Online Data Supplement. Blood eosinophils and exacerbations in COPD: the Copenhagen General Population. Study

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1 Online Data Supplement and exacerbations in COPD: the Copenhagen General Population Study Signe Vedel-Krogh, MD, Sune F. Nielsen, PhD, Peter Lange, DMSc, Jørgen Vestbo, DMSc, and Børge G. Nordestgaard, DMSc

2 Methods All participants completed a questionnaire, underwent a physical examination, and provided blood samples. The study was conducted according to the declaration of Helsinki and approved by the Regional ethics committee (H-KF /01). Spirometry FEV 1 and FVC were determined without the use of a bronchodilator. For the first 14,624 participants of the Copenhagen General Population Study a dry wedge spirometer (Vitalograph; Maids Morton) was used, and for the remaining participants an EasyOne spirometer (nnd Medizintechnik) was used. We derived at the LLN for men and women separately for each spirometer, in a subsample of healthy, asymptomatic, never-smokers using linear regression with age and height as covariates. Individuals were considered asymptomatic if they did not report any respiratory symptoms. FEV 1 as % of the predicted value was calculated separately for each spirometer and seperately for men and women using internally derived references based on the same subsample of healthy, asymptomatic, never-smokers without self-reported asthma in a linear regression with age and height as covariates. Eosinophils White blood cell counts were measured on fresh samples using the ADVIA TM 120 Hematology system subjected to daily precision testing using internal quality control material and monthly accuracy testing using an external control quality program. Eosinophil counts were reported in total numbers ( 10 9 cells/l) together with other leukocyte sub-populations and % of total white blood cell count was calculated. Covariates E2

3 Potential confounders were included in the analyses. Smoking habits were self-reported and participants were grouped as never, former, or current smokers according to the questions Do you smoke? and Have you previously smoked?. Participants also reported on cumulative tobacco consumption and the covariate was defined as tobacco consumed through smoking and measured in terms of pack-years based on the duration of smoking and the amount consumed; 1 pack-year was 20 cigarettes or equivalent smoked daily for one year. Measured weight (kg) and height (m) were used to calculate body mass index (kg/m 2 ). Level of education was dichotomised as low level of education if the participants had none or less than less than three years of education following the Danish mandatory primary school. Weekly alcohol consumption was reported in units where 1 unit 12 grams. A history of hospitalization for ischemic heart disease (World Health Organization International Classification of Diseases: ICD ; ICD10 I20-I25), ischemic cerebrovascular disease (World Health Organization International Classification of Diseases ICD ; ICD10 I61-I69, G45), and diabetes mellitus type 2 (World Health Organization International Classification of Diseases ICD8 250; ICD10 E11) was collected using the national Danish Patient Registry. Imputation of missing characteristics was done by multivariable normal regression. Information on age, sex, height, FVC, FEV 1, and FEV 1 in % of predicted was 100% complete, whereas information on other characteristics were > 98% complete. E3

4 Figure E1. Histogram of blood eosinophils in total number ( 10 9 cells/l) and percentage of leukocytes. E4

5 Figure E2. Histogram of blood eosinophil distributions in the Copenhagen General Population Study. E5

6 Figure E3. ROC curves of levels of blood eosinophils as a marker of mean annual severe exacerbation risk of 0.5 or more and 2 or more. E6

7 Figure E4. Risk of exacerbations according to blood eosinophils and other risk factors. N is the number of individuals, events is the total number of count events. IRR is incidence rate ratio given with 95% confidence interval. All models were multivariable adjusted for age, sex, body mass index and education. All models, except when stratifying according to current smoking status, are adjusted for smoking status. All models, except when stratifying by pack-years, are adjusted for cumulative smoking. All models are adjusted for FEV 1 % of predicted except when stratifying by GOLD stages. All models except when stratifying by blood eosinophil level equal to or above cells/l are adjusted for level of blood eosinophils. E7

8 Figure E5. Risk of severe and moderate exacerbations in individuals with COPD excluding individuals with clinical COPD using the cut-points for blood eosinophils of cells/l and 3.3%. N is the number of individuals, events is the total number of count events. IRR is incidence rate ratio given with 95% confidence interval. Models were multivariable adjusted for age, sex, FEV 1 in % of predicted, smoking status, cumulative smoking, education, and body mass index. E8

9 Figure E6. Mean annual exacerbation rate in the clinical COPD population according to the use of corticosteroids in the year prior to baseline. E9

10 Figure E7. Risk of severe and moderate exacerbations in individuals with COPD and clinical COPD using the cut-points of cells/l, cells/l, 3%, and 4% for blood eosinophils. Incidence rate ratios (IRR) are given with 95% confidence intervals. All models were multivariable adjusted for age, sex, smoking status, cumulative smoking, FEV 1 % of predicted, body mass index, and education. The models marked with * are the models from the main analyses. E10

