Development and evaluation of a whole blood-based approach for flow cytometric quantification of CD154+ mould-reactive T cells
Weis P, Helm J, Page L, Lauruschkat CD, Lazariotou M, Einsele H, Loeffler J, Ullmann AJ, Wurster S (2020)
Publication Type: Journal article
Publication year: 2020
Journal
Book Volume: 58
Pages Range: 187-196
Journal Issue: 2
DOI: 10.1093/mmy/myz038
Abstract
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.
Involved external institutions
Germany (DE)How to cite
APA:
Weis, P., Helm, J., Page, L., Lauruschkat, C.D., Lazariotou, M., Einsele, H.,... Wurster, S. (2020). Development and evaluation of a whole blood-based approach for flow cytometric quantification of CD154+ mould-reactive T cells. Medical Mycology, 58(2), 187-196. https://doi.org/10.1093/mmy/myz038
MLA:
Weis, Philipp, et al. "Development and evaluation of a whole blood-based approach for flow cytometric quantification of CD154+ mould-reactive T cells." Medical Mycology 58.2 (2020): 187-196.
BibTeX: Download