Validation of total and specific IgE measurements in induced sputum.

BACKGROUND AND OBJECTIVES: Soluble components are increasingly analyzed in induced sputum supernatant. However, only a few studies have measured total or specific immunoglobulin (Ig) E in sputum and none have attempted to validate it. We aim to validate laboratory measurements of total and specific IgE in induced sputum supernatant and to evaluate the influence of sputum processing with dithiothreitol (DTT) on IgE measurements. METHODS: Total and specific IgE were measured by ImmunoCAP and the process was validated using sputum spiking experiments with total and specific IgE (to Dermatophagoides pteronyssinus and Phleum pratense) over a range of concentrations according to international recommendations.The Wilcoxon signed-ranks test was used for within-group comparisons and intraclass correlation coefficients were used to evaluate agreement between measurements. Two-tailed P values lower than .05 were considered significant. RESULTS: Samples from 18 patients (13 with interstitial lung disease, 2 with allergic asthma, and 3 healthy controls; 12 men; mean [SD] age, 45.6 [15.8] years) were evaluated. Median total IgE was 5.4 kU/L (interquartile range, 4.0-6.0 kU/L). Specific IgE levels to D. pteronyssinus and P. pratense were below 0.35 kUA/L in all samples. Recovery rates were above 80% for total and specific IgE over a wide range of values. No differences were found in total IgE measurements of sputum dispersed with DTT or phosphate-buffered saline, with a good intraclass correlation coefficient between both measurements (0.81, P=.01). CONCLUSIONS: Total and specific IgE measurements performed in induced sputum with a commercially available immunoassay are valid over a wide range of IgE levels.

Validation of Total and Specific IgE Measurements in Induced Sputum

Durante los últimos años, se están analizando de forma exhaustiva los componentes solubles del esputo inducido, no obstante pocos estudios analizan la IgE total y específica y ninguno de ellos ha intentado validar la técnica. El objetivo de este estudio fue validar la cuantificación de IgE total y específica en el sobrenadante del esputo inducido y evaluar la influencia del procesamiento del mismo con dithiothreitol (DTT). La IgE total y específica fueron determinadas mediante el fluoroenzymoinmunoensayo ImmunoCAP (Phadia ThermoFisher Scientific). Para el proceso de validación utilizamos experimentos de adición de células del esputo con IgE total y específica (frente a Dermatophagoides pteronyssinus y Phleum pratense) en un rango de concentraciones de acuerdo a las recomendaciones de la ATS/ERS. Para las comparaciones intragrupo se utilizó el test de Wilcoxon y el coeficiente de correlación intraclase para evaluar el grado de acuerdo entre ambas mediciones. Fueron considerados como significativos valores de p <0.05. En cuanto a los resultados obtenidos en las muestras de esputo procedentes de 18 pacientes (13 con enfermedad pulmonar intersticial, 2 con asma alérgica y 3 controles sanos), 12 varones, con una edad media de 45.6 ± SD 15.8 años, se obtuvo una media de IgE total de 5.4 (P25-754.0-6.0) kU/L. La IgE específica frente a Dermatophagoides pteronyssinus y Phleum pratense fueron inferiores a 0.35 kUA/L en todas las muestras. Las cifras de recuperación de la IgE total y específica estaba por encima del 80% con un amplio rango de valores. No se hallaron diferencias significativas entre la cuantificación de IgE total y específica con PBS o con DTT, con un buen coeficiente de correlación intraclase (0.81; p=0.01). En conclusión, la cuantificación de IgE total y específica en esputo inducido utilizando un imnunoensayo comercializado es válido y presenta una alta dispersión de rangos de niveles de IgE (AU)

Background and objectives: Soluble components are increasingly analyzed in induced sputum supernatant. However, only a few studies have measured total or specific immunoglobulin (Ig) E in sputum and none have attempted to validate it. We aim to validate laboratory measurements of total and specific IgE in induced sputum supernatant and to evaluate the influence of sputum processing with dithiothreitol (DTT) on IgE measurements. Methods: Total and specific IgE were measured by ImmunoCAP and the process was validated using sputum spiking experiments with total and specific IgE (to Dermatophagoides pteronyssinus and Phleum pratense) over a range of concentrations according to international recommendations. The Wilcoxon signed-ranks test was used for within-group comparisons and intraclass correlation coefficients were used to evaluate agreement between measurements. Two-tailed P values lower than .05 were considered significant. Results: Samples from 18 patients (13 with interstitial lung disease, 2 with allergic asthma, and 3 healthy controls; 12 men; mean [SD] age, 45.6 [15.8] years) were evaluated. Median total IgE was 5.4 kU/L (interquartile range, 4.0-6.0 kU/L). Specific IgE levels to D pteronyssinus and P pratense were below 0.35 kUA/L in all samples. Recovery rates were above 80% for total and specific IgE over a wide range of values. No differences were found in total IgE measurements of sputum dispersed with DTT or phosphate-buffered saline, with a good intraclass correlation coefficient between both measurements (0.81, P=.01). Conclusions: Total and specific IgE measurements performed in induced sputum with a commercially available immunoassay are valid over a wide range of IgE levels (AU)

Increased circulating platelet microparticles as a potential biomarker in asthma.

BACKGROUND: Endothelial (EMPs) and platelet microparticles (PMPs) have been studied as biomarkers in several inflammatory diseases and as central players in intercellular communication. METHODS: In this cross-sectional study, we aimed to assess microparticle levels in asthma. Circulating microparticles and inflammatory and angiogenic markers were assessed by clinical and laboratorial evaluation, flow cytometry, and immunoassays, in a group of 20 asthmatic and 15 nonasthmatic subjects. RESULTS: Circulating levels of PMPs (either CD31+/42b+ or CD31+/42b+/AnV+) were significantly increased in asthmatics (P = 0.021) even after adjustment for confounders. Apoptotic EMPs (CD31+/42b--/AnV+) were significantly increased before (P = 0.005) but not after adjustments (P = 0.117). CONCLUSIONS: We propose that PMPs may be putative asthma biomarkers, playing a role in asthma pathophysiology.

