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

Authors

  • Steven G. DuBois MD,

    Corresponding author
    1. Department of Pediatrics, University of California San Francisco School of Medicine, San Francisco, California
    • Department of Pediatrics, UCSF School of Medicine, 505 Parnassus Avenue, M646, San Francisco, CA 94143-0106.
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  • C. Lorrie Epling MS,

    1. Division of Experimental Medicine, Core Immunology Laboratory, University of California San Francisco School of Medicine, San Francisco, California
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  • Juli Teague BS,

    1. Division of Experimental Medicine, Core Immunology Laboratory, University of California San Francisco School of Medicine, San Francisco, California
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  • Katherine K. Matthay MD,

    1. Department of Pediatrics, University of California San Francisco School of Medicine, San Francisco, California
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  • Elizabeth Sinclair PhD

    1. Division of Experimental Medicine, Core Immunology Laboratory, University of California San Francisco School of Medicine, San Francisco, California
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Abstract

Background

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.

Procedure

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.

Results

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%).

Conclusions

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. Pediatr Blood Cancer 2010; 54:13–18. © 2009 Wiley-Liss, Inc.

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