Introduction
Materials and methods
Samples
Selection and shipment of Peripheral Blood Mononuclear Cells (PBMC) samples
Antigens
Assays and data acquisition
Pre-screening to identify donors with FLU- and/or CMV-specific CD8+ T cells
Design of the ICS proficiency panels
Data analysis and interpretation
Laboratory environment
Results
Phase 1 proficiency panel for ICS
Laboratory ID | Phase 1 | Phase 2 | Phase 2 central analysis |
---|---|---|---|
5 | 8 | n.t. | n.t. |
8 | 7 | 5 | 3 |
9 | 7 | 8 | 8 |
10 | 6 | 6 | 6 |
13 | 5 | n.t. | n.t. |
15 | 5 | 7 | 5 |
22 | 2 | 7 | 5 |
24 | 3 | 3 | 3 |
26 | 0 | n.t. | n.t. |
Total to be detected | 8 | 8 | 8 |
Phase 2 proficiency panel for ICS
Phase 3 in silico proficiency panel to harmonize the gating strategy
Findings/errors | Recommendation and reason |
---|---|
1. Gating was done only on CD3−, CD4− and/or CD8− high expressing cells | Include dim populations as these may contain cytokine-producing cells (cells can downregulate co-receptor molecules upon activation) |
2. Gate for cytokine-positive cells was set through the cytokine-positive population | Include plots in your gating strategy for CD3+ CD4− (in case of looking at cytokine-producing CD8+ T cells) or CD3+ CD8− (for CD4) T cells to oversee all cytokine-producing cells and be able to gate on the complete cytokine-positive population for that certain T-cell type (so including dim) |
3. Gate for cytokine-positive cells was set too close to or included cytokine-negative cells | Set the gate far enough (but not too far) from the non-responding population to decrease the background staining in the unstimulated sample and increase the signal-to-noise ratio |