A novel method to assess airway eosinophilia using sputum plugs

Background: Sputum cytology utilising routine dithiothreitol (DTT) processing is a well-established method to assess airway eosinophilia. However, the multi-step nature of this protocol requires substantial resources, thereby limiting its broader clinical applicability. To address this, we evaluated formalin-fixed, paraffin-embedded (FFPE) sputum plugs as a simplified method for measuring airway eosinophilia. Methods: Excess sputum plugs from 113 patients with complex airway disease and 16 asthma patients on corticosteroid or monoclonal antibodies were fixed in 10% formalin and embedded in paraffin (Sputum-Minimising Processing, Maximising Clinical Outcomes (SSIMPLE) method). FFPE blocks were sectioned, stained with haematoxylin and eosin, and assessed by four blinded observers, who performed a differential cell count on 400 total nonsquamous cells. Eosinophil proportions were then compared between matched FFPE and DTT-processed sputum slides. Airway eosinophilia was defined as ≥2.2% sputum eosinophils in DTT-processed samples. Results: Of the 113 FFPE slides, 96% were adequate for cellular analysis, with 90% having matched DTT-processed sputum slides. The SSIMPLE method demonstrated excellent interobserver reproducibility (consistency: 0.975; agreement: 0.976). Eosinophil proportions obtained from SSIMPLE and DTT processing were significantly correlated (ρ=0.9, p<0.0001) and showed agreement (Bland-Altman, -0.38±9.51). A cut-off of 2.6% detected airway eosinophilia with high sensitivity (85.4%) and specificity (93.0%) using the SSIMPLE method, showing strong agreement with the routine DTT method, as indicated by an area under the curve of 0.957. Additionally, the method effectively assessed treatment responsiveness to monoclonal antibody therapy. Conclusion: The SSIMPLE method is a reliable, noninferior approach to DTT processing for detecting airway eosinophilia in complex airway diseases, with high reproducibility and strong concordance in monitoring treatment responsiveness.