ABSTRACT
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. © 2014 International Clinical Cytometry Society.
Subject(s)
Ascitic Fluid/pathology , Flow Cytometry/methods , Leukocyte Count/methods , Ascitic Fluid/microbiology , Bacterial Infections/microbiology , Bacterial Infections/pathology , Eosinophils/pathology , Humans , Neutrophils/pathology , Peritonitis/microbiology , Peritonitis/pathology , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
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. © 2014 Clinical Cytometry Society.
ABSTRACT
Differential white blood cell count (dWBC) is a frequently used diagnostic tool. For most patient samples an automated blood counter produces a five-part differential count. If this dWBC does not meet pre-set criteria, microscopic dWBC is performed. Microscopy is labor intensive and requires sustained training of technicians. Inter-observer variation and statistical variation are significant, due to limited numbers of cells counted. Flow cytometry is a candidate reference method for dWBC. Advantages are immunological definitions and large number of measured cells. Our goal was to replace (part of) the microscopic dWBC by a flow cytometric dWBC, that gives additional information on blasts, myeloid precursors, and lymphocyte subsets. We designed a cocktail of antibodies (CD4, CD14, CD34, CD16, CD56, CD19, CD45, CD138, CD3, and CD71) combined with a gating strategy and flow cytometric protocol for easy identification of leukocyte populations. This assay, called Leukoflow, requires low sample volume, has few manual handling steps, and a potential turn-around-time shorter than 2 h. We determine percentages and absolute concentrations of at least 13 different cell populations. For quantification of normoblasts a second flow cytometric staining was designed. We compared microscopic dWBC with that of the automated blood counter and Leukoflow for normal and abnormal blood samples. Leukoflow results correlate well with the automated blood counter for leukocytes, neutrophils, eosinophils, monocytes, and lymphocytes. Correlation with manual dWBC is lower. Blast counts reported by Leukoflow suffer less from inter-observer variation compared to manual dWBC. In addition to microscopic or cytometric dWBC-techniques T-lymphocytes, CD4-T-lymphocytes, B-lymphocytes, NK-cells, myeloid progenitors, plasma cells, and blasts are determined by Leukoflow. These populations give potential useful clinical information and are subject for future studies focusing on the additional clinical value. Leukoflow is a highly interesting and promising technique for clinical laboratories.
