Fu Yu, Zhang Rui, Wu Qisheng, Zhang Jiawei, Bao Lihua, Li Jinming
National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, China.
Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China.
J Clin Lab Anal. 2018 Nov;32(9):e22612. doi: 10.1002/jcla.22612. Epub 2018 Jun 29.
Standards play an important role in detection of the BCR-ABL1 fusion gene (FG) transcript. However, the standards widely used in laboratories are mainly based on plasmids or cDNA, which cannot accurately reflect the process of RNA extraction and cDNA synthesis. Therefore, we aimed to develop armored RNA-based standards for p210 and p190 BCR-ABL1FG transcripts' quantification.
Using overlapping polymerase chain reaction (PCR) technology, we first linked a segment of the p210 or p190 BCR-ABL1FG transcript with four control genes (CGs; ABL1, BCR, GUSB, and B2M) to form p210FG-CG and p190FG-CG. Subsequently, using armored RNA technology, we prepared p210FG-CG- and p190FG-CG-armored RNAs and the p210FG-CG and p190FG-CG standards, the values of which were assigned by digital PCR (dPCR).
The p210FG-CG and p190FG-CG standards were stable and homogeneous, and were significantly linear with r > 0.98. A field trial including 52 laboratories across China showed that the coefficient of variation (CV%) of BCR-ABL1 values among samples was in the range of 58.6%-129.6% for p210 samples and 73.2%-194.0% for p190 samples when using local standards. By contrast, when using the p210FG-CG and p190FG-CG standards, the CV% of BCR-ABL1 values was decreased to 35.6%-124.9% and 36.6%-170.6% for p210 and p190 samples, respectively. In addition, 33.3% (3/9) of the p210 and p190 samples had CV% values <50.0%, whereas 44.4% (4/9) and 77.8% (7/9) of the samples had lower CV% values when using the p210FG-CG and p190FG-CG standards.
The overall variability of detection of BCR-ABL1 transcripts decreased significantly when using the p210FG-CG or p190FG-CG standards, especially the p190FG-CG standard.
标准在BCR-ABL1融合基因（FG）转录本的检测中发挥着重要作用。然而，实验室广泛使用的标准主要基于质粒或cDNA，无法准确反映RNA提取和cDNA合成过程。因此，我们旨在开发基于铠甲RNA的标准品，用于定量检测p210和p190 BCR-ABL1 FG转录本。
我们首先利用重叠聚合酶链反应（PCR）技术，将一段p210或p190 BCR-ABL1 FG转录本与四个对照基因（ABL1、BCR、GUSB和B2M）连接，形成p210FG-CG和p190FG-CG。随后，利用铠甲RNA技术，我们制备了p210FG-CG-和p190FG-CG-铠甲RNA以及p210FG-CG和p190FG-CG标准品，其值通过数字PCR（dPCR）确定。
p210FG-CG和p190FG-CG标准品稳定且均一，r>0.98时具有显著线性关系。一项涵盖中国52个实验室的现场试验表明，使用当地标准时，p210样本中BCR-ABL1值的变异系数（CV%）在58.6%-129.6%之间，p190样本在73.2%-194.0%之间。相比之下，使用p210FG-CG和p190FG-CG标准品时，p210和p190样本中BCR-ABL1值的CV%分别降至35.6%-124.9%和36.6%-170.6%。此外，33.3%（3/9）的p210和p190样本CV%值<50.0%，而使用p210FG-CG和p190FG-CG标准品时，分别有44.4%（4/9）和77.8%（7/9）的样本CV%值更低。
使用p210FG-CG或p190FG-CG标准品时，BCR-ABL1转录本检测的总体变异性显著降低，尤其是p190FG-CG标准品。
