This was a single center, prospective, proof of concept observational cross-sectional study including one visit. Participants attended the Clinical Pharmacology Unit clinic, Catholic University of the Sacred Heart, University Hospital Agostino Gemelli Foundation IRCCS, Roma, Italy, on one occasion for spirometry, fractional exhaled nitric oxide (F _ENO) measurement, and blood sampling for blood cell count test and measurement of serum total and specific IgE and Ig FLC concentrations. Peripheral blood neutrophil counts and serum C reactive protein (CRP) were measured in persons with severe asthma.

This study was conducted in compliance with the 1975 Declaration of Helsinki, revised in 2004, and approved by the ethics committee of the University Hospital Agostino Gemelli Foundation and Catholic University of the Sacred Heart (approval number P/1034/CE2012). Written consent was obtained from participants.

This study included 54 adults with asthma (24 with severe persistent asthma, 15 with moderate persistent asthma and 15 with mild persistent asthma) and a control group consisting of 20 healthy subjects. Diagnostic criteria for asthma and classification of asthma have been reported elsewhere (Shaw et al., 2015). Asthma severity was defined by the level of pharmacotherapy required for asthma control (Chung et al., 2014; Global Initiative for Asthma, 2023). Persons with severe persistent asthma were being treated with high dose inhaled corticosteroids (ICS) plus long-acting β₂-agonists (LABA) and/or oral corticosteroids; persons with moderate persistent asthma were on maintenance therapy with low to medium dose of ICS plus LABA; persons with mild persistent asthma were steroid-naïve. Ex-smokers had a smoking history > 5 pack-years. Atopy was assessed by measuring serum specific IgE against a panel of common aeroallergens. Subjects with asthma having serum specific IgE concentrations > 1 kU/L against the aeroallergen tested were considered atopic. Participants had no respiratory tract infections in the previous 3 weeks.

Spirometry was performed with a Pony FX spirometer (Cosmed, Rome, Italy) according to standardised international procedures (Miller et al., 2005). At least three acceptable maneuvers were performed with the two best ones having a maximal difference in FEV₁ and forced vital capacity (FVC) of 150 mL. The best of three consecutive maneuvers was chosen. To perform the bronchodilator reversibility test, participants were asked to perform a spirometry, inhale 400 μg of salbutamol and repeat the spirometry after 10 min.

We measured F_ENO using an electrochemical sensor F_ENO analyser (NIOX MINO^®, Aerocrine, Stockholm, Sweden) (Menzies et al., 2007). F_ENO was measured on-line using the single breath method at constant flow of 50 mL/s according to standardised procedures (American Thoracic Society and European Respiratory Society, 2005; Dweik et al., 2011). Measurement of F_ENO was obtained before spirometry.

A whole blood sample was obtained from participants in the study for measurement of serum Ig FLC, serum total and specific IgE concentrations, and blood cell count test. Serum CRP concentrations were measured in severe asthma participants. Blood samples were centrifuged at 4,000 g for 5 min at room temperature and serum aliquots stored at −80°C until the assays were performed.

The nephelometric measurement of FLC was performed using monoclonal antibodies in the N Latex κ and λ FLC kit (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). FLC in the serum samples were assayed in a batch mode on the BNTMII automatic analyzer (Siemens Healthcare Diagnostics Ltd., Erlangen, Germany) according to the instructions provided by the manufacturer.

This method showed high precision, freedom from a high-dose hook effect and good lot-to-lot consistency (Velthuis et al., 2011). Reproducibility of serum FLC measurements ranges from 4% to 7% (Velthuis et al., 2011). Comparisons with Freelite™, an independent analytical method, showed concordance rates for serum κ FLC, λ FLC and κ/λ FLC ratio of 99.2%, 94.2%, and 95%, respectively (Velthuis et al., 2011).

Serum total IgE were measured using ImmunoCAP Total IgE applied on Phadia instrumentation (Thermo Scientific, Uppsala Sweden). Following the Clinical and Laboratory Standards Institute (CLSI) EP28-A3C guidelines (Clinical and Laboratory Standards Institute, 2010), we tested 20 healthy blood donors from the local population and set serum total IgE ≥ 100 lU/L as cut-off positive.

Mesaurement of specific IgE in serum was carried out with the Immulite^® 2000 AlaTOP Allergy Screen chemiluminescence immunoassay (Siemens Medical Solutions-Diagnostics, New Jersey, United States) performed according to the manufacturer instructions.

