Introduction
Materials and methods
Panel design
Participants and organizational setup
PBMCs and peptides
HLA-peptide multimer staining
Statistical analysis
Results
Human auditing process of final results
Overview of assay protocols currently in use at the international level
Inter- and intra-center variation
Antigen | Donor | Median | 25th Percentile | 75th Percentile | Mean | SD | CV | Min | Max |
---|---|---|---|---|---|---|---|---|---|
Influenza-M1 | D1 | 0.07 | 0.04 | 0.15 | 0.32 | 0.67 | 210.58a
| 0.00 | 2.87 |
D2 | 0.67 | 0.50 | 0.89 | 0.73 | 0.34 | 47.16 | 0.24 | 2.05 | |
D3 | 0.16 | 0.11 | 0.32 | 0.29 | 0.35 | 122.95 | 0.00 | 1.53 | |
D4 | 0.19 | 0.17 | 0.27 | 0.28 | 0.26 | 93.66 | 0.07 | 1.44 | |
D5 | 0.35 | 0.31 | 0.41 | 0.44 | 0.25 | 57.05 | 0.21 | 1.21 | |
Melan-A/Mart-1 | D1 | 0.07 | 0.03 | 0.19 | 0.23 | 0.48 | 207.48b
| 0.00 | 2.53 |
D2 | 0.10 | 0.03 | 0.17 | 0.17 | 0.27 | 156.14 | 0.00 | 1.30 | |
D3 | 0.12 | 0.06 | 0.23 | 0.25 | 0.40 | 157.76 | 0.02 | 2.09 | |
D4 | 0.10 | 0.04 | 0.15 | 0.17 | 0.23 | 135.44 | 0.01 | 1.06 | |
D5 | 0.08 | 0.03 | 0.17 | 0.18 | 0.26 | 142.63 | 0.01 | 1.06 |
Limitations of the multimer assay
Individual laboratory performance
Antigen | Donor | Frequency | Response |
---|---|---|---|
Influenza-M1 | D1 | 0.11 | >LOD and <LOQ |
D2 | 0.76 | High | |
D3 | 0.26 | Moderate | |
D4 | 0.28 | Moderate | |
D5 | 0.39 | Moderate | |
Melan-A/Mart-1 | D1 | 0.00 | Negative |
D2 | 0.22 | Moderate | |
D3 | 0.31 | Moderate | |
D4 | 0.19 | Low | |
D5 | 0.16 | Low |
No. of detected responses | No. of labs | Percentage |
---|---|---|
0 | 2 | 7 |
1 | 1 | 3 |
3 | 3 | 10 |
4 | 1 | 3 |
5 | 2 | 7 |
6 | 5 | 17 |
7 | 5 | 17 |
8 | 7 | 24 |
9 | 3 | 10 |
Subgroup-analysis
Number of CD8-positive cells | Number of triplicates | Number of responses | Responses (%) |
---|---|---|---|
<10,000 | 32 | 4 | 13 |
10–30,000 | 40 | 24 | 60 |
30–100,000 | 144 | 105 | 73 |
>100,000 | 45 | 39 | 87 |
Number of CD8-positive cells | Number of triplicates | Number of low responses | Low responses (%) |
---|---|---|---|
<10,000 | 15 | 0 | 0 |
10–30,000 | 18 | 4 | 22 |
30–100,000 | 63 | 34 | 54 |
>100,000 | 20 | 16 | 80 |
Discussion
Initial harmonization guidelines to improve assay performance
(A) Establish lab SOP for MHC-peptide multimer staining |
A1 Count at least 100,000 CD8 T cells per staining |
A2 Introduce a background control to set gates |
A3 Use more than two colors for staining |
(B) Establish SOP for software analyses of stained samples, including |
B1 Gating strategy |
B2 Rules to set the gates |
(C) Establish a human auditing process of all final results |
C1 Are all dot plots correctly compensated? |
C2 Have the gates been set correctly? |
C3 Are the calculated frequencies of multimer-positive cells plausible? |
(D) Only let well trained personnel (per lab SOP) conduct assay |