Ishizuka et al. Allergy, Asthma & Clinical Immunology 2011, 7:15 http://www.aacijournal.com/content/7/1/15 for FENO measurements: online and offline. Exhaled gas is collected in a reservoir and subsequently analyzed for nitric oxide (NO) concentrations in offline measurements [9]. Particularly in epidemiologic studies, offline measure- ment might be superior to online measurement because it is economical and makes it easy to measure a number of samples in a short time [10,11]. Worldwide epidemiological surveys on adult asthma have been mainly performed using the European Commu- nity Respiratory Health Survey (ECRHS) questionnaire [12-17]. Asthma symptoms including wheezing, a noctur- nal feeling of tightness in the chest, a nocturnal attack of shortness of breath, or a nocturnal attack of cough in the ECRHS questionnaire are consistent with asthma, but cer- tainly not specific for asthma. Moreover, we cannot guess the degree of eosinophilic airway inflammation in each subject based on the information obtained from a ques- tionnaire. Therefore, we think that the combination of FENO measurements and the questionnaire about asthma symptoms might make it possible to develop a more objective estimate in epidemiological studies of asthma prevalence. In this study, an analysis of the prevalence of asthma symptoms in university students was attempted using both the ECRHS questionnaire and FENO measured by an offline method. Additionally, we tried to investigate the gender differences in FENO levels and asthmatic symptoms. Methods 1. Subjects This study was approved by the Institutional Review Board of Gunma University Hospital, on January 27, 2010 and registered with UMIN-CTR on February 13, 2010 (ID number: UMIN000003244). Gunma University students who participated in the study were asked to fill out an ECRHS questionnaire (Japanese version) [18], and breath samples were obtained. The study was performed from April 1 to 9, 2010 when physical examinations for students were done. The flowchart of this clinical study is shown in Figure 1. We put up a notice that asked for participation in our clinical study. Of the 3247 students who underwent physical examinations, 643 students intended to take part in this study and visited our room. These 643 students were informed of the study in detail. The 640 students consented to participate in the study and signed the con- sent form. The receiver operating characteristic (ROC) curve was produced using FENO data obtained from 504 samples out of 640 samples (Additional file 1). Samples obtained from 38 students who had suffered from asthma and had wheezing for the last year were used as samples from an asthma group, and samples obtained from 466 students who had not suffered from asthma, did not have any asthmatic symptoms, and had not experienced chronic Page 2 of 8 bronchitis-like symptoms were used as samples from a non-asthma group. When 38.0 ppb was used as the cut-off value to classify the subjects in this study, the sensitivity was 86.8% and the specificity was 74.0%. To investigate the prevalence of asthma symptoms in the specified gen- eration, we used the data obtained from 584 Gunma Uni- versity students (266 male and 318 female students; age range, 18 to 24 years; mean ± SD age, 19.6 ± 1.6 years) and did not use the data from 56 students who were ≥ 25 years old in this investigation. The prevalence of asthma symptoms was analyzed based on the previous 1-year period. 2. Methods The original 9 main questions containing sub-questions translated into Japanese in the Japanese version of the ECRHS a 1-page questionnaire were used in this study [18,19]. Breath samples were obtained using an offline kit produced by the Center for Environmental Information Science (Tokyo, Japan) to measure FENO under the same conditions as with online measurement [10,11]. In this device, the expiratory flow rate is adjusted to 50 mL/s when subjects exhale at 1.5 kPa oral pressure after inhaling deeply. The adjustment was performed with a variable flux pump (SIBATA, Soka, Japan). A disposable paper mouth- piece and a viral and bacterial filter were connected to a plastic mouthpiece adaptor with a pressure gauge, fol- lowed by a plastic T-tube with a resistance valve and a bag reservoir for collecting exhaled gas. A 1.5-L Mylar bag (Sievers, Boulder, CO) was used for the reservoir bag. Dur- ing expiration, subjects were asked to maintain a constant mouth pressure (1.5 kPa) by looking at a pressure gauge. Air exhaled in the first 5 seconds (250 mL) was discarded, and the air exhaled between 5 to 10 seconds (250 mL) was collected in the Mylar bag. Samples obtained in bags were stored at 4°C to overcome error induced by sample storage and FENO was measured within 8 hours using a chemilu- minescence analyzer (NOA 280, Sievers). 3. Statistical analysis Statistical analysis was performed using SPSS software (SPSS Japan Inc., Tokyo, Japan). Differences in percentages between two groups were analyzed using the chi-square test, and differences in means between two groups were analyzed using Student’s t-test. Results 1. Gender differences in FENO and symptoms, especially wheeze and nocturnal cough attacks The students ranged in age from 18 to 24. The mean ± SD FENO in 584 students was 36.7 ± 29.6 ppb, and was ≥ 38.0 ppb in 191 (32.7%) of 584 students. Among the 266 male students, the mean FENO was 43.6 ± 34.0 ppb, and was ≥ 38.0 ppb in 111 (41.7%) of 266 students. Among