The pivotal RCTs by Stummer et al (19) and Picart et al (11) established the efficacy of 5-ALA in improving resection rates in newly diagnosed glioblastomas (1). Numerous retrospective multicenter cohorts and single-institution series further demonstrated that 5-ALA guidance significantly increases the likelihood of GTR without compromising patient safety (1). Several additional investigations explored correlations between intraoperative 5-ALA fluorescence, tumor pathology, and patient outcomes, reinforcing its role as a reliable intraoperative tool (11).

Early evidence of fluorescein's utility was provided by small institutional series and a pilot double-blind trial (12), which demonstrated improved resection rates when using a dedicated yellow filter. A subsequent multicenter Phase II trial [(‘FLUOGLIO’, Acerbi et al, (12)] in 2014 reported complete resection rates of 82-83% in glioblastoma surgery. More recently, a large retrospective controlled study of 347 patients compared fluorescein-guided surgery with white-light resection, showing superior EOR and improved survival in the fluorescein group (13). Consistent findings across multiple single-center series, several synthesized in a 2021 meta-analysis, further confirmed that FGS is a safe and effective technique for maximizing tumor resection in HGGs (14).

Direct head-to-head comparisons between 5-ALA and fluorescein-guided surgery are limited. From an initial pool of 511 studies, 112 were considered potentially eligible (Fig. 1). After applying all exclusion criteria, 41 studies remained. Further exclusion of studies focusing exclusively on one modality reduced the final dataset to 5 comparative studies (37-41). Across these 5 studies, the mean patient age was 57.9 years. The pooled cohort consisted of 732 patients with HGGs, of whom 503 (68.7%) underwent 5-ALA-guided resection (Group A) and 229 (31.3%) underwent fluorescein-guided resection (Group B) (Table II).

All five studies provided data on neurological outcomes (37-41). Among patients treated with 5-ALA, 70 of 503 (9.5%) developed new neurological deficits, compared with 73 of 229 (9.9%) in the fluorescein group (Table II). The pooled analysis demonstrated a statistically significant difference favoring 5-ALA [(OR=0.88; 95% CI (0.17-4.57); P<0.05)]. However, heterogeneity was substantial [(I²=90%; P=0.88) (Fig. 2A)], and funnel plot analysis indicated publication bias (Fig. 2B).

Information on postoperative KPS ≥70 within 30 days was reported in all five studies (37-41). The pooled results suggested a statistically significant difference between the two groups [OR=49.46; 95% CI (-2.21-101.13); P<0.05], though heterogeneity was extremely high (I²=100%, P=0.06) (Fig. 3A). Funnel plot inspection again revealed evidence of publication bias (Fig. 3B) (Table II).

Data on OS were available from all five studies (37-41). The pooled analysis showed a significant difference in favor of 5-ALA [OR=4.05; 95% CI (1.33-6.77); P<0.05]. Nonetheless, heterogeneity was substantial (I²=98%; P<0.05) (Fig. 4A), and the funnel plot demonstrated publication bias (Fig. 4B; Table II).

Both 5-ALA and fluorescein significantly increase the extent of tumor resection in HGGs compared with conventional white-light microsurgery. The pivotal 2006 ALA-Glioma Study by Stummer et al (19) demonstrated that 5-ALA guidance nearly doubled the rate of complete resection of contrast-enhancing tumor (65% with 5-ALA vs. 36% with white-light, P<0.0001). This finding has been consistently validated in subsequent studies. The multicenter RESECT trial reported GTR rates of 79.1% with 5-ALA versus 47.8% in controls (P=0.0002) (11).

For fluorescein-guided surgery, reported GTR rates are comparably high. A prospective phase II trial in glioblastoma achieved complete resection in 82.6% of patients (12), and a meta-analysis confirmed that fluorescein significantly improves GTR rates compared with non-fluorescent surgery (12,14,16). In a large retrospective study, Schebesch et al (13) showed that fluorescein guidance led to significantly higher GTR rates and greater mean extent of resection than white-light microsurgery.

Direct comparisons between the two modalities suggest broadly equivalent resection outcomes. In the largest comparative study to date, Hansen et al (37) found no significant difference in median extent of resection between 5-ALA-guided and fluorescein-guided groups (96.9 vs. 97.4%; P=0.46) (10). Similarly, the proportion of patients with only microscopic residual tumor (<0.175 cm³) did not differ significantly (29.5 vs. 36.2%; P=0.39) (37). These results suggest that in experienced hands, fluorescein can achieve resection completeness comparable to 5-ALA. Importantly, both techniques outperform conventional microsurgery: A network meta-analysis ranked fluorescence guidance (5-ALA or fluorescein) and intraoperative MRI as superior to white-light surgery for maximizing GTR in HGGs (15). In summary, both agents reliably enable high EOR, with GTR rates frequently exceeding 80%, whereas white-light resection typically achieves ≤50% (1,12).

