ALA Fluorescence-Guided Resection of High-Grade Glioma: Dye Can See, What Eye Cannot See!

Key Message: Fluorescence-guided resection (FGR) using 5-ALA serves as a “chemical neuronavigator”, enabling maximal safe resection of high-grade gliomas. When combined with intraoperative adjuncts, FGR ensures oncofunctional balance, making it a pivotal advancement in glioma surgery. Gliomas are the most common primary malignant tumors of the central nervous system. High-grade gliomas (HGG) pose challenges to surgeons due to their aggressive and invasive tumor characteristics. The standard treatment involves surgical resection followed by adjuvant therapy. Whenever possible, achieving maximal safe resection improves overall survival, as it removes the tumor’s proliferative and potentially therapy-resistant cells.[1] However, accurately delineating tumor boundaries during surgery is challenging because diffusely infiltrating tumor cells often resemble normal brain tissue to the naked eye, even if visualization is enhanced by illumination and magnification using a microscope. Various intraoperative adjuncts facilitate precise resection, thereby helping to obtain optimal EOR. Standard intraoperative imaging methods, such as intraoperative ultrasound (iOUS), magnetic resonance imaging (MRI), and computed tomography (CT), can provide tomographic imaging[2] Alternative approaches include intraoperative optical imaging strategies, such as fluorescence-guided surgery, Raman spectroscopy, and optical coherence tomography. These are essentially surface visualization techniques and can complement the previous imaging techniques which are tomographic (cross sectional) imaging techniques [Figure 1]. When a patient ingests 5-ALA, it is converted to protoporphyrin IX (PPIX), which preferentially accumulates in the mitochondria of glioma cells. PPIX is a fluorophore being excited by light in the blue wavelength to emit fluorescence in the red spectrum, which can be visualized using a suitably modified microscope. A 5-ALA-FGR helps delineate tumor boundaries beyond the contrast-enhanced regions, providing real-time feedback during resection.[3] When used with intraoperative neuromonitoring, this technique enables maximal safe resection with minimal postoperative neurological deficits, essential for balancing oncological outcomes with functional preservation.Figure 1: Illustration showing intraoperative surface imaging phenomenon of Fluorescence imaging techniqueObjective In this article, we demonstrate the technique of 5-ALA-FGR in a right temporal high-grade glioma performed with intraoperative neuromonitoring and navigated ultrasound assistance. Clinical Presentation A 32-year-old right-handed male farmer presented with a one-month history of holocranial headaches. Neurological examination was unremarkable. Brain MRI revealed a well-defined solid cystic lesion in the right temporal lobe extending to the right amygdala and hippocampus [Figure 2]. The lesion appeared hypointense on T1-weighted images, with a central T2 hyperintense cystic area and peripheral T2 intermediate signal intensity. The solid component showed peripheral enhancement, with central necrosis, highly suggestive of a high-grade glial tumor. Diffusion tensor imaging (DTI) demonstrated the tumor’s relationship with subcortical white matter fibers, showing medial displacement of the inferior fronto-occipital fasciculus (IFOF) and uncinate fasciculus (UF), along with superior displacement of the superior longitudinal fasciculus-3 (SLF-3).Figure 2: MR Imaging showing well well-defined solid cystic lesion in the right temporal lobe. The lesion is hypointense on T1 (a), central T2 hyperintense cystic area, and peripheral. T2 intermediate signal intensity (b). The solid component showed peripheral enhancement, while the central cystic portion lacked enhancement (c)Surgery Right frontotemporoparietal craniotomy and fluorescence-guided resection (FGR) of right temporal high-grade glioma with intraoperative neuromonitoring and navigated ultrasound. Equipment used Navigated ultrasound system: Brainlab KICK navigation with ultrasound navigation software (BRAINLAB AG, Munich, Germany) coupled with an ultrasound machine (bk5000®; BK Medical, Denmark) using an N13C5 navigated curvilinear probe. Mapping and monitoring: NimEclipse (version 4.2.422) (Medtronics Inc., Minneapolis, MN, USA). Microscope – Zeiss Pentero (Carl Zeiss GmBH) – with BLUE 400 filter ALA dye (Gliolan) Medac GmBH, Germany 20 mg/kg 3–4 hours before induction Anesthesia: General Anesthesia Position: Supine, 45-degree head tilt to the left, fixed on the Mayfield three-pin system, pressure points well padded. Incision: Right frontotemporal question mark. Steps of surgery Pin site block and placement of head pins. Navigation registration and incision marking were performed. Right frontoparietotemporal