Detection of fetal platelets in maternal blood using platelet RNA biomarkers.

项目主任/首席调查员(最后、第一、中间)：Gnatenko，Dmitri V. 摘要 血小板是一种小的无核细胞，在止血、血栓形成、炎症、 伤口愈合和免疫反应。它们包含一系列不同的mRNAs和microRNAs 可以作为包括癌症在内的各种疾病的生物标志物。血小板在疾病中的重要作用(S) 妊娠相关疾病，如先兆子痫、宫内发育迟缓和 同种免疫性血小板减少症。这项提议的总体目标是发展一种敏感和稳健的 检测孕妇血液中是否存在胎儿血小板的检测，这是目前在评估中未得到满足的需求 胎盘完整性。到目前为止，胎儿母体出血是通过检测胎儿红细胞来评估的。 用Kleihauer-Betke试验或用抗Hb F的流式细胞术检测母血中的 抗体，没有可用的检测方法来评估母亲血液中的新生儿血小板。广泛使用 RNAseq数据库中的成人和脐带血血小板mRNAs，我们已经鉴定出六个血小板表达 可区分脐血和成人血小板的mRNA生物标志物-ZNF385D、CPT1A、 L3MBTL4、IGF2BP1、PAICS和COL4A5。我们建议优化数字聚合酶链式反应技术以检测 并通过测量这些生物标志物的表达水平来测量母亲血液中的胎儿血小板。 数字聚合酶链式反应分析将使用模型系统-产后立即妇女的血液进行验证 这一时期，胎儿-母体出血是最严重的-与传统的 胎儿-母体出血。数字聚合酶链式反应技术广泛应用于微创产前检查 筛查具有较高的敏感性、特异性和稳健性。不同于传统的定量RT- 这项技术测量的是目标分子的绝对数量，不需要 正常化。为了证明数字聚合酶链式反应在血小板研究中的适用性，我们制作了探针 ZNF385D和IGF2BP1的特异性并证明了基于数字PCR技术的检测 产生与RNA测序和传统的CyBR-Green RT-PCR一致的结果 技术，但具有更高的灵敏度和准确性，允许清晰地分离脐带血 来自成人血小板的血小板。第一阶段的目标是(I)开发一种定量测定 利用数字聚合酶链式反应技术检测血液中的胎儿血小板RNA生物标记物 将其与现有测试进行比较。在第二阶段，我们将采用这种分析作为一种新的风险方法- 评估与母胎出血(胎盘)相关的妊娠疾病 能力不全)，例如先兆子痫，假设母亲血液中的胎儿血小板生物标志物 可能是高危妊娠的更强有力的决定因素。 PHS 398/2590(06/09版)页面续格式页面

Program Director/Principal Investigator (Last, First, Middle): Gnatenko, Dmitri V. ABSTRACT Platelets are small anucleate cells that have a key role in hemostasis, thrombosis, inflammation, wound healing and immune response. They contain a diverse array of mRNAs and microRNAs that can serve as biomarkers of various diseases including cancer. Platelets play important role(s) in pregnancy–associated diseases such as preeclampsia, intrauterine growth retardation, and alloimmune thrombocytopenia. The overall goal of this proposal is to develop a sensitive and robust assay to detect presence of fetal platelets in maternal blood, a current unmet need in assessing placental integrity. To date, fetomaternal hemorrhage is evaluated by detection of fetal red blood cells in maternal blood by either Kleihauer-Betke test or by flow cytometry using anti-hemoglobin F antibody, with no available assay to assess neonatal platelets in maternal blood. Using extensive RNASeq database of adult and cord blood platelet mRNAs, we have identified six platelet-expressed mRNA biomarkers that can discriminate cord blood platelets from adult platelets - ZNF385D, CPT1A, L3MBTL4, IGF2BP1, PAICS and COL4A5. We propose to optimize digital PCR technology to detect and measure fetal platelets in maternal blood by measuring expression levels of these biomarkers. Digital PCR assay will be validated using model system - blood of women in immediate postpartum period, when fetomaternal hemorrhage is the highest - and compared to traditional tests for fetomaternal hemorrhage. Digital PCR technology is broadly used for minimally invasive prenatal screening due to its high sensitivity, specificity and robustness. Unlike traditional quantitative RT- PCR, this technology measures absolute number of target molecules and does not require normalization. To demonstrate applicability of digital PCR to platelet studies, we generated probes specific to ZNF385D and IGF2BP1 and demonstrated that digital PCR technology - based assay generates results concordant with RNA Sequencing and traditional CYBR-green RT-PCR technologies, but with greater sensitivity and accuracy, allowing clear separation of cord blood platelets from adult platelets. The goals of Phase I are (i) to develop an assay for quantification of fetal platelet RNA biomarkers in blood using digital PCR technology and (ii) to validate this assay and compare it to existing tests. In Phase II we will adapt this assay as a novel approach for risk- assessment of gestational diseases associated with fetomaternal hemorrhage (placental incompetence) such as preeclampsia, hypothesizing that fetal platelet biomarkers in maternal blood may serve as more robust determinants of at-risk pregnancies. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page