Double dose of 5-aminolevulinic acid and its effect on protoporphyrin IX accumulation in low-grade glioma

J Neurosurg 137:943–952, 2022

Administration of 5-aminolevulinic acid (5-ALA) does not regularly elicit fluorescence in low-grade glioma (LGG) at currently established doses and timing of administration. One explanation may be differences in blood-brain barrier (BBB) integrity compared to high-grade glioma. The authors hypothesized that for a BBB semipermeable to 5-ALA there might be a relationship between plasma 5-ALA concentration and its movement into the brain. A higher dose would elicit more 5-ALA conversion into protoporphyrin IX (PPIX). The authors present a case series of patients harboring LGG who received higher doses of 5-ALA.

METHODS Patients undergoing surgery for indeterminate glioma later diagnosed as LGG were included in this study. 5-ALA was administered at a standard dose of 20 mg/kg body weight (bw) 4 hours prior to induction of anesthesia. A subgroup of patients received a higher dose of 40 mg/kg bw. Fluorescence was evaluated visually and PPIX concentration (cPPIX) was determined ex vivo by hyperspectral measurements in freshly extracted tissue. All adverse events were recorded.

RESULTS A total of 23 patients harboring diffuse low-grade astrocytomas (n = 19) and oligodendrogliomas (n = 4) were analyzed. Thirteen patients received 20 mg/kg bw, and 10 patients received 40 mg/kg bw of 5-ALA. In the 20 mg/ kg group, 30.8% (4 of 13) of tumors harbored areas of visible fluorescence, compared to 60% of cases (n = 6 of 10) with 40 mg/kg bw. The threshold to visibility was 1 μg/ml in both groups. Measured over all biopsies, the mean cPPIX was significantly higher in the double-dose group (1.8 vs 0.45 μg/ml; p < 0.001). In non–visibly fluorescent tissue the mean cPPIX was 0.146 μg/ml in the 20 mg/kg and 0.347 μg/ml in the 40 mg/kg group, indicating an increase of 138% (p < 0.001).

CONCLUSIONS These observations demonstrate different regions with different levels of PPIX accumulation in LGG. With higher 5-ALA doses cPPIX increases, leading to more regions surpassing the visibility threshold of 1 μg/ml. These observations can be explained by the fact that the BBB in LGG is semipermeable to 5-ALA. Higher 5-ALA doses result in more PPIX conversion, an observation with implications for future dosing in LGG.

Red-light excitation of protoporphyrin IX fluorescence for subsurface tumor detection

J Neurosurg 128:1690–1697, 2018

The objective of this study was to detect 5-aminolevulinic acid (ALA)-induced tumor fluorescence from glioma below the surface of the surgical field by using red-light illumination.

METHODS To overcome the shallow tissue penetration of blue light, which maximally excites the ALA-induced fluorophore protoporphyrin IX (PpIX) but is also strongly absorbed by hemoglobin and oxyhemoglobin, a system was developed to illuminate the surgical field with red light (620–640 nm) matching a secondary, smaller absorption peak of PpIX and detecting the fluorescence emission through a 650-nm longpass filter. This wide-field spectroscopic imaging system was used in conjunction with conventional blue-light fluorescence for comparison in 29 patients undergoing craniotomy for resection of high-grade glioma, low-grade glioma, meningioma, or metastasis.

RESULTS Although, as expected, red-light excitation is less sensitive to PpIX in exposed tumor, it did reveal tumor at a depth up to 5 mm below the resection bed in 22 of 24 patients who also exhibited PpIX fluorescence under blue-light excitation during the course of surgery.

CONCLUSIONS Red-light excitation of tumor-associated PpIX fluorescence below the surface of the surgical field can be achieved intraoperatively and enables detection of subsurface tumor that is not visualized under conventional bluelight excitation. Clinical trial registration no.: NCT02191488 (

5-Aminolevulinic acid-induced protoporphyrin IX fluorescence as immediate intraoperative indicator to improve the safety of malignant or high-grade brain tumor diagnosis in frameless stereotactic biopsies

Acta Neurochir (2012) 154:585–588. DOI 10.1007/s00701-012-1290-8

Frameless stereotactic biopsies are replacing frame-based stereotaxy as a diagnostic approach to brain lesions. In order to avoid a sampling bias or negative histology, multiple specimens are often taken. This in turn increases the risk of hemorrhagic complications.

Objective We present the use of 5-aminolevulinic acid (5- ALA)-induced protoporphyrin IX fluorescence in frameless stereotaxy to improve the procedure duration and yield, and thereby reduce the risk of complications.

Methods Patients with suspected high-grade brain tumors are given 5-ALA 4 h prior to stereotactic biopsy. The biopsy needle is guided to the target using frameless stereotaxy based either on preoperative images or combined with intraoperative MRI sequences. The specimen is illuminated with blue light to look for fluorescence. In case of a positive fluorescence within the tissue sample, no frozen sections are obtained, and no further specimens are taken.

Results The samples of 13 patients revealed a positive fluorescence and were histologically confirmed as malignant or high-grade brain neoplasms. four cases were fluorescence-negative, requiring frozen section confirmation and/or multiple samples. In theses cases histology was either nonspecific gliotic changes or low-grade tumors. There were no complications related to the additional use of 5-ALA.

