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Validation of Cell Analysis by Chipcytometry in Induced Sputum Cells
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A4768 - Validation of Cell Analysis by Chipcytometry in Induced Sputum Cells
Author Block: S. Carstensen1, O. Holz2, M. Mueller1, J. M. Hohlfeld3; 1Fraunhofer ITEM, Hannover, Germany, 2Clinical Airway Research, Fraunhofer ITEM, Member of the German Center for Lung Research (BREATH), Hannover, Germany, 3Clinical Airway Research, Fraunhofer ITEM, Hannover Medical School, Member of the German Center for Lung Research (BREATH), Hannover, Germany.
Rationale: Chipcytometry combines the phenotyping ability of flow cytometry with detailed imaging and functional insight of microscopic technologies. Immobilized cells on microfluidic chips can be stored for several months and multiple cellular markers can be iteratively analyzed. The aim of the present work is the validation of cell analysis by Chipcytometry for induced sputum. Induced sputum is a far more complex matrix compared with peripheral blood due to the broader spectrum of cell morphologies, the contamination with airway epithelial cells, and the need for homogenization with reducing agents. Guideline-compliant validation of cell analysis with surface marker antibodies is a prerequisite for acceptance of the use of this methodology in clinical trials.
Methods: Validation was performed for selected cell surface markers (CD3, CD4, CD8, CD14, CD16b, CD45). Cells from sputum samples of at least 4 subjects (healthy, COPD, and asthma) were analysed. The cells were pooled, loaded onto chips and iteratively stained using the respective antibodies followed by bleaching cycles in triplicates. Variances in antibody staining were investigated and data were compared with gold standard methodology flow cytometry and standard light microscopy for immune status.
Results: Lymphocytes, neutrophils, monocytes and macrophages were determined by combining cell surface markers and morphological characteristics like cell size and nuclear staining with DAPI. Cell differentiation by Chipcytometry using surface marker expression and cell morphology was similar to data obtained using flow cytometry and light microscopy. An increased CD4:CD8 ratio in sputum cells was observed reproducibly by flow and Chipcytometry compared to the ratio in PBMCs. Lymphocyte and neutrophil numbers showed comparable percentages between all methodologies. CD14 expression and macrophage counting showed underestimating numbers.
Conclusion: Measurement of CD3, CD4, CD8, CD16b and CD45 by Chipcytometry showed reliable and reproducible results on sputum cells in line with the cellular composition shown by gold standard methodologies flow cytometry and light microscopy. Chipcytometry represents a feasible tool for assessment of the immune status. Further experimental effort has to be undertaken to develop improved sputum preparation methodology to avoid cell plaque formation on the chip because cell plaques disturb automatic identification of the macrophage compartment by the cell recognition software.
Author Block: S. Carstensen1, O. Holz2, M. Mueller1, J. M. Hohlfeld3; 1Fraunhofer ITEM, Hannover, Germany, 2Clinical Airway Research, Fraunhofer ITEM, Member of the German Center for Lung Research (BREATH), Hannover, Germany, 3Clinical Airway Research, Fraunhofer ITEM, Hannover Medical School, Member of the German Center for Lung Research (BREATH), Hannover, Germany.
Rationale: Chipcytometry combines the phenotyping ability of flow cytometry with detailed imaging and functional insight of microscopic technologies. Immobilized cells on microfluidic chips can be stored for several months and multiple cellular markers can be iteratively analyzed. The aim of the present work is the validation of cell analysis by Chipcytometry for induced sputum. Induced sputum is a far more complex matrix compared with peripheral blood due to the broader spectrum of cell morphologies, the contamination with airway epithelial cells, and the need for homogenization with reducing agents. Guideline-compliant validation of cell analysis with surface marker antibodies is a prerequisite for acceptance of the use of this methodology in clinical trials.
Methods: Validation was performed for selected cell surface markers (CD3, CD4, CD8, CD14, CD16b, CD45). Cells from sputum samples of at least 4 subjects (healthy, COPD, and asthma) were analysed. The cells were pooled, loaded onto chips and iteratively stained using the respective antibodies followed by bleaching cycles in triplicates. Variances in antibody staining were investigated and data were compared with gold standard methodology flow cytometry and standard light microscopy for immune status.
Results: Lymphocytes, neutrophils, monocytes and macrophages were determined by combining cell surface markers and morphological characteristics like cell size and nuclear staining with DAPI. Cell differentiation by Chipcytometry using surface marker expression and cell morphology was similar to data obtained using flow cytometry and light microscopy. An increased CD4:CD8 ratio in sputum cells was observed reproducibly by flow and Chipcytometry compared to the ratio in PBMCs. Lymphocyte and neutrophil numbers showed comparable percentages between all methodologies. CD14 expression and macrophage counting showed underestimating numbers.
Conclusion: Measurement of CD3, CD4, CD8, CD16b and CD45 by Chipcytometry showed reliable and reproducible results on sputum cells in line with the cellular composition shown by gold standard methodologies flow cytometry and light microscopy. Chipcytometry represents a feasible tool for assessment of the immune status. Further experimental effort has to be undertaken to develop improved sputum preparation methodology to avoid cell plaque formation on the chip because cell plaques disturb automatic identification of the macrophage compartment by the cell recognition software.
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