Astrid Crespo

Flow cytometry analysis of leukocytes in induced sputum from asthmatic patients

Inflammatory cell counts in induced sputum from asthmatic patients partially correlate with respiratory physiology data. To identify and quantify these inflammatory components, microscopy has been useful but it is not without its limitations. Flow cytometry could be an alternative but still has underlying methodological difficulties. While passing airways, leukocytes undergo morphologic cellular changes that alter their conventional phenotype. To demonstrate the usefulness of cytometry in accurately identifying cellular profiles in induced sputum of asthmatic and chronic cough patients, we introduced a new panel of monoclonal antibodies against specific subset markers. To identify neutrophils, sputum cells were stained with CD45 and CD66b. To identify eosinophils, sputum cells were stained with anti-CD45 and anti-CD125. We co-stained CD45, CD14 and CD66b to identify macrophages as CD45+CD14+CD66b- cells. Comparable results of trypan blue exclusion and annexin V-FITC suggested that cytometry manipulation did not decrease cellular viability. Range values were similar in microscopy neutrophils (median 19.9%, range 1.7-90.1%) and CD45+CD66b+ neutrophils (median 31% range 0.9-89%). After gating out CD45- non-leukocyte events, CD45+ and SSC dot-plots defined three patterns of leukocyte distribution. The eosinophil range in microscopic examination was 0-71.3% (median 2.85%) whereas CD45+CD125+ cell range in cytometry was 0-29% (median 3.7%). Since no exclusive markers were found on airways macrophages, we co-stained CD45, CD14 and CD66b to identify macrophages as CD45+CD14+CD66b- cells. Microscopy showed that macrophage and CD45+CD14+CD66b- cell counts were comparable (median 52.3 and range 6.7-94.8 vs median 61 and range 10.5-97.7 respectively). Correlations between neutrophils, eosinophils and macrophages in microscopic examination and flow cytometry were strong (R=0.725, 0.747 and 0.532, respectively p<0.001). This study validates effectiveness of combining specific antibodies and cytometry to quantify inflammatory leukocytes in induced sputum. Multiple markers at a single cell level will deepen our knowledge concerning the phenotype of airway leukocytes.

Exhaled nitric oxide fraction as an add-on to ACQ-7 for not well controlled asthma detection.

Background The measurement of fractional nitric oxide concentration in exhaled breath (FeNO), a noninvasive indicator of airway inflammation, remains controversial as a tool to assess asthma control. Guidelines currently limit asthma control assessment to symptom and spirometry based appraisals such as the Asthma Control Questionnaire-7 (ACQ-7). We aimed at determining whether adding FeNO to ACQ-7 improves current asthma clinical control assessment, through enhanced detection of not well controlled asthma. Methods Asthmatic subjects, classified as not well controlled as per ACQ-7 on regular clinical practice, were included in a prospective, multicenter fashion, and had their maintenance treatment adjusted on visit 1. On follow-up (visit 2) four weeks later, the subjects were reevaluated as controlled or not well controlled using ACQ-7 versus a combination of FeNO and ACQ-7. Results Out of 381 subjects enrolled, 225 (59.1%) had not well controlled asthma on visit 2 as determined by ACQ-7, and 264 (69.3%) as per combined FeNO and ACQ-7. The combination of FeNO to ACQ-7 increased by 14.8% the detection of not well controlled asthma following maintenance therapy adjustment. Conclusions The addition of FeNO to ACQ-7 increased the detectability of not well controlled asthma upon adjustment of maintenance therapy. Adding a measure of airway inflammation to usual symptom and spirometry based scores increases the efficacy of current asthma clinical control assessment.

