Vlems F A, Ladanyi A, Gertler R, Rosenberg R, Diepstra J H S, Röder C, Nekarda H, Molnar B, Tulassay Z, van Muijen G N P, Vogel I
Department of Surgery, University Medical Centre Nijmegen, PO-box 9101, 6500HB, Nijmegen, The Netherlands.
Eur J Cancer. 2003 Feb;39(3):388-96. doi: 10.1016/s0959-8049(02)00631-7.
Differences in methods of reverse-transcriptase (RT)-polymerase chain reaction (PCR)-based detection of tumour cells in the blood gives rise to conflicting results, and standardisation is urgently needed. This pilot study aimed to assess the variation of RT-PCR-based detection of tumour cells in blood between four different laboratories using a commercially available kit with a standardised protocol. This kit allows comparison of results from different laboratories and facilitates the investigation of the influence of pre-analytical parameters. All laboratories analysed identical sets of blood samples spiked with tumour cells in a concentration range of 1-100 tumour cells/ml. To study at which level variation was introduced, three kinds of sample sets were generated in which (i) tumour cell RNA was spiked in the RNA of mononuclear cells (MNC), (ii) tumour cells were spiked in isolated MNC, and (iii) tumour cells were spiked in blood. Real-time quantitative RT-PCR was used to detect and quantify cytokeratin 20 (CK20) expression, which is indicative for the presence of epithelial tumour cells. All laboratories were able to detect CK20 expression in all spiked-RNA samples with limited variation in expression levels between laboratories. There was a positive correlation between the amount of spiked tumour cell RNA and CK20 expression level. RT-PCR analysis of spiked-MNC samples resulted in more variation in the CK20 expression levels between laboratories, however again all spiked samples were reported to be positive by all of the laboratories. The evaluation of spiked-blood samples gave rise to considerable quantitative and qualitative variation between the laboratories. Our results underline the importance and need for standardisation and extended quality control studies in the field of pre-analytics.
基于逆转录酶（RT）-聚合酶链反应（PCR）检测血液中肿瘤细胞的方法存在差异，导致结果相互矛盾，因此迫切需要标准化。这项初步研究旨在使用标准化方案的市售试剂盒，评估四个不同实验室之间基于RT-PCR检测血液中肿瘤细胞的差异。该试剂盒允许比较不同实验室的结果，并有助于研究分析前参数的影响。所有实验室分析了相同的一组添加了肿瘤细胞的血液样本，肿瘤细胞浓度范围为1-100个肿瘤细胞/毫升。为了研究差异出现在哪个水平，生成了三种样本集，其中（i）肿瘤细胞RNA添加到单核细胞（MNC）的RNA中，（ii）肿瘤细胞添加到分离的MNC中，（iii）肿瘤细胞添加到血液中。使用实时定量RT-PCR检测和定量细胞角蛋白20（CK20）的表达，其可指示上皮肿瘤细胞的存在。所有实验室都能够在所有添加RNA的样本中检测到CK20表达，实验室之间的表达水平差异有限。添加的肿瘤细胞RNA量与CK20表达水平之间存在正相关。对添加MNC样本的RT-PCR分析导致实验室之间CK20表达水平的差异更大，然而所有实验室再次报告所有添加样本均为阳性。对添加血液样本的评估导致实验室之间存在相当大的定量和定性差异。我们的结果强调了在分析前领域进行标准化和扩展质量控制研究的重要性和必要性。
