methodology-for-sputum-induction-and-laboratory-processing

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

University of Liege

It has been claimed that exhaled nitric oxide (FeNO) could be regarded as a surrogate marker for sputum eosinophil count in patients with asthma. However, the FeNO threshold value that identifies a sputum eosinophil count ≥3% in an unselected population of patients with asthma has been poorly studied.

Exhaled nitric oxide thresholds associated with a sputum eosinophil count ≥3% in a cohort of unselected patients with asthma.

Asthma diagnosis is based on the presence of symptoms and the demonstration of airflow variability. Airway inflammation measured by fractional exhaled nitric oxide, measured at a flow rate of 50 ml/s (FE(NO50)) remains a controversial diagnostic tool.

Is FENO50 useful diagnostic tool in suspected asthma?

Inhaled corticosteroids (ICS) have led to improved asthma control and reduced asthma mortality in the Western world. ICS are effective in combating T-helper type 2-driven inflammation featuring mast cell and eosinophilic airway infiltration. Their effect on innate immunity-driven neutrophilic inflammation is poor and their ability to prevent airway remodeling and accelerated lung decline is controversial. Although ICS remain pivotal drugs in asthma management, research is needed to find drugs complementary to the combination ICS/long-acting β2-agonist in refractory asthma and perhaps a new class of drugs as a first-line treatment in mild to moderate noneosinophilic asthma.

Corticosteroids: still at the frontline in asthma treatment?

Phenotyping asthma according to airway inflammation allows identification of responders to targeted therapy. Induced sputum is technically demanding. We aimed to identify predictors of sputum inflammatory phenotypes according to easily available clinical characteristics.

Distribution of sputum cellular phenotype in a large asthma cohort: predicting factors for eosinophilic vs neutrophilic inflammation.

Systemic and airway eosinophilia are recognised features of asthma. There are, however, patients who exhibit discordance between local and systemic eosinophilia. In this study, we sought to determine the prevalence and characteristics of patients with concordant and discordant systemic and bronchial eosinophilia. We conducted a retrospective study on 508 asthmatics with successful sputum induction. We assessed the relationship between blood and sputum eosinophils by breaking down the population into four groups according to blood (≥400 cells per mm3) and sputum (≥3%) eosinophils. Then, we prospectively reassessed the link between eosinophils and asthma control (Asthma Control Questionnaire (ACQ)) and exacerbation rate in a new cohort of 250 matched asthmatics. In our retrospective cohort, asthmatics without eosinophilic inflammation were the largest group (49%). The group with isolated sputum eosinophilia (25%) was, compared with noneosinophilic asthma, associated with lower forced expiratory volume in 1 s (FEV1) and FEV1/forced vital capacity ratio and higher bronchial hyperresponsiveness and exhaled nitric oxide fraction (FeNO). Asthmatics exhibiting isolated systemic eosinophilia (7%) had similar characteristics as noneosinophilic asthmatics. The group with concordant systemic and airway eosinophilia (19%) showed remarkable male predominance, and had the lowest airway calibre, asthma control and quality of life, and the highest bronchial hyperresponsiveness, FeNO and exacerbation rate. The prospective cohort confirmed the different subgroup proportions and the higher ACQ and exacerbation rates in cases of diffuse eosinophilia compared with noneosinophilic asthmatics. Concomitant systemic and bronchial eosinophilic inflammation contribute to poor asthma control.

Importance of concomitant local and systemic eosinophilia in uncontrolled asthma.

The Belgian severe asthma registry is a web-based registry encompassing demographic, clinical, functional and inflammatory data of severe asthmatics (SA), aiming at improving awareness, knowledge on its natural history and subphenotypes, and offering tools to optimize care of this asthma population.

Heterogeneity of phenotypes in severe asthmatics. The Belgian Severe Asthma Registry (BSAR).

