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Eosinophil and Neutrophil Granulocyte Immunophenotyping Using Chipcytometry

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


American Journal of Respiratory and Critical Care Medicine 199 (2019), Abstract A2956
ISSN: 1073-449X
ISSN: 0003-0805
ISSN: 1535-4970
American Thoracic Society (ATS International Conference) <2019, Dallas/Tex.>
Fraunhofer ITEM ()

Rationale: Immunophenotyping of granulocytes in sputum from patients with inflammatory airway disease is an important goal for monitoring of disease activity and treatment success. Chipcytometry may hold great potential in this respect because it allows the characterization of cell surface marker expression after storage of cells on chips. However, processing and preparation of induced sputum may have an impact on granulocyte integrity and stainability. Therefore, we tested the feasibility of Chipcytometry to immunophenotype granulocytes by using peripheral blood granulocytes that were treated similarly like induced sputum cells and determined antibody stability by flow cytometry prior to chipcytometric analysis. Methods: Granulocytes were isolated from peripheral blood of atopic subjects using 4% dextran and density gradient centrifugation. For flow cytometry, cells were incubated with PBS, DTT or PFA prior to staining, in order to observe differences in epitope binding due to chemical treatments required during sputum processing (DTT) or cell-loading ontochips (PFA).Cell surface expression of CD45, CD15, CD16, CD16b, CD66b and CCR3 was measured using a Beckman CoulterNavios™ flow cytometer. For Chipcytometry, cells were loaded onto Zellkraftwerk chips, fixed and stained iteratively with CD45, CD15, CD16, CD16b, CD66b, CCR3, eosinophil peroxidase (EPX). Percentages of eosinophils within granulocytes were compared with both methods. Results: Feasibility of measurements on eosinophil/ neutrophil granulocytes following DTT/PFA treatment was assessed by flow cytometry. Comparison of signal intensity showed no differences between treated and untreated cells. Neutrophils showed CD15+, CD16high, CD16b+, CD66b+, and CCR3-expression. Eosinophils were CD15-, CD16low, CD16b-, CD66b+ and CCR3+. Analogue results were obtained using Chipcytometry for CD15, CD16, CD16b and CD66b indicating specific epitope binding. CCR3 staining of eosinophils was possible on unfixed cells on chips only. Comparable results were obtained for flow- and Chipcytometry discriminated percentages of eosinophils within the granulocytes (%EosFACS=9,6%, %EosChip=10,2%; n=2) Conclusion: Chipcytometry allows the iterative analysis of manifold antibody markers and discrimination on peripheral blood granulocytes after DTT/PFA treatment without influence on most epitopes. Therefore, Chipcytometry will allow to achieve a profound understanding of the immune response mechanisms of atopic asthma with focus on sputum derived granulocytes.