Flow cytometric immunobead assay for the detection of BCR-ABL fusion proteins in leukemia patients

EuroFlow Consortium, F (Floor) Weerkamp, E Dekking, Peter Ng, Vincent van der Velden, H Wai, S Böttcher, M Brüggemann, AJ van der Sluijs, A Koning, N Boeckx, N Van Poecke, P Lucio, A De Mendonca, L Sedek, Tomek Szczepanski, T Kalina, M Kovac, Patricia Hoogeveen, J Flores-MonteroA Orfao, E Macintyre, L Lhermitte, Ruoqing Chen, KAJ Brouwer - de Cock, Anne van der Linden, Rianne Noordijk, Marieke Bitter, Frank Staal, Jacques Dongen*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

66 Citations (Scopus)

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 similar to 4 h, and can be run in parallel to routine immunophenotyping. Leukemia (2009) 23, 1106-1117; doi: 10.1038/leu.2009.93; published online 23 April 2009
Original languageUndefined/Unknown
Pages (from-to)1106-1117
Number of pages12
JournalLeukemia
Volume23
Issue number6
DOIs
Publication statusPublished - 2009

Bibliographical note

This study was supported by the European Commission through the STREP grant EU-FP6, LSHB-CT-2006-018708, entitled Euro-Flow, ‘Flow cytometry for fast and sensitive diagnosis and follow-up of haematological malignancies’. Work carried out at Salamanca was supported in part by a grant (EST-08-90881) from the Fondo de Investigaciones Sanitarias (FIS) of the Instituto de Salud Carlos III (Ministerio de Ciencia e Innovacion, Madrid, Spain).

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  • EMC MM-02-72-03

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