Development and validation of a triple-LED surgical loupe device for fluorescence-guided resections with 5-ALA

Eric Suero Molina Dr med, MBA1, Sönke J. Hellwig MD1, Anna Walke MSc1,2, Astrid Jeibmann Dr med3, Herbert Stepp Dr rer biol hum4, and Walter Stummer Dr med1
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  • 1 Department of Neurosurgery, University Hospital of Münster;
  • | 2 Core Unit Proteomics, Interdisciplinary Center for Clinical Research, University of Münster;
  • | 3 Institute of Neuropathology, University Hospital of Münster; and
  • | 4 Laser-Forschungslabor, LIFE Center, University Hospital, LMU Munich, Germany
Open access

OBJECTIVE

Fluorescence-guided resections performed using 5-aminolevulinic acid (5-ALA) have been studied extensively using the BLUE400 system. The authors introduce a triple–light-emitting diode (LED) headlight/loupe device for visualizing fluorescence, and compare this to the BLUE400 gold standard in order to assure similar and not more or less sensitive protoporphyrin-IX visualization.

METHODS

The authors defined the spectral requirements for a triple-LED headlight/loupe device for reproducing the xenon-based BLUE400 module. The system consisted of a white LED (normal surgery), a 409-nm LED for excitation, a 450-nm LED for background illumination, and appropriate observation filters. The prototype’s excitation and emission spectra, illumination and detection intensities, and spot homogeneity were determined. The authors further performed a prospectively randomized and blinded study for fluorescence assessments of fresh, marginal, fluorescing and nonfluorescing tumor samples comparing the LED/loupe device with BLUE400 in patients with malignant glioma treated with 20 mg/kg body weight 5-ALA. Tumor samples were immediately assessed in turn, both with a Kinevo and with a novel triple-LED/loupe device by different surgeons.

RESULTS

Seven triple-LED/loupe devices were analyzed. Illumination intensities in the 409- and 450-nm range were comparable to BLUE400, with high spot homogeneity. Fluorescence intensities measured distally to microscope oculars/loupes were 9.9-fold higher with the loupe device. For validation 26 patients with malignant gliomas with 240 biopsies were analyzed. With BLUE400 results as the reference, sensitivity for reproducing fluorescence findings was 100%, specificity was 95%, positive predictive value was 98%, negative predictive value was 100%, and accuracy was 95%. This study reached its primary aim, with agreement in 226 of 240 (94.2%, 95% CI 0.904–0.968).

CONCLUSIONS

The authors observed only minor differences regarding spectra and illumination intensities during evaluation. Fluorescence intensities available to surgeons were 9.9-fold higher with the loupe device. Importantly, the independent perception of fluorescence achieved using the new system and BLUE400 was statistically equivalent. The authors believe the triple-LED/loupe device to be a useful and safe option for surgeons who prefer loupes to the microscope for resections in appropriate patients.

ABBREVIATIONS

5-ALA = 5-aminolevulinic acid; FGR = fluorescence-guided resection; LED = light-emitting diode; PPIX = protoporphyrin-IX; PPV = positive predictive value.

OBJECTIVE

Fluorescence-guided resections performed using 5-aminolevulinic acid (5-ALA) have been studied extensively using the BLUE400 system. The authors introduce a triple–light-emitting diode (LED) headlight/loupe device for visualizing fluorescence, and compare this to the BLUE400 gold standard in order to assure similar and not more or less sensitive protoporphyrin-IX visualization.

METHODS

The authors defined the spectral requirements for a triple-LED headlight/loupe device for reproducing the xenon-based BLUE400 module. The system consisted of a white LED (normal surgery), a 409-nm LED for excitation, a 450-nm LED for background illumination, and appropriate observation filters. The prototype’s excitation and emission spectra, illumination and detection intensities, and spot homogeneity were determined. The authors further performed a prospectively randomized and blinded study for fluorescence assessments of fresh, marginal, fluorescing and nonfluorescing tumor samples comparing the LED/loupe device with BLUE400 in patients with malignant glioma treated with 20 mg/kg body weight 5-ALA. Tumor samples were immediately assessed in turn, both with a Kinevo and with a novel triple-LED/loupe device by different surgeons.

RESULTS

Seven triple-LED/loupe devices were analyzed. Illumination intensities in the 409- and 450-nm range were comparable to BLUE400, with high spot homogeneity. Fluorescence intensities measured distally to microscope oculars/loupes were 9.9-fold higher with the loupe device. For validation 26 patients with malignant gliomas with 240 biopsies were analyzed. With BLUE400 results as the reference, sensitivity for reproducing fluorescence findings was 100%, specificity was 95%, positive predictive value was 98%, negative predictive value was 100%, and accuracy was 95%. This study reached its primary aim, with agreement in 226 of 240 (94.2%, 95% CI 0.904–0.968).

CONCLUSIONS

The authors observed only minor differences regarding spectra and illumination intensities during evaluation. Fluorescence intensities available to surgeons were 9.9-fold higher with the loupe device. Importantly, the independent perception of fluorescence achieved using the new system and BLUE400 was statistically equivalent. The authors believe the triple-LED/loupe device to be a useful and safe option for surgeons who prefer loupes to the microscope for resections in appropriate patients.

In Brief

Tumor fluorescence has greatly improved the ability of neurosurgeons to resect gliomas, a type of brain tumor. Previously, a certain neurosurgical microscope was necessary for the visualization of fluorescence. Researchers developed a new, easy-to-use device based on a unique combination of light-emitting diodes and special loupe glasses, and tested these in a study on tumor tissue. In their study they confirm equivalent performance compared to the microscope for finding tumor, whereas the overall brightness of fluorescence was superior to the microscope. The new device is an important additional tool for improving glioma surgery.

Fluorescence-guided resections (FGRs) performed using 5-aminolevulinic acid (5-ALA) have expanded the surgical armamentarium for malignant glioma.1 This method is based on the visualization of protoporphyrin IX (PPIX) fluorescence induced by 5-ALA in malignant glioma tissue. The strength of the method is the fact that PPIX fluorescence, which is emitted in the red range (peak 635 nm) can be directly observed during surgery without having to rely on cameras and image processing (e.g., as for indocyanine green–based methods2).

