1. ¹Department of Immunology, Erasmus MC, Rotterdam, The Netherlands
2. ²Dynomics, Rotterdam, The Netherlands
3. ³BD Biosciences, San Jose, CA, USA
4. ⁴2nd Department of Medicine, University Klinik Schleswig-Holstein, Kiel, Germany
5. ⁵Dutch Childhood Oncology Group, The Hague, The Netherlands
6. ⁶Department of Laboratory Medicine, Hematology, University Hospitals Leuven, Leuven, Belgium
7. ⁷Department of Hematology, Instituto Portugues de Oncologia, Lisbon, Portugal
8. ⁸Department of Pediatric Hematology and Oncology, Medical University of Silesia, Zabrze, Poland
9. ⁹Department of Pediatric Hematology and Oncology, Charles University, Prague, Czechoslovakia
10. ¹⁰Department of Medicine, Cancer Research Centre (IBMCC-CSIC-USAL) and Cytometry Service, University of Salamanca, Salamanca, Spain
11. ¹¹Department of Hematology, Hôpital Necker, Paris, France

Correspondence: Professor JJM van Dongen, Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: j.j.m.vandongen@erasmusmc.nl

Received 19 March 2009; Accepted 24 March 2009; Published online 23 April 2009.

Abstract

BCR–ABL fusion proteins show increased signaling through their ABL tyrosine kinase domain, which can be blocked by specific inhibitors, thereby providing effective treatment. This makes detection of BCR–ABL aberrations of utmost importance for diagnosis, classification and treatment of leukemia patients. BCR–ABL aberrations are currently detected by karyotyping, fluorescence in situ hybridization (FISH) or PCR techniques, which are time consuming and require specialized facilities. We developed a simple flow cytometric immunobead assay for detection of BCR–ABL fusion proteins in cell lysates, using a bead-bound anti-BCR catching antibody and a fluorochrome-conjugated anti-ABL detection antibody. We noticed protein stability problems in lysates caused by proteases from mature myeloid cells. This problem could largely be solved by adding protease inhibitors in several steps of the immunobead assay. Testing of 145 patient samples showed fully concordant results between the BCR–ABL immunobead assay and reverse transcriptase PCR of fusion gene transcripts. Dilution experiments with BCR–ABL positive cell lines revealed sensitivities of at least 1%. We conclude that the BCR–ABL immunobead assay detects all types of BCR–ABL proteins in leukemic cells with high specificity and sensitivity. The assay does not need specialized laboratory facilities other than a flow cytometer, provides results within 4 h, and can be run in parallel to routine immunophenotyping.