Detection improvement of gliomas in hyperspectral imaging of protoporphyrin IX fluorescence - in vitro comparison of visual identification and machine thresholds.

Background: 5-aminolevulinic acid (5-ALA) - precursor of protoporphyrin IX (PpIX) - is utilized in fluorescence guided surgery (FGS) of high-grade gliomas. PpIX is used to identify traces of glioma during resection. Visual inspection of the fluorescence seems inaccurate in comparison to optic techniques such as hyperspectral imaging (HSI).

Aim: To characterize the limits of PpIX fluorescence detection of (i) visual evaluation and (ii) HSI analysis and to (iii) develop a classification system for visible and non-visible PpIX fluorescence.

Methods: Samples with increasing concentrations (C) of PpIX and non-fluorescent controls were evaluated using a surgical microscope under blue light illumination. Similar samples were imaged with a HSI system tuned to PpIX fluorescence peak wavelength (635 nm) and control (RGB) channels. Samples' intensities were defined, leading to 96 analysed pixels after batching.

Results: Three expert neurosurgeons assessed the PpIX samples (n = 16) and controls (n = 8) with unanimous decisions (ICC = 0.704), resulting in 63% recognition rate, 48% sensitivity, 92% specificity, 92% positive predictive value (PPV) and 47% negative predictive value (NPV). HSI image analysis, comparing mean relative values, resulted in 96%, 100%, 86%, 94%, 100%, respectively. Minimum PpIX concentration detection for experts was 0.6-1.8 μmol/l and HSI's 0.03-0.15 μmol/l.

Conclusions: PpIX concentrations of low-grade gliomas, and those reported on glioblastoma infiltration zones, are below experts' detection threshold. HSI analysis exceeds the performance of expert's visual inspection nearly by 20-fold. Hybrid FGS-HSI systems should be investigated in parallel to long-term outcomes. Described methods are applicable as a standard for calibration, testing and development of subvisual FGS techniques.