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

使用血小板 RNA 生物标志物检测母血中的胎儿血小板。

• 批准号: 9905924
• 负责人: Dmitri V GNATENKO
• 金额: $ 27.23万
• 依托单位: BLOOD CELL TECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9905924
• 关键词: Adult; Affect; Antibodies; Biological Assay; Biological Markers; Biological Models; Blood; Blood Platelets; Cancer Patient; Cells; Chimerism; Clinical; Coagulation Process; DNA; Databases; Detection; Development; Diagnosis; Diagnostic; Disease; Early Diagnosis; Enrollment; Erythrocytes; Fetal Growth Retardation; Fetal Hemoglobin; Fetomaternal Transfusion; Fetus; Flow Cytometry; Functional disorder; Goals; Half-Life; Hemostatic function; Human; Hypertension; Immune; Immune response; Inflammation; Longevity; Malignant Neoplasms; Manuals; Measures; Mediating; Messenger RNA; Methods; MicroRNAs; Microscopic; Neonatal; Perinatal mortality demographics; Phase; Phenotype; Play; Postpartum Period; Pre-Eclampsia; Pregnancy; Pregnancy Complications; Principal Investigator; RNA; Reporting; Reproducibility; Risk Assessment; Role; Sampling; Sensitivity and Specificity; Small Business Innovation Research Grant; Technology; Testing; Third Pregnancy Trimester; Thrombocytopenia; Thrombosis; Time; Transfer RNA; Umbilical Cord Blood; Woman; Y Chromosome; at-risk pregnancies; base; cell type; clinically significant; cost effective; differential expression; digital; extracellular vesicles; fetal; fetal blood; human disease; in vivo; male; maternal morbidity; minimally invasive; novel strategies; outcome prediction; perinatal morbidity; platelet function; prenatal testing; prognostic; programs; sex; thrombocytosis; transcriptome sequencing; wound healing

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

计划主任/首席研究员（最后，第一，中间）：Gnatenko，Dmitri V. 抽象的 血小板是小的环核细胞，在止血，血栓形成，炎症中具有关键作用 伤口愈合和免疫反应。它们包含潜水员阵列的mRNA和microRNA 可以用作包括癌症在内的各种疾病的生物标志物。血小板在 妊娠相关疾病，例如先兆子痫，内蛋白内生长迟缓和 同种免疫性血小板减少症。该提议的总体目标是发展一个敏感且稳健的 测定可检测胎儿血液中胎儿血小板的存在，当前未满足的评估需求 胎盘完整性。迄今为止，通过检测胎儿红细胞评估胎儿出血 在孕产妇中，通过kleihauer-betke检验或使用抗血红蛋白F进行流式细胞仪 抗体，没有评估母乳中新生儿血小板的可用评估。使用广泛 RNASEQ成人和脐带血血小板mRNA的数据库，我们已经确定了六个血小板表达的 可以区分脐带血血小板和成人血小板的mRNA生物标志物-Znf385d，CPT1A， L3MBTL4，IGF2BP1，PAICS和COL4A5。我们建议优化数字PCR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体血液中的胎儿血小板。 数字PCR分析将使用模型系统进行验证 - 产后立即的女性血液 时期，当胎儿出血最高时 - 与传统测试相比 胎儿出血。数字PCR技术广泛用于微创产前 由于其高灵敏度，特异性和鲁棒性而筛选。与传统的定量RT-不同 PCR，该技术衡量目标分子的绝对数量，并且不需要 正常化。为了证明数字PCR对血小板研究的适用性，我们产生了问题 特定于ZNF385D和IGF2BP1，并证明了基于数字PCR技术的测定 生成与RNA测序和传统Cybr-Green RT-PCR一致的结果 技术，但具有更大的灵敏度和准确性，可以清晰地分离绳索血液 成人血小板的血小板。第一阶段的目标是（i）制定量化评估 使用数字PCR技术和（ii）验证该测定法和 将其与现有测试进行比较。在第二阶段，我们将将该测定作为一种新颖的风险方法 - 评估与胎儿出血相关的妊娠疾病（胎盘 无能），例如先兆子痫，假设母体血液中的胎儿血小板生物标志物 可以作为高危妊娠的更强大的决定者。 PHS 398/2590（修订版06/09）页面延续格式页面