Available for peripheral arterial disease and muscle weakness. The complete protocol used for CT imaging, which was based on National Emphysema Treatment Trial criteria [15], was described in a previous report [16]. Emphysema was semiquantitatively assessed by a visual scoring system. Four categories were generated yielding a four-level alveolar destruction scale (no emphysema, mild emphysema affecting ,20 , moderate emphysema between 20?0 , and severe emphysema .50 of the lung) [16]. Thickening of the bronchial walls was scored on a semi-Figure 1. Flow chart. Abbreviations: BMI: body mass index; mMRC: modified Medical Research Council; CCQ: clinical COPD questionnaire; TGV: thoracic gas volume and DLCO: diffusing capacity of the lung for carbon monoxide. doi:10.1371/journal.pone.0051048.gCOPD Phenotypes at High Risk of Mortalityquantitative three-level scale and presence or absence of Acetovanillone biological activity bronchiectasis was assessed [16]. All pulmonary function data were obtained with standardized equipment (Jaeger) according to ATS/ERS consensus guidelines. Spirometric values were post-bronchodilator values. Diffusing capacity was determined by single breath carbon monoxide gas transfer method (DLCO) and corrected for alveolar ventilation (Kco) but not for haemoglobin. All data were obtained as absolute values and expressed as percent predicted 
of reference values [17].StatisticsData are presented as median [interquartile range (IQR)] or . A P,0.05 was considered statistically significant. Analyses were performed using the SAS 9.2 statistical software (Cary, North Carolina, USA).Results Characterization of COPD Patients Based on GOLD ClassificationCharacteristics of the 527 COPD patients according to the spirometric GOLD classification are presented in Table 1. Subjects recruited in the NELSON study were mostly in spirometric GOLD stage I and II (65 and 31 , respectively), whereas subjects recruited in the LEUVEN clinic were mostly in spirometric GOLD stage II, III and IV (33 , 38 , and 24 , respectively) (also see Table S2). Increasing GOLD stage was associated with increased dyspnoea, decreased HRQoL (higher CCQ total score), a lower BMI, higher 23727046 lung hyperinflation and decreased lung diffusing capacity. Extent of emphysema, bronchial thickening and bronchiectasis were also associated with more severe airflow limitation. Muscle weakness and osteoporosis increased with GOLD stages, whereas diabetes and cardiovascular comorbidities appeared relatively unrelated to the degree of airflow limitation. The median follow-up duration was of 17.2 [10.8; 22.9] months, and was not different among GOLD stages (not shown). At that time, 8/527 (1.5 ) patients were lost to follow-up and 50/519 (9.6 ) subjects had died. Mortality rates were 0.8 , 3.0 , 14.1 and 25.8 in spirometric GOLD stage I, II, III and IV subjects, AKT inhibitor 2 cost respectively.Plan for Cluster AnalysisOur strategy was to combine both continuous and categorical data in a single cluster analysis aimed at the identification of COPD phenotypes. Based on the result of a first analysis of this database (Table S1) we made a selection of 
continuous variables to be included the cluster analysis. This analysis resulted in the selection of 7 continuous variables (see below). Because some continuous variables were correlated with each other (Table S3), we eliminated correlations between variables by performing a principal component analysis. For categorical variables, all variables were used in the analysis but these vari.Available for peripheral arterial disease and muscle weakness. The complete protocol used for CT imaging, which was based on National Emphysema Treatment Trial criteria [15], was described in a previous report [16]. Emphysema was semiquantitatively assessed by a visual scoring system. Four categories were generated yielding a four-level alveolar destruction scale (no emphysema, mild emphysema affecting ,20 , moderate emphysema between 20?0 , and severe emphysema .50 of the lung) [16]. Thickening of the bronchial walls was scored on a semi-Figure 1. Flow chart. Abbreviations: BMI: body mass index; mMRC: modified Medical Research Council; CCQ: clinical COPD questionnaire; TGV: thoracic gas volume and DLCO: diffusing capacity of the lung for carbon monoxide. doi:10.1371/journal.pone.0051048.gCOPD Phenotypes at High Risk of Mortalityquantitative three-level scale and presence or absence of bronchiectasis was assessed [16]. All pulmonary function data were obtained with standardized equipment (Jaeger) according to ATS/ERS consensus guidelines. Spirometric values were post-bronchodilator values. Diffusing capacity was determined by single breath carbon monoxide gas transfer method (DLCO) and corrected for alveolar ventilation (Kco) but not for haemoglobin. All data were obtained as absolute values and expressed as percent predicted of reference values [17].StatisticsData are presented as median [interquartile range (IQR)] or . A P,0.05 was considered statistically significant. Analyses were performed using the SAS 9.2 statistical software (Cary, North Carolina, USA).Results Characterization of COPD Patients Based on GOLD ClassificationCharacteristics of the 527 COPD patients according to the spirometric GOLD classification are presented in Table 1. Subjects recruited in the NELSON study were mostly in spirometric GOLD stage I and II (65 and 31 , respectively), whereas subjects recruited in the LEUVEN clinic were mostly in spirometric GOLD stage II, III and IV (33 , 38 , and 24 , respectively) (also see Table S2). Increasing GOLD stage was associated with increased dyspnoea, decreased HRQoL (higher CCQ total score), a lower BMI, higher 23727046 lung hyperinflation and decreased lung diffusing capacity. Extent of emphysema, bronchial thickening and bronchiectasis were also associated with more severe airflow limitation. Muscle weakness and osteoporosis increased with GOLD stages, whereas diabetes and cardiovascular comorbidities appeared relatively unrelated to the degree of airflow limitation. The median follow-up duration was of 17.2 [10.8; 22.9] months, and was not different among GOLD stages (not shown). At that time, 8/527 (1.5 ) patients were lost to follow-up and 50/519 (9.6 ) subjects had died. Mortality rates were 0.8 , 3.0 , 14.1 and 25.8 in spirometric GOLD stage I, II, III and IV subjects, respectively.Plan for Cluster AnalysisOur strategy was to combine both continuous and categorical data in a single cluster analysis aimed at the identification of COPD phenotypes. Based on the result of a first analysis of this database (Table S1) we made a selection of continuous variables to be included the cluster analysis. This analysis resulted in the selection of 7 continuous variables (see below). Because some continuous variables were correlated with each other (Table S3), we eliminated correlations between variables by performing a principal component analysis. For categorical variables, all variables were used in the analysis but these vari.
