asthma and copd have been regarded as two distinct disease entities that often overlap.13 asthma and copd overlap has a prevalence of 15%20% in patients with obstructive airway disease ( asthma or copd)46 and is associated with significant health status impairment,4 increased exacerbations,5 and increased hospitalizations.6 in 2014 , the global initiative for asthma ( gina ) and the global initiative for chronic obstructive lung disease ( gold ) issued a joint document describing that asthma - copd overlap syndrome ( acos ) is characterized by persistent airflow limitation with several features usually associated with both asthma and copd.7 the clinical phenotypes and underlying mechanisms of acos have attracted interest during recent years . however , acos remains somewhat controversial , and there is no consensus on the best definition of acos.8,9 the clinical phenotypes , including biomarkers , of acos remain elusive . inflammatory biomarkers , such as fractional exhaled nitric oxide ( feno ) , blood eosinophils , and allergen - specific immunoglobulin e ( ige ) , are sometimes used to distinguish between asthma and copd.7 typically , asthma is characterised by inflammation predominantly involving eosinophils , whereas copd is characterized by inflammation by neutrophils.1,2 feno and blood eosinophil count have been considered as biomarkers of local and systemic eosinophilic inflammation , which increase in patients with asthma.10,11 total serum ige and antigen - specific ige levels are also elevated in those with allergic asthma.12 however , the significance of these inflammatory biomarkers in diagnosis of acos remains unclear . we conducted a cross - sectional study to 1 ) analyze prospectively collected data from a japanese copd registry and 2 ) compare copd patients with and without acos , focusing on inflammatory biomarkers , to investigate clinical phenotypes of acos . we conducted a cross - sectional study to analyze the prospectively collected data of consecutive scheduled visits or newly registered patients from the ishinomaki copd network ( icon ) registry13 between may 2015 and january 2016 . briefly , icon is a regional medical liaison system aimed at comprehensive care of patients with copd and has a multi - center interdisciplinary collaboration with health care providers in ishinomaki , japan . patients registered in icon are regularly treated by general practitioners in ishinomaki and surrounding cities , and receive scheduled examinations and education at the japanese red cross ishinomaki hospital ( a 452-bed tertiary community hospital in ishinomaki ) every 612 months . this study is part of an ongoing copd cohort study registered with the university hospital information network clinical trials registry ( identifier : umin000017376 ) . all patients provided written informed consent , and the study was approved by the ethics committee of japanese red cross ishinomaki hospital ( approval number : 12 - 14 - 1 ) . patients with stable copd , who were aged 4090 years and former smokers with a smoking history of at least 10 pack - years , were included in this study . all patients were diagnosed with copd according to gold criteria.14 persistent airflow limitation , defined as postbronchodilator forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) ratio < 0.7 , was confirmed by using repeated spirometry . exclusion criteria were as follows : current smoker or never smoked , chronic bronchitis or emphysema without airflow limitation , hematologic disease , history of lung resection , use of oral corticosteroids , receiving anti - ige therapy , and exacerbation of copd in the 4 weeks preceding data collection . acos was defined according to the gina / gold joint document.7 participants in this study fulfilled three or more features of copd , namely age 40 years , postbronchodilator fev1/fvc ratio < 0.7 , and exposure to cigarette smoke . among patients with copd , we identified those with features of asthma including both history of variable respiratory symptoms and variable expiratory airflow limitation.15 respiratory symptoms included wheeze , shortness of breath , chest tightness , and cough that vary over time and in intensity . expiratory airflow limitation was defined as a postbronchodilator increase in fev1 > 12% and 200 ml from baseline , or increase in fev1 > 12% and 200 ml ( or in peak expiratory flow > 20% ) from baseline after 4 weeks of anti - inflammatory treatment , in the absence of respiratory infections . we evaluated these diagnostic features based on the medical record review prior to biomarker measurement . sociodemographic characteristics , smoking status , presence of rhinitis , and maintenance treatments were recorded for each patient . dyspnea was evaluated by using the modified medical research council dyspnea scale.16 copd - related health status was assessed by using the copd assessment test.17 frequency of severe exacerbations requiring