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Introduction: Type 2 targeting biologics have reached the market first for asthma and since 2019 also for CRSwNP. As clear guidelines and predictors for optimal biological choice are missing, patients are sometimes required to switch biologic therapy in order to find the optimal treatment result. In this paper, we evaluate reasons for switching biologics and the treatment effects after each sequential switch. Materials and methods: Ninety-four patients who switched from one biologic to another for their treatment of CRSwNP and asthma were evaluated. Results: Twenty patients experienced satisfactory control of CRSwNP, but insufficient control of severe asthma. Fifty-one patients experienced satisfactory control of severe asthma, but insufficient control of CRSwNP/EOM. Twenty-eight patients experienced insufficient control of both upper and lower airways. Thirteen patients had to switch because of side effects. Furthermore, two cases are described to clarify clinical decision-making. Discussion: For abovementioned patients, a multidisciplinary approach is mandatory to find the best suitable biologic. It seems ineffective to switch to a second anti-IL5 treatment if the first one is not successful. Most patients that failed omalizumab and/or an anti-IL-5 treatment are well controlled on dupilumab. Therefore, we suggest to use dupilumab as first choice when switching biologic agents.

Multicolor Flow Cytometry (MFC)-based gating allows the selection of cellular (pheno)types based on their unique marker expression. Current manual gating practice is highly subjective and may remove relevant information to preclude discovery of cell populations with specific co-expression of multiple markers. Only multivariate approaches can extract such aspects of cell variability from multi-dimensional MFC data. We describe the novel method ECLIPSE (Elimination of Cells Lying in Patterns Similar to Endogeneity) to identify and characterize aberrant cells present in individuals out of homeostasis. ECLIPSE combines dimensionality reduction by Simultaneous Component Analysis with Kernel Density Estimates. A Difference between Densities (DbD) is used to eliminate cells in responder samples that overlap in marker expression with cells of controls. Thereby, subsequent data analyses focus on the immune response-specific cells, leading to more informative and focused models. To prove the power of ECLIPSE, we applied the method to study two distinct datasets: the in vivo neutrophil response induced by systemic endotoxin challenge and in studying the heterogeneous immune-response of asthmatics. ECLIPSE described the well-characterized common response in the LPS challenge insightfully, while identifying slight differences between responders. Also, ECLIPSE enabled characterization of the immune response associated to asthma, where the co-expressions between all markers were used to stratify patients according to disease-specific cell profiles.

Upon activation granulocytes upregulate several adhesion molecules (CD11b) and granule proteins (CD35, CD66b) and shed surface l-selectin (CD62L). These changes in expression, as assessed by flow cytometry, can be used as markers for activation. Whereas these markers are usually studied in fresh blood samples, a new method is required when samples are collected at a field site with no direct access to a flow cytometer. Therefore, we developed and tested a field-applicable method in which fixed leukocytes were cryopreserved. Using this method, the intensity of granulocyte activation markers was compared to samples that were either stained fresh, or fixed prior to staining but not cryopreserved. In addition, the response to an in vitro stimulation with fMLF was determined. While we observed differences in marker intensities when comparing fresh and fixed granulocytes, similar intensities were found between fixed cells that had been cryopreserved and fixed cells that did not undergo cryopreservation. Although fixation using FACS lysing solution might lead to membrane permeabilization, activation markers, and the responsiveness to fMLF or eotaxin could still be clearly measured. This method will, therefore, enable future studies of granulocyte activation in settings with limited resources and will allow simultaneous analysis of samples collected at different time points. © 2018 International Society for Advancement of Cytometry.

