Reply to comment on: A novel method to assess airway eosinophilia using sputum plugs

Extract

Since the publication of the recent article on the SSIMPLE method (Study on sputum to minimise processing and maximise clinical end-points) there has been great interest from the respirology community, as also reflected by the accompanied editorial by Pignatti and Spanevello [1]. Here, we welcome the opportunity to clarify the nature of the SSIMPLE method approach in the context of induced sputum analysis.

Shareable abstract

SSIMPLE histological processing complements the cytological gold standard by enabling FFPE archival storage, immunohistochemical validation and integration into existing pathology infrastructure, expanding access to airway inflammation monitoring https://bit.ly/4vDKP27

Reply to E. Fireman-Klein and E. Fireman:

Since the publication of the recent article on the SSIMPLE method (Study on sputum to minimise processing and maximise clinical end-points) there has been great interest from the respirology community, as also reflected by the accompanied editorial by Pignatti and Spanevello [1]. Here, we welcome the opportunity to clarify the nature of the SSIMPLE method approach in the context of induced sputum analysis.

Despite the technical simplicity of sputum induction via hypertonic saline nebulisation, the widespread implementation of cytological processing remains hindered by significant organisational barriers and the lack of accessible, dedicated laboratories underscoring the urgent need for more practical approaches [1].

Several attempts have been made to simplify the cytological “gold standard” Pizzichini et al. [2] procedure with dithiothreitol (DTT) by using dimethylsulfoxide [3], paraformaldehyde [4] or direct smearing of sputum plugs, each with inherent limitations in sample preservation, inconsistent results and interpretation difficulties [5]. However, Fireman et al. [6] (including E. Fireman-Klein and E. Fireman) also proposed a simplified cytological approach for routine daily practice while using DTT processing.

Now, our study demonstrated SSIMPLE to be a noninferior approach to DTT processing, the “gold standard” technique for detecting airway eosinophilia with high reproducibility [7]. In a cohort of 113 patients with complex airway disease, 96% of formalin-fixed, paraffin-embedded (FFPE) sputum plugs were adequate for cellular analysis, with 90% having matched DTT-processed sputum slides (98 matched samples). The SSIMPLE method demonstrated excellent interobserver reproducibility (consistency: 0.975; agreement: 0.976) and eosinophil proportions were significantly correlated with DTT processing (ρ=0.880, p<0.0001), with an area under the curve of 0.957 for detecting airway eosinophilia at a cut-off of 2.6% (sensitivity: 85.4%; specificity: 93.0%) [7]. These findings were obtained alongside, not in isolation from, conventional cytology: the matched-sample correlation demonstrates that histological processing preserves the eosinophil signal across the clinically relevant range. This novel method utilises the established foundation to provide a reliable, standardised assessment of airway eosinophilia that can be highly reproducible across clinical pathology settings.

Regarding the comparison between histological and cytological approaches, as stated by E. Fireman-Klein and E. Fireman, it is important to note that immunohistochemistry commonly uses FFPE tissue precisely because it preserves architectural integrity and allows for robust biomarker expression [8]. In addition, while cytology provides excellent visualisation of individual cell borders and morphology, it is less amenable to the antigen retrieval and standardised immunostaining that FFPE permits [9], which limits its capacity to further identify specific cell types and components within sputum plugs.

Indeed, we already demonstrated the validation of eosinophil peroxidase (EPX) immunofluorescence staining in FFPE samples to confirm presence of eosinophils detected originally by haematoxylin and eosin. A strong correlation was observed between EPX⁺ cells and eosinophil percentages (ρ=0.744, p<0.001) [7], which addresses the concern about formalin-mediated effects on antigenicity. Eosinophils are also among the most morphologically distinctive cells on haematoxylin and eosin staining owing to their characteristic bright pink, highly basic granules, and their identification was not impaired in FFPE preparations.

Furthermore, the authors acknowledged that all FFPE samples stored were intended for future assessment and cellular validation of neutrophils, granules, macrophages, lymphocytes and other clinically relevant components. Notably, FFPE sputum blocks have also been used to detect Charcot–Leyden crystals, extracellular traps and immune complex deposition in the airways of severe asthma patients [10, 11], underscoring the archival and translational advantage of the histological approach.

In addition, the SSIMPLE method produces a permanent FFPE tissue block that can be archived, re-sectioned and re-stained at any future time point – a feature unavailable with conventional cytospin preparations. This archival capacity enables retrospective quality assurance, immunohistochemical validation on the same tissue block and future biomarker assessment as new clinically relevant targets emerge.

While we acknowledge the benefits of the simplified sputum processing method suggested by the correspondents, the SSIMPLE method was specifically designed to leverage existing, high-throughput hospital infrastructure – namely, the pathology laboratory. This ensures that sputum analysis can be integrated into routine clinical workflows without necessitating additional training in specialised haematological smear techniques and billing codes. In our experience, the SSIMPLE method further addresses several logistical barriers beyond reducing hands-on processing time and estimated per-sample costs compared with DTT processing (CAD 25.30 versus CAD 69.60). Moreover, the ability to collect sputum remotely and transport samples in formalin to centralised pathology sites eliminates the need for immediate processing within 2 h of collection.

We believe both cytological and histological methods serve the common goal of improving access to airway inflammation monitoring and patient care. Finally, as an alternative diagnostic tool, the histological approach using the SSIMPLE plug method offers an equitable, distinct, complementary dataset to traditional DTT processing-based cytology that can support advanced personalised care in complex airway diseases.