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本研究探讨实时定量PCR(Q-PCR)检测PML/RARαmRNA的方法学,并对46例初诊急性早幼粒细胞白血病(APL)患者骨髓标本进行检测。构建PML/RARα的bcr1型和bcr3型转录本以及内参照abl基因转录本的阳性标准品质粒;利用ABI Prism7500型Q-PCR仪对46例初诊APL患者和40例非APL患者骨髓标本进行检测,PML/RARαmRNA定量结果以校正比值(NQ)表示,NQ=PML/RARαmRNA拷贝数/ABLmRNA拷贝数;应用四色流式细胞术检测免疫表型。结果显示,Q-PCR结果的日间差和日内差平均变异系数(CV)分别为1.58%和0·88%。可重复敏感度为可以检测5copies/100ng RNA。40例非APL患者PML/RARαmRNA均为阴性。46例初诊APL患者PML/RARαmRNA表达量NQ中位值为0.450(0.084-1.082)。比较32例bcr1型和14例bcr3型两组患者的特征表明,PML/RARαmRNA NQ中位值分别为0.454(0.084-1.082)和0.386(0.151-0.848)(P>0·05)。形态学诊断M3v的患者比例分别为9.40%和48.96%(P<0.05);初诊时WBC中位数分别为2.15(0.2-59.6)和9·35(0.91-122.8)(P<0.05),而在性别、年龄、初诊时血红蛋白和血小板计数、骨髓中APL细胞比例、DIC指标等方面无差异。流式细胞仪术检测时,CD45/SSC射门情况下,APL细胞群分布可以分为两类:高侧向角(L-SSC,粗颗粒)和非高侧向角(NL-SSC,细颗粒)两类。bcr1型患者中85.70%表现为L-SSC,而bcr3型患者中64·29%表现为NL-SSC。结论:建立的Q-PCR方法稳定可靠,敏感度高;bcr1型和bcr3型APL患者的PML/RARαmRNA表达量无差异,bcr3型APL患者中形态学M3v比例和WBC数比bcr1型患者高;PML/RARα不同转录本类型和免疫表型以及细胞形态学之间有一定的相关性。
This study was to explore the methodology of real-time quantitative PCR (Q-PCR) for detection of PML / RARαmRNA and to detect bone marrow samples from 46 patients with newly diagnosed acute promyelocytic leukemia (APL). The positive standard plasmids of bcr1 and bcr3 transcripts of PML / RARα and transcripts of abl gene were constructed. The bone marrow samples from 46 newly diagnosed APL patients and 40 non-APL patients were detected by ABI Prism 7500 Q-PCR instrument, The quantitative results of PML / RARαmRNA were expressed as a calibration ratio (NQ), NQ = copy number of PML / RARαmRNA / copy number of ABLmRNA, and the immunophenotype was detected by four-color flow cytometry. The results showed that the intra-day difference and the intra-day difference (CV) of Q-PCR results were 1.58% and 0.88%, respectively. Repeatable sensitivity to detect 5copies / 100ng RNA. 40 cases of non-APL patients PML / RARαmRNA were negative. The median NQ of PML / RARαmRNA in 46 newly diagnosed APL patients was 0.450 (0.084-1.082). Comparison of the characteristics of 32 patients with bcr1 and 14 patients with bcr3 revealed that the median NQ values of PML / RARα mRNA were 0.454 (0.084-1.082) and 0.386 (0.151-0.848), respectively (P> 0.05). The proportions of patients diagnosed with M3v by morphology were 9.40% and 48.96%, respectively (P <0.05). The median WBC was 2.15 (0.2-59.6) and 9.35 (0.91-122.8), respectively (P <0.05) There were no differences in gender, age, hemoglobin and platelet count at initial diagnosis, APL cell ratio in bone marrow, DIC index and so on. In the case of CD45 / SSC shooting, the distribution of APL cells can be divided into two groups by flow cytometry: high lateral angle (L-SSC, coarse particle) and non-high lateral angle (NL-SSC, Two categories. 85.70% of patients with bcr1 showed L-SSC, whereas 64.29% of patients with bcr1 showed NL-SSC. Conclusion: The established Q-PCR method is stable and reliable with high sensitivity. The expression of PML / RARαmRNA in bcr1-type and bcr3-type APL patients has no difference. The ratio of morphological M3v and WBC in bcr3-type APL patients is higher than that in bcr1 type. / RARα There is a correlation between different transcript types and immunophenotypes and cell morphology.