Nilsson C, Aboud S, Karlén K, Hejdeman B, Urassa W, Biberfeld G
Dept. of Immunology and Vaccinology, Swedish Institute for Infectious Disease Control and Karolinska Institute, 171 82 Solna, Sweden.
Clin Vaccine Immunol. 2008 Apr;15(4):585-9. doi: 10.1128/CVI.00161-07. Epub 2008 Feb 20.
Determination of antigen-specific T-cell responses is an important part of vaccine assessment. High levels of recovery, viability, and functionality of peripheral blood mononuclear cells (PBMCs) are essential for reliable assessment of cell-mediated immune responses. Here, we sought to find the cell preparation technique best suited for two clinical vaccine trial sites: Stockholm, Sweden, and Dar es Salaam, Tanzania. Standard Ficoll-Paque gradient centrifugation, BD Vacutainer cell preparation tube (CPT), and Greiner Bio-One LeucoSep tube techniques were tested. Cell yield and viability were recorded. Gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) testing was used to assess cell functionality. No differences in mean recovery or mean viability of fresh PBMCs were observed between Ficoll-Paque gradient centrifugation and CPT techniques as used in Stockholm. In Dar es Salaam, recovery of PBMCs isolated by use of the Ficoll-Paque gradient technique was higher than that seen with CPT (1.58 +/- 0.6 versus 1.34 +/- 0.4 million cells/ml of blood [P = 0.0469]), and the viability of PBMCs processed by Ficoll-Paque gradient was higher than that seen with CPT-purified cells (95.8% +/- 2.3% versus 92.6% +/- 4.8% [P = 0.0081]). Furthermore, LeucoSep cell separation gave higher levels of yield (1.10 +/- 0.3 versus 0.92 +/- 0.3 million cells/ml of blood [P = 0.0022]) and viability (95.7% +/- 2.0% versus 93.4% +/- 3.2% [P = 0.0012]) than Ficoll-Paque cell separation. The cells purified by the different techniques at the two sites performed equally well in IFN-gamma ELISPOT assays. Both techniques generated cell preparations with excellent yield, viability, and functionality in Stockholm. In Dar es Salaam, CPT did not perform as well as Ficoll-Paque separation. In a subsequent comparison, LeucoSep performed better than Ficoll-Paque separation. Our findings emphasize the need for on-site assessment of PBMC purification techniques for optimal evaluation of cell-mediated immune responses.
抗原特异性T细胞反应的测定是疫苗评估的重要组成部分。外周血单核细胞（PBMC）的高回收率、活力和功能对于可靠评估细胞介导的免疫反应至关重要。在此，我们试图找到最适合两个临床疫苗试验地点的细胞制备技术：瑞典斯德哥尔摩和坦桑尼亚达累斯萨拉姆。对标准Ficoll-Paque梯度离心法、BD Vacutainer细胞制备管（CPT）法和Greiner Bio-One LeucoSep管法进行了测试。记录细胞产量和活力。采用γ干扰素（IFN-γ）酶联免疫斑点（ELISPOT）试验评估细胞功能。在斯德哥尔摩，Ficoll-Paque梯度离心法和CPT法在新鲜PBMC的平均回收率或平均活力方面未观察到差异。在达累斯萨拉姆，使用Ficoll-Paque梯度技术分离的PBMC回收率高于CPT法（1.58±0.6对1.34±0.4×10⁶个细胞/ml血液[P = 0.0469]），且Ficoll-Paque梯度处理的PBMC活力高于CPT纯化的细胞（95.8%±2.3%对92.6%±4.8%[P = 0.0081]）。此外，LeucoSep细胞分离法的产量（1.10±0.3对0.92±0.3×10⁶个细胞/ml血液[P = 0.0022]）和活力（95.7%±2.0%对93.4%±3.2%[P = 0.0012]）均高于Ficoll-Paque细胞分离法。在两个地点通过不同技术纯化的细胞在IFN-γ ELISPOT试验中的表现同样良好。在斯德哥尔摩，两种技术都能产生产量、活力和功能俱佳的细胞制剂。在达累斯萨拉姆，CPT的效果不如Ficoll-Paque分离法。在随后的比较中，LeucoSep的表现优于Ficoll-Paque分离法。我们的研究结果强调了对PBMC纯化技术进行现场评估以优化细胞介导免疫反应评估的必要性。