hospitalization in the previous year was evaluated based on direct patient interviews , diaries kept by patients or caregivers , and medical record review . pulmonary function tests were conducted by a well - trained technician following the guidelines under a stable condition.18 severity of airflow limitation was classified in accordance with gold staging.14 feno level was measured by using the niox mino device ( aerocrine , morrisville , nc , usa ) according to the standard operating procedures recommended by the manufacturer . feno level was classified as follows : normal , < 25 parts per billion ( ppb ) ; intermediate , 2550 ppb ; and high , > 50 ppb.19 blood samples were obtained to determine blood eosinophil count and percentage and total serum ige level . presence of antigen - specific ige was determined by using the immunocap phadiatop test ( thermo fisher scientific , waltham , ma , usa ) , an in vitro assay for antigen - specific ige antibodies to common inhalant allergens . antigen - specific ige was considered to be present when the results were positive for at least one of the following : house dust mite , cat , dog , alternaria tenuis , aspergillus fumigatus , or ragweed . the cutoff value of high blood eosinophil count was set at 500 cells / mm,3,20 while the cutoff value of total serum ige level was set at 173 iu / ml according to the reference range of the clinical laboratory at japanese red cross ishinomaki hospital . data are shown as median ( interquartile range ) or mean  standard deviation unless otherwise specified . differences between groups were assessed by using the student s t - test or mann  whitney u - test for continuous variables , while associations between categorical variables were evaluated by using fisher s exact test . the value of total serum ige level was log transformed because the variables were not normally distributed ; the results are expressed as geometric mean values . distribution of gold staging between patients with and without acos was analyzed by using the kruskal receiver operating characteristic ( roc ) curves were plotted in order to estimate the diagnostic cutoff values . all statistical analyses were performed by using ezr ( saitama medical center , jichi medical university , saitama , japan ) , a graphical user interface for r ( the r foundation for statistical computing , vienna , austria).21 
 p - values < 0.05 were considered as significant . we conducted a cross - sectional study to analyze the prospectively collected data of consecutive scheduled visits or newly registered patients from the ishinomaki copd network ( icon ) registry13 between may 2015 and january 2016 . briefly , icon is a regional medical liaison system aimed at comprehensive care of patients with copd and has a multi - center interdisciplinary collaboration with health care providers in ishinomaki , japan . patients registered in icon are regularly treated by general practitioners in ishinomaki and surrounding cities , and receive scheduled examinations and education at the japanese red cross ishinomaki hospital ( a 452-bed tertiary community hospital in ishinomaki ) every 612 months . this study is part of an ongoing copd cohort study registered with the university hospital information network clinical trials registry ( identifier : umin000017376 ) . all patients provided written informed consent , and the study was approved by the ethics committee of japanese red cross ishinomaki hospital ( approval number : 12 - 14 - 1 ) . patients with stable copd , who were aged 4090 years and former smokers with a smoking history of at least 10 pack - years , were included in this study . all patients were diagnosed with copd according to gold criteria.14 persistent airflow limitation , defined as postbronchodilator forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) ratio < 0.7 , was confirmed by using repeated spirometry . exclusion criteria were as follows : current smoker or never smoked , chronic bronchitis or emphysema without airflow limitation , hematologic disease , history of lung resection , use of oral corticosteroids , receiving anti - ige therapy , and exacerbation of copd in the 4 weeks preceding data collection . acos was defined according to the gina / gold joint document.7 participants in this study fulfilled three or more features of copd , namely age 40 years , postbronchodilator fev1/fvc ratio < 0.7 , and exposure to cigarette smoke . among patients with copd , we identified those with features of asthma including both history of variable respiratory symptoms and variable expiratory airflow limitation.15 respiratory symptoms included wheeze , shortness of breath , chest tightness , and cough that vary over time and in intensity . expiratory airflow limitation was defined as a postbronchodilator increase in fev1 > 12% and 200 ml from baseline , or increase in fev1 > 12% and 200 ml ( or in peak expiratory flow > 20% ) from baseline after 4 weeks of anti - inflammatory treatment , in the absence of respiratory infections . we evaluated these diagnostic features based on the medical record review prior to biomarker measurement . sociodemographic characteristics , smoking status , presence of rhinitis , and maintenance treatments were recorded for each patient . dyspnea was evaluated by using the modified medical research council dyspnea scale.16 copd - related health status was assessed by using the copd assessment test.17 frequency of severe exacerbations requiring hospitalization in the previous year was evaluated based on direct patient interviews , diaries kept by patients or caregivers , and medical record review . pulmonary function tests were conducted by a well - trained technician following the guidelines under a stable condition.18 severity of airflow limitation was classified in accordance with gold staging.14 feno level was measured by using the niox mino device ( aerocrine , morrisville , nc , usa ) according to the standard operating procedures recommended by the manufacturer . feno level was classified as follows : normal , < 25 parts per billion ( ppb ) ; intermediate , 2550 ppb ; and high , > 50 ppb.19 blood samples were obtained to determine blood eosinophil count and percentage and total serum ige level . presence of antigen - specific ige was determined by using the immunocap phadiatop test ( thermo fisher scientific , waltham , ma , usa ) , an in vitro assay for antigen - specific ige antibodies to common inhalant allergens . antigen - specific ige was considered to be present when the results were positive for at least one of the following : house dust mite , cat , dog , alternaria tenuis , aspergillus fumigatus , or ragweed . the cutoff value of high blood eosinophil count was set at 500 cells / mm,3,20 while the cutoff value of total serum ige level was set at 173 iu / ml according to the reference range of the clinical laboratory at japanese red cross ishinomaki hospital . data are shown as median ( interquartile range ) or mean  standard deviation unless otherwise specified . differences between groups were assessed by using the student s t - test or mann  whitney u - test for continuous variables , while associations between categorical variables were evaluated by using fisher s exact test . the value of total serum ige level was log transformed because the variables were not normally distributed ; the results are expressed as geometric mean values . distribution of gold staging between patients with and without acos was analyzed by using the kruskal receiver operating characteristic ( roc ) curves were plotted in order to estimate the diagnostic cutoff values . all statistical analyses were performed by using ezr ( saitama medical center , jichi medical university , saitama , japan ) , a graphical user interface for r ( the r foundation for statistical computing , vienna , austria).21 
 p - values < 0.05 were considered as significant . a total of 334 consecutive patients with copd were recruited , and 257 eligible patients ( 236 males , 21 females ; median age , 75 years ) were identified . characteristics of the study participants are shown in table 1 . among the 257 patients with copd , 48 ( 18.7% ) had a history of variable respiratory symptoms , 57 ( 22.2% ) had variable expiratory airflow limitation , and 37 ( 14.4% ) had both features ; these 37 patients were diagnosed as having acos . among the 37 patients with acos , no patient had respiratory symptoms in childhood , but two ( 5.4% ) had symptoms before the age of 40 years . marked reversibility with postbronchodilator increase in fev1 > 12% and 400 ml from baseline was observed in four patients ( 10.8% ) . patients with acos tended to be younger ( p=0.003 ) and have a shorter smoking history ( p=0.019 ) . sex , bmi , spirometric results , dyspnea , copd - related health status , and incidence of severe exacerbations requiring hospitalization in the previous year were not significantly different between the groups . patients with acos tended to use more respiratory medications , especially inhaled corticosteroids ( ics ) and theophylline , compared with patients without acos ( 86.5% vs 36.8% , 27.0% vs 9.5% , respectively ; both p<0.001 ) . mean feno levels were significantly higher in patients with acos than in those without acos ( 38.528.6 ppb vs 20.311.0 ppb , p<0.001 ) ( figure 1a ) . high feno level were significantly present in those with acos , while low feno level were significantly present in those without acos ( table 3 ) . no significant association with feno levels and ics treatment was observed in patients with acos ( p=0.22 ) and without acos ( p=0.55 ) . mean blood eosinophil count and percentage were significantly higher in patients with acos than in those without acos ( 295160/mm vs 212223/mm , p=0.032 ; 4.73.0% vs 3.22.7% , p=0.003 , respectively ) ( figure 1b and c ) . high percentage of blood eosinophils ( > 2% ) was observed in