Analysis of induced sputum by Chipcytometry

Introduction: In Chipcytometry immobilized cells on microfluidic chips can be iteratively analyzed for multiple biomarkers. The technology is established for blood and BAL, but needs to be adapted for sputum analysis due to the required homogenization with reducing agents. We investigated whether standard treatment of sputum samples affects cell adhesion, the stainability with surface marker antibodies and the storage time. Methods: Sputum samples, homogenized with DTT, were stored for 10 d and up to 4 m prior to analysis. The slides were stained with 10 antibodies for surface marker expression of immune cells. Results: Sputum cells adhered in acceptable numbers to the microfluidic slides and their composition did not differ from non-adhered cells. Chipcytometry after 10 d storage (4°C) showed that 99.8% of the sputum cells remained on the chip following 11 staining and bleaching cycles. Some antibodies showed insufficient binding in the concentration used for PBMCs and we observed unspecific binding of CD3 on bronchial epithelial cells. Autofluorescence of sputum macrophages also requires the some methodological adaptation. During 3 m of storage the cell density decreased but was still sufficient in 75% of slides. Poor sputum quality with a higher proportion of degraded cells complicated the analysis. Conclusion: These first experiments show that the cellular analysis of sputum cells by Chipcytometry is feasible, however, high quality samples and careful preparation with low squamous cell contamination are prerequisite for the analysis. Short term storage is possible allowing multicenter trials with centralized Chipcytometry analysis. Further adaptation and fit-for-purpose validation of the method for induced sputum samples is required.