The benefit and safety of 5-ALA–mediated FGR38 as well as a high positive predictive value (PPV) for disclosing tumor9 has been repeatedly demonstrated. In most studies, the same specific filters for exciting and observing fluorescence were used (BLUE400; Carl Zeiss Meditec)4,8 (Fig. 1). The filter combination did not simply excite and detect fluorescence, but rather allowed exploitation of remitted excitation light and autofluorescence for background illumination. Additionally, nontumor-associated autofluorescence and ambient light backscatter in the red range were cloaked by remitted blue light, minimizing unspecific red tissue fluorescence. The original filter specifications have not been significantly modified since their introduction in 1998, and other microscopes using the same technology are available (Leica Microsystems and Haag-Streit Surgical). Limitations include low background brightness or porphyrin fluorescence yields, especially in deep cavities.

FIG. 1.
FIG. 1.

Established BLUE400 (Kinevo) and triple-LED light source normalized fluorescence excitation and background illumination spectra in comparison to the PPIX excitation spectrum, measured from a glioblastoma tissue specimen (black curve). The right-hand graph depicts a detail of the left-hand graph with an enlarged scale to better illustrate the emission of the background illumination at approximately 450 nm. Blue curve (Kinevo excitation): spectrum of Kinevo blue mode illumination measured at the site of the tissue; light brown curve: spectrum measured at the ocular from a white surface after having passed the long-pass 440-nm observation filter. Purple curves depict the emission spectra of the two LED beams, with the 450-nm beam driven with 2 mA (low setting) or 5 mA (high setting), respectively. Note overlap of both curves at the 409-nm LED emission band. PpIX-exc = PPIX excitation. Figure is available in color online only.

However, given that not only the efficacy but also the safety of 5-ALA was demonstrated initially by using BLUE400 or comparable systems, new devices with higher fluorescence yields would need to qualitatively provide discrimination similar to the established system. Devices more sensitive for fluorescence would be at risk for reduced specificity, and would have to be tested for equivalence of fluorescence discrimination. Such a validation study has previously been performed on a novel filter system10 based on earlier recommendations.9 Optimally, next-generation devices should have greater fluorescence brightness while maintaining an equivalent contrast between tumor fluorescence and background.

Currently, many operations for gliomas are performed using surgical loupes. Despite disadvantages (lower magnification and inferior visualization within deep cavities), advantages are greater speed, maneuverability, and better portability. Thus, we suggest that such a loupe system, which is similar to the surgical microscope in that it provides direct visualization without an intervening video chain, should be adapted with an appropriate excitation light source, mimicking the original illumination and filter characteristics.

In the present study we describe technical requirements for such a system in which multiple light-emitting diode (LED) illumination is used. We have recently educated the industry regarding such requirements, and now provide a technical and clinical validation study. This system is presently being marketed in the United States (Reveal FGS; Designs for Vision, Inc.).

Methods

Triple-LED Illumination/Loupe Device Concept

We aimed to reproduce the spectral properties of excitation light and fluorescence filters used in the BLUE400 system, as previously described1,8 and used in various studies.5,7,1118 This system is based on a xenon light filtered to 375–440 nm (violet-blue) and a small shoulder in the 1% range up to 460 nm to provide blue light background illumination (blue curve, Fig. 1). The observation filter is a 440-nm long-pass filter, overlapping the transmission shoulder of the excitation filter, allowing remitted blue light (light brown curve, Fig. 2) to pass for visualizing nonfluorescent tissue and simultaneously allowing green tissue autofluorescence with a typical peak at 520 nm and the PPIX peak to be visualized. Furthermore, remitted excitation light cloaks unspecific red autofluorescence (see Fig. 3).

FIG. 2.
FIG. 2.

Spectral transmission of filters inserted in the excitation (Exc) and detection (Det) light paths. Exc-1 is mounted in front of the 409-nm LED, Exc-2 is mounted in front of the 450-nm LED. The detection light path comprises two similar filters, together blocking the excitation beam (green curve). The blue curve at 0 transmission, recorded with all filters in series, proves that no light leaks from the excitation into the detection light path. GBM (yellow curve): spectrum obtained from glioblastoma containing porphyrins and using the triple-LED device. Figure is available in color online only.

FIG. 3.
FIG. 3.

A–D: Loupe white LED (A); 450-nm LED only (5-mA setting, B); 409-nm LED only (C); and 450-nm combined with 409-nm LEDs as observed with an iPhone 12 Pro fitted with a 435-nm long-pass filter (D). Inset in panel C: note unspecific reddish autofluorescence at bone margin. Inset in panel D: 450-nm background illumination cloaks unspecific autofluorescence in the red range. E and F: White light (E) and BLUE400 (F) images as recorded using the Kinevo microscope. Figure is available in color online only.

The novel triple-LED/loupe device, in comparison, consists of a white LED that can be toggled to violet-blue light from two separate LEDs, one with 409 ± 7 nm for eliciting PPIX fluorescence, and one with 450 nm (Fig. 4). The 409-nm LED is filtered to not allow a longer wavelength than 420 nm to pass, in order to avoid ill-defined blue excitation light from contaminating the observation light path (Fig. 2). Background illumination is accomplished by adding light from the 450-nm LED to emulate background light provided by BLUE400. This 450-nm LED is equipped with a 475-nm short-pass filter (Fig. 2). Loupes with various magnifications and a working distance of 45 cm were fitted with custom 435-nm long-pass filters, as used in the original system. In addition, the user could toggle between a low and a high setting for the 450-nm LED (2 mA and 5 mA) to enable more or less sensitive PPIX fluorescence detection; e.g., for tumors with lower PPIX accumulation.

FIG. 4.
FIG. 4.

A: Intensity profiles of the 409-nm (black curve) and 450-nm (red curve) beams in 45-cm working distance. Green curve shows the ratio of the 409-nm and 450-nm profiles. All curves in panel A are normalized to 100%. B and C: Contour plots at both wavelengths, normalized to 101%. Figure is available in color online only.

