Kagina Benjamin M, Mansoor Nazma, Kpamegan Eloi P, Penn-Nicholson Adam, Nemes Elisa, Smit Erica, Gelderbloem Sebastian, Soares Andreia P, Abel Brian, Keyser Alana, Sidibana Mzwandile, Hughes Jane E, Kaplan Gilla, Hussey Gregory D, Hanekom Willem A, Scriba Thomas J
South African Tuberculosis Vaccine Initiative (SATVI), Institute of Infectious Disease and Molecular Medicine and School of Child and Adolescent Health, University of Cape Town, South Africa; Vaccines for Africa Initiative, Division of Medical Microbiology & Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa.
South African Tuberculosis Vaccine Initiative (SATVI), Institute of Infectious Disease and Molecular Medicine and School of Child and Adolescent Health, University of Cape Town, South Africa; BD Biosciences, Johannesburg, South Africa.
J Immunol Methods. 2015 Feb;417:22-33. doi: 10.1016/j.jim.2014.12.003. Epub 2014 Dec 16.
Qualified or validated assays are essential in clinical trials. Short-term stimulation of whole blood and intracellular cytokine staining assay is commonly used to measure immunogenicity in tuberculosis vaccine clinical trials. Previously, the short-term stimulation process of whole blood with BCG was optimized. We aimed to qualify the intracellular cytokine staining process and assess the effects of long-term cryopreservation. Our hypotheses were that the assay is robust in the measurement of the mycobacteria-specific T cells, and long-term cryopreservation of fixed cells from stimulated whole blood would not compromise reliable measurement of mycobacteria induced CD4 T cell immunity.
Whole blood from healthy adults was collected in sodium heparinized tubes. The blood was left unstimulated or stimulated with mycobacterial antigens or mitogens for 12h. Cells were harvested, fixed and multiple aliquots from each participant cryopreserved. Later, mycobacteria-specific CD4 and CD8 T cells expressing IFN-γ, TNF-α, IL-2 and IL-17 were quantitated by flow cytometry. Assay performance characteristics evaluated included limit of quantification and detection, reproducibility, precision, robustness, specificity and sensitivity. To assess the effects of long-term cryopreservation, fixed cells from the stimulated bloods were analysed one week post-cryopreservation and at 3-month intervals over a 3-year period.
The limit of quantification for the different cytokines was variable: 0.04% for frequencies of IFN-γ- and IL-2-expressing T cells and less than 0.01% for TNF-α- and IL-17-expressing T cells. When measurement of the mycobacteria-specific T cells was assessed at levels above the detection limit, the whole blood intracellular cytokine assay showed high precision that was operator-independent. The assay was also robust: variation in staining conditions including temperature (4 °C or 20-23 °C) and time (45, 60 or 90 min) did not markedly affect quantification of specific T cells. Finally, prolonged periods of cryopreservation also did not significantly influence quantification of mycobacteria-specific CD4 T cells.
The whole blood intracellular cytokine assay is robust and reliable in quantification of the mycobacteria-specific T cells and is not significantly affected by cryopreservation of fixed cells.
在临床试验中，合格或经过验证的检测方法至关重要。全血短期刺激和细胞内细胞因子染色检测常用于结核病疫苗临床试验中的免疫原性检测。此前，已对卡介苗对全血的短期刺激过程进行了优化。我们旨在验证细胞内细胞因子染色过程，并评估长期冷冻保存的影响。我们的假设是，该检测方法在检测分枝杆菌特异性T细胞方面具有稳健性，并且长期冷冻保存受刺激全血中的固定细胞不会影响对分枝杆菌诱导的CD4 T细胞免疫的可靠检测。
将健康成年人的全血采集到肝素钠抗凝管中。血液不进行刺激或用分枝杆菌抗原或丝裂原刺激12小时。收集细胞，固定，并将每个参与者的多个等分试样冷冻保存。随后，通过流式细胞术对表达IFN-γ、TNF-α、IL-2和IL-17的分枝杆菌特异性CD4和CD8 T细胞进行定量。评估的检测性能特征包括定量限和检测限、重现性、精密度、稳健性、特异性和敏感性。为了评估长期冷冻保存的影响，在冷冻保存一周后以及在3年期间每隔3个月对受刺激血液中的固定细胞进行分析。
不同细胞因子的定量限各不相同：表达IFN-γ和IL-2的T细胞频率的定量限为0.04%，表达TNF-α和IL-17的T细胞的定量限小于0.01%。当在高于检测限的水平评估分枝杆菌特异性T细胞的检测时，全血细胞内细胞因子检测显示出高度的精密度，且与操作人员无关。该检测方法也具有稳健性：染色条件的变化，包括温度（4°C或20 - 23°C）和时间（45、60或90分钟），并未显著影响特异性T细胞的定量。最后，长时间的冷冻保存也未显著影响分枝杆菌特异性CD4 T细胞的定量。
全血细胞内细胞因子检测在定量分枝杆菌特异性T细胞方面具有稳健性和可靠性，并且不受固定细胞冷冻保存的显著影响。
