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Sputum Analysis by Chipcytometry Following LPS Inhalation Challenge

: Carstensen, Saskia; Holz, Olaf; Müller, Meike; Hohlfeld, Jens M.


American Journal of Respiratory and Critical Care Medicine 199 (2019), Abstract A5742
ISSN: 1073-449X
ISSN: 0003-0805
ISSN: 1535-4970
American Thoracic Society (ATS International Conference) <2019, Dallas/Tex.>
Fraunhofer ITEM ()

Rationale: The LPS inhalation challenge is a model for standardized inflammatory immune response within the lung. It´s mainly characterized by the influx of neutrophilic granulocytes, which is detectable in induced sputum. While differential cell count and flow cytometry have limitations confining some sub populations, Chipcytometry combines the immunological phenotyping capability of flow cytometry with morphological information of standard microscopy. We currently adapt the technology for induced sputum analysis. Here we tested the performance of Chipcytometry after biobanking of sputum cells in the LPS challenge model by an iterative analysis of multiple cellular markers. Methods: Induced sputum of 10 healthy non-smokers was collected 2-7 days before and 6h after 2µg LPS inhalation (Janssen et al. 2013). We performed a differential cell count by microscopy (M, n=10), flow cytometry (F, n=9) and Chipcytometry analysis (C, n=9). Flow cytometry was performed immediately following sputum processing. Cells were incubated with a CD45, CD14, CD3, CD8 and CD4 antibody panel. For Chipcytometry, cells were loaded onto specific chamber slides (Zellkraftwerk), fixed with formalin and stored at 4 °C for 8-13 months prior to iterative staining with CD14, CD66b, CD3, CD45, HLA-DR, CD8, CD4 to show signal stabilities. Results: As known from previous studies, LPS induced a clear increase in sputum neutrophils detectable by all 3 methodologies. The percentage of neutrophils in the differential cell count increased from a median of 37% to 79%. Flow cytometry detected an increase from 62 % to 72 % and Chipcytometry (CD66b) from 45% to 73%. At baseline we observed a significant correlation for the percentages of neutrophils between M and C (r=0.97) and F and C (r=0.90). After LPS challenge the coefficients were r=0.97 and r=0.90. The coefficients of reliability at baseline were 0.95 (M vs. C) and 0.71 (F vs. C), and following LPS 0.96 and 0.75, respectively. CD8 and HLA-DR were not reliably detectable by Chipcytometry showing a donor dependent variant stability of signal after that storage period. Conclusion: The LPS induced increase in sputum neutrophils was detectable by Chipcytometry and showed comparable results to flow cytometry and the standard differential cell count. Despite a longer storage period than currently recommended for sputum samples we observed a good agreement between methodologies. Our data provides further evidence for the suitability of Chipcytometry for antibody based sputum cell analysis after storage or shipment to a clinical core facility e.g. in multi-center trials overcoming the disadvantage of flow cytometer measurements.