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<p>Maximal safe tumor resection is crucial for the treatment of high-grade gliomas (HGG). 5-aminolevulinic acid (5-ALA)-mediated fluorescence-guided surgery enhances tumor visualization by inducing protoporphyrin IX (PpIX) accumulation. However, current fluorescence-based observation devices lack the sensitivity for detecting tumor cells in low-density infiltrative zones. Hyperspectral imaging (HI) offers a potential solution. In this study, HI-derived PpIX measurements were compared to those obtained from reversed-phase liquid chromatography coupled to mass spectrometry (LC–MS), a method that delivers accurate concentrations. Additionally, we investigated coproporphyrins (Cp) I and III, since they potentially interfere with PpIX determination. Pig brain was used as a surrogate for protocol development and acquisition of comparative HI and LC–MS reference data, which were subsequently used to evaluate the results obtained from 27 biopsies from nine patients undergoing 5-ALA-mediated tumor resection. During sample preparation for LC–MS, 80% PpIX and 45% combined Cp I &amp; III were recovered from brain tissue. For LC–MS quantification of PpIX, accuracy ranged from 98 to 137%, and coefficient of variation was 5–14%, indicating sufficient precision. For HI, the values were 77–121% and 11–31%, respectively. Notably, HI significantly overestimated PpIX concentrations compared to those determined by LC–MS. This study highlights LC–MS as a reliable method for porphyrin quantification and suggests that HI workflows need further optimization for accurate tumor delineation in HGG.</p>

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Integrating mass spectrometry and hyperspectral imaging for protoporphyrin IX detection in malignant glioma tissue

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月4日

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  • Fabio D’Alessandro,
  • Anna Walke,
  • Nora Maren Kiolbassa,
  • Walter Stummer,
  • Simone König,
  • Eric Suero Molina

摘要

Maximal safe tumor resection is crucial for the treatment of high-grade gliomas (HGG). 5-aminolevulinic acid (5-ALA)-mediated fluorescence-guided surgery enhances tumor visualization by inducing protoporphyrin IX (PpIX) accumulation. However, current fluorescence-based observation devices lack the sensitivity for detecting tumor cells in low-density infiltrative zones. Hyperspectral imaging (HI) offers a potential solution. In this study, HI-derived PpIX measurements were compared to those obtained from reversed-phase liquid chromatography coupled to mass spectrometry (LC–MS), a method that delivers accurate concentrations. Additionally, we investigated coproporphyrins (Cp) I and III, since they potentially interfere with PpIX determination. Pig brain was used as a surrogate for protocol development and acquisition of comparative HI and LC–MS reference data, which were subsequently used to evaluate the results obtained from 27 biopsies from nine patients undergoing 5-ALA-mediated tumor resection. During sample preparation for LC–MS, 80% PpIX and 45% combined Cp I & III were recovered from brain tissue. For LC–MS quantification of PpIX, accuracy ranged from 98 to 137%, and coefficient of variation was 5–14%, indicating sufficient precision. For HI, the values were 77–121% and 11–31%, respectively. Notably, HI significantly overestimated PpIX concentrations compared to those determined by LC–MS. This study highlights LC–MS as a reliable method for porphyrin quantification and suggests that HI workflows need further optimization for accurate tumor delineation in HGG.