Different Relationships between F_ENO and COPD Characteristics in Smokers and Ex-Smokers

Abstract

Exhaled nitric oxide (F_ENO) is a marker of type-2 inflammation in asthma and is used in its management. However, smokers and ex-smokers have lower F_ENO values, and the clinical use of F_ENO values in COPD patients is unclear. Therefore, we investigated if F_ENO had a relationship to different COPD characteristics in smoking and ex-smoking subjects. Patients with COPD (n = 533, 58% females) were investigated while in stable condition. Measurements of F_ENO₅₀, blood cell counts, IgE sensitisation and lung function were performed. Medication reconciliation was used to establish medication usage. Smokers (n = 150) had lower F_ENO₅₀ 9 (8, 10) ppb (geometric mean, 95% confidence interval) than ex-smokers did (n = 383) 15 (14, 16) ppb, p < 0.001. F_ENO₅₀ was not associated with blood eosinophil or neutrophil levels in smokers, but in ex-smokers significant associations were found (r = 0.23, p < 0.001) and (r = –0.18, p = 0.001), respectively. Lower F_ENO values were associated with lower FEV₁% predicted in both smokers (r = 0.17, p = 0.040) and ex-smokers (r = 0.20, p < 0.001). Neither the smokers nor ex-smokers with reported asthma or IgE sensitisation were linked to an increase in F_ENO₅₀. Ex-smokers treated with inhaled corticosteroids (ICS) had lower F_ENO₅₀ 14 (13, 15) ppb than non-treated ex-smokers 17 (15, 19) ppb, p = 0.024. This was not found in smokers (p = 0.325). F_ENO is associated with eosinophil inflammation and the use of ICS in ex-smoking COPD subjects, but not in smoking subjects suggesting that the value of F_ENO as an inflammatory marker is more limited in smoking subjects. The association found between low F_ENO values and low lung function requires further investigation.

Keywords:

• Exhaled NO
• asthma and COPD
• inflammatory mediators
• lung function

Introduction

Exhaled nitric oxide (F_ENO) is a marker of type-2 inflammation [1, 2] and it has a role in asthma management. It can be used to titrate anti-inflammatory medication, identify individuals at risk for exacerbation [3, 4] and study the effects of new biological treatments affecting the IL-13 pathway [5]. A high F_ENO of more than 50 ppb, measured at an exhalation flow of 50 mL/s (F_ENO₅₀) [6, 7], can be used to indicate the possible presence of type-2 inflammation in symptomatic asthmatic patients and the likely location of the response to inhaled corticosteroids (ICS) [8]. The clinical use of F_ENO in stable COPD patients is unclear as summarised by a recent scoping review [9]. Finding a relevant F_ENO cut-off level for determining the use of ICS in COPD is challenging since many studies have not reported on the smoking status, which lowers F_ENO levels in both the general population and the asthma and COPD population [10–12]. In GOLD 2019, https://goldcopd.org, ICS is recommended to group D patients, or those with an eosinophil blood count ≥300 cells/μL. In COPD, the F_ENO value during an exacerbation can be elevated and correlates well to the eosinophil count [13]. However, ICS treatment has been reported to reduce F_ENO in COPD subjects [14], but the effect does not appear to be dose dependent.

Asthma is a relatively common comorbidity in COPD, which is recognised as asthma-COPD overlap (ACO). According to recent publications, both increased F_ENO values [13, 15, 16], and non-increased F_ENO values [17] are reported in ACO. IgE sensitisation is an important determinant of F_ENO₅₀ both in population-based studies [18] and among subjects with asthma [19], yet it has been sparsely studied in COPD [20].

In a Swedish study, Tools Identifying Exacerbations in COPD (TIE-study), stable COPD subjects were investigated. The present objective was to explore the role of F_ENO among the smokers and ex-smokers with COPD, and correlate the F_ENO to COPD characteristics and inflammatory markers.

