Diagnostic Assay for Thrombocytosis

血小板增多症的诊断测定

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：我们的目标是开发体外诊断多变量测定（IVDMIA），以区分基本血小板 - ET）与非共旋转反应性血小板病（RT）病因。 ET代表骨髓增生性疾病，血小板病的独特亚型，其特征是巨核细胞增殖的增加和循环血小板的升高。迄今为止，区分血小板病的各种原因的血液学标准在其从RT中划定克隆ET的能力仍然有限。 JAK2基因测试提供了一定的诊断价值，但没有足够的特异性或选择性。因此，一系列临床测试被用于排除RT和其他疾病。目前，ET诊断本质上是通过排除。我们的PI和合作者开创了对人血小板转录组的研究。我们发现并使用Affymetrix Genechips在血小板和血小板特异性的mRNA上发表，并使用定量的实时反向转录PCR（Q-PCR）创建自定义的微阵列，调查，发现和验证，ET，RT和正常血小板之间的基因表达差异。一项对95名受试者的自定义微阵列研究（同类I：ET [n = 24]; RT [n = 23];健康对照[n = 48]）确定了一个11基因生物标志物子集，该子集在三组中歧视了86.3％的精度。双向类预测（ET与RT）精度为93.6％。使用Q-PCR的一组独立的患者（et [n = 16]; rt [n = 15]）对判别功率进行了验证，并给出了同类II，RT与ET患者的准确（87.1％）表型分类。单独的4个生物标志物基因子集使用微阵列或Q-PCR分析预测了> 85％的患者样品中的JAK2野生型ET类型。对于诊断测试，初步研究表明，基于微球的新型信号放大平台（Panomics and Luminex，Inc。）是首选的。使用简化的过程，它允许同时分析多达33个单独的成绩单。这个新颖的平台还使用了体外裂解的完整血小板，从而跳过RNA操作，并允许精确的血小板转录物分析，从少于5 x 107完整的血小板4，相当于0.1毫升的全血（其他mRNA分析技术需要20 ml血液或根据血小板水平））。我们建议开发一种简化，敏感且可靠的诊断测定法，以使用常规的静脉切开术，然后进行血小板转录分析区分RT。第一阶段将重点介绍（i）基于微球的技术来衡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。第二阶段将侧重于验证该测定的功率（结合了类预测算法），以区分大量的血小板与ET和RT患者的样本。在这项大型研究中将分析其他因素，例如性别和其他疾病。如果成功，该项目将为基于血小板转录分析的ET提供诊断工具。公共卫生相关性：该项目旨在为称为基本血小板病（ET）的血液/血液学疾病进行临床实验室诊断测试。 ET是所有血小板的重要子集，血液中的高血小板水平。这是一种相对罕见的疾病（每100,000人2-3人），但是如果未治疗的50％的患者将遭受血栓性并发症。这些包括严重的神经系统，心脏或周围动脉表现，例如中风或心脏病发作。早期确定的诊断将允许早期治疗，我们有数据支持进行诊断测试的潜力。