DOI 10.1515/cclm-2012-0549 Clin Chem Lab Med 2013; 51(6): 1285–1290 Jeffrey F.W. Keuren, Johannes J.M.L. Hoffmann and Mathie P.G. Leers* Analysis of serous body fluids using the CELL-DYN Sapphire hematology analyzer Abstract Background: Correct cell enumeration and differential analysis of body fluids are important in the diagnosis and management of several diseases. Currently, microscopic analysis is still considered the “gold standard”. The aim of the present study was to evaluate the analytical perfor- mance of the CELL-DYN Sapphire hematology analyzer for automated differentiation of cells in serous fluids and to explore whether manual analysis of the raw data files could improve the differential count compared with refer- ence microscopy. Methods: A total of 105 serous fluids (39 peritoneal and 66 pleural effusions) were analyzed by the CELL-DYN Sapphire using standard whole-blood algorithm. Addi- tionally, we performed optimized manual gating of the Sapphire raw data file using standard flow cytometry software. Results: The standard Sapphire algorithm showed substantial deviations from the reference microscopic differentiation: polymorphonuclear cell counts were too high because they contained some monocytic cells. However, when optimized manual gating strategy is used, a good correlation and negligible bias were found. Conclusions: We have demonstrated that with a modified algorithm, CELL-DYN Sapphire will provide reliable iden- tification and enumeration of blood cells in peritoneal and pleural fluids. Keywords: pleural effusion; mesothelial cells; microscopy. *Corresponding author: Mathie P.G. Leers, PhD, Department of Clinical Chemistry and Hematology, Atrium Medical Center, Henri Dunantstraat 5, 6401 CX Heerlen, the Netherlands, Phone: +31-45-576-7503, Fax: +31-45-576-6575, E-mail: m.leers@atriummc.nl Jeffrey F. W. Keuren: Department of Clinical Chemistry and Hematology, Atrium Medical Center, Heerlen, the Netherlands Johannes J.M.L. Hoffmann: Abbott Diagnostics Division, Wiesbaden- Delkenheim, Germany Introduction Hematology laboratories frequently perform cellular analy- sis of serous body fluids such as peritoneal and pleural fluids. Usually, the total nucleated cells are enumerated and a differential leukocyte count is done. The latter is impor- tant for diagnosis and therapy. For example, in peritoneal fluid, a polymorphonuclear (PMN) cell count > 250 cells/ μL is diagnostic for bacterial peritonitis, and this finding requires immediate antibiotic treatment [1]. Microscopic cell differentiation and cytological assessment of cytospin preparations are still considered the gold standard for analyzing body fluids. Because many laboratories need to increase their process efficiency, auto- mated hematology analyzers are increasingly being used for analyzing body fluids. However, these instruments are specifically designed for measuring cells in whole blood and may not be suited for analyzing cells in other body fluids without modifications [2, 3]. Serous fluids have a different matrix from whole blood, which can affect the properties of blood cells in a body fluid. Moreover, it is not unusual that these fluids contain cells of nonhematologi- cal origin, such as macrophages, mesothelial cells, and tumor cells, which cannot be accurately classified by the standard algorithms of hematology analyzers. This was recently demonstrated for the first time in a study using the CELL-DYN Sapphire [4]. These authors found that the analyzer could be used to determine the concentration of total nucleated cells with a functional sensitivity limit of 50 cells/ μL. However, it appeared that CELL-DYN Sapphire included substantial amounts of epithelial cells and mac- rophages in the PMN count, making the automated differ- entiation unreliable [4]. This PMN overestimation could lead to false-positive results in the diagnosis of bacterial peritonitis and pleural cavity infection. The aims of the present study were to evaluate the CELL-DYN Sapphire for automated cell differentiation in serous fluids compared with cytospin microscopy as a reference and to explore the modifications of the stand- ard gating strategy for optimizing the automated differ- ential counts. For this purpose, we analyzed the raw data files of CELL-DYN Sapphire off-line using a standard flow cytometry software. Brought to you by | Abbott Labs Library Authenticated | hans.hoffmann@abbott.com Download Date | 5/24/13 8:38 AM