Safe separation of sputum cells from mucoid glycoprotein.
OBJECTIVE
To compare the safety and efficacy of homogenizing sputum with deoxyribonuclease I (DNAse I), dithiothreitol (DTT), N-acetyl-L-cysteine, sodium EDTA and trypsin against standard mechanical blending to provide mucus-free, single-cell suspensions for quantitative analysis.


METHODS
Clinical sputum specimens or cultured human bronchogenic carcinoma cells were preserved in 2% polyethylene glycol/50% ethanol, divided into aliquots, counted and stained (Papanicolaou and avidin-biotin complex immunostained) at baseline. Cells of each aliquot were 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 mixture, aliquots were again counted and stained.


RESULTS
Cell counts, Papanicolaou staining and immunostaining showed that homogenization of induced, preserved sputum with 0.5 mM DTT is safe and provides mucus-free 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 resulted in up to a 24% reduction in specimen cellularity.


CONCLUSIONS
Homogenization with low-concentration DTT will probably facilitate the exploration of sputum for protein and gene markers of carcino genesis.
D001984:Bronchial Neoplasms / Q000473:pathology; D002452:Cell Count; D002469:Cell Separation / Q000379:methods*; D003850:Deoxyribonuclease I; D004229:Dithiothreitol; D005434:Flow Cytometry; D006023:Glycoproteins / Q000302:isolation & purification*; D006801:Humans; D007150:Immunohistochemistry; D009077:Mucins; D013183:Sputum / Q000166:cytology*; D014407:Tumor Cells, Cultured
D006023:Glycoproteins; D009077:Mucins; D003850:Deoxyribonuclease I; D004229:Dithiothreitol