Technical Validation

Light irradiances were measured with a power meter (Fieldmaster, photodiode VIS; Coherent) and respective spectra with a fiber-based spectrometer (Ocean Optics, USB 2000+) using a 50-µm quartz fiber providing a spectral resolution of approximately 1 nm. The spectral sensitivity of the spectrometer was corrected with a calibrated halogen lamp. Filter transmission spectra were recorded using a collimated beam generated from a halogen light source and measured with the same fiber-based spectrometer. Wavelength was calibrated with an Ar-Hg lamp (Ocean Optics) by using the lines at 404.66 nm and 435.83 nm. Homogeneity and overlap of the excitation and background illumination beams at a distance of 45 cm were assessed by scanning a bare fiber in close distance over the illuminated field in 6-mm increments, simultaneously recording backscattered intensities of both beams, represented by the areas under peak.

We tested 7 headlight/loupe systems and a Kinevo (Carl Zeiss Meditec) surgical microscope. Fluorescence intensities of the loupe system available to surgeons in comparison to the microscope were measured by illuminating a fluorescent phantom at the respective working distances (45 cm and 35 cm) with maximum intensity and recording spectra directly from the microscope oculars and behind the loupe telescope, respectively. The phantom was made of silicone with embedded scatterers and dispersed fluorescent powder, with an excitation maximum in the blue and emission peak at 600 nm. Light from the microscope oculars was focused with a lens (7-mm diameter) onto a 400-µm quartz fiber, representing a fixed area of the retina of a potential observer, and detected with the spectrometer. Photon counts of the spectrometer from 630 nm to 640 nm (which are linear to irradiance) were integrated, corrected for integration time, and then compared between BLUE400 (at minimal zoom) and the loupe systems (×2.5 and ×3.5 magnification).

Prospective Clinical Validation Study

Design

To confirm comparable performance of the BLUE400 filter system compared to the triple-LED/loupe device, we designed a prospective, randomized, and blinded study. Multiple fluorescent and nonfluorescent biopsies from viable (nonnecrotic) tumor margins were assessed in turn immediately after their collection, both with a Kinevo and the triple-LED/loupe device. Microscope assessors were unaware of the loupe assessors and vice versa, and all were blinded to intraoperative fluorescence findings.

This study was approved by the institutional review board, and all patients gave informed consent. Given that this study was based on the ex vivo assessments, and all tissue was submitted for pathology, this was a diagnostic study without burden or disadvantages to patients.

Procedure

Patients (n = 26, Table 1) with high-grade glioma received 20 mg/kg body weight Gliolan (medac GmbH) in tap water 4–5 hours prior to surgery. During surgery, a prespecified number of fluorescing and nonfluorescing biopsies as judged with a Kinevo microscope (Carl Zeiss Meditec) were collected for a total of 240 biopsies, with prespecified 10 biopsies per patient. Tissue biopsies that were not evaluable due to drying or fragmentation were replaced.

TABLE 1.

Demographic data in 26 patients with malignant glioma

CharacteristicValue%
Total no. of pts26
Sex
 Male1765%
 Female935%
Age in yrs, mean ± SD60.8 ± 9.5
Histology
 AA; IDH mutant, MGMT positive14%
 GBM; IDH wild type, MGMT positive1350%
 GBM; IDH wild type, MGMT negative1038%
 GBM; IDH mutant, MGMT positive28%
ECoG score
 11662%
 21038%
 300%
Primary tumor1662%
Recurrent tumor1038%

AA = anaplastic astrocytoma; ECoG = electrocorticography; GBM = glioblastoma; pts = patients.

Three surgeons were involved (see Fig. 5): surgeon 1 harvested fluorescing and nonfluorescing tissue biopsies from resected tissue, and surgeons 2 and 3 in turn assessed samples for fluorescence by using either the novel system (Fig. 6) or a Kinevo microscope with BLUE400. Surgeons 2 and 3 were blinded to each other’s results and were unaware of the frequency of fluorescing samples in the original biopsy bin collected by surgeon 1. Surgeon 1 was asked to follow a randomization list of previously defined numbers of fluorescing and nonfluorescing samples to ensure that surgeons 2 and 3 were not able to make assumptions regarding fluorescence in a given patient.

FIG. 5.
FIG. 5.

Prespecified biopsy collection and evaluation algorithm. Surgeons 1, 2, and 3 were unaware of each other’s assessments. Especially regarding nonfluorescing marginal biopsies, sufficient numbers were not always available. The patients’ bins were filled with fluorescing biopsies. Figure is available in color online only.

FIG. 6.
FIG. 6.

Image of the triple-LED/loupe combination. A: Counterclockwise from left, ×2.5 loupes, wireless foot paddle, power pack, and triple-LED device mounted on headband. B: Device toggled to white light. C: Device toggled to 409-nm/480-nm LEDs. Figure is available in color online only.

If surgeon 1 could not find enough evaluable material from fluorescent or nonfluorescent tissue as predefined, he was asked to fill the bin with available samples of the other fluorescence type. Fluorescence was graded as present or absent. If present, fluorescence was also graded according to its qualities (i.e., weak or strong fluorescence).

Biometry

The primary study aim was to determine the level of agreement between biopsy fluorescence status using BLUE400 compared to the triple-LED/loupe device. The aim was to demonstrate an agreement rate of > 90%, tested by a 1-sided exact binominal test with a level of significance set to 2.5%. The 1-sided test problem was defined as H0: p ≤ p0 versus H1: p > p0, with an assumed reference rate p0 = 0.9.

Assuming the level of agreement as > 95% and the independence of samples, 231 samples were calculated as necessary for obtaining a significant result with a power of 80%. With an anticipated 10 samples per patient, 24 patients were planned.

The exact 2-sided 95% confidence interval was calculated according to Clopper-Pearson. The McNemar test was used to test whether there was a tendency for one system to give a different result for fluorescence than the other. Cohen’s kappa coefficient was calculated for the observation of fluorescence with either system, as well as to evaluate subjective fluorescence intensities (i.e., weak or strong). All calculations were performed using commercial software (SPSS, version 25; IBM Corp.).

Results

Technical Evaluation

The absolute excitation irradiance measured at a focal distance of 45 cm from the front face of the 7 triple-LED devices was 6.4 mW/cm2 (SD 1 mW/cm2) at the 409-nm excitation wavelength. The absolute excitation irradiance measured at the same distance was 91 µW/cm2 (SD 7 µW/cm2) at the background illumination wavelength of 450 nm for the high setting (5 mA), and 36 µW/cm2 for the low setting (2 mA). The diameter of the illuminated field was 8.2 cm.