Maximal resection is a well-established determinant of survival in glioblastoma, and the rationale for fluorescence guidance is to increase GTR rates and thereby improve PFS and OS. The original 5-ALA trial demonstrated a significant improvement in 6-month PFS (41 vs. 21% with white-light surgery) (1), though no significant OS difference was observed, likely due to crossover and subsequent adjuvant therapies. Similarly, the RESECT trial did not show a significant OS benefit (24-month OS: ~30% with 5-ALA vs. 37% in controls, P=0.89), nor a significant PFS difference (11). Notably, in RESECT, both arms achieved higher-than-expected PFS rates (~69-70%), and multivariate analysis identified GTR, not the choice of fluorophore, as the independent predictor of survival (11).

For fluorescein, most evidence derives from non-randomized studies. Schebesch et al (13) reported that fluorescein-guided resection was associated with significantly longer median PFS and OS compared with white-light surgery, with fluorescein use emerging as an independent prognostic factor in multivariate analysis. A network meta-analysis by Naik et al (15) similarly found that fluorescein guidance was associated with improved OS versus no fluorescence, whereas pooled data for 5-ALA did not show a statistically significant OS advantage. However, this apparent difference must be interpreted cautiously, as the fluorescein data come predominantly from observational cohorts subject to selection bias.

Head-to-head comparisons again suggest broadly similar outcomes. In the study by Hansen et al (37), median OS was longer in the fluorescein group (19.7 months) compared with the 5-ALA group (14.8 months), though this difference did not reach statistical significance (P=0.06). PFS was marginally higher with fluorescein (9.2 vs. 8.7 months, P=0.03) (37), but the retrospective design limits firm conclusions.

Taken together, current evidence does not support a definitive survival advantage of one fluorophore over the other. Both 5-ALA and fluorescein contribute to improved PFS and OS indirectly, insofar as they enable higher rates of complete resection. Importantly, neither technique has been associated with increased neurological morbidity; instead, both are intended to facilitate maximal safe resection, which remains the key determinant of long-term outcomes (11).

Oral 5-ALA is generally well tolerated. In the 5-ALA arm of the RESECT trial (88 patients), adverse events related to 5-ALA were rare: Only 4.6% of patients experienced transient photosensitivity reactions and 1.1% had transient elevated liver enzymes (‘hepatic cytolysis’) (19). No significant differences in postoperative Karnofsky performance or neurological deficits were observed between 5-ALA and control groups indicating that fluorescence guidance did not lead to higher neurological risk (11,19). However, 5-ALA does have known side effects and practical precautions. Because it can cause cutaneous photosensitivity for ~24 h, treated patients must avoid direct bright light exposure post-operatively (11). Some patients report mild nausea or hypotension related to 5-ALA ingestion, and very rarely allergic reactions have been noted (though none was severe in reported trials). Overall, the incidence of serious adverse reactions to 5-ALA is low, and no long-term toxicities have been identified with its intermittent use in surgery.

Modern fluorescein-guided neurosurgery typically uses a low-dose regimen (3-5 mg/kg IV) at induction of anesthesia, which has an excellent safety profile. Across numerous series totaling hundreds of patients, there have been no significant adverse reactions attributed to fluorescein at this dosage (12). For instance, in the FLUOGLIO trial (46 patients), no fluorescein-related toxic events were recorded (12). Similarly, Martinez et al (44) reported no complications in 47 patients; importantly, they did not observe any cases of anaphylaxis, hypotension, or end-organ toxicity (14). Older literature on fluorescein (using higher doses of ~20 mg/kg without filtered microscopy) noted rare side effects such as transient skin discoloration, nausea, or very infrequently anaphylactic shock, but such issues have not been reported with the current low-dose, filtered technique. As with 5-ALA, fluorescein guidance has not been associated with increased neurological deficits, in fact, one study noted that the fluorescein-guided group had a slightly shorter operative time and trend toward fewer complications than controls (16). A practical point is that fluorescein will turn the patient's urine bright yellow for ~1 day as it is excreted, but this is harmless.

