Induced sputum eosinophil cationic protein (ECP) measurement in asthma and chronic obstructive airway disease (COAD)
BACKGROUND
Induced sputum is a useful way to monitor airway inflammation in asthma, but cell counts are time-consuming and labour intensive.


OBJECTIVE
The aim of this study was to evaluate a novel processing method using eosinophil cationic protein (ECP) as a biochemical marker of sputum eosinophil number and activation in subjects with asthma and other airway diseases.


METHODS
Sputum was dispersed with dithiothreitol and centrifuged to yield cell free supernatant and a cell pellet. The pellet was treated with a cellular lysis buffer to release cell-associated ECP. ECP was measured in sputum supernatant and in the lysed cell pellet and was compared with sputum eosinophil counts in 31 adults with asthma, chronic obstructive airway disease (COAD), bronchiectasis and healthy controls. The ratio of supernatant to pellet ECP was evaluated as an index of eosinophil degranulation. The effect of sputum processing reagents and storage time on ECP measurement was also evaluated.


RESULTS
ECP measured in the cell pellet lysate correlated closely with sputum absolute eosinophil counts across a range of subject groups (r = 0.72, P = 0.004). Sputum eosinophil counts were less well correlated with supernatant ECP levels (r = 0.54, P < 0.05). Incubation with dithiothreitol or lysis buffer did not influence ECP measurement and sputum ECP levels were stable over a 6-9 month period. Sputum supernatant and pellet lysate ECP concentrations were increased in stable asthma, asthma exacerbations and COAD/bronchiectasis (P < 0.05). The ratio of supernatant to pellet ECP was used as an index of eosinophil degranulation and found to be elevated in asthma exacerbations, COAD and bronchiectasis, but not in stable asthma.


CONCLUSIONS
The measurement of ECP in the sputum cell pellet provides a reliable and efficient estimate of sputum eosinophil counts which can potentially be used in clinical trials and epidemiological surveys. The ECP ratio may be a useful marker of eosinophil activation, and was increased in asthma exacerbation and COAD. The increased ECP in COAD reflects a non-selective accumulation of eosinophils in this condition.
Airway Research Centre, Respiratory Medicine Unit, John Hunter Hospital, Newcastle, NSW, Australia.
P G
PG
Gibson
K L
KL
Woolley
K
K
Carty
K
K
Murree-Allen
N
N
Saltos
D000328:Adult; D001249:Asthma / Q000175:diagnosis* / Q000378:metabolism; D015415:Biomarkers / Q000032:analysis; D001798:Blood Proteins / Q000032:analysis*; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000166:cytology; D005260:Female; D006801:Humans; D018836:Inflammation Mediators / Q000032:analysis*; D007958:Leukocyte Count; D008173:Lung Diseases, Obstructive / Q000175:diagnosis / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D012260:Ribonucleases; D012462:Saline Solution, Hypertonic / Q000008:administration & dosage; D013048:Specimen Handling; D013183:Sputum / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism
D015415:Biomarkers; D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D018836:Inflammation Mediators; D012462:Saline Solution, Hypertonic; D012260:Ribonucleases