Difficult asthma is a heterogeneous disease of the airways including various types of bronchial inflammation and various degrees of airway remodeling. Therapeutic response of severe asthmatics can be predicted by the use of biomarkers of Type2-high or Type2-low inflammation. Based on sputum cell analysis, four inflammatory phenotypes have been described. As induced sputum is timeconsuming and expensive technique, surrogate biomarkers are useful in clinical practice. Eosinophilic phenotype is likely to reflect ongoing adaptive immunity in response to allergen. Several biomarkers of eosinophilic asthma are easily available in clinical practice (blood eosinophils, serum IgE, exhaled nitric oxyde, serum periostin). Neutrophilic asthma is thought to reflect innate immune system activation in response to pollutants or infectious agents while paucigranulocytic asthma is thought to be not inflammatory and characterized by smooth muscle dysfunction. We currently lack of user-friendly biomarkers of neutrophilic asthma and airway remodeling. In this review, we summarize the biomarkers available for the management of difficult asthma.

Biomarkers in the Management of Difficult Asthma.

Blood eosinophil count to predict bronchial eosinophilic inflammation in COPD.

The technique of induced sputum has allowed to subdivide asthma patients into inflammatory phenotypes according to their level of granulocyte airway infiltration. There are very few studies which looked at detailed sputum and blood cell counts in a large cohort of asthmatics divided into inflammatory phenotypes. The purpose of this study was to analyze sputum cell counts, blood leukocytes and systemic inflammatory markers in these phenotypes, and investigate how those groups compared with healthy subjects.

Detailed analysis of sputum and systemic inflammation in asthma phenotypes: are paucigranulocytic asthmatics really non-inflammatory?

Asthma is classified according to severity and inflammatory phenotype and is likely to be distinguished by specific microRNA (miRNA) expression profiles.

Asthma inflammatory phenotypes show differential microRNA expression in sputum.

Increases in eosinophil numbers are associated with infection and allergic diseases, including asthma, but there is also evidence that eosinophils contribute to homeostatic immune processes. In mice, the normal lung contains resident eosinophils (rEos), but their function has not been characterized. Here, we have reported that steady-state pulmonary rEos are IL-5-independent parenchymal Siglec-FintCD62L+CD101lo cells with a ring-shaped nucleus. During house dust mite-induced airway allergy, rEos features remained unchanged, and rEos were accompanied by recruited inflammatory eosinophils (iEos), which were defined as IL-5-dependent peribronchial Siglec-FhiCD62L-CD101hi cells with a segmented nucleus. Gene expression analyses revealed a more regulatory profile for rEos than for iEos, and correspondingly, mice lacking lung rEos showed an increase in Th2 cell responses to inhaled allergens. Such elevation of Th2 responses was linked to the ability of rEos, but not iEos, to inhibit the maturation, and therefore the pro-Th2 function, of allergen-loaded DCs. Finally, we determined that the parenchymal rEos found in nonasthmatic human lungs (Siglec-8+CD62L+IL-3Rlo cells) were phenotypically distinct from the iEos isolated from the sputa of eosinophilic asthmatic patients (Siglec-8+CD62LloIL-3Rhi cells), suggesting that our findings in mice are relevant to humans. In conclusion, our data define lung rEos as a distinct eosinophil subset with key homeostatic functions.

Lung-resident eosinophils represent a distinct regulatory eosinophil subset.

Inhaled corticoids (ICS) made a dramatic breakthrough in the management of asthma in the late eighties resulting in a sharp reduction in morbidity and mortality in the following decades. Soon after, the association between ICS and long acting β2 agonists (LABA) soon became the gold standard of maintenance asthma treatment. With the advent of sputum induction it has become clear that asthma could not be considered as a unique entity but rather a display of several inflammatory phenotypes. Eosinophilic phenotype shows good response to ICS while non-eosinophilic, and in particular the neutrophilic phenotype, seems to be more resistant. Severe asthmatics show insufficient asthma control despite ICS/LABA. Those who are allergic and eosinophilic may benefit from add-on treatment with anti-IgE or anti-IL-5. Severe neutrophilic asthma could benefit from maintenance treatment with macrolides while thermoplasty offers some promise to those in whom airway smooth muscle hypertrophy contributes to disease instability.

A historical perspective: Are inhaled corticoids sufficient to control asthma?

Longitudinal trials have suggested that asthma control may be influenced by fluctuations in eosinophilic inflammation. This association has however never been confirmed in daily practice.