Conclusion 5-ALA fluorescence in stereotactic biopsies can increase the safety and accuracy of these procedures by reducing sampling errors and eliminating the need for multiple samples and/or frozen section verification, creating a more accurate, faster and safer procedure for cases of suspected malignant or high-grade brain tumors situated in deep or eloquent areas.

Delta-aminolevulinic acid–induced protoporphyrin IX concentration correlates with histopathologic markers of malignancy in human gliomas: the need for quantitative fluorescence-guided resection to identify regions of increasing malignancy

Neuro-Oncology 13(8):846–856, 2011. doi:10.1093/neuonc/nor086

Extent of resection is a major goal and prognostic factor in the treatment of gliomas. In this study we evaluate whether quantitative ex vivo tissue measurements of d-aminolevulinic acid–induced protoporphyrin IX (PpIX) identify regions of increasing malignancy in low- and high-grade gliomas beyond the capabilities of current fluorescence imaging in patients undergoing fluorescence-guided resection (FGR).

Surgical specimens were collected from 133 biopsies in 23 patients and processed for ex vivo neuropathological analysis: PpIX fluorimetry to measure PpIX concentrations (CPpIX) and Ki-67 immunohistochemistry to assess tissue proliferation. Samples displaying visible levels of fluorescence showed significantly higher levels of CPpIX and tissue proliferation. CPpIX was strongly correlated with histopathological score (nonparametric) and tissue proliferation (parametric), such that increasing levels of CPpIX were identified with regions of increasing malignancy. Furthermore, a large percentage of tumor-positive biopsy sites (∼40%) that were not visibly fluorescent under the operating microscope had levels of CPpIX greater than 0.1 mg/mL, which indicates that significant PpIX accumulation exists below the detection threshold of current fluorescence imaging.

Although PpIX fluorescence is recognized as a visual biomarker for neurosurgical resection guidance, these data show that it is quantitatively related at the microscopic level to increasing malignancy in both low- and high-grade gliomas. This work suggests a need for improved PpIX fluorescence detection technologies to achieve better sensitivity and quantification of PpIX in tissue during surgery.

Coregistered fluorescence-enhanced tumor resection of malignant glioma: relationships between δ-aminolevulinic acid–induced protoporphyrin IX fluorescence, magnetic resonance imaging enhancement, and neuropathological parameters

J Neurosurg 114:595–603,2011.DOI: 10.3171/2010.2.JNS091322.

The aim of this study was to investigate the relationships between intraoperative fluorescence, features on MR imaging, and neuropathological parameters in 11 cases of newly diagnosed glioblastoma multiforme (GBM) treated using protoporphyrin IX (PpIX) fluorescence-guided resection.

Methods. In 11 patients with a newly diagnosed GBM, δ-aminolevulinic acid (ALA) was administered to enhance endogenous synthesis of the fluorophore PpIX. The patients then underwent fluorescence-guided resection, coregistered with conventional neuronavigational image guidance. Biopsy specimens were collected at different times during surgery and assigned a fluorescence level of 0–3 (0, no fluorescence; 1, low fluorescence; 2, moderate fluorescence; or 3, high fluorescence). Contrast enhancement on MR imaging was quantified using two image metrics: 1) Gd-enhanced signal intensity (GdE) on T1-weighted subtraction MR image volumes, and 2) normalized contrast ratios (nCRs) in T1-weighted, postGd-injection MR image volumes for each biopsy specimen, using the biopsy-specific image-space coordinate transformation provided by the navigation system. Subsequently, each GdE and nCR value was grouped into one of two fluorescence categories, defined by its corresponding biopsy specimen fluorescence assessment as negative fluorescence (fluorescence level 0) or positive fluorescence (fluorescence level 1, 2, or 3). A single neuropathologist analyzed the H & E–stained tissue slides of each biopsy specimen and measured three neuropathological parameters: 1) histopathological score (0–IV); 2) tumor burden score (0–III); and 3) necrotic burden score (0–III).

Results. Mixed-model analyses with random effects for individuals show a highly statistically significant difference between fluorescing and nonfluorescing tissue in GdE (mean difference 8.33, p = 0.018) and nCRs (mean difference 5.15, p < 0.001). An analysis of association demonstrated a significant relationship between the levels of intraoperative fluorescence and histopathological score (χ2 = 58.8, p < 0.001), between fluorescence levels and tumor burden (χ2 = 42.7, p < 0.001), and between fluorescence levels and necrotic burden (χ2 = 30.9, p < 0.001). The corresponding Spearman rank correlation coefficients were 0.51 (p < 0.001) for fluorescence and histopathological score, and 0.49 (p < 0.001) for fluorescence and tumor burden, suggesting a strongly positive relationship for each of these variables.

Conclusions. These results demonstrate a significant relationship between contrast enhancement on preoperative MR imaging and observable intraoperative PpIX fluorescence. The finding that preoperative MR image signatures are predictive of intraoperative PpIX fluorescence is of practical importance for identifying candidates for the procedure. Furthermore, this study provides evidence that a strong relationship exists between tumor aggressiveness and the degree of tissue fluorescence that is observable intraoperatively, and that observable fluorescence has an excellent positive predictive value but a low negative predictive value.