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 commer cial 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 ob served by flow cytometry. The labeled cells had no other features of apopto sis. The labeling was observed regardless of die fixative used and was evid ent in control samples lacking terminal deoxynucleotidyltransferase. Common to all the kits that generated this labeling pattern was the presence of f luorescein (f) conjugated reagents, f-dUTP, f-avidin, or f-antibody. Methods: Laser scanning cytometry was used to identify the labeled cells an d 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 cel ls was studied. Gel electrophoresis was used to detect the presence of unco njugated FITC in the reagents. Results: After staining with Giemsa, the strongly fluorescent objects were identified as eosinophils with normal morphology and no evidence of apoptos is. The fluorescence was localized exclusively within the cytoplasmic granu les. Labeling of eosinophils was observed at 2 nM concentration of FITC, wh ich was over three orders of magnitude lower than that needed to label neut rophils, 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 reage nts are adequate to strongly label eosinophils thereby introducing experime ntal bias in analysis of apoptosis and in other studies on blood cells util izing f-labeled antibodies, e.g., in detecting cytokines. (2) FITC at conce ntration 2-500 nhl can be used as a marker of eosinophiles; (3) Because of high affinity to FITC, eosinophiles (or the protein from these cells) may s erve as a means of removing traces of unconjugated FITC from the reagents d uring their manufacture or prior to use. Cytometry 36:77-82, 1999. (C) 1999 Wiley-Liss, Inc.