Automatic leukocyte differentiation in bronchoalveolar lavage fluids of guinea pigs and Brown-Norway rats

Asthma is considered to be a chronic inflammatory response of the airways characterized by a leukocyte infiltration into the lungs. Whereas lymphocytes and macrophages are involved in the initiation and propagation of inflammation, both neutrophils and in particular eosinophils are considered to play major effector roles. Therefore, allergic animal models in various species have been established to assess leukocyte infiltration by bronchoalveolar lavage (BAL) of antigen-sensitized and antigen-challenged animals as an inflammatory parameter in asthma pharmacology. Differential leukocyte counts in BAL fluids are routinely assessed by visual microscopic analysis of stained slides after cytocentrifugation. This procedure is very time-consuming, and the underlying standard morphological criteria may vary between different observers. In the present paper, we propose an alternative automatic method for leukocyte differentiation in BAL fluids from ovalbumin-treated guinea pigs and Brown-Norway rats using Cobas Helios 5Diff from Hoffmann-La Roche. BAL samples are directly applied to the analyzer and are automatically mixed with “Eosinofix”, which stabilizes leukocyte membranes and specifically stains eosinophils. By a combination of electric (resistance) and optical (light scatter) analysis, the lymphocytes, monocytes/macrophages, neutrophils, and eosinophils are discriminated and the total leukocyte numbers are obtained. For both animal species we found high correlations for all leukocyte populations by comparing the results obtained with Cobas Helios 5Diff and conventional microscopic analysis. The major advantage of the automatic method is the much lower (about one-third) time requirement.