Development and evaluation of a whole blood-based approach for flow cytometric quantification of CD154+ mould-reactive T cells
| Autor: | Lukas Page, Sebastian Wurster, Juergen Loeffler, Hermann Einsele, Philipp Weis, Maria Lazariotou, Johanna Helm, Andrew J. Ullmann, Chris D. Lauruschkat |
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| Rok vydání: | 2018 |
| Předmět: |
CD4-Positive T-Lymphocytes
Antigens Fungal CD40 Ligand Major histocompatibility complex Peripheral blood mononuclear cell Sensitivity and Specificity Aspergillus fumigatus Fungal Proteins 03 medical and health sciences medicine Blood test Humans CD154 030304 developmental biology Whole blood 0303 health sciences CD40 biology medicine.diagnostic_test 030306 microbiology Chemistry Fungi General Medicine biology.organism_classification Flow Cytometry Molecular biology Healthy Volunteers Infectious Diseases biology.protein Mucorales Antibody Invasive Fungal Infections |
| Zdroj: | Medical mycology. 58(2) |
| ISSN: | 1460-2709 |
| Popis: | CD154+ mould-reactive T cells were proposed as a novel biomarker in the diagnosis of invasive mycoses. As PBMC-based protocols for flow cytometric quantification of these cells are logistically challenging and susceptible to preanalytic delays, this study evaluated and optimized a whole blood-based method for the detection of mould-reactive T cells. Blood collection tubes containing costimulatory antibodies and Aspergillus fumigatus mycelial lysates were inoculated with heparinized whole blood from healthy adults, and detection rates of CD154+/CD4+A. fumigatus reactive T cells were compared with PBMC-based detection using samples from the same donors. In contrast to the PBMC-based method, double costimulation with αCD28 and αCD49d was crucial for reliable whole blood stimulation. Optimizing stimulation schemes for both matrixes, significantly higher specific T-cell detection rates were achieved by the whole blood-based method, whereas the unspecific background stimulation remained low. MHC II-dependent CD154+ upregulation was demonstrated for both matrixes. Excellent correlation and reproducible conversion factors between whole blood and PBMC-based results were observed. Using frozen ready-to-use test tubes containing costimulatory antibodies and lysates, detection rates of specific T cells were comparable to freshly prepared blood collection tubes. The optimized whole blood-based protocol was also used to detect Rhizopus arrhizus and Rhizomucor pusillus reactive T cells, resulting in 1.5- to 2.7-fold higher detection rates compared with PBMC-based measurement. In summary, the whole blood protocol is a robust, highly sensitive, and cost-effective method for mould-reactive T-cell quantification, allowing for point-of-care sample stimulation and contributing to better assay standardization in multi-centre evaluation of mould reactive T-cell quantification. |
| Databáze: | OpenAIRE |
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