The Kinevo excitation irradiance (working distance 35 cm) was 5.1 mW/cm2 with an illuminated diameter of approximately 9 cm independent of zoom settings. Background illumination irradiance could only be achieved through a color glass filter (GG455; Schott) and was 25.5 µW/cm2. The relative areas under the spectrum of the peak measured with the GG455 filter versus the peak measured through the microscope ocular (with the original long-pass filter in place) were 0.645, resulting in an effective irradiance of background illumination of 43.6 µW/cm2, assuming reflection losses of 10% at the color glass filter surfaces.

In order to compare background illumination irradiances between Kinevo and triple-LED/loupe devices, the different excitation spectra were accounted for. The spectral distributions of a representative triple-LED/loupe device and the Kinevo are depicted in Fig. 1. Spectra were first normalized to the peak intensity and then multiplied with the PPIX excitation spectrum to adjust for the relative efficiencies for PPIX fluorescence excitation of the two systems. Due to the narrower band emission of the 409-nm LED in the loupe device, this was more effective per mW/cm2 irradiance by a factor of 1.14 in exciting PPIX fluorescence. The irradiances of 450-nm background illumination in relation to 409-nm PPIX excitation were therefore as follows: 9.7 × 10−3 for BLUE400, 14.2 × 10−3 for the high setting, and 5.6 × 10−3 for the low setting of the 450-nm LED.

On the other hand, emission light intensities available to the surgeon—normalized to the BLUE400 main observer optics—were 9.9-fold higher for the loupe system equipped with a ×3.5 telescope lens and 12.9-fold for the ×2.5 telescope lens. If the field of view reduction switch at the microscope oculars of the BLUE400 was activated ("−" position), the loupe system was still brighter by a factor of 7.0 (×3.5 telescope) and 9.2 (×2.5 telescope).

Beam profiles and overlaps over the field of view were tested. Figure 2 shows a diagonal scan of the beam profiles at both wavelengths and their ratio. The 2D representations are provided with contour plots, showing sufficient overlap and homogeneity at the working distance of 45 cm; the absolute intensities vary by less than ± 10% over a diameter of 6 cm, and the 409-/450-nm ratio varies even less.

Clinical Performance

With the triple-LED/loupe device, background and tumor fluorescence subjectively appeared much brighter than with BLUE400. The transition zones between fluorescent and adjacent nonfluorescent tissue appeared the same when inspecting the cavity, although there was no objective way of measuring this. Images were captured using either the intrinsic Kinevo camera or a draped Apple iPhone 12 Pro camera custom-fitted with a 435-nm long-pass filter. Images were recorded using the white LED, the 409-nm and the 450-nm LED separately, and then the 409-nm and 450-nm LED combined (Fig. 3). Color hues and fluorescence distributions were similar, as far as could be assessed using the different angles and magnifications. The 450-nm illumination provided no fluorescence, whereas the 409-nm illumination excited PPIX fluorescence, but outside the tumor area also resulted in a greenish-gray background with instances of unspecific red autofluorescence. The combination of both illumination modes gave a very similar color impression compared to BLUE400.

Overall, 240 evaluable biopsies were obtained for clinical validation. In the course of the study it became obvious that the predefined number of nonfluorescent, nonnecrotic biopsies were not available from the extracted tissues in all cases (i.e., from marginal, possibly noninfiltrated brain), and in many cases had to be replaced by fluorescing samples. Furthermore, we found 8% of samples to be unevaluable due to tissue damage; e.g., from drying or fragmentation. Therefore, 2 additional patients were included. The final biopsy sample bin contained 182 fluorescing and 58 nonfluorescing samples, as determined by the microscope BLUE400 assessment, which was the reference standard.

This study reached its primary aim with agreement in 226 of 240 biopsies (94.2%, 95% CI 0.904–0.968). McNemar’s test demonstrated no significant tendency for one method to show a different frequency of fluorescent or nonfluorescent samples (p > 0.99); i.e., the proportion of samples that demonstrated fluorescence, as assessed by this nonparametric test for paired dichotomous variables, was consistent with equivalence.

When using the BLUE400 results as reference, we determined the sensitivity for reproducing fluorescence findings to be 100%, the specificity to be 95%, the PPV to be 98%, the negative predictive value to be 100%, and the overall accuracy to be 95%.

Cohen’s kappa (κ) for interrater reliability for comparing fluorescence, either as a binary outcome (fluorescence, no fluorescence) or as a 3-tiered outcome (no fluorescence, weak fluorescence, strong fluorescence), was determined as κ = 0.841 (95% CI 0.760–0.921), indicating excellent agreement, or as κ = 0.778 (95% CI 0.715–0.841), indicating good agreement.

Discussion

We provide the background and present a technical and clinical validation study of a novel triple-LED headlight/loupe combination device for detecting PPIX fluorescence. The advantage of this system is the appeal to surgeons preferring loupes to the microscope for glioma surgery.

Given that approval of 5-ALA for fluorescence-guided resections was based on tumor selectivity but also neurological safety, we believed it to be important for the system to reproduce the original specifications to ensure a comparable performance, now using LED technology. Any improvements beyond this basic requirement (e.g., greater intensities of fluorescence and background light) would be welcome and expected, given the less complex construction and associated light absorption of loupes compared to the microscope.

The technical evaluation of an optical device with respect to a reference system, in which both rely on subjective visual interpretation and in which both systems comprise different light sources and optics, cannot be fully objective and exhaustive. Spectral properties can be measured, but differences will be inevitable, the impact of which can only be judged in real tissue. We have therefore assessed technical performance (i.e., light intensities, spectral blocking, background illumination), but additionally, the clinical comparability of the LED system with the gold standard on freshly resected tissue in an operating room environment.

Technical Comparison

The spectra of the excitation light sources (filtered microscope xenon light, 409-nm LED), as depicted in Fig. 1, show distinct differences. The LED emission with its narrower band revealed a better fit to the PPIX excitation spectrum. Given the completely different types of light sources, the relative difference in PPIX fluorescence excitation efficiency was astonishingly small—only 14%.

Because autofluorescence excitation has a broader peak than PPIX excitation, filtered xenon light will lead to comparably stronger autofluorescence in the green range. Accordingly, the loupe system will show somewhat stronger red PPIX fluorescence and somewhat less green autofluorescence. Together, this might result in a color shift with respect to background light remission. The recognition threshold of the color contrast of red PPIX fluorescence versus greenish-blue background should not be influenced, however.

