Lymphocytic Subpopulation Profiles in Bronchoalveolar Lavage Fluid and Peripheral Blood From Tobacco and Marijuana Smokers: BAL and Peripheral Blood Samples
The cells recovered by BAL were washed and resuspended in RPMI-1640 medium (Grand Island Biological Co, Grand Island, NY) containing 5 percent fetal calf serum. An aliquot was taken for the following: (1) a hemocytometer count of the total number of cells recovered; (2) determination of cell viability by trypan blue exclusion; and (3) cytocentrifuge slide preparations stained with Giemsa and counted manually by examination of 200 cells. White blood and differential cell counts were performed on each blood sample by the UCLA Clinical Laboratory in the standard manner.
Identification of PB and BAL lymphocyte phenotypes was performed using the following fluorescein-labeled murine monoclonal agents (Becton-Dickinson, Mountain View, Calif): Leu-4 (CD3 or mature circulating T cells); Leu-3 (CD4 or helper/ inducer T cells); Leu-2 (CD8 or suppressor/cytotoxic cells); and Leu-16 (CD20 or mature circulating В cells). The whole blood or BAL cell suspension was diluted with an equal volume of phosphate-buffered saline solution containing 2 percent newborn calf serum and 0.1 percent sodium azide, and incubated with medium containing fluorescein-labeled monoclonal antibody at 4°C for 30 min in the dark. The cells were then washed twice. The PB cells were suspended in ammonium chloride lysing solution, and 5 to 7 min were allowed for erythrocyte lysis. canada health and care mall
The PB and BAL cell preparations were analyzed in a flow cytometer (Spectrum III, Ortho Diagnostic Systems, Westwood, Mass). Lymphocyte populations were distinguished from monocytes and granulocytes by correlated analysis of forward and wide-angle scatter. On several occasions, a Leu-M-3 marker was used to detect monocyte-macrophage lines and to determine the degree of contamination in the window analyzed. Leu-M-3 positive cells comprised 0 to 1 percent of cells in these areas. Fluorescence data from 500 to 2,000 lymphocytes were analyzed to determine percentage of positive cells. Blood specimens from normal control subjects were analyzed with each daily run.
Total cell concentrations in the recovered BAL fluid were calculated and expressed as number per milliliter of recovered BAL fluid. Using the cell differential results, absolute concentrations of alveolar macrophages, lymphocytes, and neutrophils in the BAL samples were calculated. Concentrations of CD3-, CD4-, CD8-, and CD20-positive cells were calculated from the percentages determined by the fluorescein-labeled monoclonal assays. Data are expressed as mean value ± SEM.