craniotomy and complete scalp block for intra- and immediate postoperative analgesia. Predurotomy 3D navigated intraoperative ultrasound (3D NUS) delineated the tumor extent. A cortical strip was placed subdurally along the motor cortex, and baseline strip motor evoked potential (MEP) was recorded. Circumferential pial coagulation and dissection of tumor-bearing gyrus with subpial dissection. Using a BLUE filter, viable fluorescent tumor tissue appeared dark pink, red, or orange, while non-fluorescent tissue appeared blue. Dissection was carried out in a plane between fluorescent and non-fluorescent tissue by alternately toggling between the white light and BLUE filter. Pial-to-pial boundary resection was performed in the anteroposterior direction. Superior resection in the subcortical plane deep to the insula was assisted by dynamic subcortical mapping using a monopolar suction device. En-masse removal of the specimen. Residual fluorescent tissue was checked and excised as needed. Post resection 3DNUS was conducted to confirm the resection status. Link for video https://youtu.be/1qxT_e_oh0Q QR Code Video timeline with audio transcript 00.00 to 00.36 – In this video, we describe the technique of ALA-based fluorescence-guided resections in malignant gliomas, highlighting the nuances and practical tips. A young male presented with a history of headaches and no neurological deficits. The MRI showed a solid cystic necrotic peripherally enhancing mass in the right temporal lobe with the deep white matter fiber tracts being displaced medially. 00.37 to 01.19 – He was planned for an ALA-guided resection using intraoperative monitoring with MEPs. In the supine position with the head turned, a question mark temporal flap was marked under navigation guidance. After completing the craniotomy, a predurotomy intraoperative ultrasound was acquired and combined with navigation to confirm the location of the lesion and also for subsequent resection control. The dura was opened based on the Sylvian fissure. 01.20 to 01.51 – As you can see, there was no obvious telltale sign of a tumor and the ultrasound helped localize the cyst if needed for aspiration in case, the brain was bulging, which in this case it was not. Doppler power angiography intraoperatively is useful in delineating the important sylvian veins and arteries at the base of the tumor, helping gauge its relationship to the deep tumor boundaries. 01.52 to 02.41 – The tumor-bearing gyrus in the middle and inferior temporal gyri was then coagulated to uncap the tumor, which as you can see now under the blue filter is evidently fluorescing pink. The idea is to do a circumferential resection from subpial dissection, including all fluorescing tissue as is seen in this section of the video. In the tumor, the high-grade component generally is strongly fluorescing dark pink or red and orange, whereas the peripheral infiltrating zone becomes gradually more faint. 02.42 to 3.14 – A subpial resection ensures that all tumor-bearing tissue, even if it is non-fluorescing and in non-eloquent areas can be removed as is being done in this case. While using fluorescence, one needs to toggle between the white and blue light because anatomical delineation under the blue filter is usually a little suboptimal, though one can adapt to it over a period of time. 03.15 to 03.59 – As you can see, the technique is to identify the peripheral tumor zone right in the beginning. After having delineated the superficial superior and posterior boundaries, the anterior polar extension is more generously resected far beyond the fluorescing tumor because that is completely non-eloquent. Then, it is extended to the base coagulating the basal pia and encircling the entire tumor circumference. 04.00 to 04.46 – Here, one can appreciate in the tumor cavity which has been opened that the necrotic central part is non-viable and non-fluorescing, whereas the fluorescing tumor indicates viable high-grade tumor components. The dissection then carries on in a circumferential manner, trying to achieve en-block resection of the tumor as far as possible. The combination of suction and graduated ultrasonic aspiration in this white matter region is important. 04.47 to 05.34 – Always try to remain at the transition between the fluorescing and non-fluorescing tumor because there are no other anatomical landmarks subcortically. At this point, it is also important to keep in mind the relationship to deep white matter structures, and thereby, subcortical mapping, in this case using a monopolar and dynamic subcortical mapping, is performed in the depths of the tumor where it enters the inferior limiting sulcus of the insula. Continuing the dissection in the different quadrants circumferentially, the tumor is gradually resected. 