In order to exclude any uncontrolled emission of the 409-nm LED into the background illumination wavelength band around 450 nm, a short-pass filter was mounted in front of this beam. Filtering the light from the 450-nm LED by another short-pass filter ensures that no light from the headlight illuminates the tissue in the transmission range of the detection filters mounted on the loupes (Fig. 2). This is crucial, because any broadband light shining onto the tissue will be filtered by tissue absorption. Any backscattered red component would thus easily be mistaken as red PPIX fluorescence, which must be avoided.

Fluorescence Brightness

Subjectively, the fluorescence impression was much stronger when using the loupes as compared to the microscope. We quantified the fluorescence available to surgeons by using a fluorescent phantom simulating PPIX in tissue. This measurement indicated that fluorescence conveyed by the loupe systems was brighter by a factor of approximately 10. Stronger fluorescence can be explained by a somewhat greater intensity of 409-nm LED excitation light and its narrow band with optimal excitation, but more obviously by the fact that fluorescence emission passes through the loupes directly to the surgeon’s eyes rather than being split into multiple light paths for cameras or microscope observer oculars.

Necessity for Validation

The distinction between solid tumor, invasive tumor, and adjacent brain during FGR relies on the selectivity of red PPIX fluorescence excited and displayed to the surgeon’s eye by an appropriately equipped microscope. Established microscopes use wide field broadband visible light excitation, optical filters, and a camera for documentation.19 Various studies (as reviewed by Hadjipanayis et al.20) have confirmed the high PPV of red fluorescence as indicating malignant glioma tissue, which is observed beyond what would be expected based on gadolinium enhancement on MRI.21,22

In theory, red autofluorescence and especially ambient light might be mistaken for tumor PPIX fluorescence. The purpose of the specifically adjusted blue background light at 450 nm is to minimize such misinterpretation.23 In general, PPIX accumulation in high-grade glioma is stronger than in low-grade glioma.24 Reducing blue background illumination might increase the sensitivity of PPIX recognition. However, this would probably be associated with decreased specificity and would also require more careful dimming of ambient light. A similar experience has been described by Valdés et al., who used spectrography to increase sensitivity for PPIX, but observed a decrease in specificity.19

Because PPIX accumulation varies among high-grade gliomas, it appeared adequate to account for this by offering a higher and a lower setting for blue background light intensity, by driving the 450-nm LED with a lower (2 mA) or higher (5 mA) current, activated by a foot switch. These two settings compare well with the Kinevo’s background illumination intensity, which is in between both settings, as far as can be determined by a purely spectroscopic analysis and measurements of irradiances with a power meter.

However, due to the influence of blood absorption, tissue scattering, and autofluorescence, all with characteristic wavelength dependencies, a final judgment whether both systems are sufficiently similar and equally well suited for FGR can only be made on real tissue.

We experienced the triple-LED/loupe device on a subjective basis to provide comparable color impression and fluorescence distribution as compared to the microscope. However, because there is no way to record or objectively assess visual impressions from a loupe image, we created a validation study based on fluorescence assessment. Given that our study was a validation of a visual method, it appeared particularly challenging to avoid bias. This led to our study design, with independent assessments of fluorescence from multiple tissue samples either by the microscope or by the loupe system by different assessors acting independently.

This study not only confirmed an almost complete equivalence of the number of samples considered fluorescent overall. Also, by determining Cohen’s kappa coefficient for assessing interobserver results not only for fluorescence on a binary basis but also for assessing fluorescence on a 3-tiered scale (i.e., strong, weak, no fluorescence), results were highly consistent.

Possible Device Advantages and Limitations

With this study we show that fluorescence discrimination achieved using the LED/loupe system appears equivalent to the microscope. However, both devices differ fundamentally regarding their practical use, and a number of general aspects should be discussed that are not being tested here. Individual preferences regarding the use of loupes versus the microscope will also depend on a surgeon’s training and experience.

The LED/loupe combination will allow greater maneuverability for inspecting complex cavities, and is easier to set up (e.g., no draping). With a working distance of 45 cm, the surgeons might have to move in close for deep-seated tumors, and the assistant surgeon would not have the same view of the cavity as when both surgeons are using the microscope. The microscope has superior magnification and variable focus distances. The principal limitation of the loupes in deep-seated tumors might be mitigated by the superior brightness of fluorescence (approximately 10-fold). Other drawbacks of the loupe system are the lack of integrated cameras for documentation, and a smaller illumination diameter. Due to the greater maneuverability of the loupes, we did not find the latter to impair perception.

Illumination intensity using the LED/loupe device was slightly stronger (by 43%), with a narrower spectral bandwidth. Both factors might accelerate PPIX photobleaching. We have previously determined the time necessary for photobleaching to 36% fluorescence as 25.7 minutes (range 15.9–64.7 minutes) by using a greater intensity of 8 mW/cm2 of filtered xenon light.25 Therefore, photobleaching by the triple-LED/loupe devices lies within an acceptable range, with little concern that the loupes would make any noticeable difference.

Regarding stability of light sources, the LED’s power packs have a run time of 4.5 hours each, with light intensity remaining absolutely stable throughout this time, according to the manufacturer. They automatically shut down after issuing a “battery empty” warning. On the other hand, xenon light sources age and lose intensity over time, requiring replacement after approximately 500 hours. Needless to say, the loupe device comes at a lower cost than a fully equipped microscope but may be preferred by surgeons accustomed to using loupes for glioma surgery; by those not yet in possession of a microscope with fluorescence; or as an adjunct—e.g., for the initial phase of surgery, in which magnification is not as important.

Study Limitations

Although this study was designed to reduce bias arising from the test of a visual method, bias could not be completely avoided. The number of biopsies from fluorescing as opposed to nonfluorescing tissue per patient was predefined. However, in the course of the study it became evident that nonfluorescent biopsies (i.e., noninfiltrated brain) were less commonly available due to less nonfluorescent tissue being resected, thus leading to more fluorescent biopsies. Although this would not affect the statistical calculations for accuracy, the statistical power for claims regarding specificity (true negative rate) would be reduced.