Material and methods

Study design and subjects

The study subjects were recruited for the multi-disciplinary, prospective TIE-study from the central Swedish regions of Dalarna, Gävleborg and Uppsala [21]. Patients from primary and secondary care settings with a diagnosis of COPD (ICD code J44.0, J44.1, J44.8 and J44.9) were included. The present analysis is based on the data acquired from the inclusion visits made between September 2014 and September 2016. The Regional Review Board in Uppsala, Sweden (Dnr 2013/358) approved the study on 28 April 2014. All subjects gave written informed consent.

Methods

The COPD diagnosis was confirmed with spirometry using the post-bronchodilator (400 µg salbutamol) forced expiratory volume measured in the 1 second (FEV₁) divided by the highest vital capacity (VC) value from a slow (SVC) or forced manoeuvre (FVC) with a ratio of <0.70 (SpiroPerfect spirometer, Welch Allyn, Skaneateles Falls, NY, USA or Jaeger Masterscreen PFT, Erich Jaeger GmbH, Würzburg, Germany). FEV₁ and FVC are presented as a predicted percent using Swedish reference values [22, 23]. Height and weight were taken at the visit to calculate the body mass index (BMI).

Exhaled nitric oxide (NO) at an exhalation flow of 50 mL s⁻¹ (F_ENO₅₀) was measured at the different study locations with NIOX VERO (Circassia AB, Solna, Sweden), NIOX Flex (Aerocrine AB, Solna, Sweden) or Eco Medicks CLD 88 (Eco Medicks, Duernten, Switzerland). The measurements were performed in accordance with current ATS/ERS 2005 recommendations for NO measurements. Blood cell counts analysing neutrophils (B-Neu) and eosinophils (B-Eos) were performed (Cell-Dyn 4000, Abbott, Abbott Park, IL, USA and Sysmex XN-10, Sysmex America Inc., Lincoinshire, IL, USA).

Questionnaires including the COPD Assessment Test (CAT) and modified Medical Research Council Scale (mMRC) were used together with questions regarding subject demographics, smoking habits, contacts with the healthcare system and medication use. Having ever asthma was self-reported. The research nurse reviewed the answers to the questionnaires with the subjects. Medication reconciliation was used to establish pharmacological treatment. When a subject did not answer the questions regarding smoking habits in the questionnaire (n = 2), measurements of exhaled carbon monoxide were used to define their smoking status (Smoke Check, Intramedic AB, Sollentuna, Sweden).

The presence of IgE sensitisation was assessed with a multi-allergen test against a mix of aeroallergens (birch, timothy, mugwort, Dermatophagoides pteronyssinus, Dermatophagoides farinae, Cladosporium herbarum, cat, dog, and horse) using the ImmunoCAP system (Immunodiagnostics, Thermo Fischer Scientific, Uppsala, Sweden). A subject was considered sensitised if IgE levels to the Phadiatop were ≥0.35 kUA/L.

Analysis

To classify the COPD subjects with regard to severity of the disease, the GOLD risk assessment version 2017 A-D with symptoms (CAT scale) and number of exacerbations/hospitalizations was used. An exacerbation was defined as an unscheduled health care visit, and/or a course of oral corticosteroids and/or a course of antibiotics due to COPD deterioration (questionnaire assessed). Frequent exacerbations were defined as having at least two exacerbations and/or at least one hospital admittance (information retrieved from hospital records) due to a COPD exacerbation during the previous year.