05.35 to 06.16 – Medially, the tissue over the roof of the temporal horn is removed to achieve an en-bloc resection of the tumor. As you can see the tumor specimen shows patchy areas of fluorescence. Following this, the cavity needs to be meticulously inspected including all overhangs to identify residual islands of fluorescence which are then gradually resected. 06.17 to 07.09 – Remember to always respect the eloquent substrates and also keep in mind that sometimes non-fluorescing low-grade infiltrative tumors may still be present and therefore a complementary adjunct as in this case, an ultrasound is also very helpful. The navigated ultrasound in this case confirmed a complete resection of all visible tumors following which hemostasis was achieved and then the dura was closed meticulously in a watertight fashion. An immediate 24-hour postoperative scan confirms the complete resection of contrast-enhancing tumor as well as the flair component around it. 7.10 to 7.41 – Patient is normally kept 24 hours in a shielded room though no practical photosensitivity is observed in our experience. Histopathology was an IDH mutant astrocytoma, and he was subjected to a Stupp regimen and followed up with a three-month scan which showed that there were stable changes in the operative cavity. Thank you. Outcome Postoperatively, the patient exhibited no neurological deficits and was monitored in an isolation room for 24 hours (away from direct sunlight or red/yellow light). MRI showed complete resection of the contrast enhancement and surrounding T2/FLAIR tumor. He was discharged on the 4th postoperative day. Histopathological analysis confirmed IDH R132H mutant astrocytoma (WHO Grade 4). The patient’s treatment plan included radiotherapy and concurrent and adjuvant chemotherapy (Stupp protocol). Pearls and Pitfalls While FGR is very useful, to ensure the technique’s safety and effectiveness, necessary precautions must be taken throughout the preoperative, intraoperative, and postoperative stages. Preoperative precautions Appropriate Case Selection: Ensure the patient is suitable for ALA-FGR. It generally is best for enhancing HGG. Exclude patients with porphyria or other contraindications to ALA. Discontinue the use of drugs such as tetracyclines, fluoroquinolones, or other porphyrogenic medications. Logistics check: Coordinate with the nursing team and anesthesiologist to ensure the operating theater is available and prepared. Verify the microscope filter and light source, it is recommended to use a new lamp with optimal luminosity. This is essential as once administered it is preferable to complete surgery within a stipulated time. Fluorescence is best seen within 6 to 8 hours of the administration of the dye. Administer 20 mg/kg of ALA orally at least 3 hours before induction. Aim to expose the tumor between 4 and 6 hours after ALA administration. The window can be extended to 8–10 hours if necessary. Intraoperative precautions Reduce operating theater lighting to the lowest level necessary. Plan the incision and draping carefully, utilizing navigation and other adjuncts as needed. Switch between white and blue light filters as required throughout the procedure. Keep white light at the minimum level needed to allow the surgeon’s eyes to adjust when toggling between filters. Rely primarily on the image through the microscope eyepiece rather than the screen for accuracy. Avoid bleeding, blind corners, and overhanging edges to reduce the chance of false negatives. Blood clots and hemostatic agents may obscure fluorescence; carefully remove this layer-by-layer to expose any missed fluorescence. Use patties to cover areas outside the working field to prevent photo-bleaching (loss of fluorescence due to prolonged exposure to excitation light) and photosensitization (potential toxicity to normal brain tissue). Clinical experience suggests these issues are generally of minimal concern. Incorporate anatomical (IOUS) and functional tools (IONM) as appropriate. Follow the fluorescence signal carefully, but do not pursue it injudiciously. Respect functional boundaries. Postoperative precautions Protect the patient from direct sun/yellow light for 24 hours. Follow standard postoperative management and imaging protocols. (RANO 2.0 guidelines).[4] Discussion The extent of resection (EOR) is one of the most important modifiable prognostic factors in HGGs. However, achieving this maximal resection is fraught with challenges. HGG often extend microscopically beyond the visible tumor margin, making it difficult intraoperatively for neurosurgeons to discern tumor tissue from healthy brain tissue using standard techniques. More recently, it has been amply demonstrated that the T2-FLAIR tumor regions should also be resected to extend the prognostic benefit.[5] This is where FGR has garnered significant attention. Fluorescence-guided surgery was first described in 1948 by Moore et al.