A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.

定量检测CD3Z基因去甲基化CpG启动子位点的拷贝数，可用于估算人体血液及组织中T细胞的数量与占比。甲基化特异性定量PCR（qPCR）虽可用于T细胞相关研究，但需进行大量校准，且在低拷贝数情况下检测精度不足。本研究以157份来自门诊对照人群及原发性胶质瘤患者的亚硫酸氢盐转化DNA为样本，将新型数字PCR平台（液滴数字PCR，droplet digital PCR，ddPCR）的检测性能与常规qPCR进行对比。我们同时将ddPCR与qPCR的检测结果，与常规流式细胞术（Fluorescence-Activated Cell Sorting，FACS）对CD3阳性T细胞的检测结果进行了比对。对同一血液样本的重复检测结果显示，ddPCR的检测变异度低于qPCR。qPCR与ddPCR的检测结果均与外周血CD3细胞计数及CD3/总白细胞比值的FACS检测结果呈显著相关。然而，一致性统计分析结果显示，ddPCR的线性一致性优于qPCR，且其检测绝对值与FACS结果更为接近。qPCR与ddPCR均可区分T细胞占比的临床显著性差异，且检测性能与FACS相当。鉴于ddPCR具备更高的精密度、准确性以及更简便的操作流程，相较于常规qPCR，该方法是一种更优异的基于DNA甲基化的T细胞评估手段。