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 approximately 4 h, and can be run in parallel to routine immunophenotyping.
authors.affiliation: Department of Immunology, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: F
authors.initials: F
authors.lastname: Weerkamp
authors.forename: E
authors.initials: E
authors.lastname: Dekking
authors.forename: Y Y
authors.initials: YY
authors.lastname: Ng
authors.forename: V H J
authors.initials: VH
authors.lastname: van der Velden
authors.forename: H
authors.initials: H
authors.lastname: Wai
authors.forename: S
authors.initials: S
authors.lastname: Böttcher
authors.forename: M
authors.initials: M
authors.lastname: Brüggemann
authors.forename: A J
authors.initials: AJ
authors.lastname: van der Sluijs
authors.forename: A
authors.initials: A
authors.lastname: Koning
authors.forename: N
authors.initials: N
authors.lastname: Boeckx
authors.forename: N
authors.initials: N
authors.lastname: Van Poecke
authors.forename: P
authors.initials: P
authors.lastname: Lucio
authors.forename: A
authors.initials: A
authors.lastname: Mendonça
authors.forename: L
authors.initials: L
authors.lastname: Sedek
authors.forename: T
authors.initials: T
authors.lastname: Szczepański
authors.forename: T
authors.initials: T
authors.lastname: Kalina
authors.forename: M
authors.initials: M
authors.lastname: Kovac
authors.forename: P G
authors.initials: PG
authors.lastname: Hoogeveen
authors.forename: J
authors.initials: J
authors.lastname: Flores-Montero
authors.forename: A
authors.initials: A
authors.lastname: Orfao
authors.forename: E
authors.initials: E
authors.lastname: Macintyre
authors.forename: L
authors.initials: L
authors.lastname: Lhermitte
authors.forename: R
authors.initials: R
authors.lastname: Chen
authors.forename: K A J
authors.initials: KA
authors.lastname: Brouwer-De Cock
authors.forename: A
authors.initials: A
authors.lastname: van der Linden
authors.forename: A L
authors.initials: AL
authors.lastname: Noordijk
authors.forename: W M
authors.initials: WM
authors.lastname: Comans-Bitter
authors.forename: F J T
authors.initials: FJ
authors.lastname: Staal
authors.forename: J J M
authors.initials: JJ
authors.lastname: van Dongen
mesh_terms: D000911:Antibodies, Monoclonal; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D016044:Fusion Proteins, bcr-abl / Q000032:analysis*; D006801:Humans; D007118:Immunoassay / Q000379:methods* / Q000592:standards; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis*; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D011480:Protease Inhibitors; D016044:Fusion Proteins, bcr-abl