Posttransplantation Bone Marrow Assessment by Quantifying Hematopoietic Cell-Derived mRNAs in

BACKGROUND: Bone marrow (BM) aspiration often can be a painful medical procedure. It is unavoidable, however, because hematopoietic precursor cells (HPC) exist only inBMand few escape to peripheral blood (PB). We hypothesized that HPCs might release exosomes and microvesicles (EMV) in BM, and the resulting EMV would penetrate into PB. Such BM-derived EMV might be identified in PB by measuring specific mRNAs produced by HPC. METHODS: Human plasma was applied to an EMVcapture filter plate. After centrifugation, captured EMV were lysed on the filter plate. Resulting lysates were transferred to an oligo(dT)-immobilized microplate for mRNA isolation followed by reverse transcription PCR (RT-PCR). Using this system, myeloid-, erythroid-, and megakaryocyte-lineage-specific poly(A)(+) mRNAs were quantified in plasma obtained from 18 patients who had undergone hematopoietic stem cell transplantation (HSCT). RESULTS: When fluorescent liposomes were applied to the filter plate, more than 95% of applied liposomes were absorbed. When human plasma was applied, a scanning electron microscope showed EMV-like particles on the membrane of the filter plate. After RT-PCR, various HPC-specific mRNAs were detected, and the results were equivalent to those derived from the standard ultracentrifugation method. The levels of these mRNAs were undetectable after HSCT and became detectable 1-2 weeks after HSCT, a substantially earlier time point than with traditional hematological analysis. The recovery of EMV mRNA at day 15 corresponded to the final clinical outcome at day 180. `CONCLUSIONS: HPC-derived mRNAs in plasma EMV may represent new biomarkers for the assessment of BM condition and could reduce the necessity for frequent BM aspiration. (C) 2013 American Association for Clinical Chemistry