Flow cytometry analysis of leukocytes in induced sputum from asthmatic patients

Inflammatory cell counts in induced sputum from asthmatic patients partially correlate with respiratory physiology data. To identify and quantify these inflammatory components, microscopy has been useful but it is not without its limitations. Flow cytometry could be an alternative but still has underlying methodological difficulties. While passing airways, leukocytes undergo morphologic cellular changes that alter their conventional phenotype. To demonstrate the usefulness of cytometry in accurately identifying cellular profiles in induced sputum of asthmatic and chronic cough patients, we introduced a new panel of monoclonal antibodies against specific subset markers. To identify neutrophils, sputum cells were stained with CD45 and CD66b. To identify eosinophils, sputum cells were stained with anti-CD45 and anti-CD125. We co-stained CD45, CD14 and CD66b to identify macrophages as CD45+CD14+CD66b- cells. Comparable results of trypan blue exclusion and annexin V-FITC suggested that cytometry manipulation did not decrease cellular viability. Range values were similar in microscopy neutrophils (median 19.9%, range 1.7-90.1%) and CD45+CD66b+ neutrophils (median 31% range 0.9-89%). After gating out CD45- non-leukocyte events, CD45+ and SSC dot-plots defined three patterns of leukocyte distribution. The eosinophil range in microscopic examination was 0-71.3% (median 2.85%) whereas CD45+CD125+ cell range in cytometry was 0-29% (median 3.7%). Since no exclusive markers were found on airways macrophages, we co-stained CD45, CD14 and CD66b to identify macrophages as CD45+CD14+CD66b- cells. Microscopy showed that macrophage and CD45+CD14+CD66b- cell counts were comparable (median 52.3 and range 6.7-94.8 vs median 61 and range 10.5-97.7 respectively). Correlations between neutrophils, eosinophils and macrophages in microscopic examination and flow cytometry were strong (R=0.725, 0.747 and 0.532, respectively p<0.001). This study validates effectiveness of combining specific antibodies and cytometry to quantify inflammatory leukocytes in induced sputum. Multiple markers at a single cell level will deepen our knowledge concerning the phenotype of airway leukocytes.