11 Table E1. Number of participants using inhaled medication at baseline and corresponding Anatomic Therapeutic Chemical codes in the entire COPD population. Medication COPD Clinical COPD (n=7,225) (n=203) Corresponding ATC codes Short-acting β2- R03AC02, R03AC03, R03CC02, 369 (5%) 71 (35%) agonists R03CC03 Long- acting β2- R03AC12, R03AC13, R03AC18, 117 (2%) 15 (7%) agonists R03CC12 Short-acting anticholinergics 21 (0.3%) 7 (3%) R03BB01 Long-acting anticholinergics 235 (3%) 54 (27%) R03BB04 Inhaled corticosteroids 182 (2%) 27 (13%) R03BA01, R03BA02, R03BA05, R03BA07 Combination products 382 (5%) 93 (46%) R03AK03, R03AK04, R03AK06, R03AK07 Number of participants (percentage of all participants) with use of inhalation medication in the year prior to examination. Some participants are using more than one type of medication and therefore the total number is larger than any medication in Table 1. Corresponding codes are Anatomic Therapeutic Chemical codes used in the Danish Registry of Medicinal Products Statistics. E11

12 Table E2. Baseline characteristics of the chronic obstructive pulmonary disease (COPD) and clinical COPD population with a cut-point for total number of blood eosinophils of cells/l. COPD (n=7,225) Clinical COPD (n=203) < cells/l (n=6,205) Blood eosinophils cells/l (n=1,020) P-value < cells/l (n=165) cells/l (n=38) P-value Men (%) 2,984 (48%) 626 (61%) (46%) 33 (87%) Age (years) 63 (54-72) 65 (56-74) (64-77) 72 (66-78) 0.69 (10 9 cells/l) 0.16 ( ) 0.42 ( ) ( ) 0.43( ) (%) 2.2 ( ) 5.2 ( ) ( ) 5.2 ( ) FEV 1 (%) of predicted 79 (65-90) 76 (61-88) (40-61) 48 (43-56) 0.32 FEV 1/FVC 63 (59-66) 63 (58-66) (48-61) 53 (48-61) 0.80 Body-mass index, kg/m ( ) 25.3 ( ) ( ) 25.3 ( ) 0.95 Current smokers (%) 2,282 (37%) 421 (41%) (40%) 10 (26%) 0.12 Pack-years 29 (15-44) 32 (20-48) (26-50) 45 (39-63) 0.11 Familial disposition to asthma (%) 1,935 (31%) 333 (33%) (41%) 16 (42%) 0.87 Asthma, hay fever, or eczema during childhood (%) 623 (10%) 115 (11%) (4%) 4 (11%) 0.08 Allergy (%) 1,218 (20%) 231 (23%) (16%) 6 (16%) 0.93 Low level of education (%) 3,341 (54%) 564 (55%) (70%) 28 (74%) 0.63 Occupational exposure to dust or fumes (%) 962 (16%) 195 (19%) (30%) 12 (32%) 0.82 History of respiratory infections (%) 1,930 (31%) 338 (33%) (68%) 29 (76%) 0.33 Wheezing during a cold (%) 1,030 (17%) 237 (23%) (35%) 18 (47%) 0.14 mmrc 2 (%) 1,042 (17%) 198 (19%) (57%) 26 (68%) 0.20 Sputum for 3 months 1,085 (17%) 250 (25%) (41%) 21 (55%) 0.10 Weekly alcohol consumption (units) 9 (4-17) 10 (4-18) (3-20) 12 (4-21) 0.78 Degree of air flow limitation FEV1 (%) of predicted 80 (%) 2,917 (47%) 427 (42%) FEV1 (%) of predicted <80 (%) 2,761 (45%) 471 (46%) 84 (51%) 16 (42%) FEV1 (%) of predicted <50 (%) 527 (8%) 122 (12%) 81 (49%) 22 (58%) Any inhalation medication 648 (10%) 153 (15%) (61%) 27 (71%) 0.26 Corticosteroids inhalation medication 400 (6%) 94 (9%) (45%) 20 (53%) 0.42 Co-morbidities Ischemic heart disease 499 (8%) 148 (15%) (20%) 11 (29%) 0.23 Ischemic cerebrovascular disease 230 (4%) 52 (5%) (9%) 3 (8%) 0.82 Diabetes mellitus type (2%) 30 (3%) (5%) 3 (8%) 0.57 Hypertension 1,641 (27%) 242 (24%) (27%) 9 (24%) 0.71 Data are n (%) for categorical values and median (interquartile range) for continuous values. COPD = chronic obstructive pulmonary disease. FEV 1 = Forced expiratory volume in 1 second. FVC = Forced vital capacity. mmrc = modified Medical Research Council dyspnea scale. Baseline characteristics were at the date of examination except for inhalation medication which was up to 1 year prior to baseline. Pack-years were calculated for only former and current smokers. Familial disposition to asthma were the reporting of a mother and/or father with asthma. Low education was none or less than 3 years of education following the mandatory primary school. One alcohol unit 12 grams of alcohol. Co-morbidities are hospitalization with a diagnosis of ischemic heart disease, ischemic cerebrovascular disease, or diabetes mellitus type 2 prior to baseline. Hypertension was defined as systolic blood pressure 140 mmhg and diastolic blood pressure 90 mmhg at baseline. P-values are calculated using Wilcoxon s rank-sum test, Pearson's chi-squared tests, or Kruskal Wallis test. E12