Flow Cytometry is an analytical technology to simultaneously measure multiple markers per single cell. Ten thousands to millions of single cells can be measured per sample and each sample may contain a different number of cells. All samples may be bundled together, leading to a ‘multi-set’ structure. Many multivariate methods have been developed for Flow Cytometry data but none of them considers this structure in their quantitative handling of the data. The standard pre-processing used by existing multivariate methods provides models mainly influenced by the samples with more cells, while such a model should provide a balanced view of the biomedical information within all measurements. We propose an alternative ‘multi-set’ preprocessing that corrects for the difference in number of cells measured, balancing the relative importance of each multi-cell sample in the data while using all data collected from these expensive analyses. Moreover, one case example shows how multi-set pre-processing may benefit removal of undesired measurement-to-measurement variability and another where class-based multi-set pre-processing enhances the studied response upon comparison to the control reference samples. Our results show that adjusting data analysis algorithms to consider this multi-set structure may greatly benefit immunological insight and classification performance of Flow Cytometry data.

Background: Severe asthma with persistent airway eosinophilia despite high-intensity inhaled corticosteroids is a phenotype associated with high exacerbation risk. A major barrier to progress is poor understanding of the pathogenic mechanisms and cell types contributing to the disease. Aims and objectives: To determine the function of type-2 cytokine producing CD8+ T cells in severe eosinophilic asthma, and investigate their contribution to pathogenesis of the disease. Methods: Profiles of type-2 CD8+ T cells (Tc2) and type-2 T helper (Th2), in different asthma phenotypes were analysed with flow cytometry. Tc2 cells were isolated and cultured in vitro. Effects of prostaglandin D2 (PGD2) and leukotriene E4 (LTE4) on Tc2 cells were defined using chemotaxis assays, Luminex, quantitative RT-PCR, PrimeFlow RNA assay and microarray. Effects of Tc2 supernatants were determined on eosinophil shape change and apoptosis, and on eosinophil chemokine production from airway epithelial cells. Results: In two independent patient cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8+CRTH2+ Tc2 cells are enriched in blood and airways, and concentrations of prostaglandin D2 (PGD2) and cysteinyl leukotriene E4 (LTE4) are also increased in the airways in severe eosinophilic asthma. In vitro PGD2 and LTE4 function synergistically to enhance Tc2 cell recruitment and activation. They regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines which could contribute to eosinophilia. Conclusions: These finding are consistent with an important role for human Tc2 cells in severe eosinophilic asthma.

Endurance exercise is associated with a transient increase in neutrophil counts in the peripheral blood. Here we investigate the impact of intensified endurance exercise on the neutrophil compartment. We hypothesized that intensified endurance exercise leads to mobilization of neutrophil subsets, which are normally absent in the blood. Furthermore, we followed the potential build-up of neutrophil activation and the impact on overnight recovery of the neutrophil compartment during a seven-day cycling tour. The neutrophil compartment was studied in 28 healthy amateur cyclists participating in an eight-day strenuous cycling tour. Blood samples were taken at baseline, after 4 days and after 7 days of cycling. The neutrophil compartment was analyzed in terms of numbers and its phenotype by deep phenotyping of flow cytometry data with the multi-dimensional analysis method FLOOD. Repeated endurance exercise led to a gradual increase in total neutrophil counts over the days leading to a 1.26 fold-increase (95%CI 1.01-1.51 p = 0.0431) in the morning of day 8. Flow cytometric measurements revealed the appearance of 2 additional neutrophil subsets: CD16brightCD62Ldim and CD16dimCD62Lbright. A complex change in neutrophil phenotypes was present characterized by decreased expression of both CD11b and CD62L and marked increased expression of LAIR-1, VLA-4 and CBRM1/5. The changes in expression were found on all neutrophils present in the blood. Strikingly, in strong contrast to our findings during acute inflammation evoked by LPS challenge, these neutrophils did not upregulate classical degranulation markers. In fact, our FLOOD analysis revealed that the exercise induced neutrophil phenotype did not overlap with the neutrophil subsets arising upon acute inflammation. In conclusion, during multiple days of endurance exercise the neutrophil compartment does not regain homeostasis overnight. Thereby our study supports the concept of a build-up of inflammatory cues during repeated endurance exercise training, causing a prolonged change of the systemic neutrophil compartment.

