Reliability of 400-cell and 5-field leukocyte differential counts for equine bronchoalveolar lavage fluid

Background Reliable enumeration of mast cells and eosinophils in equine bronchoalveolar lavage ( BAL) fluid is important because small increases in the percentages of these cells support the clinical diagnosis of inflammatory airway disease ( IAD). Increases in BAL neutrophils also occur with IAD but are not specific due to overlap between IAD and recurrent airway obstruction ( RAO). Objectives The objectives of this study were to evaluate the reliability of a standard 400-cell leukocyte differential count and an alternate method evaluating 5 microscopic fields at 500× magnification in equine BAL fluid cytocentrifuged preparations. Methods BAL samples from 60 horses with and without pulmonary inflammation were evaluated using 400-cell and 5-field leukocyte differential counting methods. Reliability of enumeration of each leukocyte type was assessed by calculating and comparing intraclass correlation coefficients ( ICC). Reliability of mast cell enumeration was further evaluated by comparing ICCs of slides with different cell densities. Results Reliability was higher for all cell types with the 5-field method; however, overall the difference between methods was not statistically significant. Neutrophil reliability was high ( ICC > 0.90) with both methods. Adequate reliability ( ICC > 0.85) for mast cells was achieved only with the 5-field method on slides with higher cell density. Conclusion Enumeration of mast cells is unreliable when the standard 400-cell differential counting method is used, whereas the 5-field method on slides with higher cell density reached acceptable reproducibility. Neutrophil percentages were highly reliable with both methods.