DRAQ5-based, no-lyse, no-wash bone marrow aspirate evaluation by flow cytometry

Abstract

Flow cytometry (FC) is a powerful tool for objective phenotyping of hematolymphoid neoplasia. Analysis of bone marrow aspirates and peripheral blood specimens by FC typically requires an erythrocyte lysis or gradient separation method to remove erythrocytes prior to analysis, which may result in the loss of certain populations, in particular nucleated erythroid cells. We developed a method to analyze bone marrow aspirates (BMAs) by FC without erythrocyte lysis or washing to minimize cell loss by exploiting the nuclear DRAQ5 fluorescence as a gating parameter (DRAQ5 protocol). We analyzed a total of 31 BMAs from patients with a variety of diagnoses utilizing the DRAQ5 protocol in combination with CD71 and CD45 antibodies to determine the marrow differentials. These were compared with differential counts obtained by morphologic study and erythrocyte lysis FC. The DRAQ5 protocol preserved the nucleated erythrocytes, allowing calculations of the myeloid to erythroid ratio and of blasts/abnormal cells that better reflect the morphologic nucleated cell differential than erythrocyte lysis FC.