Quality control methods for optimal BCR-ABL1 clinical testing in human whole blood samples.
Quality control methods for optimal BCR-ABL1 clinical testing in human whole blood samples.
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DOI：
10.1016/j.jmoldx.2013.02.004
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发表时间：
2013-05
期刊：
影响因子：
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通讯作者：
Lauren M. Stanoszek;E. Crawford;Thomas M. Blomquist;Jessica A Warns;P. F. Willey;J. Willey
中科院分区：
文献类型：
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作者：
Lauren M. Stanoszek;E. Crawford;Thomas M. Blomquist;Jessica A Warns;P. F. Willey;J. Willey
可靠的断点簇区(BCR) -Abelson (ABL) 1测量对于慢性粒细胞白血病的最佳管理至关重要。有必要优化用于测量主要分子反应和/或治疗失败的方法的质量控制、灵敏度和可靠性。在K562细胞混合全血样品中，研究室温保存时间、不同引物和逆转录（RT）反应中RNA输入对BCR-ABL1和β-葡萄糖醛酸酶（GUSB） cDNA产率的影响。BCR-ABL1相对于GUSB进行测量，以控制样品装载，每个基因相对于已知数量的各自内部标准分子进行测量，以控制试剂质量和数量的变化，热循环器条件和PCR抑制剂的存在。临床样品和参考物质的测量结果与其他实验室报告的结果一致。储存48小时后，BCR-ABL1 / 103GUSB值显著降低（P= 0.004）。在每个RNA输入下，基因特异性引物比随机六聚体产生更多的BCR-ABL1 cDNA。此外，增加的RNA抑制随机六聚体的RT反应，而不抑制基因特异性引物。因此，在所有测试的RNA输入中，基因特异性RT引物的BCR-ABL1产量更高，增加到多达158倍。我们得出结论，在RT中使用基因特异性引物并在采集血液后24小时内分析样品时，可以获得全血中每103GUSB BCR-ABL1的最佳测量值。
Reliable breakpoint cluster region (BCR)–Abelson (ABL) 1 measurement is essential for optimal management of chronic myelogenous leukemia. There is a need to optimize quality control, sensitivity, and reliability of methods used to measure a major molecular response and/or treatment failure. The effects of room temperature storage time, different primers, and RNA input in the reverse transcription (RT) reaction on BCR-ABL1 and β-glucuronidase (GUSB) cDNA yield were assessed in whole blood samples mixed with K562 cells. BCR-ABL1 was measured relative to GUSB to control for sample loading, and each gene was measured relative to known numbers of respective internal standard molecules to control for variation in quality and quantity of reagents, thermal cycler conditions, and presence of PCR inhibitors. Clinical sample and reference material measurements with this test were concordant with results reported by other laboratories. BCR-ABL1 per 103GUSB values were significantly reduced (P= 0.004) after 48-hour storage. Gene-specific primers yielded more BCR-ABL1 cDNA than random hexamers at each RNA input. In addition, increasing RNA inhibited the RT reaction with random hexamers but not with gene-specific primers. Consequently, the yield of BCR-ABL1 was higher with gene-specific RT primers at all RNA inputs tested, increasing to as much as 158-fold. We conclude that optimal measurement of BCR-ABL1 per 103GUSB in whole blood is obtained when gene-specific primers are used in RT and samples are analyzed within 24 hours after blood collection.