5-氨基乙内酰胺（5-ALA）显著改善了高级别胶质母细胞瘤的切除手术。然而，对5-ALA的神经生物学特性并不完全了解。具体而言，尽管最近的报告表明免疫细胞可能起到一定作用，但对免疫渗透的潜在差异尚未得出明确的结论。因此，我们在这里提供了对不同5-ALA荧光水平的免疫渗透的系统映射。通过三种方法对肿瘤相关巨噬细胞（CD68，CD163），细胞毒性T细胞（CD8）和调节性T细胞（FoxP3）进行了定量分析。首先，从The Cancer Genome Atlas（TCGA）的172名患者中检查了与5-ALA荧光相关的mRNA表达标记物与免疫标记物之间的相关性。其次，按照经典组织学，对39名高级别胶质母细胞瘤患者的508张染色玻片进行了两名独立评审者的半定量分析，共产生了1016个数据点。第三，采用自动扫描和基于算法的细胞定量方法进行了数字图像分析。来自172名患者的TCGA mRNA数据显示5-ALA标志和免疫标志物之间存在直接且显著的相关性（p < 0.001）。然而，在本研究的39例初次组织学研究中，我们无法证实这一发现，我们发现不同荧光水平中的免疫渗透相当。数字图像分析与标准组织学的结果非常一致。通过对不同5-ALA类别的免疫渗透模式进行映射，我们为5-ALA和胶质母细胞瘤生物学领域增加了基本见解。TCGA数据中的显著相关性未能完全转化为初步组织学数据中的可检测的免疫渗透差异，这需要在更大的队列中进行进一步的研究。© 2023. 作者。

Resection of high-grade gliomas has been considerably improved by 5-aminolevulinic acid (5-ALA). However, not all neurobiological properties of 5-ALA are fully understood. Specifically, potential differences in immune infiltration have not been conclusively examined, despite recent reports that immune cells might play a role. Thus, we here provide a systematic mapping of immune infiltration of different 5-ALA fluorescence levels.Tumor-associated macrophages (CD68, CD163), cytotoxic T cells (CD8), and regulatory T cells (FoxP3) were quantified via three methods. First, data from The Cancer Genome Atlas (TCGA) of 172 patients was examined for correlations between 5-ALA fluorescence-related mRNA expression signatures and immune markers. Second, as classical histology, 508 stained slides from 39 high-grade glioma patients were analysed semi-quantitatively by two independent reviewers, generating 1016 data points. Third, digital image analysis was performed with automated scanning and algorithm-based cell quantification.TCGA mRNA data from 172 patients showed a direct, significant correlation between 5-ALA signatures and immune markers (p < 0.001). However, we were not able to confirm this finding in the here studied initial set of 39 patient histologies where we found a comparable immune infiltration in different fluorescence levels. Digital image analysis correlated excellently with standard histology.With mapping the immune infiltration pattern of different 5-ALA categories, we are adding fundamental basic insights to the field of 5-ALA and glioma biology. The observation that a significant correlation in TCGA data did not fully translate to detectable differences in immune infiltration in first histology data warrants further investigation in larger cohorts.© 2023. The Author(s).