In terms of surgical safety, both methods are intended to improve the surgeon's ability to resect tumor while avoiding eloquent areas, and neither has shown a detriment to functional outcomes. The field of fluorescence-guided neurosurgery is advancing rapidly, with several emerging technologies poised to expand its utility. One promising area is next-generation fluorophores, particularly near-infrared (NIR) agents operating at 700-1,000 nm, which allow deeper tissue penetration and reduced background autofluorescence, enabling visualization of subcortical tumor (23-25). Another frontier is tumor-targeted molecular probes. Unlike 5-ALA and fluorescein, which mark metabolic activity or BBB disruption, newer probes aim at specific tumor markers. For example, an EGFR-targeted probe (cetuximab-IRDye800) has been tested in EGFR-positive glioblastomas (21,36), while antibodies and peptides against IL13Rα2 and integrins are under early investigation. Dual-modality agents, labeled for both PET and NIR fluorescence, have also been developed, such as GRPR-targeting probes that guide resection of otherwise non-enhancing gliomas (24).

Combined modalities may further enhance resection safety. Integration with intraoperative MRI (26) or quantitative fluorescence analysis has shown promise; for instance, Valdés et al (33) demonstrated that spectroscopic measurement of protoporphyrin can identify residual tumor below visual detection thresholds (27,28). Theranostic approaches are also being explored, including nanoparticles capable of dual imaging and therapy, with some NIR-responsive platforms combining tumor visualization with drug delivery or photothermal ablation (39).

There were also systematic reviews and network meta-analyses using three-way comparison of three modalities (intraoperative MRI with 5 ALA or FGS) leading to greater rates of GTR in HGGs (6,15). However, the present meta-analysis focusing on correct comparative studies may constitute valuable research in this field, as more clearly explicit the real usefulness of these two modalities (5-ALA and FGS) separately.

The present systematic review has several limitations. First, direct head-to-head trials comparing 5-ALA and fluorescein remain scarce. Most included studies differed in patient populations, tumor grades and outcome definitions, resulting in significant heterogeneity and limiting quantitative synthesis. Numerous fluorescein studies were small, single-arm series without control groups, raising the risk of publication bias and overestimation of benefit (17,18,20). Blinding is inherently impossible in this field, introducing observer bias, and surgeon behavior may be influenced by the presence of fluorescence itself.

Second, available data were limited, particularly regarding long-term survival with fluorescein, which is largely inferred from indirect evidence. Most studies emphasized short-term endpoints such as extent of resection or early postoperative outcomes, rather than durable oncological control. Unpublished data, conference abstracts and non-English studies were also excluded, which may omit emerging trial results.

Third, methodological quality varied. While 5-ALA has strong Phase III trial evidence (1), most fluorescein studies remain observational with intermediate to high risk of bias. Although it was attempted to focus on objective MRI-confirmed resection rates, the lack of uniform methodology across studies restricts comparability. Also, according to the fifth edition of the WHO Classification of Tumors of the Central Nervous System, published in 2021, new tumor types and subtypes are introduced, some based on novel diagnostic technologies such as DNA methylome profiling, and these may be helpful for the management of HGMs (45). In addition, gliomas exhibit a broad range of molecular alterations, including gene mutations, amplifications and deletions, that influence tumor development and patient prognosis. Key biomarkers include IDH1/2 mutations, as well as alterations in TP53, ATRX, PTEN and EGFR. Additional important features include 1p/19q codeletion, TERT promoter mutations and CDKN2A/B deletions. Thus, further analysis and data are needed for more accurate results regarding HGMs prognosis (45). Last but not at least, another limitation was that both 5-ALA-guided resection and other FGS methods have limited sensitivity and specificity, including the false-positive and false-negative rates (46).

Despite these limitations, current evidence consistently supports the role of both 5-ALA and fluorescein in enhancing the extent of glioma resection. Each technique has distinct advantages and trade-offs, and both can be applied safely in routine neurosurgical practice. Ongoing comparative trials, standardized outcome reporting, and cost-effectiveness analyses will be crucial to clarify their relative roles and optimize fluorescence-guided strategies in malignant glioma surgery.

In conclusion, in HGG surgery, fluorescence guidance has become an indispensable strategy to maximize tumor resection. 5-ALA and fluorescein each have well-demonstrated benefits in this regard. 5-ALA offers tumor-selective visualization and a robust evidence base, whereas fluorescein provides versatility and cost-effectiveness with comparable resection outcomes. Neurosurgeons should be familiar with both modalities and their respective pros and cons. In practice, usage may be tailored to the clinical scenario and resource setting. Ongoing innovations promise to further refine fluorescence-guided resection, but at present, 5-ALA and fluorescein remain the two cornerstone options that have measurably improved surgical outcomes for patients with malignant gliomas. The ultimate goal is to achieve the maximal safe resection for every patient, a goal that, thanks to these fluorescence techniques, is more attainable than ever before.