Development and evaluation of a whole blood-based approach for flow cytometric quantification of CD154+ mould-reactive T cells

Med Mycol. 2020 Feb 1;58(2):187-196. doi: 10.1093/mmy/myz038.


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.

Keywords: Aspergillus; Mucorales; T cells; biomarker; blood test; immunology.

Publication types

  • Evaluation Study

MeSH terms

  • Antigens, Fungal / immunology*
  • Aspergillus fumigatus / immunology
  • CD4-Positive T-Lymphocytes / immunology*
  • CD40 Ligand / blood
  • Flow Cytometry*
  • Fungal Proteins / immunology
  • Fungi / chemistry
  • Fungi / immunology*
  • Healthy Volunteers
  • Humans
  • Invasive Fungal Infections / blood
  • Invasive Fungal Infections / diagnosis*
  • Mucorales / immunology
  • Sensitivity and Specificity


  • Antigens, Fungal
  • Fungal Proteins
  • CD40 Ligand