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Biotechnical Methods Section (BTS)

Transcription-mediated amplification and hybridisation protection assay to determine BCR-ABL transcript levels in patients with chronic myeloid leukaemia

Abstract

Detection of BCR-ABL transcripts in chronic myeloid leukaemia (CML) is used to confirm the diagnosis and to monitor residual disease. Quantitative techniques are required to predict response to therapy or early relapse. We have evaluated an assay in which transcription-mediated amplification (TMA) of BCR-ABL and ABLtranscripts is achieved using reverse transcriptase and RNA polymerase. The products are quantified in the hybridisation protection assay (HPA) using acridinium ester-labelled DNA probes and chemiluminescence. The method is a single tube procedure which uses small amounts of RNA (<500 ng/triplicate analysis), is technically simple (requiring just two waterbaths and a luminometer), rapid (total assay time <4 h) and sensitive (capable of detecting one BCR-ABL-positive K562 cell in the presence of 104–105 BCR-ABL-negative cells). BCR-ABL signals from patient RNA samples were quantified relative to known amounts of K562 RNA and normalised to levels of ABL. BCR-ABL/ABL ratios ranged from 0.15 to 1.59 (median 0.65) in RNA from diagnostic blood or bone marrow of 18 CML patients and were 0.0001 in 20 normal controls. Sequential samples analysed from six CML patients post-allogeneic bone marrow transplantation who relapsed and received donor lymphocyte infusions showed BCR-ABL/ABL ratios which reflected patient status or treatment. A BCR-ABL/ABL ratio of 0.01 served as a useful arbitrary indicator value, with results above and below this value generally correlating with relapse or remission, respectively.

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Acknowledgements

The authors are grateful to Bill Hanna for critical review of the manuscript. This work was supported by the Kay Kendall Leukaemia Fund.

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Langabeer, S., Gale, R., Harvey, R. et al. Transcription-mediated amplification and hybridisation protection assay to determine BCR-ABL transcript levels in patients with chronic myeloid leukaemia. Leukemia 16, 393–399 (2002). https://doi.org/10.1038/sj.leu.2402392

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  • DOI: https://doi.org/10.1038/sj.leu.2402392

Keywords

  • CML
  • BCR-ABL
  • ABL
  • TMA
  • HPA
  • diagnosis

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