Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands.
Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands.
Methods Mol Biol. 2022;2453:191-208. doi: 10.1007/978-1-0716-2115-8_12.
An accurate T cell quantification is prognostically and therapeutically relevant in various clinical applications, including oncology care and research. In this chapter, we describe how T cell quantifications can be obtained from bulk DNA samples with a multiplex digital PCR experiment. The experimental setup includes the concurrent quantification of three different DNA targets within one reaction: a unique T cell DNA marker, a regional corrector, and a reference DNA marker. The T cell marker is biallelically absent in T cells due to VDJ rearrangements, while the reference is diploid in all cells. The so-called regional corrector allows to correct for possible copy number alterations at the T cell marker locus in cancer cells. By mathematically integrating the measurements of all three markers, T cells can be accurately quantified in both copy number stable and unstable DNA samples.
准确的 T 细胞定量在各种临床应用中具有预后和治疗相关性，包括肿瘤学护理和研究。在本章中，我们将描述如何使用多重数字 PCR 实验从批量 DNA 样本中获得 T 细胞定量。实验设置包括在一个反应中同时定量三种不同的 DNA 靶标：独特的 T 细胞 DNA 标记物、区域校正物和参考 DNA 标记物。由于 VDJ 重排，T 细胞中的 T 细胞标记物呈双等位基因缺失，而参考物在所有细胞中均为二倍体。所谓的区域校正物允许校正癌细胞中 T 细胞标记物基因座可能的拷贝数改变。通过对所有三个标记物的测量进行数学整合，可以在拷贝数稳定和不稳定的 DNA 样本中准确地定量 T 细胞。