The analyses were performed separately for current smokers and ex-smokers. There are no established cut-off values for elevated F_ENO₅₀ in COPD, as opposed to asthma [8]. Therefore, to study the clinical value of F_ENO₅₀ in COPD and the influence of current smoking, the ex-smokers and smokers were divided into three groups or tertiles of about equal numbers and given the labels low, intermediate and high F_ENO₅₀. Log transformation was performed on data with a skewed distribution. Data in the results are given as n (%), mean ± SD and geometric mean (CI_95% lower and upper limit). For the statistical calculations Binominal test, Pearson χ²-test and non-parametric test, i.e. Mann–Whitney U test, Kruskal-Wallis test, Spearman’s rho and a post hoc test Tukey HSD, (SPSS, v. 24 for Windows, SPSS Inc., Chicago, MI, USA) were used. A p-value of <0.05 was considered significant. In the boxplots the horizontal line in each box corresponds to the median value, the upper and lower margins correspond to the 25th and 75th percentiles, and the whiskers correspond to the 10th and 90th percentiles.

Results

F_ENO₅₀ was measured in 533 subjects (58% female), 13 (12, 14) ppb. Women had lower F_ENO₅₀ values 12 (11, 13) ppb than men 15 (13, 16) ppb, p = 0.001. Smokers (n = 150, 65% female) had lower F_ENO₅₀ values 9 (8, 10) ppb than ex-smokers (n = 383, 55% female) 15 (14, 16) ppb, p < 0.001. Smoking women had lower F_ENO₅₀ than smoking men. In addition, ex-smoking women had lower F_ENO₅₀ than ex-smoking men (Figure 1).

Figure 1. The F_ENO₅₀ values in ex-smokers and smokers according to sex. Females had lower F_ENO₅₀ values than men did.

A lower F_ENO₅₀ was associated with lower lung function. In smokers there were correlations between F_ENO₅₀ and FEV₁% predicted (r = 0.17, p = 0.04) and FVC% predicted (r = 0.22, p = 0.007). In ex-smokers corresponding values were r = 0.20, p < 0.001 and r = 0.17, p = 0.001. The lung function values found in the different F_ENO₅₀ groups of smokers and ex-smokers are presented in Tables 1 and 2.

Table 1. Characteristics of the ex-smoking subjects divided into tertiles with regard to F_ENO₅₀.

	Low FENO50 <11.5 ppb (n = 126)	Intermediate FENO50 ≥11.5– ≤19 ppb (n = 129)	High FENO50 >19 ppb (n = 128)	p-value
Females, n (%)	76 (60)	72 (56)	64 (50)	0.253
Age (years)	70 ± 7	69 ± 8	70 ± 6	0.682
BMI	26 ± 5	28 ± 5	27 ± 5	0.131
Lung function
FEV1 % predicted	53 ± 17^a	57 ± 17	60 ± 19	0.005
FVC % predicted	67 ± 16^a	70 ± 16	73 ± 20	0.024
FEV1/FVC	0.49 ± 0.12	0.52 ± 0.11	0.51 ± 0.12	0.109
Asthma and IgE sensitisation
Asthma	47 (37%)	43 (33%)	41 (32)	0.606
IgE sensitised	5 (8%)	16 (21%)	19 (21%)	0.068
Inflammatory markers
B-Neu 10^9/L	4.9 (4.6, 5.2)^b	4.4 (4.1, 4.6)	4.2 (4.0, 4.5)	0.006
B-Eos 10^9/L	0.15 (0.13, 0.17)	0.16 (0.14, 0.18)	0.20 (0.18, 0.23)^c	0.003
Symptom burden
CAT $\ge$ 10	78 (62%)	82 (64%)	77 (60%)	0.854
mMRC $\ge$ 2	64 (51%)	68 (53%)	52 (41%)	0.115
Exacerbations
Frequent exacerbations	36 (29%)	31 (24%)	33 (26%)	0.708
Hospital admissions	12 (10%)	8 (6%)	6 (5%)	0.293
Treatments
ICS alone or in combination	87 (69%)	78 (61%)	73 (57%)	0.127
GOLD 2017
A/B/C/D	29/43/9/19%	33/43/3/21%	34/42/6/18%	0.623

Data given as n (%), mean ± SD, geometrical mean (CI_95%). IgE sensitisation performed in 227 subjects. p-values derived by Kruskal-Wallis test.

a Post hoc test Tukey HSD: low F_ENO₅₀ vs. high F_ENO₅₀.

b Post hoc test Tukey HSD: low F_ENO₅₀ vs. both intermediate and high F_ENO₅₀.

c Post hoc test Tukey HSD: high F_ENO₅₀ vs. both low p = 0.002 and intermediate F_ENO₅₀.

