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-Montero; A Orfao; E Macintyre; L Lhermitte; Ruoqing Chen; KAJ Brouwer - de Cock; Anne van der Linden; Rianne Noordijk; Marieke Bitter; Frank Staal; Jacques Dongen

doi:10.1038/leu.2009.93

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

Immunology

Research output: Contribution to journal › Article › Academic › peer-review

67 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 language	Undefined/Unknown
Pages (from-to)	1106-1117
Number of pages	12
Journal	Leukemia
Volume	23
Issue number	6
DOIs	https://doi.org/10.1038/leu.2009.93
Publication status	Published - 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

Access to Document

10.1038/leu.2009.93

http://hdl.handle.net/1765/24568

Cite this

EuroFlow Consortium, Weerkamp, F., Dekking, E., Ng, P., van der Velden, V., Wai, H., Böttcher, S., Brüggemann, M., van der Sluijs, AJ., Koning, A., Boeckx, N., Van Poecke, N., Lucio, P., De Mendonca, A., Sedek, L., Szczepanski, T., Kalina, T., Kovac, M., Hoogeveen, P., ... Dongen, J. (2009). Flow cytometric immunobead assay for the detection of BCR-ABL fusion proteins in leukemia patients. Leukemia, 23(6), 1106-1117. https://doi.org/10.1038/leu.2009.93

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

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",

author = "{EuroFlow Consortium} and Weerkamp, {F (Floor)} and E Dekking and Peter Ng and {van der Velden}, Vincent and H Wai and S B{\"o}ttcher and M Br{\"u}ggemann and {van der Sluijs}, AJ and A Koning and N Boeckx and {Van Poecke}, N and P Lucio and {De Mendonca}, A and L Sedek and Tomek Szczepanski and T Kalina and M Kovac and Patricia Hoogeveen and J Flores-Montero and A Orfao and E Macintyre and L Lhermitte and Ruoqing Chen and {Brouwer - de Cock}, KAJ and {van der Linden}, Anne and Rianne Noordijk and Marieke Bitter and Frank Staal and Jacques Dongen",

note = "This study was supported by the European Commission through the STREP grant EU-FP6, LSHB-CT-2006-018708, entitled Euro-Flow, {\textquoteleft}Flow cytometry for fast and sensitive diagnosis and follow-up of haematological malignancies{\textquoteright}. 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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doi = "10.1038/leu.2009.93",

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EuroFlow Consortium, Weerkamp, F, Dekking, E, Ng, P, van der Velden, V, Wai, H, Böttcher, S, Brüggemann, M, van der Sluijs, AJ, Koning, A, Boeckx, N, Van Poecke, N, Lucio, P, De Mendonca, A, Sedek, L, Szczepanski, T, Kalina, T, Kovac, M, Hoogeveen, P, Flores-Montero, J, Orfao, A, Macintyre, E, Lhermitte, L, Chen, R, Brouwer - de Cock, KAJ, van der Linden, A, Noordijk, R, Bitter, M, Staal, F & Dongen, J 2009, 'Flow cytometric immunobead assay for the detection of BCR-ABL fusion proteins in leukemia patients', Leukemia, vol. 23, no. 6, pp. 1106-1117. https://doi.org/10.1038/leu.2009.93

TY - JOUR

T1 - Flow cytometric immunobead assay for the detection of BCR-ABL fusion proteins in leukemia patients

AU - EuroFlow Consortium

AU - Weerkamp, F (Floor)

AU - Dekking, E

AU - Ng, Peter

AU - van der Velden, Vincent

AU - Wai, H

AU - Böttcher, S

AU - Brüggemann, M

AU - van der Sluijs, AJ

AU - Koning, A

AU - Boeckx, N

AU - Van Poecke, N

AU - Lucio, P

AU - De Mendonca, A

AU - Sedek, L

AU - Szczepanski, Tomek

AU - Kalina, T

AU - Kovac, M

AU - Hoogeveen, Patricia

AU - Flores-Montero, J

AU - Orfao, A

AU - Macintyre, E

AU - Lhermitte, L

AU - Chen, Ruoqing

AU - Brouwer - de Cock, KAJ

AU - van der Linden, Anne

AU - Noordijk, Rianne

AU - Bitter, Marieke

AU - Staal, Frank

AU - Dongen, Jacques

N1 - 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).

PY - 2009

Y1 - 2009

N2 - 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

AB - 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

U2 - 10.1038/leu.2009.93

DO - 10.1038/leu.2009.93

M3 - Article

C2 - 19387467

SN - 0887-6924

VL - 23

SP - 1106

EP - 1117

JO - Leukemia

JF - Leukemia

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