Severe interference between retinal angiography and automated four-color flow cytometry analysis of blood mononuclear cells

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. 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. 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. 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. (C) 2007 International Society for Analytical Cytology