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Protein markers unique to particular types of inflammatory cells may help to diagnose certain respiratory diseases in future. Researchers have found that eosinophil peroxidase (EPO) is unique to eosinophils and human neutrophil lipocalin (HNL) unique to neutrophils by immunocytochemical staining of inflammatory cells in induced sputum and bronchiolar lavage (BAL) samples. By comparison, eosinophil cationic protein (ECP) was specific for eosinophils and neutrophils and myeloperoxidase (MPO) for neutrophils and monocytes. EPO and HNL did not show up in other cell types—lymphocytes, monocytes, macrophages, squamous cells, and ciliated epithelial cells.
Sputum and BAL samples were obtained from four healthy controls and 10 patients with a range of inflammatory respiratory disease—acute respiratory infection, asthma, chronic obstructive pulmonary disease, and prolonged cough. Washed cell pellets from each sample were spun onto microscope slides. The slides were incubated with monoclonal primary antibody—a cocktail of six antibodies for HNL and two antibodies separately each for EPO, MPO, and ECP—then with alkaline phosphatase followed by antibody to alkaline phosphatase and counterstained. Care was taken to ensure optimum conditions for staining. Cell viability was shown by vital staining of pelleted cells to be consistently above 90%. Negative control slides were included, and a reference slide with differential staining with May-Grünwald-Giemsa was prepared for each sample.
ECP and MPO have previously been used extensively as markers for determining pathogenetic processes in respiratory disease according to the profile of inflammatory cells, though their specificity is not absolute.