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Laboratory evaluation of a flow cytometric BCR-ABL immunobead assay
Zsuzsanna Hevessy1, Renáta Hudák, Valéria Kiss-Sziráki
1Department of Laboratory Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
Background:
A new flow cytometric (FC) BCR-ABL immunobead assay has been developed recently. Here we present the laboratory evaluation of the commercially available kit.
Methods:
Mononuclear cells were isolated, lysed and processed according to the instructions of the manufacturer. Anti-BCR antibodies adsorbed to capture beads bind the BCR-ABL fusion proteins of the lysed cells, a phycoerythrin (PE)-conjugated anti-ABL antibody is the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Detection of t(9;22)(q34;q11) translocation was carried out with a quantitative PCR assay.
Results:
MFI results of 20 normal peripheral blood samples were 88±8 (mean±SD), CV 9%. K562 cells were used as positive control. Within-batch imprecision was excellent (3.7% in the normal and 10% in the pathological range). Cut-off was chosen at MFI 112, where both sensitivity and specificity were 100%. Altogether 17 chronic myeloid leukemia (CML) and 16 acute leukemia samples were analyzed. All PCR positive samples (n=14) were positive with the FC method and negative results were also concordant (n=15). Frozen cell lysates can be stored up to 4 weeks without significant decrease of MFI signal.
Conclusions:
The FC BCR-ABL assay is a fast, reproducible and reliable method that may be incorporated into standard flow cytometric protocols to help clinical decision-making.