AlaTOP represents a screening kit for the major inhalant allergens: Dermatophagoides pteronyssinus, cat dander, dog dander, Cynodon dactylon, Phleum pretense L., birtch, Cryptomerica japonica, Penicillium notatum, Alternaria tenuis, Ambrosia artemisifolia L., Plantago lanceolata L., and Parietaria officinalis.

Qualitative results were expressed as signal/cut-off ratio (s/co) and deemed positive if s/co ≥ 1.1 or negative if s/co < 1.1 as follows: s c o = c p s   s a m p l e   o r   c o n t r o l c p s   C a l i b r a t o r   x   P 1

cps = average count per second

There are no data in the literature to inform a power calculation, so the sample size was opportunistic. Statistical analysis was performed using GraphPad Prism 6.0 and MetaboAnalyst 5.0. For continuous data, values are expressed as mean ± SEM or median and interquartile range (25th and 75th percentiles), after assessing for normality with D'Agostino-Pearson omnibus normality test. Depending on data distribution, ANOVA or Kruskal-Wallis test was used for between group comparisons and overall p values are shown. To reduce the chance of type I error, post hoc Tukey’s multiple comparisons test or Fisher’s LSD post hoc test (MetaboAnalyst 5.0 available at https://www.metaboanalyst.ca/) or post hoc Dunn’s multiple comparisons test was used and post hoc test p values are reported. Adjusted p values were calculated by false discovery rate (FDR) at a cut-off value of 0.05. Categorical data are expressed as numbers. Fisher’s exact test was used for comparing two groups; Chi square test was used for comparing more than two groups. Significance was defined as a value of p < 0.05. The correlation matrix and relative p values used for generating the heatmap are provided in the Supplementary Material (correlation table.xlsx and p values correlation table.xlsx files).

We used the Debiased Sparse Partial Correlation (DSPC) network (Basu et al., 2017) to visualise the correlation network. The nodes are input variables, while the edges represent the association measures. For better visualization, the default DSPC network only shows the top correlations (edges) based on their p-value rankings (top 20% when the total number of edges is less than 1000).

Due to the exploratory nature of the study, a formal sample size calculation was not required.

A total of 74 persons 39 women and 35 men, aged 18–82 years, participated in this study, including 24 adults with persistent severe asthma, 15 adults with persistent moderate asthma, 15 adults with persistent mild asthma, and 20 healthy control adults (Table 1). Participants with severe persistent asthma were on maintenance treatment with inhaled fluticasone propionate > 500 μg/day combined with a LABA and/or oral corticosteroids; participants with moderate persistent asthma were on maintenance treatment with inhaled fluticasone propionate ≤ 500 μg/day, 13 out of 15 in combination with a LABA; participants with mild persistent asthma were steroid naive or not being received ICS for at least 3 months.

Participants were matched for gender and smoking habit, but not for age (p < 0.0001) and prevalence of atopy (p = 0.008) (Table 1). Age was elevated in severe asthma adults compared with mild asthma adults (p < 0.0001) and healthy control subjects (p < 0.001), but not compared with moderate asthma adults (Table 1). All adults with mild asthma were atopic (Table 1). The prevalence of atopy was similar across the other study groups (p = 0.65).

Supplementary Table S1 shows important features identified by one-way ANOVA and Fisher’s LSD post hoc test.

There was no difference in serum FLC concentrations in women and men in each study groups (Supplementary Table S2).

Severe asthma adults (15.9 ± 5.5 mg/L, ANOVA p-value = 0.02), but not moderate asthma adults (12.8 ± 3.7 mg/L) showed higher serum κ FLC concentrations than mild asthma adults (12.1 ± 3.7 mg/L, p < 0.05) and healthy control adults (12.3 ± 3.6 mg/L, p < 0.05) (Table 2; Figure 2A). Serum λ FLC concentrations were elevated in severe asthma individuals (22.6 ± 9.7 mg/L, ANOVA p-value = 0.02) compared with healthy control individuals (16.8 ± 4.3 mg/L, p < 0.05) (Table 2; Figure 2B). Compared with mild asthma adults (28.6 ± 9.8 mg/L, p < 0.05) and healthy control individuals (29.1 ± 7.2 mg/L, p < 0.05), the sum of κ and λ FLC concentrations in serum was elevated in severe asthma adults (38.6 ± 14.9 mg/L, ANOVA p-value 0.01), but not in moderate asthma adults (29.8 ± 9.1 mg/L) (Table 2; Figure 2C). κ/λ serum FLC concentration ratio was similar across study groups (Table 2; Figure 2D). Using one-way ANOVA and Fisher’s LSD post hoc test, instead of Tukey’s post hoc test, both types of FLC in serum were significantly elevated in severe asthma adults compared with all the other study groups (κ: f value = 3.6169, p-value = 0.017266, adjusted p values (FDR) = 0.020463; λ: f value = 3.6184, p-value = 0.017235; FDR = 0.020463) (Supplementary Table S1, anova_posthoc.xlsx supplementary file).