Human type-2 CD8+ T cells are a cell population with potentially important roles in allergic disease. We investigated this in the context of severe asthma with persistent airway eosinophilia—a phenotype associated with high exacerbation risk and responsiveness to type-2 cytokine-targeted therapies. In two independent cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8+CRTH2+ (Tc2) cells are enriched in blood and airways in severe eosinophilic asthma. Concentrations of prostaglandin D2 (PGD2) and cysteinyl leukotriene E4 (LTE4) are also increased in the airways of the same group of patients. In vitro PGD2 and LTE4 function synergistically to trigger Tc2 cell recruitment and activation in a TCR-independent manner. These lipids regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines, which could contribute to eosinophilia. These findings are consistent with an important innate-like role for human Tc2 cells in severe eosinophilic asthma and suggest a potential target for therapeutic intervention in this and other diseases.

The functions and in vivo roles of type-2 CD8+ T cells in humans have not been well defined and this cell type has been largely overlooked in models of disease. We investigated this in the context of severe asthma with persistent airway eosinophilia - a phenotype associated with high exacerbation risk and responsiveness to type-2 cytokine-targeted therapies. In two independent cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8+CRTH2+ (Tc2) cells are enriched in blood and airways in severe eosinophilic asthma. Concentrations of prostaglandin D2 (PGD2) and cysteinyl leukotriene E4 (LTE4) are also increased in the airways of the same group of patients. In vitro PGD2 and LTE4 function synergistically to trigger Tc2 cell recruitment and activation in a TCR-independent manner. These lipids regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines which could contribute to eosinophilia. These findings are consistent with an important innate-like role for human Tc2 cells in severe eosinophilic asthma and suggest a potential target for therapeutic intervention in this and other diseases.

Carbon-based nanoparticles such as fullerenes have been widely applied in personal care products, drug delivery systems, and solar cells. The properties of nanoparticles have been increasingly studied because of their applications and their potential risks to the environment and human health. Many studies have focused on the environmental fate and properties of C60. However, there is limited information available on the environmental properties of functionalised fullerenes. This study focuses on the colloidal stability of two fullerenes (C60 and [6,6]-diphenyl-C62-bis(butyric acid methyl ester)) in water in the presence of dissolved organic carbon (DOC) and different electrolytes (NaCl and CaCl2). Suspended fullerene concentrations were determined with high resolution Orbitrap mass spectrometry. The size was determined by multi-angle light scattering, dynamic light scattering and for the first time by flow cytometry. The suspended concentrations of the fullerenes were stabilised by low concentrations of DOC (2 mg C per L) in the presence of NaCl. However, sedimentation of DOC occurred at low concentrations of CaCl2 (> 2 mM) which caused removal of (functionalised) fullerenes. The results show that (functionalised) fullerenes can be rapidly removed in natural aqueous systems in the presence of low concentrations of DOC and multivalent inorganic electrolytes.

Background The identification of inflammatory asthma phenotypes, using sputum analysis, has proven its value in diagnosis and disease monitoring. However due to technical limitations of sputum analysis, there is a strong need for fast and noninvasive diagnostics. This study included the activation state of eosinophils and neutrophils in peripheral blood to phenotype and monitor asthma. Objectives To (i) construct a multivariable model using the activation state of blood granulocytes, (ii) compare its diagnostic value with sputum eosinophilia as gold standard and (iii) validate the model in an independent patient cohort. Methods Clinical parameters, activation of blood granulocytes and sputum characteristics were assessed in 115 adult patients with asthma (training cohort/Utrecht) and 34 patients (validation cohort/Oxford). Results The combination of blood eosinophil count, fractional exhaled nitric oxide, Asthma Control Questionnaire, medication use, nasal polyposis, aspirin sensitivity and neutrophil/eosinophil responsiveness upon stimulation with formyl‐methionyl‐leucyl phenylalanine was found to identify sputum eosinophilia with 90.5% sensitivity and 91.5% specificity in the training cohort and with 77% sensitivity and 71% specificity in the validation cohort (relatively high percentage on oral corticosteroids [OCS]). Conclusions The proposed prediction model identifies eosinophilic asthma without the need for sputum induction. The model forms a noninvasive and externally validated test to assess eosinophilic asthma in patients not on OCS.