Induced sputum in interstitial lung diseases - A pilot study.

INTRODUCTION: Induced sputum with hypertonic saline has been suggested as a safer and cheaper alternative to bronchoalveolar lavage for evaluation of patients with interstitial lung diseases (ILD). OBJECTIVE: To evaluate the safety and feasibility of sputum induction in ILD and to compare sputum cellular profiles with paired bronchoalveolar lavage fluid results. MATERIAL AND METHODS: Twenty patients underwent sputum induction with 4.5% saline within 2 weeks of bronchoalveolar lavage. Total, differential cell counts and cellular viability were assessed. Wilcoxon test and Spearman's rank correlation coefficient were used and a p<0,05 was considered statistically significant. RESULTS: From a total of 20 subjects (mean age 49.4±16.4 years, 70% male) a satisfactory sputum sample was obtained in 15 subjects (75%). Induction was stopped in one subject, due to a significant decrease in PEF. The cell profiles for induced sputum and bronchoalveolar lavage fluid (BALF) were different (P <.05), except for eosinophils, and there were no significant correlations between the two methods. Compared to sputum reference values there was an increase of lymphocytes (3.2% vs 0.5%) and eosinophils (1.4% vs 0.0%). Comparing sarcoidosis and hypersensitivity pneumonitis sputum, both diseases had an increase in lymphocytes (4.4 vs 3.9%), with a significant higher neutrophil count in hypersensitivity pneumonitis (65.4% vs 10.6% P <0.05), a finding also seen in BALF. CONCLUSION: Induced sputum is feasible and safe in interstitial lung diseases. Although sputum cellular counts are not correlated with bronchoalveolar lavage fluid, sputum cellular profiles may help to distinguish different ILD.

Substance P antagonist improves both obesity and asthma in a mouse model.

BACKGROUND: Evidence suggests a causal relationship between obesity and asthma; however, the underlying mechanisms remain unknown. Substance P (SP), involved in neurogenic inflammation by acting through its receptor NK1-R, seems to participate in obese-asthma phenotype in mice. OBJECTIVES: To evaluate the effect of a selective substance P receptor antagonist on a mouse model of diet-induced obesity and asthma. METHODS: Diet-induced obese Balb/c mice were sensitized and challenged with ovalbumin (OVA) and treated with a selective NK1-R antagonist or placebo. Serum glucose, insulin, IL-6, resistin, and OVA-specific IgE levels were quantified. A score for peribronchial inflammation in lung histology was used. Cells were counted in bronchoalveolar lavage fluid. Adipocyte sizes were measured. RESULTS: Ovalbumin-obese mice treated with NK1-R antagonist had lower weight (P = 0.0002), reduced daily food intake (P = 0.0021), reduced daily energy intake (P = 0.0021), reduced surface adipocyte areas (P < 0.0001), lower serum glucose (P = 0.04), lower serum insulin (P = 0.03), lower serum IL-(P = 0.0022), lower serum resistin (P = 0.0043), lower serum OVA-specific IgE (P = 0.035), and lower peribronchial inflammation score (P < 0.0001) than nontreated OVA-obese mice. We observed an interaction between obesity, allergen sensitization, and treatment with NK1-R antagonist for metabolic and systemic biomarkers, and for allergen sensitization and bronchial inflammation, showing a synergy between these variables. CONCLUSION & CLINICAL RELEVANCE: In an experimental model of obesity and asthma in mice, NK1-R blockade improved metabolic and systemic biomarkers, as well as allergen sensitization and bronchial inflammation. These positive effects support a common pathway in the obese-asthma phenotype and highlight SP as a target with potential clinical interest in the obese-asthma epidemics.

Induced sputum in children: success determinants, safety, and cell profiles.

BACKGROUND: Sputum induction is a noninvasive method for the assessment of airway inflammation. OBJECTIVES: To evaluate the safety of the procedure and the clinical predictors of successful induction, and to analyze the relationship between sputum cell counts and clinical features in asthmatic and nonasthmatic children. METHODS: We reviewed sputum inductions performed in our department between 2006 and 2008 in individuals under 18 years; 34 asthmatic and 24 nonasthmatic children were included. Sputum induction was performed with 4.5% saline for 5-minute periods with salbutamol pretreatment. The most viscid portions were selected for processing. Inductions which were tolerated for less than 4 minutes or which produced a sample volume of less than 1 mL or a sample with a squamous cell percentage of over 80% were considered unsuccessful. RESULTS: Sputum induction was successful in 43 (74%) of the 58 children studied.The total median induction time was 15 minutes (interquartile range, 10-15 minutes). Only 7 individuals (12%) experienced mild symptoms, which were easily reversed with salbutamol inhalation in all cases. The mean (SD) overall PEF variation with induction was -2.5% (7%), with no significant differences between asthmatics and nonasthmatics. Asthmatics had significantly higher total cell counts (P = .007), macrophages (P = .033), and relatively fewer neutrophils (P = .003) than nonasthmatics; metachromatic cells were rare and seen only in asthmatics (P = .026). We found a positive correlation between exhaled nitric oxide and sputum eosinophil count (r = 0.363, P = .017). CONCLUSIONS: Sputum induction is a safe, noninvasive, and feasible procedure that allows the direct assessment of airway inflammation in most children.