Enumeration and phenotypic assessment of human plasmacytoid and myeloid dendritic cells in whole blood
Enumeration and phenotypic assessment of human plasmacytoid and myeloid dendritic cells in whole blood
Traditionally, flow cytometry analysis of dendritic cells (DC) has followed a negative selection procedure, often limiting the characterization of individual DC subsets to enumeration. We demonstrate the development, evaluation, and clinical application of a novel 6 color/8 parameter flow cytometry panel to allow enumeration and monitoring of activation status of circulating human myeloid (MDC1) and plasmacytoid (PDC) dendritic cells in human whole blood. Enumeration showed a trend of greater numbers of MDC1s and PDCs being collected for fresh whole blood than frozen PBMCs, with this difference being statistically significant (P = 0.04) for unstimulated PDC enumeration. Intra-assay variation had a coefficient of variation <10% and interassay results between operators showed good correlation (r > 0.95). Our results on fresh whole blood showed a significant up regulation of CD83 on both MDC1 and PDC at 4 h post Toll-like ligand stimulus and this activity was comparable in frozen PBMC samples. Comparison for the late activation marker CCR7 showed a significant difference (P <0.05) in expression between fresh and frozen samples, precluding its use for batch analysis of frozen samples. In addition, the level of activation is dependent on the anticoagulant used for sample collection. For CD83 expression at 4 h both EDTA and lithium heparin samples are comparable for MDC1 and PDC populations. Whereas for CCR7 expression, lithium heparin is preferable as EDTA increases the background expression in PDC, preventing further functional assessments. We demonstrate the importance of establishing the kinetic profile of activation marker expression and the importance of evaluating sample collection tubes and sample type before application of novel cytometry panels to a clinical study. We have shown that this DC enumeration flow cytometry panel is a robust analysis system that allows the flexibility of including activation markers.
dendritic cell, validation, enumeration, activation, flow cytometry
Chowdhury, Ferdousi
0af499d4-17c5-40cf-9426-0d509ab82595
Johnson, Peter
3f6068ce-171e-4c2c-aca9-dc9b6a37413f
Williams, Antony Peter
973ff46f-46f1-4d7c-b27d-0f53221e4c44
6 February 2010
Chowdhury, Ferdousi
0af499d4-17c5-40cf-9426-0d509ab82595
Johnson, Peter
3f6068ce-171e-4c2c-aca9-dc9b6a37413f
Williams, Antony Peter
973ff46f-46f1-4d7c-b27d-0f53221e4c44
Chowdhury, Ferdousi, Johnson, Peter and Williams, Antony Peter
(2010)
Enumeration and phenotypic assessment of human plasmacytoid and myeloid dendritic cells in whole blood.
Cytometry Part A.
(doi:10.1002/cyto.a.20872).
Abstract
Traditionally, flow cytometry analysis of dendritic cells (DC) has followed a negative selection procedure, often limiting the characterization of individual DC subsets to enumeration. We demonstrate the development, evaluation, and clinical application of a novel 6 color/8 parameter flow cytometry panel to allow enumeration and monitoring of activation status of circulating human myeloid (MDC1) and plasmacytoid (PDC) dendritic cells in human whole blood. Enumeration showed a trend of greater numbers of MDC1s and PDCs being collected for fresh whole blood than frozen PBMCs, with this difference being statistically significant (P = 0.04) for unstimulated PDC enumeration. Intra-assay variation had a coefficient of variation <10% and interassay results between operators showed good correlation (r > 0.95). Our results on fresh whole blood showed a significant up regulation of CD83 on both MDC1 and PDC at 4 h post Toll-like ligand stimulus and this activity was comparable in frozen PBMC samples. Comparison for the late activation marker CCR7 showed a significant difference (P <0.05) in expression between fresh and frozen samples, precluding its use for batch analysis of frozen samples. In addition, the level of activation is dependent on the anticoagulant used for sample collection. For CD83 expression at 4 h both EDTA and lithium heparin samples are comparable for MDC1 and PDC populations. Whereas for CCR7 expression, lithium heparin is preferable as EDTA increases the background expression in PDC, preventing further functional assessments. We demonstrate the importance of establishing the kinetic profile of activation marker expression and the importance of evaluating sample collection tubes and sample type before application of novel cytometry panels to a clinical study. We have shown that this DC enumeration flow cytometry panel is a robust analysis system that allows the flexibility of including activation markers.
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Published date: 6 February 2010
Keywords:
dendritic cell, validation, enumeration, activation, flow cytometry
Identifiers
Local EPrints ID: 73509
URI: http://eprints.soton.ac.uk/id/eprint/73509
ISSN: 1552-4922
PURE UUID: 79b465b9-0596-42ba-92ea-f2ddfd9cff2f
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Date deposited: 08 Mar 2010
Last modified: 14 Mar 2024 02:41
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Author:
Ferdousi Chowdhury
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