those with acos ( p=0.014 ) ( table 3 ) . no significant association with ics use and blood eosinophil count or percentage was observed in patients with ( p=0.42 and p=0.78 , respectively ) and without acos ( p=0.11 and p=0.61 , respectively ) . total serum ige level was significantly higher in patients with acos than in those without acos ( 2989 iu / ml vs 451 iu / ml , p=0.004 ) ( figure 1d ) . presence of antigen - specific ige also was observed more frequently in those with acos ( 56.8% vs 28.6% , p=0.001 ) . roc curve analysis demonstrated that 23 ppb was the best diagnostic cutoff value of feno level for acos ( area under the curve [ auc ] 0.74 ; 95% confidence interval [ ci ] 0.630.84 ) . sensitivity and specificity of 23 ppb for the diagnosis of acos were 73.0% and 68.2% , respectively . in addition , roc curve analysis showed that 156.2/mm was the best diagnostic cutoff value of eosinophil count ( auc 0.70 ; 95% ci 0.610.78 ) and that 434 iu / ml was the best diagnostic cutoff value of total serum ige level ( auc 0.64 ; 95% ci 0.540.75 ) . sensitivity and specificity of 156.2/mm and 434 iu / ml for the diagnosis of acos were 83.8% and 49.5% , and 45.9% and 85.9% , respectively . combination of feno > 23 ppb and ige > 434 iu / ml showed 94.1% specificity , 37.8% sensitivity , 51.9% positive predictive value ( ppv ) , and 90.0% negative predictive value ( npv ) . combination of feno > 23 ppb and eosinophil count > 156.2/mm showed 85.5% specificity , 59.5% sensitivity , 40.7% ppv , and 92.6% npv . combination of ige > 434 iu / ml and eosinophil count > 156.2/mm showed 92.3% specificity , 40.5% sensitivity , 46.9% ppv , and 90.2% npv . triple combination ( feno > 23 ppb , ige > 434 iu / ml , and eosinophil count > 156.2/mm ) showed 96.8% specificity , 37.8% sensitivity , 66.7% ppv , and 90.3% npv . a total of 334 consecutive patients with copd were recruited , and 257 eligible patients ( 236 males , 21 females ; median age , 75 years ) were identified . characteristics of the study participants are shown in table 1 . among the 257 patients with copd , 48 ( 18.7% ) had a history of variable respiratory symptoms , 57 ( 22.2% ) had variable expiratory airflow limitation , and 37 ( 14.4% ) had both features ; these 37 patients were diagnosed as having acos . among the 37 patients with acos , no patient had respiratory symptoms in childhood , but two ( 5.4% ) had symptoms before the age of 40 years . marked reversibility with postbronchodilator increase in fev1 > 12% and 400 ml from baseline was observed in four patients ( 10.8% ) . patients with acos tended to be younger ( p=0.003 ) and have a shorter smoking history ( p=0.019 ) . sex , bmi , spirometric results , dyspnea , copd - related health status , and incidence of severe exacerbations requiring hospitalization in the previous year were not significantly different between the groups . patients with acos tended to use more respiratory medications , especially inhaled corticosteroids ( ics ) and theophylline , compared with patients without acos ( 86.5% vs 36.8% , 27.0% vs 9.5% , respectively ; both p<0.001 ) . mean feno levels were significantly higher in patients with acos than in those without acos ( 38.528.6 ppb vs 20.311.0 ppb , p<0.001 ) ( figure 1a ) . high feno level were significantly present in those with acos , while low feno level were significantly present in those without acos ( table 3 ) . no significant association with feno levels and ics treatment was observed in patients with acos ( p=0.22 ) and without acos ( p=0.55 ) . mean blood eosinophil count and percentage were significantly higher in patients with acos than in those without acos ( 295160/mm vs 212223/mm , p=0.032 ; 4.73.0% vs 3.22.7% , p=0.003 , respectively ) ( figure 1b and c ) . high percentage of blood eosinophils ( > 2% ) was observed in those with acos ( p=0.014 ) ( table 3 ) . no significant association with ics use and blood eosinophil count or percentage was observed in patients with ( p=0.42 and p=0.78 , respectively ) and without acos ( p=0.11 and p=0.61 , respectively ) . total serum ige level was significantly higher in patients with acos than in those without acos ( 2989 iu / ml vs 451 iu / ml , p=0.004 ) ( figure 1d ) . presence of antigen - specific ige also was observed more frequently in those with acos ( 56.8% vs 28.6% , p=0.001 ) . roc curve analysis demonstrated that 23 ppb was the best diagnostic cutoff value of feno level for acos ( area under the curve [ auc ] 0.74 ; 95% confidence interval [ ci ] 0.630.84 ) . sensitivity and specificity of 23 ppb for the diagnosis of acos were 73.0% and 68.2% , respectively . in addition , roc curve analysis showed that 156.2/mm was the best diagnostic cutoff value of eosinophil count ( auc 0.70 ; 95% ci 0.610.78 ) and that 434 iu / ml was the best diagnostic cutoff value of total serum ige level ( auc 0.64 ; 95% ci 0.540.75 ) . sensitivity and specificity of 156.2/mm and 434 iu / ml for the diagnosis