Neutrophils with Toxic Granulation Show High Fluorescence with Bis(Zn2+-dipicolylamine) Complex

Abstract

Although blood neutrophils with toxic granulation provide an excellent means of evaluating acute bacterial infections, the methods are labor-intensive and their reproducibilities depend on the staining technique and the observer&apos;s judgment. We measured the flavin adenine dinucleotide (FAD) content of normal neutrophils and neutrophils with toxic granulation by flow cytometry after incubating them with bis(Zn2+-dipicolylamine) complex. A total of 122 blood samples (78 with neutrophils with toxic granulation, 44 with normal neutrophils without toxic granulation) were analyzed. The mean autofluorescence levels of neutrophils in the toxic granulation (+) group and toxic granulation (-) group were both 1.9 x 10(3) MESF (mean equivalent soluble fluorochrome) values. However, after incubating neutrophils with bis(Zn2+-dipicolylamine) complex for 15 min, the mean fluorescence intensities of neutrophils in the toxic granulation (+) and toxic granulation (-) groups were significantly enhanced by 71-and 19-fold to 138 +/- 78 x 10(3) MESF and 37 +/- 37 x 10(3) MESF, respectively (p < 0.001). An MESF cutoff value of 79 x 103 showed a sensitivity of 93.2% and a specificity of 81.8% for neutrophils with toxic granulation. Similarly, the MESF level ratio, defined as the ratio of the MESF value after incubation with bis(Zn2+-dipicolylamine) complex to the autofluorescence MESF value, at a cutoff value of 41, showed a sensitivity of 92.3% and a specificity of 81.8% for the detection of neutrophils with toxic granulation. The MESF values of neutrophils after incubation with bis(Zn2+-dipicolylamine) complex did not correlate with the leukocyte (p > 0.05) or neutrophil counts (p > 0.05). In conclusion, measurement of FAD fluorescence intensity in neutrophils by flow cytometry after incubation with bis(Zn2+-dipicolylamine) complex is an easy, objective, and reliable method of detecting neutrophils with toxic granulation