A new flow cytometric method for differential cell counting in ascitic fluid

Background: Cell counts in bodyfluids such as ascitic fluid can be difficult to perform and report rapidly. The current gold standard for cell counting in body fluids is a suitable automated cell counter or a manual counting chamber, combined with differential counting on a cytospin. This technique has several disadvantages, so we designed a new flow cytometric test for cell counting in ascites. We compared this with an automatic cell counter (LH750, Beckman Coulter) and manual counting of cytospins. Methods: Ascitic samples (n=53) from 38 patients were studied. Polymorphonuclear neutrophils (PMN), lymphocytes, eosinophils, and macrophages were defined by flow cytometry. We compared this with our reference method: the absolute cell concentration calculated from the leukocyte concentration of the LH750 combined with a differential cell count performed manually on a cytospin. Results: The outcomes of validation experiments (linearity, reproducibility, and detection limit) of the flow cytometric assay prove it is well suited for cell counting in ascitic fluid. Conclusions: Based on analytical performance, flow cytometry is suited for cell counting in ascitic fluid. An ascitic fluid cell count is frequently ordered to detect spontaneous bacterial peritonitis (SBP). If the PMN count is ≥250 cells/mm3, SBP is highly suspected. Using our reference method, we calculated the sensitivities and specificities to detect ≥250 PMN cells/mm3 for the LH750 (100% and 65%, respectively) and flow cytometric assay (100%, 100%). As flow cytometry is easier and faster we recommend this method for rapid cell counting in ascitic fluid.

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