Eosinophil peroxidase in sputum represents a unique biomarker of airway eosinophilia

Abstract

Background: Sputum eosinophilia has been shown to be a predictor of response to anti-eosinophil therapies in patients with airway diseases. However, quantitative cell counts and differentials of sputum are labor intensive. The objective of this study was to validate a novel ELISA-based assay of eosinophil peroxidase (EPX) in sputum as a rapid and reliable marker of airway eosinophils.

Methods: The utility of EPX-based ELISA as an eosinophil-specific assay was achieved through comparisons with sputum eosinophil differential counts in freshly prepared and archived patient samples from a variety of clinical settings.

Results: EPX levels in sputum correlated with eosinophil percentage (r(s) = 0.84) in asthma patients with varying degrees of airway eosinophilia. Significantly, unlike assays of other eosinophil granule proteins (e.g., ECP and EDN), which often detect the presence of these proteins even in asthma patients with neutrophilic bronchitis, EPX-based ELISA levels are not increased in this subset of asthma patients or in COPD patients lacking evidence of an airway eosinophilia. Moreover, sputum EPX was a surrogate marker of airway eosinophilia in other patient studies (e.g., allergen inhalation and treatment trials the anti-(IL-5) therapeutic Mepolizumab™). Finally, EPX levels in cytocentrifuged prepared sputum supernatants correlated with those from rapidly prepared noncentrifuged filtrates of sputum (r(s) = 0.94).

Conclusion and clinical implication: EPX-based ELISA is a valid, reliable, repeatable, and specific surrogate marker of eosinophils and/or eosinophil degranulation in the sputum of respiratory patients. The novel EPX assay is a valid and reproducible eosinophil-specific assay that can potentially be developed into a point-of-care assessment of eosinophil activity in airway secretions.

Keywords: COPD; ELISA; EPX; asthma; sputum eosinophils.

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Figures

Figure 1. Sputum EPX levels increase in respiratory patients as a function of airway eosinophils and/or eosinophil-associated activities

EPX-based ELISA was used to determine sputum EPX levels in cohorts of patients participating in clinical studies as part of their care in a pulmonary clinic. The clinical details of patient cohorts examined are described in the Materials and Methods section (Study Subjects) and Table 1. The data are presented as a scatter plot of the individual patients, presenting the mean with the error bars corresponding to the standard error of the mean for the data set in question. The airway eosinophilia of each cohort (percent (%) of total sputum leukocytes) is noted above the data derived from each patient cohort. The number of granules in asthma patients whose sputum cell evaluations failed to identify intact eosinophils were quantified as containing few (+), moderate (++), or many (+++) granules by a technologist who was blinded to the clinical details. †Significant difference (p < 0.05) relative to healthy controls. †† Significant difference (p < 0.05) relative to mild asthmatics with a 1–2% sputum eosinophilia.

Figure 2. EPX-based ELISA represents a reproducibly robust assay capable of assessing EPX levels from archived sputum samples

Sputum EPX levels derived from the patients of our study cohorts were assessed following consecutive freeze-thaw cycles (freeze-thaw 1 and 2) separated by a 4 month archive period at −70°C. The congruence of the repeated measurements (ICC = 0.9) also attested to the stability of the archived samples.

Figure 3. ELISA assessments of the prominent eosinophil secondary granule protein levels in the sputum of respiratory patients demonstrated that EPX-based ELISA is the only “eosinophil-specific” assay capable of unambiguously identifying subsets of respiratory patients based on eosinophil involvement

(A) Sputum levels (ng of eosinophil secondary granule protein/mL of supernatant/gram of sputum (ng/mL-g)) of ECP and EDN were determined using commercially available ELISA kits and compared to the data obtained with our EPX-based ELISA from three patient cohorts: Control subjects (i.e., healthy patients with no evidence of respiratory and/or allergic disease (<10 × 106 total cells/gram of sputum, <1% eosinophils, <3% neutrophils), neutrophilic bronchitis patients who displayed nominal improved airflow in response to a short acting beta-agonist (<12% increase in FEV1) and were unresponsive to steroid intervention (>25 × 106 total cells/gram of sputum, <3% eosinophils, >80% neutrophils), and eosinophilic bronchitis patients who displayed improved airflow in response to a short acting beta-agonist (>15% increase in FEV1) and also had symptomatic improvement following steroid intervention (<10 × 106 total cells/gram of sputum, ≥3% eosinophils, <3% neutrophils. The number of subjects (n) is shown above the histograms derived from each patient cohort. *Significant increase (p<0.0001) relative to levels observed in neutrophilic bronchitis patients. (B) Sputum EPX levels increased as a function of sputum (i.e., airway) eosinophilia independent of the diagnosed respiratory disease. Manual cell counts and differentials of sputum from asthma and COPD patients were used to stratify each cohort on the basis of a sputum eosinophilia (< or >2% of total of sputum leukocytes). The number of subjects (n) is shown above the data derived from the stratified patients within each cohort. *p<0.001.

Figure 4. Sputum EPX levels represented a reliable biomarker for the diagnostic evaluation of clinical study subjects

(A) Post hoc assessments of asthma patients (n = 10) undergoing aeroallergen provocation as part of an investigatory study in a pulmonary clinic setting (6) demonstrated that relative to pre-challenge levels the post-challenge increase in sputum EPX accurately reflected the pre- vs. post-challenge increase in sputum eosinophil levels observed in these same patients. *p<0.01. (B) Sputum EPX levels (ng/mL-g) from post hoc assessments of asthma patients participating in a clinical study evaluating the anti-IL-5 therapeutic Mepolizumab™ (6). Patients receiving placebo (n = 8) were compared at the start of the study (baseline) and the first exacerbating event leading these patients to return to a clinical setting (exacerbation). *p<0.01. (C)Post hoc assessments of sputum EPX levels (ng/mL-g) from a cohort of asthma patients treated with Mepolizumab™ (n = 5) were also compared at the start of the study (baseline) and to the levels observed 24 weeks after the initiation of treatment. *p<0.01.

Figure 5. EPX levels in sputum filtrates recovered from a syringe-based filtration unit (Accufilter®) is not significantly different relative to the levels observed in sputum supernatants collected using established laboratory-based methods that include cyto-centrifugation

Sputum was recovered by filtration vs. laboratory-based cyto-centrifugation from each patient prior to the determination of EPX levels (ng/mL-g). The resulting EPX levels were plotted relative to one another with (●) representing data from an individual patient and the line derived from a linear regression of the collective data set comparing these sputum processing strategies (rs = 0.94, p = 0.001).