Multicolour Flow Cytometry (MFC) produces multidimensional analytical data on the quantitative expression of multiple markers on single cells. This data contains invaluable biomedical information on (1) the marker expressions per cell, (2) the variation in such expression across cells, (3) the variability of cell marker expression across samples that (4) may vary systematically between cells collected from donors and patients. Current conventional and even advanced data analysis methods for MFC data explore only a subset of these levels. The Discriminant Analysis of MultiAspect CYtometry (DAMACY) we present here provides a comprehensive view on health and disease responses by integrating all four levels. We validate DAMACY by using three distinct datasets: in vivo response of neutrophils evoked by systemic endotoxin challenge, the clonal response of leukocytes in bone marrow of acute myeloid leukaemia (AML) patients, and the complex immune response in blood of asthmatics. DAMACY provided good accuracy 91–100% in the discrimination between health and disease, on par with literature values. Additionally, the method provides figures that give insight into the marker expression and cell variability for more in-depth interpretation, that can benefit both physicians and biomedical researchers to better diagnose and monitor diseases that are reflected by changes in blood leukocytes.

Asthma is a heterogeneous disease that can be classified into different clinical endotypes, depending on the type of airway inflammation, clinical severity, and response to treatment. This article focuses on the eosinophilic endotype of asthma, which is defined by the central role that eosinophils play in the pathophysiology of the condition. It is characterized by persistently elevated sputum and/or blood eosinophils and by a significant response to treatments that suppress eosinophilia. Eosinophil activity in the airway may be more important than their numbers and this needs to be investigated. Transcriplomic or Metabolomic signatures may also be useful to identify this endotype.

In 2003, anti-immunoglobulin E (omalizumab) was registered for the treatment of mild to moderate allergic asthma. In 2015, anti-interleukin-5 (mepolizumab) followed with registration for the treatment of severe eosinophilic asthma. These biologicals are medicinal products made partially or totally of naturally occurring molecules, such as antibodies, cytokines, or receptors and have a favorable safety profile and low incidence in adverse events compared to conventional asthma treatments. Therefore, anti-IgE and anti-IL-5 treatment fulfills an unmet need of specific subgroups of patients that do not respond to the classical inhaled steroid-based treatment many of whom need on systemic glucocorticoid treatment. The severe long-term effects of systemic glucocorticoid use in these patients make biologicals a promising alternative.There are other biologicals for the treatment of allergies and asthma in the process of clinical testing among which anti-interleukin-4 receptor α-blockers, anti-interleukin-13, chemoattractant homologous receptor 2 antagonists, and thymic stromal lymphopoietin blockers. Every biological affects the immune system in a different way and the key message learned from the undertaken randomized controlled trials has been to select the right patient for the right treatment. This leads to a continuous search for biomarker-biological associations. In this article we evaluate biologicals registered for the treatment of allergies and asthma and review the clinical effects of biologicals that are in the process of clinical testing.

Summary Inflammatory phenotypes of asthma are associated with differences in disease characteristics. It is unknown whether these inflammatory phenotypes are reflected by the activation status of neutrophils in blood and sputum. We obtained peripheral blood and induced sputum from 21 asthma patients and stratified our samples based on sputum eosinophilia resulting in two groups (>3% eosinophils: n = 13

