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Proceedings Paper

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
Author(s): Georg Hennig; Michael Vogeser; Lesca M. Holdt; Christian Homann; Michael Großmann; Herbert Stepp; Christian Gruber; Ilknur Erdogan; Stephan Hasmüller; Uwe Hasbargen; Gary M. Brittenham
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Paper 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 rs = 0.943, p < 0.0001; PPIX: rs = 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.

Paper Details

Date Published: 28 February 2014
PDF: 10 pages
Proc. SPIE 8951, Optical Diagnostics and Sensing XIV: Toward Point-of-Care Diagnostics, 89510J (28 February 2014); doi: 10.1117/12.2039279
Show Author Affiliations
Georg Hennig, Klinikum der Univ. München (Germany)
Michael Vogeser, Klinikum der Univ. München (Germany)
Lesca M. Holdt, Klinikum der Univ. München (Germany)
Christian Homann, Klinikum der Univ. München (Germany)
Michael Großmann, Klinikum der Univ. München (Germany)
Herbert Stepp, Klinikum der Univ. München (Germany)
Christian Gruber, Klinikum der Univ. München (Germany)
Ilknur Erdogan, Klinikum der Univ. München (Germany)
Stephan Hasmüller, Klinikum der Univ. München (Germany)
Uwe Hasbargen, Klinikum der Univ. München (Germany)
Gary M. Brittenham, Columbia Univ. College of Physicians and Surgeons (United States)

Published in SPIE Proceedings Vol. 8951:
Optical Diagnostics and Sensing XIV: Toward Point-of-Care Diagnostics
Gerard L. Coté, Editor(s)

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