Preparation of bronchoalveolar lavage fluid with microscope slide smears

Abstract

The method of preparation of bronchoalveolar lavage fluid (BALF) for cytological examination can significantly affect the results of cellular quantitation. Investigations have shown that cytocentrifugation leads to an underestimation of the number of lymphocytes and membrane filter preparation to an underestimation of the number of neutrophils. As a simple alternative to these two techniques, BALF cells could be prepared by the microscope slide smear technique, which is familiar as the means for preparing peripheral blood for differential counts. In order to compare cell differentials determined by microscope slide technique with differentials resulting from cytocentrifugation, cells were isolated from 35 BALF samples using standard methods, and counted using a haematocytometer. Forty thousand cells in 200 μL were prepared by cytocentrifugation (3 min, 57xg; Cytospin 2) and 5x105 cells in 5 μL by microscope slide smear. Both samples were air-dried, stained using May-Grünwald Giemsa stain, and 600 cells were counted to obtain differentials. To test the adequacy of sampling by the microscope slide smear technique, known quantities of lymphocytes or neutrophils were added to fixed numbers of BALF cells, microscope slide smears prepared, and differentials determined on 600 cells. The resulting differentials were compared to the calculated differentials. Preparation of BALF cells with the microscope slide smear technique yielded well-preserved cell morphology. Compared to cytocentrifugation, microscope slide smear preparations had significantly higher percentages of lymphocytes. The microscope slide smears for the samples with predetermined numbers of cells yielded lymphocyte and neutrophil percentages which did not differ from the calculated differentials (59.6±1.5 vs 59.6±5.2% and 54.6±6.0 vs 53.1±6.0%, respectively). Varying the number of cells counted from 100 to 800 confirmed the reproducibility of the counts for counting 600 cells. Using 5x105, 2.5x105, or 1x105 cells per preparation demonstrated that adequate specimens could be obtained from as few as 1x105 cells. Thus, microscope slide smear preparation is a simple and accurate method for the quantitation of bronchoalveolar lavage fluid cytology. ©ERS Journals Ltd 1996.