Zhu Hong-Hu, Liu Yan-Rong, Qin Ya-Zhen, Li Jin-Lan, Chang Yan, Wang Ya-Zhe, Shan Fu-Xiang, Jiang Bin, Lu Dao-Pei
Institute of Hematology, People Hospital, Peking University, Beijing 100044, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2007 Feb;15(1):1-5.
In order to explore the application of real-time quantitative reverse-transcription polymerase chain reaction (Q-PCR) for detecting PML/RARalpha gene transcripts in patients with acute promyelocytic leukemia (APL), the bone marrow samples from 46 newly diagnosed APL patients were collected for analysis. Three plasmids containing cDNA fragments of the bcr1-, bcr3-form PML/RARalpha and ABL control gene were constructed respectively. The ABI Prism 7500 Sequence Detection System using Taqman fluorogenic probes was used to quantify target gene. PML/RARalpha mRNA was detected by Q-PCR in 46 APL patients and 40 non-APL patients. The normalized quotient (NQ) of PML/RARalpha mRNA was calculated as followings: NQ = PML/RARalpha mRNA copy numbers/ABL mRNA copy numbers. Immunophenotype of acute promyelocytic leukemia was determined by four-color flow cytometry. The results showed that the coefficients of variation (CV) of inter-day assay and intra-day assay by using Q-PCR were 1.58% and 0.88% respectively. Q-PCR could detect reproducibly 5 copies of target gene per 100 ng RNA and no pseudopositive results were found. The median NQ of PML/RARalpha mRNA was 0.450 (0.084 - 1.082) in 46 APL patients. There was no indication of any correlation of PML/RARalpha mRNA type with age, sex, hemoglobin, platelet count, percentage of promyelocytes in bone marrow detected by morphology or flow cytometry, PML/RARalpha NQ, or signs of clinically diagnosed coagulation/bleeding disorders. Compared with bcr1-form cases, bcr3-form cases had more M(3v) phenotype (42.9% vs 9.4%, P = 0.015) and higher WBC count (9.35 x 10(9)/L vs 2.15 x 10(9)/L, P = 0.038). APL cells could be classified into large side scatter population (L-SSC) and non-large side scatter population (NL-SSC) in CD45/SSC histogram of flow cytometry. 87.50% patients with bcr1-form showed L-SSC phenotype and 64.29% patients with bcr3-form showed NL-SSC phenotype. It is concluded that a sensitive Q-PCR method is established. The median NQ of PML/RARalpha mRNA was 0.450 in newly diagnosed APL patients. There was no significant difference about PML/RARalpha mRNA expression of both bcr3-form and bcr1-form APL patients. Type of PML/RARalpha transcripts is related with the morphology and immunophenotype.
为探讨实时定量逆转录聚合酶链反应（Q-PCR）检测急性早幼粒细胞白血病（APL）患者PML/RARα基因转录本的应用价值，收集46例新诊断APL患者的骨髓样本进行分析。分别构建了含bcr1-、bcr3-型PML/RARα及ABL对照基因cDNA片段的三种质粒。采用Taqman荧光探针的ABI Prism 7500序列检测系统对靶基因进行定量。用Q-PCR检测46例APL患者及40例非APL患者的PML/RARα mRNA。PML/RARα mRNA的标准化商（NQ）计算如下：NQ = PML/RARα mRNA拷贝数/ABL mRNA拷贝数。采用四色流式细胞术检测急性早幼粒细胞白血病的免疫表型。结果显示，Q-PCR法日间和日内检测的变异系数（CV）分别为1.58%和0.88%。Q-PCR可重复性检测到每100 ng RNA中5个拷贝的靶基因，未发现假阳性结果。46例APL患者中PML/RARα mRNA的NQ中位数为0.450（0.084 - 1.082）。未发现PML/RARα mRNA类型与年龄、性别、血红蛋白、血小板计数、形态学或流式细胞术检测的骨髓早幼粒细胞百分比、PML/RARα NQ或临床诊断的凝血/出血障碍体征之间存在相关性。与bcr1-型病例相比，bcr3-型病例具有更多的M(3v)表型（42.9%对9.4%，P = 0.015）和更高的白细胞计数（9.35×10⁹/L对2.15×10⁹/L，P = 0.038）。在流式细胞术的CD45/SSC直方图中，APL细胞可分为大侧向散射群体（L-SSC）和非大侧向散射群体（NL-SSC）。87.50%的bcr1-型患者表现为L-SSC表型，64.29%的bcr3-型患者表现为NL-SSC表型。结论是建立了一种灵敏的Q-PCR方法。新诊断APL患者中PML/RARα mRNA的NQ中位数为0.450。bcr3-型和bcr1-型APL患者的PML/RARα mRNA表达无显著差异。PML/RARα转录本类型与形态学和免疫表型相关。
