Development of Flow Cytometry-Fluorescent In Situ Hybridization (Flow-FISH) 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 at 42°. 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.