Heijnen Ingmar A F M, Barnett David, Arroz Maria J, Barry Simon M, Bonneville Marc, Brando Bruno, D'hautcourt Jean-Luc, Kern Florian, Tötterman Thomas H, Marijt Erik W A, Bossy David, Preijers Frank W M B, Rothe Gregor, Gratama Jan W
Division of Immunology, Department of Laboratory Medicine, Kantonsspital Aarau, Aarau, Switzerland.
Cytometry B Clin Cytom. 2004 Nov;62(1):1-13. doi: 10.1002/cyto.b.20028.
HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8(+) T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic assay. Hence, the European Working Group on Clinical Cell Analysis set out to develop and evaluate a single-platform tetramer-based method that used cytomegalovirus (CMV) as the antigenic model.
Absolute numbers of CMV-specific CD8(+) T cells were obtained by combining the percentage of tetramer-binding cells with the absolute CD8(+) T-cell count. Six send-outs of stabilized blood from healthy individuals or CMV-carrying donors with CMV-specific CD8(+) T-cell counts of 3 to 10 cells/microl were distributed to 7 to 16 clinical sites. These sites were requested to enumerate CD8(+) T cells and, in the case of CMV-positive donors, CMV-specific subsets on three separate occasions using the standard method.
Between-site coefficients of variation of less than 10% (absolute CD8(+) T-cell counts) and approximately 30% (percentage and absolute numbers of CMV-specific CD8(+) T cells) were achieved. Within-site coefficients of variation were approximately 5% (absolute CD8(+) T-cell counts), approximately 9% (percentage CMV-specific CD8(+) T cells), and approximately 17% (absolute CMV-specific CD8(+) T-cell counts). The degree of variation tended to correlate inversely with the proportion of CMV-specific CD8(+) T-cell subsets.
The single-platform MHC tetramer-based method for antigen-specific CD8(+) T-cell counting has been evaluated by a European group of laboratories and can be considered a reproducible assay for routine enumeration of antigen-specific CD8(+) T cells.
HLA I类肽四聚体是检测和监测抗原特异性CD8(+) T细胞的强大诊断工具。如果要将四聚体流式细胞术作为一种常规诊断检测方法实施，当前多中心研究的动力在于迫切需要对其进行标准化。因此，欧洲临床细胞分析工作组着手开发和评估一种基于四聚体的单平台方法，该方法使用巨细胞病毒（CMV）作为抗原模型。
通过将四聚体结合细胞的百分比与绝对CD8(+) T细胞计数相结合，获得CMV特异性CD8(+) T细胞的绝对数量。将来自健康个体或携带CMV的供体的6份稳定血液样本（CMV特异性CD8(+) T细胞计数为3至10个细胞/微升）分发给7至16个临床站点。要求这些站点使用标准方法在三个不同时间点对CD8(+) T细胞进行计数，对于CMV阳性供体，还要对CMV特异性亚群进行计数。
实现了位点间变异系数小于10%（绝对CD8(+) T细胞计数）和约30%（CMV特异性CD8(+) T细胞的百分比和绝对数量）。位点内变异系数约为5%（绝对CD8(+) T细胞计数）、约9%（CMV特异性CD8(+) T细胞百分比）和约17%（CMV特异性CD8(+) T细胞绝对计数）。变异程度往往与CMV特异性CD8(+) T细胞亚群的比例呈负相关。
欧洲一组实验室对基于单平台MHC四聚体的抗原特异性CD8(+) T细胞计数方法进行了评估，该方法可被视为一种用于常规计数抗原特异性CD8(+) T细胞的可重复检测方法。
