Method for DNA Ploidy Analysis Along with Immunophenotyping for Rare Populations in a Sample using FxCycle Violet

Area of Science:

• Clinical laboratory science
• Immunology
• Molecular biology

Background:

• Flow cytometric DNA ploidy assays are crucial for disease risk stratification, including monoclonal gammopathies and B cell acute lymphoblastic leukemia.
• Detecting rare cell populations, such as circulating tumor cells, necessitates sensitive analytical methods.

Purpose of the Study:

• To describe a protocol for simultaneous DNA ploidy determination and surface immunophenotyping in fixed samples.
• To provide an easy, one-step method for multicolor immunophenotyping and DNA content analysis using FxCycle Violet (FCV) dye.

Main Methods:

• The protocol involves a single-step modification of routine multicolor immunophenotyping: surface staining, fixation, and DNA staining with FCV.
• Mature lymphocytes serve as internal controls for DNA index determination.
• The method utilizes 6- to 8-color immunophenotyping and DNA content analysis.

Main Results:

• The assay is sensitive, enabling DNA ploidy and cell cycle analysis in rare tumor populations as low as 100 events.
• It allows for DNA ploidy determination in various hematopoietic cell subsets within the same sample, identified by their immunophenotype.

Conclusions:

• This protocol offers a sensitive and efficient approach for DNA ploidy and cell cycle analysis in rare cell populations.
• The method facilitates simultaneous immunophenotyping and DNA content analysis, aiding in disease diagnosis and risk stratification.