of acos were 83.8% and 49.5% , and 45.9% and 85.9% , respectively . combination of feno > 23 ppb and ige > 434 iu / ml showed 94.1% specificity , 37.8% sensitivity , 51.9% positive predictive value ( ppv ) , and 90.0% negative predictive value ( npv ) . combination of feno > 23 ppb and eosinophil count > 156.2/mm showed 85.5% specificity , 59.5% sensitivity , 40.7% ppv , and 92.6% npv . combination of ige > 434 iu / ml and eosinophil count > 156.2/mm showed 92.3% specificity , 40.5% sensitivity , 46.9% ppv , and 90.2% npv . triple combination ( feno > 23 ppb , ige > 434 iu / ml , and eosinophil count > 156.2/mm ) showed 96.8% specificity , 37.8% sensitivity , 66.7% ppv , and 90.3% npv . we identified patients with acos presenting features of asthma , including both variable respiratory symptoms and variable expiratory airflow limitation , in a copd outpatient cohort , and demonstrated that airway inflammatory biomarkers , including feno , blood eosinophil count , and ige , are increased in those with acos . the results of the present study demonstrated that inflammatory biomarkers can be used to support the diagnosis of acos . previous studies have attempted to identify the acos phenotypes by using various criteria.8 thus , there is an increased awareness of the importance of recognizing acos by using biomarkers.2225 recent studies reported that increased feno levels were identified in a subset of patients with copd.23,24 our findings are congruent with the observation by donohue et al , who reported increased feno levels in patients previously clinically diagnosed with both copd and asthma.23 in addition , the findings of our study are compatible with those of kitaguchi et al , who observed increased blood and sputum eosinophil counts in copd patients with asthmatic symptoms.25 the results of this study indicated that inflammatory biomarkers provide additional diagnostic information for acos . although the sensitivity and specificity of these biomarkers were relatively low , combinations of these biomarkers showed high specificity for acos diagnosis . total serum ige level was not affected by ics therapy ; however , treatment with ics was reported to decrease feno level26,27 and blood eosinophil count.28 half of the patients in this study used ics . furthermore , airway eosinophilia is not exclusive to asthma and is present in patients with copd.29 therefore , we propose that overconfidence in such biomarkers may lead to diagnostic errors . our finding that 14% of patients with copd fulfilled the acos phenotype is in line with the results of well - characterized copd cohorts , such as the copdgene study5 and chain cohort.30 in this study , patients with acos tended to be younger , have a shorter smoking history , and use more respiratory medications , which is compatible with previous studies.46,30,31 however , discrepancies between this and previous studies may have been caused by the older age of the patients in this study , since the participants were enrolled from a regional copd registry in japan , one of the most rapidly aging societies in the world.13 to date , a standardized treatment for acos has not been established because patients with asthma and copd overlap have been excluded from randomized controlled trials . treatment with ics is provisionally recommended by the gina / gold joint document.7 high feno level32 and blood eosinophilia33,34 have been identified as surrogate markers of the response to steroids in patients with copd . the findings of our study support the view that ics treatment may be beneficial in patients with acos and encourage the development of a standardized treatment for acos . the primary strength of our study is that we applied precise diagnostic criteria to identify patients with acos . previous reports included patients with a medical history of physician - diagnosed asthma as the main inclusion criterion . in this study , we confirmed the diagnosis in all patients , with or without features of asthma , according to both asthmatic symptoms and documented reversibility , because we found that some patients had received an inappropriate diagnosis of asthma by a nonrespiratory specialist . in addition , we excluded patients who had characteristics possibly affecting biomarker measurement , including current smokers and oral corticosteroid users . a potential weakness of our study is that the results were not confirmed by a validation cohort . thus , the results can not be generalized directly to a different setting , such as an asthma cohort . further studies are required to evaluate the diagnostic value of inflammatory biomarkers in untreated patients . the results of this study provide evidence that inflammatory biomarkers , including feno , blood eosinophil count , and ige , can be used to support the diagnosis of acos . the results of our study may guide toward better recognition of acos and encourage development of specific interventions for acos .