Detection of t(9;22) b2a2 fusion transcript by flow cytometry

Area of Science:

• Molecular Biology
• Hematology
• Cytogenetics

Background:

• Chromosomal translocations are hallmarks of hematologic malignancies.
• Accurate detection of these translocations is crucial for diagnosis and treatment.
• Traditional methods like FISH can be time-consuming and labor-intensive.

Purpose of the Study:

• To evaluate the feasibility of a flow cytometry-fluorescent in situ hybridization (FC-FISH) technique.
• To detect translocated mRNA in human peripheral blood nucleated cells.
• To assess FC-FISH as a diagnostic tool for chromosomal translocations in hematologic malignancies.

Main Methods:

• KCL-22 cell line and peripheral blood from 21 CML patients were used.
• Cells were isolated, fixed, permeabilized, and hybridized with mRNA-specific probes.
• Analysis was performed using a flow cytometer, with results compared to qRT-PCR and fluorescent microscopy.

Main Results:

• FC-FISH achieved 97 ± 2.1% labeling of KCL-22 cells with b2a2 mRNA probes.
• Seven CML patients tested positive for the t(9;22) b2a2 translocation, with positive cell percentages ranging from 3.26% to 97.8%.
• No false positives were observed in negative controls, and results were confirmed by qRT-PCR and fluorescent microscopy.

Conclusions:

• FC-FISH demonstrates high specificity, sensitivity, and rapidity in detecting translocated mRNA.
• The technique accurately quantifies the percentage of malignant cells.
• FC-FISH offers a significant improvement over traditional FISH methods for diagnosing hematologic malignancies.