Vaccinology is confronted with diseases for which the control of T-cell responses by the vaccine is essential. Among the assays that have been designed to assess T-cell responses, intracellular cytokine staining (ICS) combined with flow cytometry is well-suited in the frame of clinical trials. This assay can be used starting from isolated peripheral blood mononuclear cells (PBMC) or from whole blood (WB), but firm equivalence between the two sample preparation methods has yet to be established. Therefore, we compared both methods by analyzing the frequency of antigen-specific CD4⁺ T cells expressing at least two of four immune markers in human samples taken from two independent clinical trials (NCT00397943 and NCT00805389) with a qualified ICS assay. In the first study, M72-specific CD4⁺ T-cell responses were analyzed using WB-ICS and PBMC-ICS in 293 samples. Of these, 128 were double positive (value ≥ lower limit of quantification [LLOQ] with both methods), 130 were double negative and only 35 sample results were discordant, leading to an overall agreement of 88.05%. When analyzing the 128 double positive samples, it was found that the geometric mean of ratios (GMR) for paired observations was 0.98, which indicates a very good alignment between the two methods. The Deming regression fitted between the methods also showed a good correlation with an estimated slope being 1.1085. In the second study, HBsAg-specific CD4⁺ T cell responses were analyzed in 371 samples. Of these, 100 were double positive, 195 were double negative and 76 sample results were discordant, leading to an overall agreement of 79.51%. The GMR for paired observations was equal to 1.20, caused by a trend for overestimation in favor of the WB samples in the very high frequencies. The estimated slope of the Deming regression was 1.3057.

In conclusion, we demonstrated that WB and PBMC methods of sample collection led to statistically concordant ICS results, indicating that WB-ICS is a suitable alternative to PBMC-ICS to analyze clinical trial samples.

疫苗学面临的疾病需要通过疫苗来控制 T 细胞反应。在旨在评估 T 细胞反应的分析中，细胞内细胞因子染色 (ICS) 与流式细胞术相结合非常适合临床试验的框架。该测定可从分离的外周血单核细胞 (PBMC) 或全血 (WB) 开始使用，但两种样品制备方法之间的确切等效性尚未确定。因此，我们通过使用合格的 ICS 测定分析取自两项独立临床试验（NCT00397943 和 NCT00805389）的人类样本中表达至少四种免疫标记物中的两种的抗原特异性 CD4 ⁺ T 细胞的频率来比较两种方法。在第一项研究中，M72 特异性 CD4 ⁺使用 WB-ICS 和 PBMC-ICS 在 293 个样本中分析了 T 细胞反应。其中，128个为双阳性（两种方法均≥定量下限[LLOQ]），130个为双阴性，仅有35个样本结果不一致，总体一致性为88.05%。在分析 128 个双阳性样本时，发现配对观察的几何平均比率 (GMR) 为 0.98，这表明两种方法之间的一致性非常好。拟合方法之间的戴明回归也显示出良好的相关性，估计斜率为 1.1085。在第二项研究中，HBsAg 特异性 CD4 ⁺在 371 个样本中分析了 T 细胞反应。其中，双重阳性100份，双重阴性195份，不一致76份，总体一致率为79.51%。配对观测的 GMR 等于 1.20，这是由于高估的趋势有利于非常高频率的 WB 样本。Deming 回归的估计斜率为 1.3057。

总之，我们证明 WB 和 PBMC 样本收集方法导致 ICS 结果在统计上一致，表明 WB-ICS 是 PBMC-ICS 分析临床试验样本的合适替代品。