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
Gnatenko, Dmitri V. NARRATIVE Today accumulating evidences indicate that human blood thrombocytes (platelets) contain biomarkers of many human diseases and can be used for diagnostic purposes. We propose to adapt sensitive digital PCR technology to detect and quantify platelet RNA biomarkers in human blood. Final goal of this proposal is to develop an assay to detect and evaluate presence of fetal platelets in maternal blood during pregnancy. We anticipate that this assay will perform better than existing tests. Once developed, such assay can be applied to predict and diagnose various complications of pregnancy. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page
