Effect of blood sample handling and reverse transcriptase-polymerase chain reaction assay sensitivity on detection of CK20 expression in healthy donor blood
Effect of blood sample handling and reverse transcriptase-polymerase chain reaction assay sensitivity on detection of CK20 expression in healthy donor blood
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DOI：
10.1097/00019606-200206000-00005
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发表时间：
2002-06-01
影响因子：
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通讯作者：
van Muijen, G
中科院分区：
文献类型：
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作者：
Vlems, F;Soong, R;van Muijen, G
关于细胞角蛋白20（CK 20）作为逆转录-聚合酶链反应分析（RT-PCR）标记物检测血液中播散性肿瘤细胞的特异性的数据是相互矛盾的。需要确定这些差异的根本原因，以阐明CK 20检测的意义。由于RT-PCR检测和血液样本处理的差异可能很重要，因此研究了它们对CK 20检测的影响。使用一系列掺入结肠肿瘤细胞的健康供体血液样本，作者比较了具有不同引物组的两种常规PCR和定量LightCycler PCR（Roche Diagnostics GmbH，Penzberg，德国）的灵敏度。此外，通过检查从健康供体采集的第一份和第二份血液的单核细胞组分（MNC）以及粒细胞组分中的CK 20表达，研究了样品采集和制备对测定特异性的影响。在一个加标肿瘤细胞/mL血液的浓度下，CK 20检测频率从常规PCR的17%和67%变化到LightCycler PCR的78%。在未加标的样本中。CK 20在0%和8%的常规PCR检测和11%的LightCycler PCR检测中检出。定量分析显示，CK 20在粒细胞样品中以高水平表达。结果表明，检测灵敏度和样品处理的差异影响血液中CK 20的检测。
Data concerning the specificity of cytokeratin 20 (CK20) as a reverse transcriptase-polymerase chain reaction analysis (RT-PCR) marker to detect disseminated tumor cells in blood are conflicting. Underlying causes for these discrepancies need to be determined to clarify the significance of CK20 detection. Because differences in RT-PCR assays and blood sample handling may be important, their influence on CK20 detection was studied. Using a series of healthy donor blood samples spiked with colon tumor cells, the authors compared the sensitivities of two conventional PCRs with different primer sets and a quantitative LightCycler PCR (Roche Diagnostics GmbH, Penzberg, Germany). Additionally, the influence of sample collection and preparation on assay specificity was studied by examining CK20 expression in the mononuclear cell fraction (MNC) of the first and the second aliquot of blood drawn from healthy donors and in the granulocyte cell fraction. At the concentration of one spiked tumor cell/mL blood, the CK20 detection frequency varied from 17% and 67% for the conventional to 78% for LightCycler PCR. In the unspiked samples. CK20 was detected in 0% and 8% of the conventional and 11% of the LightCycler PCR tests. Quantitative analysis revealed that CK20 was expressed at a high level in the granulocyte samples. The results demonstrate that differences in assay sensitivity and sample handling influence CK20 detection in blood.