Comparison of sputum processing techniques in cystic fibrosis

Abstract

Sputum analysis is a useful technique for the study of airway inflammation. In asthma, dithiothreitol (DTT) is used to disperse cells from surrounding mucus; however, the applicability of these processing methods 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 galactosidase. Outcomes assessed were sample quality, cell viability (percent cells excluding trypan blue), total cell count (TCC), neutrophil count, and elastase immunoreactivity (percent cells positive). Sample quality (enzymes vs. DTT, 8.3 ± 0.3 vs 7.6 ± 0.4, mean ± SEM) and cell viability (enzymes vs. DTT, 75.0% vs. 68.0%, median) were similar for both methods. Sputum total cell count (20.5 × 106/ml vs. 12.0 × 106/ml, median, P = 0.01) and neutrophil count (13.4 × 106/ml vs. 5.5 × 106/ml, median; P = 0.02) were significantly higher with E. Elastase immunoreactivity was lost after processing with E (19.0% vs. 39.5%, median; P = 0.04). When purified peripheral blood neutrophils were incubated with DTT and E, there was no reduction in neutrophil viability, suggesting that the reduced neutrophil number in CF sputum was not due to a toxic effect of DTT, but rather incomplete dispersal. We conclude that published sputum processing methods for asthma using DTT give false results when applied to CF sputum, which should be processed using an enzyme mixture. Pediatr Pulmonol. 1996; 22:402–407. © 1996 Wiley‐Liss, Inc

Airway inflammation after treatment with aerosolized deoxyribonuclease in cystic fibrosis

“…In the current study we processed sputum samples by an improved methodology for the assessment of airway inflammation in CF. 6 Earlier processing techniques, as used to process sputum in patients with asthma, result in artefactually low total cell counts and high neutrophil counts. Airway inflammation, as assessed by total cell counts in sputum, was a prominent feature in these subjects.…”

“…A sample of sputum was obtained either by spontaneous expectoration or after sputum induction with hypertonic saline as previously described. 6 Because the patients all had chronic suppurative lung disease, sputum collection was always successful. Patients received rhDNase 2.5 mg daily for 1 month delivered by means of an Allersearch nebulizer (Allerserch, Vitalair), activated by either a Linicon LC920 (Mede/ Nitto Kohki) or a CR50 (Fisher Pakeyl) compressor pump.…”

“…A mixture of three enzymes (Sigma, New South Wales, Australia) was prepared containing deoxyribonuclease (2,550 U/mg), hyaluronidase (3,400 U/mg), and ␤-galactosidase (1,000 U/ mg), each made up to a final concentration of 10 U/mL. 6 The 300 L sputum aliquot was mixed with 3 mL of the enzyme mixture and incubated at 37°C for 20 minutes. A further 3 mL aliquot of the enzyme mixture was added for another 20 minutes of incubation.…”

“…2 This primary abnormality in CF is associated with small airways obstruction, with bacterial persistence and with the chronic presence of a massive neutrophil dominated host inflammatory response in the airways. [3][4][5][6] Large amounts of deoxyribonucleic acid (DNA) are present in purulent CF sputum. This DNA comes mainly from the nuclei of degenerating polymorphonuclear neutrophils and contributes to the increased viscidity of CF sputum.…”

Induced sputum in the investigation of airway inflammation of COPD

“…It might also interfere with the capture antibodies of immunoassays. Various studies have shown the effect of DDT on cell viability, 25 on lymphocyte, eosinophilic, neutrophilic markers [26][27][28][29] and on soluble mediators. 30,31 Consecutively, the DDT effect on the marker of interest should always be considered.…”