[6] using fluorescein. Since then, numerous fluorophores have been described in the literature; however, 5-ALA remains the most widely used exogenous fluorophore proven in a randomized control trial. Stummer et al. compared 5-ALA-guided surgery to conventional white-light surgery in patients with malignant gliomas. The study showed that 5-ALA fluorescence-guided surgery resulted in a significantly higher rate of complete tumor resection compared to the control group (65% vs. 36%). Also, patients in the 5-ALA group had longer progression-free survival (PFS), which correlated with the higher EOR.[2] Aminolevulinic acid (ALA) is an endogenous natural precursor to heme. When large doses are administered exogenously, in glioma cells, the metabolic pathway gets overwhelmed and halts at the level of protoporphyrin IX (PPIX, which is an endogenous fluorophore) due to the relative deficiency in the ferrochelatase enzyme. When excited by blue light (370–440 nm), PPIX emits red fluorescence at wavelengths of 635 and 704 nm. A specially modified operating microscope/visualization device can help visualize this fluorescence.[7] Fluorescence correlates with the grade of the tumor with strong fluorescence showing an exceptionally high positive predictive value (PPV up to 95–100%) for identifying solid, cellular, and mitotically active tumors.[8] As a result, 5-ALA serves as a “chemical neuronavigator” and can even show tumor cells beyond the contrast-enhancing regions permitting radical resections. This dynamic feedback offers an advantage over conventional neuronavigation systems, which are inherently static and may require frequent updates due to brain shifts during surgery. This was seen in our case too. Resecting this, additional tissue has prognostic benefits.[9] In eloquent regions, Della Puppa et al.[10] reported a CRET rate of 74% using ALA with IONM techniques. Although gross total resection was not achieved in 26% of patients, more than 90% resection of tumor was done overall. This highlights the importance of using ALA judiciously, in combination with other available adjuncts, to achieve the best possible neurological outcomes for the patient. For enhanced functional mapping, a monopolar suction device was utilized due to its real-time adaptability in evaluating subcortical pathways. While subdural strip electrodes and MEPs offer a broader functional assessment, their primary role is in monitoring motor function at the cortical level. In contrast, monopolar suction stimulation provides precise subcortical mapping, aiding in the identification of critical motor pathways and facilitating safe tumor resection. This technique enhances maneuverability in detecting eloquent structures within deep white matter tracts. The combined use of these modalities supports both functional preservation and maximal safe resection, ensuring an optimal balance between oncological effectiveness and neurological safety. Despite its significant benefits, some limitations are noted with 5 ALA. Fluorescence may not be uniform across all tumors. The intensity of PPIX fluorescence depends on several factors, including tumor cell density, vascularization, and metabolic activity. Some areas of the tumor may exhibit faint or no fluorescence. ALA-FGR requires the surgeon to switch between white and blue light modes, which can be disruptive to the flow of surgery, sometimes difficult to adapt, and lead to a longer learning curve. Rarely, there may be contraindications to 5-ALA, such as porphyrias precluding its use. Phototoxicity, though theoretically possible, has rarely been reported in clinical experience, including ours. The need for a specific microscope filter is often associated with significant costs. Recently introduced loupe-based visualization systems can mitigate this concern and make the technique more widely used.[11] Conclusion FGR stands as a remarkable advancement in the surgical treatment of high-grade gliomas. By enhancing the surgeon’s ability to distinguish tumor tissue from normal brain parenchyma, this technique has significantly improved the EOR and patient outcomes. As the field of neurosurgery progresses, fluorescence-guided techniques are poised to play a pivotal role in glioma management, bridging the gap between current visualization limitations and the goal of achieving maximal safe resection. Declaration of patient consent A full and detailed consent from the patient/guardian has been taken. The patient’s identity has been adequately anonymized. If anything related to the patient’s identity is shown, adequate consent has been taken from the patient/relative/guardian. The journal will not be responsible for any medico-legal issues arising out of issues related to the patient’s identity or any other issues arising from the public display of the video. Financial support and sponsorship Nil. of are no of