COMPARISON OF SPUTUM PROCESSING TECHNIQUES IN CYSTIC-FIBROSIS

Sputum analysis is a useful technique for the study of airway inflamma tion. In asthma, dithiothreitol (DTT) is used to disperse cells from s urrounding mucus; however, the applicability of these processing metho ds to cystic fibrosis (CF) sputum is unknown. In order to compare two methods for processing sputum of patients with CF, sputum was obtained from 11 subjects with CF (8 female, aged 9-21 years). The sample was split into 2 portions and sputum dispersal using DTT was compared with an enzyme mixture (E) of deoxyribonuclease, hyaluronidase, and galact osidase. Outcomes assessed were sample quality, cell viability (percen t cells excluding trypan blue), total cell count (TCC), neutrophil cou nt, and elastase immunoreactivity (percent cells positive). Sample qua lity (enzymes vs. DTT, 8.3 +/- 0.3 vs 7.6 +/- 0.4, mean +/- SEM) and c ell viability (enzymes vs. DTT, 75.0% vs. 68.0%, median) were similar for both methods. Sputum total cell count (20.5 x 10(8)/ml vs. 12.0 x 10(6)/ml, median; P = 0.01) and neutrophil count (13.4 x 10(6)/ml vs. 5.5 x 10(8)/ml, median; P = 0.02) were significantly higher with E. El astase immunoreactivity was lost after processing with E (19.0% vs. 39 .5%, median; P = 0.04). When purified peripheral blood neutrophils wer e incubated with DTT and E, there was no reduction in neutrophil viabi lity suggesting that the reduced neutrophil number in CF sputum was no t due to a toxic effect of DTT, but rather incomplete dispersal. We co nclude that published sputum processing methods for asthma using DTT g ive false results when applied to CF sputum, which should be processed using an enzyme mixture. (C) 1996 Wiley-Liss, Inc.