This retrospective study using a large clinical database demonstrates that eosinophilia in sputum may be underestimated by as much as 28% if free eosinophil granules are not taken into consideration. Limiting examination to a single time point assessment that does not consider the changes in eosinophilia associated with exacerbations, resolution of concomitant neutrophilia or change in corticosteroid dosages may lead to further underestimation by as much as 60%. This has important clinical relevance, not only to endotype for selection of patients into clinical trials, but also to make therapeutic decisions about escalating or decrease steroid dosage and initiation of eosinophil-specific biologic therapies.
It is important, for number of reasons, to recognize free eosinophil granules in sputum. Eosinophil cytolysis and release of cationic proteins are a marker of severity and contribute to bronchial epithelial injury and impairs repair (10). Eosinophil peroxidase contributes to the bromination of tyrosine residues (11) and is associated with epithelial dysfunction. Peroxidase activity also triggers an autoimmune reactive process in the airway (12) that has implications for disease severity and response to treatment of asthma with biologics (13). Further, cytolysis and release of extracellular traps may lead to crystal formation within the airways (14, 15). Indeed, recent evidence suggests that Charcot Leyden Crystals, that are the products of auto-crystallization of galectin-10 is regulated independent of IL-5 (16), and the failure to recognize this in sputum may further underestimate the eosinophilic activity in the airways. This needs further investigation. A further underestimation of airway eosinophilic activity may result from failure to recognize these granular proteins within airway macrophages (17).
A second important reason for underestimating airway eosinophilia is when conclusions are drawn from a single time point assessment. The three most clinically relevant factors that might affect eosinophil % are whether sputum was sampled during an exacerbation, the dose of corticosteroids at the time of sampling, and if there was a concomitant neutrophilia that may mask an underlying eosinophilia. Certain airway infections may also directly induce an eosinophilic response that may resolve over time (18). Particularly in patients with COPD, neutrophilic and eosinophilic exacerbations may interchange over time (19).
A limitation of our study is its retrospective nature that is associated with all the inherent biases of such a study design. However, the strengths include the large sample size, robust laboratory methods, and regular stringent external quality control. Another limitation is that we did not characterize patients into specific disease categories such as asthma, COPD, overlap, bronchiectasis etc. This is because of the likely imprecision in the data coding based on a physician diagnosis. Given the large sample size, scrutiny of individual charts to confirm the physician diagnosis was not possible and we did not have approval from our Research Ethics Board for chart review. However, the purpose of this manuscript is not to relative eosinophilia to a particular disease state, rather to report fallacies in estimation of eosinophilia in sputum analysis. Although our analysis was limited to assessment of eosinophilic activity, there might be other aspect of cellular inflammation that are often not taken into consideration leading to inaccurate characterization as a non-T2 endotype. These include lymphocyte (20) and mast cell numbers (21) in sputum that can be identified particularly with more advanced microscopy, flow cytometry, and mass cytometry. They could also be markers of steroid responsiveness and it remains to be seen how often these endotypes may occur in the absence of eosinophilia.
In summary, we highlight the relevance of recognizing free eosinophil granules, and the importance of multiple examinations to identify sputum eosinophilia. This is likely relevant to select patients for anti-eosinophil clinical trials, to interpret treatment responses, and to guide the use of corticosteroids to treat eosinophil-responsive airway diseases.