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Comprehensive comparison of FISH, RT-PCR, and RQ-PCR for monitoring the BCR-ABL gene after hematopoietic stem cell transplantation in CML

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Kim Y-J, Kim D-W, Lee S, Kim H-J, Kim Y-L, Hwang J-Y, Oh I-H, Park Y-H, Lee Y-K, Min C-K, Kim T-G, Han T-H, Min W-S, Kim C-C. Comprehensive comparison of FISH, RT-PCR, and RQ-PCR for monitoring the BCR-ABL gene after hematopoietic stem cell transplantation in CML.

Eur J Haematol 2002: 68: 272–280. © Blackwell Munksgaard 2002. Abstract:

The reverse transcriptase-polymerase chain reaction (RT-PCR) was compared with fluorescence in situ hybridization (FISH) and real-time quantitative RT-PCR (RQ-PCR) for minimal residual disease (MRD) monitoring in 266 post-transplant bone marrow samples from 78 patients with chronic myelogenous leukemia (CML). The sensitivities of FISH to BCR-ABL positive samples determined by first-round (1st) RT-PCR, second-round (2nd) RT-PCR, and RQ-PCR were 64.2%, 25.8%, and 20.7%, respectively. The BCR-ABL/ABL ratio by RQ-PCR had a mean of 0.000 13 in the 1st RT-PCR-negative samples and 1.42 in the 1st RT-PCR-positive samples (P<0.001), and means of 0.000 39 and 0.51 in the 2nd RT-PCR-negative and -positive samples (P< 0.001). The mean ratios of BCR-ABL/ABL by RQ-PCR were significantly different in N/N (1st/2nd RT-PCR) or N/P and P/P (P<0.001), but not in N/N and N/P, which showed that the discriminative power of RQ-PCR is confined to the 1st RT-PCR level. In this respect, monitoring of the 1st RT-PCR might be useful for estimating normalized BCR-ABL levels after transplantation. Nested RT-PCR was of limited use, as RQ-PCR quantified the BCR-ABL transcripts in 60 (91%) of 66 samples determined to be negative by 2nd RT-PCR. FISH was significantly correlated with RQ-PCR in FISH-positive samples (n=24, r=0.79, P=0.001). An increase of FISH preceded that of RQ-PCR in a few cases with molecular relapse. By analyzing a large number of samples post-transplant, we found that RQ-PCR might be the most useful assay for MRD monitoring; however, FISH and RT-PCR were found to be useful complementary tools.
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Keywords: BCR-ABL; chronic myelogenous leukemia; fluorescence in situ hybridization; hematopoietic stem cell transplantation; minimal residual disease; polymerase chain reaction; real-time quantitative PCR

Document Type: Research Article

Affiliations: 1: Catholic Hemopoietic Stem Cell Transplantation Center and 2: Research Institute of Medical Science, The Catholic University of Korea; 3: Department of Clinical Pathology, College of Medicine, Soonchunhyang University; 4: Department of Microbiology, The Catholic University of Korea; 5: Department of Microbiology and Immunology, College of Medicine, Sungkyunkwan University, Seoul, Korea

Publication date: May 1, 2002

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