Clinical and inflammatory features of asthma with dissociation between fractional exhaled nitric oxide and eosinophils in induced sputum

Abstract Background: Measurement of the fractional exhaled nitric oxide (FeNO) and eosinophils in induced sputum are noninvasive markers for assessing airway inflammation in asthma. The clinical usefulness of the correlation between raised FeNO and sputum eosinophilia is controversial. We aimed to examine dissociation between FeNO and sputum eosinophils in a clinical series of asthma patients and to determine whether dissociation between these noninvasive markers was associated with clinical and inflammatory differences in these patients. Methods and findings: A total of 110 patients with asthma were included in a cross-sectional study. All of them were on maintenance treatment for asthma. All patients underwent the following on the same day: FeNO, induced sputum, spirometry, serum total IgE levels and skin prick test. The level of asthma control was determined by the Asthma control Test Questionnaire. In 46 (41.8%) patients, a discrepancy between FeNO and sputum eosinophil count was observed, of those, 34 (73.9%) had a FeNO <50 ppb and high eosinophil count, and were characterized by having a predominance of nonallergic asthma with bronchial eosinophilic inflammatory phenotype. Also, 12 (26.1%) patients had FeNO ≥50 ppb and sputum eosinophilia within the normal reference values, and were characterized by having a predominance of atopy with a paucigranulocytic inflammatory phenotype. Conclusions: A high percentage of patients with dissociation between results of FeNO and sputum eosinophils was observed. These patients showed differential clinical and inflammatory features.

Variabilidad del fenotipo inflamatorio del asma en el esputo inducido. Frecuencia y causas

INTRODUCTION Recent studies have found variability in asthma inflammatory phenotypes determined by the inflammatory cells in induced sputum (IS). The aim of this study was to determine the frequency and factors affecting inflammatory phenotype variability in IS. METHODS Retrospective observational study that included 61 asthmatic patients who underwent at least two IS tests over a period of 5 years. They were classified according to their baseline inflammatory phenotype and subsequently grouped according to phenotype variability (persistent eosinophilic, persistent non-eosinophilic and intermittent eosinophilic). Demographic, clinical and functional data and factors potentially influencing IS variability were collected in all cases. RESULTS Of the 61 patients, 31 (50.8%) had a change with respect to baseline inflammatory phenotype. Of these, 16 (51.6%) were eosinophilic, 5 (16.1%) neutrophilic, 1 (3.2%) mixed and 9 (29.1%) paucigranulocytic. According to phenotype variability, 18 patients (29.5%) were classified as persistent eosinophilic, 17 (27.9%) non-persistent eosinophilic, and 26 (42.6%) intermittent eosinophilic. Smoking and recent asthma exacerbation were significantly associated with increased risk of variability of the IS inflammatory phenotype (OR=6.44; p=.013; 95% CI=1.49-27.80 and OR=5.84; p=.022; 95% CI=1.29-26.37, respectively). CONCLUSION Half of asthma patients, predominantly those with eosinophilic phenotype, present a change in IS inflammatory phenotype. This variability is associated with smoking and recent asthma exacerbation. Data suggest these factors can modify the classification of IS inflammatory phenotype in clinical practice.

Utilidad del esputo inducido en la práctica clínica habitual

OBJECTIVE To determine the general and specific utility in diagnosis and/or treatment of induced sputum (IS) inflammatory cell counts in routine clinical practice. METHODS Retrospective study of 171 patients referred for clinical sputum induction over a 1-year period in the pulmonology department of a referral hospital. Independent observers established whether the information provided by IS inflammatory cell count was useful for making diagnostic and therapeutic decisions. RESULTS The most frequent reasons for determination of IS inflammatory cell count were: asthma 103 (59.20%); uncontrolled asthma 34 (19.54%); chronic cough 19 (10.9%), and gastroesophageal reflux 15 (8.6%). In 115 patients (67.3%) it was generally useful for diagnosis and/or treatment; in 98 patients (57.3%) it provided diagnostic information and in 85 patients (49.7%) it assisted in therapeutic decision-making. In asthma, uncontrolled asthma, chronic cough and gastroesophageal reflux, the results were useful in 71.8%, 67.6%, 47.4% and 60%, respectively. CONCLUSION The information provided by IS inflammatory cell count is extremely useful in clinical practice, especially in asthma and chronic cough. These results may justify the inclusion of the IS technique in pulmonology departments and asthma units of referral centers.