Background: The Chemoattractant Receptor-homologous molecule expressed on T-Helper type 2 cells (CRTH2) is not only expressed on Th-2 cells but also on eosinophils, basophils and T-cytotoxic cells (Tc2) cells. Its ligand PGD2 is produced in the lung by mast cells upon activation and subsequently activates cells that express CRTH2. CRTH2 antagonists have shown an effect on lung function and airway eosinophilia in one RCT with severe asthma patients and could be more effective in a targeted patient population with high levels of peripheral blood cells that express CRTH2 receptor. Methods: Blood was taken from 8 patients with severe asthma (according to ERS/ATS guidelines) and airway eosinophilia (by sputum analysis) and from 5 healthy controls. CRTH-2 expression was measured on eosinophils, basophils, Th2 cells and Tc2 cells by flow cytometry. Results: Severe asthma patients have higher levels of CRTH-2 positive cells (P Conclusion: Overall, our preliminary data shows that patients with severe asthma have high levels of CRTH2 positive cells in peripheral blood irrespective of their allergic status. Eosinophil levels are significantly higher in asthma patients and other populations show interesting higher trends.

Background: The pathogenesis and the inflammatory cell profile of chronic obstructive pulmonary disease (COPD) are subject to a continuous debate. Recent studies demonstrate that eosinophils contribute to inflammation, which suggests involvement of Th2 lymphocytes. A classification of COPD into different immunophenotypes, depending on the inflammatory cell profile, may facilitate the development of targeted treatments Aims: To investigate the peripheral blood T-lymphocyte subset quantities in COPD phenotypes Method: Whole blood samples obtained from COPD patients and healthy controls were incubated with fluorochrome-conjugated antibodies (CD3+ FITC, CD8+ PE and CD4+ APC) and prepared with Ficoll-paque density gradient centrifugation for immunophenotypical analysis by flow cytometry Results: 3 different T-lymphocyte profiles in COPD patients are seen. Group A) normal CD4+ CD8+ (mean CD4+ 60.88 %; CD8+ 35.94%; %sputum eosinophils 3.75) comparable to the control group; Group B) higher than normal CD8+ and low CD4+ count (mean CD4+ 39.13%; CD8+ 57.35;sputum eosinophils 5% ); Group C) high CD4+ and low CD8+ count (mean CD4+ 81.44; CD8+ 17.88; sputum eosinophils 0.7%) ![Figure][1] Conclusion: There are distinct subgroups of COPD patients identifiable by their T- lymphocyte profiles. Whether this infers a separate pathoimmunobiology in COPD needs further investigation. [1]: pending:yes

Asthma is a heterogeneous airway disease characterized by typical symptoms in combination with variable airway obstruction. Most patients with asthma have well controlled symptoms and a low risk of asthma attacks with inhaled corticosteroid (ICS) treatment. However, a clinically important subgroup (~ 10%) remains symptomatic and/or at risk of asthma attacks despite maximum inhaled therapy. Patients with severe asthma are responsible for a significant proportion of healthcare costs attributable to asthma and have a large unmet need for better treatments. An important advance in recent years has been the recognition that severe asthma is heterogeneous with respect to clinical problems and the pattern of lower airway inflammation. Identification of eosinophilic inflammation in the airways has become an important priority as novel biologicals that target Th2 cytokines, such as anti-IL5, anti-IL-13 and combined anti-IL-4/13 are showing considerable promise as treatments for this subgroup. It has also become clear that anti-IgE (Omalizumab), the first monoclonal antibody registered for treatment of severe asthma, is only active in patients with active eosinophilic airway inflammation. The future will be identification of potentially responsive patients on the basis of raised biomarkers and, as suggested by the title of this review, targeted treatment with specific cytokine blockade that has a direct effect on the biomarkers. In this review, we outline an approach to the clinical assessment of patients potentially suitable for biological treatment and describe in detail the likely clinical impact of established and new biological treatments.