Furthermore, we could not completely control for how the harvesting surgeon proceeded with regard to the exact position of the biopsy, and biopsies might have been biased toward more strongly fluorescent tissue. However, the final tissue bins contained 50.0% weakly fluorescing samples as opposed to 24.1% strongly fluorescing tissue (as later assessed by the separate surgeons testing the devices), which supported a harvest site mostly associated with the weakly fluorescing margin.

Finally, the comparison between devices was performed in an optimized ex vivo environment. Although we are confident that our results are representative for surgery, a tumor cavity is complex, with overhanging edges, small corticotomies reducing the field of view, and blood covering fluorescence. These factors might impair discrimination with either method.

Conclusions

Our technical evaluation confirms that our proposed emulation of excitation light and background illumination resulted in only minor differences regarding excitation and emission characteristics. We did, however, observe and measure a much stronger fluorescence and background illumination brightness. Importantly, our clinical validation on freshly extracted tissues confirmed that our interpretation of what was considered as fluorescing was equivalent, based on our predefined study goal. Considering these findings together, we believe the triple-LED/loupe device to be a useful option regarding fluorescence detection for surgeons who prefer loupes to the microscope for surgery in certain brain tumors for various reasons.

Acknowledgments

We acknowledge statistical planning and support by Dr. rer. medic. Raphael Koch, of the Institute of Biostatistics and Clinical Research, University of Münster, Germany. We further acknowledge technical and hardware support by Designs for Vision, Inc.

Disclosures

Walter Stummer reports past consultant and lecture fees from SBI ALA Pharma, NXDC, medac GmbH, and Carl Zeiss Meditec. Herbert Stepp reports past lecture fees from SBI ALA Pharma and research support of his institution by Photonamic AG. None of the authors have any financial relationship whatsoever with Designs for Vision, Inc.

Author Contributions

Conception and design: Stummer, Suero Molina, Stepp. Acquisition of data: Stummer, Suero Molina, Hellwig, Walke. Analysis and interpretation of data: Stummer, Suero Molina, Stepp. Drafting the article: Stummer, Stepp. Critically revising the article: Stummer, Suero Molina, Hellwig, Walke, Stepp. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: Stummer. Statistical analysis: Stummer, Suero Molina. Administrative/technical/material support: all authors. Study supervision: Stummer.

References

  • 1

    Stummer W, Suero Molina E. Fluorescence imaging/agents in tumor resection. Neurosurg Clin N Am. 2017;28(4):569583.

  • 2

    Zeh R, Sheikh S, Xia L, Pierce J, Newton A, Predina J, et al. The second window ICG technique demonstrates a broad plateau period for near infrared fluorescence tumor contrast in glioblastoma. PLoS One. 2017;12(7):e0182034.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 3

    Stummer W, Pichlmeier U, Meinel T, Wiestler OD, Zanella F, Reulen HJ. Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial. Lancet Oncol. 2006;7(5):392401.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 4

    Stummer W, Novotny A, Stepp H, Goetz C, Bise K, Reulen HJ. Fluorescence-guided resection of glioblastoma multiforme by using 5-aminolevulinic acid-induced porphyrins: a prospective study in 52 consecutive patients. J Neurosurg. 2000;93(6):10031013.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 5

    Nabavi A, Thurm H, Zountsas B, Pietsch T, Lanfermann H, Pichlmeier U, Mehdorn M. Five-aminolevulinic acid for fluorescence-guided resection of recurrent malignant gliomas: a phase II study. Neurosurgery. 2009;65(6):10701077.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 6

    Stummer W, Stepp H, Wiestler OD, Pichlmeier U. Randomized, prospective double-blinded study comparing 3 different doses of 5-aminolevulinic acid for fluorescence-guided resections of malignant gliomas. Neurosurgery. 2017;81(2):230239.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 7

    Stummer W, Tonn JC, Goetz C, Ullrich W, Stepp H, Bink A, et al. 5-Aminolevulinic acid-derived tumor fluorescence: the diagnostic accuracy of visible fluorescence qualities as corroborated by spectrometry and histology and postoperative imaging. Neurosurgery. 2014;74(3):310320.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 8

    Stummer W, Stepp H, Möller G, Ehrhardt A, Leonhard M, Reulen HJ. Technical principles for protoporphyrin-IX-fluorescence guided microsurgical resection of malignant glioma tissue. Acta Neurochir (Wien). 1998;140(10):9951000.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9

    Stummer W, Koch R, Valle RD, Roberts DW, Sanai N, Kalkanis S, et al. Intraoperative fluorescence diagnosis in the brain: a systematic review and suggestions for future standards on reporting diagnostic accuracy and clinical utility. Acta Neurochir (Wien). 2019;161(10):20832098.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10

    Suero Molina E, Stögbauer L, Jeibmann A, Warneke N, Stummer W. Validating a new generation filter system for visualizing 5-ALA-induced PpIX fluorescence in malignant glioma surgery: a proof of principle study. Acta Neurochir (Wien). 2020;162(4):785793.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11

    Suero Molina E, Ewelt C, Warneke N, Schwake M, Müther M, Schipmann S, Stummer W. Dual labeling with 5-aminolevulinic acid and fluorescein in high-grade glioma surgery with a prototype filter system built into a neurosurgical microscope: technical note. J Neurosurg. 2020;132(6):17241730.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12

    Hadjipanayis CG, Widhalm G, Stummer W. What is the surgical benefit of utilizing 5-aminolevulinic acid for fluorescence-guided surgery of malignant gliomas?. Neurosurgery. 2015;77(5):663673.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 13

    Sanai N, Snyder LA, Honea NJ, Coons SW, Eschbacher JM, Smith KA, Spetzler RF. Intraoperative confocal microscopy in the visualization of 5-aminolevulinic acid fluorescence in low-grade gliomas. J Neurosurg. 2011;115(4):740748.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14

    Tsugu A, Ishizaka H, Mizokami Y, Osada T, Baba T, Yoshiyama M, et al. Impact of the combination of 5-aminolevulinic acid-induced fluorescence with intraoperative magnetic resonance imaging-guided surgery for glioma. World Neurosurg. 2011;76(1-2):120127.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15

    Widhalm G, Wolfsberger S, Minchev G, Woehrer A, Krssak M, Czech T, et al. 5-Aminolevulinic acid is a promising marker for detection of anaplastic foci in diffusely infiltrating gliomas with nonsignificant contrast enhancement. Cancer. 2010;116(6):15451552.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16

