Wells S J, Phillips C N, Winton E F, Farhi D C
Emory University School of Medicine, Atlanta, Georgia, USA.
Am J Clin Pathol. 1996 Jun;105(6):756-60. doi: 10.1093/ajcp/105.6.756.
Reverse transcriptase-polymerase chain reaction (RT-PCR) has shown promise as a means of detecting low levels of cells bearing the Philadelphia chromosome (Ph1) and for detecting cytogenetically inapparent ("masked") Ph1 in patients with chronic myelogenous leukemia (CML). For detection by karyotyping, dividing cells must be used, precluding use of peripheral blood samples in cases with low peripheral blood blast counts. Reverse transcriptase-polymerase chain reaction was performed in 83 bone marrow and 30 peripheral blood samples from patients with CML to compare results with karyotyping and to evaluate utility of this test on peripheral blood samples. Using isolated total cellular RNA and a single primer pair, cDNA was transcribed, amplified, electrophoresed, and probed for bcr/abl fusion involving M-bcr exons 2 and 3 of the bcr gene. Fifty-three samples were from untreated or conventionally treated patients (pre-BMT), and 60 were from patients who had undergone bone marrow transplantation (post-BMT). Fifty of 53 pre-BMT samples were positive by RT-PCR. Two samples, negative by RT-PCR, had complex translocations, t(9;16;22) and t(4;14;22). One case was indeterminate by RT-PCR, but positive on retesting. Forty-five of 53 had Ph1 by karyotyping; 8 were negative, including 5 peripheral blood samples, 2 bone marrow samples with "masked" Ph1, and 1 bone marrow sample with poor growth. Thirty-five of 60 post-BMT samples were positive by RT-PCR. Fourteen of 60 post-BMT samples had Ph1 by karyotyping. Of the RT-PCR+/Ph1- cases, most showed a weak but definite band by RT-PCR, suggesting a low level of the bcr/abl fusion gene. Nineteen patients had concurrent peripheral blood and bone marrow samples analyzed by RT-PCR and karyotyping. Of 16 patients with satisfactory RNA extraction, 15 had concordant results by RT-PCR. Five patients had adequate metaphase cells for karyotypic analysis. All had Ph1 in bone marrow, but were negative in peripheral blood. Our results indicate that RT-PCR for detection of bcr/abl fusion is more sensitive than karyotyping in pre- and post-BMT samples. Furthermore, RT-PCR can be successfully performed on peripheral blood, yielding excellent correlation with bone marrow samples.
逆转录聚合酶链反应（RT-PCR）已显示出有望成为检测携带费城染色体（Ph1）的低水平细胞以及检测慢性粒细胞白血病（CML）患者中细胞遗传学上不明显（“隐匿性”）Ph1的一种方法。对于通过核型分析进行检测，必须使用正在分裂的细胞，这就排除了在外周血原始细胞计数低的情况下使用外周血样本的可能性。对83例CML患者的骨髓样本和30例外周血样本进行了逆转录聚合酶链反应，以将结果与核型分析进行比较，并评估该检测在外周血样本中的实用性。使用分离的总细胞RNA和一对引物，进行cDNA转录、扩增、电泳，并检测涉及bcr基因M-bcr外显子2和3的bcr/abl融合。53个样本来自未治疗或接受常规治疗的患者（移植前），60个样本来自接受过骨髓移植的患者（移植后）。53个移植前样本中有50个通过RT-PCR呈阳性。2个RT-PCR阴性的样本有复杂易位，t(9;16;22)和t(4;14;22)。1例RT-PCR结果不确定，但重新检测呈阳性。53个样本中有45个通过核型分析有Ph1；8个为阴性，包括5个外周血样本、2个有“隐匿性”Ph1的骨髓样本和1个生长不良的骨髓样本。60个移植后样本中有35个通过RT-PCR呈阳性。60个移植后样本中有14个通过核型分析有Ph1。在RT-PCR阳性/Ph1阴性的病例中，大多数通过RT-PCR显示出一条微弱但明确的条带，表明bcr/abl融合基因水平较低。19例患者同时进行了外周血和骨髓样本的RT-PCR及核型分析。在16例RNA提取满意的患者中，15例RT-PCR结果一致。5例患者有足够的中期细胞用于核型分析。所有患者骨髓中有Ph1，但外周血中为阴性。我们的结果表明，在移植前和移植后样本中，用于检测bcr/abl融合的RT-PCR比核型分析更敏感。此外，RT-PCR在外周血中可以成功进行，与骨髓样本有很好的相关性。
