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

Abstract

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. RETINAL angiography has become a routine diagnostic tool in ophthalmology. This technique allows us to observe microcirculation in the retina and the choroid and to evidence, among other abnormalities, capillary leakage as well as concomitant damage of surrounding cell layers. It necessitates the intravenous injection of fluorescent dyes such as fluorescein and indocyanin green. Flow cytometry on the other hand is the method of choice for the determination of lymphocyte subsets in blood, and is based on the use of monoclonal antibodies covalently labeled with various fluorochromes, including fluorescein isothiocyanate (FITC). Two publications have described interference of fluorescein administered for retinal angiography with the subsequent measurement of CD41 T lymphocytes using simple one-or two-color flow cytometry techniques (1,2). Here we document the effects of such an interference in a widely used four-color flow cytometry system with automated computerized delineation of gates and single platform measurement of absolute cell counts. We also discuss how this phenomenon could affect other cytometric assays as well. MATERIALS AND METHODSLymphocyte subpopulations were measured on a two-laser (488, 635 nm) FACSCalibur Becton Dickinson (BD) flow cytometer using the lyse-no wash whole blood MultiTEST procedure and reagents. Venous blood was collected in K 3 -EDTA-containing tubes. After treatment of blood with the BD FACS Lysing Solution according to

Systemic frequencies of T helper 1 and T helper 17 cells in patients with age-related macular degeneration: A case-control study

“…Blood sampling was performed prior to any fluorescein and indocyanine angiography to avoid interference 35 . The volume of blood used for each participant was equivalent to 5 × 10 5 white blood cells and calculated using a white blood cell count in a hematology analyzer (Sysmex KX-21N TM , Sysmex Corporation, Kobe, Japan).…”

Altered activation state of circulating neutrophils in patients with neovascular age-related macular degeneration

“…We excluded any participant with a plasma CRP-level > 15 mg/L to avoid participants with possible ongoing infections [ 39 ]. To avoid interference with flow cytometric analyses, we did not recruit patients with neovascular AMD within 4 or 8 weeks respectively of Ranibizumab or Aflibercept therapy or immediately after retinal angiography [ 40 ].…”

Dysregulation of CXCR3 Expression on Peripheral Blood Leukocytes in Patients With Neovascular Age-Related Macular Degeneration

“…The phlebotomy was performed prior to angiography to avoid possible interference. 20 Within 4 hours of phlebotomy the blood samples were prepared for flow cytometry in the following steps. Red blood cell lysis was performed by adding 10% red blood cell lysis buffer (BioLegend, San Diego, CA, USA) to the whole blood followed by storage for 10 minutes in the dark at room temperature.…”