BMI, body mass index; FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; B-Eos, eosinophil blood count; B-Neu, neutrophil blood count; CAT, COPD assessment test; mMRC, modified British Medical Research Council; ICS, inhaled corticosteroids.

Table 2. Characteristics of the smoking subjects divided into tertiles with regard to F_ENO₅₀.

	Low FENO50 <7 ppb (n = 48)	Intermediate FENO50 ≥$7$–11 ppb (n = 51)	High FENO50 >11 ppb (n = 51)	p-value
Females, n (%)	38 (77)^a	34 (67)	26 (51)	0.023
Age (years)	65 ± 10	64 ± 8	66 ± 6	0.210
BMI	24 ± 4	25 ± 5	26 ± 4	0.053
Lung function
FEV1 % predicted	53 ± 15^a	62 ± 17	61 ± 17	0.020
FVC % predicted	67 ± 18	72 ± 15	75 ± 16	0.028
FEV1/FVC	0.48 ± 0.11^b	0.54 ± 0.12	0.52 ± 0.11	0.020
Asthma and IgE sensitisation
Asthma	17 (35%)	13 (26%)	20 (39%)	0.317
IgE sensitised	5 (10%)	6 (12%)	6 (12%)	0.731
Inflammatory markers
B-Neu 10^9/L	4.7 (4.3, 5.1)	4.9 (4.5, 5.4)	4.9 (4.5, 5.3)	0.924
B-Eos 10^9/L	0.14 (0.11, 0.17)	0.19 (0.16, 0.23)	0.19 (0.16, 0.24)	0.066
Symptom burden
CAT $\ge$ 10	32 (67%)	30 (59%)	27 (53%)	0.379
mMRC $\ge$ 2	21 (44%)	11 (22%)	17 (33%)	0.062
Exacerbations
Frequent exacerbations	15 (31%)	9 (18%)	13 (26%)	0.288
Hospital admissions	5 (10%)	3 (6%)	3 (6%)	0.610
Treatments
ICS alone or in combination	28 (52%)	21 (46%)	24 (48%)	0.164
GOLD 2017
A/B/C/D	22/44/8/26%	41/42/4/13%	46/32/2/20%	0.226

Data given as n (%), mean ± SD, geometrical mean (CI_95%). IgE sensitisation preformed in 82 subjects. p-values derived by Kruskal-Wallis test.

a Post hoc test Tukey HSD: low F_ENO₅₀ vs. high F_ENO₅₀ group.

b Post hoc test Tukey HSD: low F_ENO₅₀ vs. intermediate F_ENO₅₀ group.

BMI, body mass index; FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; B-Eos, eosinophil blood count; B-Neu, neutrophil blood count; CAT, COPD assessment test; mMRC, modified British Medical Research Council; ICS, inhaled corticosteroids.

Subjects reported having asthma, smokers 33% and ex-smokers 34%, had no significant difference in F_ENO₅₀ values from those who did not report having asthma. IgE sensitisation was analysed in subjects (n = 309) from two of the regions. A positive IgE test was found in 21% of the smokers and in 18% in ex-smokers. There were no significant differences in the F_ENO₅₀ values for the IgE sensitised and non-sensitised subjects within the smoking and ex-smoking groups (Figure 2; Tables 1 and 2). There was a positive association between F_ENO₅₀ and B-Eos (r = 0.23, p < 0.001) and a negative association between F_ENO₅₀ and B-Neu (r = –0.18, p = 0.001) in the ex-smokers. In smokers there were no statistically significant associations found. Values of the inflammatory markers are presented for the different F_ENO₅₀ group in Tables 1 and 2.