13 Table E3. Baseline characteristics of the chronic obstructive pulmonary disease (COPD) and clinical COPD population with a cut-point for blood eosinophils of 3.3%. COPD (n=7,225) Clinical COPD (n=203) <3.3% (n=5,231) Blood eosinophils 3.3% (n=1,994) P-value <3.3% (n=142) 3.3% (n=61) P-value Men (%) 2,416 (46%) 1194 (60%) (46%) 44 (72%) Age (years) 64 (54-72) 65 (55-74) (63-76) 72 (66-78) 0.25 (10 9 cells/l) 0.14 ( ) 0.33 ( ) ( ) 0.37 ( ) (%) 1.9 ( ) 4.4 ( ) ( ) 4.6 ( ) FEV 1 (%) of predicted 79 (65-90) 78 (63-89) (41-61) 48 (40-57) 0.09 FEV 1/FVC 63 (59-66) 63 (58-66) (48-61) 54 (49-60) 0.66 Body-mass index, kg/m ( ) 25.3 ( ) ( ) 25.2 ( ) 0.84 Current smokers (%) 2,023 (39%) 680 (34%) (44%) 14 (23%) Pack-years 30 (15-44) 30 (16-45) (26-50) 40 (30-58) 0.30 Familial disposition to asthma (%) 1,607 (31%) 661 (33%) (40%) 27 (44%) 0.52 Asthma, hay fever, or eczema during childhood (%) 480 (9%) 258 (13%) (3%) 6 (10%) 0.03 Allergy (%) 992 (19%) 457 (23%) (20%) 19 (14%) 0.26 Low level of education (%) 2,814 (54%) 1,091 (55%) (70%) 44 (72%) 0.73 Occupational exposure to dust or fumes (%) 807 (15%) 350 (18%) (28%) 22 (36%) 0.22 History of respiratory infections (%) 1,645 (32%) 623 (31%) (65%) 49 (80%) 0.03 Wheezing during a cold (%) 885 (17%) 382 (19%) (37%) 22 (36%) 0.86 mmrc 2 (%) 887 (17%) 353 (18%) (55%) 42 (69%) 0.06 Sputum for 3 months 933 (18%) 402 (20%) (42%) 28 (48%) 0.43 Weekly alcohol consumption (units) 8 (4-17) 10 (4-18) (4-20) 12 (3-20) 0.58 Degree of air flow limitation FEV 1 (%) of predicted 80 (%) 2,453 (47%) 819 (45%) FEV 1 (%) of predicted <80 (%) 2,329 (45%) 903 (45%) 75 (53%) 25 (41%) FEV 1 (%) of predicted <50 (%) 449 (8%) 200 (10%) 67 (47%) 36 (59%) Any inhalation medication 541 (10%) 260 (13%) (61%) 42 (69%) 0.26 Corticosteroids inhalation medication 333 (6%) 161 (8%) (46%) 30 (49%) 0.66 Co-morbidities Ischemic heart disease 349(8%) 253 (12%) (20%) 16 (26%) 0.30 Ischemic cerebrovascular disease 187 (4%) 95 (5%) (11%) 3 (5%) 0.20 Diabetes mellitus type (2%) 49 (2%) (6%) 3 (5%) 0.69 Hypertension 1,361 (26%) 522 (26%) (27%) 15 (25%) 0.75 Data are n (%) for categorical values and median (interquartile range) for continuous values. COPD = chronic obstructive pulmonary disease. FEV 1 = Forced expiratory volume in 1 second. FVC = Forced vital capacity. mmrc = modified Medical Research Council dyspnea scale. Baseline characteristics were at the date of examination except for inhalation medication which was up to 1 year prior to baseline. Pack-years were calculated for only former and current smokers. Familial disposition to asthma were the reporting of a mother and/or father with asthma. Low education was none or less than 3 years of education following the mandatory primary school. One alcohol unit 12 grams of alcohol. Co-morbidities are hospitalization with a diagnosis of ischemic heart disease, ischemic cerebrovascular disease, or diabetes mellitus type 2 prior to baseline. Hypertension was defined as systolic blood pressure 140 mmhg and diastolic blood pressure 90 mmhg at baseline. P-values are calculated using Wilcoxon s rank-sum test, Pearson's chi-squared tests, or Kruskal Wallis test. E13

