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