Simultaneous enumeration of T-cell subsets and macrophages in bronchoalveolar lavage fluids by immunoenzyme double...

Bronchoalveolar lavage is a common research and clinical tool for the retrieval of cells from the lower respiratory tract. In addition to conventional morphologic study of these cells, the subtyping of T lymphocytes is often important for reaching a diagnosis of a disease or assessing its activity; subtyping is usually done by a standard immunofluorescence assay on cell suspensions requiring about 5 X 10(5) cells. Since the number of leukocytes in the lavage fluid from many patients is too small to obtain reliable information by this assay, a double immunoenzyme staining of T-lymphocyte subtypes on Cytospin preparations was utilized. This method, which requires a small number of cells, was compared with the standard immunofluorescence assay for the identification and quantitation of lymphocyte subtypes in the lavage fluids of patients with different disorders. Although the immunoenzyme double staining assay is somewhat more laborious, it provides important advantages: (1) simultaneous observation of two lymphocyte subsets and macrophages on the same slide; (2) a considerably smaller number of cells (2 X 10(4) instead of 5 X 10(5] is necessary; (3) the availability of permanent preparations; (4) the possibility of storing the Cytospin slides before staining; and (5) conventional light microscopy can be used. Since the reliability of both techniques appeared to be the same, the double staining assay for routine usage with bronchoalveolar lavage fluids appears to be preferable.