SAFE SEPARATION OF SPUTUM CELLS FROM MUCOID GLYCOPROTEIN

OBJECTIVE: To compare the safety and efficacy of homogenizing sputum w ith deoxyribonuclease I (DNAse I), dithiothreitol (DTT), N-acetyl-L-cy steine, sodium EDTA and trypsin against standard mechanical blending t o provide mucus-free, single-cell suspensions for quantitative analysi s. STUDY DESIGN: Clinical sputum specimens or cultured human bronchoge nic carcinoma cells were preserved in 2% polyethylene glycol/50% ethan ol, divided into aliquots, counted and stained (Papanicolaou and avidi n-biotin complex immunostained) at baseline. Cells of each aliquot wer e separated from mucus by the standard physical blending method or by chemical or enzymatic mucus liquefaction. After staining, washing and resuspending in the original volume of polyethylene glycol/ethanol mix ture, aliquots were again counted and stained. RESULTS: Cell counts, P apanicolaou staining and immunostaining showed that homogenization of induced, preserved sputum with 0.5 mM DTT is safe and provides mucus-f ree monolayers for immunocytochemistry and single-cell suspensions for flow cytometry. Mucolysis with 0.5 mM DTT resulted in a significant ( 16%) increase in cells available. In contrast, mechanical blending res ulted in up to a 24% reduction in specimen cellularity. CONCLUSION: Ho mogenization with low-concentration DTT will probably facilitate the e xploration of sputum for protein and gene markers of carcinogenesis.