    Feigl GC, Ritz R, Moraes M, Klein J, Ramina K, Gharabaghi A, et al. Resection of malignant brain tumors in eloquent cortical areas: a new multimodal approach combining 5-aminolevulinic acid and intraoperative monitoring. J Neurosurg. 2010;113(2):352357.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17

    Stockhammer F, Misch M, Horn P, Koch A, Fonyuy N, Plotkin M. Association of F18-fluoro-ethyl-tyrosin uptake and 5-aminolevulinic acid-induced fluorescence in gliomas. Acta Neurochir (Wien). 2009;151(11):13771383.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18

    Hefti M, von Campe G, Moschopulos M, Siegner A, Looser H, Landolt H. 5-aminolevulinic acid induced protoporphyrin IX fluorescence in high-grade glioma surgery: a one-year experience at a single institutuion. Swiss Med Wkly. 2008;138(11-12):180185.

    • Search Google Scholar
    • Export Citation
  • 19

    Valdés PA, Leblond F, Kim A, Harris BT, Wilson BC, Fan X, et al. Quantitative fluorescence in intracranial tumor: implications for ALA-induced PpIX as an intraoperative biomarker. J Neurosurg. 2011;115(1):1117.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 20

    Hadjipanayis CG, Stummer W, Sheehan JP. 5-ALA fluorescence-guided surgery of CNS tumors. J Neurooncol. 2019;141(3):477478.

  • 21

    Müther M, Koch R, Weckesser M, Sporns P, Schwindt W, Stummer W. 5-Aminolevulinic acid fluorescence-guided resection of 18F-FET-PET positive tumor beyond gadolinium enhancing tumor improves survival in glioblastoma. Neurosurgery. 2019;85(6):E1020E1029.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 22

    Schucht P, Knittel S, Slotboom J, Seidel K, Murek M, Jilch A, et al. 5-ALA complete resections go beyond MR contrast enhancement: shift corrected volumetric analysis of the extent of resection in surgery for glioblastoma. Acta Neurochir (Wien). 2014;156(2):305312.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23

    Stepp H, Stummer W. 5-ALA in the management of malignant glioma. Lasers Surg Med. 2018;50(5):399419.

  • 24

    Johansson A, Palte G, Schnell O, Tonn JC, Herms J, Stepp H. 5-Aminolevulinic acid-induced protoporphyrin IX levels in tissue of human malignant brain tumors. Photochem Photobiol. 2010;86(6):13731378.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 25

    Stummer W, Stocker S, Wagner S, Stepp H, Fritsch C, Goetz C, et al. Intraoperative detection of malignant gliomas by 5-aminolevulinic acid-induced porphyrin fluorescence. Neurosurgery. 1998;42(3):518526.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation

Images from Minchev et al. (pp 479–488).

  • View in gallery

    Established BLUE400 (Kinevo) and triple-LED light source normalized fluorescence excitation and background illumination spectra in comparison to the PPIX excitation spectrum, measured from a glioblastoma tissue specimen (black curve). The right-hand graph depicts a detail of the left-hand graph with an enlarged scale to better illustrate the emission of the background illumination at approximately 450 nm. Blue curve (Kinevo excitation): spectrum of Kinevo blue mode illumination measured at the site of the tissue; light brown curve: spectrum measured at the ocular from a white surface after having passed the long-pass 440-nm observation filter. Purple curves depict the emission spectra of the two LED beams, with the 450-nm beam driven with 2 mA (low setting) or 5 mA (high setting), respectively. Note overlap of both curves at the 409-nm LED emission band. PpIX-exc = PPIX excitation. Figure is available in color online only.

  • View in gallery

    Spectral transmission of filters inserted in the excitation (Exc) and detection (Det) light paths. Exc-1 is mounted in front of the 409-nm LED, Exc-2 is mounted in front of the 450-nm LED. The detection light path comprises two similar filters, together blocking the excitation beam (green curve). The blue curve at 0 transmission, recorded with all filters in series, proves that no light leaks from the excitation into the detection light path. GBM (yellow curve): spectrum obtained from glioblastoma containing porphyrins and using the triple-LED device. Figure is available in color online only.

  • View in gallery

    A–D: Loupe white LED (A); 450-nm LED only (5-mA setting, B); 409-nm LED only (C); and 450-nm combined with 409-nm LEDs as observed with an iPhone 12 Pro fitted with a 435-nm long-pass filter (D). Inset in panel C: note unspecific reddish autofluorescence at bone margin. Inset in panel D: 450-nm background illumination cloaks unspecific autofluorescence in the red range. E and F: White light (E) and BLUE400 (F) images as recorded using the Kinevo microscope. Figure is available in color online only.

  • View in gallery

    A: Intensity profiles of the 409-nm (black curve) and 450-nm (red curve) beams in 45-cm working distance. Green curve shows the ratio of the 409-nm and 450-nm profiles. All curves in panel A are normalized to 100%. B and C: Contour plots at both wavelengths, normalized to 101%. Figure is available in color online only.

  • View in gallery

    Prespecified biopsy collection and evaluation algorithm. Surgeons 1, 2, and 3 were unaware of each other’s assessments. Especially regarding nonfluorescing marginal biopsies, sufficient numbers were not always available. The patients’ bins were filled with fluorescing biopsies. Figure is available in color online only.

  • View in gallery

    Image of the triple-LED/loupe combination. A: Counterclockwise from left, ×2.5 loupes, wireless foot paddle, power pack, and triple-LED device mounted on headband. B: Device toggled to white light. C: Device toggled to 409-nm/480-nm LEDs. Figure is available in color online only.

  • 1

    Stummer W, Suero Molina E. Fluorescence imaging/agents in tumor resection. Neurosurg Clin N Am. 2017;28(4):569583.