Figure 2. The F_ENO₅₀ values in ex-smokers and smokers according to positive or negative IgE sensitisation (defined as ≥0.35 kUA/L). No significant difference was found.

Smokers had a lower usage of ICS (49%) compared to the ex-smokers (62%), p = 0.005. Ex-smokers treated with ICS had lower F_ENO₅₀ than ex-smokers not treated with ICS (Figure 3). This F_ENO₅₀ difference with ICS treatment or non-treatment was not seen in the smokers. There were no differences in ICS usage when the smokers and ex-smokers were divided into the low, intermediate and high F_ENO groups (Tables 1 and 2).

Figure 3. The F_ENO₅₀ values in ex-smokers and smokers according to usage of ICS. Ex-smokers had a significantly lower F_ENO₅₀ value statistically when treated with ICS, while no difference was found in smokers.

A CAT score ≥ 10 was found in 59% of the smokers and 62% of the ex-smokers (p = 0.588). A value of ≥ 2 on the mMRC scale was present in 33% of the smokers and 48% of the ex-smokers (p = 0.001).

Subjects who had four or more exacerbations during the year before inclusion (3 smokers and 20 ex-smokers) had a F_ENO₅₀ of 13 (4, 43) and 10 (7, 14) ppb, respectively. Those with three exacerbations (8 smokers and 13 ex-smokers) had a F_ENO₅₀ of 11 (8, 16) and 11 (8, 15) ppb, respectively. Those with one or two exacerbations (49 smokers and 99 ex-smokers) had a F_ENO₅₀ of 9 (8, 11) and 16 (14, 18) ppb, respectively; and subjects with zero exacerbations (90 smokers and 251 ex-smokers) had a F_ENO₅₀ of 9 (8,10) and 15 (14, 17) ppb, respectively. Accordingly, in smokers there was no significant difference in F_ENO₅₀ (p = 0.469), but ex-smokers did show a decrease in F_ENO₅₀ when the exacerbations increased (p = 0.008).

Subjects who had at least one hospital admission the year before inclusion (11 smokers and 26 ex-smokers) had a F_ENO₅₀ of 8 (5, 12) and 12 (9, 15) ppb, respectively; and subjects with zero hospital admissions (139 smokers and 357 ex-smokers) had 9 (8, 10) and 15 (14, 16) ppb, respectively. Thus, in smokers there was no statistical difference in F_ENO₅₀ levels (p = 0.521) nor was there a difference found in the ex-smokers (p = 0.104).

A sensitivity analysis was performed to confirmed our findings by using the 5th percentile of reference population as the lower limit of normality (LLN). Hence, a COPD diagnosis set by FEV₁/VC-ratio < LLN was found in 432 subjects, among ex-smokers 305 subjects and smokers 127 subjects. Ex-smokers had a significant lower F_ENO₅₀ when on ICS (p = 0.01) while no difference was found in smokers (p = 0.30). Regarding IgE sensitisation there was no significant difference in F_ENO₅₀ in ex-smokers and smokers (p = 0.46 resp. p = 0.64).

Discussion

This multicentre study in COPD has shown that smokers have much lower F_ENO₅₀ levels than ex-smokers, and the relationship between F_ENO₅₀ and the inflammatory markers, such as blood eosinophil and blood neutrophil counts, was only found in the ex-smokers. Similarly, the relationship of lower F_ENO₅₀ and ICS usage was only found in the ex-smokers. A low F_ENO₅₀ was associated with a lower lung function in both the smokers and ex-smokers. Neither a reported history of asthma nor IgE sensitisation was shown to have a relationship with F_ENO₅₀.