Noninvasive Home Mechanical Ventilation in Elderly Patients

Study Objective: To determine the short- and long-term benefits of noninvasive home mechanical ventilation (NIHMV) in patients aged 65 and older who were eligible for this treatment. Design and Setting: This retrospective, comparative, longitudinal study was carried out in a tertiary care hospital in Barcelona (Spain). Patients and Methods: The study included all patients in whom NIHMV with a nasal mask was established in the period from 1998 to 2001. Patients were divided into 3 groups according to age: group 1 (n = 10) ≧75; group 2 (n = 40) 65–74, and group 3 (n = 41) <65 years old. Clinical characteristics, pulmonary function results, and arterial blood gas findings were assessed in all patients before starting ventilation and after 6 months. Measurements and Results: Statistically significant improvements in PaO2 (13.4, 10, and 15.3 mm Hg for groups 1–3) and PaCO2 (–17.6, –9.6 and –12.8 mm Hg for groups 1–3) were found at 6 months (p < 0.001 in all cases). There were no significant differences between the groups in blood gas parameters and treatment compliance. The incidence of related adverse events was not statistically different between the study groups (40%, 35% and 32% for groups 1–3; p = 0.859). Conclusions: NIHMV is effective in all patients for whom it is indicated, regardless of their age.

Mucus hypersecretion in asthma is associated with rhinosinusitis, polyps and exacerbations.

BACKGROUND Bronchial hypersecretion is a poorly studied symptom in asthma. The aim of the study was to determine the specific characteristics of asthmatics with bronchial hypersecretion. METHODS A total of 142 asthmatics (21.8% men; mean age 49.8 years) were prospectively followed for one year. Mucus hypersecretion was clinically classified into two severity categories: daily sputum production and frequent expectoration but not every day. Clinical and pulmonary function variables associated with mucus hypersecretion were assessed by multiple logistic regression analysis. RESULTS Daily cough was recorded in 28.9% of patients and sputum production daily or most of the days in 52.1%. Patients with mucus hypersecretion had more dyspnoea, poorer asthma control and quality of life, had suffered from more exacerbations and showed anosmia associated with chronic rhinosinusitis and nasal polyposis more frequently. Factors associated to mucus hypersecretion were anosmia, one exacerbation or more in the previous year and FEV1/FVC <70% (AUC 0.75, 95% CI 0.66-0.85) for the first definition of hypersecretion, and anosmia, poor asthma control and age (AUC 0.75, 95% CI 0.67-0.83) for the second definition. CONCLUSIONS Mucus hypersecretion is frequent in patients with asthma, and is associated with chronic upper airways disease, airway obstruction, poor asthma control and more exacerbations.

Estudio de los mecanismos implicados en la génesis y evolución del asma (proyecto MEGA): creación y seguimiento a largo plazo de una cohorte de pacientes asmáticos

The general aim of this study is to create a cohort of asthma patients with varying grades of severity in order to gain greater insight into the mechanisms underlying the genesis and course of this disease. The specific objectives focus on various studies, including imaging, lung function, inflammation, and bronchial hyperresponsiveness, to determine the relevant events that characterize the asthma population, the long-term parameters that can determine changes in the severity of patients, and the treatments that influence disease progression. The study will also seek to identify the causes of exacerbations and how this affects the course of the disease. Patients will be contacted via the outpatient clinics of the 8 participating institutions under the auspices of the Spanish Respiratory Diseases Networking System (CIBER). In the inclusion visit, a standardized clinical history will be obtained, a clinical examination, including blood pressure, body mass index, complete respiratory function tests, and FENO will be performed, and the Asthma Control Test (ACT), Morisky-Green test, Asthma Quality of Life Questionnaire (Mini AQLQ), the Sino-Nasal Outcome Test 22 (SNOT-22), and the Hospital Anxiety and Depression scale (HADS) will be administered. A specific electronic database has been designed for data collection. Exhaled breath condensate, urine and blood samples will also be collected. Non-specific bronchial hyperresponsiveness testing with methacholine will be performed and an induced sputum sample will be collected at the beginning of the study and every 24 months. A skin prick test for airborne allergens and a chest CT will be performed at the beginning of the study and repeated every 5 years.