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Background  Cigarette smoke causes both acute and chronic changes of the immune system. Excluding recent smoking is therefore important in clinical studies with chronic inflammation as primary focus. In this context, it is common to ask the study subjects to refrain from smoking within a certain time frame prior to sampling. The duration of the smoking cessation is typically from midnight the evening before, i.e. 8 hours from sampling. As it has been shown that a proportion of current smokers underestimates or denies smoking, objective assessment of recent smoking status is of great importance. Our aim was to extend the use of exhaled carbon monoxide (CO breath ), a well-established method for separating smokers from non-smokers, to assessment of recent smoking status.    Methods and Findings  The time course of CO breath  decline was investigated by hourly measurements during one day on non-symptomatic smokers and non-smokers (6+7), as well as by measurements on three separate occasions on non-smokers (n = 29), smokers with normal lung function (n = 38) and smokers with chronic obstructive pulmonary disease (n = 19) participating in a clinical study. We used regression analysis to model the decay, and receiver operator characteristics analysis for evaluation of model performance. The decline was described as a mono-exponential decay (r 2  = 0.7) with a half-life of 4.5 hours. CO decline rate depends on initial CO levels, and by necessity a generic cut-off is therefore crude as initial CO breath  varies a lot between individuals. However, a cut-off level of 12 ppm could classify recent smokers from smokers having refrained from smoking during the past 8 hours with a specificity of 94% and a sensitivity of 90%.    Conclusions  We hereby describe a method for classifying recent smokers from smokers having refrained from smoking for >8 hours that is easy to implement in a clinical setting.

Assessing Recent Smoking Status by Measuring Exhaled Carbon Monoxide Levels