The interesting finding from this study is that there were no associations found between the exhaled NO and the eosinophil and neutrophil blood counts in the current smokers with COPD. However, there was a positive correlation of F_ENO₅₀ with the eosinophil blood count and a negative correlation with the neutrophil blood count found in the ex-smokers. F_ENO is known to correlate with eosinophils in many asthma studies [24–26]. As far as we know, relationships of this kind have not been reported in COPD patients in stable condition, but a positive relationship between F_ENO₅₀ and blood eosinophil levels has been found in COPD patients experiencing exacerbations [13, 27]. Additionally, there was no difference in the F_ENO values of current smokers with or without ICS treatment. As this study is cross-sectional, we cannot infer that ICS treatment will not reduce F_ENO levels in our smokers, which is suggested by current data concerning the impaired action of ICS in smokers [28–30].

A low F_ENO was associated with low lung function in this study. This has been reported in subjects with cystic fibrosis [31] where a negative correlation was also found with neutrophils, which is similar to our findings. A reduction of NO by superoxide released from neutrophils has been presented as an alternative explanation for the low F_ENO [32]. Cystic fibrosis and primary ciliary dyskinesia (two diseases with low levels of F_ENO) and COPD share some common features such as chronic bacterial colonisation and repeated inflammatory periods leading to lung function decline [33, 34]. NO has many biological functions in the respiratory system. It is bacteriostatic, it halts viral replication and eliminates various pathogens [35]. It has also been suggested that the antiproliferative effects of NO on airway smooth muscle might become an important clue in the quest for future strategies to prevent airway remodelling [36]. In this study, 25% of the subjects had F_ENO₅₀ values under 8 ppb which can be compared to the lower normal limit for healthy subjects in the same age group [37]. Even though the lower level of F_ENO₅₀ did not reach statistical significance in smokers, we did note that ex-smokers with frequent exacerbations had significantly lower F_ENO₅₀, and when they were hospitalised for exacerbations their F_ENO₅₀ was more likely to be on the low side. Everything considered, a functional level of NO in the airways is a necessity.

One third of our COPD subjects reported having asthma, but only one fifth had atopy. In the reported subjects, there was no significant difference in the F_ENO₅₀ values of the IgE sensitised and non-sensitised subjects among the smokers or ex-smokers. There are few studies on allergic sensitisation to aeroallergens among COPD subjects. A recent Polish study reports that one third of the investigated COPD subjects had a positive skin prick test to at least one of the investigated aeroallergens [38]. In a recent Japanese study, based on the definition of total IgE above 173 kU/L, one third of the COPD subjects were considered atopic [39]. Kobayashi et al. identified participants with ACO as variable respiratory symptoms and variable expiratory airflow limitation and they found that about half of the ACO participants had a total serum IgE level above 173 kU/L [20]. The differences in prevalence of IgE sensitisation in our study might be due to different methodologies used to assess IgE sensitisation, type of patients included in the study and regional differences. Our study has tried to relate the presence of IgE aeroallergen sensitisation to the levels of exhaled NO. We were unable to find this relationship in COPD, as opposed to studies with healthy [18] and asthmatic subjects [19]. Accordingly, allergic sensitisation appears to be less important in the interpretation of F_ENO levels in COPD subjects.

A self-reported asthma diagnosis, not physician diagnosed, was found in about one third of the subjects, and this was not related to F_ENO values in the present material. There are divergent results reported in the literature regarding levels of F_ENO in subjects with both COPD and asthma, and the definitions of asthma-COPD overlap used in these studies are also heterogeneous.

In this study, women had lower F_ENO₅₀ values than men did, and this was the case for both smokers and ex-smokers. This differs from what was reported for the same age group in one study of healthy individuals [37], but is in line with results from other studies [40, 41]. The F_ENO₅₀ values in our study are slightly lower than values from healthy individuals, which is a known effect of smoking [10].

