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

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/mm , SBP is highly suspected. Using our reference method, we calculated the sensitivities and specificities to detect ≥ 250 PMN cells/mm 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.

Clinical relevance and contemporary methods for counting blood cells in body fluids suspected of inflammatory disease

“…For CSF samples, the LH 750 misclassified 64% of samples according to clinical threshold (WBC > 5 × 10 6 /L) [136]. The LH 750 was compared with the FC500 flow cytometry (Beckman Coulter) for counting WBCs (differential) in ascitic fluids [144]. The methods correlated well for WBCs (r = 0.99) and moderately for neutrophils (r = 0.82).…”

“…Though these cells are generally larger (15-21 μm) than WBCs (10-20 μm), there is a window of overlap which causes them to be mistakenly counted as WBCs. The presence of macrophages and mesothelial cells in serous fluids can lead to falsely elevated WBCs, and furthermore, mistakenly assigned to MNs and/or PMNs [55,128,135,144]. The acquisition of these false positive data can result in misdiagnosis, and have adverse implications for therapy in SBP, for example.…”

Spontaneous bacterial peritonitis: update on diagnosis and treatment

“…Although it is an independent operator and can provide inconsistent results, it has proven to be more reliable than automatic counting [10]. An alternative may be flow cytometry, a new form of technology that has a sensitivity and specificity of approximately 100% for PMN detection> 250 mm 3 [3,10,11]. The culture of ascites fluid is necessary to guide antibiotic treatment but not to confirm the diagnosis [3].…”