Development and evaluation of a whole blood-based approach for flow cytometric quantification of CD154+ mould-reactive T cells. (16th May 2019)

• Record Type: Journal Article
• Authors: Weis, Philipp; Helm, Johanna; Page, Lukas; Lauruschkat, Chris D; Lazariotou, Maria; Einsele, Hermann; Loeffler, Juergen; Ullmann, Andrew J; Wurster, Sebastian
• Is Part Of: Medical mycology. Volume 58:Number 2(2020)
• Page Start: 187
• Page End: 196
• Publication Date: 2019-05-16
• Subjects: Aspergillus -- Mucorales -- biomarker -- immunology -- T cells -- blood test; Medical mycology -- Periodicals; Veterinary mycology -- Periodicals; Mycology -- Periodicals; Mycoses -- Periodicals; Pathogenic fungi -- Periodicals; 616.969005
• DOI: 10.1093/mmy/myz038
• Languages: English
• ISSNs: 1369-3786

Abstract

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.