High affinity binding of fluorescein isothiocyanate to eosinophils detected by laser scanning cytometry: A potential source of error in analysis of blood samples utilizing fluorescein‐conjugated reagents in flow cytometry

BACKGROUND: In samples of peripheral blood cells processed using the commercial kits for detection of apoptosis based on DNA strand break labeling, a subpopulation of cells characterized by high green fluorescence, similar in intensity to that of apoptotic cells but more uniform, was consistently observed by flow cytometry. The labeled cells had no other features of apoptosis. The labeling was observed regardless of the fixative used and was evident in control samples lacking terminal deoxynucleotidyltransferase. Common to all the kits that generated this labeling pattern was the presence of fluorescein (f) conjugated reagents, f-dUTP, f-avidin, or f-antibody. METHODS: Laser scanning cytometry was used to identify the labeled cells and study the mechanism of labeling. Because it was suspected that the traces of unconjugated f-isothiocyanate (FITC) that may contaminate the reagents were responsible for the labeling, FITC binding affinity to white blood cells was studied. Gel electrophoresis was used to detect the presence of unconjugated FITC in the reagents. RESULTS: After staining with Giemsa, the strongly fluorescent objects were identified as eosinophils with normal morphology and no evidence of apoptosis. The fluorescence was localized exclusively within the cytoplasmic granules. Labeling of eosinophils was observed at 2 nM concentration of FITC, which was over three orders of magnitude lower than that needed to label neutrophils, monocytes, or lymphocytes. Gel electrophoresis of the f-conjugated reagents revealed only minor contamination with FITC. CONCLUSIONS: (1) Trace amounts of unconjugated FITC contaminating the reagents are adequate to strongly label eosinophils thereby introducing experimental bias in analysis of apoptosis and in other studies on blood cells utilizing f-labeled antibodies, e.g., in detecting cytokines. (2) FITC at concentration 2-500 nM can be used as a marker of eosinophiles; (3) Because of high affinity to FITC, eosinophiles (or the protein from these cells) may serve as a means of removing traces of unconjugated FITC from the reagents during their manufacture or prior to use.