The strength of the present study is in the large number of patients investigated. Another strength comes from the inclusion of subjects with different levels of care and disease severity, which leads to a higher external validity of the present findings. However, the study does have limitations that stem from the fact that the subjects already had their diagnoses and had already received treatment. This means that we cannot answer the question regarding the value of F_ENO in predicting the response to ICS in ICS-naïve subjects. Furthermore, the cross-sectional analysis does not allow us to study the relationship that low F_ENO has to lung function decline. This study has used both electrochemical sensors and chemiluminescence technique to measure F_ENO and the difference between them are considered by most users to be clinically acceptable [42].

In conclusion, smoking decreases F_ENO in COPD subjects and the value of F_ENO as inflammatory marker appears to be limited in current smokers. Higher levels of F_ENO in our COPD subjects did not relate to the presence of reported asthma and IgE sensitisation. Ex-smokers that had frequent exacerbations had low F_ENO when in a stable condition. This is perhaps what we must be observant for and mindful of in clinical practice. In many COPD studies smokers are analysed together with ex-smokers. This study has shown that smokers need to be analysed separately from ex-smokers. Future studies are needed to investigate if the lower F_ENO values, seen in subjects with poor lung function, can be used to identify individuals at risk for rapid lung function decline.

Abbreviations
ACO	=	asthma – COPD overlap
B-Eos	=	eosinophil blood count
B-Neu	=	neutrophil blood count
BMI	=	body mass index
CAT	=	COPD assessment test
COPD	=	chronic obstructive pulmonary disease
FENO50	=	fraction of exhaled nitric oxide at the flow of 50 mL/s
FEV1	=	forced expiratory volume in 1 second
FVC	=	forced vital capacity
GOLD	=	Global Initiative for Chronic Obstructive Lung Disease
ICS	=	inhaled corticosteroids
mMRC	=	modified British Medical Research Council
NO	=	nitric oxide
SVC	=	slow vital capacity
TIE-study	=	Tools Identifying Exacerbations in COPD
VC	=	vital capacity

Acknowledgments

The authors are grateful to the research nurses/technicians at the three study sites: Dalarna, Gävle and Uppsala. The authors wish to thank Robin Quell for proofreading and editing this manuscript.

Additional information

Funding

This study was funded by the Uppsala-Örebro Regional Research Council; Centre for Research & Development, Uppsala University/Region Gävleborg; Centre for Clinical Research, Uppsala University; County Council Dalarna; The Swedish Heart-Lung Foundation; and The Swedish Heart and Lung Association.

Notes on contributors

M. Högman

MH, BS, KL, KB, CJ and AM participated in the study design and together with AT collected and analysed the data. MH prepared the initial draft. All authors have discussed the draft, and have read and approved the final manuscript.

A. Thornadtsson

MH, BS, KL, KB, CJ and AM participated in the study design and together with AT collected and analysed the data. MH prepared the initial draft. All authors have discussed the draft, and have read and approved the final manuscript.

K. Bröms

MH, BS, KL, KB, CJ and AM participated in the study design and together with AT collected and analysed the data. MH prepared the initial draft. All authors have discussed the draft, and have read and approved the final manuscript.

C. Janson

MH, BS, KL, KB, CJ and AM participated in the study design and together with AT collected and analysed the data. MH prepared the initial draft. All authors have discussed the draft, and have read and approved the final manuscript.

K. Lisspers

MH, BS, KL, KB, CJ and AM participated in the study design and together with AT collected and analysed the data. MH prepared the initial draft. All authors have discussed the draft, and have read and approved the final manuscript.

B. Ställberg

MH, BS, KL, KB, CJ and AM participated in the study design and together with AT collected and analysed the data. MH prepared the initial draft. All authors have discussed the draft, and have read and approved the final manuscript.

H. Hedenström

MH, BS, KL, KB, CJ and AM participated in the study design and together with AT collected and analysed the data. MH prepared the initial draft. All authors have discussed the draft, and have read and approved the final manuscript.

A. Malinovschi

MH, BS, KL, KB, CJ and AM participated in the study design and together with AT collected and analysed the data. MH prepared the initial draft. All authors have discussed the draft, and have read and approved the final manuscript.