title: Induced sputum cell and fluid-phase indices of inflammation: comparison of treatment with dithiothreitol vs phosphate-buffered saline.
abstract: Treatment of sputum with dithiothreitol (DTT) gives reliable measurements of cellular and fluid-phase markers of airway inflammation. We investigated the extent to which DTT treatment influences these measurements as compared with phosphate-buffered saline (PBS). Hypertonic saline-induced sputum, collected from 20 asthmatic subjects, was examined within 2 h. All portions which looked more solid (less fluid) than saliva were collected from the expectorate. The selected sputum was then divided into two portions: one treated with one volume of DTT and one volume of PBS, the other with two volumes of PBS. The filtrates were assessed blind for total and differential cell count, viability, and fluid-phase eosinophil cationic protein (ECP), fibrinogen, interleukin (IL)-5 and IL-8. Sputum treated with DTT compared with PBS had lower proportions of viable cells (median 66 versus 74%; p=0.003). In contrast, DTT-treated sputum had higher total cell counts (median 8.8 vs 2.8 x 10(6) mL(-1); p<0.001) and levels of ECP (median 1340 vs 584 mg x L(-1); p<0.001) The measurements were similar with respect to the proportion of eosinophils, neutrophils, lymphocytes, macrophages, and fluid-phase fibrinogen, IL-5 and IL-8. We conclude that dithiothreitol disperses cells more effectively and that this might account for the higher levels of eosinophil cationic protein. Dithiothreitol may affect cell viability, but the changes are not relevant with respect to cell counts. Additionally, dithiothreitol does not seem to influence the other measurements performed.
authors.affiliation: Dept of Medicine, St. Joseph's Hospital and McMaster University, Hamilton, Ontario, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Efthimiadis
authors.forename: M M
authors.initials: MM
authors.lastname: Pizzichini
authors.forename: E
authors.initials: E
authors.lastname: Pizzichini
authors.forename: J
authors.initials: J
authors.lastname: Dolovich
authors.forename: F E
authors.initials: FE
authors.lastname: Hargreave
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D001249:Asthma / Q000378:metabolism / Q000473:pathology*; D001798:Blood Proteins / Q000032:analysis; D002452:Cell Count / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002648:Child; D004229:Dithiothreitol / Q000494:pharmacology*; D047091:Eosinophil Granule Proteins; D005100:Expectorants / Q000494:pharmacology*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007249:Inflammation; D018836:Inflammation Mediators / Q000032:analysis; D008297:Male; D008875:Middle Aged; D012260:Ribonucleases; D012462:Saline Solution, Hypertonic / Q000494:pharmacology*; D013183:Sputum / Q000737:chemistry / Q000166:cytology / Q000187:drug effects*
chemical_list: D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D005100:Expectorants; D018836:Inflammation Mediators; D012462:Saline Solution, Hypertonic; D012260:Ribonucleases; D004229:Dithiothreitol