development of flow cytometryfluorescent in situ hybridization (flowfish) method for detection of pml/rara chromosomal translocation in acute promyelocytic leukemia cell line

Background: acute promyelocytic leukemia (apl) is a subclass of acute myeloid leukemia. the chromosomal aberration in 95% of apl cases is t(15; 17) (q22; q21), which prevents cell differentiation. characterization of the underlying molecular lesion is valuable in determining optimal treatment strategy. the goal of this study was to develop a new and powerful flow- fish technique to detect the long isoform (l) of pml-rara fusion transcript in nb4 cell line. methods: to achieve the best condition for fixation, two different fixatives including 2% paraformaldehyde and 75% ethanol were used. 0.2% triton x-100 and 0.2% saponin were used for the permeabilization step .in hybridization, a wide range of times and temperatures were used and probe was designed in fret system. results were confirmed by fluorescent microscope assay and reverse transcription pcr. results: in the present study, a novel technique was successfully optimized that combines in situ hybridization with flow cytometry to detect the presence of pml-rara transcript. using standard fixation and permeabilization protocol of 2% pfa and 0.2% saponin gave the best fluorescent results in flow cytometry. also, results indicated that the optimum time and temperature for hybridization was 2 hr at 42c. the results of reverse transcription pcr and fluorescent microscopy confirmed the presence of pml-rara transcript. conclusion: the concordance between the results of flow-fish and those of two other techniques including reverse transcriptionbackground: acute promyelocytic leukemia (apl) is a subclass of acute myeloid leukemia. the chromosomal aberration in 95% of apl cases is t(15; 17) (q22; q21), which prevents cell differentiation. characterization of the underlying molecular lesion is valuable in determining optimal treatment strategy. the goal of this study was to develop a new and powerful flow- fish technique to detect the long isoform (l) of pml-rara fusion transcript in nb4 cell line. methods: to achieve the best condition for fixation, two different fixatives including 2% paraformaldehyde and 75% ethanol were used. 0.2% triton x-100 and 0.2% saponin were used for the permeabilization step .in hybridization, a wide range of times and temperatures were used and probe was designed in fret system. results were confirmed by fluorescent microscope assay and reverse transcription pcr. results: in the present study, a novel technique was successfully optimized that combines in situ hybridization with flow cytometry to detect the presence of pml-rara transcript. using standard fixation and permeabilization protocol of 2% pfa and 0.2% saponin gave the best fluorescent results in flow cytometry. also, results indicated that the optimum time and temperature for hybridization was 2 hr at 42c. the results of reverse transcription pcr and fluorescent microscopy confirmed the presence of pml-rara transcript. conclusion: the concordance between the results of flow-fish and those of two other techniques including reverse transcription pcr and fish indicated that this method would be applicable as a diagnostic test for apl in clinical samples and mrd monitoring. pcr and fish indicated that this method would be applicable as a diagnostic test for apl in clinical samples and mrd monitoring.