Thursday, 20 October 2016

Malignant cells in CSF: diagnostic & prognostic value in Leptomeningeal Carcinamatosis

Clinical researchers from centres of excellence across Spain have described a flow cytometric method to robustly detect tumour epithelial cells in cerebrospinal fluid (CSF) of patients with leptomeningeal carcinamatosis (LC).

Subirá et al. used DRAQ5(TM) to label nucleated cells in CSF and therefore discriminate these cells from debris, avoiding the need for sample processing that might otherwise impact cell integrity, cell loss or absolute enumeration.

CSF samples were collected in EDTA with Transfix (Cytomark) for preservation and transportation.

In parallel steps one fraction of CSF was labeled with DRAQ5 and supplemented by counting beads to enumerate total nucleated cell burden.  A further CSF fraction was centrifuged to pellet the cells, firstly stained with a pair of epithelial-cell adhesion molecule (EpCAM) antibodies, separately labeled with FITC and PE, and then labeled with DRAQ5.  This combination allowed discrimination of malignant (EpCam-positive) epithelial cells from the non-malignant (EpCam-negative) inflammatory cells; lymphocytes, monocytes, polymorphonuclear cells identified according to their relative FSC/SSC scatter properties.

Use of DRAQ5 is simple, ideal for such procedures.  Being supplied in aqueous solution, preparation for use is straightforward. DRAQ5 is entirely cell permeant, labeling nucleated cells in complex samples within a few minutes, and without any subsequent wash steps.  Its spectral properties (far-red fluorescent) mean it has no spectral overlap with FITC/PE antibody pairs avoiding compensation issues.

In summary, the authors suggest that a CSF burden of EpCam+ cells of above 8% was a useful further diagnostic parameter and was clinically significant in terms of both overall survival and in identifying patients that may benefit from additional therapy.  This should be the subject of further validations studies.


Diagnostic and prognostic significance of flow cytometry immunophenotyping in patients with leptomeningeal carcinomatosis.

D. Subirá, M. Simó, J. Illán, C. Serrano,S. Castañón, R. Gonzalo, J. J. Granizo,M. Martínez-García, M. Navarro,J. Pardo, et al.

Clin Exp Metastasis (2015) 32: 383-391    DOI 10.1007/s10585-015-9716-3

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