Asthma Control and Sputum Eosinophils: A Longitudinal Study in Daily Practice.

The concept of asthma inflammatory phenotypes has proved to be important in predicting response to inhaled corticosteroids. Induced sputum, which has been pivotal in the development of the concept of inflammatory phenotypes, is however not widely available. Several studies have proposed to use surrogate exhaled or blood biomarkers, like fractional exhaled nitric oxide (FENO), blood eosinophils and total serum immunoglobulin E (IgE). However, taken alone, each of these biomarkers has moderate accuracy to identify sputum eosinophilia. Here, we propose a new approach based on the likelihood ratio to study which thresholds of these biomarkers, taken alone or in combination, were able to rule in or rule out sputum eosinophils ≥3%. We showed in a large population of 869 asthmatics that combining FENO, blood eosinophils and total serum IgE could accurately predict sputum eosinophils ≥ or <3% in 58% of our population.

Is it possible to claim or refute sputum eosinophils ≥ 3% in asthmatics with sufficient accuracy using biomarkers?

The impact of inhaled corticosteroids (ICS) on eosinophilic inflammation in asthma is well established, but their effect in a real-life setting has not been extensively studied. Our purpose was to investigate the effect of ICS on airway and systemic inflammation as well as on clinical outcomes in patients with asthma from clinical practice.

Effectiveness of inhaled corticosteroids in real life on clinical outcomes, sputum cells and systemic inflammation in asthmatics: a retrospective cohort study in a secondary care centre.

Article	Year
Exhaled nitric oxide thresholds associated with a sputum eosinophil count ≥3% in a cohort of unselected patients with asthma. Thorax\| PubMed ID: 20671307	2010
Is FENO50 useful diagnostic tool in suspected asthma? International journal of clinical practice\| PubMed ID: 22257040	2012
Corticosteroids: still at the frontline in asthma treatment? Clinics in chest medicine\| PubMed ID: 22929100	2012
Distribution of sputum cellular phenotype in a large asthma cohort: predicting factors for eosinophilic vs neutrophilic inflammation. BMC pulmonary medicine\| PubMed ID: 23442497	2013
Importance of concomitant local and systemic eosinophilia in uncontrolled asthma. The European respiratory journal\| PubMed ID: 24525441	2014
Importance of concomitant local and systemic eosinophilia in uncontrolled asthma. The European respiratory journal\| PubMed ID: 25271232	2014
Heterogeneity of phenotypes in severe asthmatics. The Belgian Severe Asthma Registry (BSAR). Respiratory medicine\| PubMed ID: 25456708	2014
Biomarkers in the Management of Difficult Asthma. Current topics in medicinal chemistry\| PubMed ID: 26467509	2016
Blood eosinophil count to predict bronchial eosinophilic inflammation in COPD. The European respiratory journal\| PubMed ID: 26846827	2016
Detailed analysis of sputum and systemic inflammation in asthma phenotypes: are paucigranulocytic asthmatics really non-inflammatory? BMC pulmonary medicine\| PubMed ID: 27044366	2016
Asthma inflammatory phenotypes show differential microRNA expression in sputum. The Journal of allergy and clinical immunology\| PubMed ID: 27155035	2016
Lung-resident eosinophils represent a distinct regulatory eosinophil subset. The Journal of clinical investigation\| PubMed ID: 27548519	2016
A historical perspective: Are inhaled corticoids sufficient to control asthma? Journal of translational internal medicine\| PubMed ID: 27847898	2015
Asthma Control and Sputum Eosinophils: A Longitudinal Study in Daily Practice. The journal of allergy and clinical immunology. In practice\| PubMed ID: 28389300	2017
Is it possible to claim or refute sputum eosinophils ≥ 3% in asthmatics with sufficient accuracy using biomarkers? Respiratory research\| PubMed ID: 28673351	2017
Effectiveness of inhaled corticosteroids in real life on clinical outcomes, sputum cells and systemic inflammation in asthmatics: a retrospective cohort study in a secondary care centre. BMJ open\| PubMed ID: 29183929	2017

Florence Schleich

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