Flow cytometric bead assay of BCR-ABL fusion proteins in leukemia patients: An Indian experience.

Abstract

6595 Background: The BCR-ABL fusion gene results from the translocation t(9;22). It is the hallmark of chronic myeloid leukemia (CML) and is present in a poor-risk subgroup of precursor B cell acute lymphoblastic leukemia (ALL), which represents 25% to 30% of adult ALL and 3% to 5% of childhood ALL. So far, the BCR-ABL aberration has been detected by cytogenetics, FISH or PCR, which are time consuming and require special facilities. We have used a simple flow cytometric bead assay (BD) for detection of the BCR-ABL fusion protein in cell lysates, The aim of our study was to see the effectiveness of Flowcytometric detection of BCR-ABL. Methods: Test Specimens: 100 patients, CML 74 and Adult ALL 20, Paediatric ALL 6. BD-BCR-ABL Protein Kit Protocol: Cell Lysate was prepared as per manufacturer's protocol using Ficoll-Paque medium and several washing with PBS and 5% FBS plus IX pretreatment A and IX pretreatment B. CBA Assay: In BD Falcon 12 × 75 mm polystyrene tube 50 μl each of lysate, BCR-ABL Capture Beads and BCR-ABL detection reagent were added. Tube contents were resuspended with 300μl of BD CBA washed buffer. Samples were acquired with the BD FACS Canto II Flow Cytometer. FISH: Abbott Visys LSI BCR-ABL, Dual Fusion Translocation Probe. Results: Amongst 100 cases concordance was best obtained in CML cases where 65 cases were positive for BCR ABL by flow cytometric bead assay and 68 cases were positive with FISH. In case of adult and pediatric ALL 14 cases showed positivity with flowcytometric bead assay. In case of FISH, however, 16 cases were having BCR-ABL. Also bead assay is far quicker than FISH assay. Conclusions: We conclude that the flow cytometry immunobead assay is a fast and easy technique for specific detection of BCR ABL proteins in leukemic cells. The main advantaged of the immunobead assay are: 1) Not dependent on the breakpoint position in the BCR gene; 2) Can be run in parallel to routine immunophenotyping. No significant financial relationships to disclose.