Background CRTH2 antagonism has been shown to reduce eosinophilic airway inflammation and improve lung function in patients with severe eosinophilic asthma. To better understand the role of CRTH2 in the pathogenesis of this asthma phenotype, we have carried out a cross-sectional study to investigate the CRTH2 positive cell counts in peripheral blood of patients with severe eosinophilic asthma. Methods Blood was taken from 12 controls and 33 patients with asthma, 21 of whom met the 2014 ERS/ATS guideline criteria for severe asthma and had historical evidence of eosinophilic airway inflammation as defined before (Pavord et al . Lancet 2012;380:651–9). Th2 were detected as CD3 + CD4 + CRTH2+, Tc2 as CD3 + CD8 + CRTH2+, eosinophils as SSC high CRTH2+, and basophils as CD123 + CRTH2+ by flow cytometry, and numbers presented as total cell counts in peripheral blood. Data were analysed using one-way ANOVA followed by the Newman-Keuls test. Results CRTH2 cell counts were reasonably repeatable within patients (ICC 0.84; n = 9). Mean ± SD CRTH2+ cell counts were 168 ± 81, 322 ± 191, 710 ± 322 and 290 ± 179 × 10 6 cells/L in normal controls (n = 12), patients with mild to moderate asthma (n = 12), patients with severe asthma at BTS step 4 (n = 10), and patients with severe asthma at BTS step 5 (n = 11) respectively (Figure 1). Most CRTH2 + cells were eosinophils (Figure 1). Conclusion Blood CRTH2+ cells are increased in subjects with severe eosinophilic asthma, mainly because of increased CRTH2+ eosinophils. Eosinophils and basophils numbers are significantly increased in severe eosinophilic asthma at step 4 but not step 5. Th2 and Tc2 cell numbers are less clearly associated with severe asthma. CRTH2+ cell numbers are lower in patients treated with prednisolone.

Asthma is a chronic disease characterized by airway inflammation, bronchial hyperresponsiveness, and recurrent episodes of reversible airway obstruction. The disease is very heterogeneous in onset, course, and response to treatment, and seems to encompass a broad collection of heterogeneous disease subtypes with different underlying pathophysiological mechanisms. There is a strong need for easily interpreted clinical biomarkers to assess the nature and severity of the disease. Currently available biomarkers for clinical practice - for example markers in bronchial lavage, bronchial biopsies, sputum, or fraction of exhaled nitric oxide (FeNO) - are limited due to invasiveness or lack of specificity. The assessment of markers in peripheral blood might be a good alternative to study airway inflammation more specifically, compared to FeNO, and in a less invasive manner, compared to bronchoalveolar lavage, biopsies, or sputum induction. In addition, promising novel biomarkers are discovered in the field of breath metabolomics (eg, volatile organic compounds) and (pharmaco)genomics. Biomarker research in asthma is increasingly shifting from the assessment of the value of single biomarkers to multidimensional approaches in which the clinical value of a combination of various markers is studied. This could eventually lead to the development of a clinically applicable algorithm composed of various markers and clinical features to phenotype asthma and improve diagnosis and asthma management.

BackgroundPostoperative pain is one of the main reasons for a prolonged hospital stay after laparoscopic cholecystectomy (LC). Reduced postoperative pain might result in faster recovery and establish LC as a day-care surgical procedure. Peroperative local anaesthesia has been suggested to reduce postoperative pain. The aim of this study was to determine the effect of combined subcutaneous infiltration and intraperitoneal instillation of levobupivacaine before the start of LC on postoperative abdominal pain up to 24 h after surgery.MethodsPatients eligible for elective LC were randomized to receive preincisional infiltration and preoperative intraperitoneal instillation of 80 ml of either 0·125 per cent levobupivacaine (experimental group) or normal saline (placebo group). The primary outcome measure was abdominal pain estimated by means of a visual analogue scale at 0·5, 2, 4, 8 and 24 h after surgery.ResultsEighty of the 101 patients assessed for eligibility were randomized. There was no significant reduction in postoperative abdominal pain with levobupivacaine compared with placebo during the 24-h follow-up; the overall difference in pain score was 2·2 (95 per cent confidence interval − 4·9 to 9·3; P = 0·540). The duration of operation, use of anaesthesia, use of rescue analgesia, shoulder pain, duration of hospital stay and time to resumption of normal daily activities did not differ between the two groups.ConclusionCombined subcutaneous and intraperitoneal administration of levobupivacaine did not influence postoperative abdominal pain after LC. Registration number: NCT01199406 (http://www.clinicaltrials.gov).

