Flow Cytometry Detection of BCR-ABL Fusion Proteins in Leukemia Patients- An Indian Experience.

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, all techniques that are time consuming and require special facilities. We developed a simple flow cytometric bead assay for detection of the BCR-ABL fusion protein in cell lysates,using a bead bound catching antibody against one side of the fusion protein and a fluorochrome-conjugated detection antibody against the other side of the fusion protein. Flow cytometry allows for the discrimination of particles on the basis of attributes such as, size and fluroscence. BDTM Cytometric Bead. The aim of our study was to see the effectiveness of Flowcytometric detection of BCR-ABL and cost effectiveness of the method.

Test Specimens: 50 patients, CML 37 and Adult ALL 10, Paediatric ALL.

Reagents: BD BCR-ABL Protein Kit (BD Cat No: 643939) BD BCR-ABL buffer Kit (BD Cat No: 560272), Detection Reagent.

BD BCR-ABL Protein Kit Protocol: Lysate preparation from samples (PBMCs) : Blood samples were processed through Ficoll-Paque TM medium to collect the mono nuclear cell layer as PBMCs. Cell collected were washed with PBS with 5% FBS and resuspended with PBS with 5% FBS before cell counting. PBMC s were resuspended in pretreatment buffer (PBS with 5% FBS plus IX pretreatment A and IX pretreatment B) at 10 million cells/ml and incubated for 10 mins. on ice. The pretreated cells were washed ones, then resuspended in lysing solution (BD Pharmingen TM cell lysis buffer plus IX BD lysate treatment reagent )at 25 million cells/ ml and incubated for 15 mins. on ice. The resulting supernatant (cell lysate) was Colleted after centrifugation.

CBA Assay: to a BD Falcon TM 12 × 75mm polystyrene tube 50μl each of lysate, BCRABL Capture Beads and BCRABL detection reagent were added and mixed carefully. The tube was incubated for 2hrs at room temperature in the dark with constant mixing, and then contents were washed with 1 ml of BD CBA wash Buffer. Tube contents were resuspended with 300μl of BD CBA washed buffer. Samples were acquired with the BD FACS Canto II Flow Cytometer. The Cytometer flow rate was set to medium and data were acquired from 20 negative samples. The PE MFI for each sample was determining, and the mean of at least 20 values was calculated. The limit of detection was set at the mean plus two standard deviation.

Karyotyping: The Kaiser Home-brew reagent was used and related protocol was followed.

FISH: Abbott Visys LSI BCR/ABL, Dual Fusion Translocation Probe.

QRT PCR Detection: The Kaiser Home-brew reagent was used and related protocol was followed.

Detection of all variants of the BCR ABL fusion protein. The anti BCR antibody was developed against a nonhomologus region of appox. 80 amino acids encoded by exon 1, which allowed us to detect all known BCR aBL variants, including p190, p 210, p230 as demonstrated by analysis of a series of well defined cell lines. The assay appeared to be specific and sufficiently sensitive to detect BCR ABl proteins in Lysates of Leukocyte samples of Leukemia patients at diagnosis.

Protease inhibition needed in cell samples with many mature myeloid cells. Protein stability problems were encounter when cells samples contain high frequencies of mature myeloid cells with high levels of protease activity, such as CML cells and granulocyte fractions. This problem could be significantly encountered by adding protease inhibitors to several steps of the immunobead assay.

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 :

§ Not dependent on the breakpoint position in the BCR gene; does not need special laboratory facilities other than a Dual laser(4 colour) flow Cytometer and Provides results within few hours. The result is semiquantitative depending on the Mean Fluroscence Intensity. (MFI)

§ Can be run in parallel to routine immunophenotyping

§ Easy diagnosis and classification of leukemias with fusion genes.

No relevant conflicts of interest to declare.