Fluorescent energy transfer causing misleading signal in multicolor flow cytometry

Area of Science:

• Immunology
• Biotechnology
• Analytical Chemistry

Background:

• Multicolor immunolabeling in flow cytometry risks fluorochrome interference.
• Accurate T cell clonality analysis requires precise multicolor panel design.
• Unexpected signals can mimic disease states, complicating diagnosis.

Purpose of the Study:

• To identify and prevent artifact signals arising from fluorochrome interactions.
• To investigate an unexpected signal observed on Vbeta subsets during T cell clonality analysis.
• To optimize multicolor flow cytometry panel design for T cell analysis.

Main Methods:

• A seven-color flow cytometry panel was designed for T cell clonality analysis using fresh whole blood.
• Specific fluorochromes included CD3-APC Alexa750, CD4-Pac Blue, CD8-Krome Orange, CD56-PE-Cy7, and Vbeta clonotypes (FITC and PE).
• Data analysis was performed using Navios Cytometer (Beckman Coulter) and Kaluza software, with systematic testing of fluorochrome combinations and Full Minus One controls.

Main Results:

• A significant CD56-PE-Cy7 signal was observed on Vbeta1 T cell subsets, mimicking a lymphoproliferative disorder.
• This artifact persisted even after removing the CD56-PE-Cy7 antibody (Full Minus One control).
• Replacing CD3-APC Alexa750 with CD3-APC or CD3-FITC eliminated the artifact, confirming fluorescent energy transfer (FRET) between PE and Alexa750.

Conclusions:

• Fluorescent energy transfer (FRET) between PE and Alexa750 caused misleading signals in the T cell clonality assay.
• FRET can lead to reduced fluorescence intensity of Vbeta PE and falsely elevated signals on other detectors.
• Careful consideration of FRET is essential during multicolor flow cytometry panel design, especially when labeling closely related markers with complementary fluorochromes.