Granulocyte markers in induced sputum in patients with respiratory disorders and healthy persons obtained by two sputum-processing methods

Abstract 

Induced sputum is increasingly used to detect and monitor airway inflammation in respiratory diseases. However, the processing of sputum has been rather laborious for clinical practice.

The aim of this study was to improve the practicality of induced-sputum studies by simplifying sample processing. Eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), neutrophil lipocalin (HNL) and myeloperoxidase (MPO) were used as biochemical markers of airway inflammation and the results of the study method were compared with a previously validated (reference) method.

Induced sputum was obtained from 42 healthy controls, 10 subjects with acute respiratory infection, eight patients with chronic obstructive pulmonary disease (COPD) and 17 asthmatics. The sputum sample was divided into two parts and treated either: (i) by the reference method (released markers), where sputum was homogenized with dithiotreitol and centrifuged to yield cell-free supernatant and a cell pellet, or (ii) by the study method (total markers), where the cells were lysed after homogenization so that cell-associated markers were released and solubilized. For comparison, the four biochemical markers were measured in sputum supernatant and in sputum lysate. The differential cell count was performed from the cell pellet in the reference method. Repeatability was assessed in a group of 16 subjects. The effect of reagents and the recovery of assays were also evaluated.

Released and total markers correlated well (ECP r_s=0·80, P<0·0001; EPOr_s =0·49, P<0·0001; HNLr_s =0·87, P<0·0001; MPOr_s =0·71, P<0·0001). Incubation with dithiotreitol and lysis reagent had no negative influence on marker assays. The within-subject variability of total ECP, MPO and HNL in both methods was small in two measurements taken 1 week apart.

The study method, measuring total inflammatory markers, gave comparable results to the reference method, measuring released markers. In the study method the sputum processing was simplified, which may improve its applicability.

Keywords: asthma, chronic obstructive pulmonary disease, eosinophil cationic protein, eosinophil peroxidase, induced sputum, infection, human neutrophil lipocalin, myeloperoxidase.

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• f1 Correspondence should be addressed to: Tuula Metso, Skin and Allergy Hospital, Division of Allergy, Helsinki University Central Hospital, Meilahdentie 2, FIN-00250 Helsinki, Finland. Fax: +358-9-47186500; E-mail: tuula.metso@sci.fi