Underestimation of airway luminal eosinophilia by quantitative sputum cytometry

Abstract

Rationale On Wright-stained sputum cytospins, eosinophil differential of ≥ 1.2% is considered abnormal, and ≥ 2.3% identifies an eosinophilic endotype. We hypothesized that failure to consider free eosinophil granules (FEG), and the re-emergence (unmasking) of eosinophilia due to various reasons underestimate the prevalence of the eosinophilic endotype. Methods This is a retrospective analysis of our Institutional Review Board-approved clinical sputum database. Of the 24,176 examinations of sputa from patients with various airway diseases, 17,693 were viable cell counts from 9570 patients (6604 on a single occasion, 2967 from multiple occasions). The prevalence of intact eosinophil % at 1.2 and 2.3% thresholds was first examined. Then, additional evidence of eosinophilia was assessed by semi-quantitative enumeration of FEGs. In those patients whose sputa were examined on multiple occasions (at the time of an exacerbation or after corticosteroid dose was reduced), re-emergence (unmasking) of eosinophilia was assessed . Results Using the threshold of eosinophilia ≥ 1.2%, 6289/17693 (35.6%) of sputa were classified as eosinophilic. This increased to 7850/17693 (44.4%) when the presence of FEGs was considered. Using the threshold of eosinophilia ≥ 2.3%, 4647/17693 (26.3%) of sputa were classified as eosinophilic. This increased to 5435/17693 (30.7%) when the presence of FEG were considered. Extrapolating from the prevalence of re-emergence observed in the 2967 patients who had sputa examined on multiple occasions to the whole sample, we estimated that eosinophilia at 1.2% threshold would be observed in at least 60% of the samples, and a clinically relevant eosinophilia at 2.3% threshold would be observed in at least 48.5% of the samples. Conclusions Using a large sputum cytometry clinical database (17,693 viable cell counts), we demonstrate that a single time point intact cell count underestimates the prevalence of eosinophilia in a variety of airway diseases. The prevalence of eosinophilia increases from 35.6 to 60% (40% underestimation) at the 1.2% threshold, and from 26.3 to 48.5% (45% underestimation) at the 2.3% clinically relevant threshold, when free granules and a second examination are considered. This has important implications to identify the eosinophilic and Th2 high endotype both for clinical trials of anti-eosinophil therapies, and to select patients who may respond well to glucocorticosteroids and anti-IL5 therapies.

How reliably can algorithms identify eosinophilic asthma phenotypes using non‐invasive biomarkers?

“…Kjarsgaard et al demonstrated the presence of free eosinophil granules in the airway in the absence of intact eosinophils 11 . Detecting intact eosinophils only, as in our study, may give misleading information on the real prevalence of eosinophilic sputum, marking one limitation of our study.…”

“…Kjarsgaard et al demonstrated the presence of free eosinophil granules in the airway in the absence of intact eosinophils. 11 Detecting intact eosinophils only, as in our study, may give misleading information on the real prevalence of eosinophilic sputum, marking one limitation of our study. Further limitations include a possible bias toward recruiting predominantly eosinophilic patients in tertiary centres and allergy clinics, which could be confounding given the limitations in finding significant differences in unequally sized groups.…”

Validation and further insight into the International Severe Asthma Registry (ISAR) eosinophil gradient algorithm in the Wessex AsThma CoHort of difficult asthma (WATCH) using historical blood eosinophil counts and induced sputum

“…Particularly if this trait is defined by limited timespan assessment in this variable disease. 1 , 3 , 4 Consistent with this, we recently reported that repeated blood eosinophil counts (BECs) over time can help unmask the underlying T2 asthma status not revealed by a single snapshot measure. 3 This longitudinal approach demonstrated a higher burden of T2 asthma status than previously reported.…”

“…There are, however, limitations in defining patients as eosinophilic or not, as severe asthma patients have high oral or inhaled corticosteroid burden which may suppress blood eosinophils and mask the underlying eosinophilic nature of the disease. Particularly if this trait is defined by limited timespan assessment in this variable disease 1,3,4 . Consistent with this, we recently reported that repeated blood eosinophil counts (BECs) over time can help unmask the underlying T2 asthma status not revealed by a single snapshot measure 3 .…”

“…Our sputum findings emphasize the value of longitudinal repeat measures in truly understanding the underlying phenotypic propensity and are consistent with a recent publication that also highlighted that single sputum measures underestimate the likelihood of being classified as eosinophilic. 4 Variable granulocyte measures are also associated with poorer disease outcome, 3 , 8 highlighting the prognostic and diagnostic advantages of longitudinal repeat measurements. In addition, we are yet to fully understand the longitudinal stability of granulocyte phenotypes, the frequency of phenotype switching and how such events influence factors like disease severity and remission.…”