  • 2

    Zeh R, Sheikh S, Xia L, Pierce J, Newton A, Predina J, et al. The second window ICG technique demonstrates a broad plateau period for near infrared fluorescence tumor contrast in glioblastoma. PLoS One. 2017;12(7):e0182034.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 3

    Stummer W, Pichlmeier U, Meinel T, Wiestler OD, Zanella F, Reulen HJ. Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial. Lancet Oncol. 2006;7(5):392401.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 4

    Stummer W, Novotny A, Stepp H, Goetz C, Bise K, Reulen HJ. Fluorescence-guided resection of glioblastoma multiforme by using 5-aminolevulinic acid-induced porphyrins: a prospective study in 52 consecutive patients. J Neurosurg. 2000;93(6):10031013.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 5

    Nabavi A, Thurm H, Zountsas B, Pietsch T, Lanfermann H, Pichlmeier U, Mehdorn M. Five-aminolevulinic acid for fluorescence-guided resection of recurrent malignant gliomas: a phase II study. Neurosurgery. 2009;65(6):10701077.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 6

    Stummer W, Stepp H, Wiestler OD, Pichlmeier U. Randomized, prospective double-blinded study comparing 3 different doses of 5-aminolevulinic acid for fluorescence-guided resections of malignant gliomas. Neurosurgery. 2017;81(2):230239.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 7

    Stummer W, Tonn JC, Goetz C, Ullrich W, Stepp H, Bink A, et al. 5-Aminolevulinic acid-derived tumor fluorescence: the diagnostic accuracy of visible fluorescence qualities as corroborated by spectrometry and histology and postoperative imaging. Neurosurgery. 2014;74(3):310320.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 8

    Stummer W, Stepp H, Möller G, Ehrhardt A, Leonhard M, Reulen HJ. Technical principles for protoporphyrin-IX-fluorescence guided microsurgical resection of malignant glioma tissue. Acta Neurochir (Wien). 1998;140(10):9951000.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9

    Stummer W, Koch R, Valle RD, Roberts DW, Sanai N, Kalkanis S, et al. Intraoperative fluorescence diagnosis in the brain: a systematic review and suggestions for future standards on reporting diagnostic accuracy and clinical utility. Acta Neurochir (Wien). 2019;161(10):20832098.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10

    Suero Molina E, Stögbauer L, Jeibmann A, Warneke N, Stummer W. Validating a new generation filter system for visualizing 5-ALA-induced PpIX fluorescence in malignant glioma surgery: a proof of principle study. Acta Neurochir (Wien). 2020;162(4):785793.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11

    Suero Molina E, Ewelt C, Warneke N, Schwake M, Müther M, Schipmann S, Stummer W. Dual labeling with 5-aminolevulinic acid and fluorescein in high-grade glioma surgery with a prototype filter system built into a neurosurgical microscope: technical note. J Neurosurg. 2020;132(6):17241730.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12

    Hadjipanayis CG, Widhalm G, Stummer W. What is the surgical benefit of utilizing 5-aminolevulinic acid for fluorescence-guided surgery of malignant gliomas?. Neurosurgery. 2015;77(5):663673.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 13

    Sanai N, Snyder LA, Honea NJ, Coons SW, Eschbacher JM, Smith KA, Spetzler RF. Intraoperative confocal microscopy in the visualization of 5-aminolevulinic acid fluorescence in low-grade gliomas. J Neurosurg. 2011;115(4):740748.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14

    Tsugu A, Ishizaka H, Mizokami Y, Osada T, Baba T, Yoshiyama M, et al. Impact of the combination of 5-aminolevulinic acid-induced fluorescence with intraoperative magnetic resonance imaging-guided surgery for glioma. World Neurosurg. 2011;76(1-2):120127.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15

    Widhalm G, Wolfsberger S, Minchev G, Woehrer A, Krssak M, Czech T, et al. 5-Aminolevulinic acid is a promising marker for detection of anaplastic foci in diffusely infiltrating gliomas with nonsignificant contrast enhancement. Cancer. 2010;116(6):15451552.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16

    Feigl GC, Ritz R, Moraes M, Klein J, Ramina K, Gharabaghi A, et al. Resection of malignant brain tumors in eloquent cortical areas: a new multimodal approach combining 5-aminolevulinic acid and intraoperative monitoring. J Neurosurg. 2010;113(2):352357.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17

    Stockhammer F, Misch M, Horn P, Koch A, Fonyuy N, Plotkin M. Association of F18-fluoro-ethyl-tyrosin uptake and 5-aminolevulinic acid-induced fluorescence in gliomas. Acta Neurochir (Wien). 2009;151(11):13771383.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18

    Hefti M, von Campe G, Moschopulos M, Siegner A, Looser H, Landolt H. 5-aminolevulinic acid induced protoporphyrin IX fluorescence in high-grade glioma surgery: a one-year experience at a single institutuion. Swiss Med Wkly. 2008;138(11-12):180185.

    • Search Google Scholar
    • Export Citation
  • 19

    Valdés PA, Leblond F, Kim A, Harris BT, Wilson BC, Fan X, et al. Quantitative fluorescence in intracranial tumor: implications for ALA-induced PpIX as an intraoperative biomarker. J Neurosurg. 2011;115(1):1117.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 20

    Hadjipanayis CG, Stummer W, Sheehan JP. 5-ALA fluorescence-guided surgery of CNS tumors. J Neurooncol. 2019;141(3):477478.

  • 21

    Müther M, Koch R, Weckesser M, Sporns P, Schwindt W, Stummer W. 5-Aminolevulinic acid fluorescence-guided resection of 18F-FET-PET positive tumor beyond gadolinium enhancing tumor improves survival in glioblastoma. Neurosurgery. 2019;85(6):E1020E1029.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 22

    Schucht P, Knittel S, Slotboom J, Seidel K, Murek M, Jilch A, et al. 5-ALA complete resections go beyond MR contrast enhancement: shift corrected volumetric analysis of the extent of resection in surgery for glioblastoma. Acta Neurochir (Wien). 2014;156(2):305312.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23

    Stepp H, Stummer W. 5-ALA in the management of malignant glioma. Lasers Surg Med. 2018;50(5):399419.

  • 24

    Johansson A, Palte G, Schnell O, Tonn JC, Herms J, Stepp H. 5-Aminolevulinic acid-induced protoporphyrin IX levels in tissue of human malignant brain tumors. Photochem Photobiol. 2010;86(6):13731378.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 25

    Stummer W, Stocker S, Wagner S, Stepp H, Fritsch C, Goetz C, et al. Intraoperative detection of malignant gliomas by 5-aminolevulinic acid-induced porphyrin fluorescence. Neurosurgery. 1998;42(3):518526.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation

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