Related Experiment Video
Updated: Jul 15, 2026
Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation
Published on: July 3, 2017
Severe interference between retinal angiography and automated four-color flow cytometry analysis of blood mononuclear
Philippe Bürgisser1, Jean Vaudaux, Pierre-Alexandre Bart
1Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. Philippe.Burgisser@chuv.ch
Background:
Retinal angiography has become a widely used diagnostic tool. It requires the intravenous administration of the fluorescent dyes fluorescein and indocyanin green. We recently received blood taken 8 h after retinal angiography, without our knowing it. We describe the failure of an automated flow cytometry system in the enumeration of lymphocyte subpopulations in this sample.
Methods:
Cell enumeration was achieved by the use of the lyse-no wash MultiTEST procedure (Becton-Dickinson) together with the FACSCalibur cytometer. Absolute cell counts were obtained using TruCount beads. Data were analyzed automatically by the MultiSET and manually with the CellQuest softwares.
Results:
The dot plots obtained with this sample looked quite abnormal. All monuclear cells stained brightly in the FITC channel irrespective of anti-CD3-FITC conjugate binding. This resulted in a major undercompensation for the increased spillover of the fluorescein emission into the PE-channel. PE-labeled cell and TruCount bead events coalesced. The MultiSET software failed to draw proper gatings and proved useless. Alternative manual gatings could partially rescue the analysis.
Conclusions:
Clinicians and cytometrists should be aware that, because of dye entry or binding, blood mononuclear cells collected shortly after retinal angiography are not suitable even for common cytometry applications.
