In vitro detection of bcr/abl fusion protein immunologically with flow cytometry in k562 cell line and comparison of results with rt-pcr

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disease arising from a neoplastic transformation of hematopoietic progenitor cells, usually characterized with a specific chromosomal abnormality called Philadelphia (Ph) chromosome. Ph chromosome occurs with reciprocal translocation of BCR and ABL genes located on chromosomes 9 and 22, respectively. Follow up of minimal residual disease in CML patients is based on the detection of BCR/ABL mRNA by RT-PCR. Showing BCR/ABL oncogene by RT-PCR is a difficult and technician dependent method. Development and use of novel methods for diagnosis and follow up of CML patients are of importance in routine practice due to the difficulties in RT-PCR technique and its standardization. In this study, we aimed to standardize detection of BCR/ABL protein using a flow cytometric method in K562 cells, and to compare the results with the molecular method which is the gold standard. The study consisted of two arms. In the first arm, RT-PCR and flow cytometry methods were compared using nine different cell groups with gradually increasing amounts (1x103, 1x104, 5x104, 7.5x104, 1x105, 5x105, 1x106, 1.5x106, 2x106) in K562 cells. In the second arm, 1x107 K562 cells were incubated for 72 hours with each of the 6 groups (1 control group and 5 different concentration of imatinib applied groups). Afterwards, cell viability analysis was performed and the results of the RT-PCR and flow cytometric analyses were compared. While BCR-ABL/ABL ratio was found to be positive in all groups with RT-PCR in the first arm of the study, BCR/ABL fusion protein could not be detected by flow cytometry in groups with 1.000 and 10.000 cells. BCR/ABL protein was found to be positive in the groups that contain 50.000 or more cells (p<0.05). Specifity and sensitivity were 100% when flow cytometry results of the groups thatcontain 50.000 or more cells as compared with RT-PCR. RT-PCR and flow cytometry results were positive in all groups in the second arm. The mean fluorescence intensity was observed to decrease in flow cytometry methods in those groups with reduced viability. However, RT-PCR and BCR-ABL/ABL ratios were not affected by the viability. In conclusion, BCR/ABL fusion protein can be detected by using flow cytometry method, when a minimum of 50.000 viable cells are studied.