14 Table E4. Baseline characteristics of the chronic obstructive pulmonary disease (COPD) and clinical COPD population with a cut-point for blood eosinophils of 2%. COPD (n=7,225) Clinical COPD (n=203) <2% (n=2,618) Blood eosinophils 2% (n=4,607) P-value <2% (n=69) 2% (n=134) P-value Men (%) 1,124 (43%) 2,486 (54%) (36%) 84 (64%) Age (years) 64 (54-72) 64 (54-73) (63-77) 71 (65-76) 0.40 (10 9 cells/l) 0.10 ( ) 0.24 ( ) ( ) 0.26 ( ) (%) 1.4 ( ) 3.1 ( ) ( ) 3.3 ( ) FEV 1 (%) of predicted 79 (65-90) 78 (64-89) (40-62) 50 (41-60) 0.51 FEV 1/FVC 63 (59-66) 63 (59-66) (47-62) 55 (49-61) 0.60 Body-mass index, kg/m ( ) 25.2 ( ) ( ) 26.0 ( ) 0.13 Current smokers (%) 1,042 (40%) 1,661 (36%) (46%) 44 (33%) 0.06 Pack-years 30 (15-44) 30 (15-45) (24-50) 40 (28-55) 0.56 Familial disposition to asthma (%) 798 (30%) 1,470 (32%) (39%) 56 (42%) 0.71 Asthma, hay fever, or eczema during childhood (%) 220 (8%) 518 (11%) (4%) 7 (5%) 0.78 Allergy (%) 457 (17%) 922 (22%) (20%) 19 (14%) 0.26 Low level of education (%) 1,437 (55%) 2,468 (54%) (81%) 87 (65%) 0.02 Occupational exposure to dust or fumes (%) 388 (15%) 769 (17%) (29%) 41 (31%) 0.81 History of respiratory infections (%) 826 (32%) 1,442 (31%) (67%) 95 (71%) 0.48 Wheezing during a cold (%) 438 (17%) 829 (18%) (38%) 49 (37%) 0.88 mmrc 2 (%) 439 (17%) 801 (17%) (54%) 83 (62%) 0.25 Sputum for 3 months 469 (18%) 866 (19%) (51%) 53 (40%) 0.13 Weekly alcohol consumption (units) 9 (4-17) 10 (4-18) (4-20) 10 (3-20) 0.58 Degree of air flow limitation FEV 1 (%) of predicted 80 (%) 1,222 (47%) 2,122 (46%) FEV 1 (%) of predicted <80 (%) 1,170 (45%) 2,062 (45%) 34 (49%) 66 (49%) FEV 1 (%) of predicted <50 (%) 226 (8%) 423 (9%) 35 (51%) 68 (51%) Any inhalation medication 262 (10%) 539 (12%) (57%) 89 (66%) 0.17 Corticosteroids inhalation medication 158 (6%) 336 (7%) (45%) 67 (50%) 0.20 Co-morbidities Ischemic heart disease 185 (7%) 462 (10%) (17%) 32 (24%) 0.29 Ischemic cerebrovascular disease 95 (4%) 187 (4%) (10%) 11 (8%) 0.65 Diabetes mellitus type 2 67 (3%) 92 (2%) (4%) 9 (8%) 0.50 Hypertension 671 (26%) 1,212 (26%) (20%) 39 (29%) 0.18 Data are n (%) for categorical values and median (interquartile range) for continuous values. COPD = chronic obstructive pulmonary disease. FEV 1 = Forced expiratory volume in 1 second. FVC = Forced vital capacity. mmrc = modified Medical Research Council dyspnea scale. Baseline characteristics were at the date of examination except for inhalation medication which was up to 1 year prior to baseline. Pack-years were calculated for only former and current smokers. Familial disposition to asthma were the reporting of a mother and/or father with asthma. Low education was none or less than 3 years of education following the mandatory primary school. One alcohol unit 12 grams of alcohol. Co-morbidities are hospitalization with a diagnosis of ischemic heart disease, ischemic cerebrovascular disease, or diabetes mellitus type 2 prior to baseline. Hypertension was defined as systolic blood pressure 140 mmhg and diastolic blood pressure 90 mmhg at baseline. P-values are calculated using Wilcoxon s rank-sum test, Pearson's chi-squared tests, or Kruskal Wallis test. E14

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