Meijerink J, Mandigers C, van de Locht L, Tönnissen E, Goodsaid F, Raemaekers J
Department of Pediatrics, Division of Oncology/Hematology, Sophia Children's Hospital, Erasmus University Rotterdam, Rotterdam, The Netherlands.
J Mol Diagn. 2001 May;3(2):55-61. doi: 10.1016/S1525-1578(10)60652-6.
Quantification of residual disease by real-time polymerase chain reaction (PCR) will become a pivotal tool in the development of patient-directed therapy. In recent years, various protocols to quantify minimal residual disease in leukemia or lymphoma patients have been developed. These assays assume that PCR efficiencies are equal for all samples. Determining t(14;18) and albumin reaction efficiencies for sixteen follicular lymphoma patient samples revealed higher efficiencies for blood samples than for lymph node samples in general. However, within one sample both reactions had equivalent efficiencies. Differences in amplification efficiencies between patient samples (low efficiencies) and the calibrator in quantitative analyses result in the underestimation of residual disease in patient samples whereby the weakest positive patient samples are at highest error. Based on these findings for patient samples, the efficiency compensation control was developed. This control includes two reference reactions in a multiplex setting, specific for the beta-actin and albumin housekeeping genes that are present in a constant ratio within DNA templates. The difference in threshold cycle values for both reference reactions, ie, the Ct(2) value, is dependent on the amplification efficiency, and is used to compensate for efficiency differences between patient samples and the calibrator. The beta-actin reference reaction is also used to normalize for DNA input. Furthermore, the efficiency compensation control facilitates identification of patient samples that are so contaminated with PCR inhibitory compounds that different amplification reactions are affected to a different extent. Accurate quantitation of residual disease in these samples is therefore impossible with the current quantitative real-time PCR protocols. Identification and exclusion of these inadequate samples will be of utmost importance in quantitative retrospective studies, but even more so, in future molecular diagnostic analyses.
通过实时聚合酶链反应（PCR）对残留疾病进行定量分析将成为患者导向治疗发展中的关键工具。近年来，已经开发出各种用于量化白血病或淋巴瘤患者微小残留疾病的方案。这些检测方法假定所有样本的PCR效率相同。对16例滤泡性淋巴瘤患者样本的t(14;18)和白蛋白反应效率进行测定后发现，总体而言，血液样本的效率高于淋巴结样本。然而，在同一样本中，两种反应的效率相当。在定量分析中，患者样本（低效率）与校准物之间的扩增效率差异会导致对患者样本中残留疾病的低估，其中最弱的阳性患者样本误差最大。基于对患者样本的这些发现，开发了效率补偿对照。该对照在多重设置中包括两个参考反应，分别针对DNA模板中以恒定比例存在的β-肌动蛋白和白蛋白管家基因。两种参考反应的阈值循环值之差，即Ct(2)值，取决于扩增效率，并用于补偿患者样本与校准物之间的效率差异。β-肌动蛋白参考反应还用于对DNA输入进行标准化。此外，效率补偿对照有助于识别那些被PCR抑制性化合物严重污染的患者样本，不同的扩增反应受到的影响程度不同。因此，使用当前的定量实时PCR方案无法对这些样本中的残留疾病进行准确定量。在定量回顾性研究中，识别和排除这些不合格样本至关重要，但在未来的分子诊断分析中更是如此。
