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 fluorochromeconjugated 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 B4 h, and can be run in parallel to routine immunophenotyping.

Detection of fusion genes at the protein level in leukemia patients via the flow cytometric immunobead assay

“…However, when we used lysates from mature myeloid cells, the fluorescent signals in the BCR-ABL immunobead assay were clearly weaker than expected [29]. Additional experiments showed that the lower signals were caused by protein degradation via protease activity from the granules in more mature myeloid cells, such as granulocytes as well as CML cells.…”

“…in the ABL-SH2 domain. By using these two antibodies against "outer" epitopes, the BCR-ABL immunobead assay was expected to recognize all types of BCR-ABL fusion proteins, independent of the breakpoint position [29,30].…”

“…However, delayed processing of CML samples might still result in some protein degradation. Therefore, it is advised to perform the BCR-ABL immunobead assay, particularly in cells from chronic phase CML, within 24-36 h after sampling [29].…”

“…4) [29]. Importantly, the BCR-ABL immunobead assay detected the fusion proteins of all types of BCR breakpoints: p190, p210, p225, and p230 [29,30].…”

Biosensing of BCR/ABL fusion gene using an intensity-interrogation surface plasmon resonance imaging system

“…Determination of BCR/ABL fusion gene is important for early diagnosis, prognosis, and even better for CML patients during the treatment [18]. Nowadays, several methods have been applied to recognize BCR/ABL fusion gene, such as flow cytometry (FCM) [19], fluorescence in situ hybridization (FISH) [20] and real-time Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/optcom quantitative reverse transcription polymerase chain reaction (RT-PCR) [18]. These attractive methods are able to efficiently and accurately detect BCR/ABL fusion gene.…”