于微球的信号放大平台（Panomics和Luminex，Inc.）会更好。使用简化的程序，它允许同时分析多达33个单独的成绩单。这种新型平台还使用体外裂解的完整血小板，从而跳过RNA操作，并允许从少至5 x 107个完整血小板4（相当于0.1 ml全血）进行准确的血小板转录本分析（其他mRNA分析技术需要20 ml血液或更多，具体取决于血小板水平）。我们建议开发一种简化的，敏感的，可靠的诊断方法来区分ET从RT使用常规放血血小板转录谱。第一阶段将重点关注（i）基于微球的技术的生成和优化，以测量血小板中生物标志物的表达，以及（ii）将该技术与传统Q-PCR进行全面比较。第二阶段将重点验证该检测方法（结合类别预测算法）区分ET和RT患者的大量血小板样本的能力。其他因素，如性别和其他疾病等，将在这项大型研究中进行分析。如果成功，该项目将导致基于血小板转录谱的ET诊断工具。 公共卫生关系：该项目旨在创建一个临床实验室诊断测试的血液/血液疾病称为原发性血小板增多症（ET）。ET是所有血小板增多症的一个重要子集，血液中血小板水平高。这是一种相对罕见的疾病（每年每10万人中有2-3例），但如果不及时治疗，近50%的患者将出现血栓出血并发症。这些包括严重的神经、心脏或外周动脉表现，如中风或心脏病发作。早期明确的诊断将允许早期治疗，我们有数据支持诊断测试的潜力。