Supplementary Figure S1 shows ROC curves for serum κ and λ FLC measurements in severe asthma versus healthy control individuals and severe asthma versus mild asthma individuals. Measurement of serum κ FLC concentrations distinguished severe asthma individuals from either healthy individuals (Supplementary Datasheet S1) or mild asthma individuals (Supplementary Datasheet S2) with an area under the ROC curve (AUROC) of 0.71 for both, with p values of 0.02 and 0.03, respectively (Supplementary Figures S1A, B). Measurement of serum λ FLC concentrations distinguished severe asthma individuals from either healthy individuals (Supplementary Datasheet S3) or mild asthma individuals (Supplementary Datasheet S4) with an area under the ROC curve (AUROC) of 0.66 (p = 0.066) and 0.69 (p = 0.046), respectively (Supplementary Figures S1C, D).

Severe asthma adults with peripheral blood eosinophil cell counts > 300 cells/µL showed higher serum κ (p < 0.001) and λ FLC concentrations (p < 0.01) than severe asthma adults with peripheral blood eosinophil cell counts ≤ 300 cells/µL (Table 3; Figure 3A), whereas serum κ and λ FLC concentrations were similar across study groups with high or low serum total IgE (Table 3; Figure 3B), with or without atopy as measured by serum specific IgE (Table 3; Figure 3C), or high or low F_ENO concentrations (Table 3; Figure 3D).

Adults with severe asthma had lower pre-bronchodilator (Supplementary Figure S2) and post-bronchodilator lung function test values (Supplementary Figure S3) than moderate asthma, mild asthma and healthy control adults (Table 1). Compared with mild asthma and healthy control adults (Table 1), moderate asthma adults showed lower pre-bronchodilator (Supplementary Figure S2) and post-bronchodilator lung function test values (Supplementary Figure S3), Persistent mild asthma and healthy control adults showed similar lung function test values (Table 1, Supplementary Figures S2, S3).

Figure 5 shows the correlations of the study outcomes as a heatmap. Figure 6 shows the correlation network. Serum κ and λ FLC concentrations were highly correlated and correlated with age (Figure 5; Figure 7; Figure 8, Supplementary Figure S4; Supplementary Tables S3, S4; correlation matrix and anova_posthoc Excel files, online supplement). A negative correlation was observed between serum κ and λ FLC concentrations and most pulmonary function testing outcomes (Figure 5; Figure 7; Figure 8, Supplementary Figure S4; Supplementary Tables S3, S4; correlation table and p values correlation table supplementary files).

In adults with persistent severe asthma, serum κ and λ FLC were highly correlated with inflammatory outcomes reflecting T2 high endotype, including peripheral blood eosinophils as percentage of total counts and absolute values, with peripheral blood neutrophils as percentage of total counts and absolute values, and with serum CRP concentrations (Figure 7; Figure 8, Supplementary Figure S4, Supplementary Tables S3, S4; correlation table and p values correlation table supplementary files). By contrast, serum FLC concentrations were not correlated with other surrogate markers reflecting T2 high airway inflammation, including F_ENO, serum total IgE or specific IgE (Supplementary Tables S3, S4; correlation table and p values correlation table supplementary files). Correlations of κ FLC or λ FLC serum concentrations with peripheral blood lymphocytes and monocytes are shown in Supplementary Figures S5, S6, respectively. Linear regression analyses of κ FLC or λ FLC serum concentrations and peripheral blood lymphocytes and monocytes are shown in Supplementary Figures S7, S8, respectively. Serum FLC concentrations were correlated with peripheral blood lymphocyte absolute values (κ FLC: Pearson r = 0.45, p < 0.05, n = 23 (Supplementary Figure S5A); λ FLC: Pearson r = 0.53, p < 0.05, n = 23 (Figure 6A), but not with lymphocytes as percentage of total cell counts (Supplementary Figures S5B, S6B), monocyte absolute values (Supplementary Figures S5C, S6C) or monocytes as percentage of total cell count (Supplementary Figures S5D, S6D).