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Quantification of A. fumigatus-specific CD154+ T-cells-preanalytic considerations
1University Hospital of Wuerzburg, Department of Internal Medicine II, Infectious Diseases, Oberduerr-bacher Str. 6, 97080 Wuerzburg, Germany.
Abstract:
Fungal specific CD154+ T-cells have been described as a biomarker in invasive aspergillosis. The influence of sample storage on the detection of these cells was assessed. Six-hour delay prior to PBMC isolation is associated with an 18% decrease of cell viability and alterations of the cellular composition of the sample. This results in 87% reduction of CD154+ A. fumigatus specific cells due to reduced assay sensitivity and increased background values in unstimulated samples. If prompt cell measurement is not feasible, isolated PBMCs can be frozen (at -20°C and -80°C) and processed later with comparable assay reliability (mean value fresh vs. thawing: 0.126, 0.133; Pearson-Coefficient: 0.962).
Insights
Delayed sample processing significantly reduces detection of fungal specific CD154+ T-cells, a biomarker for invasive aspergillosis. Freezing peripheral blood mononuclear cells (PBMCs) preserves assay reliability for later analysis.
Area of Science:
- Immunology
- Clinical Microbiology
- Medical Diagnostics
Background:
- CD154+ T-cells are recognized as a biomarker for invasive aspergillosis.
- The impact of sample storage conditions on detecting these specific immune cells requires investigation.
Purpose of the Study:
- To evaluate the effect of sample storage delays on the detection of fungal-specific CD154+ T-cells.
- To determine if freezing peripheral blood mononuclear cells (PBMCs) is a viable alternative for preserving assay reliability.
Main Methods:
- Assessing cell viability and composition after a six-hour delay in PBMC isolation.
- Quantifying CD154+ Aspergillus fumigatus specific cells using flow cytometry.
- Comparing assay results from fresh PBMCs versus those stored at -20°C and -80°C.
Main Results:
- A six-hour delay before PBMC isolation decreased cell viability by 18% and altered cellular composition.
- Delayed processing led to an 87% reduction in detectable CD154+ A. fumigatus specific cells.
- Freezing PBMCs at -20°C or -80°C allowed for later processing with comparable assay reliability (Pearson-Coefficient: 0.962).
Conclusions:
- Prompt PBMC isolation is crucial for accurate detection of fungal-specific CD154+ T-cells.
- Freezing PBMCs offers a reliable method for preserving these cells for later analysis when immediate processing is not possible.
