INTRODUCTION: We assessed the feasibility of flow cytometry-fluorescent in situ hybridization technique in the detection of translocated mRNA in the cytoplasm of human peripheral blood nucleated cells. It is assumed that this assay can be applied as a diagnostic method in the detection of chromosomal translocation which commonly occurs in hematologic malignancies. METHODS: KCL-22 cell line and white blood cells from 21 CML patients were recruited in the study. Cells were isolated and fixed. After permeabilization, cells were resuspended in hybridization buffer and probes were added to the mixture. Subsequently, cells were washed and analyzed on the flow cytometer instrument. The flow cytometry results were compared with qRT-PCR and fluorescent microscope outcomes. RESULTS: Using the current principle, 97 ± 2.1% of the KCL-22 cells were labeled with b2a2 mRNA-specific probes. In addition, seven patients were recognized positive for t(9;22) b2a2. The percentage of cells containing abovementioned translocation in these patients was varied from 3.26% to 97.8%. There was no false-positive result in negative controls (K562 with BCR-ABL1 b3a2, NB4, and Jurkat cell lines) along with blood samples of normal controls. All the results obtained by flow-FISH were confirmed by qRT-PCR and fluorescent microscope. CONCLUSION: This strategy benefits from appropriate specificity, sensitivity, rapidity, and ability in the determination of malignant cell percentage. Therefore, it can improve traditional time-consuming and labor-intensive FISH method.
INTRODUCTION: We assessed the feasibility of flow cytometry-fluorescent in situ hybridization technique in the detection of translocated mRNA in the cytoplasm of human peripheral blood nucleated cells. It is assumed that this assay can be applied as a diagnostic method in the detection of chromosomal translocation which commonly occurs in hematologic malignancies. METHODS: KCL-22 cell line and white blood cells from 21 CMLpatients were recruited in the study. Cells were isolated and fixed. After permeabilization, cells were resuspended in hybridization buffer and probes were added to the mixture. Subsequently, cells were washed and analyzed on the flow cytometer instrument. The flow cytometry results were compared with qRT-PCR and fluorescent microscope outcomes. RESULTS: Using the current principle, 97 ± 2.1% of the KCL-22 cells were labeled with b2a2 mRNA-specific probes. In addition, seven patients were recognized positive for t(9;22) b2a2. The percentage of cells containing abovementioned translocation in these patients was varied from 3.26% to 97.8%. There was no false-positive result in negative controls (K562 with BCR-ABL1 b3a2, NB4, and Jurkat cell lines) along with blood samples of normal controls. All the results obtained by flow-FISH were confirmed by qRT-PCR and fluorescent microscope. CONCLUSION: This strategy benefits from appropriate specificity, sensitivity, rapidity, and ability in the determination of malignant cell percentage. Therefore, it can improve traditional time-consuming and labor-intensive FISH method.