## 2 Materials and Methods
### Bias Minimization  
#### 2.3.1 Sources of Bias  
Study surgeons collect tissue and make initial assessments regarding tissue characteristics (likely tumor or unlikely tumor) and fluorescence status, and these assessments are  
Figure 1: Biopsy acquisition from bulk tumor. As soon as surgeons encounter bulk tumor during surgery, they change to blue light, determine whether any fluorescence is visible, and, if so, take a biopsy from fluorescing tissue after taking a neuronavigation screenshot. The biopsy procedure is recorded for later assessment by the review panel. (**a**) Brain with visible bulk meningioma under elevated dura in a patient with tumor in the superior sagittal sinus infiltrating the adjacent brain. Insert shows pointer tip location marking the intended biopsy location. (**b**) Strong fluorescence of tumor under blue light, which is biopsied. (**c**) Neuronavigation screenshot (”F”: footwards; “H”: headward; “A”: anterior; “P”: posterior; “L”: left). all documented in detail. However, unintentional bias cannot be completely ruled out in the unsupervised setting of individual surgeons in the operating room. For instance, while surgeons are interrogating one area with uncertain tissue characteristics (indeterminate tissue) they might encounter a different area in the same field of view with fluorescence which immediately leads them to think that the second area might show tumor. Once fluorescence has been observed, it cannot simply be unseen, and this will influence any further decisions. Secondly, fluorescence is perceived by the surgeon but it is not objectively quantified. Therefore, a study requires mechanisms for ensuring that any fluorescence or lack thereof in the surgeon's perception is objectively verified. Figure 2: Cont. An important aspect of the study is calling out areas of tissue where the surgeon is uncertain about tissue type (likely or unlikely tumor) as \"indeterminate\" tissue. A non-compliant surgeon might simply call out an area of bulk tumor as being \"indeterminate\" in the unsupervised setting to expedite his study activities. If such tissue then also shows fluorescence, this will favor endpoints regarding diagnostic accuracy and the capability of 5-ALA-induced fluorescence for identifying tissue better than the surgeon using conventional light. The same might be true for any clearly normal tissue areas unrelated to tumor and without fluorescence. While interrogating regions of the surgical cavity for residual tumor based on fluorescence (end-of-surgery assessment), the surgeon must initially state that he or she sees no residual tumor under white light. This statement must formally be considered as subjective and therefore requires confirmation by an independent reviewer. A surgeon might unintentionally overlook obvious residual tumor which would then fluoresce and be biopsied, again skewing the study data in favor of 5-ALA. Finally, if a surgeon interrogates a specific area for fluorescence and these findings are recorded, the surgeon is required to take a biopsy from the questionable area. If he or she takes the biopsy from a slightly different area (inadvertently), then obviously such a biopsy would not be a valid representation of the underlying tissue diagnosis or the surgeon's assessment of tissue type (tumor likely or unlikely). Figure 2: Biopsy from “indeterminate” tissue: In this study the surgeon might encounter tissue which appears uncertain (tumor or not, “indeterminate” tissue) during surgery and where fluorescence might be helpful. The location of a respective region is documented by the neuronavigation screenshot. The illumination is changed to blue light to determine whether the tissue fluoresces and a biopsy is then taken. The tissue interrogation procedure is recorded by video. (**a**) In this example a portion of the sagittal sinus infiltrated by meningioma has been resected. The anterior resection margin appears inconclusive as to whether it still contains tumor and is therefore regarded as being indeterminate. The location is documented using neuronavigation.