Flow cytometric detection of Ewing sarcoma cells in peripheral blood and bone marrow

Abstract

A new method for detecting circulating Ewing sarcoma cells using flow cytometry is described. This strategy exploits the nearly universal expression of CD99 and the lack of expression of CD45 by Ewing sarcoma cells. Ewing sarcoma cell line A673, peripheral blood mononuclear cells (PBMCs), and bone marrow mononuclear cells (BMMCs) were stained for CD99 and CD45 in order to detect CD99+CD45- cells by flow cytometry. Known quantities of A673 Ewing sarcoma cells were spiked into control PBMCs to test the accuracy of this method. Control PBMCs were evaluated to assess the level of background staining. Flow cytometry was accurate at frequencies as low as one A673 cell per 500,000 PBMCs. The background rate of CD99+CD45- cell detection was low in PBMCs from nine healthy volunteers (median 0.0001% of total cells; range 0-0.00046%) and was further reduced by incorporating stains to exclude dead cells, progenitor cells, and monocytes. In one subject with newly diagnosed localized Ewing sarcoma, CD99+CD45- cells were detected in both blood (0.0021%) and bone marrow (0.048%). Multicolor flow cytometry for CD99+CD45- cells provides a new strategy for detecting circulating Ewing sarcoma cells. Clinical evaluation and validation of this method is ongoing.