Background A subgroup of patients with severe asthma have persistent eosinophilic airway inflammation despite treatment with high intensity corticosteroid treatment. One possible explanation for this pattern of disease is involvement of a type 2 innate lymphoid cells (ILC2s) dependant and relatively corticosteroid resistant pathway generating type 2 cytokines such as IL-5 and Il-13. The presence of high numbers of ILC2s in the nasal polyps commonly associated with severe eosinophilic asthma supports this view. We have carried out a cross-sectional study testing the hypothesis that ILC2 counts are increased in peripheral blood of patients with severe eosinophilic asthma. Methods Blood was taken from 9 controls and 33 patients with asthma, 23 of whom met the 2014 ERS/ATS guideline criteria for severe asthma and had historical evidence of eosinophilic airway inflammation as defined before (Pavord et al . Lancet 2012;380:651–9). ILC2 were measured as lineage- CD45 + CD127 + CRTH2+ by flow cytometry and numbers presented as total cell counts and% peripheral blood mononuclear cells. Results ILC2 c ounts were repeatable within patients (ICC 0.97; n = 6). Mean ± SD ILC2 counts were 566 ± 379, 323 ± 224, 437 ± 628 and 429 ± 421cells/mL (Figure 1A) and 0.034 ± 0.022, 0.02 ± 0.017, 0.020 ± 0.028 and 0.019 ± 0.014% of total lymphocytes (Figure 1B) in normal controls (n = 9), patients with mild to moderate asthma (n = 10), patients with severe asthma at BTS step 4 (n = 12), and patients with severe asthma at BTS step 5 (n = 11) respectively. Conclusion Type 2 innate lymphoid cells are scarce in peripheral blood but can be measured consistently. We found no evidence of increased counts in peripheral blood from patients with severe eosinophilic asthma.

Method We obtained peripheral blood and induced sputum from 21 asthma patients and defined two patient groups based on sputum eosinophilia (cut-off 3%). Eosinophils and neutrophils from blood and sputum were analysed by flow cytometry for expression of activation and degranulation markers. Data were analysed by both classical, non-parametric statistics and a more advanced multi-dimensional approach, using principal component analysis (PCA).

Method Clinical parameters, activation of blood granulocytes and sputum phenotypes were assessed in 115 adult asthma patients (NCT01611012). Blood granulocytes were stained with antibodies against the active FcgRII receptor (CD32, clones A17/A27) and a-chain of MAC-1 (CD11b) in the presence or absence of fMLF, and analysed by flow cytometry. NLPCA was used to reduce dimensions in a combined cellular and clinical dataset, followed by discriminant analysis to generate a prediction model. Phenotypes identified by the model were cross-validated by inflammatory sputum phenotypes based on sputum induction (reference test). Results Peripheral blood eosinophil count, FeNO (Fraction of Exhaled Nitric Oxide), ACQ (Asthma Control Questionnaire), medication use, nasal polyposis, aspirin sensitivity and neutrophil/eosinophil responsiveness upon stimulation, are important parameters to differentiate between eosinophilic and non-eosinophilic phenotypes. The combinations of these parameters lead to an accurate prediction model for eosinophilic asthma with 90.5% sensitivity and 91.5% specificity.

Background Identification of an inflammatory asthma phenotype currently requires sputum induction. This technique is invasive, has a high variability, is time-consuming and a burden for patients. Therefore, there is a strong need for a routine blood test to establish the inflammatory phenotype of asthma. We designed a clinical cohort study (AIR-study, NCT01611012) in 115 asthma patients visiting the outpatient clinic to compare the results of such a blood test to sputum analysis. This abstracts focuses at the preliminary results in 21 patients.