Dual-wavelength excitation to reduce background fluorescence for fluorescence spectroscopic quantitation of erythrocyte zinc protoporphyrin-IX and protoporphyrin-IX from whole blood and oral mucosa

Georg Hennig; Michael Vogeser; Lesca M. Holdt; Christian Homann; Michael Großmann; Herbert Stepp; Christian Gruber; Ilknur Erdogan; Stephan Hasmüller; Uwe Hasbargen; Gary M. Brittenham

doi:10.1117/12.2039279

28 February 2014 Dual-wavelength excitation to reduce background fluorescence for fluorescence spectroscopic quantitation of erythrocyte zinc protoporphyrin-IX and protoporphyrin-IX from whole blood and oral mucosa

Georg Hennig, Michael Vogeser, Lesca M. Holdt, Christian Homann, Michael Großmann, Herbert Stepp, Christian Gruber, Ilknur Erdogan, Stephan Hasmüller, Uwe Hasbargen, Gary M. Brittenham

Author Affiliations +

Proceedings Volume 8951, Optical Diagnostics and Sensing XIV: Toward Point-of-Care Diagnostics; 89510J (2014) https://doi.org/10.1117/12.2039279
Event: SPIE BiOS, 2014, San Francisco, California, United States

Abstract

Erythrocyte zinc protoporphyrin-IX (ZnPP) and protoporphyrin-IX (PPIX) accumulate in a variety of disorders that restrict or disrupt the biosynthesis of heme, including iron deficiency and various porphyrias. We describe a reagent-free spectroscopic method based on dual-wavelength excitation that can measure simultaneously both ZnPP and PPIX fluorescence from unwashed whole blood while virtually eliminating background fluorescence. We further aim to quantify ZnPP and PPIX non-invasively from the intact oral mucosa using dual-wavelength excitation to reduce the strong tissue background fluorescence while retaining the faint porphyrin fluorescence signal originating from erythrocytes. Fluorescence spectroscopic measurements were made on 35 diluted EDTA blood samples using a custom front-face fluorometer. The difference spectrum between fluorescence at 425 nm and 407 nm excitation effectively eliminated background autofluorescence while retaining the characteristic porphyrin peaks. These peaks were evaluated quantitatively and the results compared to a reference HPLC-kit method. A modified instrument using a single 1000 μm fiber for light delivery and detection was used to record fluorescence spectra from oral mucosa. For blood measurements, the ZnPP and PPIX fluorescence intensities from the difference spectra correlated well with the reference method (ZnPP: Spearman’s rho r_s = 0.943, p < 0.0001; PPIX: r_s = 0.959, p < 0.0001). In difference spectra from oral mucosa, background fluorescence was reduced significantly, while porphyrin signals remained observable. The dual-wavelength excitation method evaluates quantitatively the ZnPP/heme and PPIX/heme ratios from unwashed whole blood, simplifying clinical laboratory measurements. The difference technique reduces the background fluorescence from measurements on oral mucosa, allowing for future non-invasive quantitation of erythrocyte ZnPP and PPIX.

Citation Download Citation

Georg Hennig, Michael Vogeser, Lesca M. Holdt, Christian Homann, Michael Großmann, Herbert Stepp, Christian Gruber, Ilknur Erdogan, Stephan Hasmüller, Uwe Hasbargen, and Gary M. Brittenham "Dual-wavelength excitation to reduce background fluorescence for fluorescence spectroscopic quantitation of erythrocyte zinc protoporphyrin-IX and protoporphyrin-IX from whole blood and oral mucosa", Proc. SPIE 8951, Optical Diagnostics and Sensing XIV: Toward Point-of-Care Diagnostics, 89510J (28 February 2014); https://doi.org/10.1117/12.2039279

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